scholarly journals The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum

1981 ◽  
Vol 195 (2) ◽  
pp. 471-480 ◽  
Author(s):  
K J Gadd ◽  
K B M Reid
Keyword(s):  
Human Serum ◽  
Heavy Chain ◽  
Alternative Pathway ◽  
Complement Component ◽  
Amide Bond ◽  
Alpha Chain ◽  
Complement Component C3 ◽  
Alternative Pathway Of Complement ◽  
Immune Aggregates ◽  
Covalently Bound

Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.

scholarly journals Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes.

1990 ◽  
Vol 172 (4) ◽  
pp. 1201-1210 ◽  
Author(s):  
C Bellinger-Kawahara ◽  
M A Horwitz
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Legionella Pneumophila ◽  
Alternative Pathway ◽  
Major Outer Membrane Protein ◽  
Complement Component ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Monocytes ◽  
Complement Component C3

Legionella pneumophila is a facultative intracellular bacterial pathogen that parasitizes human monocytes and alveolar macrophages. Previous studies from this laboratory have shown that monocyte complement receptors CR1 and CR3 and complement component C3 in serum mediate L. pneumophila phagocytosis. In this study, we have explored C3 fixation to L. pneumophila. We developed a whole-cell enzyme-linked immunosorbent assay (ELISA) to measure C3 fixation to the bacterial surface. By this assay, C3 fixes to L. pneumophila that are opsonized in fresh nonimmune serum, and C3 fixation takes place via the alternative pathway of complement activation. Immunoblot analysis of opsonized L. pneumophila indicated that C3 fixes selectively to specific acceptor molecules of L. pneumophila. Consistent with this, when nitrocellulose blots of whole L. pneumophila or bacterial components are incubated in fresh nonimmune serum, C3 fixes exclusively to the major outer membrane protein (MOMP) of L. pneumophila, a porin; C3 does not fix to L. pneumophila LPS on these blots. To further explore the role of MOMP in C3 fixation and phagocytosis, we reconstituted purified MOMP into liposomes. By the ELISA, MOMP-liposomes, but not plain liposomes lacking MOMP, avidly fix C3. Consistent with a dominant role for MOMP in C3 fixation, MOMP-liposomes form a C3 complex of the same apparent molecular weight as whole L. pneumophila in nonimmune serum. Opsonized radioiodinated MOMP-liposomes avidly adhere to monocytes, and adherence is dose dependent upon serum. By electron microscopy, opsonized MOMP-liposomes are efficiently phagocytized by human monocytes, and phagocytosis takes place by a conventional appearing form of phagocytosis. This study demonstrates that C3 fixes selectively to the MOMP of L. pneumophila, and that, in the presence of nonimmune serum, MOMP can mediate phagocytosis of liposomes and, potentially, phagocytosis of intact L. pneumophila by human monocytes.

Hematin Promotes Complement Alternative Pathway-Mediated Deposition of C3 Activation Fragments on Human Erythrocytes: Potential Implications for the Pathogenesis of Anemia in Malaria.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 839-839
Author(s):  
Ronald P. Taylor ◽  
Andrew W. Pawluczkowycz ◽  
Margaret A. Lindorfer ◽  
John W. Waitumbi
Keyword(s):  
Monoclonal Antibody ◽  
B Cells ◽  
Alternative Pathway ◽  
Complement Alternative Pathway ◽  
Western Blots ◽  
Marginal Zone B Cells ◽  
Immune Adherence ◽  
Alternative Pathway Of Complement ◽  
Childhood Malaria ◽  
Covalently Bound

Abstract Childhood malaria caused by Plasmodium falciparum (pf) is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported that erythrocyte (E) CR1, the immune adherence receptor specific for C3b, is reduced during anemia in childhood malaria, suggesting a possible role for complement in E destruction. Intravascular lysis of infected E by pf leads to release of E breakdown products hemoglobin and hematin, which have inflammatory properties. Free hematin can bind to E, and we find that in serum and in whole blood anti-coagulated with lepirudin, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on E. We documented C3 deposition by flow cytometry, and additional fluorescence microscopy studies revealed that most of the deposited C3 fragments are located in close juxtaposition to CR1. Western blots confirmed that the C3 fragments are indeed covalently bound to the E, and immunoprecipitation experiments indicated that a fraction of the deposited C3 is covalently bound to CR1. The degree of C3 fragment deposition is directly correlated with E CR1 levels, both within a given donor’s E population and when E from different donors are compared. E opsonized with complement in the presence of hematin form rosettes with Raji cells, through interaction with CR2, the C3dg receptor expressed on several types of B cells including splenic marginal zone B cells. Thus, hematin-mediated complement activation and C3 fragment deposition on E may promote accelerated splenic (or liver) clearance of the youngest E, which have the most CR1, leading to sudden onset of anemia along with reduction of mean CR1 on surviving E. A monoclonal antibody specific for C3b, mAb 3E7, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this monoclonal antibody in non-human primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of therapies based on inhibiting the alternative pathway of complement.

scholarly journals Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937

1989 ◽  
Vol 261 (2) ◽  
pp. 407-413 ◽  
Author(s):  
C M Maison ◽  
C L Villiers ◽  
M G Colomb
Keyword(s):  
Gel Electrophoresis ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Component ◽  
Polyacrylamide Gel ◽  
Classical Pathway ◽  
U937 Cells ◽  
Complement Component C3 ◽  
Thioester Bond ◽  
Membrane Bound

Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.

scholarly journals C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

1989 ◽  
Vol 257 (3) ◽  
pp. 831-838 ◽  
Author(s):  
L C Antón ◽  
J M Alcolea ◽  
P Sánchez-Corral ◽  
G Marqués ◽  
A Sánchez ◽  
...  
Keyword(s):  
Complement Activation ◽  
Gel Electrophoresis ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biological Properties ◽  
Antibody Molecule ◽  
Peptide Profile ◽  
Normal Human ◽  
Alternative Pathway Of Complement ◽  
Immune Aggregates

Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

scholarly journals Absence of complement component 3 does not prevent classical pathway–mediated hemolysis

2019 ◽  
Vol 3 (12) ◽  
pp. 1808-1814 ◽  
Author(s):  
Lingjun Zhang ◽  
Yang Dai ◽  
Ping Huang ◽  
Thomas L. Saunders ◽  
David A. Fox ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Membrane Attack Complex ◽  
Complement Component ◽  
Classical Pathway ◽  
In Vivo Experiments ◽  
Human Sera ◽  
Alternative Pathway Of Complement ◽  
Complement Component 3

Abstract Complement component 3 (C3) is emerging as a potential therapeutic target. We studied complement-mediated hemolysis using normal and C3-depleted human sera, wild-type (WT) and C3-deficient rat sera, and WT and C3 knockout rat models. In all of the in vitro and in vivo experiments, we found that the loss of C3 did not prevent classical pathway–mediated hemolysis, but it did almost abolish alternative pathway–mediated hemolysis. Experiments using preassembled classical pathway C3 convertases confirmed that C4b2a directly activated complement component 5 (C5), leading to membrane attack complex formation and hemolysis. Our results suggest that targeting C3 should effectively inhibit hemolysis and tissue damage mediated by the alternative pathway of complement activation, but this approach might have limited efficacy in treating classical pathway–mediated pathological conditions.

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