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PubMed2006

The inflammatory process of gout and its treatment.

Gouty arthritis is a characteristically intense acute inflammatory reaction that erupts in response to articular deposits of monosodium urate (MSU) crystals. Important recent molecular biologic advances in this field have given us a clear picture of the mechanistic basis of gouty inflammation. The innate immune inflammatory response is critically involved in the pathology of gout. Specifically, MSU crystals promote inflammation directly by stimulating cells via Toll-like receptor signaling and by providing a surface for cleavage of C5 and formation of complement membrane attack complex (C5b-9), culminating in secretion of cytokines, chemokines, and other inflammatory mediators with a dramatic influx of neutrophils into the joint. Despite the detailed mechanistic picture for gouty inflammation, there are no placebo-controlled, randomized clinical studies for any of the therapies commonly used, although comparative studies have demonstrated that many nonsteroidal anti-inflammatory drugs are equivalent to indomethacin with respect to controlling acute gouty attacks. In general, the first line of anti-inflammatory therapy for acute gout is nonsteroidal anti-inflammatory drugs, and the selective cyclo-oxygenase-2 inhibitor celecoxib can be used where appropriate. The second line of treatment is glucocorticosteroids, given systemically (oral, intravenous, or intramuscular) or intra-articularly. Alternatively, synthetic adrenocorticotropic hormone is effective, partly via induction of adrenal glucocorticosteroids and partly via rapid peripheral suppression of leukocyte activation by melatonin receptor 3 signaling. The third line of treatment is oral colchicine, which is highly effective when given early in an acute gouty attack, but it is poorly tolerated because of predictable gastrointestinal side effects.

Adrenal Cortex Hormones↗
PubMed2004

Heparin-level-based anticoagulation management during cardiopulmonary bypass: a pilot investigation on the effects of a half-dose aprotinin protocol on postoperative blood loss and hemostatic activation and inflammatory response.

UNLABELLED: Cardiac surgery involving cardiopulmonary bypass (CPB) leads to activation of the hemostatic/inflammatory system. We compared the influence of a half-dose aprotinin regimen on postoperative blood loss and the activation of the hemostatic/inflammatory system during CPB, when used during a heparin-level-based heparin management for cardiac surgery. Two-hundred patients (n = 100 in each group) were enrolled in this randomized prospective study. In Group I only heparin was given according to the results of the Hepcon HMS Plus. In Group II aprotinin was added with a bolus of 1 x 10(6) kallikrein inhibiting units (KIU) for the patient immediately before initiation of CPB, 1 x 10(6) KIU in the priming solution of the CPB, and a continuous infusion of 250,000 KIU/h during CPB. Postoperative blood loss was determined after 12 h. Heparin and antithrombin activity were evaluated by an anti-Xa assay and measurement of antithrombin III activity. Hemostatic activation was evaluated by adenosine diphosphate-stimulated platelet aggregometry and by measurements of the generation/release of beta-thromboglobulin (beta-TG), soluble P-selectin (sPS), thrombin (TAT), prothrombin 1 and 2 fragments (PTF1+2), factor XIIa (FXIIa), plasmin (PAP), and D-dimers. Inflammatory response was evaluated by measuring complement factors 5b-9 (C5b-9), interleukin (IL)-6, and neutrophil elastase (NE). There were no differences in the pre-CPB values or duration of CPB between the two groups. There were no differences in the post-CPB values for platelet count, platelet aggregation, beta-TG, sPS, TAT, PTF1+2, C5b-9, NE, or IL-6. The additional use of aprotinin resulted in a significant decrease of PAP, D-dimers, and 12 h postoperative blood loss, whereas generation of the contact factor XIIa was increased. The administration of aprotinin significantly reduced postoperative blood loss after cardiac surgery and CPB. This most likely has to be attributed to the antifibrinolytic effects of aprotinin. No effects on thrombin generation, platelet activation, inflammatory response, or clinical outcome were noted. IMPLICATIONS: The use of half-dose aprotinin and heparin-level-based anticoagulation management during cardiopulmonary bypass leads to a significant reduction of postoperative blood loss after cardiac surgery. This effect can most likely be attributed to the antifibrinolytic effects of aprotinin, as we did not observe effects on other variables of activation of the hemostatic/inflammatory system.

Adult↗
PubMed2000

A small N-terminal 60-kD fragment of gp600 (megalin), the major autoantigen of active Heymann nephritis, can induce a full-blown disease.

Active Heymann nephritis of rat, an autoimmune glomerular disease, is an immunohistological, ultrastructural, and clinical model of human membranous glomerulonephritis. Both diseases in their full-blown form are characterized by (1) the formation of large, subepithelial glomerular immune deposits, which stain for IgG, C3, and membrane attack (C5b-9) components of complement and (2) the excretion of large amounts of protein in the urine (proteinuria). The target autoantigen of active Heymann nephritis is a large transmembrane renal glycoprotein with a molecular weight of approximately 600 kD, variously named gp600, gp330, LRP-2, or "megalin." This study was performed to identify the region in this enormously large glycoprotein that would produce full-blown active Heymann nephritis. A stable, small (60-kD) proteolytic fragment of gp600 was isolated and localized to the N-terminal end of the molecule using Western blot, sequencing, and amino acid analyses. Based on its primary structure, this fragment contains approximately 60 cysteine residues, the cross-linking of which to each other probably explains its stability. Immunization of rats with this fragment induced a full-blown disease that was comparable to the disease induced by a preparation containing the whole protein. These results indicate that this small fragment, retaining the natural disulfide bonds and probably its overall structure, contains those B and T cell epitopes that are sufficient to produce this organ-specific autoimmune disease.

Animals↗
PubMed2000

Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease.

Age-related macular degeneration (AMD), a blinding disorder that compromises central vision, is characterized by the accumulation of extracellular deposits, termed drusen, between the retinal pigmented epithelium and the choroid. Recent studies in this laboratory revealed that vitronectin is a major component of drusen. Because vitronectin is also a constituent of abnormal deposits associated with a variety of diseases, drusen from human donor eyes were examined for compositional similarities with other extracellular disease deposits. Thirty-four antibodies to 29 different proteins or protein complexes were tested for immunoreactivity with hard and soft drusen phenotypes. These analyses provide a partial profile of the molecular composition of drusen. Serum amyloid P component, apolipoprotein E, immunoglobulin light chains, Factor X, and complement proteins (C5 and C5b-9 complex) were identified in all drusen phenotypes. Transcripts encoding some of these molecules were also found to be synthesized by the retina, retinal pigmented epithelium, and/or choroid. The compositional similarity between drusen and other disease deposits may be significant in view of the recently established correlation between AMD and atherosclerosis. This study suggests that similar pathways may be involved in the etiologies of AMD and other age-related diseases.

Aging↗
PubMedDate not supplied

[Classic/amyopathic dermatomyositis].

Dermatomyositis (DM) is an inflammatory myopathy characterized by a typical cutaneous rash and proximal weakness. DM results from endothelium deposition of the complement membranolytic attack complex C5b-9, followed by inflammation. It is thus possible to have cases of DM with the typical rash, but with no associated myositis--amiopathic DM. DM represents a higher risk of association with malignancy and sometimes behaves as a paraneoplastic syndrome. As DM can be the alarm sign, once diagnosed, exclusion of malignancy should be done. In this article we also discuss the treatment and prognosis of dermatomyositis, according to the literature.

Adolescent↗
PubMed1985

Immunofluorescence studies of cardiac valves in infective endocarditis.

Mitral and aortic valves removed at emergency cardiac surgery from a patient with infective endocarditis caused by Streptococcus viridans were studied by immunofluorescence to ascertain the extent and pattern of various immune reactants within the large valvular vegetations. Heavy intravalvular deposits of IgG as well as bacterial antigen were present. Much more focal interstitial IgM and C3 deposits were noted within vegetations and valve substance. Diffuse endocardial and subendocardial deposition of C5b-C9 and C9 complement neoantigens was present. Direct staining of valvular tissues and vegetations for rheumatoid factor showed extensive interstitial tissue deposition. These findings emphasize the large amounts of immune reactants and constituents of immune complexes present in valves and vegetations of patients with infective endocarditis.

Adult↗
PubMed2006

JNK1 activation mediates C5b-9-induced P0 mRNA instability and P0 gene expression in Schwann cells.

The protein zero (P0) glycoprotein is an important component of compact peripheral nerve myelin produced by the glial cells of the mammalian peripheral nervous system. P0 mRNA expression is reduced following exposure of Schwann cells to sublytic C5b-9, the terminal activation complex of the complement cascade. Sublytic complement treatment decreased P0 mRNA by 81% within 6 h and required C5b-9 assembly. C5b-9 induced a threefold increase in both JNK1 activity and c-jun mRNA within 20 and 30 min, respectively, compared with cells treated with either human serum depleted of complement component C7 (C7dHS) or medium alone. Sublytic C5b-9 stimulation, in the presence of the transcription inhibitor Actinomycin D, decreased P0 mRNA expression by 52%, indicating that mRNA was selectively destabilized. This effect was prevented by pretreatment with L-JNK inhibitor 1 (L-JNKI1). To study a potential inhibition of P0 gene transcription, we transfected Schwann cells with a P0 promoter-firefly luciferase construct. Sublytic C5b-9 stimulation of the transfected cells decreased luciferase activity by 82% at 6 h, and this effect was prevented by pretreatment with L-JNKI1 inhibitor. Our results indicate that the ability of C5b-9 in vitro to affect P0 gene expression is mediated via JNK1 activation that leads to enhanced mRNA decay and transcriptional repression of P0.

Animals↗
PubMed1988

Terminal complement complex (TCC) and S-protein (vitronectin) on follicular dendritic cells in human lymphoid tissues.

Follicular dendritic cells (FDC) present in germinal centres trap considerable amounts of C3-containing immune complexes (IC). Activation of the terminal pathway of complement (C) on biological membranes normally generates C5b-9(m), which is the membranolytic form of the terminal complement complex (TCC). By contrast, when C activation takes place in the extracellular fluid phase, S-protein binds to C5b-7 and the non-lytic soluble SC5b-9 is formed. In this study deposits of C3d, C5, C9 TCC neoepitope and S-protein were demonstrated by immunohistochemistry on FDC in human tonsils, lymph nodes, spleens, appendices and colonic mucosae. TCC and S-protein were likewise observed on FDC in imprints from tonsils. The identical spatial distribution revealed by paired staining suggested that TCC had been generated in situ. The staining intensity for TCC and S-protein varied in parallel, which suggested that the S-protein may be incorporated in the membrane-bound TCC--perhaps rendering it non-lytic. However, the actual mechanism and function of the observed TCC and associated S-protein deposition needs further elucidation.

Complement Membrane Attack Complex↗
PubMed1991

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm.

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.

Autoantibodies↗
PubMed1997

The severity of clinical symptoms in ragweed-allergic patients is related to the extent of ragweed-induced complement activation in their sera.

We have previously reported a correlation between the extent of ragweed allergen (RWA)-induced in vitro serum complement activation and the symptom scores registered daily during the ragweed (RW)-blooming season in RW-allergic patients. The present study was performed in 22 15-17-year-old RW-allergic adolescents. Serum samples were incubated with 100 micrograms/ml RWA, and the generation of different complement activation products was measured by ELISA or RIA. Symptom scores were registered for 4 weeks during the RW-blooming season. The patients were divided according to the extent (low or high) of the generation of complement activation products, and symptom scores registered in the two groups were compared by two-way ANOVA. Significantly higher symptom scores were obtained in the high than in the low complement activation group (P values: 0.049 for C1rC1sC1inh, 0.022 for C3bBbP, 0.015 for C5b-9, 0.0001 for C3a, and 0.0008 for C5a). Similar results were obtained at the measurement performed in the sera obtained from the same patients half a year before the season (P values: 0.022 for C3bBbP, and 0.005 for C5b-9). These findings indicate that complement activation induced by the allergen may enhance the clinical symptoms of RW allergy.

Adolescent↗
PubMed2002

Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.

Decay-accelerating factor (DAF) and CD59 are 2 glycosylphosphatidylinositol-anchored membrane proteins that inhibit complement activation at the C3 and C5b-9 step, respectively. CD59 is considered critical for protecting erythrocytes from spontaneous complement attack, as deficiency of CD59 or CD59/DAF, but not of DAF alone, on human erythrocytes renders them sensitive to complement lysis in paroxysmal nocturnal hemoglobinuria syndrome. To evaluate the relative roles of CD59 and DAF in vivo, we have generated and studied a CD59 knockout and a CD59/DAF double-knockout mouse. CD59-deficient and CD59/DAF-double-deficient mouse erythrocytes were highly sensitive to antibody-induced complement lysis in vitro, yet neither CD59 knockout nor CD59/DAF double-knockout mouse developed spontaneous hemolytic anemia. Consistent with the latter observation, erythrocytes from the 2 strains of mutant mice were shown to have a normal lifespan in vivo. In contrast, mouse erythrocytes deficient in complement receptor 1 (CR1)-related gene y (Crry), a membrane C3 inhibitor with DAF and membrane cofactor protein activities, were rapidly eliminated from the circulation by a complement-dependent mechanism. Compared with DAF-deficient erythrocytes, Crry-deficient erythrocytes incurred higher levels of spontaneous C3 deposition in vivo. These findings demonstrate that CD59 and DAF are not indispensable on murine erythrocytes. Rather, effective C3 regulation on the cell surface, provided by Crry rather than DAF, is necessary for mouse erythrocytes to resist spontaneous complement attack. Our results raise the possibility that proper control of C3 activation may also be critical on human erythrocytes, where CR1 but not DAF could be the principal regulator of spontaneous C3 activation.

Animals↗
PubMed2006

Classical pathway complement activation on human endothelial cells.

Endothelial cells regulate vascular integrity and express complement binding proteins including gC1qR/p33 (gC1qR), which recognize C1q, a subunit of the first component of the classical complement pathway. Experiments were performed to investigate classical complement pathway activation on resting endothelial cells and endothelial cells exposed to shear stress. C1q deposition and C4 activation (C4d) were demonstrated by solid phase ELISA and flow cytometry on human microvascular and umbilical vein endothelial cells after exposure to serum or plasma. C4d deposition was accompanied by downstream complement activation including C3b and C5b-9 deposition. C4 activation failed to occur in C1q depleted serum, but was not affected by Factor B depleted serum, confirming classical complement pathway activation. Moreover, C4 activation occurred following exposure of endothelial cells to purified C1 and C4, in the absence of other plasma proteins, and in the absence of detectable cell surface IgG and IgM. Shear stress (18 dynes/cm2) increased C1q (n=9, p<0.05) and C4d (n=9, p<0.05) deposition approximately two-fold, and enhanced endothelial cell gC1qR expression (n=7, p<0.05). Treatment of endothelial cells with anti gC1qR monoclonal antibody F(ab')2 fragments reduced C4d deposition by approximately 20% (n=5, p<0.05). These data demonstrate direct classical complement pathway activation on endothelial cells. gC1qR appears to play a minor but definable role, whereas cell surface IgG or IgM are not required.

Animals↗
PubMed1998

Distribution of A beta-associated proteins in cerebrovascular amyloid of Alzheimer's disease.

Senile plaques and cerebrovascular amyloidosis (CA) are two of the major neuropathological lesions in brains of patients with dementia of the Alzheimer type. We studied the expression of a number of amyloid beta (A beta)-associated proteins in CA, which have previously been identified in senile plaques and which were suggested to play an important role in the pathogenesis of these lesions. Our findings show that involvement of inflammatory components in CA is restricted to activation of the complement system, resulting in deposition of the complement factors C1q, C3c, C4d and the membrane attack complex C5b-9 as well as of the complement inhibitor clusterin. Furthermore, we observed expression of apolipoprotein E, amyloid P component and heparan sulfate proteoglycans in CA, whereas expression of lactoferrin was almost absent. Other inflammatory proteins, known to be present in senile plaques, such as alpha1-antichymotrypsin, alpha2-macroglobulin and intercellular adhesion molecule-1, were absent or detectable only in small amounts. These data suggest that an incomplete inflammatory response occurs in CA as compared to senile plaques. This was confirmed by the finding that the number of cells of the monocyte/macrophage lineage around CA was not increased compared to unaffected vessels. Based on their expression patterns, complement factors, apolipoprotein E and heparan sulfate proteoglycans may be produced early in the process of CA formation and may play an important role in the formation of A beta fibrils in CA. The absence of a number of A beta-associated proteins in CA in comparison to senile plaques is in support of a different pathogenesis for these two lesions.

Aged↗
PubMed1995

Complement-mediated neurotoxicity is regulated by homologous restriction.

The ability of beta-amyloid peptides to activate the classical complement cascade and the presence of various complement proteins including the membrane attack complex (C5b-9) on dystrophic neurites in Alzheimer's disease brains, raises the possibility that the complement system may contribute to this neurodegenerative disorder. To address this issue, we have studied the effect of complement activation on nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells, and on retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells. Although incubation of both cell types with human serum resulted in activation of complement, as indicated by iC3b formation, only PC12 but not SH-SY5Y cells were killed by human serum treatment. In contrast, heat-inactivated serum (56 degrees C, 45 min) was not neurotoxic. On SH-SY5Y cells, both PCR amplification and immunocytochemistry demonstrated the presence of CD59, a glycosylphosphatidylinositol-anchored protein that restricts homologous complement activation by inhibiting the formation of the membrane attack complex. The presence of CD59 probably accounts for the inability of human complement to lyse the human cell lines. Indeed, removal of glycosylphosphatidylinositol (GPI)-anchored proteins with phosphatidylinositol-specific phospholipase C (PI-PLC) rendered SH-SY5Y cells vulnerable to complement attack and eventually led to serum-medicated cell death. Reconstituted C5b-9 was also toxic to both PC12 and PI-PLC-pretreated SH-SY5Y cells. These observations suggest that complement activation can cause neuronal cell death and that this process is regulated by homologous restriction.

Animals↗
PubMed2005

Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis.

The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.

Complement C8↗
PubMed2005

Stress proteins in glomerular epithelial cell injury.

Glomerular visceral epithelial cells (GEC) or podocytes are highly differentiated, specialized cells that play a key role in the maintenance of glomerular permselectivity. Injury of GEC, leading to proteinuria, contributes to the pathogenesis of human and experimental glomerulopathies. Recent studies have demonstrated that stress proteins may be induced and may be involved in the modulation of GEC injury. The C5b-9 membrane attack complex of complement induces GEC injury and proteinuria in the passive Heymann nephritis (PHN) model of membranous nephropathy. C5b-9 induces upregulation of the endoplasmic reticulum (ER) stress proteins, bip and grp94, in part, via activation of cytosolic phospholipase A2. These ER stress proteins limit complement-mediated GEC injury. In experimental nephropathy associated with hyperlipidemia, and in experimental diabetic nephropathy, there is an upregulation of the ER heat shock protein (Hsp) 47, a chaperone protein involved in the synthesis or assembly of collagens. Hsp47 expression in GEC is associated with increased deposition of collagen, and glomerulosclerosis. Hsp27, a stress protein involved in actin polymerization, is localized in GEC, and its expression and activation are increased in the rat puromycin aminonucleoside model of focal segmental glomerulosclerosis, and in PHN. Hsp27 may preserve actin structure, and facilitates survival in the context of injury. Development of methods to induce expression/activation of stress proteins may eventually lead to novel approaches to the therapy of GEC injury and proteinuria.

Animals↗
PubMed1989

Urinary excretion of C5b-9 reflects disease activity in passive Heymann nephritis.

Passive Heymann nephritis (PHN) is a model of membranous nephropathy in rats in which glomerular injury is mediated by the terminal C5b-9 membrane attack complex of complement. This model has been shown to be associated with markedly elevated urinary excretion of C5b-9, compared to other experimental models of glomerulonephritis To determine if urinary C5b-9 excretion could serve as an index of disease activity by correlating with the formation and quantity of glomerular subepithelial immune deposits in PHN, we measured urinary excretion of C5b-9 in PHN under several experimental conditions. In the heterologous phase a direct correlation was demonstrated between levels of urinary C5b-9 excretion and the amount of anti-Fx1A IgG deposited in glomeruli (r = 0.85). In the autologous phase, C5b-9 excretion correlated with the amount of deposit forming antibody present in the serum and resolved when antibody disappeared, despite persistence of glomerular deposits of antigen, antibody, C5b-9 and heavy proteinuria. Glomerular C3 deposits paralleled urinary C5b-9 excretion. Re-initiation of active deposit formation by a second injection of anti-Fx1A produced new C3 deposits and a marked rise in C5b-9 excretion. Finally, complete abrogation of deposit formation by transplanting PHN kidneys into normal recipients also halted C5b-9 excretion. Our findings demonstrate that urinary excretion of C5b-9 is a sensitive index of on-going immunologic disease activity in the PHN model of membranous nephropathy.

Animals↗
PubMed1983

Studies of the association of the eighth and ninth components of human complement within the membrane-bound cytolytic complex.

The association of the eighth (C8) and ninth (C9) components of human complement within membrane-bound C5b-9 was investigated using the photosensitive cross-linking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Reaction of this reagent with either the purified alpha-gamma or beta subunit of C8 resulted in the introduction of 6-8 mol/mol of photosensitive 6-(4'-azido-2'-nitrophenylamino)hexanoate (ANH) as an intrinsic ligand on each protein. The resulting ANH-(alpha-gamma) or ANH-(beta) was capable of recombining with equimolar amounts of beta or alpha-gamma, respectively, to yield ANH-C8. Parallel modifications of purified C9 resulted in incorporation of 3-4 mol/mol of ANH-ligand. Both ANH-C8 and ANH-C9 retained their ability to incorporate into C5b-9. Two approaches were used to determine the proximity of C8 subunits to C9 within C5b-9. In one, the complex was assembled on erythrocytes by incubating EAC1-7 cells separately with each form of ANH-C8 and subsequently saturating with 125I-C9. After lysis, membranes were irradiated, solubilized, and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of 125I-C9 to a higher molecular weight. Results using either form of ANH-C8 in C5b-9 showed that, although at least 30% was involved in cross-linking, none was cross-linked to C9. Similar results were obtained using a second approach in which cross-linker and radiolabel were transposed between C8 and C9. Here, EAC1-7 cells were incubated first with 125I-C8 containing either 125I-(alpha-gamma) or 125I-(beta) and subsequently with ANH-C9. Although at least 48% of ANH-C9 in C5b-9 was involved in cross-linking in these experiments, no cross-linking to either subunit of C8 was detected. These results suggest that C8 is not in close physical association with C9 within membrane-bound C5b-9.

Azides↗