Introduction

disease will develop significant ophthalmic problems. There are, however, indications that smoking tobacco and radioactive iodine treatment for Graves’ hyperthyroidism can increase the risk of ophthalmopathy.

Advances in imaging technology have greatly improved the ability of ophthalmologists to differentiate inflammatory orbital changes associated with Graves’ ophthalmopathy from orbital tumors, and have reduced the need for surgical biopsy to confirm the diagnosis. Ultrasonography, computed tomography, and magnetic resonance imaging each play a specific role in the diagnosis and monitoring of patients with ophthalmopathy, and each modality has specific strengths and limitations. Despite these advances in imaging technology, the diagnosis and management of these patients still depend primarily on a thorough clinical examination.

While the therapy for the hyperthyroidism associated with Graves’ disease is fairly successful, the current treatment options for the ophthalmological manifestations have numerous shortcomings and are frustrating at times for the treating physician. The treatments are mostly supportive, addressing primarily the symptoms of the disease and not its causes. Most patients with Graves’ ophthalmopathy have external eye complaints that are self-limited and usually managed with lubrication and topical therapies.

Patients who develop severe orbital inflammation, proptosis, diplopia, and compressive optic neuropathies require more aggressive therapy. Many ophthalmologists use systemic steroids, either oral or intravenous, for short-term control of the disease. Most patients show some response to the steroids, but a subset of patients either do not respond adequately or have significant side effects that warrant stopping the steroids. There are different options for treatment of these patients, but there is no definite consensus on the most appropriate. Immunosuppressive medications have also been used, some with limited success, and others, like cyclosporine, with potential benefits. Surgical orbital decompression or external beam radiation therapy may be warranted in patients with compressive optic neuropathies to prevent significant loss of vision.

Another important issue in the management of these patients is the control of the sequelae after the inflammation has subsided. The management of diplopia and strabismus secondary to extraocular muscle inflammation or fibrosis can be challenging. The cosmetic effect and corneal exposure resulting from eyelid retraction are often a major concern of these patients and may require surgical correction. In the patient with complex disease, it is often recommended that orbital decompression be performed before proceeding to extraocular muscle surgery. Eyelid surgery is often deferred until both of these other procedures are done.

The aim of this book is to present an updated and comprehensive appraisal of the basic and clinical aspects of Graves’-associated ophthalmopathy. We do not expect to settle all the issues that surround Graves’ disease, but this multidisciplinary review will at least be a step in that direction.

REFERENCES

1.Coday M, Netland P, Dallow R. Thyroid-associated ophthalmopathy (Graves’ disease). In: Albert D, Jakobiec F, Azar D, Gragoudas E, eds. Principles and Practice of Ophthalmology. Philadelphia: WB Saunders, 2000:4742–4759.

2.Duke-Elder S, MacFaul PA. Orbital involvement in general disease. In: Duke-Elder S, ed. System of Ophthalmology, Vol. XIII. St. Louis: CV Mosby, 1974:935–968.

3.Char D. Thyroid Eye Disease, 3rd ed. Boston: Butterworth–Heinemann, 1997.

London, Go¨ttingen, Berlin, Copenhagen, and Edinburgh. So impressive was his fluency and mastery of German that he was taken for a German spy in Austria and imprisoned for a few weeks.

Graves returned to Ireland and was elected physician to the Meath Hospital in Dublin in 1821. Within 2 years, he was elected a Fellow of the Irish College of Physicians. In Dublin, he introduced the continental system of clinical teaching, which required the students to examine patients and write clinical histories rather than rely almost entirely on lectures and book knowledge. Graves also made contributions to our understanding of angioneurotic edema, intermittent pallor of the fingers and toes (a decade before Raynaud), scleroderma, the importance of timing the human pulse with a watch, pinpoint pupils in pontine hemorrhage, and abandoning the practice of bleeding and starving patients with fevers. He was quoted, “Lest when I am gone you may be at a loss for an epitaph for me, let me give you one—he fed fevers.” In 1827, he became King’s Professor of the Institute of Medicine, the first full-time Chair of Medicine in Ireland. His most important work, Clinical Lectures on the Practice of Medicine, was published in 1848. Graves described the disease with which we associate his name in the London Medical and Surgical Journal for Saturday, May 23, 1835 and in his clinical lectures at the Meath Hospital in the 1834–35 session (2,3,6,7).

A lady, aged twenty, became affected with some symptoms which were supposed to be hysterical . . . the eyes assumed a singular appearance, for the eyeballs were apparently enlarged, so that when she slept or tried to shut her eyes, the lids were incapable of closing. When the eyes were open, the white sclerotic could be seen to the breadth of several lines, all around the cornea . . . a tumour, of a horseshoe shape, appeared on the front of the throat and exactly in the situation of the thyroid gland. This was at first soft but soon attained a greater hardness though still elastic (8).

Graves died of cancer of the liver on March 20, 1853, just short of his 57th birthday. Graves’ paper was slow to draw a response but figured prominently in the later publications of his colleague at the Meath Hospital, William Stokes. Graves’ name was first used in association with the disease in 1860 by the French physician Trousseau, a great admirer of Graves’ lectures (2).

III.VON BASEDOW’S DISEASE, BASEDOW’S DISEASE, OR THE MERSEBURG TRIAD

Karl Adolph von Basedow (1799–1854) was born in Dessau to an aristocratic German family (Fig. 4). He studied medicine at the University of Halle. After surgical training in France, he returned to Germany in 1822 to practice in Merseburg, near Leipzig. He practiced as a surgeon and general practitioner and had a strong interest in pathology. His 1840 description of the occurrence of exophthalmic goiter and palpitations in hyperthyroidism is named, in his honor, the Merseburg triad (2,3).

Madame F., brunette, well built, of a decided phlegmatic temperament . . . In the neck there appeared a strumous swelling of the thyroid gland . . . As far as the eyes were concerned, they were pushed out so far that one could see below and above the Cornea, the Albuginea, three lines wide; the eyelids were pushed wide for one another; could not be closed with every effort. The patient slept with the eyes entirely open (9,10).

Figure 4 Karl A. von Basedow (1799–1854). (From Ref. 12.)

Von Basedow recognized that the exophthalmos was due to an increase in tissues behind the eye. He hypothesized that dyscrasia of the blood caused this swelling as well as the goiter (11).

Von Basedow’s paper was considered to be the most comprehensive description of the disease at the time and was accompanied by a thorough review of the literature. His work went ignored for some time. In a publication in the same journal 8 years later, the German physician, Henoch, proclaimed “Nowhere do German physicians mention this striking symptom-complex.” Von Basedow’s contribution was more enthusiastically received to the west, where the French physician Charcot first suggested the name “maladie de Basedow” in 1859 (5). Unfortunately, von Basedow died 5 years before this honor could be bestowed, after contracting typhus during an autopsy.

IV. OTHER EPONYMS

By 1908, Dock (5) had catalogued 21 different appellations, and to this day the name taught to medical students depends greatly upon the location of their school.

Among the names not commonly used today is “morbido di Flajani,” a tribute to the paper published in 1802 by Giuseppe Flajani of Ascoli (12). The name was suggested in the late 1880s by several Italian physicians, claiming that Flajani was the first to publish a case of the later recognized syndrome. Many sources credit Flajani’s paper as describing

exophthalmic goiter. However, in his careful evaluation of the paper, George Dock notes that neither of Flajani’s patients had exophthalmos. One of the two patients had goiter associated with dyspnea, palpitation, and weight loss, but Flajani attributed these to respiratory compromise caused by the tumor and by frequent bleedings. The other patient had goiter with no other symptoms described. It is concluded by Dock that “it does not seem rational to continue to refer to Flajani as a contributor to the knowledge of exophthalmic goiter” (5).

In the early part of the 20th century, the disease was known to some in America as “Parsons’ disease,” as reflected by the use of that title in four American dictionaries of the time. The attribution is to James Parsons (1705–1770) who lived earlier than any of the other physicians associated with this malady. While he was the author of 31 papers, there is no evidence that any of these dealt with thyroid disease (5).

V.CONCLUSION

Parry, Graves, and von Basedow each provided elegant and dramatic descriptions of exophthalmic goiter. Other early 19th-century physicians also may be credited with descriptions. For those who feel that eponyms add to the romance and history of medicine, those associated with Parry, Graves, and von Basedow are equally appropriate.

REFERENCES

1.Poster MF. Thyroid eponymy. N Engl J Med 1973; 288:422.

2.Major RH. Classic Descriptions of Disease with Biographical Sketches of the Authors. 3d ed. Springfield, IL: Charles C Thomas, 1978.

3.Sebastian A. A Dictionary of the History of Medicine. New York: Parthenon Publishing, 1999.

4.Parry CH. Collections from the Unpublished Medical Writings. Vol. II. London: Underwoods, 1825.

5.Dock G. The development of our knowledge of exophthalmic goiter. JAMA 1908; 14:1119– 1125.

6.Taylor S. Graves of Graves’ disease, 1796–1853. J R Coll Physicians London 1986; 20:298– 230.

7.Havard CWH. Medical eponyms updated: 2, Graves’ disease. Br J Clin Pract 1990; 44:409– 410.

8.Graves RJ. Newly observed affection of the thyroid gland in females. London Med Surg J 1835; 7(2):516–517.

9.von Basedow K. Exophthalmos durch hypertrophic des zellgewebes in der augenhohle. Wochens Ges Heilkd 1840; 13:197.

10.von Basedow K. Die glotzaguen. Wochens Ges Heilkd 1848; 49:770.

11.Hennemann G. Historical aspects about the development of our knowledge of morbus Basedow. J Endocrinol Invest 1991; 14:617–624.

12.Rolleston, HD. The Endocrine Organs in Health and Disease with an Historical Review. London: Oxford University Press, 1936.

13.Herrick JB. A Short History of Cardiology. Springfield, IL: Charles C Thomas, 1942.

lish specific relationships as a result of their embryological development. When anatomical variants are encountered, the surgeon who has an understanding of the embryology will also understand the implications of, and ramifications for, other associated structures.

The thyroid gland originates from epithelial cells in the midline of the floor of the pharyngeal anlage´ (9,10). Early on, the pharyngeal anlage´ descends in the midline and divides into two lobes. By around the seventh week, it comes to rest anterior to the trachea below the cricoid cartilage. In the normal course of development, the thyroid gland will weigh about 20 g. It appears soft and uneven in outline. A smaller bridge, the isthmus, connects the two lateral lobes. The isthmus can be palpated over the trachea, between the cricoid cartilage and the sternal notch as a rubbery transverse ridge approximately 1–11/2 cm wide. The lateral lobes are 4–5 cm in height, 1–2 cm in thickness, and 2–3 cm in anterior/posterior width. Abnormalities of thyroid descent result either from a persistence of the thyroglossal pathway (11) or from a failure to descend properly (12). Thyroglossal duct cysts are the most commonly encountered midline neck masses. Ectopic thyroid secondary to nondescent is extremely rare. When it occurs, ectopic thyroid gland can be located anywhere from the foramen cecum (the remnant of the pharyngeal analogue) to the midline of the upper trachea (9,10). Understanding both of these anatomical abnormalities is important when evaluating patients with midline neck masses to ensure that removal of a neck mass thought to be a thyroglossal duct cyst does not result in removal of the entire thyroid gland.

Preservation of parathyroid gland function represents one of the major challenges in thyroid surgery. The incidence of permanent hypoparathyroidism following total thyroidectomy is 2–3% on average (5,7,8). Patients who experience permanent hypoparathyroidism have significant morbidity and require lifelong supplementation with vitamin D and calcium. An understanding of embryology is therefore of the utmost importance, as it determines the anatomical relationship between the parathyroid glands and the thyroid capsule and helps in their identification when operating on the thyroid gland (9,10). The parathyroid bodies arise as endodermal cell proliferations at the lateral tips of the third and fourth pharyngeal pouches. The third pharyngeal pouch gives rise to the third parathyroid and to the thymus. As the fetus develops, the thymus migrates inferiorly to its resting place in the upper mediastinum. Because of its relationship and attachments to the thymus, the third parathyroid descends along with the thymus. Its final resting place can be anywhere from the posterior thyroid capsule to the upper mediastinum. The fourth branchial pouches give rise to the ultimobranchial bodies. These develop into the parafollicular C cells that are eventually incorporated into the lateral lobes of the thyroid gland. The relationship between the parathyroid derived from the fourth branchial arch and the ultimobranchial body is responsible for the close association between the parathyroid and the superior pole of the thyroid gland. This developmental pathway is also responsible for the more consistent location of the superior parathyroid (Fig. 1a, 1b).

The recurrent laryngeal nerves are the other major structures at risk during thyroid surgery (2). Although they do not provide any innervation to the thyroid gland itself or impact on its homeostatic function, their function is integral to the larynx. Injury to the recurrent laryngeal nerves is a major and feared complication of thyroidectomy. Temporary recurrent laryngeal nerve paralysis occurs at a rate of between 0.5 and 3.9%, while the incidence of permanent recurrent laryngeal nerve paralysis varies between 0 and 3% (3–7). An understanding of the embryology and development process of the vagus nerve allows the surgeon to be prepared for the various anatomical abnormalities that may be present.

pulmonary artery, whereas the distal part degenerates. Because the recurrent laryngeal nerves hook around the sixth pair of aortic arches on their way to the developing larynx, their course becomes different on the two sides. On the right side, as the distal part of the right sixth and fifth aortic arches degenerate, the right recurrent laryngeal nerve moves superiorly to hook around the right subclavian artery. By contrast, the left recurrent laryngeal nerve cannot migrate superiorly as it is held by the ductus arteriosus, which becomes the ligamentum arteriosus after birth. Because of these differences, the anatomic relationships of the inferior thyroid artery may be variable. The recurrent laryngeal nerve enters the larynx through the inferior constrictor muscles. A variable branching pattern can be present, with most nerves bifurcating or trifurcating more than 5 mm inferior to the cricoid cartilage (2,3,5,6) (Fig. 2).

Malformations of the aortic arches that result from the persistence of parts of the aortic arches that normally disappear or from the disappearance of parts that normally persist may alter the position of the recurrent laryngeal nerve (11–14). An abnormal origin of the right subclavian artery will cause the right recurrent laryngeal nerve to arise in the neck and pass directly to the larynx (13). The incidence of right nonrecurrent laryngeal nerves varies from 0.35 to 1.5% (13). When this occurs, the nerve can form a deep horizontal notch in the posterior surface of the thyroid gland and become intimately associated with the capsule of the gland. In some cases of situs inversus viscerum, the left recurrent laryngeal nerve may also arise in the neck.

III. SURGICAL ANATOMY AND TECHNIQUE

A. Incision Placement

Concern about the scar associated with thyroidectomy is an issue of primary importance to the patient. This concern has led to descriptions of so-called minimally invasive approaches by which thyroidectomy is performed through a very small incision or in a closed manner using laparoscopic instrumentation (16,17). In reality, a small (3–5 cm) incision, appropriately placed and repaired, results in minimal cosmetic deformity and sensory loss. It affords adequate exposure for safe performance of the operation.

The sensory nerve supply to the skin of the lower neck comes from below and laterally. The lower an incision is placed, the more minimal the sensory deficit will be. We have found that a horizontal incision, centered on the midline and placed at approximately the level of the cricoid in a skin crease with the patient’s head extended, works well. After elevation of the skin flaps, the incision is retracted from side to side to afford adequate exposure. Gland size and the location of the abnormality within the thyroid gland must be considered in incision design rather than using a “one-size-fits-all” approach.

Skin flaps are elevated to the level of the hyoid bone superiorly, sternal notch inferiorly, and to the anterior border of the sternocleidomastoid muscle (SCM) laterally. Elevation beyond these points does not increase exposure; the limiting factor then becomes the strap muscles. The plane of elevation is the superficial layer of the deep cervical fascia, which is best identified by ensuring that one is deep to the platysma laterally and that the anterior jugular veins are deep to the plane of elevation. Sensory innervation to the upper midline skin of the neck is through the sensory branches of the cervical plexus that run over the anterior border of the sternocleidomastoid muscle at approximately the level of the thyroid cartilage notch. Elevation beyond this at the lateral aspects of the incision does not help exposure.

B.Gland Exposure

Although exposure of the thyroid gland is predominantly a “midline” exercise, certain maneuvers can facilitate this. The superficial layer of the deep cervical fascia tethers the lateral attachments of the strap muscle. This restricts the degree to which they can be retracted laterally. We therefore begin by incising the fascia along the anterior border of the SCM from the level of the thyroid notch to the sternum, avoiding the sensory branches that cross superiorly. The sternohyoid muscles are then split in the midline from the level of the hyoid to the sternum. We begin inferiorly where they are more dehiscent. They are elevated off the underlying sternothyroid muscle. The fascia at the lateral border of the sternohyoid is then divided. This circumferentially skeletonizes the muscle and allows it to be retracted laterally with the SCM muscle.

The superior attachment of the sternothyroid muscle to the thyroid cartilage restricts exposure of the superior thyroid pedicle if the muscle is only retracted laterally. Because this places the external branch of the superior laryngeal nerve at risk for injury, we prefer to divide the sternohyoid muscle between clamps, completely incising its medial and lateral fascial attachments and elevating it off the thyroid capsule all the way to its insertion superior in the thyroid cartilage and inferior in the upper sternum. This facilitates superior thyroid pedicle exposure.

This degree of strap muscle elevation usually results in division of the ansa cervicalis/hypoglossi nerve to these muscles as it inserts into the lateral border of the muscles. This has not been found to be a significant problem in terms of voice or swallowing and is considered to be a reasonable trade off for the exposure and safety it affords (18,19). In patients with a long thin neck or a small thyroid gland, a more limited elevation with ansa preservation may be performed (Fig. 3).

The descent of the thyroid gland and its “fusion” to the laryngotracheal complex as it develops means that the gland is tethered superiorly, medially, and posteriorly by fascial attachments. Systematic and appropriate sequential division of these attachments affords the best exposure and delivery of the gland without its surrounding structures such as the parathyroids and recurrent laryngeal nerves. There are three possible fascial planes of dissection when working around the thyroid. The middle layer of the deep cervical fascia, which surrounds the entire thyroid and laryngotracheal complex, represents the first and most lateral plane. While the easiest to work in, it should be avoided, as it will result in the inclusion of the superior and often the inferior parathyroid glands. The recurrent laryngeal nerve and inferior thyroid artery run in this fascia and may be damaged. Another plane, the most medial, is within the thyroid capsule itself. It is also best avoided because it represents a more difficult and vascular plane. The preferred plane of dissection is between these two. This plane is on the thyroid capsule itself but leaves the parathyroids and recurrent nerves behind. It is entered by dividing the superior and inferior venous pedicles of the thyroid at the gland capsule and connecting the openings that this creates. When working in this plane, small venous pedicles must be divided. Generally the inferior thyroid artery and its branches, the parathyroids, and the recurrent laryngeal nerves are easily identified and left undisturbed (20).

C.Superior Pedicle Exposure

After sternothyroid muscle elevation, the larynx is retracted medially and the plane between it and the medial aspect of the superior pole of the thyroid is opened. Working

Figure 3 This anatomical drawing depicts the relationship of the thyroid, parathyroid, and recurrent laryngeal nerve to the major vascular structures that run nearby.

from superior to inferior the fascia between the thyroid and larynx is divided and the external branch of the superior laryngeal nerve is visualized where it crosses from lateral to medial to innervate the cricothyroid muscle. The relationship of this nerve to the superior thyroid pedicle is variable and its identification is necessary prior to division of the pedicle. This is accomplished just inferior to it where it crosses (21–23). After superior pedicle division, the lateral fascial attachments of the thyroid lobe and (if present) the middle thyroid vein are divided immediately adjacent to the capsule.

D. Inferior Pedicle Division

Beginning medially just below the isthmus, the fascia that attaches the thyroid to the trachea is divided. The window thus created in the fascial attachment of the thyroid to the laryngotracheal complex is then expanded laterally, dividing the venous pedicles right on the thyroid capsule itself. The inferior parathyroid gland often sits immediately deep to these veins and derives its venous blood supply from them. These same veins often drain into the upper thymus, reflecting the embryological connection of these two structures (20). If the veins are divided too low and away from the thyroid capsule, the inferior parathyroid gland can be inadvertently removed with the thyroid. Once the inferior aspect of the thyroid lobe is freed up, this plane of dissection is connected to the one created by division of the superior pedicle and middle thyroid vein, and the thyroid lobe is rolled forward to expose its posterior capsule.

thick fibrous band always lies anterior to the nerve (26,27). However, small distal branches of the inferior thyroid artery often run deep to the nerve and can cause troublesome bleeding or nerve injury if cautery is attempted. In addition, in large goiters the suspensory ligament can on occasion be infiltrated by thyroid tissue that extends deep to the nerve, making total thyroidectomy without nerve injury difficult.

With sectioning of the ligament just anterior to the nerve, the thyroid lobe will roll forward and release itself from the trachea. The recurrent laryngeal nerve will fall back as the trachea rotates back into its normal position. Superior parathyroid, if not already identified, can be found superior and lateral to the point of nerve entry into the larynx.

If total thyroidectomy is planned, the above procedure is repeated on the contralateral side. The isthmus and its superior fascial attachments now suspend the gland. These reflect its embryological descent and any residual thyroglossal duct tract. This tract is followed from inferior to superior up to the hyoid bone where it is amputated. Complete dissection of this tract from inferior to superior will ensure removal of any residual thyroid tissue in a pyramidal lobe or thyroglossal duct tract.

IV. CONCLUSION

Familiarity with the embryology and anatomy of the thyroid gland allows for its safe removal and avoidance of complications, regardless of the disease process or anatomical variations that are encountered.

REFERENCES

1.Halsted WS. The operative story of goiter: the author’s operation. Johns Hopkins Hosp Rep 1920; 19:71–257.

2.Steinberg JL, Khane GJ, Fernandes CMC, Nel JP. Anatomy of the recurrent laryngeal nerve: a redescription. J Laryngol Otol 1986; 100:919–927.

3.Martensson H, Terins J. Recurrent laryngeal nerve palsy in thyroid gland surgery related to operations and nerves at risk. Arch Surg 1985; 120:475–477.

4.Riddell V. Thyroidectomy: prevention of bilateral recurrent nerve palsy. Results of identification of the nerve over 23 consecutive years (1946–69) with description of an additional safety measure. Br J Surg 1970; 57:1–11.

5.Karlan MS, Catz B, Dunkelman D, Uyeda RY, Gleischman S. A safe technique for thyroidectomy with complete nerve dissection and parathyroid preservation. Head Neck Surg 1984; 6: 1014–1019.

6.Premachandra DJ, Radcliffe GJ, Stearns MP. Intraoperative identification of the recurrent laryngeal nerve and demonstration of its function. Laryngoscope 1990; 100:94–96.

7.Lore´ JM, Banyas JB, Niemiec ER. Complications of total thyroidectomy. Arch Otolaryngol Head Neck Surg 1987; 113:1238.

8.Schwartz AE, Friedman EW. Preservation of the parathyroid glands in total thyroidectomy. Surg Gynecol Obstet 1987; 165:327–332.

9.Moore KL. The Developing Human: Clinically Oriented Anatomy. 4th ed. Philadelphia: WB Saunders, 1988.

10.Gray SW, Skandalakis JE. Embryology for Surgeons. Philadelphia: WB Saunders, 1972.

11.Haddad A, Frenkiel S, Costom B, Shapiro R, Tewfik T. Management of the undescended thyroid. J Otolaryngol 1986; 15:373–376.

12.Ellis PDM, Van Nostrand AWP. The applied anatomy of thyroglossal tract remnants. Laryngoscope 1977;87:765–770.

13.Mra Z, Wax MK. Nonrecurrent laryngeal nerves: anatomic considerations during thyroid and parathyroid surgery. Am J Otolaryngol 1999; 20:91–95.

14.Sanders G, Uyeda RY, Karlan MS. Nonrecurrent inferior laryngeal nerves and their association with a recurrent branch. Am J Surg 1983; 146:501–503.

15.Henry JF, Audiffret J, Denizot A, Plan M. The nonrecurrent inferior laryngeal nerve: review of 33 cases, including two on the left side. Surgery 1988; 104:977–984.

16.Bellantone R, Lombardi CP, Raffaelli M, Rubino F, Boscherini M, Perilli W. Minimally invasive, totally gasless video-assisted thyroid lobectomy. Am J Surg 1999; 177:342–343.

17.Miccoli P, Berti P, Bendinelli C, Conte M, Fasolini F, Martino E. Minimally invasive videoassisted surgery of the thyroid: a preliminary report. Langenbecks Arch Surg 2000; 385:261– 264.

18.Jaffe V, Young AE. Strap muscles in thyroid surgery: to cut or not to cut? Ann R Coll Surg Engl 1993; 75(2):118.

19.Hong KH, Kim YK. Phonatory characteristics of patients undergoing thyroidectomy without laryngeal nerve injury. Otolaryngol Head Neck Surg 1997; 117:399–404.

20.Gray SW, Skandalakis JE, Akin JT Jr. Embryological considerations of thyroid surgery: developmental anatomy of the thyroid, parathyroids, and the recurrent laryngeal nerve. Am Surgeon 1976; 42(9):621–628.

21.Cernea CR, Ferraz AR, Nishio S, Dutra A Jr, Hojaij FC, dos Santos LR. Surgical anatomy of the external branch of the superior laryngeal nerve. Head Neck 1992; 14:380–383.

22.Teitelbaum BJ, Wenig BL. Superior laryngeal nerve injury from thyroid surgery. Head Neck 1995; 17:36–40.

23.Sun SQ, Chang RW. The superior laryngeal nerve loop and its surgical implications. Surg Radiol Anat 1991; 13:175–180.

24.Moreau S, Goullet de Rugy M, Babin E, Salame E, Delmas P, Valdazo A. The recurrent laryngeal nerve: related vascular anatomy. Laryngoscope 1998; 108:1351–1353.

25.Sato I, Shimada K. Arborization of the inferior laryngeal nerve and internal nerve on the posterior surface of the larynx. Clin Anat 1995; 8:379–387.

26.Sasou S, Nakamura S, Kurihara H. Suspensory ligament of Berry: its relationship to recurrent laryngeal nerve and anatomic examination of 24 autopsies. Head Neck 1998; 20:695–698.

27.Leow CK, Webb AJ. The lateral thyroid ligament of Berry. Int Surg 1998; 83:75–78.

Figure 1 Schematic representation of TSHR. Holoreceptor (left) and two-subunit receptor (right) are shown.

84.5 kilodalton (kDa). It was anticipated before molecular cloning that TSHR would be structurally closely related to other glycoprotein hormone receptors (lutropin receptor [LHR] and follitropin receptor [FSHR]) and would belong to the G-protein-coupled receptor (GPCR) superfamily. The homology search indeed revealed that TSHR together with LHR and FSHR constitute a subgroup in the GPCR superfamily. Thus, the amino (N)- terminal half of TSHR (397 aa, 45.2 kDa) corresponds to the large extracellular domain (ectodomain), a unique feature of the glycoprotein hormone receptor subfamily. The carboxyl (C)-terminal half (346 aa) is made up of transmembrane/cytoplasmic regions, including seven transmembrane segments, three extraand intracellular loops, and a cytoplasmic tail, which is a characteristic of the GPCR superfamily (Fig. 1).

The predicted aa sequence of TSHR shares very high homology among different species (85–90%). TSHR ectodomain is 35–45% homologous to those of LHR and FSHR and the transmembrane/cytoplasmic regions share 70–75% homology with other members of the GPCR superfamily. The middle region of TSHR ectodomain (aa 58–277) comprises nine leucine-rich repeats (LRRs) with a consensus sequence of x(Leu)xx(Thr)xx (Leu)(Thr)x(Leu)(Pro)xx(Ala)(Phe)xx(Leu)xx(Leu)xxx(Leu) (where x is any aa) and is

relatively homologous ( 50%) to LHR and FSHR. LRRs are reported to play a significant role in protein–protein interaction, presumably for TSH binding in case of TSHR (see below). The N- and C-terminal extreme ends of the ectodomain are less conserved and contain two unique insertions (aa 38–45 and 317–366) compared to other glycoprotein hormone receptors.

There are 13 cysteines in the extracellular region of TSHR (11 in the ectodomain and two in the extracellular loops); cysteines are clustered in N- and C-terminal extreme ends of the receptor ectodomain. There are also six potential Asn-linked glycosylation sites ([Asn]x[Ser/Thr], where x is any aa except Pro).

II. TSHR STRUCTURE AND FUNCTION

A. Protein Expression

The expression of functional, conformationally intact full-length TSHR is so far limited to eukaryotic mammalian cells, including Chinese hamster ovary (CHO) cells, COS cells, 293HEK cells, SP2/0 myeloma cells, and others (7). Prokaryotic bacterial cells, eukaryotic insect cells, and in vitro transcription/translation are not adequate for this purpose, presumably because of lack of disulfide formation, glycosylation, and/or correct protein folding in these systems.

The truncated form of the entire ectodomain of recombinant TSHR anchored to cell membrane through either a hydrophobic transmembrane segment, the CD8 transmembrane region, or a glycosylphosphatidylinositol tag, is reported to be expressed as a functional membrane protein (7). Furthermore, the recombinant truncated TSHR ectodomain, similar to TSHR A subunit (see below) rather than the whole ectodomain, can be produced as a soluble, secreted protein, which is highly potent in neutralizing TSHR autoantibodies, although it is unable to bind TSH (7). These recombinant proteins may be valuable for future analysis of TSHR structure and function.

B.Post-Translational Modifications

Like many other membranous and secreted proteins, TSHR protein undergoes a series of post-translational processing events in the endoplasmic reticulum (ER) and the Golgi apparatus, many of which are crucial for cell surface expression of functional TSHR. These modifications include disulfide-bonding, protein-folding, glycosylation, palmitoylation, proteolytic cleavage, and others.

1. Disulfide Bonding and Protein Folding

Thirteen cysteines in the extracellular region of the receptor likely form six disulfide bonds with one orphan cysteine. Two cysteines in the extracellular loops (aa 494 and 569) likely bond with each other. There are three clusters of cysteines in the ectodomain; aa 24, 29, 31, and 41 at the N-terminus of the ectodomain; aa 283, 284, and 301 at the end of A subunit (see below); and aa 390, 398, and 408 at the N-terminus of B subunit. Mutagenesis studies demonstrate that 9 of 13 (aa 41, 283, 284, 301, 390, 398, 408, 494, and 569) reduce or abolish TSH binding (7). Although at present it is unclear which cysteines pair with each other to form disulfide bonds, there must be some disulfide bonding between A and B subunits, presumably between the second and third clusters (7). No doubt that these disulfide bonds significantly contribute to the very complex nature of TSHR ectodomain structure.

In the deduced three-dimensional structural model of TSHR ectodomain–TSH complex estimated from crystallization and x-ray diffraction analysis of ribonuclease inhibitor, another LRR-containing protein, the LRRs of TSHR appear nonglobular and horseshoeshaped, and its concave surface likely interacts with TSH. Binding area appears more extensive than standard protein–protein interaction (8) (Fig. 1).

2. Asn-Linked Glycosylation

TSHR is a glycoprotein with Asn-linked carbohydrates. TSHR holoreceptor can be detected as a 100 and 120 kDa doublet in CHO cells in Western blotting and immunoprecipitation studies. The 100 kDa protein is a precursor with high-mannose-type carbohydrates located in the ER and does not bind TSH. The 120 kDa protein is a mature receptor with complex-type carbohydrates, expressed on the cell surface and is capable of binding to TSH (7,9). TSHR A subunit, which is mainly produced on the cell surface (see below), also has complex-type carbohydrates; 40% of its molecular mass is carbohydrates. These findings are in agreement with the general concept for the processing of glycosylation, that is a dolichol pyrophosphate precursor is first attached to Asn residue of the consensus sequence for Asn-linked glycosylation site in the ER. This is processed to high-mannose-type carbohydrates in the ER and then to a complex type in the Golgi to give rise to structurally and functionally mature glycoprotein (10).

A recent study with CHO-Lec cells shows that the addition of Asn-linked carbohydrates to TSHR in the ER is indispensable for completion of protein folding. Processing of high-mannose-type carbohydrates to a complex type plays a role in intracellular trafficking and cell surface expression of the receptor. Of interest, TSHR with high-mannose- type carbohydrates whose folding is accomplished in the ER is able to bind to TSH, but needs to be processed in the Golgi to be expressed on the cell surface (11), suggesting that complex-type carbohydrates are not necessary for TSH binding. Furthermore, the carbohydrates on TSHR may not be part of the TSH binding site and may not be necessary to maintain the correct folding once TSHR folds correctly, because deglycosylation of native TSHR is reported not to alter the receptor function (12).

Of six potential Asn-linked glycosylation sites, two sites (aa 77 and 113) were originally reported to be critical for cell surface expression of the functional TSHR. However, a recent study clearly demonstrates that any single glycosylation site has no effect on receptor function and expression (13). The data indicate that at least four glycosylation sites are necessary for cell surface expression of the functional receptor. Indeed all six potential Asn-linked glycosylation sites appear to be actually glycosylated.

3. Palmitoylation

As for many members of the G protein-coupled receptor superfamily, TSHR has a cysteine residue in the membrane proximal region of the C-terminal cytoplasmic tail (Cys at aa 699). Palmitic acid is shown to be covalently attached to this by thioesterification, presumably forming the fourth cytoplasmic loop (Fig. 1). The palmitoylation on this residue plays a role in controlling the rate of intracellular trafficking and cell surface expression of TSHR (14).

4. Subunit Formation by Intramolecular Cleavage

Even before molecular cloning, TSHR had been demonstrated to undergo intramolecular cleavage into two subunits: TSH-binding “A subunit” and membrane-spanning “B subunit” (2). This was confirmed by eukaryotic cells expressing recombinant TSHR (6,7).

Two types of mechanisms for cleavage are proposed. The first model proposed by de Bernard et al. (15) is that the primary cleavage site is near aa 300–320, and several additional cleavage (or degradation) sites occur at the N-terminus of the B subunit up to aa370. The second is from Rapoport and his associates (7), whose original thesis involved two cleavage sites (upstream site 1 is between aa 305 and 316; downstream site 2 is around aa 370), resulting in the release of a small peptide called “C peptide.” However, their recent study (16) indicates that the cleavage at upstream site 1 appears to be followed by rapid disintegration of the C-peptide region that stops downstream site 2. Degradation of the N-terminus of the B subunit then reaccelerates to the vicinity of the plasma membrane. Continued degradation would lead to A subunit shedding (see below). In either case, 50 aa insertion can be removed from the subunit structure. The cleavage does not involve a specific amino acid motif and appears to be influenced by tertiary structure near the cleavage sites (for example, 50 aa insertion for upstream site 1) (17). The enzyme involved in and functional significance of the cleavage remains to be determined.

There is evidence to suggest that TSHR on the cell surface sheds A subunit into the circulation (7), following cleavage and reduction by protein disulfide isomerase (15), or disintegration of N-terminus of B subunit (16). The in vivo pathophysiological significance of TSHR ectodomain shedding is at present unclear, although A subunit is reportedly detectable in human sera (7).

C.Ligand Binding Sites

The binding sites for TSH and autoantibodies have been extensively studied with mutagenesis with homologous and nonhomologous substitutions and synthetic peptides. Unfortunately, many data are unconvincing and there still exist substantial amounts of controversy. For example, 200–300 aa have so far been reported to be ligand binding sites (7). Relatively reliable data include the following two findings. First, the binding sites for TSH and autoantibodies are situated in the ectodomain of the receptor and span the entire ectodomain; that is, the sites are very conformational and consist of discontinuous aa sequences

(6). These data are consistent with the hypothetical TSH–TSHR complex model mentioned above, although the contribution of the extracellular loops cannot be completely excluded. The second is that the binding sites for TSH and autoantibodies appear to overlap with, but may not be identical to, each other. Thus, stimulating and blocking autoantibody binding seems to involve the N- and C-termini of the TSHR ectodomain, respectively (6,7). The middle region of the ectodomain, particularly aa 201–211 and 222–230, participates in TSH binding (6–8).

III. STRUCTURE AND EXPRESSION OF TSHR GENE

The human TSHR gene is located on chromosome 14q31 and consists of 10 exons spanning over 60 kb. Exons 1–9 encode the ectodomain and exon 10 encodes the transmembrane/cytoplasmic regions. LRRs 1–7 correspond to exons 2–8, and LRRs 8 and 9 to exon 9. Therefore, it is suggested that exon 10 encodes the prototype GPCR to which nine exons were evolutionarily added to shape the very complex form of TSHR ectodomain.

TSHR mRNAs in human thyroid tissue include major transcripts of 4.6 and 3.9 kb in length and minor ones of 1.8 and 1.2 kb (1). The former is likely the full-length receptor, whereas the latter is identified by molecular cloning as a truncated protein (6).

The extrathyroidal expression of TSHR is of interest because of its possible involve-

ment in the pathogenesis of extrathyroidal manifestations of autoimmune thyroid disease such as Graves’ ophthalmopathy and pretibial myxedema. Detection of TSHR transcripts or protein by Northern blot or immunohistochemical methods, not by reverse transcriptionpolymerase chain reaction (RT-PCR), which may represent illegitimate transcription, has so far been reported in fibroblasts/adipose tissues of not only retro-orbital origin but also abdominal and mammary origin, thymus, and cardiac muscle (3,7).

IV. AUTOIMMUNITY

A.Autoantibodies

TSHR autoantibodies have been categorized into two types: one is the stimulatory type (thyroid-stimulating immunoglobulin [TSI] or antibody [TSAb]), which binds to and stimulates TSHR; and the other is the blocking type, which inhibits TSH binding and/or TSHmediated cAMP synthesis. To detect these autoantibodies, two types of assays have been developed; one is a bioassay for TSI, and the other is a competition of antibodies for radiolabeled TSH binding to TSHR (TSH-binding-inhibiting immunoglobulin [TBII] or antibody [TBIAb]). The TSI bioassay detects stimulating-type antibody, whereas the TBII assay cannot differentiate between stimulating and blocking antibodies. However, because of its simplicity, the TBII assay is now being used as a method for TSHR antibody measurement in most clinical laboratories.

For the TSI assay, although human, porcine, or rat thyroid cells have been long utilized, molecular cloning of hTSHR cDNA made it possible to use eukaryotic mammalian cells stably expressing recombinant hTSHR, whose sensitivity is equivalent or superior to thyroid cells (18). Furthermore, a new chemiluminescent TSI assay has been developed in which cAMP response to TSH and TSI stimulation can be detected by measuring light output in a luminometer in a CHO cell line stably transfected with both TSHR cDNA and cAMP-responsive firefly luciferase gene. This new method can eliminate cAMP measurement by radioimmunoassay (RIA), but awaits further studies to confirm its clinical value.

For the TBII assay, a crude detergent extract of porcine thyroid membrane and highly potent, affinity-purified TSH has long been available as a commercial kit (2). TBII assays using recombinant hTSHR protein have also recently been reported (19,20), one of which is now commercially available (19). Their specificity and sensitivity are reportedly superior to the conventional assay.

In both cases, use of hTSHR is an advantage, although the results obtained with hTSHR are generally closely correlated with those with other species of the receptor, and the poor correlation between TSI and TBII in Graves’ disease is still observed in assays with hTSHR (18).

New assays by which TSHR autoantibodies can be detected by direct binding to TSHR have also been reported with TSHR fused to firefly luciferase (21), TSHR fused to the biotin carboxyl carrier subunit of E. coli (22), purified full-length TSHR (23), and purified truncated TSHR (24). These assays can detect many types of antibodies including TSI, TBII, and “neutral” antibody that show neither stimulatory nor inhibitory activities. A relatively close correlation between the direct binding assay and TBII is reported (22– 24).

Some chimeric TSH/LHRs are reported to be useful to predict patients’ response to antithyroid drug treatment (25).

B.Animal Models

Unlike TG and TPO, the traditional immunological approach, that is, immunization of the animal with soluble TSHR ectodomain in adjuvant, has not worked well to induce TSHR-mediated thyroid autoimmunity. Furthermore, transfer of Graves’ pathology into mice with severe combined immunodeficiency proved inefficient (26).

Recently, however, successful induction of experimental hyperthyroidism has been reported by several laboratories (26). First, immunization of fibroblasts stably expressing TSHR and class II antigen induced TSI, hyperthyroidism, and diffuse goiter in a fraction of the immunized mice (27). However, the hyperplastic glands lack lymphocyte infiltration, which is a characteristic of autoimmune thyroid disease. Of interest, Th2 (pertussis toxin) and Th1 complete Freund adjuvant (CFA) adjuvants increased the incidence of hyperthyroidism up to 50% and delayed the disease onset, respectively (28). Second, DNA immunization of outbred mice (29), not inbred mice, proved valuable for inducing TSHR antibodies in almost all the immunized mice and hyperthyroidism and goiter with lymphocyte infiltration in a fraction of the immunized mice. The infiltrates are CD4 T lymphocytes and B cells, which are characteristic of Th2 humoral immune response. Furthermore, extraocular lesions such as edema, fibrosis, and cellular infiltrate, with resemblance to Graves’ ophthalmopathy, were also seen in the extraocular muscles. Of interest, similar eye signs are observed in inbred mice immunized by passive transfer of T cells from the mice that received DNA vaccination (30). More recently, generation of hyperthyroidism at the higher incidence in a mouse strain by immunization with a syngeneic B lymphoma cell line stably expressing TSHR has been reported (31). A difference in the incidence may be due to higher expression of costimulatory molecules on B cells than fibroblasts, which is crucial for T-cell stimulation.

V. NATURALLY OCCURRING MUTATIONS

It is logical to assume that gain-of-function mutations in any step of the Gs-cAMP cascade can be oncogenic in tissues in which cAMP is a growth factor. This was first verified in growth hormone–secreting pituitary adenomas, followed by other functional endocrine tumors including hyperfunctional thyroid adenomas. The similar constitutively activating mutations were later found in nonautoimmune congenital hyperthyroidism, toxic multinodular goiters, and some thyroid carcinomas (4,5). All the mutations except aa 281 are localized in the transmembrane/cytoplasmic regions, particularly in the third cytoplasmic loop and the sixth transmembrane segment. TSHR is known to display significant constitutive activity even in the absence of agonist. Identification of a constitutively activating mutation in the receptor ectodomain (aa 281) may suggest that the unliganded ectodomain of wt-TSHR appears to constrain the receptor activity. There appears to be a geographical difference in the incidence of toxic adenomas and multinodular goiters.

It is also a reasonable assumption that loss-of-function mutations in any step of the Gs-cAMP cascade can result in hypothyroidism associated with TSH unresponsiveness. Thus, congenital hypothyroidism associated with resistance to TSH was found in homozygotes or compound heterozygotes (different mutations in each allele) with loss-of-function mutations of TSHR (4,5). The mutations can be seen in any region of the receptor.

A unique TSHR mutation (K183R) is also identified in a familial gestational hyperthyroidism, which is reported to be more sensitive to human choriogonadotropin than the wt-receptor (32).

The TSHR mutation data base is now available on the World Wide Web at http:/ / www.uni-leipzig.de/ innere/TSH/frame_en.htm.

REFERENCES

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3.Rapoport B, Alsabeh R, Aftergood D, McLachlan SM. Elephantiasic pretibial myxedema: insight into and a hypothesis regarding the pathogenesis of the extrathyroidal manifestations of Graves’ disease. Thyroid 2000; 10:685–692.

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5.Russo D, Arturi F, Chiefari E, Filetti S. Molecular insights into TSH receptor abnormality and thyroid disease. J Endocrinol Invest 1997; 20:36–47.

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12.Atger M, Misrahi M, Young J, Jolivet A, Orgiazzi J, Schaison G, Milgrom E. Autoantibodies interacting with purified native thyrotropin receptor. Eur J Biochem 1999; 265:1022–1031.

13.Nagayama Y, Nishihara E, Namba H, Yamashita S, Niwa M. Identification of the sites of asparagine-linked glycosylation on the human thyrotropin receptor and studies on their role in the receptor function and expression. J Pharmacol Exp Ther 2000; 295:404–409.

14.Tanaka K, Nagayama Y, Nishihara E, Namba H, Yamashita S, Niwa M. Palmitoylation of the human thyrotropin receptor. Slower intracellular trafficking of the palmitoylation-defective mutant. Endocrinology 1998; 139:803–806.

15.de Bernard S, Misrahi M, Huet JC, Beau I, Desroches A, Loosfelt H, Pichon C, Pernollet JC, Milgrom E. Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain. J Biol Chem 1999; 274:101–107.

16.Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B. Subunit structure of thyrotropin receptors expressed on the cell surface. J Biol Chem 1999; 274:33979–33984.

17.Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B. Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion. J Biol Chem 1998; 273:1959–1963.

18.Gupta MK. Thyrotropin-receptor antibodies in thyroid diseases: advances in detection techniques and clinical applications. Clin Chim Acta 2000; 293:1–29.

19.Costagliola S, Morgenthaler NG, Hoermann R, Badenhoop K, Struck J, Freitag D, Poertl S, Weglohner W, Hollidt JM, Quadbeck B, Dumont JE, Schumm-Draeger PM, Bergmann A,

Mann K, Vassart G, Usadel KH. Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves’ disease. J Clin Endocrinol Metab 1999; 84:90– 97.

20.Kakinuma A, Chazenbalk GD, Jaume JC, Rapoport B, McLachlan SM. The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies. J Clin Endocrinol Metab 1997; 82:2129–2134.

21.Minich WB, Loos U. Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves’ patients sera by luminescent immunoprecipitation analysis. Exp Clin Endocrinol Diabetes 2000; 108:110–119.

22.Minich WB, Weymayer JD, Loos U. Immunoprecipitation analysis of pathological autoantibodies in Graves’ patients’ sera using biotinated human thyrotropin receptor labeled with 125I- neutravidiny. Exp Clin Endocrinol Diabetes 1999; 107:555–560.

23.Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J, Mcgrath V, Walters S, Jaskolski D, Furmaniak J, Smith B. The interaction of TSH receptor autoantibodies with 125I- labelled TSH receptor. J Clin Endocrinol Metab 1999; 84:3797–3802.

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25.Kim WB, Cho BY, Park HY, Lee HK, Kohn LD, Tahara K, Koh CS. Epitopes for thyroidstimulating antibodies in Graves’ sera: a possible link of heterogeneity to differences in response to antithyroid drug treatment. J Clin Endocrinol Metab 1996; 81:1758–1767.

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27.Shimojo N, Kohno Y, Yamaguchi K, Kikuoka S, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K. Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 1996; 93:11074–11079.

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31.Kaithamana S, Fan J, Osuga Y, Liang SG, Prabhakar BS. Induction of experimental autoimmune Graves’ disease in BALB/c mice. J Immunol 1999; 163:5157–5164.

32.Rodien P, Bremont C, Raffin Sanson ML, Parma J, Van Sande J, Costagliola S, Luton J-P, Vassart G, Duprez L. Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensitive to human chorionic gonadotropin. N Engl J Med 1998; 339:1823– 1826.

II. IS THE PATIENT BIOCHEMICALLY HYPERTHYROID?

The first step in the laboratory evaluation of Graves’ disease is to determine if the patient is hyperthyroid by using basic laboratory tests. A TSH level and a free thyroxine level (free T4) or equivalent (free thyroxine index, FTI) must be obtained. A suppressed TSH level is consistent with the diagnosis of hyperthyroidism, in the presence of normal pituitary function. If the TSH is suppressed but the T4 is normal, a tri-iodothyronine (T3) level should be obtained to evaluate for T3 toxicosis, which is more common in early Graves’ disease or recurrence of hyperthyroidism. Initial evaluation should start with a TSH measurement and proceed to a free T4 and free T3 if the TSH value is abnormal (3).

A.TSH

A low or suppressed TSH value is seen in Graves’ disease and other causes of hyperthyroidism. Measurements of TSH have improved greatly since Odell first measured TSH in 1965 (4). TSH was initially measured using a radioimmunoassay (RIA) technique, which used a single antibody and competitive binding. Although this method was adequate for measuring high levels of TSH seen in hypothyroidism, the lower limit of detection was 1.0 mU/L, making the determination of hyperthyroidism impossible. This type of TSH assay, referred to as a first-generation TSH assay, was capable only of differentiating hypothyroid from euthyroid patients, but was not useful in differentiating euthyroid from hyperthyroid patients.

In the mid-1980s, second-generation TSH assays were developed using an immunometric assay (IMA) technique, with either a monoclonal or polyclonal antibody (5,6). In this method, two anti-TSH antibodies are used to create a sandwich configuration, thus improving the sensitivity of the assay (Fig. 1). One antibody is attached to a solid-phase substrate, such as a test tube, plastic beads, or ferromagnetic particles. The second antibody is labeled with a radioisotope, enzyme, fluorescent marker, or chemiluminescent molecule. Since two sites on the TSH molecule must be recognized, the sensitivity is greatly improved. Immunometric assays are noncompetitive, with the label being directly proportional to the TSH concentration. The increased sensitivity and specificity of this method allow measurement of TSH down to 0.1 mU/L. These TSH assays are capable of differentiating euthyroid from hyperthyroid patients and are termed “sensitive TSH” assays (7– 12). These assays are known by a variety of other names, depending on the label found on the second antibody (Table 2).

Figure 1 Second-generation, so-called sensitive TSH assay, using two antibodies: one antibody attached to solid substrate and one antibody with a label attached. The TSH molecule is sandwiched between the two antibodies.

Finally, in 1990, a third-generation immunochemiluminometric assay (ICMA) was reported with improved analytical and functional sensitivity and a lower limit of detection of 0.01 mU/L (13). This type of assay can differentiate mild TSH suppression due to nonthyroidal illness ( 0.01 mU/L) from profound TSH suppression ( 0.01 mU/L) due to hyperthyroidism and is termed ultrasensitive TSH assay. A variety of third-generation TSH assays are now commercially available (14). Other third-generation TSH assay techniques include two-site chemiluminescent immunoassays and time-resolved immunofluorometric assays (TR-IFMA) (15,16).

In summary, the lower limits of detection are 1.0 mU/L for first-generation TSH assays, 0.1 mU/L for second-generation TSH assays, and 0.01 mU/L for third-generation TSH assays (Table 3). In hyperthyroidism due to Graves’ disease, the TSH is usually less than 0.1 mU/L. Despite these significant improvements in TSH measurements, there are several situations in which TSH does not reflect the patient’s thyroid status. Hospitalized patients frequently have abnormal TSH levels without thyroid disease, because of nonthyroidal illness or use of medications (Table 4) (17–21). TSH is also unreliable with recent changes in thyroid medications before the patient reaches steady state, which can take 6– 8 weeks. A subnormal TSH level can also be seen in the first trimester of pregnancy and in patients with acute psychiatric illness (22). Finally, the unusual situations of central hypothyroidism, TSH-secreting pituitary tumors, and central resistance to thyroid hormone result in TSH levels that do not correspond to the clinical status of the patient.

Table 2 Second-Generation TSH Assays

B.Thyroid Hormone Measurements

In addition to TSH, measurements of the thyroid hormones T4 and T3 should be obtained. These hormones are found circulating in the bound and unbound states. Thyroid hormones are bound to serum proteins, including albumin, prealbumin, and thyroid-binding globulin (TBG). The total thyroid hormone levels include both the bound and unbound (free) fractions. Both free and total thyroid hormone levels will be elevated in patients with Graves’ disease and other causes of hyperthyroidism.

C.Free Thyroxine

Free T4 is the portion of thyroxine not bound to serum proteins and reflects tissue hormone levels and the patient’s metabolic status. By measuring free T4 directly, the variations in protein serum and thyroxine-binding globulin levels do not affect the measured hormone level. In order for measurements of free T4 to be valid, the equilibrium between free and bound hormone must be preserved (23).

Free T4 can be measured by four methods: direct immunoassays (DIA), “indirect” tracer equilibrium dialysis, “direct” equilibrium dialysis with sensitive radioimmunoassay of dialysate, or ultrafiltration. Direct equilibrium dialysis, available in larger clinical laboratories, is considered to be the reference method for measuring free hormone levels, while ultrafiltration of undiluted sera is a research method (24–26). Direct immunoassays are employed when free T4 is measured in most clinical laboratories, due to ease and cost considerations, despite potential bias due to binding of labeled tracer to the patient’s serum proteins (10,23,25,27). DIA can be “one-step” (analogue) or “two-step” (back titration), and over 20 kits are commercially available (28–30). In the one-step method, labeled

Table 4 Drugs That Affect TSH

tracer (hormone or hormone analogue) and solid-phase antibody are added to the patient’s serum at the same time. The patient’s free hormone and labeled tracer compete for sites on the solid phase antibody. In the two-step method, solid-phase antibody is added to the patient’s serum first. Then the patient’s serum is removed and labeled tracer is added, which binds to the remaining solid-phase antibody sites. Since the patient’s serum and labeled tracer do not mix directly, binding of tracer to serum proteins cannot occur. With both methods, the amount of labeled tracer bound to the solid-phase antibody indirectly indicates the free hormone level. Although there is conflict in the literature, two-step DIA methods seem to be more accurate and reliable (29,31).

Although equilibrium dialysis and ultrafiltration are generally considered to be more accurate methods than DIA, Liewendahl et al. showed good correlation between free T4 measured by equilibrium dialysis and by DIA (32). In the setting of critical nonthyroidal illness, free T4 is often low when measured by two-step DIA. Equilibrium dialysis with undiluted serum or ultrafiltration may be better able to differentiate euthyroid from hypothyroidism in critically ill patients (33). In patients with familial dysalbuminemic hyperthyroxinemia, antithyroxine antibodies, or triiodothyronine antibodies, interference with free T4 assays can occur and give falsely high results (34).

D. Total Thyroxine

In 1965, measurement of total thyroxine (TT4) by thin-layer chromatography was reported by West (35). Although free (unbound) T4 is now the preferred test for assessing thyroid hormone production, measurements of TT4 are still frequently performed. Total T4 includes free thyroxine and thyroxine bound to proteins, including albumin, prealbumin, and TBG. The standard method for measuring TT4 is radioimmunoassay (36). Total T4 is usually increased in hyperthyroidism, including hyperthyroidism due to Graves’ disease. Other causes of elevated TT4 include increased TBG, increased binding of thyroxine to albumin or prealbumin, medications, pregnancy with hyperemesis gravidarum, endogenous antibodies to T4, peripheral resistance to thyroid hormone, and familial dysalbuminemic hyperthyroxinemia (37–39). Nonthyroidal illness can cause elevation or suppression of TT4. Although TT4 does not always reflect the metabolic status of the patient, TT4 in conjunction with the triiodothyronine resin uptake (T3RU) can be used to calculate the free thyroxine index (FTI), which does correlate with metabolic status.

E.Triiodothyronine Resin Uptake

The T3RU is an indirect measurement of the thyroid-binding globulin (Fig. 2). In this test, radiolabeled T3 is added to the patient’s serum, which contains T3, T4, and binding proteins. The radiolabeled T3, patient’s T3, and patient’s T4 bind to the binding proteins found in the patient’s serum. A portion of the radiolabeled T3 is not bound to the patient’s binding proteins. The unbound radiolabeled T3 is absorbed onto an ion exchange resin. The amount of radiolabeled T3 bound to the ion exchange resin (T3 resin uptake) can be measured. This value indirectly measures the amount of serum-binding proteins in the patient’s serum. When TBG is low, T3RU increases; when TBG is high, T3RU decreases.

F. Free Thyroxine Index

When a direct measurement of free T4 is not available, the TT4 and T3RU can be used to calculate an estimate of the free T4, which is called free thyroxine index (FTI). The

Figure 2 T3 resin uptake test. (1) Patient’s serum with T4 bound to TBG. Labeled T3 is added. (2) Labeled T3 fills empty TBG. (3) Ion exchange resin is added. (4) Free labeled T3 binds to ion exchange resin. (5) Amount of labeled T3 bound to ion exchange resin is measured. If TBG levels are low, T3RU is high. If TBG levels are high, T3RU is low.

FTI reflects the free thyroxine level because the T3RU accounts for the changes in proteins to which T4 binds while circulating in the body. The FTI is obtained by multiplying the TT4 and T3RU.

FTI TT4 (T3RU measured)/(T3RU normal)

When extremes of TBG exist or nonthyroidal illness occurs, the FTI may not accurately reflect the actual free T4 (37).

G.Free Triiodothyronine

Free triiodothyronine (free T3) is the unbound portion of T3. Free T3 is usually measured when there is evidence of thyrotoxicosis (low TSH) with a normal thyroxine level. Measuring the free T3 directly is preferred, since the variations in protein binding and TBG levels do not affect the free hormone level. The reference method for measuring free T3 is equilibrium dialysis; however, two-step radioimmunoassays, labeled analog competitive immunoassays, labeled antibody immunoassays, and time-resolved fluoroimmunoassays are also available (16,29,30,40,41). As with free T4 assays, the measurement of free T3 by a two-step DIA has been shown to correlate well with results from equilibrium dialysis (40). Although a variety of thyroid hormone changes can be seen with nonthyroidal illness, a low free T3 due to decreased peripheral conversion of T4 to T3 is the most common (33,42).

H. Total Triiodothyronine

TT3, like TT4, is measured by radioimmunoassay. TT3 is a combination of free T3 and T3 bound to serum proteins. T3 binds to serum proteins such as TBG with less affinity than T4. The amount of metabolically active T3 is affected by binding to serum proteins and peripheral conversion of T4 to T3. Total or free T3 can be used to assess for T3 toxicosis in patients with undetectable TSH and normal T4.

III. WHAT IS THE UPTAKE OF IODINE BY THE THYROID GLAND?

Radioactive iodine scanning and uptake measurements are very helpful in differentiating Graves’ disease from other causes of hyperthyroidism. In a survey of members of the American Thyroid Association in 1990, 92.3% of respondents would obtain a radioiodine uptake as part of the initial evaluation of Graves’ disease (43). In this test, the patient swallows a capsule containing radioactive iodine, usually 3–5 Ci 131I or 123I, and the uptake of the radioactive iodine by the thyroid gland is measured at 24 h, using nuclear medicine imaging techniques. Measurements of radioiodine uptake can also be made at 3–6 h with comparable results (44). Iodine is the preferred isotope because it is both transported and organified by the thyroid follicular cell. 131I is effective whether the thyroid is in the chest or neck; however, technetium pertechnetate (99 mTc) and 123I are more effective when the thyroid is in the neck (45). The normal range of iodine uptake is 10– 30% and is inversely proportional to dietary iodine intake. A low-iodine diet may increase radioiodine uptake by the thyroid (46).

Low uptake of radioactive iodine by the gland indicates thyroiditis or excessive thyroid hormone ingestion as the cause of hyperthyroidism. High uptake is caused by Graves’ disease, resulting in diffuse uptake; single toxic or hyperfunctioning nodule, with focal uptake; and toxic multinodular goiter, with diffuse or multiple focal areas of increased uptake. In addition to providing information about the cause of hyperthyroidism, the amount of uptake of iodine by the thyroid gland determines the dose of radioiodine needed for radioactive iodine (RAI) ablation therapy.

IV. ARE STIMULATING THYROID ANTIBODIES PRESENT?

Although the presence of antibodies may help to confirm the diagnosis of Graves’ disease, a measurement of thyroid-stimulating antibodies is not required in order to make the diagnosis of Graves’ disease. The nomenclature of thyroid-stimulating antibodies is confusing. The terms thyroid-stimulating immunoglobulin or antibody (TSI, TSAb); TSH-binding- inhibiting immunoglobulin or antibody (TBII, TBIA); and thyroid receptor antibody or TSH receptor antibody (TSHRAb) all refer to the same molecule, but measured with different techniques (47). The American Thyroid Association (ATA) recommends the use of the term TSH receptor autoantibody (TRAb), followed by a description of the assay used (48). TSI is present in 85% of patients with Graves’ disease; by measuring both TSI and TBII, 98% of patients with Graves’ disease will be detected. TSI is present in 92% of patients with hyperthyroid Graves’ disease, while TBII is present in 92% of patients with hyperthyroid Graves’ disease. By measuring both TSI and TBII, 99% of patients with hyperthyroid Graves’ disease will be detected (49).

Other thyroid antibodies include antithyroid peroxidase antibody (TPOAb), also called antimicrosomal antibody (AMA), and antithyroglobulin antibody (TgAb). TPOAb

is useful in the diagnosis of Hashimoto’s thyroiditis, postpartum thyroiditis, and polyglandular autoimmune disease. TgAb interferes with measurement of thyroglobulin, which is used as a tumor marker in patients with thyroid cancer. These antibodies are not useful in the evaluation of Graves’ disease (50).

There are several methods for measuring TRAb, although no standardization exists (50). Current antibody assays do not use intact animals, but instead use thyroid cell membranes or thyroid slices to measure antibody activity. The TBII assay measures inhibition of binding of labeled TSH to its receptor by the antireceptor antibody. The TSI assay demonstrates an increase in thyroid function after attachment of the antibody to the TSH receptor and is dependent on generation of cAMP. Both of these tests are measuring the same antibody, TRAb, via different techniques (51). Some experts advocate the measurement of both TSI and TBII activity for accurate clinical correlation (52).

A variety of new techniques have been developed to measure TRAb. A secondgeneration assay for TRAb, using a murine monoclonal antibody in conjunction with radioactive and nonradioactive coated tube technology, demonstrates inhibition of TSH binding to its receptor by sera of patients with Graves’ disease, with specificity of 99.6% and sensitivity of 98.8% (53). Several techniques use a CHO cell line with a luciferase reporter plasmid to demonstrate TSH receptor stimulation, caused by sera of Graves’ disease patients (49,54–56).

Measurement of thyroid antibodies is not always necessary to make or confirm the diagnosis of Graves’ disease in routine cases. A positive result of TRAb testing does not prove that the patient is hyperthyroid or has Graves’ disease. TRAb usually continues to increase for several months after 131I therapy for Graves’ disease. TRAb usually declines with antithyroid drug therapy. A meta-analysis of 18 studies demonstrated that the absence of TRAb is protective against relapse of Graves’ disease after antithyroid drug therapy (57–59).

There are several situations in which TRAb measurements are indicated. In a patient with ophthalmopathy who is clinically and biochemically euthyroid, high levels of TRAb favor a diagnosis of euthyroid Graves’ disease over orbital tumor (60). Kazuo et al. compared TBII and TSI in 62 patients with thyroid-associated ophthalmopathy and showed that TSI is a better marker for euthyroid ophthalmopathy than TBII (60). In patients with known Graves’ thyroid disease who are euthyroid, the titers of TSI and TBII correlate with the severity, but not the duration, of the eye disease (61).

In a pregnant patient, high TRAb levels measured during weeks 28–30 predict fetal or neonatal Graves’ disease, regardless of the mother’s thyroid status (47,62). The presence of TRAb in a baby may confirm the diagnosis of transient neonatal hyperthyroidism due to transplacental passage of blocking TRAb (62). When RAI uptake cannot be performed, such as during pregnancy or recent iodine exposure, positive results of TRAb testing favor the diagnosis of Graves’ disease over other causes of hyperthyroidism.

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defense against infectious agents. It is a rapid, nonspecific response and consists of a variety of cellular components including nonspecific phagocytes such as neutrophils and macrophages, granulocytes that release chemical mediators, and natural killer cells. The innate response also includes the acute phase proteins, complement, and numerous cytokines (1). However, the innate response lacks immunological memory and therefore does not change in response to repeated encounters with an antigen.

The acquired immune response, on the other hand, is capable of recognizing and eliminating specific foreign antigens. Acquired immunity has several features that differentiate it from innate immunity. First is the ability to discriminate differences among a large number of antigens. It also allows for the generation of extensive diversity within its recognition molecules. Acquired immunity also has the ability to differentiate self from nonself antigens. Finally, the acquired immune response has the ability to develop immunological memory whereby the response improves with repeated exposure to an antigen.

Several phases characterize the acquired immune response. The afferent phase involves the recognition of the antigen by an antigen-presenting cell (APC), transport to the peripheral lymph node, and presentation to lymphocytes within the lymph node. During the next phase, lymphocytes are activated within the lymphoid tissues. Finally, during the effector phase, lymphocytes help other inflammatory cells including macrophages and B cells to eliminate the antigen.

II. COMPONENTS OF THE IMMUNE SYSTEM

The immune system consists of a variety of cells and soluble mediators. Cells of the innate response include neutrophils, macrophages, monocytes, basophils, eosinophils, mast cells, and natural killer cells. Neutrophils are nonspecific phagocytes that function to remove most extracellular antigens as well as the body’s own dying or dead cells (2). Basophils, eosinophils, and mast cells participate in the innate response by releasing the contents of cytoplasmic granules as well as secreting inflammatory mediators including prostaglandins, leukotrienes, histamine, and cytokines. Natural killer cells, or large granular lymphocytes, destroy virus-infected cells as well as tumor cells (3). Macrophages and dendritic cells participate in both innate and acquired immune responses. As participants in the innate response, both cell types phagocytose or endocytose foreign antigens. Macrophages also secrete inflammatory cytokines that are responsible for the recruitment of other inflammatory cells to sites of inflammation. Macrophages and dendritic cells also have important roles in the acquired immune response where they serve as APCs.

The acquired immune response utilizes two major groups of cells: the mononuclear phagocytes and lymphocytes. The mononuclear phagocytes consist of monocytes, macrophages, and dendritic cells. All of these cells originate from a common stem cell precursor in the bone marrow. Monocytes circulate in the peripheral blood and mature into macrophages after arriving in various tissues. In the acquired immune response, macrophages serve as antigen-presenting cells by displaying foreign antigen on their cell surface. These antigens can then be recognized by antigen-specific T lymphocytes. The macrophages also function as one of the primary effector cells of the acquired immune response. During the effector phase of the acquired immune response, macrophages become activated, thereby allowing more efficient destruction of phagocytosed pathogens. Macrophages also participate in humoral immunity by binding and phagocytosing antigens that have been coated with antibody with greater affinity than uncoated antigens.

Dendritic cells are important in the initiation of most immune responses. There are two types of dendritic cells: the interdigitating dendritic cells (usually called ‘‘dendritic cells’’) and follicular dendritic cells (4). Dendritic cells are located in the skin and most organs as well as the T-cell regions of the lymph nodes and spleen. They have several functions including the endocytosis of extracellular antigens and, if activated, serve as APCs (5,6). Activated dendritic cells serve as APCs by migrating to regional lymph nodes where they present antigen to T cells. In contrast, follicular dendritic cells are not related to the interdigitating dendritic cells and are located within the germinal centers of lymph nodes and the spleen as well as mucosal-associated lymphoid tissues. The function of these cells is to present antigen to B cells.

The other major group of cells that participate in the acquired immune response are the lymphocytes. These are unique in that they are the only cells that have the ability to recognize and discriminate between the different antigens they encounter. Three major groups of lymphocytes exist: T lymphocytes, B lymphocytes, and natural killer cells. Similarly to the mononuclear phagocytes, these cells arise from a common stem cell precursor in the bone marrow. B lymphocytes mature in the bone marrow and T lymphocytes mature in the thymus. Both B and T lymphocytes participate in clonally specific immune responses while natural killer cells are responsible for the recognition and killing of abnormal cells (e.g., virus-infected cells and tumor cells). Both B cells and T cells are activated by the binding of antigen to specific cell surface receptors.

The secreted products and membrane receptors of B cells are immunoglobulin (antibody) molecules. The B-cell receptor consists of a cell-surface immunoglobulin that binds antigen to initiate clonal expansion and differentiation of B cells into antibody-producing plasma cells. Immunoglobulins are glycoproteins containing two identical heavy chains and two identical light chains (Fig. 1). The two heavy chains are attached to each other by disulfide bonds and one light chain is attached to each heavy chain (7,8). The amino terminal domains of each chain form the antigen-binding variable region of the immunoglobulin while the carboxy terminal domains form the constant region of the immunoglobulin. The constant region specifies the class (IgA, IgD, IgE, IgG, and IgM) and subclass of each immunoglobulin.

Mature B lymphocytes leave the bone marrow and enter the peripheral circulation and lymphoid tissues. Activation of these mature cells can then occur by two different mechanisms. Direct activation occurs when certain antigens are recognized by the B-cell receptor. These antigens are typically polysaccharides or other antigens with repeating epitopes such as the capsular polysaccharides of certain bacteria. The other mechanism involves T lymphocyte–dependent antigens that have been processed by the B cell and expressed on the cell membrane complexed with class II major histocompatibility complex (MHC) molecules. T helper cells recognize the antigen–MHC class II molecule complex and bind to the B cell, resulting in activation. Activation of mature B lymphocytes promotes terminal differentiation of the cell into an antibody-secreting plasma cell or a memory cell. Memory cells produce antibody as part of the secondary response that occurs with repeated exposure to an antigen.

Following antigenic stimulation, B cells also undergo two processes: immunoglobulin class switching and affinity maturation. In class switching, mature B cells are stimulated by T helper cells and cytokines to produce different classes of antibodies. This heavy chain class switching occurs as a result of several DNA recombination events and allows a B cell that produces one immunoglobulin to differentiate into a cell that can produce a different immunoglobulin (9). As a result, the immunoglobulin retains its antigen specific-

Figure 1 Schematic diagram of a membrane-bound IgG molecule. Each molecule consists of two heavy and two light chains connected by interchain disulfide bonds (S–S). Each heavy chain has four domains: three constant (CH ) and one variable (VH ). The light chains contain one constant (CL ) and one variable (VL ) domain. The antigenbinding site is located at the amino (NH2) terminal region and formed by the variable domains of both the heavy and light chains.

ity while the effector mechanisms of the molecule vary depending upon the specific class of antibody produced. The second process of affinity maturation occurs as a result of somatic hypermutation. Within the germinal centers of secondary lymphoid tissues, B cells undergo this somatic hypermutation in which a large number of somatic mutations occur, especially within the variable region genes of the heavy and light chains (10). These mutations will generate increased antibody diversity and determine the strength of antibody binding (affinity). As the concentration of an antigen decreases, those B cells with a higher affinity for the antigen will have a greater chance for survival than those with low affinity (11). The surviving clones of B cells will produce higher-affinity antibodies with re-exposure to the antigen. This affinity maturation occurs only in antibody responses to T-helper-cell-dependent antigens (12).

The T-cell receptor (TCR) is a transmembrane heterodimer that functions to recognize antigen presented in association with an MHC molecule. The domain structure of the TCR is similar to that of the immunoglobulins and B-cell receptor (Fig. 2). Most T-

Figure 2 Diagram of a T-cell receptor. The T-cell receptor is a cell-membrane-bound heterodimer. Each polypeptide chain is linked by disulfide bonds and contains a constant (C) and variable (V) domain similar to those of the B-cell receptor.

cell receptors are composed of an α and β chain (αβ TCR), although some T cells express the γδ TCR (13). Each polypeptide chain contains one constant and one variable domain that are each structurally homologous, respectively, to the constant and variable domains of the B-cell receptor and immunoglobulins. However, the TCR differs from the B-cell receptor in two ways. First, the TCR is produced only as a membrane-bound molecule and does not have a circulating form similar to the immunoglobulins. Second, the TCR is not specific for antigen alone but instead recognizes the specific antigen only if it is associated with an MHC molecule.

There are two major types of T lymphocytes: CD4 T cells (T helper) and CD8 T cells (T suppressor). The CD4 and CD8 cell-surface molecules help T cells to recognize specific MHC molecules and serve as coreceptors for the TCR. T helper cells recognize antigen combined with MHC class II molecules and function primarily by secreting cytokines, thereby helping other cells mediating the immune response. T suppressor cells recognize antigen combined with an MHC class I molecule and function as cytotoxic killer cells and eliminate virally infected cells (11). T helper cells can be further classified into two subsets of cells: type 1 and type 2 (Th1 and Th2) helper T cells (14). Th1 helper T cells secrete interleukin-2 and interferon-γ and promote cell-mediated immunity, including the activation of macrophages and T cell-mediated cytotoxicity. Th2 helper T cells secrete interleukin-4, -5, -6, and -10 and function to help B cells produce antibodies, stimulate mast cell development, and activate eosinophils. Th2 cells also may act as regulators of immune responses by antagonizing the effects of Th1 cells.

III. MAJOR HISTOCOMPATIBILITY COMPLEX

The major histocompatibility complex is a region of highly polymorphic genes located along a region of chromosome 6 in humans. The products of the MHC play a central role

Figure 4 Schematic diagram of the class II MHC molecule. Class II molecules are membrane-bound glycoproteins containing two polypeptide chains that are noncovalently linked. Each chain contains an α and β domain. The interaction of the α1 and β1 domains forms the peptide-binding cleft of the class II molecule.

IV. ACQUIRED IMMUNE RESPONSES

All immune responses can be classified into three basic phases: recognition, activation, and the effector phase. The acquired immune response occurs as a result of proliferation of antigen-specific T and B cells and typically begins with the uptake of antigen at the site of inflammation. At these sites, antigen-presenting cells phagocytose antigen and then migrate to the regional lymph nodes or spleen. Extracellular antigens enter APCs by phagocytosis or endocytosis, are processed by lysosomes or endosomes, and the resulting peptides are loaded into class II MHC molecules. The peptide–class II MHC complex is expressed at the cell surface where it can be recognized by CD4 T cells. Intracellular antigens produced by virally infected cells and other intracellular microbes are processed within the cytoplasm. These peptide fragments are loaded into class I MHC molecules and transported to the cell surface for presentation to CD8 T cells.

The recognition phase occurs within specific regions of the lymph nodes and spleen where APCs present antigen to both T and B cells. The B-cell receptor recognizes native (unprocessed) antigens while the αβ TCR recognizes processed antigens complexed with MHC molecules. Activation of both B and T lymphocytes requires binding of the appropriate receptor as well as additional signals from a variety of costimulatory molecules on APCs and soluble mediators including interleukin-6, interleukin-1, and tumor necrosis factor α (11,16–18). Activation of T lymphocytes results in clonal proliferation and differentiation into effector and memory cells. B lymphocyte activation occurs in the germinal centers of lymphoid tissues resulting in affinity maturation, class switching, and differentiation into antibody-producing plasma cells and memory cells.

During the effector phase, activated lymphocytes perform functions that ultimately result in the elimination of specific antigens. This phase includes a variety of effector responses such as antibody binding to antigens, antibody activation of the complement cascade, antibody-enhanced phagocytosis, and antibody-dependent cellular cytotoxicity. Type 1 T helper cells produce cytokines that activate macrophages and T-cell-dependent cytotoxicity while type 2 T helper cells stimulate B cells to produce antibodies (11). The CD8 T cells destroy virally infected cells and produce cytokines that protect adjacent cells from infection (19). Therefore, the effector phase is characterized by an interaction of both innate and acquired immune responses to eliminate foreign antigens.

V.AUTOIMMUNITY

The immune system does not normally recognize and respond to self antigens, thereby preventing harmful reactions against an individual’s own antigens. A critical property of the immune system is this ability to differentiate self from nonself antigens, also known as self-tolerance. Self-tolerance is a process involving several active mechanisms that prevents the maturation or activation of self-reacting lymphocytes. The two principal mechanisms involved in tolerance are clonal deletion, in which antigen-specific clones are deleted by apoptosis; and clonal anergy or induction of unresponsiveness against selfreactive cells within the thymus and peripheral tissues (20). Autoimmunity occurs when there is a failure in the normal mechanism of self-tolerance, resulting in an immune reaction against self antigens. Several factors may contribute to the development of autoimmunity including genetic susceptibility, gender, immunological abnormalities affecting antigen-presenting cells or lymphocytes, microbial infections, and environmental factors.

Autoimmune disease occurs when an immune response is directed against self antigens, resulting in chronic tissue damage. Graves’ disease is an autoimmune disorder of the thyroid gland that is caused by thyroid-stimulating antibodies (21). The thyroid cells are both the source and target of these autoantibodies. The disease appears to occur in genetically susceptible individuals, although environmental and endogenous factors may contribute to the development of the disease (22). Patients with Graves’ disease exhibit a variety of immunological abnormalities that are highly suggestive of autoimmunity, including diffuse lymphocytic infiltration of the thyroid gland and sensitization to several thyroid antigens as well as the thyrotropin receptor (20,23). One of the important autoantigens in this disorder is the thyrotropin receptor. Autoantibodies to this receptor probably stimulate the production of excessive amounts of glycosaminoglycans in a variety of cells of the orbit (20,21,24). As a result, the orbital fatty tissues expand and the extraocular muscles enlarge, leading to the familiar clinical signs of Graves’ ophthalmopathy.

The TSH receptor is thought to be the major autoantigen in patients with Graves’ disease, although it remains unclear if it contributes to the pathogenesis of Graves’ ophthalmopathy (23,25). However, several studies have shown that TSH receptors are expressed by fibroblasts and other orbital tissues from patients with Graves’ ophthalmopathy (21,26,27). Recently, an animal model has been developed that has features similar to Graves’ ophthalmopathy (28). This model utilizes the transfer of TSH receptor-primed T cells to naı¨ve mice to induce thyroiditis and orbital pathology. The orbital changes include orbital infiltration by lymphocytes, edema, TSH receptor immunoreactivity, and periodic acid–Schiff-positive material between muscle fibers. Although these findings are similar

to those in patients with Graves’ disease, it remains unclear if this model reflects the true pathogenesis of Graves’ ophthalmopathy.

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terms of Burnet’s clonal selection theory (4). Antigen encountered before birth would delete the clones which Burnet termed ‘‘forbidden clones,’’ whereas antigen encountered after birth would activate specific clones to proliferate and respond.

Implicit in this theory is the requirement for prenatal generation of the entire immune repertoire. This is, of course, not the case since lymphocyte differentiation continues in postnatal life and probably throughout most of life, and somatic mutation generates new B-cell specificities after antigenic stimulation. Thus, the key factor in determining responsiveness, whether tolerance or immunity, cannot be the development stage of the individual but the state of maturity of the lymphocyte at the time it encounters antigen. This was pointed out by Lederberg in 1959 (5) in his modification of Burnet’s clonal selection theory. Immature lymphocytes encountering antigen would be deleted, whereas mature lymphocytes would be activated to respond. Although strong evidence has been obtained to support this idea, this simple and elegant scheme cannot accommodate many experimental situations, as will be seen below.

In Burnet’s laboratory, Nossal (6) failed to induce tolerance in mice even after injection of influenza virus in utero, a failure presumably due to rapid antigen clearance. By contrast, tolerance to the immunogenic form of a protein could be induced by preinoculation of the protein in deaggregated form in adult mice (7), which of course possess many mature lymphocytes. Likewise, tolerance to synthetic polypeptides could easily be achieved in the adult (8). Hence, contact of mature lymphocytes with antigen does not always lead to an immune response.

Although lymphocytes in neonatal mice were thought to be immature, because of the ease with which tolerance to allografts could be induced at that age, it is clear that neonatal mouse T cells are perfectly able to mount an immune response to antigen, as was shown more than 20 years ago when the T cells of 1-day-old mice reacted strongly to foreign antigens (9).

II. INTRATHYMIC TOLERANCE

What about immature T cells in the thymus? Are they always deleted when they encounter antigen?

The first hint that the thymus may be involved in tolerance induction came from experiments in which neonatally thymectomized mice were grafted with syngeneic or allogeneic thymus tissue. Whereas nongrafted mice were severely immunodeficient (10), those given syngeneic thymus grafts were perfectly immunocompetent, while those receiving allogeneic grafts were also competent except insofar as they were specifically tolerant to tissues from the donor of the thymus graft (11) (Fig. 2). It was suggested that the tolerance caused by injecting allogeneic cells at birth induced a so-called immunological thymectomy, that is, it deleted those host thymus lymphocytes specifically reactive to the antigens on the donor cells (11).

It is now well established that immature T cells in the thymus can either be deleted or selected for survival by the same self antigen (peptide) (12–14). Developing T cells are positively selected for survival only if they can express an antigen-specific receptor (T-cell receptor [TCR]) that enables them to bind with a certain degree of strength (avidity) to molecules encoded by the major histocompatibility complex (MHC) and encountered on thymic cortical epithelial cells (Fig. 3). It is probable that such binding protects the cells from programmed cell death. Positive selection thus ensures that the mature T cell will recognize antigenic epitopes (peptides) accommodated in the binding cleft of self

Figure 2 Restoring immune function to immunodeficient neonatally thymectomized mice was achieved by grafting thymus tissue. If the tissue was derived from syngeneic mice, the grafted mice were perfectly immunocompetent. If derived from allogeneic mice, the recipients were competent except that they were specifically tolerant of tissues from the strain of mice providing the foreign thymus graft. (From Ref. 11.)

MHC molecules, and hence will be self-MHC restricted. This selection will not, however, prevent the differentiation of T cells with high-avidity TCR for self peptides and MHC molecules. Some form of negative selection must therefore operate to prevent the autoimmune potential of such self-reactive cells. This generally occurs by the physical deletion of those clones of T cells that have high-avidity TCR directed to self-antigens present within the thymus, on thymus dendritic cells, and also on some thymic medullary epithelial cells (14). Thus, the avidity of the TCR for the target self peptide and self MHC molecule dictates whether the selection will be positive or negative.

III. EXTRATHYMIC TOLERANCE

Self antigens expressed only outside the thymus may not provoke an immune response if they are sequestered in privileged sites away from the circulating routes of naı¨ve T cells, or exposed on certain cell types that do not express MHC molecules and hence cannot present peptides derived from those antigens to T cells. It may also be the case if the autoantigens are present in amounts too low to be detected by T cells, or if the avidity of the combined TCR and accessory molecules is not high enough for T cells to establish effective contact with the autoantigen-presenting cells. Under these conditions, the naı¨ve T cells ignore the existence of the autoantigens (15) but the resulting lack of T cell activation is not equivalent to tolerance induction since presentation of the autoantigen by professional APCs would immunize. Fail-safe mechanisms inducing postthymic tolerance must, however, exist since molecules may be released from dying cells and hence processed and presented by professional APCs, such as macrophages or dendritic cells. Because

Figure 3 Development pathways of thymocytes expressing the αβ TCR and the CD4 (4 ) and CD8 (8 ) coreceptors showing the stages at which positive and negative selection operate (see text). Here, avidity is towards self peptide accommodated in the cleft of self MHC molecules.

these APCs have costimulatory function, they are well equipped to activate T cells (see below).

If contact does occur between postthymic mature T cells and antigens, the result may not always be a productive immune response. Thus, under conditions of antigen persistence, T-cell apoptosis results and operational tolerance may follow. For example, high doses of a particular strain of the lymphocytic choriomeningitis virus caused the differentiation of presumably all virus-specific CD8 T cells to cytolytic lymphocytes, which reached maximum levels at 6 days and then declined to undetectable levels at 15 days as a result of apoptosis. The virus was not cleared and persisted (16).

Figure 4 shows three pathways in which antigen may be presented to T cells. When parenchymal tissue self antigens are cross-presented above a certain critical level in the draining lymph nodes, apoptosis of any self-reactive CD8 T cells present there at the time will occur (17). This was demonstrated in work using mice transgenic for a TCR directed to the major peptide of the ovalbumin (OVA) molecule. When these transgenic T cells (OT-I cells) were confronted in the periphery of other transgenic mice expressing the target antigen as a self antigen in the β cells of the islets of the pancreas, the following events were observed (17):

Figure 4 Pathways of antigen presentation to extrathymic, mature CD4 and CD8 T lymphocytes. CD4 T cells generally recognize antigenic determinants in association with MHC class II molecules on the surface of APCs that have taken up the antigen exogenously and processed it intracellularly. CD8 T cells recognize antigen synthesized within the APC and presented in association with MHC class I molecules (endogenous pathway). They can also recognize antigen taken up by the APC exogenously, processed intracellularly by an as yet undetermined pathway, and presented with MHC class I molecules (cross presentation pathway).

1.The transgenic self antigen migrated from the islets to the draining lymph nodes where it was recognized by OT-I cells on the surface of APCs. It is presumed that this must be occurring spontaneously and continuously, and in the absence of any inflammatory response or harmful influence.

2.The APCs were derived from bone marrow, had a short life span, and antigen cross-presented by these cells was not only sufficient but also essential for OT-I cell activation.

3.Following activation, the OT-I cells proliferated.

4.After some proliferation, activated OT-I cells disappeared within 4–5 weeks, presumably as a result of activation-induced cell death.

5.Only when 1 million or more OT-I cells were given did diabetes occur. Such a high number of cells of a particular clone is, of course, not physiological.

6.Only relatively high doses of self-antigen were cross-presented; low doses were not unless autoantigen-expressing cells had been damaged.

The situation just described could well mimic the following scenario. Self-reactive T cells that have escaped thymus censorship for one reason or another find their way to the lymph nodes draining a healthy tissue that releases the target self antigen at a certain rate. If this rate is below some threshold level, the naı¨ve T cells (which do not normally circulate into nonlymphoid tissues) (18) will not be activated and hence will ignore their target. If the rate is above that level, the T cells will be activated as soon as they enter the nodes and will eventually succumb to activation-induced cell death. Autoimmune damage will thus not take place under these conditions.

(18), that is, the very tissues whose cells do not possess costimulatory activity. How, then, could naive T cells be anergized in vivo, since they enter such tissues only after being activated in the draining lymph nodes?

V.THE DANGER CONCEPT

Matzinger (24) has argued that the immune system, rather than discriminating between self and nonself, does so between harmless and dangerous entities. Of the several examples that are difficult to explain in accordance with the danger hypothesis, two will be singled out for special mention. When one immunizes mice with mouse cytochrome c, one gets no response, but a comparable immunization with pigeon cytochrome c does give a response (25). In both cases, adjuvant containing dangerous and highly immunostimulatory mycobacteria was used. A similar situation is seen in allograft rejection. Allografts do not normally occur in nature and so the immune response could not have evolved to regard these as dangerous. Yet they trigger powerful immune responses (26). The danger from the trauma of surgically implanting the graft cannot account for the rejection, since syngeneic grafts do not provoke a response and are accepted. Yet the inflammation following the surgical procedures would be expected to stimulate the many dendritic cells expressing costimulatory molecules and MHC molecules carrying peptides. Clearly, in both these cases the clonally individuated T cells with high affinity receptors for self MHC and self peptides have been deleted in the thymus (14). Hence at least in the thymus, there is discrimination between self and nonself epitopes rather than between harmless and harmful entities. Yet since, in these examples, there are no self-reactive T cells in the periphery, does the danger concept apply only to situations where there are specific T cells circulating? In the classic experiments of Billingham and colleagues, longstanding (up to 144 days) allografts in healthy immunologically tolerant mice were destroyed following an injection of purified naive lymphocytes from normal unsensitized donors of the same strain as the tolerant host (27). It would be stretching credulity to argue that an intravenous injection mimics a danger signal, particularly as an injection of lymph node cells immune to the tolerated graft, but foreign to the host, did not lead to skin graft rejection. Further doubts concerning the validity of the danger hypothesis have been admirably expressed in a recent article (28).

Danger is not excluded as playing any role whatsoever in immune responses. It certainly does in the response to pathogens, which, as mentioned before, possess molecular structures recognized by invariant pattern recognition receptors of the innate defense mechanisms (1).

VI. T-CELL-DEPENDENT SUPPRESSION

Evidence has steadily been mounting for the tolerogenic importance of some type of T- cell-dependent suppression of potentially autoaggressive T cells (29). The idea of suppressor T cells was first suggested in 1974 by Gershon (30), but the failure to isolate a distinct subset of suppressor T cells has led many to question their existence (31). Nevertheless, one way in which T cells can suppress immune response is by the inhibitory effects of cytokines. The release of TGF-β by T cells after some forms of antigen stimulation is one example (32). Furthermore, the evidence obtained by Mosmann and Coffman (33) for two types of helper T cells, Th1 and Th2 with distinct antagonistic lymphokine profiles

Figure 6 Two subsets of Th cells, with distinct patterns of cytokine production, have been identified: Th1 and Th2. Through their production of IL4 and IL10, the Th2 cells may interfere with the helper function of Th1 cells. Other cytokines, such as γ -interferon (IFN-γ) (e.g., synthesized by CD8 T cells) can enhance ( ) or diminish ( ) the activities of Th1 and Th2 cells, respectively.

(Fig. 6), strongly suggests that T-cell-dependent immunoregulation of immune responses is a reality that needs further exploration at both cellular and molecular levels.

VII. TOLERANCE IN B CELLS

There are many ways in which T-cell tolerance or lack of T-cell responses to self antigens can be achieved. What happens in the case of self-reactive B cells that may be circulating? The production of high-affinity antibodies of the IgG class is known to be T-cell dependent (34). Such antibodies are usually responsible for tissue damage associated with autoimmune disease. For this reason, and since the threshold of tolerance for T cells is lower than for B cells (35), the lack of self-reactivity in the B-cell repertoire is most likely to result simply from the absence of T-cell help (36,37), the self-reactive helper T cells having been subjected to tolerance induction by one of the mechanisms discussed above (Fig. 7).

Nevertheless circumstances do exist in which B cells may become self-reactive. For example, exposure to antigens derived from micro-organisms, expressing both foreign T- cell epitopes and B-cell epitopes cross-reacting with self antigens, will result in a vigorous antibody response (38) (Fig. 8). Furthermore, in contrast to the TCR, the Ig receptor on mature, antigenically stimulated B cells has been shown to undergo hypermutation (39), which could lead to antiself reactivity. Mechanisms inducing tolerance in B cells must thus operate both during their development and following antigenic stimulation in secondary lymphoid tissues (40).

Figure 7 Self-reactive B cells may in many cases simply fail to react because of the absence of T-cell help, the self-reactive Th cells having been deleted intrathymically.

Experiments in transgenic models indicated that, with the caveat concerning the tolerance threshold (35), tolerance mechanisms operating in the B-cell lineage are similar to those for T cells. Thus, encounter of B cells with multivalent cell-membrane-associated self antigens, able to crosslink the Ig receptors on these cells, led to their deletion from secondary lymphoid tissues. This type of tolerance occurred with self antigens, irrespective of whether they were expressed on cells located within the bone marrow or elsewhere. On the other hand, self-reactive B cells exposed to oligovalent, soluble antigen, giving a receptor occupancy of 25%–30%, were not deleted immediately from secondary lymphoid tissues and became anergic. The anergic state was associated with persistent downregulation of the membrane IgM receptor, a failure to upregulate the B7 complex, and death of the B cells within 3–4 days in the T-cell zone, where they had migrated in search of T- cell help. Thus anergic B cells did not persist for significant periods of time after antigenic stimulation (41). However, they could be rescued if given a strong T-cell-help stimulus within 24 h of antigen, once again pointing to the decision between activation and tolerance in B cells being largely T-cell dependent.

No evidence for the presence of suppressor T cells or of anti-idiotypic B cells was found in these transgenic models.

Figure 8 Self-reactive B cells may be able to respond to self-antigenic determinants if the antigens bear foreign T-cell epitopes and self B-cell epitopes. Mechanisms must therefore exist to induce B-cell tolerance as discussed in the text.

ACKNOWLEDGMENTS

I am grateful to Professor A. Basten and to Dr. M. Lenardo for useful suggestions and comments.

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molecules including intercellular adhesion molecule-1 and vascular cell adhesion mole- cule-1. Soluble mediators such as interleukin (IL)-12 that can be produced by APC also play a major role in multimolecular interactions between APC and T cells (3). Together, the nature of all signals and their duration will be integrated by T cells and translated into a comprehensive response (4,5).

Of crucial importance is the fact that APC do not express the costimulatory sets of ligands required by T cells in a constitutive manner. Almost all costimulatory molecules are inducible and produced only by the APC when it first receives specific signals to do so (6,7). These signals are given by proinflammatory mediators. Essentially, these mediators provide a signal to APC that homeostasis is disrupted, infection or stress occurs, and that a specific immune response may be in order. As a consequence, APC set the scene for T cells to probe the site. MHC molecules are upregulated, phagocytic activity of APC is stimulated to sample the antigenic microenvironment, surface ligands appear for engagement of supramolecular TCR complexes, and soluble mediators and chemotactic gradients are formed to recruit T cells actively and prepare them for activation. These elements together not only control whether or not T cells can become activated at all but they also play important roles in determining the quality of the ensuing specific immune response. By secreting IL-12, for example, APC can polarize specific immune responses into predominantly proinflammatory type-1 responses. Very complex interactions determine whether or not an APC will direct T cells into type 1, type 2, or downregulatory responses (3).

Apart from activating APC, as sentinels that translate changes in their microenvironment into molecular signals for T cells, inflammatory mediators can also directly interact with T cells and influence their function. Thus, inflammatory mediators control autoimmune responses at various levels and via interactions with different cell types. The molecular mechanisms involved are highly complex and are only beginning to be unraveled. Inflammatory mediators include mediators such as cytokines, chemokines, lipids, hormones, and low-molecular-weight (toxic) chemicals; physicochemical parameters including temperature, osmolarity, radiation, and pH; and a whole range of microbial products. These mediators or conditions activate intracellular signaling pathways that integrate the inflammatory signals and regulate gene transcription, enzyme cascades, and structural reorganizations within cells. The wide range of inflammatory mediators precludes their comprehensive discussion in a single chapter. In this chapter, I have limited myself to discussing three elements in the story of how inflammatory mediators affect (auto)immune responses.

First, the recently discovered toll-like receptors will be discussed. These receptors form part of an ancient defense system against microbial pathogens and specifically recognize microbial molecules alien to the mammalian body. Thus, the discovery of toll-like receptors essentially redefines a wide range of microbial molecules as bona fide inflammatory mediators. Understanding the role of these receptors, and the consequence of their engagement, sheds new light on mechanisms via which microbial pathogens can influence the development of (auto)immune responses. Toll-like receptors represent a completely new level of immune responsiveness to pathogens, in addition to the well-known level of specific immune responses mediated by T and B cells. Next, the p38 mitogen-activated protein kinase (MAPK) pathway is reviewed, which is one of the major intracellular regulatory pathways activated by inflammatory mediators, including those that act via the above-mentioned toll-like receptors. This review is primarily intended to highlight the complexity of intracellular mechanisms that translate the signals given by inflammatory

mediators into changes in gene expression. To some extent, members of the p38 MAPK pathway may be regarded as inflammatory mediators themselves, and they have recently attracted much attention as novel targets for selective anti-inflammatory drugs. Finally, the role of stress proteins (or heat-shock proteins) in autoimmune disease will be discussed. Stress proteins have been under investigation for a long time as potential antigenic triggers for autoimmune disease. Yet novel data reveal that stress proteins can also act as inflammatory mediators, independent from any specific T- or B-cell response. They can, for example, engage toll-like receptors and directly activate inflammatory responses. Again, however, the level of differentiation in such mechanisms is high and no uniform rules apply.

II. TOLL-LIKE RECEPTORS

Over the past 3 years, remarkable progress has been made in our understanding of how microbial pathogens can influence (auto)immune responses. Previously, attempts to understand the well-recognized impact of viruses or bacteria on autoimmune disease were primarily focused on specific immune reactivity. Structural similarities between pathogens and self proteins, for example, have been exhaustively examined for their potential to induce cross-reactive responses and, thus, to influence autoimmune disease. It has now become clear, however, that pathogens can also influence autoimmune reactions at a completely different level. This level includes activation of specific sets of receptors designed to detect the presence of microbial structures.

In insects such as Drosophila, no specific immune system exists but innate responses allow the production of antimicrobial peptides. In 1996, it was found that certain Drosophila receptors important for embryonic development also control the production of such antimicrobial peptides and that these receptors were activated by microbial structures, or by the products of proteolytic pathways activated by pathogens (8,9). The first receptor of this kind to be identified was termed ‘‘toll,’’ German slang for ‘‘great’’ or ‘‘far out.’’ Key to the ability of the toll receptor to control antimicrobial responses in Drosophila is its intracellular domain, which is strikingly homologous to the intracellular portion of the mammalian IL-1 receptor (IL-1R). Thus, the question arose whether perhaps specific receptors for microbial products also exist in mammals. Indeed, the first human toll-like receptor was discovered in 1997 (10). Several toll-like receptors (TLRs) have since been identified and found to be essential components of the innate immune response in vertebrates (11–13). For example, both in mice and humans, TLRs participate as key receptors in the response against bacterial endotoxins (14).

It has now become clear that TLRs represent a family of homologous proteins characterized by an extracellular leucine-rich repeat domain and a cytoplasmic domain responsible for intracellular signaling. This intracellular IL-1R-like domain binds to the protein myeloid differentiation factor-88 (MyD-88) that links TLRs to downstream signaling pathways similar to those involved in IL-1 signaling, involving the IL-1R-associated kinase (IRAK) (15,16). Downstream, IRAK activation by TLRs primarily triggers activation of NF-κB, which induces expression of molecules such as interferons α and β, IL-1, IL-6, IL-8, and also CD80, an essential costimulatory molecule for the onset of specific T-cell responses (17). Since MyD-88 can also link TLRs to the Fas-associated death domain (FADD) and caspase-8 activation pathways, TLR signaling can also lead to the induction of apoptosis (18). Although NF-κB activation appears to be a major consequence of TLR signaling, activation of other signaling pathways may also occur, for example, of the p38

mammals, DNA sequences typical of bacterial (CpG-DNA), and microbial carbohydrates. As such, TLRs function as sensors for the presence of potentially pathogenic microbial structures. In addition to microbial structures, TLRs can also recognize self-structures that are abnormally modified or expressed as the consequence of stress, damage, or disease. Recognition of the body’s own stress proteins, for example, reflects this function of TLRs.

By triggering innate immune responses, TLRs induce immediate antimicrobial responses and, in a more general sense, prepare the site for local activation of specific immune responses by T cells. This seems to be an appropriate response when microbial infection occurs or when the tissue is stressed, damaged, or diseased by some other cause. Although TLRs generally appear to perform their sensor function on the surface of cells, they may also probe structures within phagosomes of macrophages and monocytes in which extracellular molecules, including microbial structures, are taken up. For TLR-2, this specific way of operating has recently been described (20). Figure 1, shows a schematic overview of TLR function in innate immune responses. It is not unreasonable to expect that in the near future more TLRs will be identified, polymorphisms will emerge, and much more will become clear of the specific ligand recognition patterns of these TLRs.

III. p38 MITOGEN-ACTIVATED PROTEIN KINASE

The human genome encodes more than a thousand protein kinases. These enzymes phosphorylate a wide range of targets, making up as much as 30% of all cellular proteins in eukaryotic cells. Many inflammatory mediators including cytokines and growth factors, bacterial products, and physicochemical stress activate intracellular MAPK cascades. These cascades consist of tiered signaling molecules, each phosphorylating and thereby activating another downstream mediator. They culminate in the activation of transcription factors that regulate gene expression by direct binding to specific DNA sequences (21– 23). MAPK family members are characterized by a protein loop that contains two sites for phosphorylation in a common threonine-x-tyrosine motif. Dual-specificity MAPK kinases (MKKs) are responsible for their phosphorylation at both sites and these MKKs are in turn activated by other upstream kinases in the cascade, termed MKK kinases (MKKKs). At the same time, sets of specific phosphatases are active in rapidly and selectively removing phosphate groups from activated kinases, thus regulating their activity. This strategy allows the cell to respond rapidly to signals, often within minutes, without having to synthesize or degrade entire protein molecules.

Although MAPK signaling cascades are still not fully mapped, three major and one minor pathway have been defined to date. The major ones are referred to as the epidermal growth factor-regulated kinase (ERK) pathway, the c-Jun N-terminal kinase (JNK) pathway, and the p38 MAPK pathway. Their activation affects the function of several transcriptional factors notably activator protein-1 (AP-1), the nuclear factor of activated T cells (NFAT), and members of the signal transducer and activator of transcription (STAT) family, which are intimately involved in cytokine regulation. Activation of MAPK pathways has been repeatedly documented in conditions of inflammation (22). For example, the activation of p38 MAPK, a pathway typical for higher eukaryotes, has been documented to lead to production of proinflammatory cytokines such as IL1-β and tumor necrosis factor alpha (TNF-α); chemokines such as monocyte chemotactic protein-1; enzymes involved in inflammation and remodeling such as inducible nitric oxide synthase, cyclo-

oxygenases, and collagenases; and adhesion molecules including vascular cell adhesion molecule-1. Like other MAPK pathways, the p38 MAPK pathway is activated by a variety of triggers including stress, UV light, osmotic shock, engagement of toll-like receptors, and certain proinflammatory cytokines such as IL-1β and TNF-α (24,25). As a typical stress-activated kinase, p38 MAPK is known to be involved in a wide range of cellular functions including growth, development and differentiation, apoptosis and migration, but a large part of p38 MAPK functions certainly also has an impact on inflammatory processes, cytokine production, and leukocyte recruitment and migration.

In order to understand p38 MAPK functions, it is important to realize that there are, in fact, several different isoforms of p38. The four known p38 MAPK isoforms, α, β2, γ, and δ, are structurally similar, but not identical (26,27). Each of these isoforms is expressed at different levels in different types of cells, they respond to different triggers, and they are sensitive to different inhibitors (28). The major p38α isoform, for example, is highly expressed in leukocytes and endothelial cells; the γ isoform is only found at high levels in skeletal muscle (26,29); and the δ isoform is primarily found in lung, kidney, testis, pancreas, and small intestine (28). The ability of upstream MKKs to activate p38 MAPKs is different for each of the isoforms. For example, p38 MAPKα can be phosphorylated by MKK-3, -4, and -6, but p38 β2 is preferentially phosphorylated by MEK-6 and cannot be activated by MKK-3 (30,31). A range of different phosphatases exist for the selective inactivation of p38 MAPK (32,33). Every different type of cell may have its own p38 MAPK and phosphatase profile and therefore respond in its own individual way to defined stimuli (22). In neutrophils, for example, TNF-α activates p38 MAPKα and δ, whereas LPS leads to the selective activation of p38δ (34). Phorbol esters instead lead to the preferential phosphorylation of ERK1/2 and not p38 MAPKs (21). In other cells, this situation may very well be different again. Also in vivo, defined forms of stress trigger markedly different p38 activation profiles in the different cell types present in one organ (35).

A variety of signals converge in the p38 MAPK pathway, and a variety of possible activation cascades diverge again downstream of p38 (36). The predominant flow of activation via selective phosphorylation is determined in part by levels of regulatory ‘‘chemostats’’ inside cells, which are members of other signaling cascades that modulate the MAPK cascade. Ceramide is a well-known example of a substance that has a substantial impact on the activation flow in MAPK-signaling cascades (37). Ceramide can promote MAPK-regulated growth and differentiation in some cells, while it stimulates MAPKinduced apoptosis in others. ‘‘Chemostats’’ such as ceramide determine to a large part the exact way that specific signals flow through signaling cascades and, thus, the exact way in which individual signals are translated into changes in gene expression. This complexity renders it very difficult to compile a generally applicable list of potential triggers for each of the MAPK family members. Each time, not only the trigger but also the responding type of cell (and even its developmental stage) must be defined. Table 1 shows some of the triggers that have been described for activation of p38 MAPKs in different cell types. (The examples are derived from a more complete summary given in Ref. 22.) The notion that specific kinases control inflammatory signals has inspired a search for specific inhibitors for members of the MAPK cascades. Such inhibitors could potentially be useful as anti-inflammatory agents and in autoimmune diseases. A series of pyridinyl imidazole compounds has been identified that are very specific and effective inhibitors of p38 MAPK. Both in vitro and in vivo such inhibitors reduce cytokine production and have been found effective in reducing the severity of experimental autoimmune disease in animal models (38). Side effects such as hepatotoxicity, however, have so far precluded applica-

tion of MAPK inhibitors in humans. The development of MAPK inhibitors as novel antiinflammatory agents, however, will no doubt continue.

IV. STRESS PROTEINS

Stress proteins (or heat shock proteins [HSP]) are a remarkable group of proteins whose expression can be rapidly modulated under the influence of a variety of triggers. Stress proteins are generally grouped into families of similar molecular mass. The families of 60 kDa (HSP60), 70 kDa (HSP70), and 90 kDa (HSP90) stress proteins are the best studied (39,40). Much less is known about the smaller stress proteins with molecular masses between 20 and 27 kDa (HSP27) (41). Most stress proteins share the property of being upregulated in many types of cells in response to elevated temperatures (hence the term ‘‘heat shock proteins’’). Although this may perhaps suggest a common regulatory pathway for stress proteins, they are in fact regulated by highly differentiated signaling cascades that are usually different from one cell type to another. Most stress proteins perform housekeeping functions under normal conditions and are expressed in a constitutive man-

ner in almost all cells. HSP60 and HSP70, for example, play important roles in normal protein biosynthesis and intracellular protein trafficking.

Small stress proteins appear to have important functions in maintaining the integrity and motility of cytoskeletal elements, which may explain why several small stress proteins are not present in prokaryotes. For some time, stress proteins have been implicated in the development of autoimmune disease (40,42). The remarkable immunogenicity of some stress proteins, combined with the fact that they readily accumulate in diseased tissues, fueled interest in stress proteins as potential autoimmune targets in human diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis. This idea was originally supported by the observation that adjuvant-induced arthritis in rats was primarily mediated by responses against the mycobacterial HSP60 included in the adjuvant. It was believed that cross-reactivity between the mycobacterial HSP60 in the adjuvant and the rat’s own HSP60 was the major factor that led to the precipitation of autoimmune disease (39). Subsequent studies, however, have revealed that the relationship between anti-HSP60 responses and autoimmunity are not that simple. Contrary to the original expectations, evidence has accumulated that anti-HSP60 T-cell responses are more likely involved in the downregulation of specific autoimmune responses than in stimulating them. HSP60 immunization protects animals from the subsequent development of experimentally induced autoimmune disease in several different models (43,44) T cells from both mice and autoimmune patients also appear to respond to self-HSP60, primarily by producing downregulatory cytokines such as IL-4 and IL-10, rather than by secreting proinflammatory cytokines such as interferon-γ (45,46).

A downregulatory response by T cells to HSP60 certainly makes sense in the light of the fact that it is a true self-antigen that is constitutively expressed in lymphoid organs. Such a condition usually tolerizes the immune system by leading to deletion from the immune repertoire of strongly reactive T cells and selecting for regulatory qualities in mildly reactive ones. The regulatory quality of the T-cell response to HSP60 therefore meets this expectation but innate responses to HSP60 do not. Several reports have documented that an encounter with HSP60 (or HSP70) protein triggers several different types of cells to mount an innate response involving the production of proinflammatory factors. Mouse or human macrophages, as well as endothelial or smooth muscle cells, respond to HSP60 by upregulating the expression of adhesion molecules and the release of proinflammatory mediators including IL-6, IL-12, IL-15, and TNF-α (47,48). It was recently discovered that HSP60 mediates these effects by binding to the toll-like receptor-4 and/or CD14, the LPS receptor, which is often closely linked to TLRs (49,50). In human macrophages, HSP60 stimulation via CD14 or TLRs leads to activation of the p38 MAPK pathway, the consequences of which are discussed above. These findings reveal that HSP60 must not only be considered as a self-antigen for specific autoimmune T-cell responses but also as a truly inflammatory mediator itself.

It is still unclear what controls the balance between proinflammatory innate responses to HSP60 and regulatory adaptive responses. Both factors may play a role in responses to other HSP as well, but the balance may be different for each HSP. Tissue distribution of HSP and individual regulatory pathways is of crucial importance. Recent data for the small stress protein alpha B-crystallin highlight the impact of tissue distribution on the nature and quality of adaptive anti-HSP immune responses. In humans, alpha B-crystallin is restricted in its expression to a limited number of tissues and, most importantly, it is not expressed in any lymphoid organ under normal conditions (51). Because the human immune system is not tolerized for self-alpha B-crystallin, it is potentially

Figure 2 Stress proteins not only represent self antigens for specific autoimmune responses but also act as inflammatory mediators that directly trigger innate immune responses. Stress proteins have been under investigation for a long time as important self antigens in autoimmunity. Primarily dependent on whether or not stress proteins are constitutively expressed in lymphoid organs, the specific immune repertoire may or may not be tolerant for self stress proteins. In the case of heat-shock protein 60 (HSP60), constitutive lymphoid expression renders the specific immune repertoire of vertebrates functionally tolerant for the protein. Uptake of HSP60 by routine phagocytosis by an APC will lead to presentation of HSP60-derived antigenic determinants to T cells via the trimolecular complex of major histocompatibility complex molecules (MHC), processed antigen (Ag), and the T-cell receptor (TCR). An ensuing specific T-cell response by the tolerized immune system will largely lead to a regulatory response, characterized by the production of cytokines such as IL-4 and IL-10.

In the case of the small stress protein alpha B-crystallin (αB), the exceptional situation exists in humans that this protein is absent from lymphoid organs. Not being tolerant, a specific immune response to self-alpha B-crystallin in humans is proinflammatory, leading to secretion of large amounts of IFN-γ. In addition to these specific immune responses, both stress proteins are likely to provoke proinflammatory innate immune responses since they can also directly interact with TLR on the surface of APC. In the case of HSP60, the aggregate of innate and specific immune responses includes both proand anti-inflammatory components. The response to alpha B-crys- tallin tends to be uniformly directed toward proinflammatory responses.

highly responsive to the protein. For this reason alpha B-crystallin, which sometimes accumulates at high levels in central nervous system myelin, is currently under investigation as a potential trigger for autoimmune responses in multiple sclerosis (52,53).

The potential responsiveness of the human immune system against alpha B-crys- tallin is activated when viral infections occur. Under such conditions, alpha B-crystallin appears in lymphoid cells concomitant with the pathogen, and a strong proinflammatory autoimmune response against the protein is mounted (51). Other mammals show a radically different tissue distribution of alpha B-crystallin. In normal rodents and primates, alpha B-crystallin is constitutively expressed in all lymphoid tissues (as is the case for most HSP) and, consequently, immune tolerance exists (54–56). No T-cell or antibody responses are triggered when the animals are immunized with self-alpha B-crystallin. In humans, alpha B-crystallin can act as a potent proinflammatory factor in specific autoimmune responses, but it fails to do so in other mammals. Recent data indicate that alpha B-crystallin, like HSP60, can also induce direct innate responses. In humans, the innate response to alpha B-crystallin appears to amplify the adaptive immune response: both are proinflammatory. Microglia cells, for example, produce elevated levels of nitric oxide and TNF-α in response to exposure in vitro to the small HSP (57). It is unknown what mechanism mediates innate responses to alpha B-crystallin. Whether or not these also involve signaling via toll-like receptors remains to be established.

The above two examples again illustrate the complexity and diversity of both adaptive and innate immune responses to HSP (Fig. 2). Adaptive immune responses may be regulatory, or proinflammatory, dependent on the nature of the HSP and the species. Innate responses to HSP also exist and, to date, only proinflammatory innate responses have been documented. Much is still to be learned about the role that HSP may play in autoimmune diseases as either general inflammatory mediators for innate responses or as antigenic targets of specific autoimmunity.

V.CONCLUDING REMARKS

Recent developments in the field of inflammatory mediators have started to uncover the dazzling complexity of the collection of molecules and mechanisms involved in inflammation and innate immune responses. Not long ago, inflammation and stress were widely perceived as conditions that would activate signaling pathways and production of certain molecules in a fairly uniform and straightforward manner. It is now becoming increasingly clear that every individual type of cell responds in its own way to inflammatory mediators and that myriad molecules and pathways are involved in translating the precise type of stress or inflammatory signal into a well-defined individualized cellular response. The levels of complexity and differentiation in these processes are much higher than was once imagined. Sometimes a single inflammatory mediator can trigger one type of cell to proliferate while leading another type of cell into apoptosis. Novel inflammatory mediators are emerging, such as microbial molecules and stress proteins that can be added to the already impressive list of different inflammatory mediators. This growing complexity certainly poses new challenges to our understanding of autoimmune diseases and the impact of microbial infection on their development. Yet, the more complex the mechanisms of inflammation turn out to be, the more options may emerge for intervention using selective anti-inflammatory agents, to the ultimate benefit of patients with autoimmune disorders.

ACKNOWLEDGMENTS

These studies were supported by the Dutch Foundation for the support of MS Research. The authors are grateful to Drs. K. Havenith and J.M. te Koppele for critical reading of the manuscript.

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Figure 1 T-cell subsets, cytokines, and autoimmune diseases.

been identified as a cytokine that acts in synergy with IL-12 by upregulation of IL-12 receptor β2 subunits (6). T-helper subsets and the cytokines they produce play a major role in a wide variety of immunological diseases. Given the fact that Th1 and Th2 cells mutually inhibit each other’s activities, it is generally thought that an imbalance between Th1 and Th2 cells may result in a decreased threshold facilitating loss of tolerance to selfpeptides and the subsequent development of autoimmune disease (Fig. 1).

Various experimental autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE), experimental autoimmune thyroid disease, and collagen-induced arthritis, but also the spontaneous development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic mice (7) are considered to include a pathogenic role of autoreactive Th1 cells. On the other hand, autoimmune diseases that demonstrate a strong involvement of autoantibodies are considered to be associated with an increased activity of Th2 cells. These considerations are taken into account in the development of intervention strategies based on the modulation of cytokines. However, as discussed below, treatment of experimental autoimmune disease employing neutralizing antibodies and studying disease models in cytokine knock-out mice have shown that selective targeting of cytokines might be ineffective or even harmful.

II. LESSONS FROM ANIMAL MODELS

The vast majority of studies concerning the role of cytokines and regulatory mechanisms have been performed in EAE, a model for multiple sclerosis. The advantage of this model is that disease can be induced by peripheral sensitization of T cells with defined myelin peptides, and that the pathology is confined to the central nervous system. The induction of EAE requires the presence of Freunds’ complete adjuvant, most likely because the Mycobacterium component strongly induces the Th1-polarizing cytokine IL-12. The dis-

ease is dependent on autoreactive Th1 cells and can be transferred by such cells to naive recipients. Th1 cells recruited to the central nervous system in response to locally secreted chemokines become involved in the activation of macrophages, which in turn are responsible for the destruction of oligodendrocytes and myelin by the secretion of proteolytic enzymes and release of reactive oxygen intermediates. This process results in the loss of nerve conduction and paralytic symptoms. Full consensus with regard to the precise role of individual cytokines in EAE has not yet been reached in view of many contradictory findings. Unexpected was that neutralization of IFN-γ with specific antibodies—aimed to limit the activity of Th1 cells—was found to exacerbate EAE (8). On the other hand, IL-4 (which is regarded as a Th2-derived anti-inflammatory cytokine) was found both to improve (9) and aggravate the severity of EAE (10). Moreover, myelin basic proteinspecific Th2 cells appeared to have the potential to induce EAE rather than providing for protection (11). These unexpected findings may be explained by the fact that these cytokines may have a broad spectrum of activities and may play different roles during different stages of the disease.

Recent studies have demonstrated that the effect of IL-12, which is regarded as one of the key factors in the development of Th1-mediated autoimmunity, is highly dependent on the timeframe of treatment and apparently on the stage of the disease. In experimental autoimmune uveitis and in experimental autoimmune thyroiditis, both regarded as Th1mediated diseases, IL-12 may even be protective (12,13).

III. AUTOIMMUNITY IN CYTOKINE KNOCK-OUT MICE

Tumor-necrosis-factor-α (TNF-α) is presumed to play an important role as a cytokine in the effector phase of inflammatory responses. Indeed, neutralizing antibodies block much of its deleterious effects in EAE (14). Nevertheless, this cytokine might play a dual role: TNF knock-out mice immunized with myelin–oligodendrocyte glycoprotein develop more severe EAE than wild-type mice, suggesting that TNF may also limit the extent of the inflammatory response in the central nervous system (15).

Although an important role has been attributed to IFN-γ as one the major products of Th1 cells, early experiments have shown that anti-IFN-γ antibodies exacerbate EAE. The use of IFN-γ and IFN-γ receptor knockouts made clear that IFN-γ can suppress the severity of disease in EAE (16,17) and collagen-induced arthritis (18,19). Accordingly, although initial studies suggested that Th2 cells may control the activity of EAE by the secretion of IL-4, IL-4 knockouts do not show an increased severity of EAE (20), suggesting that the potential of this cytokine as a therapeutic may have been overestimated.

After the identification of IL-12 as a bridge between innate and adaptive immunity and a key factor in Th1-mediated responses, many studies have provided evidence for an important role for this cytokine in autoimmunity as well. Because IL-12 is strongly inhibited by IL-10, it has been postulated that an IL-10/IL-12 immunoregulatory circuit determines the development of autoimmunity (21). Early studies showed inhibition of EAE (22) and collagen-induced arthritis (23) by anti-IL-12 antibodies or by IL-10 (10). IL-10- deficient mice develop more severe EAE (21,24) whereas IL-10 transgenic mice are resistant (25). IL-10-deficient mice are also more sensitive to colitis than wild-type mice and this sensitivity is abrogated by anti-IL-12 not by anti-IFN-γ (26). On the other hand, IL- 12-deficient mice are resistant to EAE; IFN-γ-deficient mice are sensitive to EAE unless treated with anti-IL-12 (21).

Although increasing evidence supports a major role for a proper balance between IL-10 and IL-12 in the development and progression of autoimmune disease, the exact roles of these cytokines still need further attention.

IV. CYTOKINES IN HUMAN AUTOIMMUNE DISEASE

Whereas animal models provide evidence (although sometimes conflicting) for the importance of a proper Th1/Th2 balance in the control of autoimmunity, the situation is less clear in humans. This is due in part to the fact that the availability of pathological material is mostly limited and consequently many studies are performed employing peripheral blood. Lack of consensus can thus in part be due to the fact that cytokine levels in body fluids do not always give the right impression of local autoimmune processes. Indeed, it is often difficult to find a correlation between continuing inflammatory processes in tissues, cytokine levels in body fluids, or cytokine production by circulating immunocompetent cells. Moreover, clinical trials aimed at the neutralization of harmful cytokines may sometimes be associated with unexpected, severe side effects.

A.TNF-

TNF-α is considered to be one of the most important cytokines involved in the pathology of many autoimmune diseases. Genetic studies have identified several TNF-region markers that are associated with susceptibility to rheumatoid arthritis (RA) and the severity of the disease (27). TNF-α levels are increased in plasma, synovial fluid, and rheumatoid joint tissues of such patients. TNF-α receptors are simultaneously expressed by synoviocytes, thereby suggesting a pathogenic role for TNF-α in cartilage destruction (28). TNF-α levels in blood are also correlated with the severity of RA and joint destruction (29). In view of its deleterious effects, clinical trials have been designed aiming at the neutralization of this cytokine. Two agents for neutralizing TNF-α are currently available: humanized anti-TNF antibodies and soluble human TNF receptors. Randomized phase II and III clinical trials with these anti-TNF reagents have demonstrated an acceptable safety profile and marked clinical efficacy in cases of RA that have not responded adequately to conventional therapy (30). Whether anti-TNF therapy also protects joints from structural damage is under investigation.

A pathogenic role for TNF-α has also been demonstrated in Crohn’s disease; antiTNF therapy was reported to have beneficial effects in moderate to severe disease, although some side effects were observed (31,32). The role of TNF-α in multiple sclerosis (MS) is less certain. The expression of TNF-α and its receptors is increased in acute MS lesions (33). Likewise, increased TNF-α mRNA expression has been found both in peripheral blood mononuclear cells (PBMC) and cells derived from cerebrospinal fluid (34,35). Moreover, increased levels of this cytokine are correlated with the severity and progression of the disease (36). A recombinant TNF receptor p55 immunoglobulin fusion protein has been used in a double-blind, placebo-controlled phase II clinical trial in MS patients to evaluate whether it would reduce the formation of new lesions. However, treatment failed to be beneficial: treated patients experienced more exacerbations than placebo controls (37). These contradictory observations might be explained by assuming a local beneficial role for TNF-α, as has been suggested by studies in EAE (16). An involvement of TNF-α has particularly been found in the pathogenesis of autoimmune diseases with a proinflammatory compound, such as those mentioned above: IDDM and autoimmune

thyroiditis. However, it might have some impact as well on Th2-mediated disease like myasthenia gravis (MG) or disease presumed to have both a Th1 and Th2 involvement such as systemic lupus erythematosus (SLE).

B.IFNand IL-12

These two cytokines are regarded as representative for Th1-mediated immune responses and they may play an early role in the pathogenesis of many autoimmune diseases. For instance, IFN-γ polymorphisms are associated with susceptibility to IDDM (38), whereas increased expression of IFN-γ has been found in MS, RA, and IDDM. Evidence for a harmful role for IFN-γ in MS comes from an early trial employing this cytokine as an antiviral agent for the treatment of MS; this trial had to be interrupted in view of serious aggravation of the disease (39). This, however, supports the idea that MS is largely Th1mediated.

Much attention has recently focused on IL-12, a heterodimer including p35 and p40 subunits, which are encoded by unrelated genes and regulated separately. RA patients show an increased expression of IL-12p40 mRNA and an increased production of both IL-12p70 and IL-12p40 by synovial fluid mononuclear cells and PBMC, compared to healthy controls; levels of IL-12 reflect disease activity in these patients (40). In Crohn’s disease, lamina propria mononuclear cells show an increased capacity to release bioactive IL-12 (41), whereas such cells are rare or undetectable in patients with noninflammatory gut disorders.

Increased expression of IL-12p40 mRNA has also been found in acute MS plaques (42). Furthermore, the IL-12p40 subunit is strongly increased in cerebrospinal fluid and serum of MS patients with a progressive course of the disease (43). A longitudinal study by our group has demonstrated that PBMC of MS patients express increased levels of IL12p40 mRNA during the development of active lesions; these increased levels precede the clinical relapses (44).

Altogether, cumulative data show that in particular IL-12 is an important determinant of Th1-mediated autoimmunity. As discussed below, experimental trials employing recombinant IL-10 largely support this idea.

An imbalance between IL-12 and IL-10 may also play a role in SLE. PBMC from SLE patients produce low levels of IL-12p40 in response to polysaccharides, which correlates negatively with disease activity (45). Dependent on the disease stage, serum levels of IL-12 may be increased in these patients (46).

C.IL-10

As an endogenous inhibitor of IL-12, low levels of IL-10 are considered to be unfavorable for Th1-mediated autoimmune diseases. Evidence has been obtained that these diseases are associated with impaired IL-10 production and an exaggerated production of proinflammatory cytokines. A decreased expression of IL-10 mRNA has been demonstrated in MS patients compared with controls; this decrease is even more pronounced in secondary progressive MS (44). The remission phase of the disease appears to be associated with increased IL-10 mRNA levels (47).

A significant negative correlation between IL-10 production and clinical activity is also found in RA (48). IL-10 inhibits the antigen-presenting capacity of synovial macrophages, even when they are efficiently activated, which further emphasizes the anti-in- flammatory potential of IL-10 in RA (49). IL-10 is a potent inhibitor of proinflammatory

cytokines, and is one of the candidate therapeutics for Th1-mediated diseases. However, initial clinical trials employing IL-10 in RA are not encouraging: unfortunately, so far no clinical improvement was achieved by treatment with different IL-10 dosages. Microscopic analysis of synovial tissue revealed no significant change in the extent of infiltration by inflammatory cells or the expression of cytokines in response to treatment (50).

Whereas IL-10 may have the potential to control Th1-mediated disease, it is suggested to promote Th2-mediated autoimmunity. IL-10 is involved in the pathogenesis of pemphigus vulgaris and bullous pemphigoid, which are both considered to be driven by Th2-cytokines. Blister fluid from patients with bullous pemphigoid contains elevated amounts of IL-10 (51). Serum levels of IL-10 are increased during the active stage of pemphigus vulgaris and correlate with increased titers of autoantibodies and disease severity.

IL-10 is also increased in patients with Sjo¨gren’s syndrome, in whom it may account for the overproduction of autoantibodies (52). Unexpectedly, IL-10 is more frequently increased in the incomplete (possible) form of Sjo¨gren’s syndrome than the complete (definite) form. Therefore, elevated IL-10 levels may either characterize an early stage of exocrine dysfunction or may be upregulated to contribute to limiting the severity of disease (53).

PBMC of patients with SLE and their first-degree relatives show increased numbers of cells that spontaneously produce IL-10. Moreover, cells have higher basal and induced IL-10 levels (54). This supports the hypothesis that IL-10 production may be genetically determined and predisposes toward the development of SLE. High innate IL-10 production underlies susceptibility for SLE, but not the severity of the disease (55).

The first studies employing IL-10 antagonists in SLE patients show that such a treatment may be beneficial in the management of refractory SLE and underline the involvement of IL-10 in the pathogenesis of this disease (56,57).

D.IL-4 and IL-13

IL-4 and IL-13 are two closely related Th2 cytokines, which share a common receptor component. Both inhibit the production of proinflammatory cytokines and chemokines by monocytes and promote human B-cell proliferation and activation. These cytokines are deficient in most of the Th1-driven diseases and overexpressed in Th2-type autoimmunity triggering aggravation of the diseases.

PBMC from IDDM patients have a decreased capacity of IL-4 production in comparison to healthy controls (58). Moreover, a downregulated production of IL-4 combined with a normal Th1-type cytokine secretion has been found in prediabetic humans, suggesting that the early stage of the autoimmune process in type-I diabetes in humans is associated with decreased function of Th2 cells rather than overactivation of Th1 cells (59). In RA, the levels of IL-4 and IL-13 are low both in synovial fluid and peripheral blood (60), whereas the expression of IL-4 mRNA in synovial fluid mononuclear cells and PBMC from these patients is below the detection level. The expression of IL-4 mRNA is almost absent from cerebrospinal fluid derived cells in MS patients (35). However, MS lesions show an increased expression of IL-4 mRNA by microglial cells and astrocytes and this possibly reflects an attempt to control the inflammatory response (61).

The immunomodulatory properties of IL-4, in particular its potential to polarize T- helper-cell responses to a Th2 phenotype, suggest that it may be of benefit in the treatment of Th1-mediated autoimmune diseases. Indeed, in several autoimmune models IL-4 was

shown to have inhibitory effects. Both the systemic administration of IL-4 and transgene expression of IL-4 in β-cells of the islets of Langerhans prevent the onset of diabetes in NOD mice (62). In adjuvant arthritis intra-articular retroviral gene treatment results in a significant reduction in paw swelling; moreover, treatment resulted in decreased bone destruction, as evidenced by x-ray (63). The therapeutic use of IL-4 in humans has been limited, in part due to dose-limiting side effects observed in clinical human trials. IL-4 together with IL-10 is currently being tested for the local liposome-mediated gene transfer in patients with severe IBD of the rectum. Local administration of cytokines has the advantage that it avoids systemic toxic side effects and is not associated with systemic inhibition of proinflammatory cytokines. Moreover, it allows for increased local concentrations over a prolonged period of time (64). Recently, it has also been demonstrated that gene therapydelivered IL-13 decreases the production of proinflammatory cytokines by synovial fluid mononuclear cells in humans, suggesting its therapeutic potential in the treatment of RA patients (65). Although IL-4 and IL-13 may be of benefit for Th1-mediated diseases, they appear to promote the progression of Th2-mediated autoimmune diseases. Perilesional skin biopsies from patients with bullous pemphigoid are characterized by the deposition of IL-4 and IL-13, which are relevant in the recruitment and adhesion of eosinophils within the dermal infiltrates. Thus these cytokines may play a role in the pathogenesis of blister formation in these patients (66).

V. CYTOKINES IN GRAVES’ OPHTHALMOPATHY

Although the pathology of Graves’ ophthalmopathy (GO) comprises the involvement of thyroid-specific antibodies that specifically bind to, or cross-react with, antigens of retroorbital tissues, an important local role for cytokines is increasingly appreciated (67,68). Locally produced cytokines, derived from infiltrating T cells or from the tissue itself, are responsible for the enhanced expression of MHC molecules, heat shock proteins and adhesion molecules in retro-orbital fibroblasts, adipocytes, myocytes, and endothelial cells. Infiltrating retro-orbital T cells from GO patients recognize autologous retro-orbital fibroblasts in an MHC-class I restricted manner. On the other hand, eye muscle and retroorbital fat tissue are also considered as two major targets of the autoreactive response, based on evidence that lymphocyte infiltration in these tissues is a prominent histological feature of GO (69). There are contradictory data about the exact role that cytokines play in GO. Some authors consider GO to be Th1-dependent, some Th2-mediated, whereas other data suggest the involvement of both Th1 and Th2 cytokines in the pathogenesis of this disease (70). In orbital tissues of GO patients a variety of proinflammatory cytokines has been found (71), either in frozen sections or in primary cultures of orbital fibroblasts. However, the cytokine expression pattern varies in time, which may reflect the course of the disease. The few available studies performed in a limited number of patients suggest a Th1 cytokine profile in the early stages, whereas Th2 cytokines might be involved in the progression of the disease (72). It has recently been shown that Th1 cytokines are mainly found in eye muscle tissue, whereas Th2-cytokines are detected mostly in orbital fat tissue. Moreover, a correlation between the expression of IL-6 mRNA and the orbital volume has been demonstrated (69). These results suggest that both Th1-like and Th2like immune responses may play a role in the development of ophthalmopathy. Accordingly, both Th1 and Th2 cytokines are increased in the serum of GO patients (70). However, cultures of PBMC from patients with GO produce significantly less IL-12 and significantly more IL-10 and IL-4 than PBMC cultures from healthy controls (64). This

suggests a Th2-bias in GO patients; however, it should be taken into account that the cells responsible for the pathology have migrated from the periphery into the target organ.

VI. CONCLUDING REMARKS

Many studies in experimental mouse models have pointed to a pivotal role of certain cytokines in the pathogenesis of autoimmune diseases. However, although cytokinedeficient mice have illustrated that certain cytokines are key to the pathogenesis of autoimmunity, they have also made clear that redundancy exists that may limit the success of targeted therapy. Moreover, many cytokines are pleiotropic in nature and may display disease-inhibiting as well as disease-promoting activities dependent on the stage of the disease. As a consequence, selective treatment with cytokines or their antagonists may result in a shift from Th1-associated to Th2-associated pathology and vice versa.

It is uncertain to what extent evidence from (inbred) animal models can be extrapolated to the clinical situation. Nevertheless, several human autoimmune diseases show a Th1-biased cytokine profile, whereas others show a Th2-biased cytokine profile, although this may depend on the stage/progression of the disease. Altogether, there is ample evidence that certain cytokines do not only reflect the pathogenesis of autoimmune disease but can also act as targets for therapy, provided that harmful side effects are eliminated.

ACKNOWLEDGMENTS

Dr. Lopatinskaya is supported by grant 940-33-047 from the Dutch Organization for Scientific Research. Dr. Nikolaeva is supported by a Du-Pre´ fellowship from the International Federation of Multiple Sclerosis Societies.

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Table 1 Glossary of Terms

Adhesion molecule

A cell surface protein that functions in leukocyte circulation, migration, attachment, or signal transduction.

α (alpha) 4

The alpha chain component of the heterodimeric integrin molecule with the number designation ‘‘4’’ as opposed to other distinct alpha chains (e.g., α4β7 as VLA-4); other distinct α chains include α5, α6, αL, and αM.

Avidity

Adhering strength. Extracellular matrix (ECM)

Loose milieu of glycoproteins and mucopolysaccharides (e.g., fibronectin and laminin) suspending and supporting cells and tissue structure.

Heterodimer

A molecule consisting of two different chain structures joined together. Immunoglobulin supergene family (IGSF)

A family of cell surface molecules whose structural motifs are similar to that of immunoglobulin chains.

Integrin

An adhesion receptor with a heterodimeric structure that binds to counterreceptors or extracellular matrix.

ICAM-1—Intercellular adhesion molecule-1

An IGSF adhesion molecule that binds LFA-1. LFA-1—Lymphocyte function antigen

An integrin adhesion receptor that binds ICAM-1. Ligand

The binding partner of a specified molecule. Selectin

An adhesion receptor with an N-terminal lectin-binding structure. Soluble adhesion receptor

An adhesion receptor that is solubilized in fluids such as serum or cerebrospinal fluid. VLA-4—Very late antigen 4

An integrin adhesion molecule receptor that binds to VCAM-1 or extracellular matrix. VCAM-1—Vascular cell adhesion molecule-1

An IGSF receptor expressed primarily on endothelium binding VLA-4.

II. CLASSIFICATION

Selectins are expressed on lymphocytes (L-selectins), platelets (P-selectins), and endothelium (E-selectins) and have the common structural motif of an N-terminal lectin binding domain. Rapid association and disassociation binding to glycosylated and sialylated ligands of the endothelium or extracellular matrix mediates leukocyte ‘‘rolling’’ and localization of leukocytes to inflammatory sites. This function facilitates leukocyte response to additional chemoattractants and cytokines, resulting in upregulation of additional integrin and IGSF adhesion molecules that serve as costimulatory molecules for leukocyte activation as well as transendothelial migration. L-selectin is expressed constitutively on all leukocytes and is critical to leukocyte adherence to peripheral lymph nodes and activated endothelium, through glycosylated molecules, glycam-1, CD34, and MadCAM-1. P-selectin also serves to bind leukocytes to endothelium and induces upregulation of inte-

grin molecule expression, leading to further adhesive events. E-selectin is upregulated on endothelium by interleukin-1 (IL-1) and tumor necrosis factor (TNF-α) and binds leukocytes to inflamed endothelium through sialylated mucin-like molecules. Adhesive actions of selectin are transient but chronologically essential to the formation of the stronger and more persistent adhesions of integrins and their ligands.

Integrin adhesion molecules are heterodimeric (α and β) molecules classified on the basis of their β subunit. Subunit recombination forms different receptors with different binding specificities. Integrins may be identified by a descriptive name (e.g., very late antigen-4 [VLA-4]), heterodimeric combination name (α4β1 ), or cluster designation name (CD49d/CD29). Following initial adhesive actions of selectins, integrins noncovalently bind their respective ligand and lead to transendothelial migration and more cell/cell or cell/extracellular matrix (ECM) interactions. These receptors also ‘‘integrate’’ or transduce extracellular information into the inside of the cell and serve as costimulatory signals, providing a secondary level of stimulation to effector cells. Integrins bind to a variety of extracellular matrix proteins or specific IGSF family members (see Table 2), are crucial to persistence of the inflammatory response, and have been associated with several autoimmune inflammatory diseases. Blockade of these receptors clearly abrogates the resultant damage of effector cells in many experimental autoimmune diseases (1). The most common integrin/ligand pairs are VLA-4/VCAM, LFA-1/ICAM-1 or ICAM-2, α4β7 / MadCAM-1 or VCAM, all of which mediate primarily lymphocyte tissue interactions and progressive inflammatory responses. VLA-4 and LFA-1 are prototypical integrin adhesion molecules and have been found to play crucial proinflammatory adhesive roles in several immune and autoimmune inflammatory responses. Both are expressed on leukocytes, bind to endothelial or target cell IGSF receptors and fibronectin, and are upregulated by activation.

Table 2 Primary Adhesion Molecules and Their Ligands

Members of the IGSF of receptors share structural amino acid immunoglobulin-like motifs and serve as ligands to integrin receptors. ICAM-1 and ICAM-2, VCAM, and LFA-2 and LFA-3 are the primary IGSF receptors, with ICAM-1 and VCAM-1 being expressed on a wide variety of cells and found predominantly in inflammatory responses, binding leukocyte LFA-1 and VLA-4, respectively. An additional unclassified adhesion molecule includes CD44, which adheres to hyaluronate in extracellular matrix, serves as a memory T-cell marker, and has a multitude of other functions involved in lymphocyte adherence, activation, and formation of memory responses (1).

In summary, selectins, integrins, and IGSF adhesion molecules are expressed by a wide variety of cells. Adhesion molecule expression and changes in avidity, in concert with humoral inflammatory mediators and effector cells, coordinate the initiation, progression, and intensity of a localized inflammatory response.

III. REGULATION OF ADHESION MOLECULE EXPRESSION

Homeostatic balance between adhesive forces determines leukocyte circulation, homing, and transendothelial migration to inflammatory sites, and persistence of the immune response. Expression of adhesion molecules is essential for cell/endothelial, cell/cell, and cell/extracellular matrix adhesion and their expression may be constitutive, regulated, or both. Cytokines such as TNF-α and IL-1 typically upregulate expression of adhesion molecules; however, adhesion molecule persistence may be dependent upon other cytokines such as interferon-gamma (INF-γ), IL-4, and IL-6, which further induces or prolongs expression (1). Furthermore, activation of cells by cytokines, chemoattractants, or costimulatory molecules upregulates either the expression of adhesion molecules or their avidity for their respective ligands. Additional pathophysiological attractants modulating effector cell adhesion and migration in various immune and autoimmune reactions include, but are likely not limited to, leukotriene B4, C5a, histamine, thrombin, substance P, vasoactive intestinal peptide, calcitonin, and endotoxin. Lack of cytokine or activation stimulation leads to transient adhesion interactions and the effector cells. Adhesion molecules are also found in circulating, soluble forms. Soluble adhesion molecules are elevated and circulate in inflammatory disease, but their pathophysiological or theoretical blockade of adhesive interactions or their subsequent clinical significance have not been clearly established. An additional level of adhesive control likely occurs at the level of cell-specific distribution of receptors (e.g., CD4 vs. CD8 lymphocytes). Hence, regulation of adhesive interactions is a complex orchestration of several cascading events leading to persistence and accumulation of effector cells prior to significant inflammatory response and clinical and disease presentation. Elucidation of the temporal events leading to tissue effects and disease presentation will likely identify pathophysiological targets of therapeutic importance.

IV. ADHESION MOLECULE EXPRESSION IN THYROID

AUTOIMMUNITY

The development of autoimmune thyroid disease (AITD) is very complex and appears to involve the expression and adhesive interactions of adhesion molecules. Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations in AITD.

Since initiation of a specific immune response also involves antigen-receptor independent interactions between accessory molecules, such as adhesion molecules, the expression of adhesion molecules on thyroid epithelial cells (TEC) has been examined. TEC derived from patients with Graves’ disease expressed ICAM-1 and LFA-3 in vitro after stimulation with recombinant human interferon-γ (IFN-γ) or human tumor necrosis factor-alpha (TNF- α). However, TEC from nontoxic goiter could be induced to express ICAM-1, but not LFA-3 under similar conditions. Both ICAM-1 and LFA-3 were highly expressed in vivo in Graves’ disease, but not in nontoxic goiter. These findings suggest that TEC are able to express adhesion molecules and support the concept that adhesion molecules play a role in the TEC-specific immune response in autoimmune thyroiditis (3).

In similar studies, examination of thyroid specimens of Graves’ disease (GD) thyroid glands and control thyroid glands demonstrated that patients with GD also had enhanced expression of intercellular adhesion molecule-1 (ICAM-1) on capillary endothelial cells around the thyroid follicles and on postcapillary endothelial cells in lesions with aggregates of mononuclear cells. ICAM-1, LFA-1, and VLA-4 were found on thyroid-infiltrating mononuclear cells. Postcapillary vascular endothelial cells also expressed increased ELAM-1, but not VCAM-1. VCAM-1 and ELAM-1 were, however, detected on the den- dritic-like cells in the germinal centers of lymphoid follicle-like areas. No significant expression of these adhesion molecules was detected on normal thyroid glands. These results suggest that the LFA-1/ICAM-1 and ELAM-1 pathways may be responsible for the migration of mononuclear cells into the thyroid glands of patients with GD. Furthermore, the VLA-4/VCAM-1 adhesive interactions may play a critical role in the cellular interactions that lead to the formation of B-memory cells and the excess production of antibodies in Graves’ disease (4). These results are further supported by the finding that in Graves’ disease thyroid samples, infiltrating memory CD4 cells expressed high levels of LFA-1 and LFA-2, but expression levels of VLA-4 and VLA-5 did not differ significantly from controls (5).

In a separate study, a high proportion of GD intrathyroidal T lymphocytes expressed increased LFA-1, VLA-1, VLA-4, VLA-5 and integrin receptors compared with peripheral blood T lymphocytes from the same patients. The expression of ICAM-1 was increased in EC from GD thyroids. In addition, an upregulated expression of VCAM-1 was found in EC in GD thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM-1 and VCAM-1. In addition, most intrathyroidal mononuclear cells expressed the ICAM-3 adhesion molecule. This enhanced expression of ICAM-1 and VCAM-1 by thyroid EC in GD likely reflects their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. These data further imply that the LFA-1/ICAM-1, ICAM-3, and VLA-4/VCAM-1 adhesion pathways are relevant in localizing and perpetuating the autoimmune response in GD thyroids (6). The complex chronological and cell-specific nature of adhesion molecule expression and interaction are likely responsible for the development of Graves’ thyroiditis. However, the dynamic progression of this disease remains to be elucidated.

V. ADHESION MOLECULES IN GRAVES’ OPHTHALMOPATHY

The role of adhesion molecules in GD suggests a similar role in Graves’ ophthalmopathy, although such participation remains to be elucidated. The immunological perspectives of GD opthalmopathy are discussed in subsequent chapters of this book. Nevertheless, the inflammatory process and tissue proliferation within the orbit in GD ophthalmopathy sug-

gest that adhesion and cell/cell or cell/extracellular matrix interaction are crucial to the pathogenesis of orbital disease. Work described in this book has recently extended our knowledge of the evolution and perpetuation of this orbital immune process, including orbital T-cell repertoires, candidate orbital antigens, potential target and effector cells, and their role in the extrathyroidal manifestations of this autoimmune thyroid disease (7,8). It is possible that adhesion receptors discussed in this chapter are pivotal to GD orbital pathogenesis, but their expression, regulation, and role have not been thoroughly characterized. Immunotherapy of this disorder may involve blockade of cell/cell or cell/extracellular matrix adhesive interactions. For example, immunoglobulin superfamily member VCAM-1, which recognizes VLA-4 integrin, is expressed on all leukocytes except neutrophils. Blockade or inhibition of VCAM-1/VLA-4 interaction is expected to have therapeutic potential in treating various inflammatory disorders and autoimmune diseases since this adhesion pathway has a major influence on eosinophil, lymphocyte, and monocyte trafficking. Strategies currently used to selectively inhibit the VCAM-1/VLA-4 adhesive pathway include soluble VCAM-Ig fusion protein, peptide antagonists, antisense oligonucleotides, natural products, and neutralizing antibodies to VCAM-1 or α4 integrin (9,10). Further characterization of adhesion molecule expression in early Graves’ ophthalmopathy (i.e., prior to the establishment of a chronic process), the development of sensitive diagnostic techniques, and the definition of effective antiadhesive immunotherapy may provide a basis for efficacious intervention in this problematic autoimmune disease.

REFERENCES

1.McMurray RW. Adhesion molecules in autoimmune disease. Semin Arthritis Rheum 1996; 25:215–233.

2.Mojcik CF, Shevach EM. Adhesion molecules: a rheumatologic perspective [see comments]. Arthritis Rheum 1997; 40:991–1004.

3.Zheng RQ, Abney ER, Grubeck-Loebenstein B, Dayan C, Maini RN, Feldmann M. Expression of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-3 on human thyroid epithelial cells in Graves’ and Hashimoto’s diseases. J Autoimmunity 1990; 3:727– 736.

4.Nakashima M, Eguchi K, Ida H, Yamashita I, Sakai M, Origuchi T, Kawabe Y, Ishikawa N, Ito K, Nagataki S. The expression of adhesion molecules in thyroid glands from patients with Graves’ disease. Thyroid 1994; 4:19–25.

5.Ishikawa N, Eguchi K, Ueki Y, Nakashima M, Shimada H, Ito K, Nagataki S. Expression of adhesion molecules on infiltrating T cells in thyroid glands from patients with Graves’ disease. Clin Exp Immunol 1993; 94:363–370.

6.Marazuela M, Postigo AA, Acevedo A, Diaz-Gonzalez F, Sanchez-Madrid F, de Landazuri MO. Adhesion molecules from the LFA-1/ICAM-1,3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves’ and Hashimoto’s thyroid glands. Eur J Immunol 1994; 24:2483–2490.

7.Heufelder AE. Retro-orbital autoimmunity. Baillieres Clin Endocrinol Metab 1997; 11:499– 520.

8.Heufelder AE, Spitzweg C. [Pathogenesis of immunogenic hyperthyroidism and endocrine orbitopathy] Pathogenese der immunogenen Hyperthyreose und endokrinen Orbitopathie. Internist (Berl) 1998; 39(6):599–606.

9.Oppenheimer-Marks N, Lipsky PE. Adhesion molecules as targets for the treatment of autoimmune diseases. Clin Immunol Immunopathol 1996; 79:203.

10.Foster CA. VCAM-1/alpha 4-integrin adhesion pathway: therapeutic target for allergic inflammatory disorders. J Allergy Clin Immunol 1996; 98(6 Pt 2):S270–S277.

TSH, thyroid stimulating hormone; TSHR, TSH receptor; hCG, human chorionic gonadotrophin.

When injected into mice this ‘‘stimulator’’ induced thyroid hormone released over a much longer time scale than TSH (5). This so-called long-acting thyroid stimulator (LATS) was subsequently shown to be an IgG (6), which provided what is still the best example of type V hypersensitivity: the production of antibodies that activate cell surface receptors

(7). TSHR antibodies have proven to be very difficult to study for a number of reasons. They are present at much lower concentrations in serum than other thyroid autoantibodies, making derivation of human monoclonal antibodies problematic (8). Also, their binding to the receptor is extremely conformation-dependent, so that attempts to study their interaction with recombinant TSHR have been difficult. Finally, animal models have only been established in the last few years (9) and information derived from these models is only just beginning to provide fresh insights into the pathogenesis of Graves’ disease.

II. PREDISPOSITION

Although it is now clear that TSHR stimulating antibodies are the proximal cause of Graves’ disease, it is far less obvious what predisposes certain individuals to the condition. In common with most autoimmune disorders, a combination of genetic and environmental factors is involved, with different relative contributions of each between individuals. Some of these factors, especially the inheritance of certain HLA alleles, are common to several autoimmune disorders, and this largely explains the frequent association of Graves’ disease with other conditions (Table 2). The alternative explanation for these associations is that certain autoantigens have cross-reactive epitopes that trigger a dual autoimmune process. The evidence for this explanation is slim at present (10), although thyroid-associated oph-

thalmopathy may be the result of an orbital antigen that is cross-reactive with, rather than identical to, a thyroid autoantigen.

The best evidence for a genetic predisposition in Graves’ disease comes from twin studies, which have shown a 20–30% concordance rate in monozygotic twins, a much higher figure than in dizygotic twins that share similar exposure to environmental factors (11). However, this concordance rate implies at best only a modest genetic contribution to Graves’ disease, supported by the relatively low frequency in siblings or other family members. A huge effort has been made to determine the genetic basis for Graves’ disease, largely using population-based association studies that have often lacked sufficient statistical power to confirm genuine associations. Moreover, such studies of candidate genes are capable of detecting only relatively small effects that may have less biological relevance than the kind of effect that might be detected by whole genome screening, or searching for new disease-specific loci.

Association studies have confirmed a role for HLA alleles in Graves’ disease, especially the HLA-DR3 specificity in white patients. Other HLA associations outside the DR region in general reflect linkage disequilibrium with HLA-DRB1 alleles rather than any additional and independent effect (12,13). The other confirmed genetic association with Graves’ disease is polymorphism of the CTLA-4 gene (14). The latter is expressed in T cells and downregulates their responses when the B7 costimulatory signal is engaged. The contribution of CTLA-4 polymorphism to susceptibility is somewhat less than HLA and the same polymorphisms are associated with other autoimmune disease, such as type 1 diabetes mellitus.

Linkage studies have demonstrated a number of new candidate genetic loci for Graves’ disease (15), but much larger numbers of families need to be studied to determine the exact importance of these loci before exact mapping and identification of the responsible genes will become worthwhile. Other candidate loci continue to be examined and may also yield insights into pathogenesis. One example is the association of Graves’ disease with a polymorphism of the IL-4 gene that encodes a key Th2 cytokine involved in regulating antibody production (16). There does not appear to be a major, consistent genetic predisposition to the development of ophthalmopathy that is separate from that seen in Graves’ thyroid disease, although more work is needed in this regard (17).

One of Caleb Parry’s original patients had suffered an accident in the weeks before her diagnosis and, since then, stress has been a leading contender among the environmental factors that could cause Graves’ disease. Recent retrospective surveys have tended to confirm this impression, with major life events and minor hassles both being more frequent in the year prior to disease onset than in matched controls (18–20). Prospective studies would provide more powerful evidence, but are logistically impossible. However, the wellknown effects of stress on the neuroendocrine system, and in turn on the immune system, provide a plausible biological explanation for this association.

Smoking weakly predisposes individuals to the development of Graves’ disease but is a major risk factor in the development of ophthalmopathy (21). Whether these two effects are linked by a single adverse action on the immune system is unknown; smoking also has direct effects on the thyroid and, via hypoxia, on fibroblasts (22). An increase in dietary iodine may also precipitate Graves’ disease and once again this is likely to be a complex effect, with both iodine-induced hyperthyroidism (the Jod-Basedow phenomenon) and initiation or exacerbation of the autoimmune process playing roles (23,24).

Drugs such as lithium and amiodarone (which contains iodine) have been associated with the development of Graves’ disease. A remarkable frequency of Graves’ disease is

seen after administration of T-cell monoclonal antibodies in the treatment of multiple sclerosis (25). This may provide an important model in which to study disease development from its earliest stage. The female predominance of Graves’ disease (and other autoimmune disorders) is most likely the result of modulation of the autoimmune response by estrogens and other hormones. These endocrine effects may account for the frequent emergence of Graves’ disease in the year after delivery (26).

III. THE AUTOIMMUNE RESPONSE

Whatever the combination of predisposing factors, the autoimmune response in Graves’ disease is far more complex than the mere production of TSHR-stimulating antibodies. Many abnormal immunological phenomena have been described and it is often unclear which are primary and how they contribute to pathogenesis. The B-cell response to TSHR is T-cell-dependent and TSHR-reactive T cells have been described in Graves’ disease, although there is no single dominant TSHR epitope that could serve as a target for therapeutic modification (27). As in many autoimmune disorders, T-cell tolerance to TSHR appears incomplete in healthy individuals (28), suggesting the operation of mechanisms in addition to central deletion or anergy in maintaining nonresponsiveness to the receptor. The existence of specific T-suppressor cells, peripheral tolerance through the presentation of TSHR by major histocompatibility complex (MHC) class II molecules in the absence of costimulation, resulting in anergy, and clonal ignorance may all be involved (29,30).

In addition to MHC class II molecules, thyroid cells in Graves’ disease express a wide array of immunologically active molecules, including cytokines, adhesion molecules, complement-regulatory proteins, and CD40. These are induced by the lymphocytic infiltrate that characterizes Graves’ disease and other types of autoimmune thyroid disease, via cytokines and sublethal complement activation. It is likely that this intrathyroidal response exacerbates and perpetuates the autoimmune responses (31), resulting in the thyroid being a major site of thyroid autoantibody synthesis, with contributions from the draining lymph nodes and bone marrow (32).

Most patients with Graves’ disease have evidence of autoimmunity to other thyroid autoantigens besides the TSHR, including autoantibodies against thyroglobulin (TG), thyroid peroxidase (TPO), and the sodium–iodide symporter. Some patients with Graves’ disease initially present with hypothyroidism or run a fluctuating course between this and hyperthyroidism, most likely due to the presence of autoantibodies to the TSHR that block rather than stimulate (33,34). It is not known why there should sometimes be rapid transit between these two antibody species. However, responses against other thyroid autoantigens may contribute to the clinical picture by modifying the thyroid responsiveness to TSHR-stimulating antibodies, and in particular may account for the late development of permanent hypothyroidism in 10–20% of patients successfully treated with antithyroid drugs.

IV. CLINICAL FEATURES AND DIAGNOSIS

A brief description of the systemic manifestations of Graves’ disease is provided in Table 2. It is helpful to differentiate between the features common to all types of thyrotoxicosis and those specific for Graves’ disease (Table 2). The latter include the presence of ophthalmopathy that can be detected clinically in 50–60% of patients with Graves’ disease, although a much larger proportion have subclinical evidence of disease detectable by orbital

Table 2 Clinical Features of Graves’ Disease

Features of thyrotoxicosis

Irritability, dysphoria

Heat intolerance, sweating, warm and moist skin

Fatigue, weakness, tremor

Palpitations, tachycardia, atrial fibrillation in the elderly

Weight loss with increased appetite

Muscle weakness, myopathy

Diarrhea, polyuria

Oligomenorrhea, loss of libido, gynecomastia

Diffuse hair loss

Features of Graves’ disease

Diffuse goiter (found in some other types of hyperthyroidism)

Ophthalmopathy

Dermopathy, especially pretibial myxedema

Acropachy

Splenomegaly, lymphoid hyperplasia

Family or personal history of a related autoimmune disease

Autoimmune hypothyroidism

Type 1 diabetes mellitus

Addison’s disease

Vitiligo

Pernicious anemia

Alopecia areata

Celiac disease, dermatitis herpetiformis

Myasthenia gravis

imaging or other methods (35,36). A minor degree of lid retraction, 1–2 mm, may occur in any type of hyperthyroidism, secondary to sympathetic overactivity, but the eyelid retraction seen in Graves’ disease is usually more extensive and due to additional pathological changes in the levator palpebrae superioris muscle (37).

Establishing the diagnosis is a two-stage process. First one must confirm that the patient with suspicious signs and symptoms is truly thyrotoxic and, second, establish that Graves’ disease, rather than an alternative process (Table 1), is the cause. The presence of primary thyrotoxicosis is usually readily verified by the combination of a suppressed circulating TSH level and elevated free T4 level. Around 2–5% of patients with the earliest stage of any type of hyperthyroidism may have an elevated free T3 but normal free T4 level (T3 toxicosis).

Once thyrotoxicosis is confirmed, the diagnosis of Graves’ disease can be made by the presence of eye signs or dermopathy; highly suggestive features include a diffuse goiter on palpation, a strong family or personal history of associated autoimmune disorders (Table 2), and positive circulating TG or TPO antibodies. It would seem most logical to confirm the diagnosis by measuring TSHR-stimulating antibodies, but commercially available assays generally measure only antibody binding to the TSHR and hence will include autoantibodies that have a blocking or neutral effect on the receptor (38). Methods to detect TSHR-stimulating antibodies require bioassays that are not widely available. Improved, second-generation assays for TSH-R-binding antibodies have recently been introduced and their role in diagnosis might increase (39), but at present it is debatable

whether measurement of these antibodies offers a real diagnostic advantage (40). In contrast, the use of TSHR antibody measurement in predicting the likelihood of neonatal thyrotoxicosis in a pregnant woman with Graves’ disease is now well established (40). Patients with apparent Graves’ disease but no detectable circulating TSHR antibodies are sometimes seen; misdiagnosis, poor assay sensitivity, and exclusively intrathyroidal production of TSHR antibodies could be responsible.

The vigor with which other diagnostic tests are pursued generally depends on local practice. In cases of doubt, radionuclide tests, showing high thyroidal uptake of radioiodine isotopes or 99m Tc and a diffuse goiter on scintiscanning, are useful in excluding the diagnosis of toxic nodular goiter or destructive thyroiditis. Perhaps the clearest indication for such testing is in the postpartum period, in which the appearance of thyrotoxicosis could be the result of destructive autoimmune thyroiditis or Graves’ disease (41).

V.RELATIONSHIP TO OPHTHALMOPATHY AND DERMOPATHY

The exact relationship between Graves’ thyroid disease and the extrathyroidal manifestations of eye disease and dermopathy remains unclear—are these separate but closely associated disorders or part of the same spectrum? In favor of the former is the presence of ophthalmopathy without Graves’ disease in up to 10% of cases (35). However, half of these patients have autoimmune hypothyroidism and many of the remainder have more subtle evidence of thyroid disease, such as the presence of thyroid autoantibodies or a goiter (42). It is also clear that Graves’ disease may occur several years after the first manifestations of ophthalmopathy (and vice versa), so that the existence of ophthalmopathy without thyroid disease over a protracted period needs to be demonstrated if one is attempting to confirm the existence of two separate processes.

At present, the balance of evidence seems to support the concept of a complex but single spectrum of disease, albeit including thyroid autoimmunity generally rather than Graves’ disease alone. For reasons yet to be established, but possibly related to autoreactivity to the TSHR, patients with Graves’ disease have an almost constant, subclinical ophthalmopathy, whereas autoimmune hypothyroidism is far less commonly associated. We still need to clarify whether some Graves’ patients escape orbital disease totally, because present indirect assessment methods may not be sufficiently sensitive to confirm minor orbital involvement (35,36). Dermopathy is much less common than ophthalmopathy and may be a more general manifestation of the same pathogenic process (43). However, generalized subclinical dermopathy in Graves’ disease does not seem to be present (44). Almost all patients with dermopathy have Graves’ disease plus ophthalmopathy, suggesting a very close relationship with this type of thyroid autoimmunity (45). Thyroid acropachy is the rarest and most extreme extrathyroidal manifestation of Graves’ disease; its pathogenesis is obscure but the condition is strongly linked with the presence of dermopathy.

VI. OVERVIEW OF TREATMENT

Although apparent remission may occur in mild cases of Graves’ disease, the duration of such improvements is uncertain. The mortality from untreated Graves’ disease is 10–30%, with considerable morbidity due to cardiovascular and neuropsychiatric complications as well as osteoporosis. Initial treatment is usually with a thionamide antithyroid drug in

Europe and Japan, whereas radioiodine (131 I) is generally preferred as first-line treatment in North America (46). Subtotal or near total thyroidectomy is a third alternative, and is particularly useful in patients in whom there is a large goiter or any suspicion of coincidental thyroid malignancy. Age and gender are important determinants of success with any treatment (47). A summary of the main benefits and side effects of these treatments is given in Table 3.

Antithyroid drugs cause a fall in TSHR and other thyroid antibody levels, an effect most likely related to an immunomodulatory action on the thyroid cells (48). As a result, permanent remission may occur in up to 50% of patients who receive a course of antithyroid drugs, lower rates being likely in those with large goiters or in areas of high iodine intake. In patients whose condition relapses after drug treatment, radioiodine is usually the treatment of choice. However, some physicians remain cautious about administering radioiodine to children and adolescents, because of the theoretical risks of malignancy, especially of the thyroid (49,50). Caution should also be exercised when radioiodine is given to patients with ophthalmopathy, especially smokers, since there can be worsening of the eye problem (51). Prophylactic corticosteroids, given for a short period immediately after radioiodine, prevent this complication.

The management of Graves’ disease in pregnancy requires particular attention, as excessive antithyroid drugs given to the mother can cause fetal hypothyroidism and goiter. The lowest possible dosage of drug, given by the titration regimen, is used to maintain maternal free T4 levels in the upper part of the reference range. The autoimmune process usually ameliorates during pregnancy and antithyroid drugs can often be stopped during

Table 3 Overview of Treatment for Graves’ Disease

the last trimester. Close follow-up is needed after delivery, as the mother is at increased risk of relapse during the year after delivery. Also, in the last trimester, the risk of neonatal thyrotoxicosis must be established (40).

Although present treatments for Graves’ disease are effective, this is often at the expense of hypothyroidism, and complications can be serious, especially in socioeconomically disadvantaged populations (52). Optimal treatment would restore euthyroidism promptly and permanently, without the need for lifelong follow-up or thyroxine therapy. Future advances in our understanding of the immunopathogenesis of Graves’ disease may make such treatment an attainable goal.

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predilection for Graves’ disease, although Asian patients are more likely to present with hypokalemic periodic paralysis.

The symptoms of hyperthyroidism relate to excessive sympathomimetic activity and increased catabolic activity: nervousness (irritability and emotional labiality), increased perspiration, heat intolerance, palpitations, weight loss, dyspnea, fatigue and weakness, increased appetite, hyperdefecation, menstrual dysfunction, and eye symptoms.

Signs of hyperthyroidism include goiter (thyroid enlargement), tremor, hyperkinesis, eye signs (exophthalmos, lid retraction, lid lag), tachycardia (resting rate 90; atrial fibrillation), smooth and velvety skin, moist and warm hands, onycholysis (‘‘Plummer’s nails’’), and thyroid bruit. In the elderly these classic signs are often missing and one sees cardiac problems (heart failure and tachydysrhythmias), weight loss, weakness, or anorexia. The striking absence of the adrenergic and hyperkinetic symptoms is sometimes called apathetic hyperthyroidism. When a patient with hyperthyroidism presents with fever, altered mental status, and acceleration of thyrotoxic signs and symptoms, the clinical diagnosis of thyroid storm is made. This is often precipitated by stress such as surgery, infection, and pneumonia in patients who have previously had undiagnosed hyperthyroidism. The diagnosis of hyperthyroidism is very easy when the clinical disease is obvious. When the signs and symptoms are minimal, the laboratory results are helpful. The serum T4(RIA)/T3U or free T4 levels are usually elevated, as is the serum level of T3 (determined by radioimmunoassay [RIA]). The T3 (RIA) or FT3 is usually elevated to a greater extent than the T4 (RIA) and is sometimes the only abnormal laboratory finding (i.e., T3thyrotoxicosis). Serum TSH levels are suppressed or not measurable ( 0.05 U/mL). Hyperthyroidism has generalized effects on various tissues producing numerous abnormal laboratory studies that revert to normal when the hyperthyroidism resolves. Such findings include hypercalcemia, abnormal liver function studies, and increased turnover and degradation of metabolites (e.g., an increase in urinary 17-OHCS, making one suspect Cushing’s syndrome). Subclinical hyperthyroidism diagnosed with low levels of TSH ( 0.1 U/ mL with normal free T4 and free T3 levels) on screening chemistry panel is usually asymptomatic.

The presenting manifestations of classic Graves’ disease are unmistakable and leave a lasting impression on the examiner. The affected young woman is anxious, nervous, and fidgety: sitting still and keeping hands and feet stationary is nearly impossible. Her speech is quick and generally she is quite talkative. She complains of weakness and fatigue, and generally likes the weight loss and the ability to eat excessively without gaining weight. Palpitations and ‘‘racing heart’’ are easily elicited symptoms, as are heat intolerance and a preference for cooler environs than other people. She often is perspiring and her skin literally radiates heat. Such patients are truly ‘‘wired.’’ Prominence of the eyes, a stare appearance, or diplopia could be symptoms. A friend or relative may notice changes such as goiter or increased nervousness well before the patient does. The history usually reports a 5–20 lb weight loss, but it can be more severe. However, about one in ten thyrotoxic patients actually gain weight because of increased caloric intake in excess of their catabolism. Frequent bowel movements and occasionally frank diarrhea are a concern.

On examination, the patient with a classic case demonstrates a diffuse enlargement of the thyroid (goiter). This occurs in over 95% of the patients who present with Graves’ defect (autoimmune process causing hyperthyroidism). On occasion, the goiter may be somewhat nodular. Eye manifestations may vary from normal with minimal eye stare and lidlag, to severe with limitation of extraocular movements, proptosis, and periorbital edema. In the author’s experience, only 5% of the people who initially present with thyro-

Figure 1 Onycholyses of 4th and 5th digits. Note recession of nail bed.

toxicosis due to Graves’ defect have the infiltrative eye disease. Cardiac bruits are important signs of Graves’ disease, but they are often absent, and thrills are even less common. Both indicate increased blood flow and signify thyrotoxicosis due to overactivity of the thyroid gland. They are not found in other causes of hyperthyroidism such as subacute thyroiditis, toxic adenoma, and multinodular goiter.

In patients with Graves’ thyrotoxicosis the skin is warm, moist, and hyperemic, particularly in the hands. Some patients flush easily. The handshake often reveals moist and warm hands. Patients have onycholysis, that is, changes in the nail bed in which the skin separates from the subungual margin leaving a concave margin on the fourth and fifth fingers (see Fig. 1). Onycholysis (Plummer’s nails) is a useful sign of chronic hyperthyroxinemia regardless of cause (seen in other causes of long-standing hyperthyroidism as well). Vitiligo can be found and occasionally some patients report hyperpigmentation. Examination of the lower legs rarely reveals pretibial nonpitting edema, thickening of the skin, accentuation of the hair follicles, and often erythema (Fig. 2a). Pretibial myxedema is uncommon in Graves’ disease and usually occurs in patients with ophthalmopathy and often after the eye disease is stable. Even more uncommon are formation of nodules or tuberous changes (Fig 2b). Other areas of trauma may rarely show the myxedematous changes. The cause of such changes remains unknown. Thyroid acropachy results in clubbing of the fingers with soft tissue changes. Radiographs may show subperiosteal new bone formation. This is also a very rare finding in Graves’ disease.

II. ORGAN SYSTEM REVIEW OF HYPERTHYROIDISM

As mentioned above, mental and nervous findings vary and are striking. Graves’ disease patients cannot stay still. They often do not sleep well and at times have been found to clean the house at night. Some patients have increased fatigue, fine tremor, and hyperkinetic reflexes. There is occasionally a significant change in personality leading to psychosis.

The muscle system is often affected, particularly when these patients lose weight. The weight loss is indiscriminate: fat and muscle loss lead to weakness and fatigue. Facial examination reveals wasting of the temporal muscles and often weakness with an inability to get up from a chair without use of the hands. Occasionally, patients will have concomi-

tant myasthenia gravis. Periodic paralysis is a rare manifestation of hyperthyroidism. In this condition there is a sudden paralysis with hypokalemia, and transport of acid to the cellular tissue leading to profound weakness and paralysis. The paralysis reverses as potassium levels return to normal. The cause of this is unknown, but has an increased frequency among people of Asian origin. The effects from thyroid hormone on the bone lead to increased absorption and calciuria. The changes of the bone are frequent and apparent with longstanding hyperthyroidism. Bone disease may present as a stress fracture. The changes seen on bone DEXA scans have been reported and are generally reversed once the hyperthyroidism has resolved.

The cardiovascular changes are due to increased sensitivity to circulating epinephrine and norepinephrine. Palpitations or tachycardia are common symptoms. The pulse is rapid and bounding and systolic blood pressure is often elevated. There is a characteristic wide pulse pressure between diastole and systole. The precordium is often dynamic and bounding in the patient with classical hyperthyroidism. Flow murmurs are often heard. A short, high-pitched murmur in the left second intercostal space has been described in the severely thyrotoxic patient. Premature beats are common. Atrial fibrillation may occur; this may be continuous or paroxysmal. Congestive heart failure is a rare manifestation today. Hyperthyroidism can precipitate stroke and risk of myocardial injury is increased. Beta-blockers produce a dramatic effect on the cardiovascular system in reducing palpitations/increased heart rate and ameliorate sweating and tremor in the symptomatic patient.

The changes in the respiratory system are manifested by shortness of breath and dyspnea on exertion.

Hematological changes of thyrotoxicosis are generally mild. The hemoglobin and hematocrit are in the lower limits of the normal range because the blood volume is increased. In some patients, there is often lymphocytosis. There may be generalized lymphadenopathy and in about 5% of patients, the spleen tip can be palpitated.

Gastrointestinal findings of thyrotoxicosis include increased appetite and weight loss. As mentioned above, there is occasional weight gain with increased caloric intake. Nausea and vomiting are rare except in hyperemesis gravidarum. The transit time for food is decreased and increased frequency of bowel movements is common. The liver may be palpable, especially when there is heart failure. There are occasionally abnormal results of liver function studies including levels of alkaline phosphatase and bilirubin.

The reproductive system, particularly in women, is affected by decrease in menstrual flow and shortening or elongation of the menstrual cycle. In men, gynecomastia may develop as a consequence of increased conversion of testosterone to estradiol while they are hyperthyroid. The other endocrine organs including the adrenal cortex have no obvious clinical symptoms. There are measurable changes to the adrenal gland, which are often hypertrophic with increased production of 17-OHCS to match the increased clearance due to hyperthyroidism. It is not uncommon to have elevated ACTH levels with slightly increased pigmentation.

Other metabolic effects include increased oxygen consumption. The basal metabolic rate is no longer available. The effects on lipid metabolism have been well recognized and the serum cholesterol level is suppressed. There appears to be increased clearance of cholesterol into bile acids and excretion of bile. Even triglyceride levels are lower.