Complement function in mAb-mediated cancer immunotherapy.
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Adjuvants, Immunologic↗
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Results for “Complement C3b”
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Early complement activation increases in the brain in some aged normal subjects.
Complement activation is increased in Alzheimer's disease (AD) and may contribute to the development and progression of this disorder. To compare early complement activation between normal and AD brain specimens, C4d and iC3b concentrations were measured in hippocampus, entorhinal cortex, temporal cortex, parietal cortex, and cerebellum from aged normal and AD subjects n=10-14 for both), and in hippocampus and entorhinal cortex from younger normal subjects (n=5-6). C4d and iC3b levels increased 2.3- to 4.6-fold in AD versus aged normal specimens (all P <0.05), with lowest concentrations of these activation proteins generally in cerebellum. No significant differences were present between aged and younger normal C4d and iC3b levels in hippocampus or entorhinal cortex. However, the concentrations of these proteins were markedly increased in several aged normal specimens. Normal subject age was moderately associated with both C4d (r=0.49) and iC3b (r=0.53) concentrations in the hippocampus. Increased brain complement activation in some elderly individuals may promote the subsequent development of AD.
Adult↗
Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21).
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Amino Acid Sequence↗
Leukotriene C4 production by normal-density and low-density eosinophils of atopic individuals and other patients with eosinophilia.
With the use of a percoll gradient separation procedure, eosinophils of individuals with asthma and with allergy could be separated into normal- and low-density cell fractions. The presence of low-density eosinophils possibly reflects an ongoing process of activation of these cells induced by the allergic reaction. Ca-ionophore-induced leukotriene (LT) C4 production, in the absence of added substrates, demonstrated a decreased potency for LT generation by low-density eosinophils compared with the LT generation of normal-density cells (57 +/- 33 ng and 103 +/- 44 ng per 10(6) cells, respectively). In contrast with the Ca-ionophore-induced LT formation, incubations with serum-treated zymosan in the presence of glutathione demonstrated higher productions of LTC4 with the low-density eosinophilic subpopulation compared with normal-density cells. This is compatible with a possibly higher expression of complement C3b receptors on the low-density eosinophils. Total arylsulfatase contents demonstrated that low-density eosinophils are not degranulated with respect to their small granules. Although release of the large granules by low-density eosinophils cannot be excluded, electron-microscopy studies indicated that degranulation is not the only (or major) factor that determines the density of the various eosinophilic subpopulations.
Cell Count↗
Studies on protein adsorption and activation of complement on hydrated aluminium surfaces in vitro.
Adsorption of human plasma and serum proteins onto hydrated aluminium was studied by ellipsometry/antibody techniques, and soluble complement components iC3b, Bb, and C4d with commercial ELISA plates. Aluminium that was incubated in plasma for 1 min bound significant amounts of anti-lipoproteins (anti-LP), no antibodies against contact activation of coagulation proteins, and no anti-fibrinogen (anti-Fib). Time course studies with serum revealed increasing deposition of anti-C3c with time. Complement factor 1q (C1q) was antibody detectable only after short-time serum incubations, but no anti-IgG and anti-properdin bound to the protein film at any time. Anti-C3c was not deposited after exposure of the surfaces to Clq-depleted serum. Intriguingly, and in spite of increasing deposition of C3 to the surface with time, the combined ellipsometry and ELISA results gave no unequivocal proof of activation of complement by hydrated aluminium.
Adsorption↗
Amino acid differences in the N-terminus of C(H)2 influence the relative abilities of IgG2 and IgG3 to activate complement.
The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C'): IgG3 and IgG1 are very active, IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain [C(H)2] is critical for C' activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C(H)2. Mutants containing the N-terminus of the C(H)2 of IgG3 were as effective as wildtype IgG3 in C1q binding, C1 activation and terminal complex (MAC) formation, but had reduced ability to effect C'-mediated lysis. IgG2 and mutants containing the N-terminal portion of the C(H)2 of IgG2 were reduced compared to IgG3 in activating C1, binding C1q and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C(H)2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind C1q and activate the C' cascade although additional residues of C(H)2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C(H)2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C(H)2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders.
Amino Acid Sequence↗
Immunohistochemical evidence for C3bi involvement in Graves ophthalmopathy.
PURPOSE: To determine by immunohistochemical methods if components of the complement system are present in Graves ophthalmopathy extraocular and periocular tissues compared with non-Graves ophthalmopathy ocular tissues, and, if so, whether a qualitative difference exists. METHODS: Orbital muscle, periorbital muscle, and adipose tissue from 10 Graves ophthalmopathy patients were studied with in situ assays using monoclonal antibodies for C3bi and C5b-9 (the terminal attack complex) complement components. Extraocular muscle, periocular muscle, and adipose tissue from 12 patients treated for unrelated orbital disorders were used as controls. RESULTS: All nine Graves extraocular and periocular muscle tissues exhibited C3bi positive staining in an intense, localized oval- to spindle-shaped reaction that appeared to represent cells on a diffuse staining background of the endomysial and perimysial connective tissues with no staining of the muscle fibers themselves. Some reactivity was seen in 6 of the 12 control muscles, but this was much less intense than that of Graves ocular muscle tissue. Only two Graves muscle samples stained minimally with the monoclonal antibody for the C5b-9 terminal attack complex while none of the control muscle samples demonstrated reactivity. Orbital fat from Graves and control patients did not demonstrate any reactivity for C3bi or C5b-9. CONCLUSION: C3bi and not C5b-9 (the terminal attack complex) is present in Graves ophthalmopathy extraocular and periocular tissues in a qualitatively greater way than in control non-Graves ophthalmopathy ocular tissue. Consequently, C3bi may contribute to the pathophysiology of Graves ophthalmopathy.
Adult↗
C1-inhibitor attenuates hyperacute rejection and inhibits complement, leukocyte and platelet activation in an ex vivo pig-to-human perfusion model.
Xenotransplantation may be a future alternative due to increased shortage of organs. Classical complement activation is central in hyperacute rejection in pig-to-human combinations. We investigated the effects of C1-inhibitor (C1-INH), a regulator of the complement and contact systems, on hyperacute rejection. Pig kidneys were perfused with fresh human blood to which either C1-INH (n = 6) or human serum albumin (n = 6) was added. The survival of the C1-INH perfused kidneys (mean 327 min) was significantly longer (p < 0.00001) than the controls (79 min). C1-INH substantially inhibited complement activation (C1rs-C1-INH complexes, C4bc, C3bc and terminal complement complex) (p < 0.001 for all) compared with the marked complement activation in the controls. No contact activation was found. Leukocytes and platelets were substantially activated (counts, myeloperoxidase, beta-thromboglobulin, thrombospondin, soluble P-selectin) in the control group, and this activation was markedly reduced by C1-INH (p < 0.02 for all). Immunohistochemistry showed less C1q, C3, TCC, IgG and fibrin deposition in the C1-INH group. C1-INH may be useful to attenuate hyperacute rejection, probably through inhibition of complement. The reduced activation of neutrophils and platelets may mainly be secondary to inhibition of complement.
Animals↗
Ragweed allergy: correlation between skin reactivity and in vitro complement activation.
Recently we have reported on several observations which indicate that allergen-induced complement activation contributes to the development of the symptoms of ragweed allergy. In the present paper a new finding that supports this assumption is summarized. In 48 ragweed-allergic patients individual skin reactivity to ragweed allergen extract (RWA) was assessed using dilution skin prick testing. Sera of these patients were incubated with 20, 100, and 400 U/ml RWA and generation of two complement activation products, alternative pathway C3-convertase (C3bBbP) and terminal pathway activation complex (C5b-9) was measured by ELISA methods. A strong positive correlation (Spearman correlation coefficient r = 0.495, P = 0.0004, and r = 0.454, P = 0.0012, respectively) was found between individual skin reactivity to RWA and C3bBbP generation induced by 20 and 100 A allergological units/ml (U/ml) RWA. This finding further supports the role of complement activation products in the aggravation of the basic IgE-mediated immunopathology of ragweed allergy.
Complement Activation↗
Intercellular adhesion molecule-1/leukocyte function associated antigen-1-mediated and complement receptor type 4-mediated infiltration and activation of glomerular immune cells in immunoglobulin A nephropathy.
Glomerular expression of intercellular adhesion molecule-1 (ICAM1) (CD54) and membrane cofactor protein (MCP; CD46) and positive infiltrating cells in leukocyte function associated antigen-1 (LFA1)alpha (CD11a) and C3bi receptors (CR3/CD11b, CR4/CD11c) were examined by the indirect immunoperoxidase method on 43 sets of repeated renal biopsy specimens from patients with immunoglobulin A nephropathy. Twenty-four-hour urine protein at the time of renal biopsy was also evaluated. Glomerular infiltration of LFA1alpha+ cells was significantly correlated with glomerular expression of ICAM1 (r = 0.494, P < 0.0001). Glomerular complement receptor type 4 (CR4)+ cells were significantly correlated with glomerular expression of MCP (r = 0.405, P < 0.0001). The glomerular expressions of ICAM1 and MCP were significantly correlated with each other (r = 0.700, P < 0.00001). The glomerular infiltrations of LFA1alpha+ and CR4+ cells were highly correlated with each other (r = 0.884, P < 0.00001), and both cell types were significantly correlated with urine protein (respectively, r = 0.426 and 0.478, P < 0.001 and 0.0001). When the change in these parameters between the time of the initial and follow-up biopsies was evaluated, there was a significant correlation between the change in glomerular expression of ICAM1 (DeltaICAM1) and MCP (DeltaMCP) as well as between the change in glomerular infiltration of LFA1alpha+ cells (DeltaLFA1alpha+) and CR4+ cells (DeltaCR4+). Both DeltaLFA1alpha+ and DeltaCR4+ were significantly correlated with the change in urine protein. These findings suggest that ICAM1/LFA1 interaction and MCP-mediated C3bi/C3biR interaction cooperate and participate in persistent glomerular infiltration of immune cells in immunoglobulin A nephropathy, and that these LFA1alpha+ and C3biR+ cells contribute to the induction of proteinuria.
Adult↗
Naturally occurring anti-band 3 antibodies.
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Anemia, Hemolytic, Autoimmune↗
Surface-modified biodegradable albumin nano- and microspheres. II: effect of surface charges on in vitro phagocytosis and biodistribution in rats.
The surface charges on biodegradable albumin nanoparticles were introduced by covalent coupling different primary amines to examine their influence on phagocytosis by macrophages under in vitro conditions. Albumin particles with a zeta potential close to zero showed a reduced phagocytic uptake in comparison with charged particles, especially nanoparticles with a positive zeta potential. The phagocytic uptake in the present study was examined using an established cell culture model based on primary mouse peritoneal macrophages and a human hematopoietic monocytic cell line (U-937) treated with phorbol-12-myristic-13-acetate to induce cell differentiation. The influence of opsonins on in vitro phagocytosis experiments was characterized using carriers pre-treated with human serum. In the presence of human serum the phagocytic activity of U-937 cells was found to be similar to primary mouse macrophages without serum. In contrast to peritoneal macrophages, U-937 cells showed no phagocytic activity in the absence of serum. In particular, only the C3b- complement deposition on the particle surface seems to promote the phagocytic process. The in vivo distribution of albumin carriers in rats was investigated using magnetic resonance imaging (MRI). No differences in blood circulation times and organ accumulation between different nanoparticle preparations with positive, neutral and negative surface charges could be observed in rats, suggesting that the in vivo fate of albumin nanoparticles is significantly influenced by factors not reflected in the in vitro cell culture models.
Albumins↗
Effect of intravenous immunoglobulin therapy on plasma complement.
The effect of the intravenous immunoglobulin preparation (Sandoglobulin) on the complement system and the formation of circulating immune complexes (CIC) was investigated in 10 patients with primary humoral immunodeficiency and in 12 normal volunteers. Infusion of 9 g of the pH4 treated preparation did not show relevant changes in total hemolytic complement activity, plasma levels of C4, C3, and factor B. C3dg fragments, however, increased markedly in some individuals in both groups immediately after infusion. CIC measured by 125 I-C1q binding were not detectable. Furthermore, the initially low C1q concentrations in immune-deficient patients correlated with the pre-infusion IgG levels (p < 0.05) and rose significantly 24 h after therapy (p < 0.001). In contrast, in vitro incubation of the immunoglobulin preparation with normal human serum did not reveal spontaneous anticomplementary activity nor did it lead to formation of immune aggregates. The results indicate that immunoglobulin infusions may moderately activate complement in vivo, as evidenced by increased C3dg fragments; in addition, partial C1q deficiency in hypogammoglobulinemia seems to be related to IgG concentration and can be corrected by immunoglobulin substitution.
Adolescent↗
Characterization of neuronal cell death induced by complement activation.
The complement system plays an important role in human immune defense mechanism. Its activation via either the classical or the alternative pathway can lead to the formation of membrane attack complex (MAC) and subsequently kills target cells. Activation of the classical pathway can be initiated with binding of C1q which is first factor of complement cascade to the Fc (fragment crystalline) region of immunoglobulin. This triggers a cascade of proteolytic events resulting in the activation of C5 convertase which cleaves C5 into C5b and C5a. The C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9, the MAC, which pore size increases as the number of C9 in the complex increases. If this membrane lesion persists and results in uncontrolled ion fluxes, the cells swell and eventually lyse. To restrict the activity of the complement system, endogenous complement inhibitors are available to regulate complement-mediated cytolysis. This enables the complement system to distinguish "self" from "foreign" and protect the host from inadvertent complement attack. Activation of the classical complement cascade has been reported in Alzheimer's disease and other neurodegenerative disorders. Recently, we demonstrated that complement activation causes neuronal cell death in vitro, and this neurodegenerative process is regulated by homologous restriction. In this article, we describe the use of two cell lines as in vitro models to evaluate cell injury/cell death induced by complement activation.
Animals↗
Covalent binding efficiency of the third and fourth complement proteins in relation to pH, nucleophilicity, and availability of hydroxyl groups.
The binding of [3H]glycerol and [3H]putrescine to C3 was studied in a fluid-phase system using trypsin as the C3 convertase. The binding of glycerol showed little variation in the pH range between 6.0 and 10.0. The binding of putrescine (pKa = 9.0) is rather ineffective below pH 7.5 but becomes more efficient as the pH of the reaction mixture increases. These results agree with the contention that the final step of the binding reaction is the transfer of the acyl group of the exposed thio ester of C3 to a nucleophile since the nucleophilicity of hydroxyl groups is rather independent of pH whereas only the unprotonated form of amino groups is nucleophilic. The inefficient reaction of amino groups with the exposed thio ester of C3 is also supported by the study of the inhibitory activity of serine and its two derivatives, N-acetylserine and O-methylserine, to the binding of [3H]glycerol to C3. N-Acetylserine showed an inhibitory activity equivalent to that of serine, whereas O-methylated serine showed only minimal activity. It can be concluded, therefore, that serine reacts with the thio ester of C3 by its hydroxyl group but not by its alpha-amino group. The ability of the alcohol group of various alkanes to inhibit the binding of [3H]glycerol to C3 was also studied. The primary alcohols inhibit the binding reaction with an efficiency that is similar to glycerol, and there are no significant differences in the binding efficiencies of methanol, ethanol, 1-propanol, and 1-butanol.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohols↗
Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.
Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.
Amino Acid Sequence↗
Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts.
Compstatin, a newly described C3-binding peptide, inhibits complement activation by blocking C3 convertase-mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Pig kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid-phase and in the tissue were examined. The survival of the Compstatin-perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs-C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immune electron microscopy showed less terminal SC5b-9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62L (L-selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P-selectin or beta-thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.
Animals↗
Structural biology of the alternative pathway convertase.
Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.
Animals↗