Deposition of immune complexes in and around the vessel wall is thought to be an important event in the genesis of vessel damage in systemic vasculitic syndromes often associated with scleritis. DeÞnitive proof of the presence of immune complexes in vessel wall deposits requires the demonstration of speciÞc antigen and antibody. However, common practice in diagnosing systemic vasculitic syndromes (often associated with scleritis) dictates that detection of immunoglobulins and complement components in vessel walls provides evidence of immune complex deposition, even if the relevant antigen is unknown, much less detected. This detection can be done by use of immunoßuorescence or immunoperoxidase techniques (Fig. 3.32). Recognizing the limitations in the interpretation of these Þndings, we therefore also deÞne inßammatory microangiopathy as immunoreactant (immunoglobulin and complement component) deposition in capillaries and postcapillary venules, such as conjunctival, episcleral, and perforating scleral vessels.

5.2.1Pathology of Episcleritis

Light and electron microscopic studies of episcleral tissue in simple and nodular episcleritis cases show chronic, nongranulomatous inßammation with lymphocytes and plasma cells, vascular dilatation, and edema [21, 22]. Some cases in association with connective tissue diseases may reveal the presence of a granulomatous inßammation with epithelioid cells and some multinucleated giant cells, with or without central necrosis, resembling the histopathological picture seen in subcutaneous rheumatoid nodules [23Ð26]. Relatively few cases of episcleritis have been studied histologically, probably because there has been little reason to biopsy episcleral tissue: episcleral inßammation is a benign entity with a tendency to disappear spontaneously over the course of several weeks, and pathological studies are usually noncontributory to the diagnosis or to the treatment.

5.2.2Pathology of Scleritis

5.2.2.1 Noninfectious Scleritis

Light and electron microscopic studies of ocular tissue in patients with noninfectious scleritis may show the same characteristic morphologic changes regardless of whether the scleral inßammation is associated with autoimmune systemic diseases, follows surgical or accidental trauma, or is idiopathic [27Ð29]. InÞltration of the sclera with inßammatory cells may derive from the superÞcial and deep episcleral vessels, the perforating scleral vessels, and the choroidal vessels. All types of scleritis are histologically similar but vary in severity of morphologic changes, necrotizing scleritis having obviously the most destructive lesions.

Because scleral inßammation is always accompanied by episcleral inßammation and often by conjunctival, uveal tract, and corneal inßammation, we describe the morphological changes in sclera, episclera, conjunctiva, uveal tract, and cornea. Occasionally, other ocular structures, including trabecular meshwork, retina, optic nerve, and extraocular muscles, may also be involved in scleritis.

Sclera

Cells

The sclera in necrotizing scleritis reveals a granulomatous inßammatory reaction, the center of which consists of an area of Þbrinoid necrosis surrounded by epithelioid cells, multinucleated giant cells, lymphocytes, plasma cells, and less often neutrophils [27Ð32]. The involved sclera is diffusely thickened. Mast cells and eosinophils can occasionally be seen throughout the granuloma and around vessels. Fibroblasts within the granuloma either are absent or display degenerative changes, including membrane disruption and loss of organelles; however, in the area outside the granuloma they appear to be metabolically active, containing active cell surfaces with peripheral pseudopodia extending into the surrounding matrix, numerous lysosomal granules and mitochondria, and prominent

rough endoplasmic reticulum and Golgi apparatus (Þbroblastic cells) [27]. Cellular inÞltrates in scleral granulomas secrete a plethora of enzymes and mediators that interact in a complex pattern to orchestrate the development and perpetuation of the inßammatory process (Table 5.1).

The sclera in diffuse and nodular scleritis shows a nongranulomatous inßammatory reaction characterized by inÞltration of mononuclear cells, such as macrophages, lymphocytes, and plasma cells. In some cases, however, especially in the most severe ones, mononuclear cells organize into granulomatous lesions. Mast cells, neutrophils, and eosinophils also may be present.

We biopsied the sclera of 26 of 34 (76%) of our patients with noninfectious necrotizing scleritis. Scleral tissue was obtained in many instances at the time of necrotic tissue removal in those cases requiring scleral grafting for structural support (13 patients) [33]. Scleral tissue was processed for histopathological studies and stained with hematoxylinÐeosin and alkaline Giemsa. HematoxylinÐeosin stains cell nuclei blue and collagen red. Alkaline Giemsa stains mast cell granules metachromatically purple and eosinophil granules red.

The most frequent histopathological Þnding in sclera consisted of a central focus of Þbrinoid necrosis surrounded by granulomatous inßammation

Fig. 5.3 Scleral biopsy, patient with necrotizing scleritis. Note the granuloma formation with perivasculitis, neutrophil margination in the venule, and the multinucleated giant cell at the edge of the venule

with epithelioid cells, lymphocytes, and plasma cells (Fig. 5.3) (Table 5.2). A moderate number of multinucleated giant cells containing a row of nuclei arranged along the periphery were also present in some cases (Fig. 5.4). In addition, scattered numbers of neutrophils, mast cells (Fig. 5.5), and eosinophils (Fig. 5.6) were dispersed throughout the inßamed tissue and around vessels. Areas outside the granuloma were inÞltrated by macrophages, lymphocytes, and plasma cells (Fig. 5.7).

Thirteen of 112 (12%) of our patients with noninfectious diffuse and nodular scleritis

Table 5.2 Analysis of hystopathology of scleritis

a%, Percentage of patients with necrotizing and nonnecrotizing scleritis with the different cell subsets

Fig. 5.4 Same patient as in Fig. 5.3: different Þeld of the specimen, showing many multinucleated giant cells (MagniÞcation, ×160; hematoxylinÐeosin stain)

Fig. 5.6 Scleral biopsy. Note the striking presence of eosinophils in this specimen (MagniÞcation, ×60; hematoxylinÐeosin stain)

Fig. 5.7 Granuloma in a scleral biopsy from a patient with necrotizing scleritis. Note the granuloma and the presence of histiocytes, lymphocytes, and plasma cells in areas surrounding the granuloma

Fig. 5.5 Scleral biopsy of a specimen from a patient with scleritis. Note the large number of purple-stained cells in the specimen, the mast cells (MagniÞcation, ×40; alkaline Giemsa stain)

underwent scleral biopsy. All patients had had recurrent attacks of active scleritis for at least 6 months. Examination under light microscopy

revealed in all cases mononuclear cell inÞltration, including macrophages and lymphocytes without Þbrinoid necrosis (Table 5.2). In addition, some specimens showed the presence of epithelioid cells characteristic of granulomatous inßammation; none had multinucleated giant cells. Neutrophils and mast cells were scattered in the tissues of some patients.

IdentiÞcation of cellular subsets and surface glycoproteins in necrotizing scleritis was accomplished with the use of the immunoperoxidase technique, using monoclonal antibodies directed against T lymphocytes (CD3), T helper/inducer lymphocytes (CD4), T cytotoxic/suppressor lymphocytes (CD8), neutrophils (CD16), B lymphocytes (CD22), macrophages (CD14), HLA-DR

Table 5.3 Mononuclear cell subset antibody panel in immunoperoxidase studies

aMean cell count/mm,2 ± standard error of the mean bSigniÞcant values, using StudentÕs t test (P < 0.05)

glycoproteins(anti-HLA-DR)(BectonDickinson, Inc., Mountain View, CA), and LangerhansÕ cells (CD1) (Ortho Pharmaceuticals Corp., Raritan, NJ) (Table 5.3). Details of tissue processing and staining have been previously published [28]. A comparison between nine scleral specimens from patients with necrotizing scleritis and four scleral specimens from normal eyes (New England Eye Bank, Boston, MA), revealed a predominance of macrophages (CD14) and T lymphocytes (CD3) in scleral tissue (Table 5.4). Although both T helper/inducer lymphocytes (CD4) and T suppressor/cytotoxic (CD8) lymphocytes were increased, a high T helper/T suppressor ratio revealed a predominance of the former. Neutrophils (CD16), Langerhans cells (CD1),

and B lymphocytes (CD22) were present in scleritis but their numbers were not signiÞcantly increased when compared with normal tissue. HLA-DR glycoproteins (anti-HLA-DR), present constitutively in macrophages, and after inßammatory stimuli in scleral Þbroblasts and endothelial cells, were markedly increased. These Þndings show the participation of macrophages and T lymphocytes, particularly T helper lymphocytes, in scleritis, and support the idea that macrophages and Þbroblasts activate T lymphocytes, which in turn may participate in granuloma formation. Damaged endothelial cells also may play a role as antigen-presenting cells. The T helper/T suppressor imbalance may contribute to the perpetuation of the highly inßammatory nature of the lesion. In addition to the predominant macrophages and T helper lymphocytes, neutrophils, B lymphocytes, and Langerhans cells may be present.

Extracellular Matrix

Collagen degradation may take place by two mechanisms, one (intracellular) involving acid proteases of macrophages, and the other (extracellular) involving neutral proteases (collagenase and elastase) of Þbroblasts and macrophages [34].

In intracellular digestion of collagen, the collagen Þbrils undergo phagocytosis into phagolysosomes (acid pH) of macrophages, where lysosomal acid proteases lead to tissue breakdown. In extracellular digestion, the collagen Þbrils appear swollen and unraveled in areas of scleral stroma (neutral pH) as a result of the release of neutral proteases by Þbroblasts and macrophages into the connective tissue matrix. Both intracellular and extracellular mechanisms of collagen degradation may occur simultaneously, distant from the granuloma, suggesting that collagen degradation may precede granuloma formation in scleral inßammation [35].

Degradation of collagen may be almost total in the center of the granuloma. Fibril fragments may be found in close apposition to the Þbroblast plasma membrane or enclosed in invaginations of the Þbroblast plasma membrane or vacuole membranes within the cytoplasm (intracellular mechanism). Derangements of collagen Þbril

structure, such as Þbril swelling and unraveling, irregular contour, and increased interÞbrillar distance, may also be seen without close apposition to active Þbroblasts (extracellular mechanism) [35]. In areas distal to the inßammatory focus, collagen appears normal by light microscopy; however, intracellular or extracellular resorptive changes in the absence of inßammatory cells can be seen by electron microscopy [35, 36].

Electron microscopy studies with cuprolinic blue staining show that proteoglycans are reduced or absent in areas of active scleral inßammation before the collagen Þbrils undergo resorptive changes; these results suggest that proteoglycan degradation precedes collagen degradation [37]. Our own studies on immunolocalization of extracellular matrix components in two diseased scleral specimens as compared with Þve normal scleral specimens show similar Þndings. An indirect immunoßuorescence technique was performed in anterior, equatorial, and posterior areas, using monoclonal antibodies against the

proteoglycans heparan sulfate, dermatan sulfate, hyaluronic acid, and chondroitin sulfate, collagen types I, II, III, IV, V, VI, VII, IX, XII, XIII, XIV, XVII, and the glycoproteins Þbronectin, vitronectin, and laminin (Table 5.5).

1.Proteoglycans. The most abundant proteoglycans in normal sclera were dermatan sulfate and chondroitin sulfate; hyaluronic acid and heparan sulfate also were present, although in small amounts. Dermatan sulfate in necrotizing scleritis was frankly decreased when compared to normal sclera. A negative background with scattered areas of mild patchy positivity in diseased sclera contrasted with an intense striped pattern in normal sclera. (Figs. 5.8 and 5.9). Chondroitin sulfate in necrotizing scleritis also showed a marked reduction in amount of staining, unlike normal sclera, which showed a generalized speckled pattern of intense positivity (Figs. 5.10 and 5.11). Because the presence of heparan sulfate and hyaluronic acid was detected in small amounts

Fig. 5.8 Immunoßuorescence microscopy: specimen is from a patient with scleritis. The antibody is antidermatan sulfate antibody. Note (in comparison to Fig. 5.9) the dramatic reduction in the presence of dermatan sulfate in the scleritis specimen

Fig. 5.9 Immunoßuorescence microscopy: biopsy of normal sclera. Antibody is antidermatan sulfate antibody. Note the large amount of bright apple-green ßuorescence, indicating rather large amounts of dermatan sulfate in normal sclera (MagniÞcation, ×100)

Fig. 5.10 Immunoßuorescence microscopy: scleral biopsy from a patient with scleritis. Note (particularly in relationship to Fig. 5.11) the relative lack of bright staining, except around the vessels, indicating a relative paucity of chondroitin sulfate in this scleral specimen (MagniÞcation, ×40)

Fig. 5.11 Immunoßuroescence microscopy: normal sclera. Antibody is anti-chondroitin sulfate antibody. Note the relative abundance of bright apple-green Þbrils, indicating a relatively large amount of chondroitin sulfate in normal sclera (MagniÞcation, ×40)

in normal sclera, comparison of these proteoglycans between normal and diseased sclera did not show obvious differences.

2.Collagens. The most abundant types of collagen in normal extravascular sclera were collagens type I and III collagens; type V and type VI also were present. Type II, VII, IX, XII, XIII, XIV, and XVII were not identiÞed. Collagen type IV also was absent except for its dramatic presence in the vessels. Comparison of staining of collagen types I, III, V, and VI between normal and necrotizing scleritis specimens did not show differences in

intensity or pattern. Collagen types I and III showed intense homogeneous Þbrillar staining; these two collagen types impart tensile strength (type I) and resilience (type III) to the scleral ÒskeletonÓ [38]. Collagen type V showed mild, delicate, patchy, granular staining, particularly associated with the edges of the collagen bundles; this pattern leads us to suspect that collagen V forms a Þne network that maintains the structural integrity of the main Þbril core (types I and III). Collagen type VI showed an intense, Þne, regional, granular staining; this pattern also leads us to