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.     

Detection of HLA class I and II antigens in rejected human corneal allografts

We compared the distribution of HLA-ABC (class I) and HLA-DR (class II) antigens on fresh human donor corneal tissue, donor corneas following a 72-hour storage in McCarey-Kaufman (M-K) medium, and corneal buttons from patients with allograft rejection and with chronic herpetic stromal keratitis. Incubation in M-K media had little or no effect on the distribution of HLA antigens as compared with fresh tissue. In contrast to control corneas, both HLA class I and II antigens were detected on corneal endothelial cells, cells in the stroma, and on basal epithelial cells in rejected allografts. Corneal endothelium in herpetic buttons did not express detectable HLA antigens. HLA-DR positive Langerhan's cells were demonstrated in the central corneal epithelium of rejected allografts, as well as in herpetic corneas, but not in control corneas except at the limbus. Based upon these observations, a theory of corneal allograft rejection in humans is proposed based upon the induction of class I HLA-ABC and class II HLA-DR antigens on cells in the donor button by a factor(s) associated with cellular inflammation.     

Expression of HLA class I, beta(2)-microglobulin and HLA class II antigens in primary orbital melanoma

Major histocompatibility antigens (MHC) play a crucial role in the recognition of tumor cells by the immune system. There is not much information on the role of MHC molecule expression in primary orbital melanomas. In the present study, the authors examined the expression of human leukocyte antigen (HLA) class I, beta(2)-microglobulin (beta(2)-m) and HLA class II antigens in primary orbital melanoma and correlated this with the clinical and pathological findings. HLA class I antigen, beta(2)-m and HLA class II antigen expression were evaluated immunohistochemically in three primary orbital melanomas and correlated with cell type and metastasis. Immunohistochemistry showed heterogeneous expression of HLA class I, beta(2)-m and HLA class II antigen in two cases with no liver metastasis and negative expression in one case with liver metastasis. This preliminary observation deserves further investigation, which may shed more light on the immune escape mechanisms of this tumor and thus make possible novel therapeutic strategies.     

水通道蛋白1在培养人眼小梁细胞中的表达及意义

目的探讨培养人眼小梁细胞水通道蛋白1(AQPl)的存在、定位及意义。方法应用逆转录聚合酶链反应(RT-PCR)检测培养人眼小梁细胞AQP1mRNA的表达。Westernblot用兔抗人AQP1多克隆抗体检测AQP1蛋白表达。免疫荧光测定AQP1在培养人眼小梁细胞的所在部位。结果RT-PCR扩增出一条347bp标志AQP1mRNA的表达产物。Westernblot可见约28000相应位置的发光条带。免疫荧光定位AQP1在培养的人眼小梁细胞胞膜。结论AQP1在培养人眼小梁细胞的细胞膜表达,可能在调节房水通过小梁网流出系统中起到重要作用。     

培养的人眼小梁细胞HGF和c-met的表达

目的　观察体外培养的人眼小梁细胞肝细胞生长因子 (HGF)和其受体 (c- m et)的表达。方法　成人眼小梁细胞原代培养 ,RT- PCR检测 HGF和 c- met m RNA的表达 ,免疫组织化学染色法检测 HGF和 c- met蛋白的表达 ,酶联免疫吸附法 (Elisa)检测小梁细胞分泌 HGF蛋白。结果　 RT- PCR产物为 2 6 2 bp和 30 4 bp的条带 ,为 HGF和 c- met m RNA表达阳性 ;免疫组织化学染色细胞浆呈棕色 ,为 HGF蛋白阳性表达 ,细胞膜呈棕色 ,为 c- m et蛋白阳性表达 ;Elisa法在培养的小梁细胞上清液中检测出 HGF。结论　培养的人眼小梁细胞表达 HGF和 c- m et m RNA和蛋白     

Expression of matrix metalloproteinases and their inhibitors in human trabecular meshwork cells

PURPOSE: Matrix metalloproteinases (MMPs) are involved in trabecular meshwork (TM) extracellular matrix metabolism and have been shown to increase aqueous outflow facility. The purpose of this study was to characterize effects of cytokines, a phorbol ester, and prostanoids on the expression of MMP-1, -2, -3, and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in cultured human TM cells. METHODS: Five human TM cell strains were treated with selected compounds. Levels of proMMPs and TIMPs in cell media were quantified by ELISA. MMP-3 activity was assayed by casein zymography. RESULTS: All human TM cell strains produced detectable basal amounts of proMMPs and TIMPs. 12-O-tetradecanoyl-phorbol-13-acetate was effective in increasing the levels of proMMP-1, -3, and -9 and TIMP-1. Its effect on proMMP-1 was concentration-dependent with an EC(50) of 2 to 3 nM. Interleukin (IL)-1alpha did not affect levels of proMMP-1 and -2 or the TIMPs, but was most efficacious in increasing proMMP-3 production with an EC(50) of 0.5 ng/mL. The IL-1alpha-induced upregulation of proMMP-3 correlated with an increase in MMP-3 activity. Tumor necrosis factor-alpha activated proMMP-3 production in some but not all cell strains. Platelet-derived growth factor-BB was generally ineffective in modulating MMP and TIMP levels. Prostaglandins E(2) and F(2alpha) at 10 micro M did not affect levels of proMMP-1 or -3. CONCLUSIONS: The expression of the different MMPs and TIMPs in human TM cells was independently regulated. Production of MMP-3 was maximally activated by IL-1alpha. The IL-1alpha-stimulated expression of MMP-3 provides a probable mechanism for IL-1alpha-enhanced aqueous outflow.     

Preliminary observations on human trabecular meshwork cells in vitro

This report presents our preliminary observations on the trabecular meshwork from human eyes up to 5 days post-mortem in tissue culture. Satisfactory primary cultures were obtained from about 20% of the 423 explants which were investigated. The period prior to growth was from 4 days to 4 weeks and from the appearance of the initial outgrowth it took 25 to 30 days to reach maximum cellular spread within the culture chambers. The progress of the explant and the spreading of the trabecular meshwork cells was monitored by phase-contrast microscopy, time-lapse cinephotomicrography, light microscopy, transmission electron microscopy, scanning electron microscopy and autoradiography (using tritiated thymidine). On the basis of their ultrastructural appearance the cultured meshwork cells seemed to be metabolically active. Their cytoplasm contained abundant rough endoplasmic reticulum, many mitochondria, a well developed Golgi apparatus and many coated and uncoated micropinosomes. However even in short-term culture the trabecular meshwork cells had adapted to the artificial environment of our system and no longer resembled normal trabecular meshwork cells as seen in vivo. Since trabecular meshwork cells can quickly adapt their morphology in a culture environment and because the adult human meshwork contains a significant population of non-trabecular cells, the value of long term culture as a means of investigating the cellular activity of the normal and glaucomatous outflow system must be open to question.This paper was presented in part at the 7th Annual Meeting of the European Club for Ophthalmic Fine Structure in Ystad, Sweden on April 20 and 21, 1979     

Characterization of beta-adrenergic receptors in cultured human trabecular cells and in human trabecular meshwork

The characterization of beta-adrenergic receptors on cultured human trabecular cells and trabecular meshwork from human autopsy eyes was carried out by radioligand binding utilizing (125I)-iodopindolol. In cultured cells, the observed binding of (125I)-iodopindolol was of high affinity (Kd = 43 pM) and saturable. Scatchard plots were linear and revealed a Bmax of 33 +/- 7 fmol/mg of protein. Competition studies with a series of agonists and antagonists revealed that human trabecular cells contain a single class of beta-adrenergic receptors of the beta 2 subtype. Similarly, the IC50 of ICI 89,406 (176 nM) in human trabecular meshwork from autopsy eyes supports the presence of beta 2-adrenergic receptors in this tissue.     

Expression of classic and nonclassic HLA class I antigens in uveal melanoma

PURPOSE: Because the expression of classic and nonclassic HLA antigens is crucial for the recognition and elimination of tumor cells by cytotoxic T and/or NK cells, we analyzed the HLA-A, -B, -C, and -G expression in uveal melanoma specimens from 18 patients. METHODS: Tumor specimens and EDTA plasma samples from 18 patients treated by primary enucleation or tumor resection for primary uveal melanoma in the University Eye Clinic, Essen, Germany, were collected immediate after surgery. After solubilization of tumor tissue and specific immunoprecipitation of classic HLA-A, -B, and -C and nonclassic HLA-G antigens the tumor samples were analyzed by one-dimensional isoelectric focusing (1D-IEF) and Western blot analysis. In parallel, patients were typed for HLA-A, -B, and -C class I antigens by PCR with sequence-specific primers (PCR-SSP). In addition, HLA-A2 and -G expression was investigated by immunohistochemistry in paraffin-embedded tumor sections from these patients. RESULTS: In 9 (50%) of 18 specimens, a full HLA-A and -B antigen expression pattern was detected by 1D-IEF. In six (33.3%) tumor specimens, an HLA class I allotype was missing (HLA-A2, -A28, -A29, -B18, -B35, and -B55), in two cases a haplotypic loss (HLA-A2, -B44 and HLA-A2, -B13) and in another case an allotype-specific loss combined with a haplotypic loss (HLA-A26, -A32, -B41) were observed. HLA-C and -G antigens were not detectable in any of the tumor samples by biochemical methods used. CONCLUSIONS: A considerable portion of the uveal melanomas tested showed a loss of classic HLA class I antigens, which may enable them to escape from the immunosurveillance of cytotoxic T cells. HLA-C and -G antigens were not found in uveal melanoma tissue implying a susceptibility for NK lysis.     

Arachidonic acid metabolism in human trabecular meshwork cells

Prostaglandins and other eicosanoids in the trabecular meshwork may play important physiological and pharmacological roles in the aqueous outflow pathway. In the present studies, we employed [14C]-arachidonic acid to explore potentially important pathways for the production of eicosanoids in cultured human trabecular meshwork cells (HTM). In these cells, we demonstrated that prostaglandin E2 (PGE2) and PGF2 alpha are major cyclooxygenase products, with some 6-keto-PGF1 alpha also detected. The amount of radiolabelled PGE2 formed was substantially higher than the PGF2 alpha formed in the early time periods. The amount of PGF2 alpha in the culture media increased at a time when the amount of PGE2 was declining, suggesting a possible metabolic conversion between the prostaglandins. HTM produced a range of products of the lipoxygenase pathway. Products co-eluting with 5, 12, and 15-hydroxyeicosatetraenoic acids (HETEs) were detected, with 12 and 15-HETEs predominating. A large amount of radiolabelled product was detected also in peaks co-eluting with leukotriene B4 (LTB4) and an LTB4 degradation product. Biosynthesis of lipoxygenase products was markedly inhibited by BW 755c and partially inhibited by dexamethasone. These data emphasize that HTM cells are capable of converting arachidonic acid into a wider variety of biologically active products than previously recognized.     

Microfilaments in the cells of the human trabecular meshwork.

In this present study the results are presented of a combined ultrastructural and immunofluorescent investigation of 'smooth muscle' elements within the cytoplasm of human trabecular meshwork cells. The cytoplasm of human meshwork cells both in vivo and in vitro is replete with 10 nm intermediate filaments and also contains smaller 6 nm filaments which are particularly prominent in the cell processes. By immunofluorescence using sera rich in antibodies to contractile proteins, particularly actin, cultured meshwork cells showed strong cytoplasmic fluorescence. On occasion the cytoplasmic fluorescence was diffuse, but more often recognisable bundless (stress fibres) or a loose fibrillar framework was found. The possible role of structural and contractile cellular proteins in trabecular function was discussed.     

Effects of dexamethasone on human trabecular meshwork cells in vitro

Purpose

To study the effects of dexamethasone sodium phosphate (Dex) on human trabecular meshwork (HTM) cells in vitro.

Methods

HTM cells were treated with Dex 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, or 0.05 mg/ml for 24 h. Cell viability was measured by a trypan blue exclusion test. Caspase-3/7, -8, -9 and -12 activities were measured by fluorochrome assays as mean signal intensity (msi) to assess apoptosis. Mitochondrial dehydrogenase activity was determined by a WST assay to quantify mitochondrial damage.

Results

Mean cell viabilities of HTM cells exposed to Dex at the higher doses of 2 mg/ml, 1 mg/ml, and 0.5 mg/ml were reduced: 11.9 %?±?3.5 (P?<?0.001), 31.2 %?±?3.2 (P?<?0.001), and 76.6 %?±?4.4 (P?<?0.01). At the lower doses of 0.25 mg/ml, 0.1 mg/ml or 0.05 mg/ml, no significant cell viability reductions were seen: 96.3 %?±?0.7 (P?>?0.05), 95.3 %?±?2.5 (P?>?0.05) and 93.8 %?±?2.3 (P?>?0.05), respectively compared to untreated HTM cells (97.0 %?±?1.9). Caspase-3/7 activity (msi) of HTM cells exposed to Dex 2, 1 or 0.5 mg/ml was 21068?±?2498 (P?<?0.001), 26994?±?3104 (P?<?0.001) and 20416?±?1150 (P?<?0.001) compared to untreated HTM cells 1148?±?803. Caspase-9 activity (msi) of HTM cells after exposure to Dex 2, 1 or 0.5 mg/ml was 14188?±?1203 (P?<?0.001), 13256?±?1564 (P?<?0.001) and 15041?±?1584 (P?<?0.001) compared to untreated HTM cells 1748?±?524. The lower doses of Dex did not significantly increase caspase-3/7 or -9 activities. There were no increases for caspase-8 or -12 activities at any of the tested Dex doses. The WST assay showed mitochondrial dehydrogenase activities of 14.3?±?0.7 (P?<?0.001), 9.6?±?0.3 (P?<?0.001) and 56.0?±?7.6 (P?<?0.001) at 2 mg/ml, 1 mg/ml and 0.5 mg/ml Dex compared to untreated HTM cells (186.1?±?15.0).

Conclusions

Dex at 0.25, 0.1 and 0.05 mg/ml clinical dose did not cause significant reduction in cell viability, increased apoptosis, or mitochondrial dysfunction of HTM cells in vitro. At high doses (2, 1 or 0.5 mg/ml) Dex caused apoptosis via mitochondrial pathways.     

Infection of endotheliotropic human cytomegalovirus of trabecular meshwork cells

Purpose

Human cytomegalovirus (HCMV) infections can cause endotheliitis which is associated with an elevation in the intraocular pressure (IOP). However, the mechanism of the IOP elevation has not been established. The purpose of this study was to determine whether HCMV strains which are capable of infecting corneal endothelial cells can also replicate and induce anti-viral responses, and can reorganize the actin cytoskeleton in trabecular meshwork cells.

Study design

Experimental study design.

Methods

Cultured primary human trabecular meshwork cells (HTMCs) were infected with the Towne or TB40/E strains of HCMV. TB40/E is trophic for vascular endothelial and corneal endothelial cells. Real-time PCR, western blot, and fluorescent immunostaining have been used to determine whether HCMV-infected HTMCs will support the expression of viral mRNA and protein, allow viral replication, and elicit anti-viral host responses. We also determined whether lytic replication was present after HCMV infection.

Results

HCMV infection led to the expression of viral mRNA and proteins of IE1, glycoprotein B(gB), and pp65. TB40/E infection induced interferon-β, a sign of host anti-viral immune response and monocyte chemotactic protein-1 (MCP-1) as IOP-related chemokine. Together with the induction of the regulators of actin cytoskeleton, myosin phosphatase Rho interacting protein (MPRIP) and MCP-1, TB40/E induced a high level of expression of viral proteins, including IE1, gB, and pp65 as well as actin stress fiber formation, and achieved pathogenically high viral titers.

Conclusions

Human trabecular meshwork cells support the replication of endotheliotropic TB40/E strain of HCMV which indicates that this strain may have high virulence for trabecular meshwork.

     

Mechanobiology of trabecular meshwork cells

Trabecular meshwork (TM) cells likely play a key role in regulating outflow facility and hence intraocular pressure. They function in a dynamic environment subjected to variations in mechanical and fluid shear forces. Because the extent of mechanical stress on the trabecular meshwork is dependent on the intraocular pressure, the behavior of TM cells under mechanical strain may suggest mechanisms for how outflow facility is regulated. Studies have demonstrated that TM cells respond in a variety of ways to mechanical loads, including increased extracellular matrix turnover, altered gene expression, cytokine release, and altered signal transduction. This review highlights some of the considerations and limitations of studying the mechanobiology of TM cells.     

Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork.

Extracellular matrix (ECM) turnover and remodeling are initiated, at least in part, by the regulated secretion of members of a family of matrix metalloproteinases. Human and bovine trabecular mesh-work in culture secrete interstitial collagenase, both the 72- and the 92-kD forms of type IV collagenase (gelatinases) and stromelysin, and the tissue inhibitor of metalloproteinases (TIMP). These proteinases and TIMP were identified by immunoblotting western transfers from sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using several specific antiprotein and antipeptide polyclonal antibodies. Gelatinase and stromelysin enzymatic activities were also analyzed by substrate SDS-PAGE, in which proteinase substrates were polymerized into the gels before electrophoresis to allow subsequent activity assays. These matrix metalloproteinases and TIMP are secreted at low basal levels into trabecular culture medium; their secretion levels are increased several-fold by treatment of the cultures with the phorbol mitogen. 12-O-tetradecanoylphorbol-13-acetate (TPA). Characteristics of the trabecular matrix metalloproteinases and TIMP are similar to those secreted by numerous other tissues, including the retinal pigment epithelium. These proteinases may serve an important role in the maintenance and regulation of the trabecular extracellular matrix and, subsequently, of the aqueous humor outflow pathway in normal and glaucomatous eyes.     

Dexamethasone induces specific proteins in human trabecular meshwork cells

Previous studies have demonstrated the presence of high-affinity, glucocorticoid-specific receptors in explants of human outflow tissue and in cultured trabecular meshwork. Glucocorticoid-induced responses of scleral fibroblasts and trabecular meshwork cells were evaluated in this study. Incubation of human trabecular meshwork (HTM) and scleral fibroblast (HS) cells with 10(-7) M dexamethasone (DEX) results in a 60% inhibition of prostaglandin production. The effects of glucocorticoid treatment on cellular and secreted proteins using [35S] methionine incorporation were evaluated. Treatment of HTM cells cultured from two normal individuals with DEX induced the expression of [35S] methionine-labelled cellular proteins of 35, 65 and 70 kD, and secreted proteins of 40, 90 and 100 kD. Under the same experimental conditions, a 70 kD molecular weight cellular protein was induced in the HS cells. There were no apparent DEX-induced alterations in HS-secreted proteins. Since a functional common response to glucocorticoid treatment in both HS and HTM cells was inhibition of prostaglandin production, the dexamethasone-induced expression of the 70 kD protein in these cells may be related to this effect. Further studies are required to elucidate specific roles of the steroid-induced proteins in the effects of glucocorticoids on HTM and HS cells.     

Decreased expression of HLA class II antigens on human uveal melanoma cells after in vivo X-ray irradiation

In order to determine the presence of HLA antigens on human uveal melanomas, we tested anti-HLA monoclonal antibodies on tissue sections of these tumors. A great variety in expression of HLA class I and II antigens was present. A significantly lower expression of HLA class II antigens was present on uveal melanomas that had been irradiated before enucleation. These tumors lacked a lymphocytic infiltrate in comparison with nonirradiated tumors. These data suggest that radiotherapy affects expression of histocompatibility antigens on tumors.     

小梁网干细胞的归巢及其在小梁创伤愈合中的作用

目的 研究胶原蛋白在小梁网干细胞（trabecular meshwork stem cells，TMSC）归巢中的作用以及小梁网（trabecular meshwork，TM）细胞和TMSC在创伤愈合中的相互作用。  设计 实验研究。研究对象 人原代TM细胞和TMSC。方法 分离、培养和传代人TM细胞和TMSC，通过定量RT-PCR和地塞米松诱导方法明确细胞类型；通过细胞贴附实验评价TM细胞及胶原蛋白对TMSC的亲和力；利用划痕损伤实验和时差显微镜观察TMSC分布及其与TM的相互作用。主要指标  TMSC和TM细胞的鉴定、TMSC穿膜数量和划痕损伤实验中TM细胞的迁移状态。 结果TMSC高表达OCT4而TM细胞高表达CHI3L1和MYOC，地塞米松刺激后TM细胞MYOC的表达显著增加。TMSC在含有TM细胞、胶原蛋白、TM细胞＋胶原蛋白的培养皿中的穿膜数分别为（13.1±5.3）个/高倍视野、（22.6±10.1）个/高倍视野、（4.8±2.2）个/高倍视野，均明显高于在单纯细胞培养皿中的穿膜数（3.7±0.5）个/高倍视野（P=0.0009，<0.0001，<0.001）。用AMD3100抑制TMSC中CXCR4的表达后，TMSC在各种状态的迁移数量差异消失。在TM细胞划痕损伤模型中加入TMSC可促进TM细胞的分化和迁移。结论  TMSC可诱导小梁网细胞分裂向受损区域迁移，从而促进组织损伤修复。胶原蛋白在TMSC归巢过程中发挥重要作用。