RESUMO
Conformation-specific Ags are ideal targets for mAb-based immunotherapy. Here, we demonstrate that the monomeric form of C-reactive protein (mCRP) is a specific therapeutic target for arthritis and nephritis in a murine model. Screening of >1800 anti-mCRP mAb clones identified 3C as a clone recognizing the monomeric, but not polymeric, form of CRP. The anti-mCRP mAb suppressed leukocyte infiltration in thioglycollate-induced peritonitis, attenuated rheumatoid arthritis symptoms in collagen Ab-induced arthritis model mice, and attenuated lupus nephritis symptoms in MRL/Mp-lpr/lpr lupus-prone model mice. These data suggest that the anti-mCRP mAb 3C has therapeutic potential against rheumatoid arthritis and lupus nephritis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proteína C-Reativa/imunologia , Imunoterapia/métodos , Nefrite Lúpica/imunologia , Peritonite/imunologia , Pleura/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Artrite Reumatoide/terapia , Modelos Animais de Doenças , Humanos , Nefrite Lúpica/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Endogâmicos MRL lpr , Peritonite/terapia , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , ToracenteseRESUMO
Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvß3 and α4ß1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvß3 and transmembrane αvß3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvß3. A peptide from site 2 of integrin ß1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvß3 through binding to site 2. sPLA2-IIA also activated integrins α4ß1 and α5ß1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.
Assuntos
Fosfolipases A2 do Grupo II/química , Integrina alfa4beta1/química , Integrina alfaVbeta3/química , Receptores de Vitronectina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetulus , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/genética , Fosfolipases A2 do Grupo II/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Células K562 , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de SinaisRESUMO
Background: C-reactive protein (CRP) is a dynamic protein that undergoes conformational changes between circulating native pentameric CRP (pCRP), pentameric symmetrical forms (pCRP*) and monomeric (or modified) CRP (mCRP) forms. mCRP exhibits strong pro-inflammatory activity and activates platelets, leukocytes, and endothelial cells. Abundant deposition of mCRP in inflamed tissues plays a role in several disease conditions, such as ischemia/reperfusion injury, Alzheimer's disease, and cardiovascular disease. Although pCRP is typically quantified rather than mCRP for clinical purposes, mCRP may be a more appropriate disease marker of inflammatory diseases. Therefore, simple methods for quantifying mCRP are needed. Methods: We developed a specific enzyme-linked immunosorbent assay (ELISA) to measure plasma levels of mCRP. Plasma mCRP concentration was measured in patients with adult-onset Still's disease (AOSD) (n=20), polymyalgia rheumatica (PMR) (n=20), rheumatoid arthritis (RA) (n=30), infection (n=50), and in control subjects (n=30) using the developed ELISA. Results: We demonstrated that mCRP is elevated in some inflammatory autoimmune diseases, particularly AOSD. The mCRP concentration was also significantly higher among AOSD patients than RA, PMR patients and controls (477 ng/ml, 77 ng/ml, 186 ng/ml, and 1.2 ng/ml, respectively). Also, the mCRP (×1,000)/pCRP ratio was significantly higher among AOSD patients than RA, PMR, and infection patients (3.5, 0.6, 1,6, and 2.0, respectively). Conclusion: The plasma mCRP levels are elevated in some autoimmune diseases, particularly AOSD. The plasma mCRP levels may therefore be a potentially useful biomarker for AOSD.