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The presence of antigen-presenting cells and hem- and lymphangiogenesis in the cornea are risk factors for the rejection of corneal transplants. We previously reported that antigen-presenting cells such as macrophages (MPs) play an important role in the induction of lymphatic endothelial cells during inflammation. This prompted us to inquire whether the existence of lymphatic vessels in the cornea is associated with the activation of MPs during inflammation. To investigate this question, we performed suture placement on the cornea to induce inflammation. We found that a large number of MPs were recruited and that lymphatic vessels were formed in response. Next, as C57BL/6 mice have a higher rejection rate after corneal transplantation than BALB/c mice, we compared the corneas of C57BL/6 and BALB/c mice under normal and inflamed conditions. We found that the number of spontaneously formed lymphatic vessels in the C57BL/6 corneas was significantly greater than in the BALB/c corneas, and that there were more activated MPs in the C57BL/6 corneas than in the BALB/c corneas. Additionally, to confirm that activated MPs induced and maintained lymphatic vessels in the cornea, we depleted the number of MPs in C57BL/6 mice via clodronate liposomes. We found that MP depletion reduced the spontaneous formation of lymphatic vessels and reduced inflammation-induced lymphangiogenesis relative to control mice. Finally, we found that mice deficient in MP markers had fewer spontaneously formed lymphatic vessels and less lymphangiogenesis than control C57BL/6 mice. The evidence gathered in this study leads us to conclude that activated MPs appear to play an important role in the formation of new lymphatic vessels and in their maintenance.

Purpose: We performed an immunohistochemical study on the development of interstitial keratitis in a rabbit model sensitized by ovalbumin (OA)., Methods: A mixture of OA (5 micrograms/ml) and Freund's complete adjuvant was initially injected subcutaneously (0.4 ml) into the rabbits' back and footpad (0.2 ml). After a week, the rabbits were sensitized by an injection of the same solution into the back (0.5 ml) again. After 4 weeks the rabbits underwent the same treatment as a booster shot. Then a week later OA solution (0.05 ml) was injected into the corneal stroma. We observed the cornea on days 1-3, 7, and 10 after the corneal treatment. Corneas on days 3 and 10 were examined by immunohistological methods using hematoxylin-eosin stain, methylgreen pyronin stain, and by immunohistochemical methods with anti-CD 4, anti-CD 8, and anti-OA antibodies. The localization of specific antibodies was identified by fluorescent-antigen method. Electron-microscopic observation was also done., Results: Rabbits developed corneal opacity and edema on day 1, corneas had on immune ring with a peak intensity on day 3, and vascularization on day 7 after corneal treatment. Microscopic examination revealed infiltration of neutrophils in to the area of the immune ring on day 3 and many plasma cells at the limbus and stroma on day 10. CD 4 positive cells were found at the limbus and stroma on days 3 and 10. CD 8 positive cells were found at the limbus and stroma only on day 10. OA positive findings were observed at the immune ring and its inner area. In the fluorescent-antigen method, specific antibodies were found in plasma cells at the limbus on days 3 and 10., Conclusions: In our experimental model of interstitial keratitis with immune ring and vascularization, the Arthus reaction was dominant. Infiltration of antigen specific plasma cells in the stroma with vascularization caused a modification of pathologic reaction in keratitis.

Corneal transplantation was performed by grafting C3H/He donor corneas into BALB/c corneal beds. The allografted mice were injected either with 0.5 mg/day of anti-very late antigen (VLA)-4 antibody, anti-leukocyte function-associated antigen (LFA)-1 antibody, or 0.25 mg/day each of both antibodies on days -2, 0, 1, 3, 5, and 7. After 3 weeks, cytotoxic T lymphocyte (CTL) responses to donor alloantigens were assessed. Splenocytes in the allografted mice without treatment demonstrated greater CTL responses that those in naive mice. CTL responses were depressed in mice treated with either anti-LFA-1 alone or a combination of anti-LFA-1 and anti-VLA-4 antibodies as compared with splenocytes from allografted mice without treatment. These data suggest that CTLs play significant roles in eliciting immune response to corneal allografts.

We have been investigating the production of cytokines in ocular tissues. In this paper, we demonstrated the in vitro production of interleukin-6 (IL-6) by human corneal epithelial, stromal and endothelial cells using enzyme-linked immunosorbent assay (ELISA). In culture supernatant of the stromal cells, the production of immunoreactive IL-6 was induced, depending upon the doses of lipopolysaccharide (LPS) or IL-1 alpha added into the culture. Detectable IL-6 activity in the supernatant of the stromal cells was found 2 hours after addition of IL-1 alpha and the activity increased to a peak level at 48 hours. On the other hand, in the supernatant of the endothelial cells, IL-6 activity was found even in unstimulated-culture, and induced further by LPS stimulation. The molecular weights (MWs) of the IL-6 produced by the epithelial, stromal and endothelial cells were calculated by gel filtration as about 30 kDa. From Western blotting analysis, the MW of IL-6 produced by the stromal cells was also determined to be 30 kDa.

Penetrating keratoplasty was performed on inbred rats (DA rat----Lewis rat) and the appearance of infiltrating cells in rejection was studied immunohistochemically using anti-rat monoclonal antibodies W3/25 (helper T cell), OX8 (suppressor/cytotoxic T cell), OX6 (Ia antigen positive cell) and OX42 (macrophage). In all cells evaluated, infiltration around the graft occurred between days 2 and 4. In OX42-positive cells infiltration was most intense in the early phase. Rejection occurred on postoperative day 12 +/- 2. Prior to the development of rejection, OX6-positive cells and W3/25-positive cells were dominant. However, during rejection, OX8-positive cells markedly increased, becoming dominant. These changes were not reflected in the peripheral blood. During rejection, the endothelial flat preparation was Ia antigen-positive which not found in normal cornea.

Cellular infiltration of the suture line was studied immunohistologically after rat corneas were sutured with various types of suture materials. The suture line was invaded by helper/inducer T lymphocytes, cytotoxic/suppressor T lymphocytes, and Ia-antigen-positive cells. Among the invading cells, Ia-antigen-positive cells were the most prominent over an extended period of time from immediately after suture placement. The helper/inducer T lymphocytes consisted predominantly of cytotoxic/suppressor T lymphocytes. The 9-0 silk suture produced a higher degree of cellular infiltration than the 10-0 nylon suture or the 10-0 prolene suture. There was no marked difference in the infiltration produced by the 10-0 nylon and 10-0 prolene sutures. After the sutures were removed, the cellular infiltration appreciably decreased. More marked invasion occurred with interrupted sutures than after the inter-lamellar insertion of suture material. These findings indicate that suture materials on the cornea promote infiltration of immunocompetent cells and that mechanical stimulation of the suture notch enhances such cellular response, which may be involved in graft rejection of the corneal transplantation.

Changes in localization of virus-associated antigens induced by F3TdR were studied using HSV-1 infected monolayer of cultured SIRC cells. A direct enzyme-antibody method was employed for preembedding for immunoelectron and light microscopy. At 3 hours post infection (h.p.i.), immunoreaction products began to appear in cytoplasm and nuclei in both the control and the F3TdR-treated group. The amounts increased during the subsequent hours of observation. In the control group, there was an intense diffuse immunoreaction in the cytoplasm and nuclei and dense accumulations of the reaction products were seen on the cell surfaces. In F3TdR-treated cells, intranuclear antigens were present as diffuse accumulations of granular deposits, and cytoplasmic stainings were very much reduced in degree; deposits on the cell surface were a rare finding. Progeny virus particles were significantly fewer in the presence of F3TdR; nucleocapsids were found but in a greatly reduced number and no enveloped particle was found. The morphology of individual nucleocapsid, however, was not significantly altered by treatment with F3TdR. A preponderance of antigenic substances in the nuclei over the cytoplasm in treatment group may reflect distorted protein synthesis and may provide morphological evidence of the biochemically assumed hypothesis of F3TdR efficacy; F3TdR exerts it's antiviral effects through abnormal protein synthesis and resultant defects in capsid assembly. Decreased antigenic substances in the membranous structures of cytoplasm may be related to reduced or abnormal production of viral antigens closely associated with envelopment.