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Background: Rejection of corneal grafts is dependent on influx of T lymphocytes and macrophages. This process is partly regulated by adhesion molecules. Earlier investigations showed that corneal graft rejection in rats could be prevented by clodronate liposomes that selectively eliminate macrophages. In the present study the effect of macrophage depletion on adhesion molecule expression after corneal allotransplantation was investigated., Methods: Orthotopic corneal allografts were performed, after which rats received subconjunctival injections with clodronate liposomes or remained untreated. On various postoperative days, grafted rats were killed and mid-eye sections were stained for expression of ICAM-1 (CD54) and beta(2)-integrins (CD18 and CD11b/c)., Results: In the clodronate liposome-treated group grafts were not rejected, while in untreated rats grafts had a mean survival time of 12 days. During the first postoperative days a slightly enhanced expression of ICAM-1 in the conjunctiva and allografted cornea of clodronate liposome-treated recipients was seen. On day 12, however, ICAM-1 expression was markedly downregulated in the allografts of this treated group. The expression of beta(2)-integrins was also significantly decreased in the allografts and recipient corneas of treated rats at this time point., Conclusion: Prolonged corneal graft survival in rats, obtained via local depletion of macrophages, correlates with diminished expression of adhesion molecules.

Purpose: In rats, corneal allograft rejection is delayed for at least 100 days by clodronate liposomes. These liposomes selectively deplete macrophages. To investigate the immunologic basis for absence of graft rejection in treated rats, the effect of these liposomes on the generation of cytotoxic T lymphocytes (CTLs) and antibody production after orthotopic corneal allotransplantation was determined., Methods: Transplantations of corneal buttons from PVG rats were performed in AO rats. After surgery, one group received clodronate liposomes subconjunctivally at five time points, and the other group remained untreated. On postoperative day (POD) 3, 7, 12, or 17, rats were killed, the presence of CTLs was investigated at three different anatomic locations, and antibodies against donor antigens were tested., Results: No significant differences were found between the groups tested 3 and 7 days after surgery. But on POD 12 (the time of onset of rejection in the untreated group) and on POD 17, the CTL activities detected in the submandibular lymph nodes (P < or = 0.008) and the spleen (P < or = 0.009) were significantly less in the treated groups compared with the untreated groups. In the untreated groups complement-independent antibodies were present only on POD 17, whereas no antibodies were found in the treated rats., Conclusions: Local treatment with clodronate liposomes was shown to downregulate local and systemic CTL responses and to prevent the generation of antibodies. Local depletion of macrophages in the initiation phase of the immune response appears to lead to a less vigorous attack on the grafted tissue and therefore to promote graft survival.

Purpose: To test the effects of clodronate liposomes on graft survival and neovascularisation after transplantation in pre-vascularised recipient corneas., Methods: Corneal neovascularisation was induced in F344 rats by injecting heat inactivated rabbit serum intrastromally. After 4 weeks F344 rats were orthotopically grafted with corneal buttons from DA rats. Directly after transplantation and on 2, 4, 6 and 8 days postoperatively clodronate liposomes were administrated subconjunctivally in one group, whereas the other group remained untreated. For 60 days grafts were observed for signs of graft rejection and neovascularisation., Results: Graft survival was significantly prolonged, but not prevented in clodronate liposome treated rats compared to untreated rats ( p =.004). Also clodronate liposome administration delays growth of corneal neovascularisation after transplantation., Conclusions: Previous studies revealed that clodronate liposomes prevent corneal graft rejection and reduce neovascularisation in orthotopic corneal allotransplantation in rats. This study shows that also in pre-vascularised recipient corneas subconjunctival administration of clodronate liposomes seems to delay corneal graft rejection and reduces neovascularisation.

Purpose: Corneal allograft rejection in rats can be prevented by subconjunctival injections of liposomes containing dichloromethylene diphosphonate (clodronate-LIP), which selectively eliminate macrophages. In this study, the effect of clodronate-LIP treatment on cytokine mRNA levels in corneal allografts was examined., Methods: AO rats received corneal grafts of PVG rats. Rats were either not treated or injected subconjunctivally with clodronate-LIP on the day of transplantation and on postoperative days (PODs) 2, 4, 6, and 8. RNA was isolated from the graft and rim of corneas at different times after transplantation and from normal controls. Interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-10, IL-12p40, tumor necrosis factor (TNF)-alpha, TNF-beta/lymphotoxin (LT), interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) mRNA levels were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR)., Results: Corneal rejection, observed in all untreated rats by POD 12, was associated with increased mRNA levels of all cytokines investigated in grafts and rims. Clodronate-LIP treatment prevented allograft rejection and strongly decreased the levels of IL-1beta, IL-1RA, IL-2, IL-4, IL-6, IL-10, IFN-gamma, TNF-beta/LT, MCP-1, and MIP-2 mRNA in grafts and IL-1 beta, IL-2, IL-4, IL-6, and IFN-gamma mRNA in rims. Interleukin-12p40 mRNA levels were unaltered in clodronate-treated rats, except for a transient increase in grafts at POD 3. TNF-alpha mRNA levels were increased by clodronate-LIP in grafts and rims early after transplantation (PODs 3 and 7). Despite a normal appearance, long-term accepted corneal grafts (POD 100) contained mRNA for IL-10, IL-12p40, TNF-alpha, MCP-1, and MIP-2., Conclusions: Clodronate-liposome treatment markedly altered the mRNA levels of all cytokines investigated in corneal allografts. These results may explain in part the mechanism by which clodronate-LIP treatment prevents corneal allograft rejection.

Purpose: Idiopathic inflammation of the cornea, keratitis, has been proposed to result from an autoimmune process, but thus far no convenient animal model of keratitis exists. An attempt was made to establish an animal model for keratitis, to investigate possible autoimmune mechanisms., Methods: T-cell lines were established from lymph node cells removed from rats immunized with bovine corneal epithelium (BCE) extract. After restimulation in vitro with BCE or a specific corneal antigen, the cells were transferred by intraperitoneal injection into naive rats, rats subjected to total body irradiation, or rats in which only one eye was irradiated., Results: Neither direct immunization with corneal antigens nor transfer of activated anti-corneal T-cells into naive rats gave any signs of keratitis. Irradiation alone did not induce corneal inflammation. Transfer of corneal-specific activated T cells into irradiated rats produced keratitis starting around day 4 and culminating around day 8. The disease was self-limiting and the severity dependent on the dose and site of radiation. Keratitis was characterized by corneal haze, conjunctival and episcleral hyperemia, episcleral hemorrhages, chemosis, corneal infiltrates, and vascularization. Immunohistochemistry showed T-cell and macrophage infiltration of epithelium and stroma in the affected corneas., Conclusions: Thus, keratitis may be produced by T cells reactive to corneal antigens, provided that the target tissue has been made susceptible by irradiation. The effectiveness of T-cell vaccination in preventing adoptive keratitis suggests that systemic as well as local tissue factors may regulate the disease process.

Purpose: To determine the effect of a fresh corneal wound or a healed corneal scar on the immunodiffusion of immunoglobulins into the cornea., Methods: F344 rats were immunized with human serum albumin (HSA) 1 week before an autologous rotational keratoplasty of the right cornea or 1 year after an autograft was performed. One group of rats also was treated with gentamicin-dexamethasone ointment in the grafted eye for 1 week after transplantation to reduce the postsurgical inflammatory signs. A serum sample was drawn every week and booster injections with HSA were given after 2 and 3 weeks. At various times after immunization, groups of rats were killed, blood and aqueous humor samples were taken, and the corneas of both eyes were removed. The corneas were divided into the graft or a 3-mm central button and the peripheral rim and weighed. The anti-HSA titer was determined in serum, aqueous humor, and both parts of the corneas., Results: Up to 5 weeks after transplantation, the grafted cornea contained more anti-HSA immunoglobulins than did the control eye. One year postgrafting, no difference was seen. In the first weeks after keratoplasty, influx of anti-HSA from the peripheral into the central cornea was, however, neither obstructed nor enhanced., Conclusions: Surgical trauma in itself causes increased influx of anti-HSA immunoglobulins into the cornea. Within the cornea, a wound or a scar does not appear to be a barrier for centripetal immunoglobulin diffusion.

Allograft rejection is the main cause of corneal graft failure. T lymphocytes and macrophages have been implied to be involved in corneal rejection, but little is known about the molecular mechanism in this process. In this study, cytokine mRNA expression in the cornea was analysed during experimental corneal transplantation. The donor and acceptor corneas of two groups of rats were studied after receiving an allo- (PVG to AO rat) or autograft (AO rat). For controls, central buttons and peripheral corneal rings of the non-transplanted contralateral eyes were used. At different post-operative days (1, 3, 7, 12 and 19), the corneas were removed and subjected to mRNA isolation. All corneal samples underwent semi-quantitative reverse transcriptase-polymerase chain reaction analysis for interleukin-1 beta, interleukin-1, receptor antagonist, interleukin-2, interleukin-4, interleukin-6, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, monocyte chemotactic protein-1 and macrophage inflammatory protein-2 mRNA expression. Corneal rejection, characterized by opaque corneas with prominent neovascularization, was always diagnosed around day 12. Contralateral, non-grafted corneas showed constitutive mRNA expression for interleukin-1 receptor antagonist and in a few samples also monocyte chemotactic protein-1 and macrophage inflammatory protein-2 mRNA was found. Both allo- and autografts expressed mRNA for the cytokines found in contralateral, non-grafted tissue, as well as for interleukin-1 beta, interleukin-6, interleukin-10 and tumor necrosis factor-alpha. In allografts, the mRNA levels for these cytokines remained constant throughout all post-operative days, with increased interleukin-6 mRNA expression after post-operative day 12. The analysis of the autografts revealed high cytokine mRNA levels until post-operative day 3 or 7, which decreased from then on, except for interleukin-1 receptor antagonist. mRNA for interleukin-2, interleukin-4 and interferon-gamma was not observed in autografts at any time point and in allografts, until post-operative day 12. Interleukin-2 and interferon-gamma mRNA showed maximal expression on POD 12, while in autografts, a marked decrease was observed after POD 3. IL-10 mRNA levels decreased immediately after POD 1 in autografted eyes. For TNF-alpha, an increased mRNA expression starting on POD 7 was found in recipient rings of allografted eyes, while in autografts a weak expression was seen in some samples. MIP-2 transcription increased on PAD 12, while in autografts, its expression was not markedly different from that detected in the contralateral, non-grafted peripheral cornea.

In order to establish the significance of circulating cornea-specific antibodies, we determined the presence of anti-corneal antibodies in the serum of 100 patients with corneal disease and in 50 healthy controls, and subsequently followed the pattern of antibody reactivity in 46 patients who underwent corneal transplantation. An indirect immunofluorescence test on cryostat sections of rabbit corneas was used for screening. The reactivity against two known bovine corneal epithelial proteins was also tested: a 54-kD protein (BCP 54) and an 11-kD protein (BCP 11/24). No significant difference in the presence and specificity of anti-corneal antibodies was observed between the group of patients with corneal disease, taken as a whole, and the healthy controls. Patients with keratoconus or non-immunological graft failure, however, were significantly more often positive for anti-corneal antibodies. Neither the presence of antibodies prior to corneal transplantation nor their appearance post-transplantation had a predictive value for corneal graft survival.

Autoimmune reactivity against corneal antigens is described in two patients with Cogan's syndrome, a nonsyphilitic deep interstitial keratitis with vestibuloauditory symptoms. In both cases corneal antibodies were found at the beginning or during an exacerbation of the disease. After administration of high doses of corticosteroids the corneal antibodies diminished. Interstitial keratitis can generally be controlled by local or systemic corticosteroids. The effect of corticosteroid therapy on the audiovestibular symptoms is variable. The possibility of an autoimmune pathogenesis of Cogan's syndrome is discussed.

The water-soluble fraction of bovine corneal epithelium was analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Next to the principal soluble protein BCP 54, which has recently been identified as a corneal aldehyde dehydrogenase (ALDH), another abundant protein was observed, which we have denoted BCP 11/24, due to its estimated molecular weight of 11 kDa in SDS-PAGE and 24 kDa in high performance gel filtration under non-denaturing conditions. This protein was isolated and characterized by biochemical and immunochemical techniques. The isolation of BCP 11/24 was initially hampered by its tendency to bind non-covalently to BCP 54. BCP 11/24 behaves identically in reduced and unreduced SDS-PAGE and is probably not a glycoprotein. Isoelectric focusing indicated microheterogeneity of BCP 11/24, yielding bands with isoelectric points of 6.1, 5.9, 5.7 and 5.6. A rabbit antiserum directed against BCP 11/24, that did not recognize BCP 54, demonstrated that the distribution of BCP 11/24 in different ocular tissues as well as its light microscopic localization in corneal epithelium is strikingly similar to that of BCP 54. Together with its tendency to interact with BCP 54 in vitro, this suggests the possibility that BCP 11/24 is associated with BCP 54 in vivo, fulfilling a function which may be related to the activity of BCP 54 as a corneal ALDH. In contrast with BCP 54, however, BCP 11/24 was not detectable in corneal endothelium. The antiserum did not detect any immunologically related molecules in corneal epithelium extracts of sheep, human or rat origin, indicating that BCP 11/24 is probably not as highly conserved as BCP 54.

We report our experiences with a patient with relapsing polychondritis, in whom circulating antibodies against corneal epithelium were determined by immunofluorescence before and after medical treatment. The results provide further evidence for an autoimmune pathogenesis of the disease.

Immunologic responses play a role in the rejection process of corneal transplants. Both histocompatibility antigens and tissue-specific antigens may be potential targets for such an immune response. To identify relevant responses, the humoral immune response against corneal tissue and the cellular immune response against one specific corneal protein were determined in patients before and after corneal transplantation. The results were compared with known risk factors for corneal transplantation, but no correlations were observed. A conversion from negative to positive in cellular immune response against the cornea-specific protein was seen in patients who had experienced an inflammatory episode during the time interval between measurements. The anticorneal protein response may therefore be the result of an intraocular inflammatory response, but not a prognostic factor to help predict the patients at risk for rejection.

Corneal epithelium antibodies were detected in patients with corneal melting disease and uveitis using an immunofluorescence technique with cryostat sections of corneas obtained from various species (man, guinea pig, rabbit, mouse, rat, cow, pig). No differences in results were found using these various substrates, indicating that the autoimmune response is directed against common non-species specific corneal epithelium antigens. The serum of a patient with corneal melting disease, containing a high antibody titer against corneal epithelium was used to identify and isolate one of the bovine corneal antigens. A 54,000 dalton protein was isolated, which was shown to be the major protein present in the corneal epithelium. Absorption studies with other tissues taken from human eyes showed that cornea epithelium, cornea devoid of epithelium, ciliary body and retina contained material which cross-reacted with the isolated bovine corneal epithelium antigen, whereas iris and sclera showed no detectable cross-reaction. The incidence of autoantibodies directed against this antigen was investigated in patients with corneal melting disease, corneal transplantion and in uveitis patients using an ELISA and comparing the results with those obtained with the immunofluorescence assay on rabbit cornea sections. A positive ELISA was always associated with a positive immunofluorescence test. The presence of antibodies against the 54 Kd antigen as detected by the ELISA could be confirmed by immunoblotting in 7 out of 9 positive sera tested. A large number of sera showed a positive immunofluorescence test but a negative ELISA against the 54 Kd corneal epithelium antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the incidence of circulating corneal epithelium antibodies in patients with uveitis. A high percentage of the patients (42%) were positive, whereas only 4% of controls had these antibodies in their serum. Significantly more patients with anterior and diffuse uveitis had corneal epithelium antibodies than did those with posterior uveitis. Subdivision of anterior uveitis into HLA-B27 positive versus negative patients showed a higher incidence of the antibodies in the HLA-B27 positive group. A previous history of uveitis may play a role in the pathogenesis of peripheral corneal thinning diseases, for which an autoimmune aetiology has been suggested.

Sera obtained from 26 patients with Fuchs' heterochromic cyclitis were examined for the presence of autoantibodies directed against the anterior segment of the eye by means of immunofluorescence techniques. Antibodies against human iris tissue could not be detected, whereas autoantibodies against corneal epithelium were found in almost 90% of cases. This is the first report describing the presence of autoantibodies in patients with Fuchs' heterochromic cyclitis and it provides a further clue that immunological mechanisms might play an important role in the aetiology of Fuchs' heterochromic cyclitis.

Immunity to a major corneal antigen was studied in 28 Fuchs' heterochromic cyclitis patients and compared with the response of 44 patients with other types of uveitis and 19 healthy controls. The highest incidence of immune response was found in patients with anterior segment involvement only (anterior uveitis and Fuchs') whereas the frequency of anti-corneal immune response in patients with posterior segment involvement only was low and not significantly different from that of healthy controls. Cellular immunity to corneal antigens was found in the majority of Fuchs' heterochromic cyclitis patients, and in one-third of the anterior uveitis patients. No correlation could be established in these patients between a positive cellular response and the chronicity of the disease or the presence of keratic precipitates. Humoral immunity to the corneal antigen was also the highest in patients with anterior segment involvement, but there was no difference in response between Fuchs' and non-Fuchs' anterior uveitis patients. This study suggests that anti-corneal immunity may be triggered in inflammatory diseases of the anterior segment, especially in Fuchs' heterochromic cyclitis.

Fresh and organ-culture preserved human corneas were examined for HLA-A,B,C (class I) and HLA-DR (class II) histocompatibility antigens using mouse monoclonal antibodies and an indirect immunofluorescence technique. HLA-A,B,C antigens were detected on epithelial cells and on keratocytes, but not on endothelial cells in fresh corneas. The expression of HLA-A,B,C antigens was not significantly altered by organ-culture for a period up to 7 days. The epithelial cell layer bearing the larger part of the HLA-A,B,C antigens decreased, however, from 6-7 layers to 2-3 during organ culture. HLA-DR antigens were not detected on any of the corneal layers, but were present on scattered dendritic cells within the corneal epithelium and on cells in the corneal stroma just beneath the Bowman's membrane. Using immunofluorescence, no cells bearing HLA-DR antigens were seen in cornea sections obtained after 1 week in organ-culture. These results demonstrate that the presence of HLA antigens, particularly of the HLA-DR antigens, is affected by the organ-culture preservation of the human cornea.

Corneal epithelium antibodies were detected in patients with corneal melting disease (55%), uveitis (42%), corneal transplantation (42%) and marginal furrow disease (20%). These antibodies were not found in herpetic keratitis patients. In control groups, consisting of ocular surgery patients (glaucoma, retinal detachment and cataract) and persons without a history of ocular disease, approximately 4% of the subjects had these antibodies. To investigate the possible role of trauma to the cornea as an initiator of corneal epithelium antibodies, these antibodies were determined in rabbits after alkaline burns were made on the cornea. These antibodies were detected one week later and disappeared after six weeks. Serum from three patients with corneal melting disease and corneal transplantation containing a high antibody titre against corneal epithelium were used to isolate corneal epithelium antigens. A 54 kD and a 17 kD corneal epithelium antigen were isolated. The incidence of autoantibodies directed against these antigens was investigated in patients with corneal melting disease, uveitis and corneal transplantation using an ELISA. 50% of the sera positive in the immunofluorescence test were positive in the ELISA.