Stressed cells undergoing necrosis release molecules that acts as endogenous danger signals to alert and activate innate immune cells. Both HMGB1 and HSP70 are induced in activated monocytes/macrophages and also are released from stressed or injured cells. We investigated whether HMGB1 and HSP70 released from necrotic monocytes/macrophages, can act as danger signals to mediate proinflammatory cytokine responses to bacterial endotoxin or lipopolysaccharide (LPS). We show that cell lysate, obtained from necrotic cells directly stimulates the proinflammatory cytokine and chemokine responses in human monocyte/ macrophage cell line, THP-1, as revealed by the induction of TNF-α, IL-6 and IL-8 mRNA expression and protein production. In the presence of LPS, necrotic cell lysate induced a more robust increase in all three proteins. We found that HMGB1 and HSP70 were indeed present in the necrotic cell lysate and were responsible for the significant induction of the proinflammatory cytokine expression, as neutralization with antibodies against both proteins blocked the increase in the cytokine production seen after incubating LPS-stimulated cells with the necrotic cell lysate. We also found that the newly identified triggering receptor expressed on myeloid cells-1 (TREM-1) was involved in mediating the HMGB1-and HSP70-induced cytokine production. Blocking TREM-1 on THP-1 cells with a recombinant chimera prevented the increase in cytokine production, while simultaneous blocking of TLR4 and TREM-1 completely abolished the proinflammatory response, suggesting that TREM-1 synergizes with TLR4 to mediate the effects of such signals from necrotic cells. In addition, blocking HMGB1 or HSP70 simultaneously with TREM-1 did not decrease the cytokine level further, confirming the involvement of TREM-1 in mediating the effect of HMGB1 and HSP70. Although the interaction of HMGB1 and HSP70 with TREM-1 induced IκBα and p38 expression, both of which are required for the inflammatory cytokine expression, blockade of TREM-1 did not affect IκBα expression but markedly reduced p38 activation, as revealed by Western blot analysis. Together, these results demonstrate that HMGB1 and HSP70 released from necrotic cells function as endogenous danger signals to augment the proinflammatory responses in monocytes/macrophage and that TREM-1 relays such signals to the cytokine expression cascade. This mechanism may contribute to the amplification and persistence of the inflammatory response to bacterial infection.

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Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

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Summary Age-associated changes in immune response increase the risk of infection and promote inflammation and auto-immunity in older adults. The newly discovered cytokine IL-23 contributes to the maintenance and expansion of Th-17 cells, which promote proinflammatory responses. Our preliminary findings suggested that Th-17 responses are increased in aged mice. IL-23 consists of p40 and p19 subunits. Expression of the p19 subunit is regulated at the transcriptional level by NF-κB p65 and c-Rel transcription factors. Using bone-marrow-derived dendritic cells (DCs) from C57BL/6 mice, we show that IL-23 protein production and p19 subunit mRNA levels are significantly increased in DCs from aged mice after activation with TLR ligands (LPS + R848) when compared with DCs of young adult mice. We found that the increase in p19 expression in aged cells is associated with chromatin remodeling characterized by di- and tri-methylation of histone H3K4 and binding of mainly c-Rel at the p19 promoter. In young DCs, the promoter is tri-methylated only at H3K4 and bound by both p65 and c-Rel. C-Rel knockdown restores p65 binding in aged cells but does not activate p19 expression, suggesting that c-Rel is critical for p19 expression. In addition, p65 knockdown significantly increases c-Rel binding and p19 expression in young DCs to levels close to those detected in old cells. Furthermore, the decrease in p65 binding at the p19 promoter in old DCs was specific to the p19 gene since p65 binding to the IL-12p40 promoter was not significantly different between old and young DCs. Our results demonstrate that selective changes in H3K4 methylation, and c-Rel and p65 binding at the p19 promoter occur in DCs and contribute to the upregulation of the p19 subunit expression and IL-23 protein production observed in aged mice. This suggests epigenetic and transcriptional mechanisms contribute to dysregulated inflammatory and autoimmune responses associated with aging.

CD4+ T cells of the Th17 subtype are over-represented in the aged immune system. Dendritic cells (DC) play a critical role in naive CD4+ T cell differentiation. However, expression of cytokines by aged DC that promote differentiation or survival of Th17 cells has not been extensively investigated. Using bone marrow-derived DC from C57BL/6 mice of different ages we compared cytokine production after DC activation by Toll-like receptor agonists for TLR4 and/or TLR7/8. DC-derived TNF-α and IL-12p70 production and expression of DC co-stimulatory molecules did not vary significantly by age indicating TLR expression, function and signal transduction were intact in aged DC. There were relatively minor age-related changes in TGF-β and IL-6 which promote Th17 differentiation, but IL-23, a Th17-suvival cytokine, increased more than 40-fold across the lifespan. DC-derived prostaglandin E2 (PGE2) also increased with age and the up-regulation of IL-23 expression by aged DC was blocked by indomethacin that prevents PGE2 production, and by antagonists of PGE2 receptors. Exogenous PGE2 added to DC cultures further enhanced IL-23 production from aged but not young DCs. These data indicate that age-related changes in DC PGE2 production are necessary, but not sufficient to induce DC IL-23 production. Such changes may play a role in the expansion of Th17 cells in the aged immune system.