CYCLOSPORINE AND ANTI-INTERLEUKIN 2 RECEPTOR MONOCLONAL ANTIBODY THERAPY SUPPRESS ACCELERATED REJECTION OF RAT CARDIAC ALLOGRAFTS THROUGH DIFFERENT EFFECTOR MECHANISMS

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Abstract
Increasing numbers of sensitized patients are either precluded from receiving an allograft or experience accelerated rejection which may be refractory to conventional therapy. Using a rat model, we have shown that accelerated (24 hr) rejection of LBN cardiac Tx in LEW rats sensitized with BN skin grafts 7 days earlier, could be prevented by treatment with cyclosporine (15 mg/kg/ day ×7 days, Tx survival about 42 days) or ART-18, an anti-IL-2R mAb (300 μg/kg/day ×10 days i.v., Tx survival about 16 days). In this study, we evaluated intragraft mechanisms responsible for these effects by immunoperoxidase localization of relevant humoral mediators (IgG, IgM, C3, cross-linked fibrin), graft infiltrating cells (GIC), and associated cytokines (IL-2, IFN-g, tumor necrosis factor [TNF], or cytokine receptors (IL-2R). Tx rejected in fulminating fashion by 24 hr in sensitized hosts showed extensive and progressive endothe-lial deposition of IgG, C3, and fibrin from 2 hr, followed by an influx of neutrophils at 3 hr, and peak numbers of GIC by 18 hr (88.8± 20.3 leukocytes/field). At 18 hr, GIC consisted of neutrophils (26%), T cells (20%, >90% of which were OX-8+), and monocytes/macrophages (53%), whereas B cells were absent. By 18 hr, up to 20% of GIC were IFN-g+, 10% were IL-2R+, and 10% were IL-2+, consistent with labeling of 20% of cells with OX-22. Widespread endothelial and mononuclear cell labeling for TNF and the procoagulant molecular tissue factor (TF) were also noted. In contrast to untreated grafts, CsA treatment essentially abolished intragraft Ig, C3, and fibrin deposition. Moreover, despite dense cell infiltration at 24 hr (total GIC 55.3±13.4/field), analysis of CsA-treated Tx showed markedly decreased neutrophils (0.5%), with increased T cells (35%) and similar proportions of macrophages (66%). In addition to the reduction in neutrophils, Ig and C3, fewer IL-2R+ (6%) and OX-22+ (3%) cells, considerably less TNF and TF, and almost no IL-2+ or IFN-g+ GIC (+ mononuclear cells to only 2% in ART-18-treated rats, up to 10% of mononuclear cells still showed binding with anti-IL-2 and anti-IFN-g mAbs, and 14% of cells were OX-22+, suggesting persistence of immune activation in this group despite prolongation of graft survival. Rats treated with a synergistic combination of ART-18 and low-dose CsA (1.5 mg/kg/day, for 7 days) showed effects intermediate between ART-18 and full-dose CsA-treated rats. In summary, accelerated rejection in untreated rats is associated with rapid and progressive endothelial deposition of Ig, C3, and fibrin, as well as dense neutrophil infiltration. In addition to these anticipated mediators, evidence of cellular immune activation was found, including progressive graft infiltration by mononuclear cells producing IL-2, IFN-g, and TNF, and expressing IL-2R. CsA and ART-18 each significantly decreased intragraft Ig and C3 deposition and prolonged graft survival. However, though both agents were able to blunt the vascular response, presumably by inhibiting terminal B cell differentiation and/or proliferation, only CsA appeared to effectively abrogate a cellular immune response. Nevertheless, based on these studies and dependent upon confirmation in subhuman primate models, treatment with anti-IL-2R mAbs may prove to be a useful additional therapeutic option in the management of highly sensitized patients, particularly when CsA therapy may be contraindicated, such as in the immediate post-Tx period in patients with poor renal function.

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