RESUMO
Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.
Assuntos
Ativação do Complemento , Complemento C3/metabolismo , Infecções por Herpesviridae/fisiopatologia , Herpesvirus Humano 4/fisiologia , Animais , Complemento C3b/metabolismo , Complemento C4/metabolismo , Complemento C4b , Fator I do Complemento , Humanos , Técnicas In Vitro , Ligação Proteica , Receptores de Complemento/metabolismo , Receptores de Complemento 3b , Serina Endopeptidases/metabolismoRESUMO
The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Complemento C3b/metabolismo , Receptores de Complemento , Complemento C3/análise , Proteínas Inativadoras do Complemento C3b/metabolismo , Eletroforese em Gel de Poliacrilamida , Eritrócitos/imunologia , HumanosRESUMO
C-reactive protein (CRP) has several properties that suggest that it may function as a bacterial opsonin. CRP shows binding reactivity with pneumococcal C-polysaccharide, the cell wall carbohydrate of Streptococcus pneumoniae. In this study we have demonstrated protection of mice against serotypes 3 and 4 of S. pneumoniae infection by a single prior injection of CRP. This effect was seen both in mice that lacked antibody to phosphocholine and in normal mice. Thus the opsonic properties of CRP previously described may be related to protection against pneumococcal infection.
Assuntos
Proteína C-Reativa/farmacologia , Infecções Pneumocócicas/prevenção & controle , Animais , Animais Recém-Nascidos , Antígenos , Feminino , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Fosforilcolina/imunologia , Infecções Pneumocócicas/mortalidade , Polissacarídeos Bacterianos/imunologiaRESUMO
C-reactive protein (CRP) is an acute phase serum protein that shares several functions with immunoglobulin (Ig)G including complement activation and binding to receptors on monocytes and neutrophils. The identity of the receptor for CRP has been the target of extensive research. We previously determined that CRP binds to the high affinity receptor for IgG, FcgammaRI (CD64). However, this interaction could not account for the majority of binding of CRP to neutrophils or monocytic cells. We now determine that CRP also interacts with FcgammaRIIa (CD32), the low affinity receptor for IgG on monocytes and neutrophils. COS-7 cells were transfected with a construct containing the human FcgammaRIIA cDNA. CRP binding and the presence of CD32 were detected by mAb and analyzed by two-color flow cytometry. Cells expressing CD32 bound CRP in a dose-dependent and saturable manner consistent with receptor binding. CRP bound to transfectants and K-562 cells with similar kinetics, and in both cases binding was completely inhibited by aggregated IgG. On monocytic cell lines, treatment with Bt(2)cAMP increased FcgammaRII expression and enhanced CRP binding. CRP also specifically precipitated FcgammaRI and FcgammaRII from the monocytic cell line, THP-1. It is suggested that the major receptor for CRP on phagocytic cells is FcgammaRII.
Assuntos
Proteína C-Reativa/metabolismo , Monócitos/metabolismo , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo , Bucladesina/farmacologia , Humanos , Imunoglobulina G/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de IgG/genética , Proteínas Recombinantes/metabolismo , Células U937RESUMO
Deoxygenation of erythrocytes from sickle cell anemia (SCA) patients alters membrane phospholipid distribution with increased exposure of phosphatidylethanolamine (PE) and phosphatidylserine (PS) on the outer leaflet. This study investigated whether altered membrane phospholipid exposure on sickle erythrocytes results in complement activation. In vitro deoxygenation of sickle but not normal erythrocytes resulted in complement activation measured by C3 binding. Additional evidence indicated that this activation was the result of the alterations in membrane phospholipids. First, complement was activated by normal erythrocytes after incubation with sodium tetrathionate, which produces similar phospholipid changes. Second, antibody was not required for complement activation by sickle or tetrathionate-treated erythrocytes. Third, the membrane regulatory proteins, decay-accelerating factor (CD55) and the C3b/C4b receptor (CD35), were normal on sickle and tetrathionate-treated erythrocytes. Finally, insertion of PE or PS into normal erythrocytes induced alternative pathway activation. SCA patients in crisis exhibited increased plasma factor Bb levels compared with baseline, and erythrocytes isolated from hospitalized SCA patients had increased levels of bound C3, indicating that alternative pathway activation occurs in vivo. Activation of complement may be a contributing factor in sickle crisis episodes, shortening the life span of erythrocytes and decreasing host defense against infections.
Assuntos
Anemia Falciforme/sangue , Via Alternativa do Complemento , Membrana Eritrocítica/química , Fosfatidiletanolaminas/sangue , Fosfatidilserinas/sangue , Antígenos CD/metabolismo , Antígenos CD55 , Via Alternativa do Complemento/efeitos dos fármacos , Diamida/farmacologia , Humanos , Glicoproteínas de Membrana/metabolismo , Lipídeos de Membrana/sangue , Oxigênio/sangue , Receptores de Complemento 3b/metabolismoRESUMO
C-reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcgammaRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcgammaRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcgammaRIIA - which encodes histidine or arginine at position 131 - strongly influences IgG2 binding, we determined this polymorphism's effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcgammaRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcgammaRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-gamma enhanced FcgammaRI expression by H-131 monocytes and increased CRP binding. FcgammaRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca(2+)](i) in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcgammaRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcgammaRIIA alleles to host defense and autoimmunity.
Assuntos
Alelos , Antígenos CD/genética , Antígenos CD/metabolismo , Proteína C-Reativa/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Células Cultivadas , Heterozigoto , Homozigoto , Humanos , Ligação ProteicaRESUMO
C3 nephritic factor is an autoantibody to the alternative-pathway C3 convertase (C3bBb) which increases the half-life of the convertase both in the presence and absence of serum regulatory proteins. Human erythrocytes contain membrane proteins which also can regulate C3bBb. One of these proteins, the C3b/C4b receptor (CR1), plays an important role in the processing of soluble immune complexes. C3b which is fixed to immune complexes binds to CR1 and is cleaved by factor I to C3c and C3dg. We have tested the effectiveness of the nephritic factor in protecting bound C3b from cleavage by factor I and human erythrocytes. Sheep erythrocyte intermediates EAC1423b were prepared using 125I-labeled C3 and incubated with factors B and D in the presence and absence of nephritic factor. Breakdown of C3b was measured by release of 125I-C3c following incubation with human erythrocytes and factor I. Purified IgG from two patients with nephritic factor prevented C3c release in a dose-dependent manner. Normal human IgG was ineffective as was nephritic factor in the absence of factor B. Factor P also inhibited the release of C3c in the presence of factor B with equivalent activity at approx. 20-fold higher concns than nephritic factor. These results indicate that nephritic factor can impair human erythrocyte dependent degradation of C3b in alternative-pathway-activating immune complexes.
Assuntos
Enzimas Ativadoras do Complemento/metabolismo , Fator Nefrítico do Complemento 3/fisiologia , Convertases de Complemento C3-C5/metabolismo , Proteínas Inativadoras do Complemento/fisiologia , Eritrócitos/imunologia , Fibrinogênio/fisiologia , Fator B do Complemento/fisiologia , Via Alternativa do Complemento , Eletroforese em Gel de Poliacrilamida , HumanosRESUMO
Hamster female protein (FP) is a member of the family of proteins known as pentraxins which share amino acid sequence homology, cyclic pentameric structure and calcium-dependent binding to ligands. Other members of this family include C-reactive protein (CRP) and serum amyloid P component (SAP), and most species synthesize both CRP and SAP. FP is unusual in that it is apparently the only pentraxin produced in hamsters, it is under hormonal control and it shares binding characteristics with both CRP and SAP. CRP has been defined and isolated by its calcium-dependent binding to pneumococcal C-polysaccharide via phosphocholine (PC) residues. SAP has been isolated by calcium-dependent binding to agarose. FP binds to both PC and agarose. Recently, both SAP and CRP have been found to bind to chromatin in a calcium-dependent manner and involvement of these proteins in the clearance of nuclear material has been proposed. In this paper we test whether FP shares the ability to bind to chromatin and histones, and compare its relative avidities for these ligands. Similar to CRP, FP bound to histones H1 and H2A, and chromatin. FP shared with SAP the ability to bind to DNA. However, FP binding was inhibited by PC for all ligands, whereas SAP binding was not. FP and SAP also failed to compete with each other for binding to DNA. By cross-inhibition FP bound much less well to PC than CRP, but was a very effective inhibitor of CRP binding to H2A. These studies demonstrate that chromatin and histone binding are conserved among these pentraxins. The role of the proposed PC binding site in these binding reactions is discussed.
Assuntos
alfa-Globulinas/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Histonas/metabolismo , alfa-Globulinas/química , Sequência de Aminoácidos , Proteína C-Reativa/metabolismo , Proteínas de Ligação a DNA/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Fosforilcolina/metabolismo , Ligação ProteicaRESUMO
This short review will highlight three areas of current research on the role of complement in bacterial infection. These are i) the use of mice with targeted disruption of complement genes to study infectious disease models; ii) studies on the interactions between bacterial virulence factors and complement regulatory proteins; and iii) the description of a novel strategy by which mycobacteria promote complement activation and use complement receptors to gain entry into macrophages.
Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , Proteínas do Sistema Complemento/imunologia , Animais , Bactérias/metabolismo , Bacteriólise/imunologia , Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Humanos , Receptores de Complemento/metabolismoRESUMO
In the studies described here we have attempted to evaluate the hypothesis that CRP may function in host defense using two systems in which CRP in the presence of C appears to have opsonic properties. In the first, CRP and C were found to stimulate ingestion of erythrocytes by human monocyte or mouse macrophages in vitro, and to alter clearance patterns in vivo. In the second, we have studied opsonization of S. pneumoniae by CRP and C. Experiments with human neutrophils indicate that although CRP and C can enhance opsonization of S. pneumoniae, this effect is more pronounced in the absence of antibody. In vivo CRP was found to protect mice against intravenous infection with S. pneumoniae.
Assuntos
Proteína C-Reativa/fisiologia , Proteínas do Sistema Complemento/metabolismo , Proteínas Opsonizantes , Fagocitose , Animais , Atividade Bactericida do Sangue , Proteína C-Reativa/metabolismo , Via Clássica do Complemento , Humanos , Infecções Pneumocócicas/imunologia , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Streptococcus pneumoniae/imunologiaRESUMO
Foreign particles and damaged host cells can activate the complement system leading to their destruction by the host defense system. Factor H (fH) plays a vital role in restricting complement activation on host cells through interactions with polyanions such as heparin, while allowing activation to proceed on foreign surfaces. Complement activation by damaged host cells is also down regulated by fH, which is localized to injured areas through interactions with C-reactive protein (CRP). A number of pathogens have developed mechanisms by which they can also bind fH and thus exploit its protective properties. One such organism is Group A Streptococcus (GAS) which mediates fH binding via its surface expressed M-protein. fH consists of 20 conserved short consensus repeat (SCR) units and mutagenesis studies indicate that the seventh repeat is responsible for interactions with heparin, CRP and M-protein. We recently performed molecular modelling of fH SCR 7 and identified a cluster of positively charged residues on one face of the domain. By alanine replacement mutagenesis, we demonstrated that these residues are involved in heparin, CRP and M protein binding, which indicates that there is a common site within fH SCR 7 responsible for multiple ligand recognition.
Assuntos
Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/imunologia , Sítios de Ligação , Proteína C-Reativa/imunologia , Proteínas de Transporte/imunologia , Ativação do Complemento , Fator H do Complemento/genética , Heparina/química , Humanos , Técnicas In Vitro , Infecções/etiologia , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/imunologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Streptococcus pyogenes/imunologiaRESUMO
Although some of the membrane glycoproteins that serve as activators or regulators of C activation have been identified, the influence of membrane lipids has not been studied extensively. A model of alternative C pathway activation was established using liposomes composed of cholesterol and synthetic phospholipids. Liposomes containing phosphatidylcholine (PC) as the sole phospholipid did not activate C as measured by C3 binding after incubation in normal human serum containing 2.5 mM MgCl2 and 10 mM EGTA. When phosphatidylethanolamine (PE) was included as 20% or more of the phospholipid, C3 binding was observed. C3 binding to liposomes was inhibited by salicylhydroxamic acid indicating binding through the C3 thioester bond. The phospholipid composition did not influence C3 binding to liposomes in an unregulated system of C3, B, D, and P indicating equivalent C3b binding sites on activating and nonactivating liposomes. When the regulatory proteins H and I were added to the other components, liposomes containing PE bound three times more C3 than PC liposomes suggesting that the phospholipid affects C3 regulation. This was tested directly in a radiolabeled H binding assay. In the presence of equal amounts of C3b, PC liposomes showed a greater number of high affinity H binding sites than PE liposomes. Using different PE derivatives, C activation could be directly related to the phospholipid polar head group. Liposomes containing PE, trinitrophenyl-PE or monomethyl-PE did activate the alternative C pathway, whereas those containing dimethyl-PE, PC, or phosphatidylserine did not. These studies provide evidence that primary and secondary amino groups on lipid membranes can decrease the interaction between H and C3b and provide sites for alternative pathway activation.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Via Alternativa do Complemento/efeitos dos fármacos , Lipídeos de Membrana/farmacologia , Fosfolipídeos/farmacologia , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento , Humanos , Lipossomos , Antígeno de Macrófago 1 , Fluidez de Membrana/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Receptores de Complemento/análise , Receptores de Complemento/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
C-reactive protein (CRP) is an acute phase serum protein found associated with damaged tissue at sites of inflammation. CRP bound to multivalent phosphocholine-containing or polycationic ligands activates C by the classical pathway. We have previously described liposomes of a particular lipid composition that are able both to activate the alternative pathway of C and to bind CRP. In addition many strains of Streptococcus pneumoniae activate the alternative pathway. We have shown CRP binding to these bacteria as well. Because antibody to an activating surface in many cases enhances alternative pathway activation, we tested CRP for a similar function in these systems. Our results indicate that, in contrast to antibody, CRP inhibits alternative pathway activation. This inhibition by CRP is apparently restricted to surfaces that bind CRP. Thus, CRP binding to membrane or bacterial surfaces can convert them from alternative pathway activation to classical pathway activation.
Assuntos
Proteína C-Reativa/farmacologia , Ativação do Complemento , Via Alternativa do Complemento , Lipossomos/farmacologia , Streptococcus pneumoniae/imunologia , Aminas/farmacologia , Sítios de Ligação , Proteína C-Reativa/metabolismo , Colesterol/farmacologia , Complemento C3/antagonistas & inibidores , Fator B do Complemento/antagonistas & inibidores , Dimiristoilfosfatidilcolina , Galactosilceramidas/farmacologia , Humanos , Miristatos/farmacologia , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/farmacologiaRESUMO
C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.
Assuntos
Proteína C-Reativa/metabolismo , Carcinoma/imunologia , Ativação do Complemento , Citotoxicidade Imunológica , Neoplasias Laríngeas/imunologia , Animais , Anticorpos Antineoplásicos/fisiologia , Sítios de Ligação de Anticorpos , Carcinoma/metabolismo , Linhagem Celular , Ativação do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Neoplasias Laríngeas/metabolismo , Fosforilcolina/farmacologia , Protaminas/farmacologia , Ligação Proteica/efeitos dos fármacos , CoelhosRESUMO
C-reactive protein is an acute phase protein in man and an important component of the innate immune system. C-reactive protein activates the classical pathway of complement, which is one of its main mechanisms in providing host defense. It has recently been recognized that C-reactive protein interacts with the cells of the immune system by binding to Fc gamma receptors. It may thus bridge the gap between innate and adaptive immunity and provide an early, effective antibacterial response. Furthermore, as it protects against the damaging inflammatory response to lipopolysaccharide and cytokines, it may prevent the lethal side-effects of bacterial products. The recent identification of the interaction of C-reactive protein with Fc gamma receptors will lead to an enhanced understanding of C-reactive protein and its role in both the innate and acquired immune systems.
Assuntos
Infecções Bacterianas/imunologia , Proteína C-Reativa/metabolismo , Imunidade Inata , Proteína C-Reativa/química , HumanosRESUMO
Reperfusion of ischaemic tissue initiates an inflammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this inflammation. This study investigated the mechanism of complement activation following reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 deposition. There was an increase in apoptotic cells in these cultures that was demonstrated by binding of fluorescein isothiocyanate-Annexin V and staining for hypodiploid nuclei. To determine if apoptotic HUVEC activate complement, uniformly apoptotic cells were produced by serum and growth factor deprivation. These cells, but not the control HUVEC, activated the classical complement pathway in the absence of antibody or other serum factors. To determine if apoptotic cells in the reoxygenated cultures were activating complement, fluorescent analysis was done. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, complement activation following reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis with exposure of phosphatidylserine. Apoptotic cells directly activate the classical pathway of complement by binding C1. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.
Assuntos
Apoptose/imunologia , Via Clássica do Complemento/imunologia , Endotélio Vascular/imunologia , Traumatismo por Reperfusão/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Técnicas de Cultura de Células , Hipóxia Celular/imunologia , Complemento C3/metabolismo , Via Clássica do Complemento/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Endotélio Vascular/patologia , Humanos , Imunoglobulina M/metabolismoRESUMO
Complement-dependent clearance of immune complexes in humans is dependent on the activation and binding of the early components of the classical complement cascade. This prevents immune complex precipitation and promotes binding of the complexes by the C4b/C3b complement receptor CR1 (CD35) found on erythrocytes. The fourth component of human complement is encoded by two closely linked genes within the MHC. These genes give rise to the isotypic forms C4A and C4B, and recent studies suggest that CR1 binds activated C4A (C4Ab) to a greater extent than activated C4B (C4Bb). To study this difference in a more quantitative way the binding reactions between CR1 and C4Ab- and C4Bb-coated immune complexes and between CR1 and soluble dimers of C4Ab (C4Ab2) and C4Bb (C4Bb2) were analysed using the native receptor on human erythrocytes. The binding reaction between immune complexes with equivalent amounts of covalently bound C4Ab or C4Bb and erythrocyte CR1 showed a two-fold higher binding of complexes coated with C4A. Furthermore, erythrocyte CR1 bound C4Ab2 with an apparent four-fold higher affinity (Kd approximately 1.4 x 10(-7) M) than C4Bb2 (Kd approximately 4.8 x 10(-7) M), indicating a preferential binding of CR1 for C4A.
Assuntos
Complemento C4a/metabolismo , Complemento C4b/metabolismo , Eritrócitos/imunologia , Receptores de Complemento 3b/metabolismo , Complexo Antígeno-Anticorpo/imunologia , Complemento C4a/imunologia , Complemento C4b/imunologia , Eritrócitos/metabolismo , Humanos , Ligação Proteica , Ensaio Radioligante , Receptores de Complemento 3b/imunologiaRESUMO
We previously studied two alternative pathway activators, Streptococcus pneumoniae and positively charged liposomes, which react with C-reactive protein (CRP). Binding of CRP to these surfaces initiates classical pathway but blocks alternative pathway activation. In this study, we investigated the mechanism of this inhibition using S. pneumoniae, R36a. R36a were pretreated with CRP (CRP-R36a) or buffer and incubated with C2-deficient human serum to which 125I-labeled C3 had been added. The amount of specific 125I-C3 binding was decreased from 8200 mol/CFU on R36a to 2200 mol/CFU on CRP-R36a. In contrast, when the same experiment was performed with purified factors B, D, P, and C3, in the absence of regulatory proteins, specific 125I-C3 uptake was slightly lower on R36a (6100 mol/CFU) than on CRP-R36a (8100 mol/CFU). The ability of the fixed C3b to inactivate factor B in the presence of factor D was equivalent on the two surfaces. The binding of the regulatory factor H to C3b fixed to R36a and CRP-R36a was compared by using purified 125I-labeled factor H. The ratio of factor H bound to C3 bound was twofold greater on CRP-R36a than on R36a. This increase was found by using C2-deficient serum or purified factors B, D, P, and C3 to fix C3b to the surfaces. The ability of CRP to inhibit C3 binding to R36a was restored by the addition of factors H and I to factors B, D, P, and C3. These results indicate that CRP inhibits alternative pathway activation by increasing regulation of bound C3.
Assuntos
Proteína C-Reativa/farmacologia , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/metabolismo , Complemento C3b/metabolismo , Via Alternativa do Complemento , Streptococcus pneumoniae/imunologia , Ligação Competitiva , Proteína C-Reativa/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Fator H do Complemento , Fator I do Complemento , Interações Medicamentosas , Endopeptidases/farmacologia , Humanos , Cinética , Antígeno de Macrófago 1 , Receptores de Complemento/análise , Streptococcus pneumoniae/metabolismoRESUMO
Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.