Molecular immunobiology of complement biosynthesis: a model of single-cell control of effector-inhibitor balance.
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Animals↗
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Patterns of circulating complement in renal diseases.
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Complement C1 Inactivator Proteins↗
Deterioration of immune complex solubilization activity of serum by increased concentration of factor D.
We investigated the effect of excess complement factor D on the immune complex solubilization activity (ICSA) of serum. First, we estimated the concentration of factor D, ICSA and the hemolytic activity via the classical complement pathway (CH50) in the sera of 16 healthy individuals and 36 patients on hemodialysis for end-stage renal failure. The serum concentration of factor D in these patients (mean +/- SD: 12.12 +/- 2.38 micrograms/ml) was significantly higher (p less than 0.001) than that in the healthy subjects (1.02 +/- 0.11 micrograms/ml). In this study, peroxidase and antiperoxidase rabbit IgG were used as immune precipitates for ICSA. The ICSA in patients (45.8 +/- 7.4 normal human serum %, NHS%) was significantly lower (p less than 0.001) than that in the healthy subjects (100.2 +/- 12.5 NHS%). There was no difference in CH50 between the sera of the patients (31.8 +/- 2.5) and that of the healthy group (32.4 +/- 2.6). Second, we determined that increasing amounts of purified factor D added to fresh serum resulted in a decrease in ICSA. This occurred in the serum of a healthy individual as well as in that of a patient. CH50 did not change regardless of the concentration of factor D used. There was an increase in C3 conversion in the sera to which purified factor D had been added, as observed by crossed immunoelectrophoresis. It is suggested that ICSA had deteriorated due to the excess of factor D, which had activated the alternative pathway of complement.
Antigen-Antibody Complex↗
Binding and activation of human precursor C1 by soluble aggregates of human and rabbit IgG.
The capacities of soluble human and rabbit IgG aggregates to bind and to activate human C1 were compared. Aggregates prepared by incubation of purified IgG at 63 degrees C were fractionated by gel filtration and hemolytic assays were used to measure the binding and activation of isolated human precursor C1. The C1 binding and activation capacities of both human and rabbit IgG aggregates were highly dependent on their size. Human IgG aggregates had a slightly higher binding avidity for human C1 than rabbit IgG aggregates of comparable size, but no clear differences were found between their capacities to activate C1. Experiments with nonaggregated IgG also indicated that although human IgG binds human C1 somewhat more avidly, human and rabbit IgG do not differ in their capacities to initiate fluid-phase activation of the human classical complement pathway.
Animals↗
Immunological studies in patients with alcoholic liver disease: evidence for the in vivo activation of helper T cells and of the monocyte-macrophage system.
Immunologic parameters were evaluated in 22 patients with alcoholic liver disease (ALD). Patients with ALD had increased levels of circulating immune complexes (CIC) and serum immunoglobulins, particularly IgA. The classical complement pathway was preferentially activated in CIC-positive patients. There was no increase in total lymphocyte or total T lymphocyte counts, but a significant rise in both the helper/inducer population (OKT4) and helper/suppressor cell ratio (T4/T8) was noted. The presence of interleukin-2 receptors and HLA-DC/DS and HLA-DR antigens suggested in vivo activation of ALD patients' T cells. The rate of differentiation of B cells to plasma cells was high in both pokeweed mitogen-stimulated and unstimulated cultures of ALD patients' B cells, whereas plasma cell generation doubled when patient T lymphocytes were added to enriched normal B cells. The above data support a role for activated helper (T4+) T cells in the immune response initiated by alcoholic hepatitis. Serum angiotensin-converting enzyme levels and lysozyme levels were increased in ALD patients, and cultured adherent mononuclear cells from ALD patients secreted more lysozyme in vitro than normal cells, suggesting the presence of an activated monocyte-macrophage system in ALD.
Adult↗
Carbohydrate-specific human heterophile antibodies in normal human sera that react with xenogeneic cells.
Human heterophile antibodies (HHA) that are present in normal human sera (NHS) play an important role in hyperacute xenograft rejection. The aim of this study was to analyze the occurrence, mode of action and molecular specificity of HHA in NHS that are directed against xenogeneic lymphocytes (isolated from mouse, rat, guinea pig, rabbit, cattle and pig) and isolated rat pancreatic islets. All sera contained variable amounts of HHA that killed the target cells via the classical complement pathway. The cytotoxic activity of these HHA was specifically inhibited by certain carbohydrates (alpha-D-melibiose, beta-lactose, beta-gentiobiose, beta-cellobiose, D-mannose, N-acetyl-beta-D-mannosamine and alpha-D-rhamnose) and by rat IgM. By means of affinity chromatography with immobilized inhibitors we obtained an antibody preparation of mainly IgG type from NHS (up to 3.5 mg/10 ml serum) that reacted strongly with rat lymphocytes and isolated rat pancreatic islets. Though thus far residual xenospecific antibody activity has remained in the sera even after multiple affinity chromatography, these data suggest that specific elimination of HHA is feasible and that it may be thus possible to overcome a major obstacle to xenotransplantation.
Antibodies, Heterophile↗
The role of complement in opsonization of GBS.
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Adult↗
A sensitive specific hemolytic assay for proenzyme C1.
The traditional hemolytic assay of the functional activity of C1, the first component of the classical complement pathway, was modified to permit differentiation between proenzyme (unactivated) C1 and the activated state of the enzyme (C1). A two-step assay was developed to quantitate proenzyme C1. The C1 sample to be assayed was first preincubated with C1 inhibitor, a process that specifically inhibits the enzymatic activity of C1 without affecting the subsequent activation of proenzyme C1 by EAC4, a model immune complex. Since the rate of reaction between C1 inhibitor, a serum regulatory protein, and C1 is concentration-dependent, this step is performed at high C1 and C1 inhibitor concentrations. Subsequent dilutions of the sample prevents C1 inhibitor-mediated inactivation of the C1 that is activated during the C1 hemolytic assay. Thus, in the presence of C1 inhibitor, the level of C1 hemolytic activity specifically reflects the activity of proenzyme C1, while in the absence of C1 inhibitor, the hemolytic activity reflects the total activity of C1. Both the absolute and the relative amounts of the proenzyme (unactivated) and activated C1 can thereby be quantitated in most samples. Furthermore, a partially purified C1 inhibitor reagent, easily prepared from serum, was shown to function identically to the purified C1 inhibitor, obviating the need for a multistep isolation procedure for this protein. Using this simple yet sensitive assay to investigate the efficiency of reconstitution of C1 activity from the purified components C1q, C1r, and C1s, we also find evidence for temperature- and concentration-dependent reaction steps in the formation of functional C1.
Animals↗
Isolation and characterization of human antioxidized LDL autoantibodies.
Autoantibodies to oxidized LDL have been reported in normal subjects and in patients with arteriosclerosis, but their possible pathogenic role is not yet well defined. One important problem is the existence of contradictory data reported by different groups concerning the associations between antioxidized LDL autoantibodies and the presence or progression of arteriosclerotic lesions. Such contradictions led us to decide to isolate and characterize antioxidized LDL antibodies by affinity chromatography with the use of oxidized LDL cross-linked to Sepharose. Antioxidized LDL antibodies were isolated from selected serum samples obtained from eight subjects. Seven of them (six patients and one control subject) had high levels of antioxidized LDL antibody during screening. The other subject, a healthy volunteer, had a low level of antibody. All purified antibodies contained IgG (of subclasses 1 and 3) as the predominant isotype and were primarily specific for oxidized LDL but showed some cross-reactivity with malondialdehyde-modified LDL and native LDL. Two of the purified antibodies cross-reacted with cardiolipin. We determined average dissociation constants for the antioxidized LDL antibodies purified from five individuals, which varied between 2.4 x 10(-7) and 7.5 x 10(-7) mol/L, whereas the average dissociation constant of rabbit hyperimmune anti-LDL antibody was determined to be 2.7 x 10(-8) mol/L. In conclusion, we have purified human autoantibodies reactive with oxidized LDL that appear to be predominantly of moderate-to-low affinity and of variable cross-reactivity. The predominance of IgG1 and IgG3 antibodies is significant from the standpoint of potential pathogenicity, since these two subclasses activate the classic complement pathway system and have the highest binding affinities for Fc gamma receptors on phagocytic cells.
Adult↗
Activation of complement and kinin systems after thrombolytic therapy in patients with acute myocardial infarction. A comparison between streptokinase and recombinant tissue-type plasminogen activator.
BACKGROUND: We have previously shown that treatment with streptokinase induces abrupt complement activation and transient neutropenia in patients with acute myocardial infarction (AMI). The purpose of this study was to compare the effects of two different thrombolytic agents--streptokinase (SK) and recombinant tissue-type plasminogen activator (rTPA)--on activation of the complement and kinin systems in plasma of patients with AMI. METHODS AND RESULTS: Forty-one patients with AMI who were eligible for thrombolytic therapy were studied. Twenty-three patients were treated with streptokinase (1.5 million IU IV over 60 minutes) and 18 were treated with rTPA (8 with bolus of 10 mg IV, followed by 50 mg infused over 60 minutes and then 40 mg infused over 120 minutes; 10 patients were administered rTPA and heparin according to the accelerated infusion protocol indicated by the GUSTO study). C4a and C3a were measured by radioimmunoassay, soluble terminal complement components (SC5b-9) and anti-SK IgG antibodies were measured by ELISA. Cleaved high molecular weight kininogen (HK) was quantitated in plasma by SDS-PAGE and immunoblotting analysis. C4a levels were significantly and similarly increased in both groups, whereas the levels of C3a and SC5b-9 after rTPA infusion were only slightly elevated and were significantly lower than after SK. No differences were observed between patients treated with slow or accelerated rTPA regimens. The titer of antibodies to SK was highly correlated with the levels of C3a and SC5b-9, whereas a lesser correlation was observed with C4a. Treatment with rTPA did not induce the transient neutropenia observed after SK infusion. The cleavage products of HK were significantly greater after SK than after rTPA infusion. CONCLUSIONS: Our results show that both thrombolytic agents activate the classic complement pathway and that plasmin could be the common trigger for this phenomenon. A significant activation of the complement common pathway (from C3 to terminal components) was observed only with SK infusion and is attributable to the rapid formation of immunocomplexes between SK and anti-SK antibodies present in plasma as a consequence of previous streptococcal infections. The minimal activation of C5 component of the common pathway explains the absence of leukopenia in patients treated with rTPA. Cleavage of HK, larger after SK than after rTPA infusion, represents a condition enhancing the generation of bradykinin by kallikrein. The recent experimental data that indicate a damaging effect of complement activation on the infarcted zone and the contrasting favorable effect consequent to bradykinin formation raise some questions about the clinical importance of the different biological consequences of SK versus rTPA.
Adult↗
The effect of Bacillus Calmette-Guerin-induced macrophage activation on the in vivo clearance of sensitized erythrocytes.
The clearance of (51)Cr-labeled guinea pig erythrocytes, sensitized with a known amount of IgM or IgG antibody, was examined in normal and BCG-infected guinea pigs. In normal animals, IgM-coated cells were rapidly sequestered in the liver. Most of these cells were then slowly released into the circulation where they survived normally as Coombs-positive erythrocytes. Neither the site nor extent of initial clearance showed major alterations in BCG-infected animals; however, there was no return of the sequestered erythrocytes into the circulation. This pattern of clearance was only seen in normals at very high levels of sensitization. In contrast to the IgM studies, the pattern of clearance of IgG-sensitized erythrocytes was not altered, but the rate and magnitude was markedly increased at all levels of sensitization. In addition, complement-independent clearance of IgG-sensitized erythrocytes was augmented in BCG-infected guinea pigs lacking classical complement pathway function. The spleen remained the organ primarily responsible for this increased clearance of IgG-sensitized erythrocytes. Sensitized cells in BCG-infected animals were removed from the circulation as if they were coated with several times the amount of antibody. Serum factors were shown not to be responsible for the increased clearance. These data suggest that increased macrophage activation in BCG-infected animals plays a critical role in determining the consequences of cell sensitization in vivo. These studies may help to explain exacerbations of hemolytic anemias and related states after intercurrent infections.
Animals↗
Studies on the in vivo effects of antibody. Interaction of IgM antibody and complement in the immune clearance and destruction of erythrocytes in man.
Purified human IgM isoagglutinins were utilized to sensitize (51)Cr-labeled erythrocytes so as to produce a known number of complement-fixing sites. These cells were then reinfused into the erythrocyte donor. A minimum of 20 C1-fixing sites/erythrocyte were required for decreased survival. As the amount of antibody coating the erythrocytes was increased, a larger percentage was sequestered. With 80 C1-fixing sites, more than 75% of the injected erythrocytes were removed from the circulation within 10 min. In each case, the clearance pattern consisted of rapid hepatic sequestration followed by a gradual return of a portion of the erythrocytes into the circulation where they survived normally. Clearance was shown to be dependent upon activation of the classical complement pathway, since sensitized cells survived normally in hereditary angioedema patients with low levels of C4 and no detectable C2. Exposure of sensitized cells to fresh serum for 15 min led to the deposition of 550-800 C3 molecules/C1-fixing site. Such cells were immune adherence positive, were agglutinated by anti-C3b, formed rosettes with human alveolar macrophages, and were sequestered in vivo, presumably because of the interaction of cell-bound C3b with the C3b receptor on hepatic macrophages. After exposure to heated serum as a source of the C3b inactivator, the cells were immune adherence negative, were agglutinated only by anti-C3d, did not form rosettes with macrophages, and survived normally in vivo despite, being Coombs positive. Cleavage of cell-bound C3b to C3d may explain the release phase of the IgM clearance pattern. Whereas erythrocytes coated with IgM antibody and complement were previously thought to be sequestered in the liver because of extensive membrane damage, these experiments suggest that clearance is determined by the interaction of erythrocyte-bound complement fragments with specific receptors on hepatic macrophages.
Adolescent↗
The immunopathology of herpes gestationis. Immunofluorescence studies and characterization of "HG factor".
Nine skin biopsies from seven herpes gestationis patients were studied by immunofluorescence (IF) techniques. Basement membrane zone (BMZ) deposition of C3 and properdin was present in all nine skin specimens, while IgG deposition was apparent in only one. With in vitro C3 IF staining, positive BMZ staining (HG factor activity) was noted with all seven of our patients' serum samples tested. By standard indirect IF staining, however, only one of these serum samples contained BMZ antibodies of the IgG type. Two cord serum samples, tested by these same methods, yielded positive in vitro C3 staining (HG factor activity) but negative indirect IF staining (IgG). HG factor activity was found to be stable at 56 degrees C for 30 min and in two of three specimens at 56 degrees C for 1 h. Treatment of the complement source (normal human serum) used in the in vitro C3 staining assay with Mg2-EGTA or use of C2-deficient serum as the complement source inhibited HG factor activity. HG factor blocked the specific staining of the BMZ of normal human skin by labeled bullous pemphigoid antibodies. By sucrose density gradient ultracentrifugation and gel chromatography (Sephadex G-200), HG factor activity eluted with IgG-containing fractions. The highly purified IgG fraction of two herpes gestationis sera was also positive for HG factor activity. Our studies suggest that HG factor is an IgG antibody that may not be demonstrable by conventional IF methods, but which activates the classical complement pathway.
Basement Membrane↗
A comparative analysis of the C1-binding ability of fragments derived from complement-fixing and noncomplement-fixing IgM proteins.
The purpose of this study was to examine the molecular parameters necessary for initiation of complement fixation by IgM proteins. To determine why some IgM molecules are capable of complement fixation while others are not, several different Waldenström IgM proteins were examined for their ability to fix total hemolytic complement in the CH(50) assay. Subsequently, the C1 fixing ability of a 56-residue fragment derived from the Cmu4 domain of each of these IgM molecules was studied with C1 fixation assay. One of the three Waldenström IgM proteins (Gr) used in the present study was found unable to consume complement in a CH(50) assay when tested at the same concentration as the two complement-consuming IgM molecules (Dau and Bus). However, when the 56-residue C(H)4 fragment from the Cmu4 domain of each IgM molecule was tested for C1-fixing ability, all three were found to bind C1. On the basis of these observations, it is proposed that a C1 binding site exists within the Cmu4 domain of both complement-fixing and noncomplement-fixing IgM molecules. Presumably, the latter molecules are unable to interact in their native state with C1 in the manner required for initiation of the classical complement pathway, possibly due to the configurational inaccessibility of the entire C1 binding site.
Complement C1↗
Platelet C1- inhibitor. A secreted alpha-granule protein.
In order to characterize which proteins of the contact phase of coagulation interact with platelets, human platelets were studied immunochemically and functionally to determine if they contain C1- inhibitor. By means of monospecific antibody to C1- inhibitor, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to measure directly platelet C1- inhibitor. With the CELISA, from 33 to 115 ng of C1- inhibitor antigen per 10(8) platelets from 15 normal donors was quantified in lysates of washed human platelets solubilized in nonionic detergent. The mean concentration in 10(8) platelets was 62 +/- 33 ng (SD). Plasma C1- inhibitor either in the platelet suspension medium or on the surface of the platelets could account for only from 6.5 to 16% of the total antigen measured in the solubilized platelets. Upon functional studies, platelets contained 84 +/- 36 ng (SD) of C1- inhibitor activity in 10(8) platelets. As assessed by the CELISA, platelet C1- inhibitor antigen was immunochemically identical to plasma and purified C1- inhibitor. In contrast, the mean concentration of platelet C1- inhibitor antigen in platelets from four patients with classical hereditary angioedema was 8.3 ng/10(8) platelets (range, 5.3 to 11.3 ng/10(8) platelets). 25 and 31% of the total platelet C1- inhibitor was secreted without cell lysis from normal platelets after exposure to collagen (20 micrograms/ml) and thrombin (1 U/ml), respectively, and this secretion was blocked by metabolic inhibitors. Platelet subcellular fractionation showed that platelet C1- inhibitor resided mostly in alpha-granules, similar to the location of platelet fibrinogen. Thus, human platelets contained C1- inhibitor, which became available by platelet secretion. The identification of platelet C1- inhibitor suggests that platelets may modulate the activation of the proteins of early blood coagulation and the classical complement pathways.
Blood Platelets↗
Immunoglobulin prevents complement-mediated hyperacute rejection in swine-to-primate xenotransplantation.
Immunoglobulins regulate the complement system by activating complement on foreign surfaces and diverting reactive complement proteins away from autologous cell surfaces. Based on this model, we explored the ability of Ig to balance complement activation versus control in a pig-to-primate cardiac xenotransplantation model in which the binding of xenoreactive antibodies of the recipient to graft blood vessels and the activation of complement cause hyperacute rejection. Human IgG added to human serum caused a dose-dependent decrease in deposition of iC3b, cytotoxicity, and heparan sulfate release when the serum was incubated with porcine endothelial cells. This decrease was not caused by alteration in antibody binding or consumption of complement but presumably reflected decreased formation of C3 convertase on the endothelial cells. Infusion of purified human IgG into nonhuman primates prevented hyperacute rejection of porcine hearts transplanted into the primates. As expected, the transplants contained deposits of recipient Ig and C1q but not other complement components. The inhibition of complement on endothelial cell surfaces and in the xenotransplantation model supports the idea that IgG regulates the classical complement pathway and supports therapeutic use of that agent in humoral-mediated disease.
Animals↗
A family with complement factor D deficiency.
A complement factor D deficiency was found in a young woman who had experienced a serious Neisseria meningitidis infection, in a deceased family member with a history of meningitis, and in three relatives without a history of serious infections. The patient and these three relatives showed a normal activity of the classical complement pathway, but a very low activity of the alternative complement pathway and a very low capacity to opsonize Escherichia coli and N. meningitidis (isolated from the patient) for phagocytosis by normal human neutrophils. The alternative pathway-dependent hemolytic activity and the opsonizing capacity of these sera were restored by addition of purified factor D. The family had a high degree of consanguinity, and several other family members exhibited decreased levels of factor D. The gene encoding factor D was found to contain a point mutation that changed the TCG codon for serine 42 into a TAG stop codon. This mutation was found in both alleles of the five completely factor D-deficient family members and in one allele of 21 other members of the same family who had decreased or low-normal factor D levels in their serum. The gene sequence of the signal peptide of human factor D was also identified. Our report is the first, to our knowledge, to document a Factor D gene mutation. The mode of inheritance of factor D deficiency is autosomal recessive, in accordance with the localization of the Factor D gene on chromosome 19. Increased susceptibility for infections in individuals with a partial factor D deficiency is unlikely.
Adult↗
Complement and disease: a review.
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Antigen-Antibody Reactions↗