Immunolocalization of prostanoid EP receptor isotypes in human trabecular meshwork.

PURPOSE: To assess the localization of the EP-type prostanoid receptors in the human trabecular meshwork (TM) and to determine their spatial distribution in relation to the contractile a-smooth muscle actin fibres. METHODS: Cryosections of human anterior segments were obtained from 17 different donors and immunostained with different EP receptor subtype specific antibodies. Double staining for the EP2 receptor and smooth muscle actin was carried out. Western blots of TM protein samples were studied. RESULTS: No specific staining for the EP1 receptor was observed. The antibodies against the EP2 receptor revealed in all donors intense staining of human trabecular cells throughout the meshwork. EP3 receptor specific staining was not detected. EP4 immunostaining was confined to the corneoscleral region near Schwalbe's line. On western blots, the EP2 receptor was detected. In the posterior TM, the EP2 receptor staining was associated with the dense network of actin fibres. CONCLUSIONS: These immunocytochemical results present evidence that the EP2 receptor is the most abundantly expressed isotype of the PGE receptors in the human TM. This conclusion is in agreement with our previous findings at the transcript level. The relaxant responses of the TM to application of EP2 receptor agonists, and flow enhancement evoked by prostaglandin PGE1, may be explained by the close spatial association of the EP2 receptor with actin fibres.

Macrophages play a role in the early phase of corneal allograft rejection in rats.

BACKGROUND: Rat corneal allograft rejection is delayed by repeated local injection of liposomes filled with clodronate (dichloromethylene diphosphonate), which selectively deplete macrophages. Various administration schedules of liposomes were tested to determine the optimum schedule for prevention of graft rejection. Cell subpopulations in the anterior segment of the eye were studied at different time points after transplantation to assess the kinetics of the immune response. METHODS: AO rats were grafted orthotopically with corneal buttons from PVG rats. Postoperatively, rats remained untreated or received clodronate liposomes subconjunctivally. Clodronate liposomes were injected five times on postoperative days (PODs) 0, 2, 4, 6, and 8; or once, on POD 0 or 6; or twice on PODs 0 and 2 or PODs 0 and 6. Grafts were examined for signs of rejection clinically and immunohistologically. RESULTS: All untreated rats rejected their grafts as did all five rats that received clodronate liposomes once on POD 6. In all the other administration schedules tested, graft survival was prolonged compared with the untreated control group (P <0.01). Injections of clodronate liposomes on PODs 0 and 2 proved to be the most effective treatment. Histologically reduced influx of virtually all cell types tested was found in this group. CONCLUSIONS: To prevent or delay graft rejection, it is necessary to administer clodronate liposomes in the early phase after corneal transplantation. These results suggest a role for macrophages in the afferent phase of corneal graft rejection.

Adhesion molecule expression in local-macrophage-depleted rats bearing orthotopic corneal allografts.

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.