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PubMed1994

Clusterin gene expression in the rat thymus is not modulated by dexamethasone treatment.

Clusterin, a multifunctional glycoprotein, characterized as a potent inhibitor of the membrane attack complex of complement, is also known to be the product of a gene that is highly up-regulated in certain tissues undergoing programmed cell death. We have studied the expression of this gene in the rat thymus after the induction of thymocyte programmed cell death (PCD) by in vivo dexamethasone administration. Northern blot analysis of clusterin mRNA 2, 4, 6 and 8 hr after dexamethasone administration in a total of 21 rats revealed no modification in the level of clusterin gene expression. In situ hybridization demonstrated that clusterin gene expression is macroscopically confined to the medullary region of the thymus, and that this distribution is not modified by dexamethasone administration. These results strongly suggest that in the rat, clusterin gene expression is not associated with the programmed cell death of thymocytes following in vivo dexamethasone administration. In situ hybridization of the clusterin cRNA to thymus cryostat sections confirmed the results obtained by Northern blot analysis. Indeed, no consistent increase in the amount of clusterin mRNA was detectable at any of the time-points studied. The macroscopic distribution of clusterin mRNA in the rat thymus was identical to that previously observed in the human thymus, clusterin message being essentially detected within the medullary regions. No modification in the macroscopic distribution of clusterin gene expression was detected after dexamethasone administration. These results suggest that, like the human thymus, medullary epithelial cells are the site of clusterin gene expression in the rat thymus. Moreover they indicate that an increase in the extent of ongoing thymocyte PCD does not significantly modify the rate or site of clusterin gene expression within the thymus.

Animals↗
PubMed1993

The temporal relationship between urinary C5b-9 and C3dg and clinical parameters in human membranous nephropathy.

We have previously reported that urinary excretion of the complement activation products C3dg and C5b-9 in human membranous nephropathy (MN) correlated with clinical outcome in a cross-sectional analysis. We report here the results of a retrospective longitudinal study of the temporal relationship between urinary C3dg and C5b-9 excretion and clinical parameters. A group of 23 adult patients with biopsy-proven MN were studied over a mean time period per patient of 3.5 years. Freshly voided urine samples were collected regularly; C3dg and C5b-9 were measured by ELISA (mean number of samples per patient = 13). During the period of the study, nine patients with declining renal function (group A) were treated with a standard steroid regimen. Serum creatinine had improved or stabilized in seven of these patients at the end of treatment. All nine patients were excreting C3dg and C5b-9 before treatment. Six other patients with declining renal function (group B) were not treated with steroids because of clinical contraindications. Serum creatinine continued to increase during the study in four of these six patients. C3dg and C5b-9 were present in the urine samples of these six patients on the majority of dates tested. Eight patients maintained stable renal function during the study (group C), either normal (6 patients) or impaired (2 patients). Of these patients, six were consistently negative for urinary C3dg and C5b-9 despite persistent proteinuria, and one patient who was initially positive became negative within 15 months, and remained negative for the rest of the study period. One patient was positive on one of 12 occasions tested. These results suggest that urinary complement activation products indicate ongoing active glomerular damage and may prove to be important determinants for the introduction and monitoring of therapy.

Complement Activation↗
PubMed1993

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.

Arthritis, Rheumatoid↗
PubMed1996

Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin).

Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the surrounding extracellular matrix.

Antigens, CD↗
PubMed1996

Mediators of perivascular inflammation in the left ventricle of renovascular hypertensive rats.

OBJECTIVE: Inflammatory cells invade the fibrotic myocardium of spontaneously hypertensive rats at the same sites as where fibroblasts are produced. The role of these inflammatory cells in myocardial fibrogenesis was studied in the present work. METHODS: The production and distribution of proteins that may be implicated in inflammation was examined by immunohistochemistry of sections of left ventricles from 1-month and 4-month renovascular hypertensive and age-matched control rats using antibodies against ICAM-1, LFA-1, TGF beta 1, PDGF-A, T and H kininogens, IgG, IgM, C3, and C5b-9. Infiltrating inflammatory cells were phenotyped by immunohistochemistry. The TGF beta 1 and PDGF-A mRNA levels were checked by RT-PCR. RESULTS: Infiltrating cells were mainly T helper lymphocytes and macrophages, and there were more inflammatory cells in hypertensive rats than in control rats, localized especially around coronary arteries and in microscars. There were more ICAM-1 and LFA-1 in the ventricles of hypertensive than in control rats at 1 month, but the ICAM-1 expressions in hypertensive and control rats were similar at 4 months. TGF beta 1 and PDGF-A mRNA steady states increased in 4-month hypertensive rats, but there was no labeling for TGF beta or PDGF by immunohistochemistry. There was only faint labeling for T and H kininogens, and it was not increased in hypertensive rats. There were deposits of IgM and C5b-9 only in hypertensive rats. CONCLUSION: Thus, inflammatory cells infiltrate the cardiac tissue of renovascular hypertensive rats as in the case of spontaneously hypertensive rats and these cells may use the ICAM-1/LFA-1 system to infiltrate, but neither TGF beta 1 and PDGF-A, nor the kininogen system seem to be associated with cardiac fibrogenesis. Otherwise, the complement system could act as arteriosclerotic and/or leukocyte mobilizing factors.

Animals↗
PubMed1997

Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.

The present work was designed to elucidate the in vivo role of complement in the proteinuria-associated tubulointerstitial injury. Rats were intravenously injected with puromycin aminonucleoside, and massive proteinuria was observed within 5 days. Prominent tubulointerstitial injury characterized by proximal tubular degeneration, tubular dilatation, and leukocyte infiltration were observed 7 days after injection. C3 and C5b-9 were observed in the luminal side of proximal tubular cells. Renal function, assessed by inulin and para-aminohippurate clearance, was significantly decreased. To-assess the role of complement in this model, rats were injected with either cobra venom factor or soluble recombinant human complement receptor type 1 starting at day 3. These manipulations significantly improved tubulointerstitial pathology and para-aminohippurate clearance without affecting the degree of proteinuria. Deposition of C3 and C5b-9 was not detected in the kidney of rats depleted of complement by cobra venom factor. In rats treated with soluble complement receptor, C3 was still detected in the tubules, but deposition of C5b-9 was not observed. Soluble complement receptor was detected at the site of C3 deposition and in the urine. These data strongly suggest that complement plays a pivotal role in proteinuria-associated tubulointerstitial injury and that systemic complement depletion or inhibition of complement in the tubular lumen may diminish the tubulointerstitial damage.

Animals↗
PubMed1997

Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.

In rat membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). To address mechanisms of PLA2 activation, GEC were stably transfected with cDNAs of wild-type cytosolic PLA2 (cPLA2-wt), or group II secretory PLA2, producing overexpression of PLA2 activity. Sublytic C5b-9 markedly increased free [3H]AA in cPLA2-wt-transfected GEC, but only trivial increases were evident in secretory PLA2-transfected, or neo (control) GEC. In cPLA2-wt-transfected GEC, reduction of extracellular free Ca2+ or down-regulation of protein kinase C inhibited [3H]AA release. To further address the regulation of cPLA2, we stably expressed a mutant cPLA2 in which the Ca2+-dependent lipid binding domain was deleted (deltaCaLB). In GEC that express cPLA2-deltaCaLB, the C5b-9-induced increase in free [3H]AA was comparable with neo, despite expression of cPLA2-deltaCaLB at levels similar to cPLA2-wt. We then stably expressed another cPLA2 mutant (cPLA2-srcmyr) in which the CaLB domain was replaced by the N-terminal myristoylation domain of c-Src. cPLA2-srcmyr is permanently membrane associated. At low extracellular free Ca2+, C5b-9 increased free [3H]AA significantly in GEC that express cPLA2-srcmyr, while in neo GEC, the change was negligible. Thus, C5b-9 activates the cPLA2 isoform. Activation is dependent on the CaLB domain, and is mediated by phosphorylation, Ca2+ influx, and membrane association.

Animals↗
PubMed1997

Clinical characterization of Dicea a new cellulose membrane for haemodialysis.

A prospective randomised clinical study comparing the functional performance and biocompatibility of a new cellulose diacetate variant (Dicea) in which the degree of hydroxyl group substitution differs, with cellulose diacetate and low flux polysulfone incorporated into commercially produced hollow fiber hemodialysers with a surface area 1.5-1.6 m2 has been undertaken. All dialysers studied demonstrated clinically acceptable performance in terms of their small molecular removal characteristics, with minor statistical but not clinical differences. Use of both cellulose diacetate membranes but not low flux polysulfone resulted in a reduction in plasma beta(2) microglobulin levels. The membranes were impermeable to albumin, but showed some permeability to low molecular weight proteins. The average protein recovery from the dialysis fluid was 3105 mg for Dicea, 2913 mg for cellulose diacetate and 2842 mg for low flux polysulfone. For Dicea the white cell count by 15 minutes had declined to 68% of pre treatment value, compared with 59% and 86% for cellulose diacetate and low flux polysulfone. The differences between Dicea and cellulose diacetate were not significant, but both cellulose based membranes differed from low flux polysulfone (p = 0.0015). There was a strong evidence of differences between the membranes in respect of C5a and C5b-9 generation (p = 0.0001) but not for C3a (p = 0.16) furthermore the levels of C5b-9 generated during dialysis also showed a significant positive correlation compared to C5a for all membranes. (Pearson's correlation coefficient = 0.856, p = 0.0001). It is concluded that the two cellulose diacetate membranes are not identical, with the differences observed being a consequence of the degree of acetyl substitution, resulting in alteration of membrane structure and the method of sterilization. The clinical significance of these differences are difficult to characterize but the modification of the cellulose structure appears to be a promising method to improve the biocompatibility of cellulose membranes. The improved biocompatibility offered by this method still falls short of that achieved with low flux synthetic membranes such as Fresenius Polysulfone.

Adult↗
PubMed1994

Paroxysmal nocturnal hemoglobinuria and complement-mediated erythrocyte damage.

The erythrocytes of paroxysmal nocturnal hemoglobinuria are abnormally sensitive to complement-mediated lysis because they are deficient in membrane proteins that regulate the functional activity of complement. All the deficient proteins in paroxysmal nocturnal hemoglobinuria share the common structural feature of being anchored to the cell surface by a glycosyl phosphatidylinositol moiety. Recent studies showed that the first intermediate in the pathway of the glycosyl phosphatidylinositol anchor synthesis is not formed in paroxysmal nocturnal hemoglobinuria cells. This observation suggests that the molecular basis of paroxysmal nocturnal hemoglobinuria is due to an abnormality involving a gene that encodes a protein essential for the normal biosynthesis of the first intermediate. By using expression cloning, the complementary DNA (called phosphatidylinositol glycan class A [PIG-A]) that corrects the abnormality in glycosyl phosphatidylinositol-anchor synthesis in paroxysmal nocturnal hemoglobinuria cells was identified. Subsequent studies showed that the PIG-A gene is located on the X chromosome. Together, these studies provided a molecular explanation for paroxysmal nocturnal hemoglobinuria.

Animals↗
PubMed2000

Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

Amino Acid Sequence↗
PubMed2004

C5b-9 regulates peritubular myofibroblast accumulation in experimental focal segmental glomerulosclerosis.

BACKGROUND: In human focal segmental glomerulosclerosis (FSGS), the tubulointerstitial deposition of the complement (C5b-9) membrane attack complex is correlated with interstitial myofibroblast accumulation and proteinuria. Here, we hypothesized that C5b-9 formation regulates renal myofibroblast accumulation in Adriamycin nephropathy. METHODS: Adriamycin nephropathy was induced in complement C6-sufficient (C6+) and C6-deficient (C6-) piebold viral glaxo (PVG) rats. Groups of animals (N= 7 to 8 each) were examined on days 21 and 42. A group of C6+ animals, injected with vehicle, served as the control group. RESULTS: C6+ and C6- rats with Adriamycin nephropathy had equivalent proteinuria. C5b-9 deposition was increased and present on the apical surface of proximal tubular epithelial cells (day 21 and 42) and peritubular region (day 42 only) in C6+ rats with Adriamycin nephropathy, and absent in C6- rats. Peritubular myofibroblast accumulation increased in a time-dependent manner in C6+ proteinuric rats (control 1.2 +/- 0.4; Adriamycin nephropathy day 21 11.0 +/- 0.7; Adriamycin nephropathy day 42 19.8 +/- 1.7 cells per high power field). In C6- rats this increase was blunted by 87% and 56% on days 21 and 42, respectively (P < 0.01), and was associated with reduced interstitial extracellular matrix (ECM) deposition. Tubulointerstitial injury, tubular vimentin and interstitial monocyte accumulation were also reduced in C6- rats with Adriamycin nephropathy on day 21, but not at day 42. In contrast, the increase in periglomerular myofibroblast accumulation and glomerulosclerosis in Adriamycin nephropathy were not altered by C6 deficiency. CONCLUSION: These data suggest that glomerular ultrafiltration of complement components and the intratubular formation of C5b-9 is a specific promotor of peritubular myofibroblast accumulation in FSGS.

Animals↗
PubMed1999

Induction of decay-accelerating factor by cytokines or the membrane-attack complex protects vascular endothelial cells against complement deposition.

Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.

Antigens, CD↗
PubMed2001

Cytolytic complement activity in otitis media with effusion.

Otitis media with effusion (OME) is a chronic inflammation persisting in the middle ear cavity of at least 8 weeks duration. Middle ear effusion (MEE; n = 38), samples from children suffering from OME were investigated for their direct cytolytic activity or an ability to enhance complement lysis of unsensitized bystander cells. Thirteen of the 38 MEEs had direct endogenous haemolytic activity and 27 samples had an ability to enhance serum-initiated lysis. Using an enzyme immunoassay, high levels of terminal complement complexes (TCC) were detected in the MEE samples (mean 34.1 microg/ml, range 5--89 microg/ml). This indicated strong local complement activation that had progressed to the terminal stage. As one potential factor promoting complement activation we identified both monomeric and trimeric properdin in MEE by Western blotting. By stabilizing C3 and C5 convertases properdin accelerates the alternative and terminal pathways of complement. On the other hand, the membrane attack complex (MAC) inhibitor CD59, which was found to be extensively shed into the MEE in a functionally active form, may control excessive cytotoxicity of the MEE. In conclusion, intense complement activation, up to the terminal level, maintains ongoing inflammation in the middle ear cavity and can pose a threat to the local epithelium.

CD59 Antigens↗
PubMed1999

Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex.

BACKGROUND: The dependence of intestinal ischemia-reperfusion injury on the classical complement pathway has been shown with the complement antagonist (sCR1) and complement-specific knockout mice. Using C5 deficient mice, we show that the membrane attack complex mediates local injury. METHODS: Mice underwent intestinal ischemia-reperfusion. Albumin leak and histologic evidence were compared in wildtype mice, wildtypes treated with sCR1, neutrophil-depleted wildtypes, C5-deficient mice, and C5-deficient mice reconstituted with wildtype serum. Neutrophil tissue levels in injured C5-deficient and wildtype intestines were compared. RESULTS: C5-deficient mice had a reduction in injury similar to mice treated with sCR1. Injury was restored by reconstitution with wildtype serum. Wildtype injury was unaffected by neutrophil depletion. Injured intestines of C5-deficient and wildtype mice had similar neutrophil levels. Immunohistochemistry of wildtype and reconstituted C5-deficient mice demonstrated injured intestinal epithelium although C5-deficient mice and sCr1-treated mice were similar to sham mice. CONCLUSIONS: C5-deficient animals are protected from local injury. Injury is unaffected by neutrophil depletion, and the presence of neutrophils in injured tissue is independent of C5. Local injury is C5 dependent, but the action of C5a on granulocytes is not required. Therefore the membrane attack complex mediates local injury.

Animals↗
PubMed1987

A conceptual view of the complement system.

The activities of the complement system encompass cell destruction either directly through the membrane attack complex or indirectly via the effects of inflammation. The effects of complement fragments on immune functioning are just now being elucidated. Recent advances in methodology have allowed recognition of complement deficiencies of both quantitative and structural varieties. Complement activation studies may detect subclinical activation and be useful guideposts for therapeutic intervention. Coupling our new understanding of the mechanisms of complement activation with new technologies to measure this activation may result in the better understanding of the pathology of inflammation and its concurrent immunologic reactions.

Complement Activation↗
PubMed2006

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

Adult↗