IMMUNOHISTOLOGICAL ANALYSIS OF SERIAL BIOPSIES TAKEN DURING HUMAN RENAL ALLOGRAFT REJECTION

Abstract

This immunohistological study investigated two aspects of the mechanisms underlying human renal allograft rejection. First, because rejection is a dynamic, complex process, we sought to delineate any changes in the types of cells mediating graft destruction by evaluating the cellular infiltrates in sequential renal biopsies from 14 patients with rejection. Second, because macrophage accumulation and fibrin deposition are major features of kidney rejection, the membrane characteristics of intragraft macrophages were analyzed to determine whether these cells could indeed cause the fibrin deposition frequently observed. Thirty-six biopsies, performed for assessment of renal failure posttransplantation (post-Tx), were studied using a panel of monoclonal antibodies and a 4-layer immunoperoxidase technique. Biopsies were divided into 3 groups depending upon the time post-Tx. Comparison of biopsies taken on days 2–3 post-Tx with those taken either at days 10–12, or later than 30 days, showed similar proportions of T cells, T cell subsets, B cells and macrophages. By contrast, the proportion of natural killer (NK) cells was significantly increased at days 2–3 (P < .01), and the proportion of activated T cells bearing interleukin-2 receptors was significantly increased at days 10–12 (P < .01). Granulocytes were restricted to biopsies that displayed areas of infarction, regardless of the time at which this occurred. In addition, various proportions of intragraft macrophages exhibited the membrane phenotype of activated macrophages, as a result of their expression of the procoagulant molecule termed human-tissue-factor-related antigen (HTF:RAg). The proportion of graft macrophages exhibiting HTF:RAg was significantly increased in biopsies on days 10–12 (P < .05) compared with biopsies on days 3–4, and remained elevated thereafter. Interstitial and perivascular collections of HTF:RAg+ macrophages were closely associated with fibrin deposits and, in two cases, mononuclear cells harvested from rejected grafts were shown to contain significant procoagulant activity in vitro. These studies demonstrate a major temporal variation in the types of cells contributing to human kidney rejection. Furthermore, the demonstration of macrophages with procoagulant activity and HTF:RAg in close association with fibrin deposits suggests that macrophage infiltration and activation is an important mechanism in the initiation of intragraft fibrin deposition. Finally, the association of macrophage expression of HTF:RAg with the entry of activated T cells may implicate delayed-type hypersensitivity (DTH) reactions in the pathogenesis of human graft rejection.