Aggregation state-dependent activation of the classical complement pathway by the amyloid beta peptide

Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid beta peptide (A beta) is a potent activator of complement, and recent evidence suggesting that the assembly state of A beta is crucial to the progress of the disease prompted efforts to determine whether the ability of A beta to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of A beta, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3. These results suggest that the classical complement pathway provides a mechanism whereby complement-dependent processes may contribute to neuronal injury in the proximity of fibrillar but not diffuse A beta deposits in the AD brain.     

Inhibition of activation of the classical pathway of complement by human neutrophil defensins

Defensins are small, cationic antimicrobial peptides that are present in the azurophilic granules of neutrophils. Earlier studies have shown that defensins may influence complement activation by specific interaction with activated C1, C1q, and C1-inhibitor. In the present study, we show that the defensin human neutrophil peptide-1 (HNP-1) is able to inhibit activation of the classical complement pathway by inhibition of C1q hemolytic activity. The binding site for HNP-1 on C1q is most likely located on the collagen-like stalks, as a clear, dose-dependent binding of HNP-1 to either intact C1q or to the collagen-like stalks of C1q was demonstrated using enzyme-linked immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a limited binding to C1 and to a mixture of C1r and C1s was observed, whereas no binding to C1-inhibitor was found. Because binding of HNP-1 to C1-inhibitor has been suggested in earlier studies, we also assessed the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s or C1. No binding was found. Using a competition ELISA, it was found that HNP-1, but not protamine, inhibited binding of biotin-labeled HNP-1 to C1q in a dose-dependent fashion. In the fluid phase, preincubation of HNP-1 with C1q resulted in complex formation of HNP-1 and C1q and generation of stable complexes. In conclusion, HNP-1 is able to bind to C1q in the fluid phase and inhibits the classical complement pathway. This mechanism may be involved in the control of an inflammatory response in vivo.     

Binding of properdin to solid-phase immune complexes: critical role of the classical activation pathway of complement

OBJECTIVE: Our objectives were to assess the clinical and microbiological aspects of septic osteoarthritis in children admitted to our center from 1987 until July 1997 and to determine the sensitivity of ultrasound in this age group. PATIENTS AND METHODS: The medical records of 36 children diagnosed as having septic osteoarthritis of the hip were reviewed retrospectively. The diagnosis had been based on clinical criteria, along with synovectomy and drainage of purulent material from the affected joint. An X-ray and/or ultrasound had been performed when the diagnosis was suspected. RESULTS: Nineteen children were diagnosed during the neonatal period, 8 between the ages of 1 and 12 months and 9 older than one year of age. Mean age at diagnosis was 16.8 +/- 6.2 months (median 29 days, range 6 days to 13 years). The hip was involved in 32 children, the ankle in 3 and the elbow in 1. A microbiological diagnosis was achieved in 22 cases (61%) by culture from blood, CSF, and/or synovial fluid. The most common isolates were Gram positive cocci (S. aureus in 9 cases and coagulase negative Staphylococcus in 3). The diagnostic value of the X-rays was very low (18%). The ultrasound was initially considered abnormal in 64.5% of the patients, with a lower sensitivity in the neonatal period. After a mean follow-up period of 36 months, the outcome was good in 86% of the cases, although three children continue with sequelae. Two preterm infants died due to sepsis associated with the osteoarthritis. CONCLUSIONS: At the time of diagnosis of septic arthritis of the hip, the ultrasound is frequently normal. Due to the poor outcome when there is a delay in surgery, we suggest immediate synovectomy and drainage when there is clinical suspicion of septic arthritis despite an apparently normal ultrasound.     

Regulation of the B cell response to T-dependent antigens by classical pathway complement

We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.     

A three-dimensional animation of the classical pathway of the complement cascade

This paper describes the creation of a three-dimensional animation showing the classical pathway of the complement cascade, made in Studio Strata Pro Blitz (v1.75+) on the Macintosh computer. An analysis of two dimensional depictions of the process from the chapters of current textbooks of immunology revealed inconsistencies; animation was used to demonstrate how these difficulties can be overcome.     

Plasma factor triggering alternative complement pathway activation by liposomes

Several plasma components, such as complement (C) components, play a role in the clearance of liposomes from the circulation. The interactions between liposomes and the C system were investigated in this study. Multilamellar vesicle (MLV) liposomes, which were damaged by activation of the complement, became susceptible depending on the density of cetylmannoside (Man) on the liposome membrane, and activation proceeded through the alternative C pathway as observed for liposomes without Man (PC-MLV) (K. Funato et al., Biochim. Biophys. Acta 1103:198-204, 1992). In addition, the capacity of Man-modified liposomes (Man-MLV) to activate the alternative C pathway was abolished by preadsorption of plasma with Man-MLV but not with PC-MLV. The results suggest that a specific plasma factor adsorbed with Man-MLV was responsible for the augmentation of the C activation and, further, that the rapid clearance of Man-MLV from the circulation is caused by both enhanced C-mediated liposome permeability and enhanced C-mediated phagocytosis of liposomes.     

Comparison of three different methods for measuring classical pathway complement activity

A 30-year-old man was referred as an acute case for keratitis. Two days earlier he had had photorefractive keratectomy for myopia at another clinic; a bandage contact lens was placed over the eye, but prophylactic antibiotics were not prescribed. The keratitis was treated with gentamicin sulfate (Garamycin) and chloramphenicol eyedrops. Scrapings from the cornea showed nonhemolytic streptococci. Two and a half months later, visual acuity was finger counting because of gross distortion of the corneal contour. The combination of a bandage contact lens and the lack of prophylactic antibiotics may have been the source of bacterial keratitis.     

Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells

Human cytomegalovirus (HCMV)-infected cells persist in the presence of anti-HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus-encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host-encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV-1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up-regulation of CD55 suppressed the activity of cell-associated C3 convertases on HCMV-infected cells. Furthermore, increased CD55 expression protected infected cells from complement-mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host-encoded complement regulator proteins may provide protection of HCMV-infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement-mediated lysis and decreasing the deposition of C3-derived products on the cell surface.     

In vitro alternative pathway activation of complement by the brush border of proximal tubules of normal rat kidney

The purpose of this study was to investigate which structures of the nephron, if any, are capable of directly activating the complement (C) system. To this end, two sets of experiments were performed. First, activation of C was assessed on sections of frozen kidney tissue, using the indirect immunofluorescence technique for the demonstration of C fixation. Second, glomerular or tubular fractions of kidney were incubated with normal fresh serum, and subsequent C consumption was measured. The data obtained support the interpretation that the brush border of proximal tubules activates the alternative pathway of the C system. This phenomenon may have pathogenic significance in conditions of aselective proteinuria.     

Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity

OBJECTIVE: The purpose of this research was to empirically develop the Cognitive Behavioral Dieting Scale (CBDS), a measure of current dieting. METHOD: The first study involved item generation and a procedure to boost internal consistency while reducing scale length. Study 2 involved a factor analysis and measures of scale reliability. The third study evaluated the ability of the CBDS to predict calorie intake and negative calorie balance from a 24-hr diet recall. Study 4 evaluated construct validity by comparing the CBDS to dietary restraint, body image, and health behavior self-efficacy. RESULTS: The CBDS is a 14-item scale which measures current dieting behavior and related thoughts within the past 2 weeks. Internal consistency was alpha = .95 and 2-day test-retest reliability was r = .95. This scale provides a method for operationalizing dieting, provides a construct that is different from restraint, and assess dieting behavior on a continuum. Additionally, this scale was able to predict calorie intake and negative calorie balance above and beyond the predictive ability of physical variables (i.e., body mass index BMI] and exercise calories). An additional study of construct validity showed the CBDS was related to poor body image esteem and dietary restraint, but minimally related to healthy eating self-efficacy. DISCUSSION: In conclusion, the CBDS shows promise as a valid and reliable measure of dieting behavior. This scale should have utility in future research on how current dieting relates to eating disorders, dietary restraint, and obesity.     

Rapid activation of the complement system by cuprophane depends on complement component C4

Hemodialysis with cuprophane dialyzer membranes promotes rapid activation of the complement system, which is thought to be mediated by the alternative pathway. Complete hereditary deficiency of complement C4, a classical pathway component, in two hemodialysis patients provided the opportunity to investigate a possible role of the classical pathway. In two hemodialysis patients with both C4 isotypes, C4A and C4B, and in one patient with C4B deficiency complement activation occurred immediately after the onset of hemodialysis, with peak levels of C3a and terminal complement complex (TCC) after ten to fifteen minutes. In patients with complete C4 deficiency, C3a and TCC remained unchanged for fifteen minutes and increased thereafter, reaching the highest level after thirty minutes. The leukocyte nadir was also delayed from fifteen to thirty minutes. In vitro incubation of normal, C4A- or C4B-deficient serum with cuprophane caused complement activation after fifteen minutes. In contrast, no activation was observed in sera of four C4-deficient patients. The addition of normal serum or purified human C4 restored the capacity for rapid complement activation. In one patient with severe immunoglobulin deficiency, C3a and TCC levels increased only moderately after 25 minutes of cuprophane dialysis. This patient's serum also exhibited delayed complement activation in vitro, which was normalized after pretreatment of cuprophane with immunoglobulins. Preincubation of normal serum with MgEGTA, a blocker of the classical pathway, inhibited rapid complement activation through cuprophane. As basal levels of C4a are markedly increased in hemodialysis patients (3450 +/- 850 ng/ml) compared to healthy controls (224 +/- 81 ng/ml), no further elevation of C4a was detectable during cuprophane hemodialysis. Incubation of normal serum with cuprophane, however, caused a slight increase in C4a after five minutes. These results indicate that the initial deposition of complement C3b on the cuprophane membrane, necessary for activation of the amplification loop of the alternative pathway, is mediated by the classical pathway C3-convertase C4b2a. We propose an extended concept of complement activation through cuprophane, which is based on four steps: (a) binding of anti-polysaccharide antibodies, (b) classical pathway activation, (c) alternative pathway activation and (d) terminal pathway activation.     

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.     

Activation of homologous complement via the alternative complement pathway by quail cells transformed by Rous sarcoma virus

After incubation of the cells with fresh quail serum, deposition of the third component of complement (C3) was demonstrated on the cell surface of various quail cell lines transformed by Rous sarcoma virus (RSV) as well as on that of primary quail embryo (QE) cells transformed by RSV. The C3 deposition occurred irrespective of virus production. On the other hand, the C-3 deposition was not observed on two quail cell lines transformed by a chemical carcinogen, QE cells infected with avian leukosis virus or normal QE cells. Moreover, QE cells infected with a temperature-sensitive mutant of RSV activated the complement at 37 degrees C but not at 41 degrees C. Since the progeny virus was generated even at 41 degrees C, viral molecules on the cell surface may not play an essential role for the activation. The activation of complement was blocked by EDTA but not by EGTA-Mg++. Therefore, the complement activation on the transformed cells appears to be mediated via the alternative complement pathway (ACP). Similar results were obtained with the complement consumption test; the residual cytolytic activity of fresh quail serum via ACP was markedly reduced by pre-incubation of the serum with transformed cells.     

Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury

Myocardial injury after ischemia (I) and reperfusion (R) is related to leukocyte activation with subsequent release of cytokines and oxygen-derived free radicals as well as complement activation. In our study, the cardioprotective effects of exogenous C1 esterase inhibitor (C1 INH) were examined in a rat model of myocardial I + R (i.e., 20 min + 24 hr or 48 hr). The C1 INH (10, 50 and 100 U/kg) administered 2 min before reperfusion significantly attenuated myocardial injury after 24 hr of R compared to vehicle treated rats (P < .001). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN [polymorphonuclear leukocyte] accumulation) in the ischemic area was significantly reduced after C1 INH treatment compared to vehicle treated animals (0.81 +/- 0.1, 0.34 +/- 0.13, 0.13 +/- 0.1 vs. 1.44 +/- 0.3 U/100 mg tissue, P < .001). In addition, C1 INH (100 U/kg) significantly attenuated myocardial injury and neutrophil infiltration even after 48 hr of reperfusion compared to vehicle treatment. Immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated activation of classical complement pathway by deposition of C1q on cardiac myocytes and cardiac vessels. In addition, expression of the endothelial adhesion molecules P-selectin and intercellular adhesion molecule 1 (ICAM-1) was observed after reperfusion of the ischemic myocardium. In this regard, C1 INH administration abolished expression of P-selectin and ICAM-1 on the cardiac vasculature after myocardial ischemia and reperfusion. Blocking the classical complement pathway by exogenous C1 INH appears to be an effective means to preserve ischemic myocardium from injury after 24 and 48 hr of reperfusion. The mechanisms of this cardioprotective effect appears to be due to blocking of complement activation and reduced endothelial adhesion molecule expression with subsequent reduced PMN-endothelium interaction, resulting in diminished cardiac necrosis.     

Bivalency is required for anticapsular monoclonal antibodies to optimally suppress activation of the alternative complement pathway by the Cryptococcus neoformans capsule

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.     

Zymosan-induced arthritis: a model of chronic proliferative arthritis following activation of the alternative pathway of complement

Zymosan particles, which are able to activate complement by the alternative pathway and to induce enzyme secretion from macrophages, were injected into knee joints of mice. After various time intervals, synovia were assessed histologically for various markers of inflammation. Within 7 days after intraarticular injection of zymosan, a chronic inflammatory arthritis with mononuclear cell infiltration, synovial hypertrophy and pannus formation was observed. Latex particles, which do not activate complement or macrophages, produced only mild, transient inflammatory reactions.     

Evidence for the presence of components of the alternative (properdin) pathway of complement activation in respiratory secretions

Activation of the alternative complement pathway by respiratory secretory IgA was demonstrated by incubating purified, aggregated preparations of serum and secretory IgA with neat human serum. No depletion of the early components (C1-4) was observed, but 63 and 70% of C3-9, respectively, were consumed. The C3-9-consuming capacity of heat-aggregated nasal secretions from an IgA-deficient volunteer was compared with heat-aggregated nasal secretions from a normal volunteer known to have secretory IgA. The deficient secretions consumed C3-9, whereas the IgA deficient secretions did not. Reconstitution of the nasal secretions from the IgA-deficient volunteer with purified secretory IgA produced alternative pathway activation. Factor B of the alternative complement pathway was found to be present in 16 of 18 bronchoalveolar lavage samples (BALF) from normal volunteers. Simultaneous measurement of lavage and serum albumin and Factor B concentrations rendered it unlikely that Factor B was merely a transudative product from serum in half the samples but rather suggested that it may be a component of lower respiratory tract secretions. The presence of an intact alternative complement pathway in BALF was indicated by showing that cobra venom factor and endotoxin cleaved functionally pure human C3 when mixed with BALF, but had no effect on C3 in the absence of BALF.     

Complement classical pathway expression by human skeletal myoblasts in vitro

Human myoblasts express immunological properties in vitro and we have previously reported that they produce Complement (C) components of the alternative pathway. Myoblasts activate the classical pathway but are fully protected against C attack by the expression of major C regulators. In order to fully understand the relationship between myoblasts and C, we here report the biosynthesis of C components of the classical pathway by skeletal muscle cells. Human myoblasts in vitro produced C1q, C1r, C1s, C2 and C4 constitutively and all syntheses were upregulated after stimulation with IFN-gamma. We suggest that human myoblasts may constitute a local source of C and therefore C could be implicated in inflammatory or physiopathological processes developed in skeletal muscle.