Recently there has been a growing awareness of the importance of the interactions of the human complement system with biomaterials which largely stems from the understanding that complement-materials interactions may result in the production of inflammatory mediators, termed C3a and C5a anaphylatoxins, that can produce significant pathopysiologic alterations in the exposed patient. This presentation will review studies of complement activation produced by various hemodialysis membranes. our current data suggests that hemodialysis membranes may be roughly divided into three categories. The first type of membrane displays a high complement-activating potential as judged by measurements of C3a antigen levels produced during dialysis. These membranes also liberate sufficient quantities of C5a to induce both significant granulocytopenia and cardiopulmonary effects. The second group of membranes exhibits a moderate capacity to activate complement, with the amount of C3a antigen produced by these devices being about half that seen with the highly activating dialyzers. Dialyzers in this intermediate group produce only enough C5a to provoke a modest degree of leukopenia and they generally fail to induce cardiopulmonary manifestations. The third type of device fails to produce significant amounts of either free C3a or C5a and does not produce significant granulocytopenia. Factors that could affect the propensity of a material to activate complement include the density and type of surface nucleophiles, and the presence of surface dcarge which may facilitate the interaction of regulatory components and the adsorbtion of both active and bystander proteins.

We studied the cardiopulmonary, hematologic, and inflammatory response to hemodialysis with seven different membranes in sheep. We also compared acetate dialysate with bicarbonate dialysate and evaluated the role of thromboxane in mediating these responses to dialysis with Cuprophan membranes (Baxter Healthcare Corp., Renal Division, Deerfield, Ill.) in sheep. The data generated in these studies indicate that dialyzer membranes can be divided into three major categories, defined by propensity to activate complement. High complement activators such as Cuprophan (low surface-area CF-1511 and high surface-area ST-25 dialyzers) produced dramatic neutropenia and hypoxemia and significant (p < 0.01) increases in the plasma concentration of thromboxane and in mean pulmonary artery blood pressure. The magnitude of these effects appeared to be surface area related. The low-flux Fresenius F-6 polysulfone membrane (Fresenius USA Inc., Concord, Calif.) also resulted in the generation of significant levels of C3a. In contrast, low complement activators such as polyacrylonitrile (AN-69; Gambro Hospal, Inc., Lakewood, Colo.) and cellulose triacetate (CT-110G; Baxter) produced little or no neutropenia, small transient increases in thromboxane, and no rise in mean pulmonary artery pressure. Dialyzers with intermediate complement-activating potential such as cellulose acetate (CA-110; Baxter) and Hemophan (HT-100; Baxter) produced small to moderate degrees of neutropenia and small increases in thromboxane and mean pulmonary artery pressure. Treatment of sheep with sodium ibuprofen before dialysis with Cuprophan CF-1511 membranes prevented the initial increases in mean pulmonary arterial pressure and thromboxane generation and the decrease in arterial oxygen tension, but did not affect the degree of complement activation or neutropenia. In sheep undergoing Cuprophan dialysis, bicarbonate dialysate did not prevent the increase in circulating complement and the associated neutropenia otherwise seen during the early portions of dialysis with acetate dialysate. Bicarbonate dialysate did, however, reduce (not prevent) the initial increases in thromboxane and mean pulmonary arterial pressure, and the magnitude of the hypoxemia seen with the use of acetate dialysate. The results of these experiments therefore indicate that (1) reactions in sheep correlate well with data collected in human beings and the model can be an effective means for comparing novel dialysis membranes and pharmacologic interventions during dialysis and (2) although complement appears to be the transducer of the hematologic and immunologic response, thromboxane appears to be the final effector of the cardiovascular responses to hemodialysis with Cuprophan membranes.

Activated polymorphonuclear leukocytes (PMNs) contribute to myocardial injury during ischemia and reperfusion. There is evidence that activation of the complement pathway may be one of the mechanisms of PMN activation during ischemia. Intracoronary infusion of complement C5a during normal perfusion pressure is associated with decreased coronary flow, contractile dysfunction, and PMN accumulation. The mechanisms responsible for these changes have not been identified. Thromboxane A2 (TXA2) is a potential mediator of this myocardial ischemic response. Activated PMNs produce TXA2, a known coronary vasoconstrictor, and TXA2 was shown to be a mediator of the pulmonary hypertensive response to activated complement. The goal of the present study was to determine if an enhanced TXA2 production is associated with the myocardial response to C5a and whether cyclooxygenase blockade would reduce the myocardial ischemia. In open-chest pigs, intracoronary C5a (500 ng) caused reversible reductions in blood flow (50.0% of control), regional contractile function (25.8% of control), leukocyte trapping (1.0 x 10(6) cells/g myocardium or a peak artery-coronary venous difference of 5.3 x 10(3) cells/microliters blood), and increased coronary venous TXB2 (the TXA2 breakdown product) from 1.6 pmol/ml to a peak of 6.9 pmol/ml. Cyclooxygenase blockade with aspirin or indomethacin, which prevented TXB2 production, did not alter the response in flow, function, or PMN trapping. Ibuprofen, a known direct inhibitor of PMNs in addition to its cyclooxygenase blockade effect, reduced the response slightly. The pig coronary vascular bed was responsive to the TXA2 agonist U46619, which reduced flow and function without PMN trapping. Mechanical reductions in coronary flow to levels equivalent to those during the C5a infusions did not increase coronary venous TXB2 nor cause PMN trapping but did cause equivalent contractile dysfunction. Incubation of whole blood with C5a at concentrations equivalent to those achieved in vivo did not cause TXB2 production. We conclude that 1) TXA2 is produced in response to intracoronary C5a and 2) cyclooxygenase blockade does not prevent the C5a-induced myocardial ischemia, contractile dysfunction, and PMN trapping. The TXA2 production likely involves a vascular site or a blood cell-vascular interaction. This model system indicates the potential for persistently activated PMNs to cause continued ischemia during myocardial reperfusion.

Granulocytes cause some of the pathophysiological effects associated with the capillary no-reflow phenomenon during ischemia and in ischemia-reperfusion injury. However, no study has examined the consequences of in vivo granulocyte activation during normal perfusion pressures. In this study, we examined the effects of intracoronary administration of the complement component C5a, which is known to be a potent granulocyte activating factor. Nine open-chest, anesthetized pigs were instrumented to monitor regional coronary blood flow and segment shortening, left ventricular dP/dt, heart rate, and pulmonary artery and aortic blood pressures and to sample arterial and regional coronary venous blood for oxygen content and complete blood counts. Intracoronary infusion of human or porcine C5a in doses ranging from 10 to 500 ng produced a significant reduction in regional coronary blood flow and myocardial function. Although perfusion pressure and heart rate remained constant, venous oxygen content fell, indicating an imbalance between myocardial oxygen supply and demand. In addition, the arteriovenous difference of white blood cells was increased significantly after anaphylatoxin infusion, indicating intravascular trapping in the myocardium. Granulocytes accounted entirely for the differences in leukocyte counts because no significant changes in platelet, lymphocyte, or hematocrit levels were observed. Injection of vehicle alone did not alter any of the monitored variables.(ABSTRACT TRUNCATED AT 250 WORDS)

Human C5a anaphylatoxin is a potent bioactive molecule that possesses both spasmogenic and leukocyte-related properties. As such, it normally serves as a local mediator of the acute inflammatory response. Additionally, C5a, through its actions of mononuclear phagocytes, may act to bridge the gap in the acute-chronic inflammatory continuum. While these properties are critical to normal host defense mechanisms, it is now apparent that this anaphylatoxin and/or its des-Arg74 derivative, may exert significant systemic effects that are manifest as cardiopulmonary abnormalities and intravascular activation of granulocytes. Knowledge of these properties is critically important for understanding the clinical sequelae exhibited by patients undergoing extracorporeal circulation since we now know that both hemodialysis and cardiopulmonary bypass [28-30] procedures promote intravascular complement activation and C5a formation. Viewed in this context, it seems reasonable to postulate that many of the immediate and delayed responses to extracorporeal circulation might be mediated by C5a formed in the extracorporeal circuit (table IV). For example, it is now recognized that a few particularly susceptible patients display adverse reactions during the initial phases of hemodialysis. The symptoms of this so-called 'first-use syndrome' may range from severe urticaria and angioedema to life-threatening bronchospasm, hypotension, and cardiopulmonary collapse. Some investigators have presented data which suggest that complement-derived products may be causative of these symptoms in some patients [31]. While this hypothesis remains to be confirmed, present evidence clearly demonstrates that C5a alone may produce many of the observed phenomena. In addition to the acute effects produced by C5a, both our own basic studies and the clinical investigations presented by others at this conference suggest that the long-term effects of repeated C5a exposure in the dialyzed patient may be considerable. Thus, there has been a great deal of interest in the role of complement-derived mediators as initiators of leukocyte degranulation and toxic oxygen radical production and an exploration of the significance of these events in the eventual development of chronic pulmonary fibrosis in the dialyzed patient. Similarly, the effects of repeated exposure to IL-1 that has been postulated to occur as a result of C5a triggering of monocytes during dialysis is currently an active area of investigation.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of standard Ficoll-Hypaque (density = 1.077 g/ml) separation profiles of peripheral white blood cells (WBC) from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked decrease in the density of polymorphonuclear leukocytes (PMN) resulting in about 50% of these cells separating with the mononuclear cells. In vitro exposure of normal control peripheral blood to HD membranes as well as to the purified chemotactic factors C5a, C5ades-Arg, and formyl-Met-Leu-Phe (fMLP) also resulted in PMN density changes which altered the Ficoll-Hypaque separation profiles of WBC. Therefore, these results imply that C5a generation, resulting from complement activation by the HD membrane, induced the density changes in the PMN from HD patients. Further studies using flow cytometry and fluorescein-labeled chemotactic factors (C5a, formyl-Met-Leu-Phe-Lys [fMLPL] and casein) indicated that HD patients had a significant reduction in the ability of their PMN and monocytes to bind C5a. This contrasted with the findings of no significant difference in the percentage or fluorescence intensity of HD patients' PMN or monocytes binding casein or fMLPL. Functional studies to analyze chemotactic-factor-mediated responses indicated that there was a decreased ability of HD patients' PMN and monocytes to generate superoxide anion, produce H2O2 and release myeloperoxidase in response to both C5a and fMLP. Additional studies evaluated the binding of chemotactic factors to PMN and monocytes from normal blood following passage through a hemodialyzer and from patients undergoing HD. Analysis of receptor binding by control cells passed through the dialyzer showed that there was a progressive decrease in the percentage of C5a-receptor-positive PMN and monocytes but no change with casein or fMLPL. In contrast, peripheral PMN and monocytes from chronic renal failure patients on HD showed no difference in C5a, casein or fMLPL receptors during the course of HD as compared to the predialysis period. This appears to be attributable to a difference in the regulation of the C5a that is generated as a result of the dialysis-membrane-induced activation of the complement system. Although C5a has been shown to be continuously generated during the course of HD, these patients show no modulation of their C5a receptors during the course of HD or when their whole blood is exposed to dialysis membrane fibers. These findings suggest that there are mechanisms functioning in chronically dialyzed patients to protect them from the effects of excessive C5a generation during HD.(ABSTRACT TRUNCATED AT 400 WORDS)

Human C5a is a complement derived inflammatory mediator that binds to specific receptors that are found in the granulocyte plasma membrane. A new photoreactive C5a analog, that can be radiolabeled in the aryl azide moiety, has been synthesized by coupling p-azidosalicyl-2-mercapto-N-ethylamide-2'-thiopyridine disulfide to a partially reduced form of C5a. This unique analog (ASAMEA-SC5a) specifically bound to the granulocyte C5a receptor with an apparent Kd of 1.8 nM, a value that is comparable to that of the native ligand. Photoactivation resulted in crosslinking of this probe to the granulocyte C5a receptor. Subsequent reductions of the complex produced a radiolabeled C5a receptor, or a subunit of this receptor that had an apparent molecular weight of 47,000 Daltons on SDS-PAGE.

A laboratory method that facilitates delineation of the complement-activating characteristics of various dialyzers under defined conditions has been developed. Results obtained by circulating reconstituted human serum through these devices and measuring time-dependent production of both C3a and C5a antigens are entirely consistent with previous clinical observations. For example, the complement-activating potential of dialyzer membranes could be described as high (cuprammonium cellulose), moderate (cellulose acetate), or low (polycarbonate or polyacrylonitrile). Furthermore, these techniques provided the opportunity to identify membrane characteristics that are not readily defined by clinical studies alone. Specifically, membranes that transported and absorbed C5a antigen were readily identified by these methods. Additionally, laboratory evaluation provided the unique ability to define the efficiency of complement activation taking place on the membrane surface. Results of these investigations are compatible with a hypothetical model that not only describes the properties of a typical dialyzer membrane but may be generally applicable to other biomaterials as well.

Clinical as well as laboratory studies have been employed to assess the complement activating potential of polycarbonate membrane hemodialyzers. Blood samples from a group of patients undergoing sequential maintenance hemodialysis with cuprophane, polyacrylonitrile and polycarbonate devices were evaluated to define plasma levels of C3a antigen and leukocyte counts during the initial phases of hemodialysis. While polyacrylonitrile dialyzers did not activate complement to a significant extent, we did observe transient elevations in the plasma concentration of C3a and corresponding diminutions in the granulocyte counts of patients dialyzed with both cuprophane and polycarbonate dialyzers. However, polycarbonate devices appeared to activate complement to a lesser degree than cellulosic dialyzers. Laboratory evaluation of these three different types of dialyzers also provided evidence that polycarbonate membranes did not appear to activate human complement as readily as cuprophane. These observations suggest that polycarbonate membranes display complement-related biocompatibility properties that are intermediate between those of cuprophane and polyacrylonitrile.

Hemodialysis with cuprophane membrane is associated with complement activation and the formation of anaphylatoxins. Frequently, it is also complicated by various adverse reactions which include hypoxemia and hemodynamic changes. This study examined the cardiopulmonary effects of cuprophane membrane on experimental animals. To support the hypothesis that these effects were mediated by complement activation products, the effects of zymosan-activated plasma and C5adesArg challenge on the same variables were compared. We showed that intravenous infusion of autologous cuprophane-activated plasma into swine produced severe pulmonary hypertension, hypoxemia and leukopenia. In addition, mean systemic arterial pressure fluctuated and cardiac output fell. Infusion of zymosan-activated plasma produced similar results, suggesting that complement activation products are responsible for these alterations. Similar responses to porcine C5adesArg infusion suggested further that this polypeptide was the mediator. When swine were subjected to extracorporeal circulation using cuprophane membrane but without dialysis, acute pulmonary hypertension was seen preceding the onset of significant leukopenia. These data suggest that blood contact with cuprophane membrane produces both pulmonary and systemic hemodynamic changes, which are mediated by complement activation products. Furthermore, these products and/or other humoral factors, but not leukoagglutination, cause the pulmonary hypertension.

cAMP and 1,25-dihydroxyvitamin D3 [1,25- (OH)2D3] have both been shown to affect cell proliferation and phenotype. Since 1,25-(OH)2D3 modulates cellular cAMP production in the U937 cell line, we have investigated the possibility that 1,25-(OH)2D3 may interact with cAMP to affect distal cellular events. The U937 cell normally contains less than 2000 receptors/cell for the chemotactic factor C5a, as measured by concentration-dependent binding assays with the ligand probe [125I]iodo-C5a; this number rises when the cell is exposed to agents that promote cellular differentiation, such as (Bu)2cAMP. U937 cells incubated with 1,25-(OH)2D3 or cAMP agonists [prostaglandin (0.03–3μm), isoproterenol (10−7–10−5m), and forskolin (5–15 μm)] alone demonstrate no increase in C5a receptor number. When the cAMP agonists are combined with 1,25-(OH)2D3 for up to 4 days, receptors are induced to levels comparable to those achieved with pharmacological doses of (Bu)2cAMP (25,000–75,000/cell), with a similar dose inducing half-maximal binding of C5a (∼l–3 nm). The synergistic effects of 1,25-(OH)2D3 and cAMP agonists are dose and time dependent; other metabolites of vitamin D are not effective. The data suggest that 1,25-(OH)2D3 may facilitate the effects of endogenous cAMP to promote certain types of differentiation. (Endocrinology118: 2540–2545, 1986)

The compartmental distribution of the human anaphylatoxins C3a and C5a has been defined during simulated hemodialysis performed with various types of hemodialyzers. New cuprophan hollow fiber dialyzers were found to activate human complement very readily in vitro, while re-used cuprophan dialyzers displayed only modest complement activating potential. The C3a and C5a antigens, formed as a result of complement activation in these dialyzers, accumulated predominantly in the blood path and were not adsorbed extensively on the membrane surface or transported into the dialysate compartment. Cellulose acetate membranes also produced complement activation in vitro, but to a lesser degree than new cuprophan hollow fibers. However, these membranes exhibited a significant capacity to bind the anaphylatoxins to their surface. Polyacrylonitrile membranes appeared to be unique in that they not only failed to activate complement significantly, but they rapidly adsorbed large quantities of C3a and C5a. These findings demonstrate that hemodialysis membranes may differ with regard to their complement activating potential as well as their ability to remove circulating anaphylatoxins from the blood path. Clinical measurements of anaphylatoxin production during hemodialysis reflect these dynamic events.