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PubMed1973

The membrane attack mechanism of complement. Verification of a stable C5-9 complex in free solution.

The membrane attack mechanism of complement, C5 to C9, has previously been postulated to associate on the target cell surface to a stable decamolecular complex with a calculated mol wt of 995,000. A soluble and stable complex consisting of C5, C6, C7, C8, and C9 has now been demonstrated to arise as a consequence of complement activation by the classical or alternate pathway. It has a sedimentation coefficient of 22.5S and a mol wt of 1 million daltons, and it migrates on electrophoresis at pH 8.6 as an alpha-globulin. The stable and soluble C5b-9 complex cannot bind to erythrocytes and has no demonstrable cytolytic activity. However, due to partially unsaturated binding sites for C9, it can bind additional C9 and thus function as an inhibitor of lysis of EAC1-8 by C9. These results support the concept according to which the membrane-bound attack system of complement represents a stable, decamolecular assembly of C5b-9. Unlike its analogue in free solution, the membrane-bound complex is cytolytically active.

Cell-Free System↗
PubMed1990

Recombinant soluble CR1 suppressed complement activation, inflammation, and necrosis associated with reperfusion of ischemic myocardium.

In summary, conversion of wild-type CR1 to a soluble form (sCR1) creates a potent inhibitor of complement activation by both the classical and alternative pathways by inhibiting the C3/C5 convertases. In the rat reperfusion infarct model, sCR1 significantly suppresses complement activation at the endothelial surface of capillaries and venules. This suppression of complement activation is accompanied by reduced accumulation of leukocytes within the infarct zone, perhaps because of reduction of the generation of C5a, which promotes expression of leukocyte adhesion receptors and leukocyte chemotaxis. In addition, formation of the C5b-9 attack complex, which may contribute to direct endothelial injury, was suppressed by sCR1. The inhibition of complement activation and leukocyte infiltration by sCR1 explains the observed significant reduction in myocardial necrosis after ischemia and reperfusion. These studies have identified sCR1 as a potential agent for therapeutic intervention in diseases associated with complement-dependent tissue injury.

Amino Acid Sequence↗
PubMed1984

Effect of erythrocyte membrane modulation by lysolecithin on complement-mediated lysis.

Alterations of the physiochemical properties of membranes, such as acyl chain length of phospholipids, cholesterol content, and disturbance of the bilayer packing, affect the efficiency of membrane attack by C5b-9. In the present study, we explored the effect of lysolecithin (LL), a naturally occurring derivative of membrane phospholipids, on membrane damage by C5b-9. Sublytic doses of the L isomer of palmitoyl-lysolecithin were incorporated into the erythrocyte membranes of guinea pig (gpE) or sheep (shE) and the cells were then lysed with C5b6-C9. Marked enhancement of complement-mediated lysis was observed with LL-treated gpE after 2 hr of incubation, whereas the lytic enhancement was either nil or only marginal in shE. Studies on the kinetics of LL incorporation and metabolism in gpE membranes with 14C-LL showed that LL was rapidly incorporated and catabolized to generate free fatty acid (FA). Maximal accumulation of labeled FA in the membrane and loss of membrane-incorporated LL, resulting in approximately equimolar amounts of LL and FA, occurred at 2 hr, concomitant with enhancement of complement-mediated lysis. In shE such a breakdown of LL occurred only minimally. This quantitative difference in LL breakdown between gpE and shE was in accord with lysophospholipase activity that was two and one-half to seven times more active in gpE than shE membranes. When an ether-linked analog of LL (1-O-hexadecyl-sn-phosphorylcholine), which is resistant to lysophospholipase, was incorporated in gpE, the lytic enhancement was not observed. The results of our experiments indicate that the membrane modulating effect of LL on lysis by C5b-9 requires the enzymatic breakdown of LL.

Animals↗
PubMed1996

Complement-induced endothelial dysfunction in rabbits: mechanisms, recovery, and gender differences.

Activation of complement and attenuation of endothelium-dependent relaxation occur in a number of pathophysiological conditions. The aim of this study was to investigate the mechanisms of human complement activation and loss of endothelium-dependent relaxation in rabbit tissue, the duration of this loss, and the effects of gender and serum concentration. In rabbit thoracic aortic rings precontracted with phenylephrine, human serum (HS) concentration dependently induced a loss of endothelium-dependent relaxation to the receptor-dependent vasodilator acetylcholine (ACh) and receptor-independent vasodilator calcium ionophore A23187. Serum-induced loss of ACh-dependent relaxation was decreased when rings were bathed in 1) HS depleted of factor B, C2, or C8, 2) heat-inactivated HS, or 3) HS with complement inhibitor sCR1 or sCR1[desLHR-A]. Superoxide dismutase had no effect on serum-induced loss of ACh-dependent relaxation. Serum-induced loss of ACh-dependent relaxation returned to control values after removal of HS. Serum-induced loss of ACh-dependent relaxation was greater in male than in female aortic rings. These results suggest that 1) complement activation directly attenuates endothelium-dependent relaxation via the classical and alternative pathways independent of superoxide anion formation, 2) this attenuation is concentration dependent, reversible, and dependent on formation of C5b-9, and 3) endothelial tissue from males is more susceptible than that from females to the acute effects of complement activation.

Acetylcholine↗
PubMed1999

Complement membrane attack complex and protectin (CD59) in liver allografts during acute rejection.

BACKGROUND/AIMS: The complement system is important in the rejection of xenografts, but very little is known about its activation in the rejection of allografts. Complement lysis is induced by the membrane attack complex (MAC), an aggregate of C5b, C6, C7, C8 and C9 molecules. The main defender against MAC is the CD59 molecule, also called protectin. In this study, the aim was to analyze the possible deposition of MAC and the fate of CD59 on distinct cell populations during liver allograft rejection. METHODS: Liver allografts were monitored by frequent fine-needle aspiration biopsies (FNAB) to demonstrate the immunoactivation of rejection. To examine MAC and CD59 in the FNAB, in relation to the activation markers of rejection, IL2-receptor, MHC class II and ICAM-1 expression, specific monoclonal antibodies and immunoperoxidase staining were used. RESULTS: Ten out of 21 consecutive liver transplants underwent a histologically confirmed episode of reversible acute rejection. In the FNAB, a significant increase of the activation markers IL2-receptor, class II and ICAM-1 correlated with the peak of inflammation during the episode. In association with inflammation, a significant deposition of MAC was recorded in neutrophils and lymphocytes infiltrating the graft and in the parenchymal cells. MAC deposition subsided together with the inflammation. A significant decrease in CD59 expression was seen in neutrophils during rejection, but CD59 expression on other inflammatory cells and hepatic tissue cells varied greatly. CONCLUSIONS: Complement activation was seen in association with acute rejection of liver allografts and it led to MAC assembly on leukocytes and tissue cells. A decrease in CD59 expression was less clear-cut, but it may predispose the cells to complement-mediated elimination.

Acute Disease↗
PubMed1996

Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.

New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.

Animals↗
PubMed1989

SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules.

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)n and (C5b-8)n, negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 less than 27 S) appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.

Avidin↗
PubMed1988

Activation of complement in IgA nephropathy.

Considerable evidence supports a role for the complement system in the pathogenesis of IgA nephropathy (IgAN). The alternative pathway components C3 and properdin (P) and the membrane attack complex (C5b-9) are generally found in the mesangial deposits in IgAN, while the classical pathway components C1q and C4 are usually absent. This pattern of immunofluorescence staining for complement components suggests activation of the alternative and terminal pathways in most patients. Despite normal serum concentrations of C3 and other complement proteins, fragments generated by activation of C3, including iC3b, C3d, and iC3b-C3d neoantigen, and sometimes C4, are often detected in plasma. We found that the severity of the histologic changes in the renal biopsy specimens correlated with plasma iC3b-C3d neoantigen concentrations as measured by an enzyme-linked immunosorbent assay. However, no other clinical feature correlated with the plasma concentrations of this neoantigen.

Complement Activation↗
PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal complement components are deposited and released from Salmonella minnesota S218 without causing bacterial death.

The mechanism of resistance of gram-negative bacteria to killing by complement was investigated. Complement consumption and uptake of purified, radiolabeled complement components on bacteria was studied using a serum- sensitive and a serum-resistant strain of Salmonella minnesota. Twice as many molecules of (125)I C3 were bound per colony-forming unit (CFU) of the smooth, serum-resistant S. minnesota S218 as were bound per CFU of the rough, serum-sensitive S. minnesota Re595 in 10 percent pooled normal human serum (PNHS), although 75-80 percent of C3 was consumed by both organisms. Hemolytic titrations documented total consumption of C9 by 5 min and more than 95 percent consumption of C5 and C7 by 15 min in the reaction with S218 with 10 percent PNHS. In contrast, negligible C5 depletion, 10 percent C7 consumption, and only a 26 percent decrease in C9 titer occurred with the serum-sensitive Re595. Binding of (125)I C5, (125)I C7, and (125)I C9 to S218 and Re595 was measured in 10 percent PNHS. A total of 6,600 molecules C5/CFU, 5,200 molecules C7/CFU, and 3,100 molecules C9/CFU bound to S218 after 5-10 min of incubation at 37 degrees C, but 50-70 percent of the C5, C7, and C9 bound to S218 was released from the organism during incubation at 37 degrees C for 60 min. Binding of 2,000 molecules C5/CFU, 1,900 molecules C7/CFU, and 9,000 molecules C9/CFU to Re595 was achieved by 20 min and was stable. The ratio of bound C9 molecules to bound C7 molecules, measured using (131)I C9 and (125)I C7, was constant for both organisms after 15 min and was 4.3:1 on Re595 and 0.65:1 on S218 in 10 percent PNHS. With addition of increasing amounts of purified, unlabeled (29 to 10 percent PNHS, there was no change in the C9:C7 ratio on Re595. However, with S218 there was a linear increase of the C9:C7 ratio, which approached the ratio on Re595. There was no (14)C release from S218 incubated in PNHS, nor was there evidence by electron microscopy of outer membrane damage to S218. Therefore, S. minnesota S218 is resistant to killing by PNHS, despite the fact that the organism consumes terminal complement components efficiently and that terminal components are deposited on the surface in significant amounts. The C5b-9 complex is released from the surface of S218 without causing lethal outer membrane damage.

Adsorption↗
PubMed1991

Human immunodeficiency virus (HIV)-infected cells and free virus directly activate the classical complement pathway in rabbit, mouse and guinea-pig sera; activation results in virus neutralization by virolysis.

Since animal models of human immunodeficiency virus (HIV) infection are being used increasingly in determining various aspects of virus/host interaction and as models for virus expression, it will be important to assess any significant differences in anti-viral immune responses between animals and humans. Previous studies have shown that incubation of HIV with non-immune sera from several animal species results in virus neutralization, and that rabbit serum can lyse HIV-infected cells. The objectives of the current study were to evaluate the animal complement pathway(s) activated by HIV and HIV-infected cells and determine the mechanism by which complement could mediate viral neutralization. Incubation of HIV-infected cells with mouse, guinea-pig or rabbit sera resulted in cell-surface deposition of C3 fragments. Deposition of C3 fragments did not occur either in the presence of C4-deficient guinea-pig serum or in the absence of Ca2+, indicating that activation by infected cells occurred via the classical pathway. Neutralization of free virus was also mediated by the classical pathway since C4-deficient guinea-pig serum and Ca(2+)-chelated sera lacked activity. Serum treatment of virus resulted in release of HIV reverse transcriptase (RT), suggesting that neutralization occurred by C5b-9-mediated virolysis. RT was also released from simian immunodeficiency virus by animal complement. Antibodies in animal sera were not responsible for the classical pathway activation by free virus or HIV-infected cells. These results define several substantial differences between animal and human complement reactivity with HIV which could significantly affect the ability of HIV to replicate in animals, and which need to be considered in the assessment of animal models of HIV infection.

Animals↗
PubMed2002

Does endotoxin-activated complement alter myocellular sodium homeostasis during sepsis?

BACKGROUND: Inappropriate complement activation is closely related to tissue injury and organ dysfunction during systemic infection. It is not clear, however, if endotoxin-induced complement activation is responsible for changes in myocellular sodium homeostasis during sepsis. METHODS: Rats underwent cecal ligation and puncture (CLP) or sham operation. Twenty-four hours after operation, fast-twitch extensor digitorum longus (EDL) muscles were isolated, incubated at 30 degrees C for 1 hour in Krebs-Henseleit buffer (KHB) (pH 7.4), and used to measure intracellular Na+ and K+ contents. Blood samples were collected to measure serum hemolytic complement activity and endotoxin levels. In addition, EDL muscles isolated from normal animals were incubated at 30 degrees C for 1 hour with zymosan-activated (10 mg/mL at 37 degrees C for 1 hour) rat sera, with lipopolysaccharide (LPS)-activated (LPS from Escherichia coli 055:B5, 10 or 200 microg/mL at 37 degrees C for 30 minutes) rat sera, with heat-inactivated (56 degrees C for 30 minutes) rat sera, with LPS (1 or 20 microg/mL), or in KHB. EDL muscles isolated from normal animals were also incubated with septic sera collected 6 or 24 hours after CLP with or without administration of soluble complement receptor type 1 (20 mg/kg, intraperitoneally). Myocellular Na+ and K+ contents ([Na+]i and [K+]i) were assayed using "washout" technique. Soluble C5b-9 complex levels in zymosan-activated or LPS-activated human sera were determined by enzyme-linked immunosorbent assay to evaluate the degree of complement activation induced by zymosan or LPS. RESULTS: Myocellular [Na+]i and [Na+]i/[K+]i ratios increased significantly 24 hours after CLP as compared with sham operation and were associated with decreased serum hemolytic complement activity and increased serum endotoxin levels. Zymosan-activated rat sera at sublytic concentrations markedly increased [Na+]i and [Na+]i/[K+]i ratios in isolated EDL muscles relative to heat-inactivated rat sera. LPS-activated rat sera, however, did not alter these two indices. In addition, myocellular [Na+]i and [Na+]i/[K+]i ratios were equivalent among normal EDL muscles incubated with septic sera, soluble complement receptor type 1-treated septic sera, or KHB. CONCLUSION: These results collectively suggest that polymicrobial sepsis, as produced by CLP, alters sodium homeostasis in fast-twitch skeletal muscles in association with changes in systemic complement activation and circulating endotoxin levels. Although endotoxin can activate the complement cascade, endotoxin-induced complement activation does not appear to be responsible for changes in myocellular sodium homeostasis observed during sepsis in rats.

Animals↗
PubMed1994

Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59.

Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.

Amino Acid Sequence↗
PubMed1994

Defective regulation of complement by the sickle erythrocyte: evidence for a defect in control of membrane attack complex formation.

A prominent clinical manifestation of sickle cell disease (SCD) is hemolytic anemia. Although complement activation can lead to intravascular hemolysis, its role in the hemolysis of SCD is not known. Because normal red blood cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement and because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte; therefore, defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. These studies show a defect in the regulation of membrane attack complex (C5b-9) formation in sickle erythrocytes, particularly in the most dense cells. The defect is characterized by increased binding of C5b-7 and of C9 to denser sickle cells and results in increased susceptibility of sickle cells to C5b-9-mediated (reactive) lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells are especially sensitive to reactive lysis. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement-mediated hemolysis could play a role in the anemia of SCD.

Adenosine Triphosphate↗
PubMed1992

Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement.

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

Amino Acid Sequence↗
PubMed1984

The mechanism of cell-mediated cytotoxicity. IV. K-76 COONa, which inhibits the activity of Factor I and of C5, inhibits early events in cytotoxic T-lymphocyte-mediated cytolysis and in T-lymphocyte activation.

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.

Cells, Cultured↗
PubMed1994

Homologous restriction factor: effect on complement C8 and C9 uptake and lysis.

Homologous restriction factor (HRF) is a complementary regulatory protein found on the surface of human erythrocytes and other cell types. It has the function of blocking the lytic action of the membrane attack complex (MAC) of complement when incorporated into a membrane. HRF was found to inhibit not only the MAC composed of the proteins C5b-9, but also the C5b-8 complex. When 125I-labelled HRF was allowed to insert into an erythrocyte target, lysis by C5b-8 was reduced by about 80%. When human erythrocytes were treated with antibody to HRF, which bound to and presumably blocked the action of HRF, lysis by C5b-8 increased. This increase was not caused by additional binding of C8 on the cell membrane. When HRF was inserted into a target cell membrane, it had the effect of reducing the amount of C9 that could bind to very low levels, in one experiment from 6000 molecules per cell to about 500 molecules per cell. When this value was compared to the number of C7 molecules that bound, it was found that HRF reduced the amount of C9 bound to the level of C7 that was bound. HRF seemed to reduce the number of molecules of C9 that could bind to the C5b-8 complex so that only one C9 bound per complex.

Animals↗
PubMed2004

Pathogenesis and pathology of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantation.

UNLABELLED: It has been recognized that delayed xenograft rejection (DXR) is the major barrier to the acceptance of xenotransplantation after overcoming hyperacute rejection. OBJECTIVES: This study sought to investigate the pathogenesis and pathology of delayed xenograft rejection following pig-to-rhesus monkey heart xenotransplantation. METHODS: Heterotopic xenogeneic heart transplants in the abdominal cavity were performed using piglet donors to four monkey recipients. Complete complement depletion was achieved in the recipients with repetitive doses of high-activity cobra venom factor (Y-CVF). The recipients were immunosuppressed with a combination of cyclosporine, cyclophosphamide, and steroids. Sera were analyzed for C3 and C4 levels and complement activity and anti-pig endothelial xenoantibody. The grafts were examined histopathologically and immunohistochemically for C3, C4;C5b-9, IgM, IgG, tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1(ICAM-1), CD57(NK cells), CD68 (macrophages), CD4, and CD8. RESULTS: Xenografts survived 8, 10, 13, and 13 days respectively, all developing DXR. Venous thrombosis was the outstanding feature within DXR xenografts, complicated by interstitial edema, local hemorrhage, myocardial necrosis, and mild to moderate cellular infiltration. The serum C3 levels and complement activity decreased to almost 0 from the day of transplantation due to treatment with Y-CVF. The C4 level began to decrease 2 to 4 days before the cardiac xenografts lost their function. Anti-pig endothelial xenoantibody also decreased after transplantation, slightly increasing during DXR. All rejected xenografts showed C3, C4, C5b-9, IgG, and IgM deposits to various degrees. Large numbers of macrophages (50% of total leukocytes) infiltrated the entire xenograft with a few natural killer cells (8% to 10%), as well as some CD4+ T cells (15%) and CD8+ T cells (25%). Upregulation of ICAM-1 on graft endothelial cells and TNF-alpha in the interstitium were also demonstrated in the rejected heart. CONCLUSION: Both humoral and cell-mediated immunologic reactions may play important roles in the pathogenesis of DXR. Besides C3, C4, C5b-9, IgM, and IgG destroying the xenograft, NK cells, macrophages, and CD4+ and CD8+ T cells may further aggravate the development of DXR.

Animals↗
PubMed1992

Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing.

The different mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes K1, K10, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed.

Blood Bactericidal Activity↗