Differences in leukocyte phenotype and interferon-gamma expression in stroma and endothelium during corneal graft rejection.

Critical to the success of human corneal transplants is prevention of corneal endothelial rejection, yet little is known about the endothelial infiltrate. To examine the endothelium, a method for removal and processing this layer as a flat sheet was used and the infiltrate was compared with stroma and epithelium. LEW or PVG strain rat corneas were transplanted to PVG strain recipients. Clinical changes after transplantation were monitored by slit lamp and animals sacrificed at a range of time points during rejection. Clinically defined rejection, accompanied by an epithelial rejection line and endothelial cell infiltration, occurred between days 10 and 15. There was some infiltration of leukocytes in the stroma of isografts at these time points, but significantly more in allografts (p<0.003 for all subsets). There was no infiltration of isograft endothelium at any time and no infiltration of allograft endothelium on day 10. On day 15, there were similar numbers of all major subsets except B cells in the stroma, while on the endothelium macrophages, MHC class II(+) cells and CD8(+) cells predominated (p<0.001 CD4(+) vs CD8(+) cells). T cells and NK cells predominated in the epithelial rejection line. While TNF-alpha and IFN-gamma-producing cells were numerous in stroma and epithelium, no IFN-gamma-producing cells were found on endothelium. Distinct differences in infiltrative profile within layers of the cornea suggest that the mechanisms of rejection may also differ. The restricted endothelial cell profile and lack of IFN-gamma suggests that the anti-endothelial response may be modulated by the anterior chamber environment.

Sorsby's fundus dystrophy mutant tissue inhibitors of metalloproteinase-3 induce apoptosis of retinal pigment epithelial and MCF-7 cells.

C-terminal domain tissue inhibitor of metalloproteinases-3 (TIMP-3) mutations cause the rare hereditary blindness Sorsby's fundus dystrophy (SFD), which involves loss of retinal pigment epithelial (RPE) cells. Since wild-type TIMP-3 causes apoptosis, we investigated whether SFD TIMP-3 might kill RPE and other cells. Plasmid-mediated overexpression of Ser-156, Gly-167, Tyr-168 and Ser-181 SFD mutant TIMP-3 decreased RPE viability to 22+/-8, 20+/-6, 32+/-5, 30+/-12% (SFD mutants all P<0.01 versus wild-type 50+/-8%) and similarly increased propidium iodide staining and in situ end labelling. Adenovirus-mediated overexpression of the Gly-167 mutant also caused RPE apoptosis dose-dependently. Apoptosis of RPE cells might therefore contribute to the pathology of SFD.

Primary herpes simplex virus type 1 infection of the eye triggers similar immune responses in the cornea and the skin of the eyelids.

Herpetic stromal keratitis (HSK) and blepharoconjunctivitis in humans are thought partly to result from immunopathological responses to herpes simplex virus type 1 (HSV-1). The corneas of NIH mice were inoculated with HSV-1 (strain McKrae) and mice were examined for signs of disease and infection on days 1, 4, 7, 10, 14 and 21. The eyes and eyelids of infected and control mice were processed for immunohistochemistry and double stained for viral antigens and one of the following cell surface markers (Gr-1, F4/80, CD4, CD8, CD45R or MHC class II) or one of the following cytokines (IL-2, IL-4, IL-6, IL-10, IL-12 or IFN-gamma). All infected mice developed signs of HSK by day 4 and blepharitis by day 7 and these both persisted until day 21, when signs of resolution where apparent. Virus was detected during the first week of infection and became undetectable by day 10. Large numbers of Gr-1(+) cells (neutrophils) infiltrated infected corneas and eyelids in areas of viral antigen and CD4(+) T cells increased significantly in number after virus clearance. In both sites, the predominant cytokines were IL-6, IL-10, IL-12 and IFN-gamma, with few IL-2(+) and IL-4(+) cells. These observations suggest that the immune responses in the cornea are similar to those in the eyelids but, overall, the responses are not clearly characterized as either Th1 or Th2. In both sites, the neutrophil is the predominant infiltrating cell type and is a likely source of the cytokines observed and a major effector of the disease process.

Corneal epithelial rejection in the rat.

PURPOSE: To investigate clinical and histologic changes in the epithelium during corneal graft rejection in the rat. METHODS: LEW (RT1(l)) or PVG (RT1(c)) strain corneas were transplanted to PVG strain recipients and examined by slit lamp for clinical signs of rejection. Recipients were killed, and corneal epithelial sheets were removed and examined by adenosine diphosphatase (ADPase) staining for Langerhans cells (LC) and by immunohistology for leukocytes and adhesion molecules (T cells, macrophages, granulocytes, major histocompatibility complex [MHC] class II, CD2 and CD54 intercellular adhesion molecule [ICAM]-1) at a range of time points before, during, and after rejection, depending on the cell type sought. Normal and contralateral eyes were examined for ADPase(+) and MHC class II(+) cells. RESULTS: Clinical rejection, as defined by stromal opacity, occurred between days 10 and 15 after transplantation. In 94% of allografts, a curved clinical epithelial rejection line was observed in which ADPase(+)/MHC class II(+), CD4(+), or CD8(+) T cells were identified. There were significantly more infiltrating cells of all types in epithelia of allografts than in those of isografts. The most numerous cells were CD4(+) and CD8(+) T cells, suggesting preferential migration of these cells into the epithelium from underlying layers. Expression of MHC class II and ICAM-1 was induced on epithelial cells. CONCLUSIONS: Epithelial rejection in rats is clinically similar to that in humans and occurs simultaneously with stromal infiltration. It may be mediated by T cells rather than macrophages. In isolation, its recognition in humans may be a useful indication that the patient is at high risk of endothelial rejection.

Adenovirus mediated gene delivery of tissue inhibitor of metalloproteinases-3 induces death in retinal pigment epithelial cells.

BACKGROUND: Sorsby's fundus dystrophy (SFD) and age related macular degeneration (ARMD) are retinal diseases associated with a high level of accumulation of mutant and wild type TIMP-3, respectively, in Bruch's membrane. The pathogenic role of TIMP-3 in these diseases is uncertain, but causative mutations have been identified in the TIMP-3 gene of patients with SFD. Recent reports that TIMP-3 causes apoptosis in certain cell types and not in others prompted the authors to investigate whether TIMP-3 causes apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: RPE and MCF-7 cells (as a positive control) were initially infected with replication deficient adenovirus, to overexpress beta-galactosidase (RAdLacZ) or TIMP-3 (RAdTIMP-3). TIMP-3 was detected by western blotting and ELISA. Cell viability was defined by cell counts. ISEL was used to investigate the mechanism of cell death. RESULTS: Cultured RPE cells produced small quantities of endogenous TIMP-3 and remained viable. However, overexpression of TIMP-3 caused a dose related death of RPE cells. The mechanism of cell death was apoptosis. CONCLUSION: The previously unreported finding of TIMP-3 induced apoptosis of RPE cells may account for some of the early features seen in SFD and ARMD.