Flow-cytometric analysis of peripheral lymphocytes in the rat following penetrating keratoplasty and immunosuppressive treatment

Abstract

Flow cytometry has been found to be a useful tool in the clinical monitoring of patients following solid organ transplantation. Despite the easier assessibility of corneal graft acceptance or rejection, flow-cytometric evaluation of peripheral lymphocytes following systemic immunosuppression is important in the evaluation of therapeutic efficacy. The systemic effect of penetrating keratoplasty (PKP) and of immunosuppression with cyclosporin A (CsA), leflunomide (LF), and the nondepleting anti-CD4 antibody, RIB 5/2, on peripheral lymphocytes was investigated in the rat model. Corneal buttons were grafted from Lewis/Brown Norway rats to Lewis recipients. Animals were randomly assigned to the following treatment groups: (1) untreated; (2) CsA, 10 mg/kg; (3) LF, 10 mg/kg; (4) LF, 5 mg/kg; (5) LF, 2.5 mg/kg; (6) LF, 10 mg/kg; combined with CsA, 10 mg/kg; and (7) RIB 5/2, 4 mg/kg, combined with CsA, 1.5 mg/kg. Controls included the following groups: (8) unoperated, CsA-treated at 10 mg/kg; (9) unoperated, LF-treated at 10 mg/kg; (10) unoperated, LF-treated at 10 mg/kg and CsA-treated at 10 mg/kg; (11) unoperated, RIB 5/2-treated at 4 mg/kg and CsA-treated at 1.5 mg/kg; (12) syngeneic grafts; and (13) normal Lewis rats. Cells from blood and spleen samples were enriched using Ficoll density centrifugation, and lymphocytic surface markers CD 3, CD 4, CD 8, and RT 1b (Ia) were analyzed by direct immunofluorescence using flow cytometry. In the untreated allogeneic PKP, a decrease in the percentage of serum CD 3+, CD 4+, CD 8+, and CD 4+ RT 1b+ lymphocytes was apparent from the 5th to the 9th postoperative day. At corneal graft rejection, a normalization of serum CD 3+ and CD 4+ levels occurred, whereas the percentage of serum CD 8+ lymphocytes remained slightly raised. Significantly enhanced decreases in levels of CD 4+ lymphocytes were observed following treatment with CsA, LF, and, particularly, therapy with RIB 5/2 and a subtherapeutic dose of CsA. Concomitant decreases were also apparent following treatment with CsA or LF. In contrast, the combination of RIB 5/2 and CsA (1.5 mg/kg) resulted in a reactive increase in levels of CD 8+ lymphocytes as well as an increased expression of RT 1b by the remaining CD 4+ lymphocytes. The latter alterations corresponded to indefinite acceptance of the corneal grafts, which was observed only in the animals treated with RIB 5/2 and CsA. PKP in the rat model is accompanied by alterations in peripheral lymphocytes. Additional influences are exerted by immunosuppressive therapy, with the most specific and predictable alterations being achieved with anti-CD4 monoclonal antibody therapy.