Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cells.

The inflammatory chemokines, monocyte chemoattractant protein (MCP)-1 and IL-8, are produced by normal trabecular meshwork cells (TM) and elevated in the aqueous humor of primary open angle glaucoma (POAG) and hypertensive anterior uveitis associated with viral infection. However, their role in TM cells and aqueous humor outflow remains unclear. Here, we explored the possible involvement of MCP-1 and IL-8 in the physiology of TM cells in the context of aqueous outflow, and the viral anterior uveitis. We found that the stimulation of human TM cells with MCP-1 and IL-8 induced significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, and the contraction of TM cells. MCP-1 and IL-8 also demonstrated elevation of extracellular matrix proteins, and the migration of TM cells. When TM cells were infected with HSV-1 and CMV virus, there was a significant increase in cytoskeletal contraction and Rho-GTPase activation. Viral infection of TM cells revealed significantly increased expression of MCP-1 and IL-8. Taken together, these results indicate that MCP-1 and IL-8 induce TM cell contractibility, fibrogenic activity, and plasticity, which are presumed to increase resistance to aqueous outflow in viral anterior uveitis and POAG.

Molecular changes in glaucomatous trabecular meshwork. Correlations with retinal ganglion cell death and novel strategies for neuroprotection.

Glaucoma is a chronic neurodegenerative disease characterized by retinal ganglion cell loss. Although significant advances in ophthalmologic knowledge and practice have been made, some glaucoma mechanisms are not yet understood, therefore, up to now there is no effective treatment able to ensure healing. Indeed, either pharmacological or surgical approaches to this disease aim in lowering intraocular pressure, which is considered the only modifiable risk factor. However, it is well known that several factors and metabolites are equally (if not more) involved in glaucoma. Oxidative stress, for instance, plays a pivotal role in both glaucoma onset and progression because it is responsible for the trabecular meshwork cell damage and, consequently, for intraocular pressure increase as well as for glaucomatous damage cascade. This review at first shows accurately the molecular-derived dysfunctions in antioxidant system and in mitochondria homeostasis which due to both oxidative stress and aging, lead to a chronic inflammation state, the trabecular meshwork damage as well as the glaucoma neurodegeneration. Therefore, the main molecular events triggered by oxidative stress up to the proapoptotic signals that promote the ganglion cell death have been highlighted. The second part of this review, instead, describes some of neuroprotective agents such as polyphenols or polyunsaturated fatty acids as possible therapeutic source against the propagation of glaucomatous damage.

ABCB1 transporter and Toll-like receptor 4 in trabecular meshwork cells.

PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 µM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 µM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 µM verapamil and 50 µM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. ß-amyloid significantly reduced ABCB1 activity, and the ß-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.

Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

Cells in the trabecular meshwork (TM), a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment). Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB). Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

sCD44 internalization in human trabecular meshwork cells.

PURPOSE: To determine whether soluble CD44 (sCD44), a likely biomarker of primary open-angle glaucoma (POAG), is internalized in cultured human trabecular meshwork (TM) cells and trafficked to mitochondria. METHODS: In vitro, 32-kD sCD44 was isolated from human sera, biotinylated, and dephosphorylated. TM cells were incubated for 1 hour at 4°C with biotinylated albumin (b-albumin), biotin-labeled sCD44 (b-sCD44), or hypophosphorylated biotin-labeled sCD44 (-p b-sCD44) in the presence or absence of unlabeled sCD44, hyaluronic acid (HA), and a selected 10-mer HA binding peptide. The slides were warmed for 1 or 2 hours at 37°C, and 125 nM MitoTracker Red was added for the last 20 minutes of the incubation. The cells were washed, fixed, incubated with anti-biotin antibody and FITC-labeled goat anti-mouse antibody, and examined under a confocal microscope. RESULTS: TM cell membranes were positive for b-sCD44 after 4°C incubation. When the temperature was raised to 37°C, b-sCD44 or -p b-sCD44 appeared in the cytoplasm. The internalization of b-sCD44 was blocked by excess unlabeled sCD44, HA, and a 10-mer HA-binding peptide. Double label experiments with b-sCD44 or -p b-sCD44 and MitoTracker Red indicated partial overlap. The percent co-localization of MitoTracker Red at 2 hours and FITC -p b-sCD44 was 17.4% (P < 0.001) and for FITC b-sCD44 was 11.7% (P < 0.001) compared with b-albumin. The influence of putative CD44 phosphorylation sites on mitochondrial trafficking was determined by TargetP 1.1. CONCLUSIONS: sCD44 is internalized by TM cells and trafficked in part to mitochondria, which may be a factor in the toxicity of sCD44 in the POAG disease process.

[Intracameral moxifloxacin: a safe option for endophthalmitis prophylaxis? In vitro safety profile for intraocular application]. / Moxifloxacin intrakameral: Eine sichere Option zur Endophthalmitisprophylaxe? In-vitro-Sicherheitsprofil zur intraokularen Anwendung.

BACKGROUND: Moxifloxacin (Vigamox), a 4th-generation fluoroquinolone, covers most isolates causing endophthalmitis. It is safe and effective for systemic and topical use; however, only very limited data are available on prophylactic intracameral administration to prevent endophthalmitis. This study investigated the safety of Vigamox for intracameral application in a cell-culture model. METHODS: The endothelial toxicity of moxifloxacin (Vigamox) was evaluated in cultured human corneas. Primary human retinal pigment epithelium cells (RPEs), trabecular meshwork cells (TMCs), lens epithelium cells (LECs), and corneal endothelial cells (CECs) were treated with concentrations of Vigamox. Toxic effects were evaluated after 24 h (MTT assay and live-dead assay). By treating TMC, CEC, and RPE cells either with oxidative stress or tumor necrosis factor-alpha (TNF-a), lipopolysaccharide (LPS), and interleukin-6 (IL-6), the effects of moxifloxacin on cellular viability under conditions of inflammation were investigated. RESULTS: No corneal endothelial toxicity could be detected after 30 days of treatment with moxifloxacin 500 microg/ml. Primary RPEs, TMCs, LECs, and CECs showed adverse effects on proliferation and viability only at concentrations higher than 150 microg/ml moxifloxacin. After preincubation with TNF-a, LPS, and IL-6 for 24 h and subsequent treatment with moxifloxacin at concentrations of 10-150 microg/ml for 24 h, no significant decrease in proliferation or viability was observed. H2O2 exposure did not increase cellular toxicity CONCLUSION: Vigamox did not show significant toxicity on primary RPEs, TMCs, LECs, CECs, or human corneal endothelium at concentrations up to 150 microg/ml. The MIC90 of moxifloxacin for pathogens commonly encountered in endophthalmitis is known to be in the range of 0.25-2.5 microg/ml. Therefore, intracameral use of Vigamox at concentrations up to 150 microg/ml may be safe and effective for preventing endophthalmitis after intraocular surgery.

[Effect of dexamethasone on adhesion and phagocytosis of bovine trebacular meshwork cells].

PURPOSE: To study the effect of dexamethasone on adhesion and phagocyosis of bovine trebacular cells (BTC). METHODS: After treated with different concentrations of dexamethasone (10(-7) M, 10(-6) M, 10(-5) M) for three days, BTC were added to 96-well plates coated with various extracellular matrix (ECM) and incubated for 90 minutes at 37 degrees C, Methyl thiazolyl tetrazolium (MTT) assay were used to evaluate the amount of the cells attach to ECM; In addition, using latex micropheries 0.8 microm in diameter as markers,we investigated the effect of dexamethasone on phagocytosis of BTC. RESULTS: All groups of Dexamethasone can inhibit BTC attach to various ECM and in a dose-depend manner; the phagocytosis of BTC were also inhibited by three concentrations of dexamethasone and in a dose-depend manner. CONCLUSIONS: Dexamethasone can inhibit adhesion and phagocytosis of BTC, it may be related with pathogenasis of glucocorticoid induced glaucoma.

[Effect of transforming growth factor beta 2 on phagocytosis in cultured bovine trabecular meshwork cells]. / Einfluss von TGF-beta 2 auf die Phagozytose im bovinen Trabekelwerk.

AIM: This study was performed to investigate the effect of transforming growth factor beta 2 (TGF-beta 2) on phagocytosis in bovine trabecular meshwork cells in vitro. METHODS: After cultured bovine trabecular meshwork cells were treated for 24 h with 0 ng/ml (control), 0.32 ng/ml, 1 ng/ml, and 3.2 ng/ml TGF-beta 2, latex beads were added to the incubation medium, and the numbers of latex beads in 20 adjacent cells were then counted under a microscope after treatment with Wright's stain. RESULTS: The average numbers of latex beads in the trabecular meshwork cells treated with TGF-beta 2 of different concentrations were 53.1+/-1.7 beads/cell, 56.4+/-2.9 beads/cell, and 77.9+/-6.5 beads/cell, respectively, compared to 45.5+/-3.3 beads/cell in the nontreated control group. Thus, TGF-beta 2 significantly increased the numbers of latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose-dependent manner. CONCLUSION: TGF-beta 2 can promote the phagocytosis of bovine trabecular meshwork cells in vitro. It may be involved in the reduced cellularity of the trabecular meshwork in patients with primary open angle glaucoma by promoting the phagocytosis of these cells.

The effect of integrin antibodies on the attachment and proliferation of human Tenon's capsule fibroblasts.

The integrins are protein heterodimers consisting of noncovalently associated alpha and beta subunits. The adhesive interactions mediated by integrins are necessary for cellular survival and proliferation. In this study we investigated the effects of three different integrin antibodies on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Human Tenon's capsule fibroblasts were cultured into 96 well plates and treated with different concentrations (ranging from 10(-6) to 1 microg ml(-1)) of three different integrin antibodies: human integrin alpha-2 antibody, human integrin alpha-3 antibody and human integrin alpha-5/FnR (fibronectin receptor) antibody. Coulter counter, hexosaminidase, and 3H-thymidine assays were used to determine the inhibitory effects of these integrin antibodies on ocular fibroblasts on days 0 (attachment), 1,3 and 7 following antibody treatment. The concentration of each antibody required to produce a proliferation 50% less than the control (ID50) was calculated for each assay. With respect to attachment, all three antibodies studied displayed some inhibitory activity. All three antibodies also displayed dose-dependent antiproliferative properties, especially at the highest concentration tested after 7 days of exposure. The integrin alpha-2 antibody was the most potent of the inhibitors, followed by the integrin alpha-3 antibody, with the integrin alpha-5 antibody being the least potent antibody tested. In addition, the anti-proliferative activities of the integrin alpha-2 and integrin alpha-3 antibodies increased with increasing incubation time. In conclusion, these integrin antibodies demonstrated some inhibitory effects on the attachment and proliferation of human Tenon's capsule fibroblasts in culture. Further investigation will be required to determine whether integrin antibodies can significantly limit scar formation in vivo without significant toxicity.

Immunoelectron microscopic localization of the HNK-1 carbohydrate epitope in the anterior segment of pseudoexfoliation and normal eyes.

PURPOSE: To study the presence of the cell-adhesion related HNK-1 carbohydrate epitope in the anterior segment of pseudoexfoliation and normal eyes by immunoelectron microscopy. METHODS: Anterior segment tissue of 6 autopsy eyes with pseudoexfoliation (PEX) syndrome (5 eyes without glaucoma and 1 with glaucoma), and 6 normal autopsy eyes without PEX syndrome were studied by an electron microscopic immunogold technique using a monoclonal antibody to the HNK-1 epitope. RESULTS: In both normal and PEX eyes, the HNK-1 epitope could be immunolocalized to the basement membranes of both ciliary epithelia and posterior iris pigmented epithelium, to the lens capsule and zonular lamella, and to the pigmented epithelial cells of iris and ciliary body. Within the inner connective tissue layer of the ciliary body, the gold label was mainly associated with the periphery of elastic fibers and microfibrillar bundles. PEX material on the surfaces of posterior iris, ciliary body, anterior lens capsule, zonular fibers, and uveal part of the trabecular meshwork reacted strongly with the HNK-1 antibody. In contrast, PEX material accumulations within the iris stroma or the juxtacanalicular tissue of the trabecular meshwork showed only weak immunoreactivity, while PEX material in the conjunctiva was totally negative. CONCLUSIONS: The wide distribution of the HNK-1 epitope in anterior segment tissues and its association with a variety of extracellular and cellular structures was ultrastructurally demonstrated. In PEX syndrome, the varying labelling density of PEX fibers indicates a deviating carbohydrate composition in different locations of the eye. The HNK-1 epitope might be involved in the adhesiveness of PEX deposits on intraocular surfaces.

[The effects of pressure on cultured bovine trabecular meshwork cells].

OBJECTIVE: To observe the effects of pressure on trabecular meshwork cells. METHODS: Bovine trabecular meshwork cells were cultured and submitted to different amounts of hydrostatic pressure. Cellular morphology and phagocytic function were observed under inverted phase-contrast microscope, light microscope and electron microscope. RESULTS: Compared with the control group, the cells under 2.0 kPa or 2.67 kPa for 48 hours had no remarkable difference in criteria observed. Those under 4.0 kPa for 24 hours showed slight changes in structure and a mild decrease in phagocytic function. The damage appeared more severe if the pressure was higher or lasted longer. CONCLUSION: Trabecular meshwork cells can only bear pressure below a certain level. They may be destroyed structurally or impaired functionally by pressure over this level.

[Application of proliferating cell nuclear antigen in the study of human trabecular cell proliferation].

OBJECTIVE: To evaluate the application of proliferating cell nuclear antigen (PCNA) in the study of human trabecular cell proliferation. METHODS: Immunohistochemical technique was used to observe the PCNA expressive level in the fourth passage of human trabecular meshwork, the effects of different concentrations of epinephrine, dexamethasone and epidermal growth factor on the level were also investigated, the results were compared with that of the normal cells cultured at the same time, and analyzed by a graphic device operation system in a computer. RESULTS: A stable proliferating curve was obtained according to the normal cell PCNA level, by which we could choose the best opportunity of drug application. Epinephrine and dexamethasone were found to significantly inhibit cell proliferation, while epidermal growth factor (EGF) could promote the proliferation. CONCLUSION: PCNA is considered to be a useful agent to observe the process of cell proliferation. The above mentioned methods are beneficial to the investigations of biochemical characteristics of trabecular meshwork cells and the pathogenic mechanisms of open-angle glaucoma.

[Establishment of in vitro culture of bovine trabecular meshwork cells and their phagocytosis].

OBJECTIVE: To study the role of abnormality of phagocytosis of trabecular meshwork cells in the pathogenesis of glaucoma. METHOD: In vitro trabecular meshwork cell culture was successfully established from excised bovine aqueous outflow pathway. Using latex microspheres 0.6 micron in diameter as markers, we investigated the phagocytosis of cultured bovine trabecular cells and its influential factors qualitatively and quantitatively. RESULTS: Immunohistochemical staining studies have shown that trabecular cells secrete fibronectin and laminin. It is shown that bovine trabecular cells in vitro had strong phagocytic ability to ingest the particles, the number of latex beads in cells was increased with the prolongation of time in culture, and by 24 hours of incubation, the average number of beads in each cell was 40.5 +/- 6.7, in the mean time marked morphological and damaging changes, even death, of trabecular cells occurred. Epinephrine and cortisone inhibited significantly the phagocytosis of cells (P < 0.05), while epidermal growth factor had no such effect. CONCLUSION: The phagocytosis of trabecular cells is important to keep aqueous humour outflow patent, and abnormal phagocytosis of trabecular cells might be related to the pathogenesis of primary glaucoma.

Distinctive distribution of HLA class II presenting and bone marrow derived cells in the anterior segment of human eyes.

Cells of bone marrow origin that normally occupy the stroma of the murine iris and ciliary body have been implicated in the immune phenomenon, anterior chamber associated immune deviation (ACAID). Following injection of antigen into the anterior chamber, cells of this type deliver an ACAID inducing signal into the systemic circulation, presumably through the outflow tract. In an effort to identify such cells in man, anterior chambers of 34 human donor eyes of different age groups were stained immunocytochemically with monoclonal antibodies directed at HLA class II molecules, CD 45 (a molecular marker of bone marrow-derived cells) and macrophage-associated membrane molecules (CD 68, CD 14). Within the outflow tissue, the cells of the filtering trabecular meshwork stained with none of those reagents. However, infrequent single, dispersed, dendritic cells were positively stained in the intertrabecular spaces. More numerous labelled cells were found in the anterior- and posterior-most portions of the non-filtering part of the trabecular meshwork. These cells were continuous with stained cells adjacent to the outer wall of Schlemm's canal and to the collector channels. Numerous labelled cells were seen in the vicinity of the intra- and episcleral vessels, the ciliary meshwork, the stroma of the ciliary muscle and epithelial processes, and the iris stroma. With advancing age, increasing numbers of CD 45+, HLA class II expressing cells appeared to accumulate in the so-called uveoscleral pathway. These results indicate that bone marrow-derived cells with the potential to function of ACAID induction reside within human eyes, and that cells of this type are located not only in the stroma of iris and ciliary body, but within the non-filtering portions of the trabecular meshwork and the uveoscleral pathway. The appearance of rare CD 45+ cells "in transit" in the filtering trabecular meshwork is compatible with the view that cells carrying ACAID-inducing signals to the systemic immune apparatus escape from the eye by this route.

[A theory on the role of immunologic factors in the pathogenesis of primary glaucoma]. / Kontseptsiia uchastiia immunnykh faktorov v patogeneze pervichnoi glaukomy.

Basing on the literature data and their own clinical, immunomorphologic, and immunochemical findings in glaucoma patients and normal subjects, the authors suggest a concept on the immune factor contribution to the pathogenesis of primary open-angle glaucoma. According to the scheme they suggest, of primary importance in the development of autoimmune reactions in glaucoma are the following factors: changed antigenic specificity of the drainage zone tissues, resultant from involution processes; impaired immune homeostasis, effects of various internal and environmental factors. The pathogenetic role of autoimmune reactions and immunity system regulatory mechanism disorders is explained.

Expression of HLA by the human trabecular meshwork and corneal endothelium.

By using immunohistochemical techniques, we demonstrated that HLA class I (A, B and C) and HLA class II (DR), the major histocompatibility antigens in man, are expressed constitutively by cells of the trabecular meshwork/Schlemm's canal system and corneal endothelium, as well as by the conjunctival epithelium, Langerhans cells, vascular endothelium and uvea. Because clinical studies indicate that these antigens are involved in mediating corneal graft rejection and possibly in glaucomatous disease of the eye, the presence of both class I and II HLA in the corneal endothelium and in the trabecular cells has important implications for an understanding of immune disorders in the anterior segment of the eye. The presence of HLA on trabecular cells raises the possibility that these antigens potentiate the recognized role of Langerhans cells at the limbus and that they participate in, and/or regulate, the maintenance and defense of the aqueous outflow pathway. Our findings also open up the possibility of using HLA as a genetic marker in the determination of susceptibility to these disorders in man.

Trabecular meshwork recovery after phagocytic challenge.

The short and long term response of the trabecular meshwork to a phagocytic challenge and the response of the meshwork to different types of foreign particles was studied by injecting one eye of 25 adult cats with a phagocytic agent (zymosan, blood, or latex microspheres) while the fellow eye received a control solution. Eyes were examined histologically at various intervals from one day to five months after infusion. Active trabecular cell phagocytosis and changes in cell shape were found with all agents. The extent of these changes varied with the agent used. Zymosan caused marked changes and inflammation, with numerous macrophages found throughout the meshwork. Trabecular cell migration and cell loss occurred, although it was often difficult to distinguish macrophages from rounded trabecular cells. The meshwork eventually recovered from this inflammatory insult, as trabecular lamellae became less edematous and once again acquired a lining of trabecular cells. Blood and latex microspheres caused less disruption, with microspheres often found in otherwise normal appearing cells. Trabecular cellularity was quantitated after the blood and the zymosan infusions. No cell loss was observed after the blood infusion, while zymosan-infused eyes had an initial 15% cell loss (p less than .04) when compared with fellow control eyes. This zymosan-associated trabecular cell loss may have been due to phagocytosis, inflammation, or a combination of both. The cell loss had recovered by the end of 150 days (p less than .02), as trabecular cell numbers in experimental eyes became comparable to fellow control eyes.

Immunohistochemical staining of the human anterior segment. Evidence that resident cells play a role in immunologic responses.

We examined human corneoscleral tissue for cells that are phenotypically similar to known antigen-presenting cell (APC) populations. Antigen-presenting cells are involved in the uptake and processing of antigen for presentation to T lymphocytes, thereby playing a central role in induction of the immune response. The recognition of antigen by T lymphocytes requires that an APC express major histocompatibility complex class II molecules. Using immunoperoxidase staining techniques, the presence of cells expressing class II glycoproteins and T-cell subsets were determined. The staining patterns of the trabecular meshwork, ciliary body, cornea/sclera, and conjunctive are described for monoclonal antibodies OKT6, OKM1, HLA-DR, and HLA-DQ, and T-cell markers OKT8, Leu-3a, and Leu-4. The results of the present study demonstrate that the anterior chamber contains a network of immunocompetent cells. The presence of a subpopulation of cells within the anterior chamber that express class II glycoproteins of the major histocompatibility complex suggests this tissue may play an important role in immune regulation within the eye.

Cultured trabecular-meshwork cells: immunohistochemical and lectin-binding characteristics.

We examined the extracellular matrix formation and lectin-binding properties of cultured trabecular-meshwork cells established from cynomolgus monkey and bovine eyes. Using an avidin-biotin complex method, we found that the extracellular matrix in both the monkey and bovine cultures stained intensely with antibodies to fibronectin, type IV collagen, and laminin. These materials were especially prominent when the cultured monkey trabecular-meshwork cells aggregated in clusters, manifesting their close anatomic relationship to trabecular beams. Cell-surface and intracellular lectin-binding properties were studied using eight biotinylated lectins: concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Dolichos biflorus agglutinin (DBA), soy bean agglutinin (SBA), Phaseolus vulgaris (PHA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin I (UEA I). Both monkey and bovine trabecular-meshwork cells showed positive cell surface and intracellular binding to Con A, WGA, and PHA. Only moderate cell-surface staining was observed with RCA, and no visible staining occurred with PNA, DBA, SBA, and UEA-1.

Expression of HLA class I and II antigens on cells of the human trabecular meshwork.

Human corneoscleral tissue containing trabecular meshwork and cultured human trabecular cells were examined for HLA-ABC (class I) and HLA-DR (class II) antigens of the major histocompatibility complex using an indirect immunofluorescence assay. Class I antigens were detected in the trabecular meshwork on frozen sections and on cultured trabecular cells. Class II antigens were constitutively expressed on some, but not all, cells within the trabecular meshwork. Many more cells could be induced to express class II antigens by pre-incubation in human gamma interferon. Cultured trabecular cells did not express class II antigens constitutively, but expression could be induced by gamma interferon. This study suggests that, in addition to Langerhans' cells at the limbus, other cell types within the anterior segment express major histocompatibility complex-encoded class II antigens either constitutively or inducibly. These cells may be important for the initiation and regulation of ocular immunity.