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.

Here we analyze the role of the angiotensinergic system in the differentiation of dendritic cells (DC). We found that human monocytes produce angiotensin II (AII) and express AT1 and AT2 receptors for AII. DC differentiated from human monocytes in the presence of AT1 receptor antagonists losartan or candesartan show very low levels of CD1a expression and poor endocytic and allostimulatory activities. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in the development of nonadherent cells with CD1a expression and endocytic and allostimulatory activities higher than control DC. Similar contrasting effects were observed in mouse DC obtained from bone marrow cultures supplemented with granulocyte-monocyte colony-stimulating factor. DC differentiated in the presence of the AT1 receptor antagonist losartan express lower levels of CD11c, CD40, and Ia and display a lower ability to endocyte horseradish peroxidase (HRP) and to induce antibody responses in vivo, compared with controls. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in cells with high levels of CD11c, CD40, and Ia, as well as high ability to endocyte HRP and to induce antibody responses in vivo. Our results support the notion that the differentiation of DC is regulated by AII.

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8+ CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration. Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA. However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both singlestranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN-γ, and the proliferation of γδ T cells induced by (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophilderived ROS. We also show that the ROS-generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi-directional cross-talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation. Keywords: γδ T cells r Neutrophils r ROSAdditional supporting information may be found in the online version of this article at the publisher's web-site Introduction γδ T cells constitute a functionally specialized subset of T lymphocytes that play an important role in linking the innate and adaptive immunity [1]. γδ T cells expressing the Vγ9Vδ2TCR are only found in higher primates and humans [2]. They represent the vast majority of γδ T cells in human peripheral blood [3]. In healthy Correspondence: Dr. Carolina C. Jancic e-mail: cjancic@hematologia.anm.edu.ar adults, they comprise about 0.5-5% of circulating T cells [4], however, the number of Vγ9Vδ2 T cells can dramatically increase during the early response to many viral, bacterial, and parasitic infections, at times comprising up to >50% of all circulating T cells within a few days [5]. Vγ9Vδ2 T cells acquire a preactivated phenotype early in their development allowing the rapid induction of a wide variety of functions upon stimulation in a non-MHC restricted manner by phosphoantigens such as (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate (HMBPP) [6][7][8][9]. These functions include a highly cytotoxic response against infected and transformed cells, the production of a range of cytokines and chemokines, the recruitment and the activation of neutrophils, the differentiation of C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 820Florencia Sabbione et al. Eur. J. Immunol. 2014. 44: 819-830 monocytes into a proinflammatory profile, the phenotypic maturation of dendritic cells (DCs), the polarization of CD4 + T cells into a Th1 profile, the promotion of B-cell activation, and the presentation of antigenic peptides to both CD4 + and CD8 + T cells [...