To investigate the effect of the terminal complement complex (TCC) on the central nervous system, we injected both the cytolytically active and the inactive complexes into the lateral ventricle of rats. Both complexes promoted accumulation of leukocytes into the cerebrospinal fluid at 4-6 h post-injection. The cells recovered at this time were mostly polymorphonuclear leukocytes (PMN) that were partially replaced by mononuclear cells at 12 h. A direct contribution of the complexes to the in-vivo migration of leukocytes was ruled out by their inability to be chemotactic for rat PMN. Contaminating C5a is unlikely to be responsible for the effect of TCC because it failed to mobilize leukocytes when injected into the lateral ventricle. Histological analysis of rat brains 6 hours after injection of TCC revealed marked leukocyte infiltration of the choroid plexus, increased expression of intercellular adhesion molecule-1 and egression of leukocytes out of the meningeal vessels. The cerebrospinal fluid of rats treated with TCC exhibited chemotactic activity for rat PMN and increased levels of growth related oncogen/cytokine-induced neutrophil chemoattractant-1 and monocyte chemoattractant protein-1 preceding the accumulation of leukocytes. Elevated concentration of IL-1beta was also found in the cerebrospinal fluid and in periventricular areas of rats treated with TCC.

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab′)2 anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8β.

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.

Intravital microscopy was used to monitor leukocyte traffic across rat mesenteric postcapillary venules induced by the inactive terminal complement (C) complex (iTCC) topically applied to ileal mesentery. Leukocytes started rolling within 15 minutes from the administration of iTCC, and by 1 hour they adhered almost completely to the endothelium emigrating from the vessels in the next 3 hours. C5a caused a similar, though less marked, effect, whereas boiled iTCC was inactive, excluding the contribution of contaminating lipopolysaccharide. The complex stimulated the migration of polymorphonuclear neutrophils (PMNs) across endothelial cells (ECs) in a transwell system after a 4-hour incubation of ECs with iTCC added to the lower chamber of the transwell, whereas a 30-minute incubation was sufficient for C5a and interleukin (IL)-8 to induce the passage of PMNs. C5a was not responsible for the effect of iTCC because this complex had no chemotactic activity and contained too small an amount of C5a to account for the transendothelial migration of PMNs. Similarly, the effect of iTCC was not mediated by IL-8 released by stimulated ECs because anti-IL-8 failed to inhibit the migration of PMNs induced by the complex. Unlike tumor necrosis factor-alpha, iTCC did not cause the redistribution of platelet-endothelial cell adhesion molecule-1 (PECAM-1), and PMN mobilization was partially blocked by anti-PECAM-1 antibodies.

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1α (HNF1α) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8β, C8γ, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1α-deficient mice showed negligible hemolytic activity of both C5 and C8α-γ subunits. The activity of C8β was severely affected despite only a 50% reduction in C8β mRNA levels in the liver. This is reminiscent of C8α-γ–deficient patients who accumulate extremely low levels of the C8β subunit. Our results demonstrate that HNF1α plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.

The complement system is an important component of innate immunity that contributes to the host defence against bacteria] infections and promotes clearance of immune complexes. Components of this system are synthesized by many cell types including hepatocytes and macrophages and are widely distributed both in the circulation and in the extravascular fluids. Complement activation products released in the fluid phase may interact with endothelial cells on the luminal and abluminal sides and activate these cells to express surface proteins (adhesion molecules, tissue factor) and to release molecules (chemokines) involved in the promotion of the inflammatory and coagulation processes as well as in modication of the vascular tone. Another effect of biologically active complement products on endothelial cells is to remove from the cell surface molecules, which, like heparan sulfate, protect from the formation of trombi. Some of the complement components bind to receptors present on the surface of endothelial cells and this is the case of C1q and C5a. Endothelial cells also interact with the terminal complement complex even in the cytolytically inactive form through a mechanism that has not been completely elucidated. The consequences of the complement-dependent activation of endothelial cells vary from a transient and reversible stimulation to a more persistent activation which may lead to endothelial cell and vascular alterations.

The function of the endothelial cells can be modulated by humoral factors present in the circulation and in the extravascular fluid, including proteins of the complement system. This review examines the multiple interactions between the complement system and the endothelial cells and their functional consequences on inflammation, coagulation and regulation of vascular tone. The implications of these interactions in the induction and progression of the vascular lesions occurring in atherosclerosis, ischemia/reperfusion and xenotransplantation and the possible therapeutic approaches in terms of complement regulation are also discussed.

The level of the terminal complement components secreted by human umbilical vein endothelial cells (HUVEC) was measured by a sensitive ELISA which allows the detection of 30-50 pg/ml of these components. C7 was the only terminal component detected in measurable amounts in the cell supernatant. The mean value was 11 ng/106 cells at 96 h and was slightly higher than that of C3 (9 ng/106 cells). HUVEC and serum C7 analysed by SDS-PAGE and immunoblot exhibited the same electrophoretic mobility. A proportion of C7 secreted by HUVEC was incorporated into the terminal complement complex (TCC) assembled spontaneously in the supernatant of cells cultured in C7-deficient human serum, and was not detected by the standard ELISA for C7 measurement. By adding the amount of C7 present in the TCC to that of free C7, the total amount of the component released by HUVEC was calculated to be approximately 35 ng/106 cells. Further TCC was produced following complement activation of the cell supernatant through the alternative pathway. Synthesis of C7 by HUVEC was confirmed by inhibition experiments in the presence of cycloheximide and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of C7 mRNA expression. Addition of IL-1alpha and tumour necrosis factor-alpha to the cell culture stimulated the secretion of C3, but had no effect on the synthesis of C7. By contrast, interferon-gamma had only a marginal effect on the production of C3, but markedly down-regulated the synthesis of C7 as assessed both by ELISA and RT-PCR.

PROBLEM:May anti-phospholipid or other autoantibodies interfere with trophoblast-endothelial cells interaction in women with unexplained pregnancy losses?METHODS OF STUDY:The sera of 72 women with recurrent spontaneous abortions (RSA) containing antibodies to endothelial cells (28), trophoblast (14), and cardiolipin (10) or lacking antibodies (25), and 26 controls were examined in an inhibition assay of trophoblast adhesion to endothelial cells using an ELISA based on the recognition of trophoblast by antibodies to cytokeratin.RESULTS:Adhesion of trophoblast to endothelial cells was time- and dose-dependent. Patients and control sera inhibited trophoblast adhesion with mean values of 37% and 7%, respectively. Inhibition above 2SD of the mean control value was still observed in 58% of the patients sera and 8% of the control sera. Sera containing antibodies to endothelial cells had higher inhibitory effect (38%) than those with antibodies to trophoblast (23%) and cardiolipin (28%) or lacking antibodies (26%).CONCLUSIONS:Antibodies and other undefined factors in the sera of women with RSA inhibit adhesion of trophoblast to endothelial cells.

The CD59 membrane protein confers protection from C5b-9-mediated cell lysis. Because evidence exists for complement (C) activation and generation of C5b-9 in the peritoneal cavity during chronic peritoneal dialysis (CPD), we investigated, on mesothelial cell (MC) lines, the expression of CD59 and the production of C components. Four MC lines were obtained from children on CPD, and two from non uremic children. CD59 expression on MCs was investigated with anti-CD59 monoclonal antibody (mAb) and polyclonal goat immunoglobulin G (IgG). MC lines were positive for staining with anti-CD59 mAb. Western blotting analysis of MC membrane demonstrated a band with the same molecular weight as CD59. Incubation of MC with anti-CD59 mAb abrogated the protective effect of CD59 (100% cytotoxicity). C3, C4, and C6 were detected in the supernatants of MC; in non uremic MC supernatants, C5, C7, C8, and C9 were also detectable, and C4 concentration was tenfold higher. CD59 expression confers to MCs protection from C5b-9-mediated lysis. MCs produce C factors. These findings suggest that production of complement components and expression of CD59 on MCs could play a role both in peritoneal cavity infection (decreased complement production) and in peritoneal membrane damage (decreased CD59 expression and reduced remesothelialization owing to MC lysis).

Sera genetically deficient in either the α−γ or the β‐subunit of complement component C8 virtually lack haemolytic activity. We have studied the formation and the structural organization of the soluble terminal complement complex (TCC) assembled in these sera following activation with cobra venom factor (CVF). The TCC concentration in the activated C8α−γ and C8β‐deficient samples was 0.2% and 4%, respectively, when compared with zymosan‐activated normal serum. TCC was purified from the activated C8β‐deficient samples by affinity chromatography and analysed by immunoblotting and enzyme immunoassay. No C8β was detected in one TCC preparation, while 7% of the normal level was present in the other. The level of the other terminal components, including that of C8α−γ, was normal. The ability of C8α−γ to promote the assembly of TCC in the presence of a limited amount of C8β or in the apparent absence of this subunit was confirmed using purified components, by mixing C5b6 and either of the purified C8 subunits together with C7 and C9. These data show that soluble TCC can be formed in C8β‐deficient sera that contain little or no C8β.

PTX3 is a prototypic long pentraxin consisting of a C-terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. The present study was designed to characterize the structure and ligand binding properties of human PTX3, in comparison with the classical pentraxins C-reactive protein and serum amyloid P component. Sequencing of Chinese hamster ovary cell-expressed PTX3 revealed that the mature secreted protein starts at residue 18 (Glu). Lectin binding and treatment with N-glycosidase F showed that PTX3 is N-glycosylated, sugars accounting for 5 kDa of the monomer mass (45 kDa). Circular dichroism analysis indicated that the protein consists predominantly of β-sheets with a minor α-helical component. While in gel filtration the protein is eluted with a molecular mass of ≅900 kDa, gel electrophoresis using nondenaturing, nonreducing conditions revealed that PTX3 forms multimers predominantly of 440 kDa apparent molecular mass, corresponding to decamers, and that disulfide bonds are required for multimer formation. The ligand binding properties of PTX3 were then examined. As predicted based on modeling, inductive coupled plasma/atomic emission spectroscopy showed that PTX3 does not have coordinated Ca2+. Unlike the classical pentraxins CRP and SAP, PTX3 did not bind phosphoethanolamine, phosphocholine, or high pyruvate agarose. PTX3 in solution, bound to immobilized C1q, but not C1s, and, reciprocally, C1q bound to immobilized PTX3. Binding of PTX3 to C1q is specific and saturable with a K d 7.4 × 10−8m as determined by solid phase binding assay. The Chinese hamster ovary cell-expressed pentraxin domain bound C1q when multimerized. Thus, as predicted on the basis of computer modeling, the prototypic long pentraxin PTX3 forms multimers, which differ from those formed by classical pentraxins in terms of protomer composition and requirement for disulfide bonds, and does not recognize CRP/SAP ligands. The capacity to bind C1q, mediated by the pentraxin domain, is consistent with the view that PTX3, produced in tissues by endothelial cells or macrophages in response to interleukin-1 and tumor necrosis factor, may act as a local regulator of innate immunity.