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PubMed1996

Adhesion molecule expression and complement activation in vessel walls in synovial tissue from patients with chronic inflammatory joint disease.

Histopathology demonstrating a manifest vasculitis is a rare event in synovial tissue (ST) from patients with chronic inflammatory joint disease. As a possibly more subtle sign of a vasculitic process, complement activation in vessels in ST was studied. In the same tissues, the expression of the adhesion molecules ICAM-1 and E-selectin in vessel walls was examined, to see if the expression was related to vasculitic processes. The study was performed by use of direct immunofluorescence technique on cryostat sections of ST, using mouse monoclonal antibodies to the terminal complement complex (TCC, C5b-9), ICAM-1 and E-selectin. Expression of ICAM-1 was found in the vessel walls in all of 28 tissues tested, whereas E-selectin was found in 4 cases and TCC in 11. E-selectin and TCC were found together in only 1 tissue. The study supports the view that ICAM-1 is always, or nearly always, present in vessel walls in synovial tissue from patients with chronic inflammatory joint disease. E-selectin and TCC may also be present, but the lack of association between these two proteins suggests that the mechanism leading to a complement mediated vasculitic process is different from that causing expression of E-selectin.

Animals↗
PubMed1996

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Amino Acid Sequence↗
PubMed1998

Preferential activation of the complement system in the lower epidermis of patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an immune-mediated blistering skin disease characterized by acantholysis of the suprabasal epidermis and by IgG autoantibodies targeting a desmosomal component, desmoglein 3. IgG alone has been demonstrated to induce acantholysis in some experimental conditions. The role of the complement system in blister formation in PV remains controversial. We describe four consecutive patients with new-onset PV. The acantholytic process occurred in the lower epidermis and colocalized with deposition of complement C3 and the membrane attack complex C5b-9. The colocalization of complement deposition with the acantholytic process in the lower epidermis supports a role for the complement system in blister formation in PV.

Adult↗
PubMed1979

Human complement in thrombin-mediated platelet function: uptake of the C5b-9 complex.

Thrombin-mediated platelet aggregation and release is enhanced by the presence of C3, C5, C6, C7, C8, and C9 of human complement. The interaction of thrombin with its receptor on the platelet membrane initiates activation of complement on the platelet surface. Trypsin-mediated platelet function is not enhanced by the addition of complement, probably because trypsin has no receptor on the platelet surface so activation of complement is triggered in the fluid phase and not on the platelet surface. Activation of complement by thrombin led to production of dimers of the C5b-9 complex on the platelet surface. These complexes were eluted from the platelet membrane and were identified physicochemically and morphologically. The mechanism of complement-induced enhancement of platelet function is not clear, however, it probably is mediated via the arachidonic acid transormation pathway because this activity was blocked by known inhibitors of cyclo-oxygenase, namely, aspirin and indomethacin.

Antithrombin III↗
PubMed1985

Possible involvement of terminal complement complex in active Heymann nephritis.

We investigated whether the appearance of various complement components in renal deposits of immune complexes correlated with the development of proteinuria in rats with active Heymann nephritis. Sequential kidney biopsy specimens and serum samples were obtained from Lewis rats immunized with Fx1A in complete Freund's adjuvant. Circulating antibodies against purified auto-antigen renal tubular epithelial glycoprotein, as measured by ELISA, were found in the circulation together with a diffuse granular deposition of IgG1, IgG2a, and IgG2b in the glomeruli within 2 weeks after immunization. Biopsy specimens taken 4 weeks after immunization showed diffuse deposition of C4 and C3, which indicated that activation of complement by the classical pathway had occurred. The detection of the C5b-9 complex of complement in glomerular deposits coincided with the development of abnormal proteinuria indicating that the glomerular damage in this autoimmune disease may be caused by complement-mediated lesions in the glomerular capillary walls.

Animals↗
PubMed1994

IgG subclass distribution and complement activation ability of autoantibodies to neutrophil cytoplasmic antigens (ANCA).

To study the IgG subclass distribution and complement activation ability of ANCA, 24 sera containing C-ANCA (cytoplasmatic) and 7 sera containing P-ANCA (perinuclear), as determined by a routine immunofluorescence test, were examined. The subclass distribution was tested by the use of immunofluorescence and ELISA technique and monoclonal antibodies to IgG subclasses. The complement activating activity was studied by the use of immunofluorescence technique and antibodies to C3c and the terminal complement complex (TCC, C5b-9), directed against a neoepitope on C9. For C-ANCA, IgG1 and IgG4 were the dominating subclasses. The subclass distribution differed from that of other autoantibodies tested and that of the total IgG subclass concentrations. For P-ANCA, the results were inconclusive, but the lack of IgG3 was striking. All of the C-ANCA-containing sera caused deposition of C3c, and 50% of the sera gave formation of TCC when reacting with ethanol-fixed granulocytes. P-ANCA-containing sera caused some C3c deposition, but not TCC formation. The unusual IgG subclass distribution for C-ANCA is possibly due to repeated antigenic stimulations and/or to T cell factors influencing the antibody isotype switching. Despite the high IgG4 activity, sera containing C-ANCA are often able to activate complement and are therefore potentially harmful.

Animals↗
PubMed1997

Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.

B-Lymphocytes↗
PubMed1976

A new activity of complement component C3: cell-bound C3b potentiates lysis of erythrocytes by C5b,6 and terminal components.

EAC4b,3b (sheep erythrocytes carrying rabbit antibody and guinea pig complement component fragments C4b and C3) adsorb human C5b,6 reversibly; the avidity of binding varies inversely with ionic strength. We believe that the receptor of C5b,6 is contributed by the cell-bound C3b because the binding capacity of EAC4b,3b varies with C3b multiplicity and can be blocked with rabbit antibody to guinea pig C3. The fixation of C5b,6 to the erythrocyte-bound C3b serves to concentrate C5b,6 on the cell surface; as a consequence, the hemolytic efficiency of C5b,6 is almost 100 times greater when assayed with EAC4b,3b than with plain erythrocytes. This potentiation represents a hitherto unrecognized function of cell-bound C3b.

Animals↗
PubMed1981

Homologous species restriction in lysis of erythrocytes by terminal complement proteins.

The cytolytic efficiency of the terminal complement protein complex, C5b-9, varies with the species of origin of C8 and C9. In the present study, we explored the susceptibility of erythrocytes from various species to lysis by C5b6,7 plus C8 and C9 from different species. EC5b6,7 intermediates were prepared on human, guinea pig, rabbit, mouse, and rat erythrocytes with human C5b6 and guinea pig C7. The degree of lysis of these intermediates by C8 and C9 was found to vary widely depending on the species of the proteins and the target cells. In all cases, lysis was least efficient when C8 and C9 were homologous with respect to the target cell species. This effect was mostly attributable to C9. The inefficient lysis in a homologous system is not due to a failure of C9 binding. Rather, the poor lysis in the homologous system may be attributable to inefficient insertion or channel formation.

Animals↗
PubMed1997

Biological responses to endovascular treatment of abdominal aortic aneurysms.

PURPOSE: To review the findings of two studies investigating the apparent differences in inflammatory responses demonstrated in patients undergoing endovascular as opposed to classic surgical treatment of abdominal aortic aneurysms (AAAs). METHODS: The clinical course of seven patients treated with an endoluminal procedure (AAA-E) and seven patients undergoing conventional surgery (AAA-C) were compared (all men; ages 52 to 80 years). Blood samples were taken pre-, intra-, and postoperatively for up to 7 days. Inflammatory responses were assessed from measurement of interleukins (IL)-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF-alpha); complement proteins C1q, C4, C5a, and terminal complement complexes, C5b-C9; and C-reactive proteins. Granulocyte and monocyte surface adhesion molecule expression was determined indirectly using a panel of monoclonal antibodies against CD11a, CD11b, CD11c, CD18, and L-selectin in donor white blood cells exposed to patient plasma. RESULTS: In six of the AAA-E patients, blood pressure decreases were recorded during the introduction of the device. Elevated body temperature was sustained for 2 to 5 days postoperatively in the AAA-E group. IL-6 levels were significantly higher in AAA-C patients (p < 0.0005), while TNF-alpha release was recorded in the AAA-E group only. CD11b, CD11c, and CD18 molecules on both granulocytes and monocytes were significantly upregulated 60 minutes after the endovascular procedure compared to conventional surgery. CONCLUSIONS: Endovascular aortic aneurysm repair apparently induces a significant inflammatory response, mainly involving TNF-alpha release, which differs from open AAA repair. These inflammatory responses, which may be related to the observed intraprocedural blood pressure decreases, could be caused by cell activation arising from intra-aneurysmal device manipulation.

Aged↗
PubMed1990

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.

Animals↗
PubMed1994

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Amino Acid Sequence↗
PubMed1996

Physical linkage and orientation of the human complement C8 alpha and C8 beta genes on chromosome 1p32.

Human C8 is one of five components (C5b, C6, C7, C8, C9) of the cytolytic C5b-9 complex of complement. It consists of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma), which are encoded in separate genes. Genetic linkage and chromosomal localization studies previously established that C8 alpha and C8 beta are closely linked on chromosome 1p32. In this study, clones with inserts containing genes for both C8 alpha and C8 beta were isolated from a yeast artificial chromosome (YAC) human genomic DNA library and characterized in an effort to determine intergenic distance and orientation. One clone with a approximately 330-kb insert yielded restriction digest patterns for C8 alpha and C8 beta that agreed with those obtained previously from digests of human genomic DNA, thereby confirming the presence of intact copies of both genes. A second clone with a approximately 280-kb insert yielded similar results; however, it was truncated at the 5' end of the C8 alpha gene. Restriction digests of both clones were subjected to PFGE and Southern blot analysis using probes specific for the terminal exons of C8 alpha (exons 1 and 11) and C8 beta (exons 1 and 12). Results indicate the genes are physically linked (< 23 kb) and in a 3'-3' orientation. This is the same orientation as the ancestrally related C6 and C7 genes, which are also physically linked on chromosome 5p13.

Chromosome Mapping↗
PubMedDate not supplied

Terminal complement complex in synovial tissue from patients affected by rheumatoid arthritis, osteoarthritis and acute joint trauma.

The C5b-9 complex (Terminal Complement Complex-TCC) is the final product of the terminal complement pathway. In this study, using the monoclonal antibody MCaE11 (specific for a C9 neoantigen) and an immunohistochemical technique, we examined the TCC deposits in synovial tissues from 4 patients affected by rheumatoid arthritis (RA) and 6 patients affected by osteoarthritis (OA). Synovial tissues from 8 patients affected by acute joint trauma were examined as controls. Furthermore, plasma TCC levels were measured in 44 RA patients and 51 controls, using the above mentioned antibody and a sandwich ELISA. Eight synovial fluids were also included in this study. Abundant TCC deposits were detected in the cytoplasm of the synovial lining cells and of large stromal mononuclear cells in all the RA and in 3 out of 6 OA synovial tissues characterized by histological signs of inflammation. No TCC deposits were found in non-inflamed synovial tissues from patients with joint trauma. In agreement with previous observations, the TCC plasma levels found were significantly higher in RA patients than in controls, but no difference was seen between patients with active and non-active disease. The mean TCC level was significantly higher in the synovial fluid than in the plasma, but no correlation emerged between these two series of values. This study shows that: a) the plasma level of TCCs cannot serve as an indicator of disease activity in RA; b) the TCC deposits in synovial tissue correlate well with the extent of inflammatory synovitis, irrespective of whether the synovitis is rheumatoid or osteoarthritic in nature.

Acute Disease↗
PubMed1985

Elimination of terminal complement intermediates from the plasma membrane of nucleated cells: the rate of disappearance differs for cells carrying C5b-7 or C5b-8 or a mixture of C5b-8 with a limited number of C5b-9.

We have previously shown that multiple complement (C) channels are required for lysis of a nucleated cell in contrast to the single channel requirement for erythrocytes. To further investigate this multichannel requirement for nucleated cells, we examined the stability of terminal C complexes in the plasma membrane of Ehrlich ascites tumor cells. Ehrlich cells bearing C5b-7 or C5b-8 with or without C9 were incubated at 37 degrees C or 0 degree C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. C5b-7, C5b-8, and C5b-8 in the presence of a limited number of C5b-9 complexes disappeared functionally from the plasma membrane at 37 degrees C, with initial half-lives of 31, 20, and 10 min, respectively. Disappearance of these complexes did not occur at 0 degree C, nor did disappearance occur at 37 degrees C when formed on sheep erythrocytes. The fate of C5b-8 complexes on the surface of Ehrlich cells was traced with colloidal gold particles bound to C5 determinants on C5b-8 with the use of immunoelectron microscopy. Colloidal gold could be seen on the cell surface after specific binding to cells carrying C5b-8 sites at 0 degree C. After incubating these cells at 37 degrees C, gold particles were internalized into the cell continuously via endocytic vesicles. It is postulated that terminal C complexes may stimulate or accelerate the removal of these complexes from the cell surface.

Animals↗
PubMed2002

Histopathology of cardiac xenograft rejection in the pig-to-baboon model.

BACKGROUND: The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed. METHODS: 44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates. RESULTS: In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells. CONCLUSIONS: The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects.

Acute Disease↗
PubMed1999

Greater increase in cytokine concentration after salvage with filtered whole blood than with washed red cells, but no difference in postoperative hemoglobin recovery.

BACKGROUND: Inflammatory mediators are released in association with intraoperative and postoperative salvage of blood. Whether these mediators (cytokines) participate in the modulation of erythropoiesis or not has been investigated. STUDY DESIGN AND METHODS: Twenty-seven patients who were to undergo total knee replacement surgery were randomly assigned to postoperative blood salvage with either filtered whole blood or washed red cells. Patients with postoperative blood loss <400 mL were considered a control group. The control group did not receive any transfusions. Plasma concentrations of the anaphylatoxin C3a, the C5b-9 terminal complement complex, and the cytokines interleukins 6 and 8, hemoglobin, reticulocytes, and red cell volume fraction in the patients were repeatedly analyzed before and after surgery. RESULTS: Significantly increased concentrations of interleukin 6 appeared in all three groups, which was interpreted as a response to the surgical trauma. The increase was significantly greater in the group that received filtered whole blood after return of shed blood. The recovery of hemoglobin levels did not differ in the groups. CONCLUSION: The transfusion of filtered whole blood leads to the formation of interleukin 6 in the circulation, but postoperative hemoglobin recovery was similar in all groups.

Aged↗
PubMed1993

Current topics in childhood lupus nephritis.

Lupus nephritis is a major predictor of the prognosis of systemic lupus erythematosus (SLE). The present paper discusses lupus nephritis from clinical and immunopathological points of view. Although recent advances in diagnosis and treatment improve the prognosis of children with SLE, there remain many unsolved clinical problems. One of the current topics in the treatment for SLE is intermittent intravenous cyclophosphamide therapy which is effective even for the steroid-resistant patients with severe lupus nephritis, at least for short-term observation. Immunopathologically, the following issues are discussed: (i) The C5b-9 terminal complement complex plays an important role in the pathogenesis of lupus nephritis. The possible interaction of vitronectin and SP-40,40 is also mentioned; (ii) A semi-quantitative analysis of the charge barrier of the glomerular basement membrane reveals that the charge barrier dysfunction plays an important role in the pathogenesis of proteinuria in lupus nephritis. This study also demonstrates that the charge of immune deposits is important for the initiation of glomerular injury in lupus nephritis; (iii) It is demonstrated that the histopathological diversity of lupus nephritis is based on biological properties of nephritogenic auto-antibodies in murine lupus models.

Adolescent↗