RESUMO
The pentraxins, C-reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C-and F-strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA-binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA-binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA-binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C'-strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC- and FG-loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.
Assuntos
Antígenos CD/química , Proteína C-Reativa/química , Imunoglobulina A/química , Receptores Fc/química , Componente Amiloide P Sérico/química , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Humanos , Mutação , Neutrófilos/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismoRESUMO
C-reactive protein (CRP) is an important biomarker for inflammatory diseases. However, its role in inflammation beyond complement-mediated pathogen clearance remains poorly defined. We identified the major IgA receptor, FcαRI, as a ligand for pentraxins. CRP recognized FcαRI both in solution and on cells, and the pentraxin binding site on the receptor appears distinct from that recognized by IgA. Further competitive binding and mutational analysis showed that FcαRI bound to the effector face of CRP in a region overlapping with complement C1q and Fcγ receptor (FcγR) binding sites. CRP cross-linking of FcαRI resulted in extracellular signal-regulated kinase (ERK) phosphorylation, cytokine production, and degranulation in FcαRI-transfected RBL cells. In neutrophils, CRP induced FcαRI surface expression, phagocytosis, and TNF-α secretion. The ability of CRP to activate FcαRI defines a function for pentraxins in inflammatory responses involving neutrophils and macrophages. It also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors.
Assuntos
Antígenos CD/imunologia , Proteína C-Reativa/imunologia , Imunidade Inata/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Receptores Fc/imunologia , Antígenos CD/química , Antígenos CD/genética , Sítios de Ligação , Biomarcadores/química , Proteína C-Reativa/química , Proteína C-Reativa/genética , Complemento C1q/química , Complemento C1q/genética , Complemento C1q/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Humanos , Imunidade Inata/genética , Inflamação , Mutação , Fosforilação/genética , Fosforilação/imunologia , Ligação Proteica , Receptores Fc/química , Receptores Fc/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
C-reactive protein (CRP) is an acute-phase protein with therapeutic activity in mouse models of systemic lupus erythematosus and other inflammatory and autoimmune diseases. To determine the mechanism by which CRP suppresses immune complex disease, an adoptive transfer system was developed in a model of immune thrombocytopenic purpura (ITP). Injection of 200 microg of CRP 24 h before induction of ITP markedly decreased thrombocytopenia induced by anti-CD41. CRP-treated splenocytes also provided protection from ITP in adoptive transfer. Splenocytes from C57BL/6 mice were treated with 200 microg/ml CRP for 30 min, washed, and injected into mice 24 h before induction of ITP. Injection of 10(6) CRP-treated splenocytes protected mice from thrombocytopenia, as did i.v. Ig-treated but not BSA-treated splenocytes. The suppressive cell induced by CRP was found to be a macrophage by depletion, enrichment, and the use of purified bone marrow-derived macrophages. The induction of protection by CRP-treated cells was dependent on FcRgamma-chain and Syk activation, indicating an activating effect of CRP on the donor cell. Suppression of ITP by CRP-treated splenocytes required Fc gamma RI on the donor cell and Fc gamma RIIb in the recipient mice. These findings suggest that CRP generates suppressive macrophages through Fc gamma RI, which then act through an Fc gamma RIIb-dependent pathway in the recipient to decrease platelet clearance. These results provide insight into the mechanism of CRP regulatory activity in autoimmunity and suggest a potential new therapeutic approach to ITP.
Assuntos
Transferência Adotiva , Proteína C-Reativa/fisiologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Púrpura Trombocitopênica/imunologia , Receptores de IgG/fisiologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/prevenção & controle , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Proteína C-Reativa/administração & dosagem , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Doenças do Complexo Imune/imunologia , Doenças do Complexo Imune/prevenção & controle , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Púrpura Trombocitopênica/metabolismo , Púrpura Trombocitopênica/prevenção & controle , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q (refs 3 and 4). More recently, members of the pentraxin family were found to interact with cell-surface Fcgamma receptors (FcgammaR) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin's binding to FcgammaR and its functional activation of FcgammaR-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and FcgammaRIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and FcgammaRIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcgammaR isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for FcgammaR binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the FcgammaR pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.
Assuntos
Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Imunidade Inata/imunologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Componente Amiloide P Sérico/imunologia , Componente Amiloide P Sérico/metabolismo , Sítios de Ligação , Ligação Competitiva , Proteína C-Reativa/química , Cristalografia por Raios X , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Modelos Moleculares , Fagocitose , Conformação Proteica , Receptores de IgG/química , Componente Amiloide P Sérico/químicaRESUMO
C-reactive protein (CRP), the prototypic acute-phase protein, increases rapidly in response to infection and inflammation. Although CRP was thought to be a passive, nonspecific marker of inflammation, recent studies indicate that CRP plays a key role in the innate immune system by recognizing pathogens and altered self determinants. Activation of complement and interaction with Fcgamma receptors by CRP provides a link between the innate and adaptive immune systems. Recent evidence suggests that CRP is a marker of atherosclerotic disease and may play a role in its induction. However, CRP has an anti-inflammatory role in autoimmune diseases, such as systemic lupus erythematosus. In this article, we review the biological mechanisms by which CRP exerts its effects on the immune system and discuss its role in infection, cardiovascular disease, malignancy and systemic lupus erythematosus.
RESUMO
C-reactive protein (CRP) is a member of the pentraxin family of proteins and an acute phase reactant. CRP modulates the response to inflammatory stimuli including LPS and C5a. We recently demonstrated that CRP prevents and reverses proteinuria in accelerated nephrotoxic nephritis (NTN). NTN is a model of active inflammatory immune complex-mediated nephritis induced by injection of antiglomerular basement membrane. CRP treatment prevented the induction of NTN in C57BL/6 (B6) mice, increased survival, and reversed ongoing nephritis. Protection was associated with a decrease in IL-1beta and chemokines in the kidney and peritoneal cells as measured by quantitative RT-PCR. However, IL-10(-/-) mice were not protected by CRP either when given before disease onset or when disease activity was maximal. FcgammaRI(-/-) mice developed NTN, but were only transiently protected by CRP treatment. This transient protection was abrogated by cobra venom factor depletion of complement from FcgammaRI(-/-) mice. However, complement depletion did not prevent CRP-mediated protection in B6 mice, and CRP was protective in C3(-/-) mice. The role of macrophages in the protection provided by CRP was tested by treating B6 mice with liposomes containing clodronate. Clodronate-containing liposomes deplete mice of splenic and hepatic macrophages for 5-7 days. Pretreatment of NTN mice with clodronate but not control liposomes completely prevented CRP-mediated protection. These studies suggest that CRP mediates protection from NTN through the induction of IL-10 and that macrophages are required. In addition, FcgammaRI plays an important role but is not the sole mediator of CRP-mediated protection.
Assuntos
Proteína C-Reativa/farmacologia , Proteínas do Sistema Complemento/fisiologia , Glomerulonefrite Membranosa/prevenção & controle , Interleucina-10/metabolismo , Macrófagos/imunologia , Receptores de IgG/fisiologia , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Ácido Clodrônico/farmacologia , Complemento C3/genética , Proteínas do Sistema Complemento/genética , Modelos Animais de Doenças , Venenos Elapídicos/farmacologia , Perfilação da Expressão Gênica , Glomerulonefrite Membranosa/imunologia , Glomerulonefrite Membranosa/patologia , Terapia de Imunossupressão , Interleucina-10/genética , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteinúria/imunologia , Proteinúria/prevenção & controle , RNA Mensageiro/metabolismo , Receptores de IgG/efeitos dos fármacos , Receptores de IgG/genéticaRESUMO
OBJECTIVE: C-reactive protein (CRP) is an acute-phase serum protein with binding reactivity to nuclear autoantigens and immunomodulatory function. The MRL/lpr mouse is an important model of human systemic lupus erythematosus (SLE). These mice develop high-titer anti-DNA antibodies and immune complex-mediated nephritis and exhibit progressive lymphadenopathy. The mortality rate among these mice is 50% by age 18-20 weeks; the most frequent cause of death is glomerulonephritis. The present study was undertaken to determine whether treatment of mice with CRP would affect the course of lupus nephritis. METHODS: MRL/lpr mice were treated with a single 200-mug injection of CRP at either age 6 weeks (before disease onset) or age 13 or 15 weeks (when proteinuria had reached high levels). Proteinuria was measured weekly, and levels of anti-double-stranded DNA autoantibodies and blood urea nitrogen were determined monthly. Glomerular immune complex deposition and renal pathology were assessed in mice ages 15 weeks and 17 weeks. RESULTS: Early CRP treatment markedly delayed the onset of proteinuria and lymphadenopathy, increased survival, and reduced levels of autoantibodies to DNA. Treatment of mice with active disease reversed proteinuria and prolonged survival. Renal disease was decreased in CRP-treated mice, with a marked suppression of glomerular pathology, tubular degeneration, and interstitial inflammation, which correlated with the decrease in proteinuria and azotemia. CONCLUSION: These findings demonstrate that systemic suppression of autoimmunity is initiated by a single injection of CRP. Long-term maintenance of CRP-mediated protection was reversed by injection of an anti-CD25 monoclonal antibody but not by macrophage depletion, suggesting that disease suppression is maintained by CD25-bearing T cells.
Assuntos
Proteína C-Reativa/administração & dosagem , Nefrite/tratamento farmacológico , Nefrite/prevenção & controle , Animais , Feminino , Injeções , Camundongos , Camundongos Endogâmicos MRL lpr , Nefrite/patologia , Indução de RemissãoRESUMO
C-reactive protein (CRP) is the prototypical acute phase serum protein, rising rapidly in response to inflammation. CRP binds to phosphocholine (PC) and related molecules on microorganisms and plays an important role in host defense. However, a more important role may be the binding of CRP to PC in damaged membranes. CRP increases clearance of apoptotic cells, binds to nuclear antigens and by masking autoantigens from the immune system or enhancing their clearance, CRP may prevent autoimmunity. CRP binds to both the stimulatory receptors, FcgammaRI and FcgammaRIIa, increasing phagocytosis and the release of inflammatory cytokines; and to the inhibitory receptor, FcgammaRIIb, blocking activating signals. We have shown that, in two animal models of systemic lupus erythematosus (SLE), the (NZB x NZW)F1 mouse and the MRL/lpr mouse, a single injection of CRP before onset of proteinuria delayed disease development and late treatment reversed proteinuria. Thus, in these models, CRP plays an anti-inflammatory role.
Assuntos
Proteína C-Reativa/fisiologia , Mediadores da Inflamação/fisiologia , Animais , Proteína C-Reativa/biossíntese , Proteína C-Reativa/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , LigantesRESUMO
Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.
Assuntos
Antígenos CD/química , Antígenos CD/genética , Complemento C1q/química , Complemento C1q/genética , Mutagênese Sítio-Dirigida , Receptores de IgG/química , Receptores de IgG/genética , Alelos , Animais , Baculoviridae/genética , Sítios de Ligação , Células COS , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/química , Insetos , Células K562 , Leucina/química , Lisina/química , Modelos Moleculares , Mutagênese , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , TransfecçãoRESUMO
OBJECTIVE: To examine the ability of injection of C-reactive protein (CRP) to treat systemic lupus erythematosus (SLE) in the (NZB x NZW)F(1) (NZB/NZW) mouse and to use a nephrotoxic nephritis (NTN) model to further examine the mechanism of this activity. METHODS: NZB/NZW mice were given a single injection of 200 mug of CRP prior to disease onset or after the onset of high-grade proteinuria. Mice were monitored weekly for proteinuria and monthly for anti-double-stranded DNA (anti-dsDNA) antibodies. NTN was induced by immunization with rabbit IgG followed by rabbit anti-mouse glomerular basement membrane. Proteinuria was measured daily, and renal pathology was scored. CRP was injected at the time of disease induction or 9 days later. RESULTS: Treatment of NZB/NZW mice with CRP prior to disease onset delayed the onset of high-grade proteinuria by 16 weeks (P < 0.0001) and prolonged survival by 13 weeks (P < 0.002). CRP treatment of NZB/NZW mice during acute disease rapidly decreased proteinuria, and the treated mice remained aproteinuric for at least 10 weeks. Control and CRP-treated mice developed similar levels of anti-dsDNA. In C57BL/6 mice, injection of CRP either before or after induction of NTN suppressed proteinuria and glomerular pathology. CRP was completely ineffective in treating NTN in interleukin-10 (IL-10)-deficient mice. CONCLUSION: CRP injection suppresses inflammation in the kidney in SLE and NTN. The requirement for IL-10 in this protection suggests that CRP must rapidly initiate an IL-10-dependent antiinflammatory process. These findings suggest that a major function of CRP during the acute-phase response is to limit tissue damage and modulate acute inflammation.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Proteína C-Reativa/uso terapêutico , Proteinúria/tratamento farmacológico , Animais , Autoanticorpos/sangue , DNA/imunologia , Feminino , Imunização , Imunoglobulina G/imunologia , Interleucina-10/fisiologia , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZBRESUMO
Large interindividual variability in urinary arsenic profiles, following chronic inorganic arsenic exposure, is well-known in humans. To understand this variability, we studied the relationship between polymorphisms in the gene for human monomethylarsonic acid (MMA(V)) reductase/hGSTO1 and the urinary arsenic profiles of individuals chronically exposed to arsenic in their drinking water. To ensure that we did not overlook rare polymorphisms, not included in the public databases, we amplified and sequenced all six exons of the gene and their flanking regions, using DNA isolated from peripheral blood samples of 75 subjects, living in the vicinity of Torreon, Mexico. Four groups, based on the levels of arsenic (9-100 microg/L) in their drinking water, were studied. We identified six novel polymorphisms and two reported previously. The novel polymorphisms were a three base pair deletion (delGGC) in the first intron; a G > C transversion, leading to a serine-to-cysteine substitution at amino acid 86; a G > T transversion and a A > T transversion in intron 5; a G > A transition resulting in glutamate-to-lysine substitution in amino acid 208; and a C > T transition producing an alanine-to-valine substitution in amino acid 236. Two subjects displayed significant differences in patterns of urinary arsenic; they had increased levels of urinary inorganic arsenic and reduced levels of methylated urinary arsenic species as compared to the rest of the study population. These two subjects had the same unique polymorphisms in hGSTO1 in that they were heterozygous for E155del and Glu208Lys. The identified SNPs may be one of the reasons for the large interindividual variability in the response of humans to chronic inorganic arsenic exposure. The findings suggest the need for further studies to identify unambiguously specific polymorphisms that may account for interindividual variability in the human response to chronic inorganic arsenic exposure.
Assuntos
Arsênio/urina , Arsenicais/metabolismo , Glutationa Transferase/genética , Adolescente , Adulto , Idoso , Arsênio/química , Mapeamento Cromossômico , Primers do DNA/genética , Éxons/genética , Feminino , Genótipo , Glutationa Transferase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Expression of the yeast mitochondrial cytochrome b gene (COB) is controlled by at least 15 nuclear-encoded proteins. One of these proteins, Cbp1, is required for COB mRNA stability. Delta cbp1 null strains fail to accumulate mature COB mRNA and cannot respire. Since Delta cbp1 null strains lack mature COB transcripts, the hypothesis that Cbp1 also plays a role in translation of these mRNAs could not be tested previously. 5'-End trimming of precursor COB RNA and other mitochondrial transcripts is dependent on Pet127. pet127 mutants accumulate high levels of precursor COB mRNA and have no mature mRNA. pet127 mutants respire well; this phenotype implies that COB precursor RNA is translated efficiently. With the expectation that a Delta cbp1 Delta pet127 strain might accumulate substantial levels of COB RNA, the double null strain was constructed and analyzed to test the hypothesis that Cbp1 is required for translation of COB RNA. The Delta cbp1 Delta pet127 strain does accumulate levels of COB precursor mRNA that are approximately 60% of the level of COB mRNA in the wild-type strain. However, cytochrome b protein is not synthesized, and thus the Delta cbp1 Delta pet127 strain does not respire. These results suggest that Cbp1 is required for translation of COB RNAs.