Immunohistological analysis of serial biopsies taken during human renal allograft rejection. Changing profile of infiltrating cells and activation of the coagulation system.

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 less than .01), and the proportion of activated T cells bearing interleukin-2 receptors was significantly increased at days 10-12 (P less than .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 less than .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.(ABSTRACT TRUNCATED AT 400 WORDS)

Preliminary characterization of the procoagulant material in human ascites.

Disseminated intravascular coagulation invariably accompanies placement of peritoneovenous (LeVeen) shunts, which suggests that ascitic fluid contains procoagulant material capable of activating blood coagulation. In this study, we identified thrombogenic activity in human ascites and the hemostatic pathway by which it acts. Peritoneal fluid was removed percutaneously from patients with ascites due to various causes. Four fractions were prepared by centrifugation: cells, a low-speed, cell-free fluid, a high-speed supernatant, and the precipitate from the high-speed centrifugation. Cellular fractions from all ascitic fluids shortened a one-stage clotting time of normal pooled plasma by 68% in comparison with saline solution and endotoxin controls. Similarly, the cell-free fluids also shortened the clotting time of normal pooled plasma by 41%. The cellular and cell-free fractions shortened the clotting time of factor VIII-deficient plasma but failed to demonstrate procoagulant activity in factor VII-deficient plasma. These fractions had no effect on platelet aggregation or the platelet release reaction. The high-speed precipitate was dissociated by ethylenediaminetetra-acetate (EDTA) into fluid phase and precipitate, both of which demonstrated procoagulant activity. Furthermore, high-speed precipitate contained protein, phospholipid, and sterol in proportions similar to those of plasma membranes and contained membrane-bound vesicles as identified by means of electron microscopy. This material could be rendered inactive by heating to 100 degrees C for 2 minutes or by incubation with phospholipase C for 15 minutes. Finally, the ability of the high-speed precipitate to shorten the clotting time was prevented by preincubation with a monoclonal antibody, which is known to inhibit the procoagulant activity of human tissue factor. We suggest that several entities contribute to the procoagulant properties of human ascites, with procoagulant material deriving at least in part from peritoneal cells. The sedimentable procoagulant factor appears to be associated with cellular membranes or membrane fragments and is thromboplastin-like in its chemical composition, immunoreactivity, and substrate specificity.

Expression of procoagulant activity in a human monocyte-like cell line.

Peripheral blood monocytes generate the potent membrane-bound procoagulant TF in response to a number of immune-related stimuli. Although the contribution of the monocyte/macrophage and its soluble mediators to the immune response has been recognized, the role of macrophage procoagulant in the pathogenesis of this response is less certain. Previous studies have suggested that MTF generation is important in the pathogenesis of fibrin deposition in the inflammatory response. In order to pursue this relationship, we have studied the activation of a procoagulant in a human monocyte-like cell line, the U937. The U937 procoagulant has been characterized as TF by the following criteria: the PCA requires factors VII and X for expression; the PCA is not due to serine protease activity; PCA is neutralized by a monospecific antibody to purified bovine TF. The expression of TF in these cells was amplified after stimulation with LPS, a potent activator of peripheral blood MTF expression, and was inhibited by actinomycin D and cycloheximide. Cytosine arabinoside, an inhibitor of cell division, failed to affect U937 TF generation. The U937 cell line appears to be a useful in vitro model for the study of the activation of MTF.

Immunohistological studies with A1-3, a monoclonal antibody to activated human monocytes and macrophages.

A wide variety of monoclonal antibodies (mAb) have now been produced which recognize cell surface antigens on human peripheral blood monocytes. However, few of these mAb demonstrate specificity for monocytes, and fewer still recognize antigens exclusively on monocytes activated by one or more stimuli and/or block specific monocyte functions. The mAb A1-3 binds to lipopolysaccharide (LPS)-stimulated monocytes but not to resting blood monocytes, and inhibits the procoagulant activity of these LPS-activated cells. By using this mAb, we examined the reactivity of monocytes/macrophages (MO) in a broad range of normal and inflammatory tissues by means of a sensitive, four-layer immunoperoxidase technique. Cells of the MO system, in addition to lymphocytes and dendritic cells resident in lymphoid tissues, liver, lung, and other organs, were nonreactive with the A1-3 mAb. In contrast, intense staining of inflammatory MO was found in biopsies from patients with renal allograft rejection, acute glomerulonephritis, or granulomatous diseases. This apparent restriction of A1-3 binding to inflammatory, "activated" MO suggests that A1-3 mAb will be useful for the analysis of MO "activation" in many pathologic processes.

Production and characterization of a monoclonal antibody (A1-3) that binds selectively to activated monocytes and inhibits monocyte procoagulant activity.

We describe the generation and characterization of a new monoclonal antibody, A1-3, which possesses two unique properties. First, A1-3 binds selectively to stimulated human monocytes. Secondly, A1-3 inhibits the procoagulant activity expressed by stimulated monocytes and by human brain tissue factor. Unstimulated human peripheral blood cells (granulocytes, lymphocytes, monocytes, red blood cells, and platelets), prepared in the absence of detectable endotoxin, express no procoagulant activity and fail to bind A1-3. Stimulation of peripheral blood monocytes. alveolar macrophages, or the monocyte-like cell line U937, however, results in the expression of procoagulant activity and the binding of A1-3. The surface antigen recognized by A1-3 was recovered from endotoxin-stimulated human monocyte vesicles by immune precipitation and demonstrated an apparent m.w. of approximately 52,000. It is proposed that the monoclonal antibody A1-3 detects a differentiation antigen on human monocytes that is expressed in response to stimuli for monocyte activation.

Mononuclear cell modulation of fibroblast procoagulant activity.

The release of procoagulant material by connective tissue cells as a sequel to cell injury can initiate blood coagulation and may thus play a role in he pathogenesis of inflammatory lesions. Human foreskin fibroblasts were shown to synthesize high levels of the procoagulant TF in vitro. Generation of TF by fibroblasts was inhibited by addition of supernatants of PHA-stimulated human mononuclear cells to fibroblast cultures. The inhibition was independent of supernatant effects on cellular proliferation and was accompanied by up to a 20-fold increases in PGE2 synthesis in the fibroblast cultures. The inhibition of TF generation by MC-SNs was reversed by adding indomethacin to the fibroblast cultures, suggesting that mononuclear cells suppress fibroblast TF generation by stimulation of endogenous fibroblast synthesis of prostaglandin. Regulation of fibroblast PCA by products of immune cells may be important in the pathogenesis of inflammatory lesions.