Effect of the late complement components C5b-9 on human monocytes: release of prostanoids, oxygen radicals and of a factor inducing cell proliferation

Recently, we reported stimulation of rat macrophages and human platelets by isolated C5b-9 to synthesize prostaglandin E (PGE) or thromboxane B2 (TXB2). In the present study, we tested whether besides prostanoids, C5b-9 also would induce the production of other mediators. We found that C5b-9 in sublytic concentrations stimulated human granulocytes (polymorphonuclear leukocytes) or monocytes to release oxygen radicals. Furthermore, monocytes release interleukin-1 in response to C5b-9. Thus, besides having a lytic capacity, C5b-9 also functions as a stimulator of various cells.     

Functional C8 associated with human platelets

Haemolytic assay for C8 revealed its association in functionally active form with washed human platelets. Platelet-bound C8 haemolytic activity was inhibited by F(ab')2 anti-C8 and was undetectable in the platelet suspension obtained from three C8 deficient patients. Incubation of platelets from C8 deficient individuals in normal plasma did not restore C8 haemolytic activity, indicating that platelets do not absorb C8 from plasma in vitro during platelet preparation. Thrombin, a mediator of the platelet release reaction, did not induce the release of C8 from normal platelets. Conversely, lysis of EAC1-7.9 by platelet bound C8 was not accompanied by release of beta-thromboglobulin or serotonin from the platelets. C8 was detected in a homogenate prepared from platelets as well as in the supernatant collected after high speed centrifugation of the homogenate. The association of C8 with platelets as an individual component rather than as part of the C5b-9 membrane-attack complex was supported by the following evidence: platelet bound C8 eluted from a Sephacryl S-200 column at the same volume as C8 from normal human serum; F(ab')2 anti-C8, but not F(ab')2 anti-C5, inhibited platelet C8 activity; the platelet homogenate, which lysed EAC1-7.9, had no effect on EAC43 which are susceptible to the lytic activity of the C5b-9 complex.     

Comparison of channels formed by poly C9, C5b-8 and the membrane attack complex of complement.

The channels formed by poly C9, C5b-8 and C5b-9 were examined using the liposome swelling assay. By plotting the relative rate of swelling of C5b-8-containing liposomes vs the molecular weight of the sugar solute and by applying the Renkin equation, the size of the C5b-8 channel was estimated to be 1.5 mm radius. As increasing amounts of C9 were added during the formation of C5b-9, in C8:C9 ratios of 1:1, 1:2, 1:6 and 1:12, the size of the function channel increased. Poly C9 had a pore that was somewhat larger than C5b-9 at a C8:C9 ratio of 1:12. Using molecular sieving experiments with four different iodinated protein size markers, the channel diameter of poly C9 was estimated at between 90 and 100 A. Monoclonal antibodies to different complement proteins were added to the liposomes to see which might inhibit the channels. C5b-8 containing liposomes could be inhibited by antibodies to C8. Liposomes containing C5b-9 could be inhibited slightly by antibodies to C9 and most strongly by antibodies to the neoantigen of poly C9.     

Secretion of the terminal complement proteins, C5-C9, by human platelets

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.     

Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

Calcium is required for PMA induced superoxide release from human neutrophils

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.     

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal poly-morphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [³H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B₄ (LTB₄), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB₄ was rapid; the initial release occurred within 4–6 min and a second rise in release coincided with cell death. Virtually all the LTB₄ produced was released as we found no evidence of retention of intracellular LTB₄ at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB₄ was completely abolished and the release of C20:4 and PGs was drastically reduced. [³H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation ofC9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.     

Aggregating and prostanoid-releasing effects of platelet-activating factor and leukotrienes on human polymorphonuclear leukocytes and platelets

Aggregating and prostanoid-releasing properties of the inflammatory mediators, platelet-activating factor (PAF) and leukotrienes B4, C4 and D4 were studied in human polymorphonuclear leukocytes (PMNL) and in human platelet-rich plasma. Leukotriene B4 (LTB4) and PAF both induced a reversible aggregation of human PMNL with concomitant stimulation of PGE2 formation, whereas LTC4 had no effect on human PMNL. Arachidonic acid (AA) caused an irreversible aggregation of PMNL which was accompanied by formation of both PGE2 and TXB2. Inhibition of TXB2 synthesis by indomethacin or by OKY-1581, a thromboxane synthetase inhibitor, had no effect on the PMNL aggregation induced by LTB4, PAF or AA. Leukotrienes B4, C4 and D4 caused neither aggregation nor TXB2 release in human platelet-rich plasma. PAF, on the other hand, induced a dose-dependent, reversible platelet aggregation which was not accompanied by TXB2 formation nor inhibited by OKY-1581. The present study indicates that in addition to inducing PMNL aggregation, LTB4 is capable of releasing arachidonate metabolites from human PMNL but not from human platelets. Also the responses of PMNL and platelets to PAF seem to differ as the PAF-induced PMNL aggregation was accompanied by increased prostanoid formation whereas the PAF-induced reversible platelet aggregation was obviously independent from arachidonate metabolism.     

Complement activation in patients with immune thrombocytopenic purpura according to phases of disease course

Immune thrombocytopenic purpura (ITP) is an autoimmune thrombocytopenia with shortened platelet survival and relative bone marrow failure. The pathogenesis involves antibody production, cytokine release, T cell impairment, complement activation and clearance of platelets. We measured plasma levels of C3, C4, C1q and sC5b-9 in 80 ITP patients in acute phase, 50 ITP patients in complete (CR) or partial (PR) remission and 50 age- and sex-matched healthy volunteers. Statistical analyses showed that acute ITP patients had higher plasma levels of sC5b-9 and C1q than CR or PR patients (median = sC5b-9: 200 versus 98 mg/dl, P-value < 0·001) (median C1q = 2·11 versus 1·00 mg/dl, P-value < 0·001). CR and PR ITP patients had sC5b-9 and C1q plasma levels comparable to those observed in healthy volunteers. There was a significant correlation between sC5b-9 and C1q plasma levels (Spearman’s rho correlation index on 130 ITP patients equal to 0·58, P-value < 0·001). We also found that sC5b-9 plasma level is inversely correlated with the number of platelets. Furthermore, we divided acute ITP patients into subjects with detectable (24 of 80, 30%) or undetectable (56 of 80, 70%) anti-platelet antibodies; patients with detectable anti-platelet antibodies have significantly higher plasma levels of C1q and sC5b-9. This research will potentially offer novel therapeutic strategies in light of new drugs affecting complement activation for monitoring therapy response.     

Mechanisms of the potentiation by adenosine of adenosine triphosphate-induced calcium release in tracheal smooth-muscle cells.

The effects of concomitant P1-receptor stimulation on peak intracellular Ca2+ release by extracellular adenosine 5'-triphosphate (ATP) and 5-hydroxytryptamine (5-HT) were investigated in cultured airway smooth-muscle (ASM) cells. The results show that peak Ca2+ release to ATP is enhanced by preincubation with adenosine (ADO) and with the specific A3 receptor agonist 1-Deoxy-1-(6-([(3-iodophenyl)methyl] amino)-9H-purin-9-yl)-N-methyl-beta-D-ribofuranuronamide (1B-MECA). The response to 5-HT, a smooth-muscle contractile agonist, was also enhanced after preincubation with ADO. Further measurements showed that this enhancement of the response to ATP was dependent on extracellular calcium because it was abolished by the removal of Ca2+ from the extracellular fluid and by incubation with the calcium channel blocker nifedipine. In addition, there was no difference between the levels of total inositol phosphates measured in the presence of ATP alone or of ADO + ATP. AACOCF3, a specific blocker of phospholipase A2, decreased the peak Ca2+ response to ATP and abolished the enhanced response to ATP and 5-HT produced by ADO. We conclude that stimulation of P1 and P2 receptors in ASM cells activates not only phospholipase C but also phospholipase A2. The enhancement of ATP-induced and 5-HT-induced Ca2+ release is due to Ca2+ influx from the extracellular fluid through a Ca2+ channel presumably modulated by arachidonic acid. These data show that endogenous ADO may modulate airway hyperresponsiveness by enhancing the ASM response to contractile agonists.     

Polymorphonuclear leucocytes as a model of Ca++ and Mg++-dependent cellular activation. Effect of calcium entry blockers

Flunarizine inhibited FMLP- and A23187-induced aggregation, enzyme release and O2- generation from human PMN as a function of its concentration. A23187-dependent PMN aggregation was also studied in media devoid of Ca++ or Mg++. Flunarizine (2.4 X 10(-5)M) significantly affected not only Ca++-supported but also Mg++-sustained PMN aggregation. The inhibiting effect of the drug was reversed by increasing the level of Ca++ (1.2 mM) or Mg++ (2 mM). Nifedipine, another Ca++-entry blocker, was shown to inhibit enzyme release and O2- generation induced by FMLP and A23187 as a function of its concentration, but only slightly affected PMN aggregation at very high concentration (10(-4)M). A role for flunarizine as a specific Mg++-entry blocker is suggested.     

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.     

补体C5b-9复合物刺激大鼠肾小球系膜细胞合成NO的机制探讨

目的:探讨人C5b-9复合物刺激大鼠肾小球系膜细胞(MC)合成一氧化氮(NO)的机制。方法:用人C5b-9复合物刺激培养的大鼠肾小球MC诱生肿瘤坏死因子α(TNFα)和白细胞介素1β(IL-1β),并用抗TNFα或抗IL-1β单克隆抗体进行处理。在上述基础上,分析处理3 h、6 h及24 h时与NO升高有关的某些指标的变化。结果:经C5b-9复合物刺激6、24 h后的MC(C5b-9组)产生TNFα明显高于对照组,并能被TNFα单抗逆转。用C5b-9复合物刺激MC未见IL-1β的产生。另用C5b-9刺激3 h时可见MC表达iNOS mRNA,而在刺激6 h和24 h时,MCiNOSmRNA表达,MC内cGMP含量及培养上清液中NO³_-/NO²_-含量均显著高于对照组。不过,C5b-9刺激时加用TNFα单抗处理,这些指标在6 h、24 h时均较C5b-9组低。结论:C5b-9复合物早期(3 h)能诱导MC表达iNOS mRNA,而6h后NO的升高则与MC释放的TNFα作用有关。     

Mechanisms of human platelet aggregation and release reaction induced by influenza virus

Mechanisms of aggregation and release reactions of human platelets induced by non-hemolytic influenza virus were studied. The influenza virus (PR/8 strain) caused aggregation of platelets with ATP release in a dose-dependent manner over the range of 640-10,240 HA titers. The aggregation was always preceded by a lag period and subsequent shape changes. The virus-induced aggregation was enhanced by exposure of the reaction mixtures to cold at 4 degrees C for 30 min and inhibited by apyrase, acetylsalicylic acid and dipyridamole. Ingestion of acetylsalicylic acid also inhibited the aggregation. Gel-filtered platelets were aggregated by influenza virus only after the reaction mixtures had been previously incubated at 4 degrees C for 30 min and then stirred at 37 degrees C. In the absence of divalent cations (Ca2+ less than or equal to 2 x 10(-5) M, Mg2+ less than or equal to 1 x 10(-5) M), gel-filtered platelets were not aggregated by influenza virus. These results suggest that influenza virus was absorbed onto the platelet surface and caused the release of ADP from platelets, which in turn, aggregated platelets.     

Thrombin, kallikrein and complement C5b-9 adsorption on hydrophilic and hydrophobic titanium and glass after short time exposure to whole blood

Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5s and 24h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively. Complement C5b-9 and expression of CD11b, CD66b, CD62P and Pan-platelets were measured by immunofluorescence. Thrombin and kallikrein were present on the surfaces during the whole investigated periods. Platelet adhesion and PMN cell adhesion and activation on all surfaces and activation of platelets on hydrophobic surfaces showed a similar pattern to thrombin adsorption. Kallikrein adsorption had a different pattern on each surface. C5b-9 was detected between 32min and 24h of blood exposure and a varying pattern of C5b-9 coverage was observed on each surface. In conclusion, our results indicate that the interaction between material and blood coagulation and kinin-activating proteins regulate the adhesion and activation of blood cells, whereas after longer time the coagulation and kallikrein-kinin system play minor roles and the complement system is decisive for mediating and elongating the inflammatory process.     

The effect of phosphatidyl serine on the activation stage of histamine release induced by neutrophil cationic protein.

Histamine release from rat mast cells induced by cationic protein (band 2) from rabbit neutrophil lysosomes occurs in Ca++-deficient medium. At higher concentrations of Ca++ the release is inhibited. Strontium not only supports, but also enhances the release of histamine in the absence of Ca++. Progressive enhancement of release occurs between 1.8 and 14.4 mM Sr++. The release of histamine from mast cells, activated at low temperature (0-4 degrees C) in the presence of 14.4 mM Ca++ and then washed prior to incubation at 37 degrees C, is inhibited. However, if phosphatidyl serine (PS) (10 microgram) is present with 14.4 mM Ca++, the inhibition is reversed. There is also inhibition of release when cells, activated in the presence of 1.8 mM Ca++, are incubated in the second stage with 14.4 mM Ca++, but this inhibition is less pronounced than when the 14.4 mM Ca++ is in the activation stage. PS enhances the release in the presence of both Ca++ and Sr++. The presence of PS in the activation stage enhances the release, but there is no significant enhancement when cells activated in the absence of PS are washed and incubated in the presence of PS. This suggests that PS enhancement of histamine release occurs at the activation stage, probably through the efficient delivery of calcium to the membrane sites, thereby increasing the efficacy of the membrane perturbation by band a protein.     

Synergistic inhibition of complement induced granulocyte margination by BW755C and calcium channel blockers

Intravenous infusion of granulocyte (PMNL) chemotactic factors including C5ades Arg present in zymosan activated plasma (ZAP), induces granulocytopenia due to PMNL margination. Since some PMNL responses are dependent on Ca++ ions and lipoxygenation of arachidonic acid, we evaluated the effects of a lipoxygenase (and cyclooxygenase) inhibitor, BW755C and Ca++ channel blocking agents, verapamil and nifedipine, on chemotactic factor induced granulocytopenia and margination in rabbits. BW755C (20 mg/kg i.v.) treatment significantly attenuated ZAP induced granulocytopenia. Verapamil or nifedipine alone were without effect. However, combined treatment with BW755C and verapamil or nifedipine (250 micrograms/kg) completely prevented ZAP-induced granulocytopenia. Ibuprofen, a cyclooxygenase inhibitor, was without effect either by itself or in combination with the calcium channel blockers. In striking contrast to the effect on ZAP-induced granulocytopenia, BW755C plus verapamil or nifedipine had virtually no effect on f-met-leu-phe, platelet activating factor or leukotriene B4 induced granulocytopenia. PMNL aggregation in vitro in response to all of the above chemotactic factors was inhibited by BW775C to similar degrees (56-75%) and was not influenced by simultaneous treatment with verapamil. We conclude that: (a) inhibitors of the lipoxygenase pathway may synergize with Ca++ channel blocking agents in inhibiting PMNL responses to complement derived chemotactic factors in vivo; (b) that in vivo PMNL margination to other chemotactic factors may be less dependent on endogenous lipoxygenation and/or Ca++ fluxes; and (c) there is a poor correlation between pharmacological inhibition of PMNL aggregation in vitro and PMNL margination in vivo in this system.     

Effect of osmotic protection on nucleated cell killing by C5b-9: cell death is not affected by the prevention of cell swelling

Formation of C5b-9 channels in the plasma membrane can lead to erythrocyte lysis or nucleated cell death. Lysis of erythrocytes by complement occurs as a result of colloid osmotic swelling and rupture of the plasma membrane, due to the unregulated flux of ions and water through C5b-9 channels. This colloid osmotic mechanism of lysis is largely based on the evidence that the extent of hemolysis is reduced, when macromolecules are placed in the medium to balance the osmotic gradient created by intracellular macromolecules, which are too large to diffuse through complement channels. The role of colloid osmotic deregulation, as a cause of nucleated cell killing by C5b-9, however, has been recently questioned [Kim S., Carney D. F. and Shin M. L. J. Immun. 138, 1530 (1987)]. In the present study, we investigated the effect of osmotic protection, with an 81,000 mol. wt dextran or bovine serum albumin, on Ehrlich cell killing by complement channels. The results indicated that prevention of cell swelling by dextran did not reduce the extent or rate of nucleated cell killing by either small (C5b-9l), or large (C5b-9m), complement channels when assessed by vital dye stain. The release of cytoplasmic lactate dehydrogenase as an alternative measure of cell death, however, was retarded and/or reduced, in the presence of dextran or albumin, at concns that prevented cell swelling. These results indicate that C5b-9 can kill nucleated cells effectively, in the absence of colloidal osmotic cell swelling, and that release of cytoplasmic macromolecules may not be a reliable indicator of cell death, when osmotic protectants are employed.     

Induction of mediator release from human glomerular mesangial cells by the terminal complement components C5b-9.

Exposure of cultured human glomerular mesangial cells (GMC) to normal human serum and an activator of the complement system results in rapid uptake of the terminal complement proteins C5b-9 by the cells. This 'innocent bystander' complement attack, however, does not result in cell killing, but in the stimulation of the GMC to release prostaglandin E (PGE), interleukin 1 (Il-1) and tumor necrosis factor (TNF). Endogenously synthesized Il-1 in turn activates PGE release, indicating that the C5b-9 attack initiates an autocrine feedback stimulation. Together with the fact that C5b-9 is found in many forms of glomerulonephritis, the data point to a role of the terminal complement proteins in the initiation and perpetuation of an inflammatory response.     

Interaction of the terminal complement components C5b-9 with synovial fibroblasts: binding to the membrane surface leads to increased levels in collagenase-specific mRNA.

The late complement components, apart from their lytic function, are known to trigger the release of various proinflammatory substances from different types of nucleated cells. In the present study, the interaction of C5b-9 with synovial fibroblast cells (SFC) was examined. It was found that incubation of SFC with activated complement components resulted in binding of C5b-9 to the cell membrane; subsequently an increase in abundance of collagenase-specific mRNA was seen, as assessed by Northern blotting. When C8-deficient serum was used as source of complement neither binding of C5b-9 nor an increase in collagenase-specific mRNA could be detected. These findings suggest that C5b-9, which might be generated during rheumatoid inflammation, may contribute to chronic joint destruction by triggering collagenolytic activity.