Inflammation in peripheral tissues is usually associated with the development of local acidosis; however, there are few studies aimed at analyzing the influence of acidosis on immune cells. We have shown previously that extracellular acidosis triggers human neutrophil activation, inducing a transient increase in intracellular Ca2+ concentration, a shape change response, the up-regulation of CD18 expression, and a delay of apoptosis. In this study, we analyzed the signaling pathways responsible for neutrophil activation. We found that acidosis triggers the phosphorylation of Akt (the main downstream target of PI3K) and ERK MAPK, but not that of p38 and JNK MAPK. No degradation of IκB was observed, supporting the hypothesis that NF-κB is not activated under acidosis. Inhibition of PI3K by wortmannin or LY294002 markedly decreased the shape change response and the induction of Ca2+ transients triggered by acidosis, whereas the inhibition of MEK by PD98059 or U0126 significantly inhibited the shape change response without affecting the induction of Ca2+ transients. We also found that acidosis not only induces a shape change response and the induction of Ca2+ transients in human neutrophils but also stimulates the endocytosis of FITC-OVA and FITC-dextran. Stimulation of endocytosis was partially prevented by inhibitors of PI3K and MEK. Together, our results support the notion that the stimulation of human neutrophils by extracellular acidosis is dependent on the activation of PI3K/Akt and ERK pathways. Of note, using mouse peritoneal neutrophils we observed that the enhancement of endocytosis induced by acidosis was associated with an improved ability to present extracellular Ags through a MHC class I-restricted pathway.

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-γ, but not IL-4, by Ag-specific CD4+ T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.

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We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-κB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-κB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-κB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9−/− but not in MyD88−/− mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.

SUMMARYWe have previously reported that human neutrophils pretreated with tumour necrosis factor-a (TNF-a) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12-myristate 13-acetate (PMA), C5a, fMLP, or granulocyte-macrophage colony-stimulating factor (GM-CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF-a is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac-1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down-regulation in expression of the anti-apoptotic protein, Mcl-1. Interestingly, we also found that the anti-apoptotic cytokine interleukin-1 (IL-1) improves the ability of TNF-a to promote apoptosis, supporting the notion than TNF-a, acting together with IL-1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation.