Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Corneal APCs in Inflammation

Microbial products stimulate the immune system by interacting with toll-like receptors (TLRs) on APCs and other cells. The interaction between TLRs and their ligands activates APCs toward maturity. In addition, the release of proinflammatory cytokines, including interleukin (IL)-1b, GM-CSF, tumor necrosis factor (TNF)-a, and lipopolysaccharides (LPS), or heat-shock proteins from dying cells, creates a microenvironment that activates immature DCs. DCs themselves are also important producers of these proinflammatory cytokines, which act in an autocrine fashion to promote DC activation and maturation. Resident immature epithelial LCs and stromal DCs in the central cornea can significantly upregulate maturation markers, including MHC-II and costimulatory markers, within 24 h after induction of inflammation (Table 2).

In addition to the resident APC population, APCs are also recruited into the cornea from the limbal areas through upregulation of IL-1 and TNF-a during inflammation (Figure 2). In general, the migration of APCs to peripheral tissues requires the concerted activity of cell adhesion molecules and chemotactic factors. Cell adhesion molecules regulate both cell–cell and cell–matrix interactions, while chemokines provide directionality to local and infiltrating APCs. Suppression of these cytokines leads to downmodulation of APC migration into the cornea. IL-1 and TNF-a can act in concert to recruit APCs from the limbus into the cornea by mediating the expression of cell adhesion molecules and chemokines. Recruitment of macrophages into the cornea plays a crucial role in inducing inflammatory neovascularization by supplying or amplifying signals essential for pathological hemangiogenesis. Macrophages, but not DCs, physically

Normal cornea

CD45+,

CD11c+, CD11b−,

CD8a−, GR-1−,

MHC class II+,

CD80+, CD86+

Mature

CD45+,

CD11c+, CD11b−,

CD8a−, GR-1−,

MHC class II−,

CD80−, CD86−

Immature

Langerhans cell

.Mallen P

contribute to lymphangiogenesis under pathological conditions and express lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions. Macrophages are capable of forming tube-like structures that express LYVE-1 and podoplanin, and are actively involved in lymphangiogenesis.

The Function of APCs in Corneal

Transplantation

The process of corneal transplant rejection includes an induction phase, called the afferent arm, and an expression phase, called the efferent arm. In the afferent arm the host becomes sensitized to the donor antigens by means of APCs , presenting antigens to T cells. This process can take place through two different pathways. The direct pathway, involving donor APCs that sensitize the host directly and the indirect pathway, involving host APCs that move toward the graft, take up donor antigens, and then present these antigens to T cells in draining LNs. While both direct and indirect alloreactive T cells can mediate graft rejection, host sensitization to donor antigens of corneal grafts occurs through both pathways of sensitization, especially in high-risk corneal grafting, where transplantation occurs in an inflamed bed. CD40 is a critical costimulatory molecule expressed by many APCs (including corneal DCs and LCs), whose ligation by CD154 leads to overexpression of other costimulatory molecules, and IL-12 – critical factors in priming a T-cell response. Blocking the interaction of CD40 with CD40 ligand/CD154 can block both the direct and indirect pathways of allosensitization, by preventing T-cell priming, but without promoting active tolerance.

Disruption of the eye–LN axis in the setting of corneal transplantation has been shown to lead to both complete prevention of host allosensitization and the indefinite survival of corneal grafts, demonstrating the functional relevance of corneal APC trafficking to draining LNs. Both donor and host-derived corneal APCs are capable of migrating efficiently to host LNs within 24 h after corneal transplantation. Since the cornea is alymphatic, this is achieved through sprouting of new lymphatic vessels into the cornea upon inflammation, and through migration of APCs toward the lymphatic-rich limbus and conjunctiva. Signaling through vascular endothelial growth factor receptor-3 (VEGFR-3) is critical for DC access to lymphatics, and selective blockade of this pathway can impair DC flow to LNs and the induction of alloimmunity, leading to reduction in the rate of graft rejection. On the one hand, migration of DC to LNs is facilitated by the interaction of the chemokine receptor CCR7 on their surface, with CCL21 secreted by the lymphatic vessels. On the other, CCR1 and CCR5 have been shown to be responsible for the recruitment of immature DCs in inflamed tissues, through their ligands

CCL4, CCL5, and CCL7, with only stromal DCs expressing CCR1. Blockade of CCR1 leads to significant reduction in the rate of graft rejection, indicating the important role of stromal DCs in the alloimmune response. Interestingly, depletion of donor APCs before transplantation, however, does not have a significant effect on promoting graft survival, even in the high-risk setting, suggesting that these cells, other than promoting immunization, may also be relevant in the induction of maintenance of tolerance.

The Function of APCs in Microbial Keratitis

Herpetic stromal keratitis (HSK) is an inflammatory disorder induced by herpes simplex virus (HSV)-1 infection and is characterized by T-cell-dependent destruction of corneal tissues. The number of MHC-IIþ LCs present in the central areas of the cornea has been shown to correlate with the degree of corneal damage. Virally induced migration or maturation of LCs in the cornea precedes the development of HSK. Induction of LC migration into the central cornea before HSV-1 infection results in an accelerated and enhanced delayed-type hypersensitivity (DTH) response to HSV-1 antigens, and in an increased severity of HSK. Contrary, depletion of DCs reduces the incidence and severity of HSK, suggesting a role for DCs in the induction of a T-cell response. These findings have led to the conclusion that HSV-1 infection results in de novo migration of LCs from the limbus, which in turn might play a role in the immunopathology of HSK though presentation of antigens to T cells in the infected cornea.

Pseudomonas aeruginosa is an opportunistic pathogen associated with sight-threatening keratitis, whose outcome is largely determined by the host inflammatory response. Specific susceptible mouse strains challenged with P. aeruginosa undergo corneal perforation, while other strains are resistant. While induction of LCs into the central cornea of already susceptible strains before infection does not alter the outcome of disease, induction of LCs into the central cornea of resistant strains converts these to a susceptible phenotype. LCs in these mice express the costimulatory molecule B7-1, enhancing their capacity to present antigen to T cells. Further, macrophages control resistance to P. aeruginosa corneal infection through regulation of neutrophil number and apoptosis, bacterial killing and balancing proand anti-inflammatory cytokine levels.

Conjunctival APCs

In the naive conjunctiva, predominantly MHC-IIþ DCs are consistently detected from birth in the subepithelial layer and substantia propria. There are species and strain-specific differences in the numbers of these cells, with 200–400 LCs mm 2 in humans as compared to 100–150 LCs mm 2 in mice, with rat and guinea pig numbers intermediate between these two. Further, the

number of LCs is not static and increases with age. Moreover, a significant variability in LC density is also found in different regions of the conjunctiva. The largest number of epithelial LCs is found in the palpebral and inferior fornical region, followed by the medial and inferior epibulbar conjunctiva. DCs of the substantia propria are distributed most densely in the superior and medial epibulbar conjunctiva. This variability within the conjunctiva is interesting, and may be related to exogenous antigenic challenge secondary to the direction of normal tear drainage or to microenvironmental differences within the conjunctiva. Finally, macrophages have a density of 6.5 cells/mm2 in the tarsal epithelium and 32.2 cells/mm2 in the tarsal substantia propria, with similar numbers in the bulbar conjunctiva.

Role of APCs in Allergic Eye Disease

Allergic eye disease is a spectrum of diseases that share a common initiating mechanism and pattern of inflammation. While B cells and T cells are the mediators of the allergic immune response, DCs are the actual initiators and modulators of this response. Mast cells mediate the effector phase of the allergic response, whereas DCs are critical in determining the nature of the allergic response. During the sensitization phase, allergens encounter DCs on the ocular surface. Following allergen challenge, there is a marked influx of conventional DCs and plasmocytoid DCs into the subepithelial layer and throughout the substantia propria. DCs then process and present the allergen to T cells in association with the MHC-II. These T cells are then polarized in favor of the development of allergen sensitivity.

APCs of the Uvea

The uveal tract, the vascularized middle layer of the eye, consists of the iris, ciliary body, and choroid. It contains rich networks of F4/80þ APCs that reside and traffic through the eye (Figure 1 and Table 3). These populations include large numbers of macrophages and to a lesser extent, immature DCs, maintaining local immunological homeostasis, and play a role in inflammatory processes and immune-mediated diseases. Dendritiform and pleiomorphic macrophages are distributed in a regular array within the rat iris and ciliary body stroma (600–700 cells/ mm2). The iris contains a network of MHC-IIþ DC

Table 3 Antigen-presenting cells of the normal uvea

(400–600 cells/mm2) within the iris stroma and ciliary epithelium, with few DCs in the uveal tract expressing costimulatory molecules. BM chimera studies demonstrate replenishment of BM-derived cells starting at 2 weeks, with almost complete turnover by 8 weeks in the iris stroma and posterior iris surface. Replenishment rates in the uveal tract are similar in the choroid, iris, and ciliary body stroma, although DCs in the ciliary epithelium replenish at a slightly slower rate starting at 4 weeks.

Intraocular DCs, after contact with aqueous humor of the anterior chamber, migrate through the trabecular meshwork to the spleen. Additionally, they begin to secret IL-10 and transforming growth factor (TGF)-b in an autocrine fashion, thereby creating a microenvironment that is rich in tolerance-inducing mediators. These DCs promote the effective suppression of T-cell-dependent inflammatory reactions in the lymphoid organs, inducing sufficient levels of tolerance. DCs in the tissues lining the anterior chamber represent a rich network of APCs and are the most likely candidates for transmitting antigenspecific signals from the anterior chamber in vivo and in experimental models such as anterior chamber-associated immune deviation (ACAID).

Anterior Chamber-Associated Immune

Deviation

The eye receives immune protection against pathogens, while avoiding inflammatory and immunological damage. The selective inability to develop delayed-hypersensitivity responses following antigen invasion into the anterior segment of the eye is highly dependent on DCs, which form the basis of an extraordinary phenomenon called ACAID. The immune response begins with intraocular capture of antigen by specialized ocular F4/80þ APCs in the iris/ ciliary body. ACAID-inducing APCs create a microenvironment rich in TGF-b and IL-10, but deficient in IL-12, thus failing to upregulate CD40. These APCs then migrate through the trabecular meshwork and the venous circulation, preferentially to the marginal zone of the spleen, where they become part of an intricate and highly specific cluster of immune cells. The end result is the emergence of a population of antigen-specific T-regulatory lymphocytes that return to the eye and suppress DTH response. A similar process has been described in the vitreous and other posterior compartments of the eye.

Role of APCs in Age-Related Macular

Degeneration

AMD is the most common cause of legal blindness in elderly individuals of industrialized countries. The presence of complement factor proteins in drusen in AMD eyes and single nucleotide polymorphisms (SNPs) for complement factor regulatory genes in individuals with AMD implicate inflammation as an important component

in this disease. Choroidal macrophages are proposed as key players in the removal of age-related accumulation of extracellular debris at the choroidal–retinal interface. Observation of aging mice deficient in CCR2 or CCL-2 indicates that defective clearance or scavenging mechanism by resident choroidal macrophages may, in part, be responsible for the presence of drusen deposits at the choroidal–retinal interface. In addition to the potential role of choroidal macrophages, the discovery of agedependent accumulation of subretinal microglia has recently implicated this population of cells as potential initiators of neovascularization and photoreceptor damage.

Role of APCs in EAU

Resident APCs of the normal human uvea are endowed with the complete LPS receptor complex and are strategically positioned in perivascular and subepithelial locations for surveying blood-borne or intraocular LPS. LPS may act as an adjuvant by activating APC maturation in the presence of the putative uveitogenic self-antigens and thus mediate the breakdown of peripheral tolerance resulting in the induction of an autoimmune response. APCs that capture self-antigens, present them to autoreactive T cells and induce T-cell tolerance by deletion or anergy, as these APCs are relatively immature. TLRs, however, can convert tolerogenic signals to activating signals by promoting APC maturation.

DCs and macrophages act as local APCs in the induction of uveoretinitis. Specifically, MHC-IIþ DCs appear at the time of disease onset and continue to be recruited during the inflammatory process, indicating their role in initiation if EAU. MHC-II macrophages expressing antigens, however, are prominent during the peak phase of tissue damage in the retina and choroid. Depletion of these cells causes a delay in the onset and a reduction in the severity of EAU.

APCs of the Retina

The presence of the blood–retinal barrier and a predominantly immunosuppressive intraocular environment contribute to the suppression of local immune responses to retinal antigens. Nevertheless, retinal inflammation is not uncommon. Retinal antigen-specific T cells must encounter cognate antigen on APCs within the retina to initiate retinal inflammation. Several distinct populations of myeloid-derived cells reside in the retina, namely, the more prevalent retinal microglia (CD11bþF4/80þCD8aþ CD80þMHC-IIþ), as well as perivascular macrophages (Figure 1 and Table 4). Perivascular macrophages have poor antigen-presenting capability and are not thought to be absolutely essential for disease induction. Further, a population of BM-derived MHC-IIþ 33D1þ DCs has been identified in mice, of which small numbers reside in

Table 4 Antigen-presenting cells of the normal retina

the peripheral margin and juxtapapillary regions. Of note, the distribution and phenotype of these DCs within the retinas differs between mouse strains exhibiting different disease susceptibility. In EAU-resistant mice, DCs are MHC-II (low/–). Conversely, DCs are MHC-IIþ in EAU-susceptible mice.

Microglia reside in the nerve fiber/ganglion cell layer, inner plexiform layer, and outer plexiform layer of the retina. Resting microglia play various roles in host defense, immunoregulation, and tissue repair and rapidly increase in numbers in response to various insults in the retina. Retinal microglia respond to photoreceptor light-induced injury or degeneration by migration from the inner retinal layers toward the photoreceptor layer and subretinal space, where they phagocytose photoreceptor debris and remain for prolonged periods. Resident host microglia residing in the inner retina are the principal source of the phagocytic microglia that accumulate in the photoreceptor layer and subretinal space during aging or retinal degeneration.

BM chimera studies demonstrate reconstitution of myeloid cells in the retina beginning at 4 weeks. Migrating cells are evident at the juxtapapillary margin and migrating deeper into the retinal layers, and almost completely replenish between 2 and 6 months, depending on the strain. Turnover of microglia within the retinal microenvironment occurs at a much slower rate than other peripheral tissue macrophages. When photoreceptor degeneration is induced, large numbers of microglia/macrophages are observed in the injured retina, starting at 12 h after injury, and peaking at 24 h. In addition, the number of MHC-IIþ cells in the retina increases greatly after retinal injury. In response to retinal damage, numerous BM-derived cells migrate to the retina from the ciliary body, optic nerve, and retinal vessels and differentiate into microglia. The higher rate of immunologic activation and the increased specificity to the damaged site appear to be the characteristic features of BM-derived microglia.

APCs of the Sclera

BM-derived cells have been described in the sclera. In BM chimeras, BM-derived cells replenish the sclera

Table 5 Antigen-presenting cells of the normal sclera

through limbal vessels and optic nerve vessels, migrating into the equatorial zone. These cells are CD11cþ or CD11bþ DCs and macrophages are found among the scleral fibroblasts (Figure 1 and Table 5). During EAU, significant infiltration of these BM-derived cells takes place into the sclera, contributing to the ocular immune response.

Conclusions

The integrity of the visual system in the face of everchanging immune challenges is vital. The unique immune homeostasis and immunological status of the eye and ocular surface is fascinating and continuously evolving. It is compelling that most of the APCs described herein were only discovered less than 10 years ago, emphasizing how much still has to be learned about APCs and their function in the eye. The constitutive presence of APCs in the ocular tissues has significant implications for a variety of infectious, autoimmune, and inflammatory responses in the eye. Since the presence of resident ocular APCs was largely unknown until very recently, many paradigms have already been shifted and many more will need to be rethought in the future. Understanding the mechanisms

that lead to APC maturation, activation, and trafficking may well lead to novel approaches in the induction of tolerance, autoimmunity, and vaccine therapy.

See also: Adaptive Immune System and the Eye: Mucosal Immunity; Adaptive Immune System and the Eye: T Cell-Mediated Immunity; Dry Eye: An ImmuneBased Inflammation; Dynamic Immunoregulatory Processes that Sustain Immune Privilege in the Eye; Immunosuppressive and Anti-Inflammatory Molecules that Maintain Immune Privilege of the Eye; Innate Immune System and the Eye; Penetrating Keratoplasty.

Further Reading

Dana, R. (2004). Corneal antigen-presenting cells: Diversity, plasticity, and disguise: The Cogan lecture. Investigative Ophthalmology and Visual Science 45: 722–727.

Hamrah, P. and Dana, R. (2007). Corneal antigen-presenting cells.

Chemical Immunology and Allergy 92: 58–70.

Hamrah, P., Huq, S. O., Liu, Y., Zhang, Q., and Dana, M. R. (2003). Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. Journal of Leukocyte Biology 74: 172–178.

Kezic, J. and McMenamin, P. G. (2008). Differential turnover rates of monocyte-derived cells in varied ocular tissue microenvironments.

Journal of Leukocyte Biology 84: 721–729.

McMenamin, P. G. (1999). Dendritic cells and macrophages in the uveal tract of the normal mouse eye. British Journal of Ophthalmology

83: 598–604.

Novak, N., Siepmann, K., Zierhut, M., and Bieber, T. (2003). The good, the bad and the ugly – APCs of the eye. Trends in Immunology 24: 570–574.

Streilein, J. W. (2003). Ocular immune privilege: Therapeutic opportunities from an experiment of nature. Nature Reviews Immunology 3: 879–889.

Xu, H., Dawson, R., Forrester, J. V., and Liversidge, J. (2007). Identification of novel dendritic cell populations in normal mouse retina. Investigative Ophthalmology and Visual Science

48: 1701–1710.

Glossary

Autoimmunity – An immune response of an organism against any of its own tissues, cells, or cellular components. Diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and dry eye are considered autoimmune based. CD4þ T cells – T helper cells (Th), a subgroup of lymphocytes that play an important role in establishing and maximizing the capacity of the immune response against invading extracellular pathogens, for example, bacteria and parasites. The CD4þ T cells bearing T-cell receptors that recognize self-antigen, that is, autoantigen, contribute to the immunopathogenesis of several autoimmune diseases, for example, rheumatoid arthritis, multiple sclerosis, and dry eye.

Dry eye – An ocular surface immune-based inflammatory disease resulting from an unstable tear film composition mediated by dysfunction of a complex Lacrimal Function Unit (LFU: cornea, conjunctiva, lacrimal glands, and meibomian glands), which causes damage to the interpalpebral ocular surface and is associated with symptoms of ocular discomfort. Dry eye is also known as lacrimal keratoconjunctivtis (LKC) and most recently, dysfunctional tear syndrome (DTS).

Desiccating stress (DS) – Following exposure to DS in low humidity (<40%) mice display similar clinical and histopathological features to human patients with dry eye, including rapid and coordinated upregulation of proinflammatory cytokines, decreased tear production and goblet cell number, surface epithelial apoptosis, and increased cellular infiltration, for example, CD4þ T cells into the LFU. Goblet cells – Glandular simple columnar conjunctival epithelial cells that function to secrete mucus. Lacrimal glands – Glands located in the upper, distal portion of the orbit of each eye that secrete the aqueous layer of the tear film.

Lacrimal function unit (LFU) – The lacrimal functional unit is composed of the lacrimal glands (both main and accessory), the ocular surface (cornea, conjunctiva, goblet cells, and meibomian glands), and the interconnecting innervation that coordinates afferent (ocular surface to the brain) and efferent (brain to the ocular surface tissues and associated glands) signals. The LFU is responsible

for maintaining the quantity and quality of the tear fluid.

Meibomian glands – Sebaceous glands at the rim of the eyelids responsible for the supply of sebum, an oily substance that prevents evaporation of the eye’s tear film.

MHC class II (major histocompatability complex class II) – This is responsible for presenting antigen fragments to T helper cells by binding exclusively to the T-cell receptor present of the surface of CD4þ T cells. The MHC class II is involved in presentation of antigen derived from extracellular pathogens, thus providing specificity for the generation of adaptive immunity. The MHC class II molecules bearing selfantigen (autoantigen) may trigger activation of autoreactive CD4þ T cells and autoimmunity.

Defining the Problem

Epidemiology of Dry Eye

In 1993 the National Eye Institute (NEI)/Industry workshop formally defined dry eye as a ‘‘disorder of the tear film due to tear deficiency or excessive evaporation, which causes damage to the interpalpebral ocular surface and is associated with symptoms of discomfort.’’ Recently, we and others proposed a more comprehensive definition of dry eye based on the increasing evidence demonstrating that ocular surface immune-based inflammation and ocular surface epithelial diseases result from dysfunction of a complex Lacrimal Function Unit (LFU) and the resultant unstable tear film. To provide guidelines for selection of treatment, the Delphi panel of experts coined the term dysfunctional tear syndrome (DTS) based on symptoms and signs (not tests) of dry eye disease.

Dry eye is a highly prevalent condition and one of the leading causes of visits to ophthalmologists and optometrists in the United States. Epidemiologic studies have reported that dry eye affects up to 11% of people 30–60 years of age and 15% of those 65 years of age or older. As many as 12 million Americans have moderate to severe dry eye and this number is likely to increase as the population ages. Dry eye affects 0.1–33% of the worldwide population; the wide range of variability is dictated by the study and diagnostic criteria used.

Dry Eye Syndrome in Periand

Post-menopausal Women

Dry eye is more common in women than men (2:1) and the incidence increases with age. The role of sex hormones in dry eye has been reported in several studies. For example, data from 36 995 female health professionals ranging from age 49 to 89 years old (estimated at least 3.2 million (or 7.8%) women aged 50 years and older) suffer from dry eye disease in the US. The incidence appears to be higher among older women (9.8%, 75 years) than women under 50 years of age (5.7%). The incidence of clinically significant rosacea is also higher among aging females and occurs in approximately 30% of menopausal women. In fact, it is predicted that up to 75% of peri-menopausal women with facial rosacea will develop ocular involvement.

Estrogen may have detrimental effects on the tear film and could influence the development of dry eye; although the ratio between estrogens and androgens may be a better indicator. It was reported that women who receive hormone replacement therapy, especially with unopposed estrogen therapy, have an increased risk of developing dry eye disease. On the other hand, androgen deficiency and/or imbalance in estrogen–androgen levels are also associated with dry eye. Along these lines, androgen deficiency as seen in Sjo¨gren syndrome and Sjo¨gren’s syndrome keratoconjunctivitis sicca (KCS) occurs almost exclusively in women. Furthermore, women who suffer from premature ovarian failure lack both estrogens and androgens and exhibit more ocular surface damage and dry eye-related symptoms than age-matched controls.

Patients on Anti-androgen Therapy

Androgenic hormones play an important role in supporting the secretory immune function of the lacrimal glands and meibomian glands. The meibomian glands are the main androgen target organs on the ocular surface. Androgen deficiency that may occur during menopause, aging in both sexes, autoimmune disorders (e.g., Sjo¨gren’s syndrome, Systemic Lupus Erythematosus, rheumatoid arthritis, RA), complete androgen insensitivity syndrome (i.e., women with dysfunctional androgen receptors, congenital androgen insensitivity syndrome), and the use of anti-androgen medications (e.g., for prostatic cancer or hypertrophy) is associated with meibomian gland dysfunction, tear film instability, and a significant increase in dry eye signs and symptoms. Studies showed that anti-androgen treatment is paralleled by significant changes in the fatty acid profiles of neutral lipid fractions in meibomian gland secretions. Conversely, treatment with androgens has been reported to alleviate dry eye conditions and stimulate tear flow in Sjo¨gren’s syndrome patients.

Clinical Features of Dry Eye

Chronic Pain

Ocular surface neuropathy in dry eye

Ocular surface pain and discomfort in severe sicca disease may partially result from the well-documented neuropathy associated with Sjo¨gren’s syndrome, which is categorized with the neuropathies associated with connective tissue disease. Indeed, clinical evidence has shown that peripheral sensory neuropathy may be an important presenting sign for Sjo¨gren’s patients. In accordance, ocular surface discomfort is often the initial motivation for dry eye patients to visit the ophthalmologist. In affected individuals, ganglioside-specific antibodies are found in peripheral nerves, dorsal root ganglia, and dorsal roots, and inflammatory cells are localized within the ganglia. Although the trigeminal system has not been studied in as much detail, the ocular surface discomfort of dry eye may be a form of sensory neuropathy; however, this theory requires confirmation. Small diameter myelinated and unmyelinated axons in the cornea are potential targets for peripheral nerve disorders, and inflammatory cells infiltrating the ocular surface are well documented in dry eye. These cells, in combination with ganglioside-specific antibodies and other neural proteins, could cause local degeneration of small diameter axons and axon terminals. Cranial neuropathies may be more common in Sjo¨gren’s syndrome than is currently recognized, and the dysthesias associated with the cornea may indicate an inflammatory neuropathy within the trigeminal system.

Comorbidities

Patients with lacrimal keratoconjunctivitis (LKC) typically experience ocular discomfort. The most common symptoms include scratchiness, grittiness, foreign body sensation, burning, and itching; these symptoms are exacerbated by prolonged visual activity (e.g., viewing a video display terminal) and environmental stresses, such as low humidity and air drafts. The LKC patients often complain of blurred and fluctuating vision that stimulates increased blink frequency, an unconscious response to clear the visual field. Together, these symptoms contribute to severe ocular fatigue and many patients report that they are unable to read or concentrate for more than a few minutes at a time.

Also, LKC can cause considerable ocular morbidity. The thinned and unstable precorneal tear layer and the altered corneal epithelial barrier function that accompany LKC are major risk factors for sterile keratolysis (loss of uppermost layer of cells in cornea) and microbial keratitis (infection of the cornea). Severe and recurrent corneal ulceration mediated by LKC can ultimately lead to reduced vision, blindness, and in severe cases, loss of the eye.

Pre-existing LKC is an important cause of complications following corneal surgery. Complications include

penetrating keratoplasty and LASIK, and may lead to decreased vision, pain, epithelial and stromal wound healing problems, haze, ulceration, and predisposition to microbial infections. Surgical amputation of the corneal sensory nerves that drive glandular secretion, a direct consequence of LASIK and other refractive procedures, negatively impacts the integrated ocular surface secretory gland functional unit. This exacerbates pre-existing LKC and most likely results in new cases of dry eye.

Quality of Life Impact

The LKC symptoms significantly impact quality of life documented by utility scores. Utility scores quantify how many years a subject would give up from the end of his/her life in exchange for avoiding a particular malady. Utility scores for dry eye were found to be similar to those from patients with angina. The chronic and unremitting nature of dry eye syndrome can lead to despair, depression, decreased productivity, and in some cases permanent job disability. The physical and psychological impact of LKC symptoms is similar to that experienced by patients with other chronic regional pain syndromes, such as those affecting the lower back.

How We Secrete Normal Tears

The Lacrimal Functional Unit

The ophthalmic pathology seen in Sjo¨gren’s syndrome and chronic dry eye surrounds an immune-based inflammatory disruption of the LFU (Figure 1). The LFU is composed of the ocular surface (cornea, conjunctiva, conjunctival blood vessels), the lacrimal glands (main and accessory (Wolfring and Krauss)), and the interconnecting innervations (V, VII). This tear secreting reflex is also modulated by hormonal and immune factors. The role of the LFU is to secrete a precise tear film composition that maintains a homeostatic environment around the epithelial cells of the ocular surface.

The general role of the LFU in homeostasis and disease

The purpose of the tightly controlled ocular surface environment is to preserve corneal clarity and vision. The main and accessory lacrimal glands, the corneal limbus, and the meibomian glands provide the vital supportive function to protect the sensitive epithelial surfaces of the conjunctival and corneal tissues from environmental

Long ciliary

nerve Nasociliary nerve

Short ciliary nerves

Lacrimal Carotid nerve artery

V Nucleus

Frontal nerve

injury that results in pain and decreased visual acuity. The function of the LFU is to control secretion of tear constituents that help sustain a stable, anti-infective, and epithelial supportive tear layer essential for optimal optical performance. Signals emanating from ocular surface sensory nerves supply continuous input into the CNS that tells the brain what changes are occurring within the ocular surface milieu. The brain then sends signals to the specialized support tissues, for example, lacrimal and meibomian glands that are programmed to secrete the optimal tear quantity and composition.

The process by which normal tears are secreted is initiated following corneal nerve stimulation. The process occurs unconsciously and in response to many stimuli; however, environmentally induced dry spot formation is thought to be among the most common. Out of necessity for survival, evolution has shaped the cornea to become the most densely sensory nerve innervated epithelial surface in the body. Conduction of pain originates from myelinated and unmyelinated nerves that terminate in the cornea, limbus, and conjunctival epithelium. Neural receptors in the cornea are free nerve endings that terminate in all of the corneal epithelial layers and are protected from direct irritation by zonula occludens and the tear mucin gel. Afferent (ocular surface to the brain) nerve traffic through the ophthalmic branch of the Trigeminal Nerve (V) enters the central nervous system in the area of the pons (midbrain) and the para spinal sympathetic tract. These signals are integrated with cortical and other inputs and are then transmitted to the efferent (brain to ocular surface tissues and associated glands) secretomotor impulses resulting in secretion of the homeostatic tear-film components.

Tear secretion by the lacrimal gland also occurs in response to neural stimulation. The acini, ducts, and blood vessels of the lacrimal gland are innervated by parasympathetic, sympathetic, and sensory nerves. The initial signal originates from the parasympathetic cholinergic nerves via acetylcholine release, which then binds to M3 muscarinic acetycholine receptors on the basolateral cell membrane of secretory epithelia. At the same time, vasoactive intestinal peptide (VIP) binds to VIPergic receptors, and norepinephrine, a sympathetic neurotransmitter binds to a1- and b-adrenergic receptors. Neural innervation of the accessory lacrimal glands has also been reported and fibers positive for CGRP and substance P are associated with secretory tubules, interlobular and excretory ducts, and blood vessels. However, the degree of neural influence over accessory lacrimal glands is still being elucidated.

Sensory, sympathetic, and parasympathetic neuropeptides are present in the ocular surface tissues and associated glands. Conjunctival goblet cells have a secretory response to the parasympathetic cholinergic muscarinic output from the pterygopalatine ganglion. Goblet cells express M3-muscarinic receptors on their membranes. The M1 and M2 receptors are located throughout the

conjunctiva. The presence of a1A- and b3-adrenergic receptors on conjunctival goblet cells suggests the presence of sympathetic innervation. In addition, transmission electron microscopy of meibomian glands revealed the presence of unmyelinated axons with granular and agranular vesicles. Substance P- and CGRP-positive axons have also been identified, but their function is uncertain, as these neurological peptides would be expected to conduct information into, rather than away from, the CNS. It is predicted that parasympathetic fibers innervating the meibomian glands are indeed present at higher levels. Parasympathetic neurotransmitters neuropeptide Y and VIP have been found around the meibomian glands, as well as tyrosine hydroxylase in sympathetic axons, implicating that both types of autonomic nerves may play an important role in stimulating lipid secretion onto the ocular surface.

Patients with LKC commonly complain of constant corneal sensations normally described as a gritty, sandy, or itchy. These complaints are usually accompanied with a pathophysiological state that indicates a chronic state of inflammation and a disadvantageous change in tear film composition. Infiltrating inflammatory cells within the ocular surface tissues have been reported in dry eye. These inflammatory cells, in addition to ganglioside-specific antibodies and other neural proteins, may result in regional degeneration of small diameter axons and their terminals. Chronic dysfunction of the LFU results in a shift toward inflammation and persistent psychological distress.

Events on the Ocular Surface

Environmental Impact on the Ocular Surface

The immune response is designed to defend against stress and/or microbial assaults on the ocular surface and paradoxically may also contribute to autoimmunity. Along these lines, regulatory mechanisms have evolved to modulate the afferent and efferent arms of the immune response to preserve tissue and limit activation of autoreactive lymphocytes following acute inflammation. The afferent events leading to cellular immunity include antigen processing and presentation by ocular surface antigen presenting cells (APCs) and migration of these cells to the draining lymph nodes. Afferent immune processes are modulated by a wide variety of anti-inflammatory factors that include cellular, for example, T regulatory cells (Tregs), and diffusible factors, for example, transforming growth factor beta (TGF-b) and interleukin (IL)-1 receptor antagonist, that favor protective immunity without breaking self-tolerance. The afferent arm of the immune response dictates the efferent response, the phase involved with antigen driven homing of primed and targeted lymphocytes to tissue-specific inflammatory sites. The efferent response is initiated in the secondary

lymphoid organs and amplified on the ocular surface via cell-to-cell interactions between lymphocytes and APCs; activation and differentiation of lymphocytes and migration to the ocular surface is tightly regulated to control the efferent immune response. Indeed, immunoregulation on the ocular surface is the result of the coordinated effort between a wide variety of immune players. However, when these mechanisms are compromised the ocular surface may become susceptible to chronic and/or autoimmunemediated ocular disease, such is the case with dry eye.

Afferent arm of the immune response: immunoregulation

Redundant mechanisms regulate the afferent immune response to guard against activation and infiltration of autoreactive lymphocytes to the ocular surface tissues. For instance, there is a predominance of intraepithelial lymphocytes, for example, CD4þ/CD8þTregs and gamma delta Tcells in the normal conjunctival epithelium; these cells are thought to harbor immunoregulatory functions similar to those found in other mucosal tissues, such as the intestine. The conjunctiva and cornea are also covered by mucin, which forms a barrier, guarding against unwarranted infiltration of inflammatory cells into the epithelium. Furthermore, the cornea lacks lymphatic and blood vessels, mature APCs, and resident Tcells, thereby reducing the incidence of chronic inflammation and bystander cell damage on ocular surface following an acute inflammatory insult.

The tear fluid also contains high concentrations of soluble immunoregulatory factors that help maintain homeostasis before, during, and after environmental challenge. For example, androgenic hormones provide an immunosuppressive umbrella to help protect the secretory function of the lacrimal and the meibomian glands. In addition, the corneal epithelium expresses vascular endothelial growth factor (VEGF) receptors that function in part to sequester soluble VEGF, which ultimately reduces the stimulus for neovascularization after ocular surface challenge. Neurotrophic factors produced by the limbal corneal epithelia, such as glial cell line-derived neurotrophic factor (GDNF) also appear to have immunoregulatory activity. Transforming growth factor beta (TGF-b), a cytokine that can inhibit the function of APCs and effector T-cell proliferation, is secreted by goblet cells and is found in high levels within the tear fluid. Indeed, the presence of TGF-b may bias conjunctival APCs to activate Tregs instead of effector T cells, thereby preventing the activation/infiltration of autoreactive T cells. In addition, interleukin 1 receptor antagonist (IL-1RA) mutes the effects of the potent proinflammatory cytokine IL-1. The action of tissue inhibitor of matrix metalloproteinase (TIMP-1) inhibits matrix metalloproteinases (MMPs), which play a dominant role in promoting immune cell infiltration into ocular surface tissues during inflammation.

Efferent arm of the immune response: immunoregulation

The efferent arm of the immune response includes several mechanisms to prevent spurious activation and infiltration of autoreactive lymphocytes to the ocular surface during acute inflammation. It is critically important that the immune response is sufficient to eliminate the current threat, and then tempered to avoid chronic inflammation and tissue destruction. Similar to the afferent arm, recent studies have shown that the efferent arm of the immune response is also regulated by the concerted effort of Tregs, anti-inflammatory cytokines, and other factors vital for preventing autoimmunity.

Activation and differentiation of CD4þ and CD8þ Tregs in the secondary lymphoid organs are critical for limiting bystander tissue damage and maintaining selftolerance and are emerging as important efferent immune modulators in the eye. Indeed, CD4þ Tregs present in C57BL/6 mice decrease clinical and histopathological disease in a mouse model of dry eye, which are exacerbated when mice are depleted of CD4þCD25þFoxP3þ Tregs. In addition, in vitro expanded CD4þCD25þFoxp3þ Tregs mute ocular surface inflammation in a Th1-mediated adoptive transfer model of dry eye disease. These data suggest that CD4þCD25þ Tregs present in the secondary lymphoid organs and ocular surface tissues inhibit the pathogenic effect of autoreactive effector T cells in an effort to maintain homeostasis.

Restricted homing of effector T cells from the regional lymphoid organs to the ocular surface also facilitates immunoregulation following acute insult. For example, the programmed death ligand-1 (PD-L1) has been implicated in protecting the ocular surface from unwanted T cell infiltration and tissue injury. The PDL-1 is a negative regulator of T-cell activation; the interaction between PDL-1 and the PD-1 receptor (expressed on activated T cells) inhibits lymphocyte proliferation and cytokine secretion. Furthermore, PD-1-deficient mice develop spontaneous autoimmunity. The PDL-1 is expressed constitutively on the ocular surface and is upregulated in both human cell lines stimulated with proinflammatory cytokines and in patients with ocular inflammation. In a mouse model of corneal allograft transplantation, PDL-1 was shown to promote apoptosis of the infiltrating CD4þ and CD8þ T cells that was associated with sustained corneal allograft survival. By contrast, PDL-1 blockade resulted in increased lymphocyte infiltration within the ocular surface tissues and enhanced allograft rejection.

Exactly where and how CD4þ and CD8þ Tregs exert their regulatory affects is a current area of intense research. Activation and differentiation of antigen-specific Tregs is mediated by interaction with APCs within the lymphoid organs and is influenced by the local cytokine milieu. It is clear that CD4þ Tregs are involved in suppressing inflammation on the ocular surface; however, the underlying