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This preliminary data indicates that kinins are activated in blood shed into a wound particularly at the beginning of an operation. Reinfusion of such blood should only be accomplished after the removal of these activated factors.
The development of acute lung inflammatory injury induced by alveolar deposition of IgG immune complexes in rats requires increased production of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) as well as the complement activation product, C5a. Transcription of TNF-alpha and IL-1beta genes are known to be regulated by the nuclear factor-kappa B (NF-kappaB). During IgG immune complex-induced lung inflammation, NF-kappaB has been shown to be activated in both alveolar macrophages and whole lung tissues. In the current studies we sought to determine whether TNF-alpha, IL-1beta, the complement system and oxidants contribute to the activation of NF-kappaB in the lung. Electrophoretic mobility shift analysis of nuclear extracts from whole lung tissues demonstrated that NF-kappaB activation induced by the presence of IgG immune complexes occurred independently of the complement system and neutrophils. Intrapulmonary instillation of TNF-alpha or IL-1beta into normal lung induced NF-kappaB, whereas C5a was incapable of causing NF-kappaB activation. In alveolar macrophages stimulated in vitro with IgG immune complexes, NF-kappaB activation was greatly attenuated in the presence of antibodies to TNF-alpha or IL-1beta. Similarly, in vivo blockade of TNF-alpha or IL-1beta suppressed lung NF-kappaB activation during IgG immune complex-induced lung injury. N-acetylcysteine, but not catalase, suppressed activation of lung NF-kappaB. These data suggest that TNF-alpha and IL-1beta function in an autocrine or paracrine manner to amplify the lung inflammatory response through activation of NF-kappaB. Oxidants not derived from neutrophils also appear to play a role in this process, whereas complement activation products are not involved in this phenomenon.
A major complication in sepsis is progressively impaired lung function and susceptibility to intrapulmonary infection. Why sepsis predisposes the lung to injury is not clear. In the current studies, rats were rendered septic by cecal ligation/puncture and evaluated for increased susceptibility to injury after a direct pulmonary insult (deposition of IgG immune complexes or airway instillation of lipopolysaccharide). By itself, cecal ligation/puncture did not produce evidence of lung injury. However, after a direct pulmonary insult, lung injury in septic animals was significantly enhanced. Enhanced lung injury was associated with increased accumulation of neutrophils in lung, enhanced production of CXC chemokines (but not tumor necrosis factor-alpha) in bronchoalveolar lavage fluids, and increased expression of lung vascular intercellular adhesion molecule-1 (ICAM-1). Complement depletion or treatment with anti-C5a abolished all evidence of enhanced lung injury in septic animals. When stimulated in vitro, bronchoalveolar lavage macrophages from septic animals had greatly enhanced CXC chemokine responses as compared with macrophages from sham-operated animals or from septic animals that had been complement depleted. These data indicate that the septic state causes priming of lung macrophages and suggest that enhanced lung injury in the septic state is complement dependent and related to increased production of CXC chemokines.
Activation of the complement system with generation of the potent neutrophil chemotactic factor C5a has been proposed to play a significant role in the neutrophil accumulation in the lungs of cigarette smokers. Chemotactic factor inactivator (CFI) can inhibit C5a-directed neutrophil chemotaxis by binding to the C5a cochemotaxin GcGlobulin (GcG), a vitamin-D-binding protein, and inhibiting the capacity of GcG to enhance the chemotactic activity of C5a. Because cigarette smoke can inhibit the function of some proteins, a loss of CFI functional activity induced by cigarette smoke would allow an increased capacity of GcG to augment C5a-directed neutrophil chemotaxis. In order to test this hypothesis, cigarette smoke was bubbled through a CFI solution, and the solution was evaluated for its ability to inhibit the chemotactic activity of C5a and GcG. Smoke-treated CFI inhibited only 36% of the C5a-GcG chemotactic activity. In contrast, a CFI solution treated with air inhibited 62% of the chemotactic activity (p less than 0.001). Consistent with these observations, smoke-treated CFI exhibited a decreased capacity to bind to GcG and a decreased capacity to inhibit the binding of C5a des Arg to GcG. CFI contained in the bronchial lavage fluids obtained from patients with chronic obstructive pulmonary disease secondary to cigarette smoking and asymptomatic smokers exhibited a decreased capacity to inhibit C5a-GcG neutrophil chemotaxis and to bind to GcG (p less than 0.05, both comparisons). Furthermore, smoke bubbled through normal bronchial lavage fluid decreased the capacity of CFI to bind to GcG.(ABSTRACT TRUNCATED AT 250 WORDS)
Eight patients with advanced liver malignancy undergoing isolated hyperthermic liver perfusion with melphalan and cisplatin were studied with regard to complement activation and formation of anaphylatoxins (C3a and C5a) and terminal C5b-9 complement complexes (TCCs). Blood samples for complement variables (C1-INH, C3, C4, C5, C3a, C5a and TCCs) were taken before surgery, 1 min before the start of perfusion, 1, 2 and 3 h after the start of perfusion, and 24 h after operation. Samples were drawn from the perfusate 1 h after the start of perfusion. Activation of complement was observed during perfusion. Raised plasma concentrations of C3a and TCCs were recorded and high levels of C3a and TCCs were found in the perfusate. In vitro tests indicated that melphalan and cisplatin may activate complement. This activation occurred at 37 and 42 degrees C but was more pronounced at 42 degrees C.
Complement has been implicated in liver repair after toxic injury. Here, we demonstrate that complement components are essential for liver regeneration, and mediate their effect by interacting with key signaling networks that promote hepatocyte proliferation. C3- or C5-deficient mice exhibited high mortality, parenchymal damage, and impaired liver regeneration after partial hepatectomy. Mice with dual C3 and C5 deficiency had a more exacerbated phenotype that was reversed by combined C3a and C5a reconstitution. Interception of C5a receptor signaling resulted in suppression of IL-6/TNFalpha induction and lack of C3 and C5a receptor stimulation attenuated nuclear factor-kappaB/STAT-3 activation after hepatectomy. These data indicate that C3a and C5a, two potent inflammatory mediators of the innate immune response, contribute essentially to the early priming stages of hepatocyte regeneration.
Activation of the complement system generates the anaphylatoxin C5a whose activities are mediated through its binding to the widely expressed C5aR. C5aR mRNA and protein expressions are known to be induced in rat hepatocytes under inflammatory conditions. However, little is known about the role of the C5a/C5aR complex in liver and its involvement during a proliferative process. We have evaluated the expression of C5aR in regenerating rat hepatocytes following a partial hepatectomy and in hepatocyte cultures. C5aR induction was observed in hepatocytes from regenerating liver, as well as in normal hepatocytes under a culture-induced stress. The effect of a stimulation by a C5a agonist upon the synthesis of a growth factor/receptor pair (hepatocyte growth factor/c-Met) was also evaluated. Our data demonstrated an up-regulated expression of hepatocyte growth factor and c-Met mRNAs, but we failed to observe a direct mitogenic effect of C5a in culture. However, a significantly increased expression of cyclin E and D1mRNA levels, as well as an increased BrdU incorporation, were observed in rats given an i.v. C5a agonist injection following an 80% partial hepatectomy. These studies demonstrate for the first time that: 1) C5aR is up-regulated during liver regeneration, 2) the binding of C5a to C5aR promotes a growth response, and 3) C5aR is involved in a cell cycle signaling pathway. Taken together, these findings point to a novel role for the hepatic C5aR implicating this complement system in the context of normal or abnormal proliferative pathways.
The effects of the same form of crystalline silica variously modified were compared to investigate the mechanisms by which silica activates C5 molecules. After incubation in human plasma, silica generated C5a-type fragments that stimulated polymorphonuclear leukocyte chemotaxis. This activity was totally abolished when plasma, adsorbed with antiserum against C5a or thermally inactivated, was used. Pretreatment of plasma with deferoxamine, 1,3 dimethyl-2-thiourea, or aprotinin markedly inhibited or totally abolished C5 activation. Finally, a significant increase in kallikrein activity was detected after incubation of silica particles in plasma. The results seem to indicate that the activation of C5 by crystalline silica occurs through a complex mechanism: the redox-active iron possibly present at the silica surface catalyzes, via Haber-Weiss cycles, the production of hydroxyl radicals, which in turn convert native C5 to an oxidized C5-like form. This product is then cleaved by kallikrein, activated by the same silica particles, yielding oxidized C5a with the same functional properties as C5a. The different types of the same form of silica exhibited different reactivity. Two separate properties of the dusts seem to contribute to C5 activation: the potential to release hydroxyl radicals and the extent of C5 adsorption at the surface. The degree of surface hydrophobicity/hydrophilicity appeared sufficient to explain the different responses.
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)
Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcgammaR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcgammaRIIB(-/-) mice, suggesting that C5aR signaling is the crucial initial event upstream of FcgammaR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcgammaRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcgammaR, which amplifies complement-induced inflammation in autoimmunity.
The present report summarizes our experience with a new polycarbonate (PC)-based dialysis membrane (Gambro Lundia PRO-5) compared with a cuprophan (CP)-based membrane (Gambro Lundia 10-5N). Platelet count decreased during dialysis with CP, but was unchanged with PC membranes. Platelet injury, reflected by decreased platelet aggregation and increase in plasma beta-thromboglobulin, occurred equally with both membranes. Complement activation (C3d and C5a), leukopenia and release of granulocyte-derived elastase was more extensive with CP than with PC membranes. The new membrane represents an improvement of biocompatibility with respect to the complement-leukocyte system, whereas the effect on platelets resembles the injury caused by CP membranes.
Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.
C5a, a product of complement system activation, causes a significant contraction of the isolated guinea pig trachea, and the antihistamine diphenhydramine does not alter the rate, amplitude, or duration of the contraction (Regal et al., 1980). The present study demonstrates that over the range of C5a concentrations investigated, the C5a-induced contractile response of the trachea maximized, whereas the contraction of lung parenchymal strips and the release of histamine from chopped lung did not. In addition, the antihistamines, diphenhydramine and pyrilamine, caused a significant delay in the onset of the C5a-induced contraction of lung parenchymal strips. When parenchymal strips from nonperfused lung were used, the maximum of the contraction was also reduced by pyrilamine. Aspirin and indomethacin effectively inhibited the C5a-induced contraction of lung parenchymal strips, and inhibition by a combination of aspirin and pyrilamine was the sum of the inhibition by aspirin and the inhibition by pyrilamine. The SRS-A antagonist, FPL 55712, caused a more rapid relaxation of the C5a-induced response over control. These studies suggest that products of the cyclo-oxygenase pathway are mediators involved in the C5a-induced contraction of lung parenchyma, with histamine and leukotrienes contributing as well.
Hypotension is one of the most common adverse effect of plasmapheresis (PP) and often is attributed to hypovolemia due to extracorporeal circulation and the vasovagal reflex. Complements are activated during PP, and the activated complements are strong anaphylatoxins and potent vasodilators. Therefore, we studied the relationship between the transient hypotension and the plasma levels of activated complements during and after PP in 8 sessions of 7 patients using the Plasmafro OP-08 as a plasma separator. Five of the patients underwent immunoadsorption PP using the IM-TR 350 or IM-PH 350 as the adsorption column. The other underwent double filtration PP using the Evaflux 4A as a second filter. In 4 of 8 sessions, patients experienced transient hypotension with significantly elevated plasma levels of activated complements C3a and C5a. In contrast, patients without hypotension showed no increases in C3a and C5a values during PP. In this report, we emphasize the critical role of activated complements for hypotension during PP.
Human C5a, a complement-derived anaphylatoxin, is a potent mediator of human leukocyte chemotaxis. Using a homogeneous preparation of C5a that was 125I-labeled, we have demonstrated the presence of a specific cellular receptor for this glycoprotein on intact human polymorphonuclear leukocytes. Cellular uptake of the radiolabeled ligand occurred rapidly and the rate of dissociation was extremely slow. Cellular binding was saturable with respect to 125I-labeled C5a, and half-saturation occurred at a concentration of 3-7 X 10(-9) M. The number of C5a binding sites per cell was estimated as 1-3 X 10(5). The ligand (C5a) displays specific structural features that are required for binding because analogs of C5a such as C5ades Arg or a yeast carboxypeptidase-digested C5a derivative C5a-(I-69) inhibited the binding but C3a anaphylatoxin, which resembles C5a chemically, did not. Both C5a-mediated leukocyte chemotaxis and C5a-induced lysosomal enzyme release from cytochalasin B-treated cells closely paralleled uptake of the ligand, clearly indicating that it is a receptor-C5a interaction that leads to stimulation of these cellular responses.
Although previous studies have demonstrated that complement (C)5a causes myocardial ischemia and mechanical dysfunction, the cardiac response of endogenously produced C5a and C5a des-Arg in zymosan-activated serum (ZAS) and the critical role of granulocytes in this process are poorly understood. Therefore, we compared the coronary and cardiac effects of ZAS and purified C5a and investigated the role of leukocyte adhesion-promoting receptors (i.e., CD11/CD18). Like purified C5a, ZAS (0.5 ml) significantly reduced coronary artery blood flow and regional segment shortening, whereas coronary venous granulocyte concentration and myocardial lactate extraction were significantly decreased. A monoclonal antibody (MoAb) to C5a/C5a des-Arg attenuated ZAS-induced cardiac alterations. Three minutes of continuous infusion of C5a or ZAS induced sustained decreases in coronary venous granulocyte concentrations, although coronary flow and segment shortening returned to control levels after 2 min. Another MoAb, IB4, directed against CD18, significantly inhibited ZAS-induced granulocyte extraction and associated cardiac effects. Thus, cardiac dysfunction occurs after activation of the complement cascade with zymosan resulting in extraction of granulocytes mediated by the CD18 adherence glycoprotein. Furthermore, intramyocardial retention of granulocytes appears necessary for the initial and full ZAS-induced cardiac dysfunction.
Promastigotes of Leishmania move progressively up a concentration gradient of: various sugars, specific sugars attracting individual species of Leishmania; serum albumin and another unidentified constituent of serum; hemoglobin; and a factor generated by promastigotes in NNN medium. The movement of promastigotes up a concentration gradient of serum is optimal at a pH of 6.4 to 6.8 and a temperature of 28 degrees C and above. Cholinergic and adrenergic agents did not affect the attraction of serum for promastigotes, and cyclic nucleotides, inflammatory mediators, and macrophage products were not chemotaxic. It is postulated that the sugar chemotaxins influence the movement of promastigotes from the sand fly midgut to the esophagus, and serum chemotaxins may play a part in the entry of promastigotes into the skin of a mammal from the proboscis. Macrophages, the host cell of the obligate intracellular Leishmania species, were not attracted to any product of promastigotes. When, however, promastigotes interact with serum, complement is activated to form C5a which is chemotaxic for macrophages. Activation of complement by promastigotes is, at least partially, by the alternate pathway. Other chemotaxins resulting from promastigote interaction with serum may also be present. Promastigotes may also produce inhibitors of C5a activity.
Cleavage of the complement protein C5 by activation of the complement system yields a low molecular weight fragment C5a. Knowledge of the alterations in blood pressure induced by C5a as well as the mediators responsible for the blood pressure changes may provide information concerning the potential role of C5a in the adverse hemodynamic responses associated with complement activation. The purpose of this study was to characterize changes in mean arterial pressure in the guinea pig after intravenous challenge with a combination of guinea pig C5a plus C5a(des-Arg) (C5a/C5a(des-Arg)) and determine the mediators responsible for the transient increase in blood pressure which was observed. Mean arterial pressure was monitored in mechanically ventilated pentobarbital-anesthetized guinea pigs. Intravenous injection of C5a/C5a(des-Arg) consistently caused a marked but transient rise in blood pressure. A transient hypotensive response was also seen with injection of markedly higher doses of guinea pig C5a/C5a(des-Arg). Various pharmacological antagonists were used to determine the mediators responsible for the increase in blood pressure induced by guinea pig C5a/C5a(des-Arg). We found that the LTD4 antagonist L-649,923 did not inhibit the transient rise in blood pressure. However, the cyclooxygenase inhibitor indomethacin inhibited the C5a/C5a(des-Arg)-induced pressor response as did the thromboxane synthetase inhibitor U-63557A and the thromboxane receptor antagonist SQ 29,548. In addition, the C5a/C5a(des-Arg)-induced pressor response was not inhibited by the H1 antagonist pyrilamine, but was inhibited in part by the alpha-adrenergic antagonist phentolamine. Also, the response was reduced in animals depleted of circulating platelets or white blood cells. Thus, the results of our studies suggest that intravenously injected guinea pig C5a/C5a(des-Arg) causes release of the vasoconstrictor thromboxane, most likely from circulating white blood cells or platelets, resulting in a transient rise in blood pressure in the guinea pig. In addition, release of catecholamines may contribute to the pressor response observed.