RESUMEN
The nuclear protein high mobility group box protein 1 (HMGB1) promotes inflammation upon extracellular release. HMGB1 induces proinflammatory cytokine production in macrophages via Toll-like receptor (TLR)-4 signaling in a redox-dependent fashion. Independent of its redox state and endogenous cytokine-inducing ability, HMGB1 can form highly immunostimulatory complexes by interaction with certain proinflammatory mediators. Such complexes have the ability to enhance the induced immune response up to 100-fold, compared with induction by the ligand alone. To clarify the mechanisms for these strong synergistic effects, we studied receptor requirements. Interleukin (IL)-6 production was assessed in supernatants from cultured peritoneal macrophages from mice each deficient in one of the HMGB1 receptors (receptor for advanced glycation end products [RAGE], TLR2 or TLR4) or from wild-type controls. The cultures were stimulated with the TLR4 ligand lipopolysaccaride (LPS), the TLR2 ligand Pam3CysSerLys4 (Pam3CSK4), noninflammatory HMGB1 or each TLR ligand in complex with noninflammatory HMGB1. The activity of the HMGB1-TLR ligand complexes relied on engagement of the same receptor as for the noncomplexed TLR ligand, since HMGB1-LPS complexes used TLR4 and HMGB1-Pam3CSK4 complexes used TLR2. Deletion of any of the intracellular adaptor molecules used by TLR2 (myeloid differentiation factor-88 [MyD88], TIR domain-containing adaptor protein [TIRAP]) or TLR4 (MyD88, TIRAP, TIR domain-containing adaptor-inducing interferon-ß [TRIF], TRIF-related adaptor molecule [TRAM]) had similar effects on HMGB1 complex activation compared with noncomplexed LPS or Pam3CSK4. This result implies that the enhancing effects of HMGB1-partner molecule complexes are not regulated by the induction of additional signaling cascades. Elucidating HMGB1 receptor usage in processes where HMGB1 acts alone or in complex with other molecules is essential for the understanding of basic HMGB1 biology and for designing HMGB1-targeted therapies.
Asunto(s)
Citocinas/metabolismo , Proteína HMGB1/metabolismo , Animales , Células Cultivadas , Proteína HMGB1/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Ratas , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The nuclear protein HMGB1 has previously been demonstrated to act as an alarmin and to promote inflammation upon extracellular release, yet its mode of action is still not well defined. Access to highly purified HMGB1 preparations from prokaryotic and eukaryotic sources enabled studies of activation of human PBMC or synovial fibroblast cultures in response to HMGB1 alone or after binding to cofactors. HMGB1 on its own could not induce detectable IL-6 production. However, strong enhancing effects on induction of proinflammatory cytokine production occurred when the protein associated with each of the separate proinflammatory molecules, rhIL-1beta, the TLR4 ligand LPS, the TLR9 ligand CpG-ODN, or the TLR1-TLR2 ligand Pam3CSK4. The bioactivities were recorded in cocultures with preformed HMGB1 complexes but not after sequential or simultaneous addition of HMGB1 and the individual ligands. Individual A-box and B-box domains of HMGB1 had the ability to bind LPS and enhance IL-6 production. Heat denaturation of HMGB1 eliminated this enhancement. Cocultures with HMGB1 and other proinflammatory molecules such as TNF, RANKL, or IL-18 did not induce enhancement. HMGB1 thus acts broadly with many but not all immunostimulatory molecules to amplify their activity in a synergistic manner.
Asunto(s)
Proteína HMGB1/inmunología , Mediadores de Inflamación/inmunología , Inflamación/inmunología , Transducción de Señal/inmunología , Animales , Artritis Reumatoide/patología , Biotinilación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Citocinas/análisis , Fibroblastos/inmunología , Fibroblastos/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/aislamiento & purificación , Proteína HMGB1/farmacología , Humanos , Interleucina-1beta/inmunología , Interleucina-6/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Ligandos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Oligodesoxirribonucleótidos/inmunología , Ratas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA), although their mechanism of action is not well defined. As the proinflammatory mediator high mobility group box chromosomal protein 1 (HMGB1) may play a role in the pathogenesis of RA, we have performed in vitro studies to investigate whether GST inhibits HMGB1 release as the basis of its mode of action. Murine RAW 264.7 or human THP-1 macrophage cells were stimulated in culture with agents causing extracellular HMGB1 release, including LPS, IFN-gamma, polyinosinic:polycytidylic acid, IFN-beta, or NO in the presence of GST, ranging from 0 microM to 250 microM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1-specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN-beta levels were analyzed by ELISPOT and/or ELISA. Supernatant NO production was analyzed by the Griess method. At pharmacologically relevant doses, GST inhibited the extracellular release of HMGB1 from activated macrophages and caused the nuclear retention of this protein; in contrast, no effects were observed on the secretion or production of TNF. Release of the key endogenous mediators of HMGB1 translocation, IFN-beta and NO, was inhibited by GST. This inhibition required gold, as sodium thiomalate did not affect the responses measured. Furthermore, gold chloride also inhibited release of HMGB1. Together, these results suggest a new mechanism for the anti-rheumatic effects of gold salts in RA and the potential of drugs, which interfere with intracellular HMGB1 transport mechanisms, as novel agents to treat RA.
Asunto(s)
Núcleo Celular/metabolismo , Tiomalato Sódico de Oro/farmacología , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/inmunología , Animales , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/inmunología , Tiomalato Sódico de Oro/uso terapéutico , Proteína HMGB1/inmunología , Humanos , Interferón beta/efectos de los fármacos , Interferón beta/metabolismo , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
High-mobility group chromosomal box protein 1 (HMGB1) is a structural nuclear protein that promotes inflammation when present extracellularly. Aberrant, extracellular HMGB1 expression has been demonstrated in human and experimental synovitis. The aim of the present study was to elucidate the temporal and spatial expression of HMGB1 compared to that of the central mediators tumor necrosis factor (TNF) and interleukin-1-beta (IL-1beta) during the course of collagen-induced arthritis. Thus, Dark Agouti rats were immunized with homologous type II collagen and synovial tissue specimens were obtained at various time points prior to and during the course of clinical arthritis. Local cytokine responses were assessed by immunohistochemistry and by in situ hybridization. We demonstrate a distinct nuclear expression of HMGB1 at early disease-preceding time points. Preceding clinical onset by a few days, cytoplasmic HMGB1 expression was evident in synoviocytes within the non-proliferative lining layer. Pronounced cytoplasmic and additional extracellular HMGB1 expression coincided with the progression of clinical disease. In advanced arthritis, the number of cells with cytoplasmic HMGB1 expression was quantitatively comparable to that of cells expressing TNF and IL-1beta. Interestingly, although HMGB1 was abundantly expressed throughout the inflamed synovium at a protein level, upregulation of HMGB1 mRNA was restricted mainly to areas of cartilage and bone destruction. In conclusion, these new findings implicate a role for HMGB1 in both inducing and perpetuating inflammatory events of significant importance in the destructive processes in chronic arthritis.