A P Dalmasso

Acute leukopenia occurs in all patients during the first hour of hemodialysis with cellophanemembrane equipment. This transient cytopenia specifically involves granulocytes and monocytes, cells which share plasma membrane reactivity towards activated complement components. The present studies document that complement is activated during exposure of plasma to dialyzer cellophane, and that upon reinfusion of this plasma into the venous circulation, granulocyte and monocyte entrapment in the pulmonary vasculature is induced. During early dialysis, conversion of both C3 and factor B can be demonstrated in plasma as it leaves the dialyzer. Moreover, simple incubation of human plasma with dialyzer cellophane causes conversion of C3 and factor B, accompanied by depletion of total hemolytic complement and C3 but sparing of hemolytic C1. Reinfusion of autologous, cellophane-incubated plasma into rabbits produces selective granulocytopenia and monocytopenia identical to that seen in dialyzed patients. Lungs from such animals reveal striking pulmonary vessel engorgement with granulocytes. The activated complement component(s) responsible for leukostasis has an approximate molecular weight of 7,000-20,000 daltons. Since it is generated in C2-deficient plasma and is associated with factor B conversion, it is suggested that activation of complement by dialysis is predominantly through the altermative pathway.

Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.

Most studies of discordant xenograft rejection have focused on the roles of recipient xenoreactive antibody and complement as mediators of hyperacute rejection; there are essentially no data from in vivo studies as to the contribution of endothelial cell responses to the pathobiology of xenograft rejection. We hypothesized that the mechanism by which xenoreactive natural antibodies and complement of the recipient are involved in rejection of a discordant, immediately vascularized xenograft involves donor organ endothelial cell activation, with the consequences of such activation contributing significantly to the rejection process. We performed a kinetic analysis of rejection of guinea pig hearts by untreated Lewis rats or recipients depleted of complement activity that underwent delayed xenograft rejection. We report that in both hyperacute rejection and delayed xenograft rejection there is widespread evidence of endothelial cell activation, including expression of P-selectin and E-selectin, upregulation of tissue factor, and downregulation of thrombomodulin and antithrombin III expression. Many of these changes occur very early posttransplantation in grafts that are not completely rejected until approximately 3 days. In delayed xenograft rejection, an intense cellular infiltrate is seen that results from progressive accumulation of activated macrophages and natural killer cells. T cell receptor alpha/beta+T cells are present only at relatively low levels. This cellular infiltrate is associated with dense expression of pro-inflammatory cytokines, including interferon gamma, interleukin 1, and tumor necrosis factor-alpha. We conclude that both endothelial cell activation and infiltration by activated macrophages and natural killer cells may play an important role in xenograft rejection. These newly described features of the xenogeneic rejection response may require targeting by future therapeutic regimens aimed at prolonging xenograft survival.

Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the surrounding extracellular matrix.

Complement plays important roles in host immune defences, and recent studies suggest that adipose tissue is an important site of production for some complement proteins. Starvation has been associated with low complement levels, but studied populations have usually had concomitant opportunistic infections or other conditions which might affect complement levels. To determine the impact of body weight and changes in body weight on serum complement, we investigated levels of complement proteins in otherwise healthy patients with a wide range of body weights, including patients with anorexia nervosa before and after treatment, obese dieters before and after weight loss, and normal weight controls. We found that complement proteins of the alternative pathway (C3, B, and D), alternative pathway haemolytic activity (AP50) and the inhibitors H and I were low in starving anorectics and normalized with weight gain. C3a levels were comparable in anorectics at low weight and after weight gain, indicating that low serum complement levels were attributable to hypoproduction and not complement cascade activation with consumption. Further, levels of C3, B, AP50, H and I, but not D, were higher than controls in obese patients and decreased toward normal after weight loss. Overall, percentage of ideal body weight, changes in body weight, and serum transferrin were each highly correlated with serum levels of complement proteins. We conclude that levels of alternative pathway complement components are determined in part by factors that influence body weight and by weight changes, possibly due to changes in production in adipose tissue or at other sites.