Gabriela Salamone

Control of dendritic cell differentiation by angiotensin II

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.

Promotion of Neutrophil Apoptosis by TNF-α

We examined the ability of TNF-α to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-α alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-α for 1–2 min at 37°C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37°C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-α for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-α enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-α, there was no stimulation of apoptosis when TNF-α was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-α promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-α plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.

Extracellular acidosis induces neutrophil activation by a mechanism dependent on activation of phosphatidylinositol 3-kinase/Akt and ERK pathways.

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.

Bacterial DNA activates human neutrophils by a CpG-independent pathway

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 responsesis 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 single‐stranded 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.

Acidosis Improves Uptake of Antigens and MHC Class I-Restricted Presentation by Dendritic Cells

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.

Extracellular Acidosis Triggers the Maturation of Human Dendritic Cells and the Production of IL-12

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.

Human Seminal Plasma Abrogates the Capture and Transmission of Human Immunodeficiency Virus Type 1 to CD4+ T Cells Mediated by DC-SIGN

ABSTRACT Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is expressed by dendritic cells (DCs) at mucosal surfaces and appears to play an important role in the dissemination of human immunodeficiency virus type 1 (HIV-1) infection. DC-SIGN binds HIV-1 gp120 and efficiently transmits the virus to T CD4+ cells, which become the center of viral replication. Semen represents the main vector for HIV-1 dissemination worldwide. In the present study we show that human seminal plasma (SP), even when used at very high dilutions (1:104 to 1:105), markedly inhibits the capture and transmission of HIV-1 to T CD4+ cells mediated by both DCs and B-THP-1-DC-SIGN cells. In contrast, SP does not inhibit the capture of HIV-1 by DC-SIGN-negative target cells, such as the T-cell line SupT-1, monocytes, and activated peripheral blood mononuclear cells. The SP inhibitor has a high molecular mass (>100 kDa) and directly interacts with DC-SIGN-positive target cells but not with HIV-1. Moreover, the inhibitor binds to concanavalin A, suggesting that it contains high-mannose N-linked carbohydrates. Of note, using biotin-labeled SP we found that the binding of SP components to DCs was abrogated by mannan, while their interaction with B-THP-1 cells was almost completely dependent on the expression of DC-SIGN. Since epithelium integrity is often compromised after vaginal or anal intercourse, as well as in the presence of ulcerative-sexually transmitted diseases, our results support the notion that components of the SP might be able to access to the subepithelium, inhibiting the recognition of HIV-1 gp120 by DC-SIGN-positive DCs.

Histamine improves antigen uptake and cross-presentation by dendritic cells.

Previous studies have shown that histamine is able to modulate the function of dendritic cells (DCs). Histamine seems to be required for the normal differentiation of DCs. Moreover, it is capable of stimulating the chemotaxis of immature DCs and of promoting the differentiation of T CD4+ cells into a Th2 profile. In this study, we analyzed whether histamine was able to modulate endocytosis and cross-presentation mediated by immature DCs. Our results show that both functions are stimulated by histamine. Endocytosis of soluble HRP and FITC-OVA and cross-presentation of soluble OVA were markedly increased by histamine. Interestingly, stimulation of endocytosis and cross-presentation appeared to be mediated through different histamine receptors. In fact, the enhancement of endocytosis was prevented by the histamine2 receptor (H2R) antagonist cimetidine, whereas the stimulation of cross-presentation was prevented by the H3R/H4R antagonist thioperamide. Of note, contrasting with the observations made with soluble Ags, we found that histamine did not increase either the uptake of OVA-attached to latex beads, or the cross-presentation of OVA immobilized on latex beads. This suggests that the ability of histamine to increase endocytosis and cross-presentation is dependent on the Ag form and/or the mechanisms through which the Ag is internalized by DCs. Our results support that histamine may favor cross-presentation of soluble allergens by DCs enabling the activation of allergen-specific T CD8+ cells, which appears to play an important role in the development of allergic responses in the airway.

Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor-α

We have previously reported that human neutrophils pretreated with tumour necrosis factor‐α (TNF‐α) 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‐α 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‐α to promote apoptosis, supporting the notion than TNF‐α, acting together with IL‐1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation.

Acidic pH increases the avidity of FcγR for immune complexes

The interaction of immunoglobulin G (IgG) antibodies with FcγR constitutes a critical mechanism through which IgG antibody effector functions are mediated. In the current work we have examined whether human neutrophil FcγR exhibit pH dependence in their association with IgG. Binding assays were performed in culture medium adjusted to different pH values. It was found that the binding of either heat‐aggregated human IgG (AIgG), soluble immune complexes (sIC) or IgG‐coated erythrocytes (IgG‐E) was markedly higher at pHu20036·5 than at pHu20037·3. This effect was not observed when saturation of FcγR was achieved, suggesting that acidic pH increases the avidity of FcγR for IC without modifying the total binding capacity. Similar results were observed for the binding of AIgG to either monocytes, natural killer (NK) or K562 cells, suggesting that acidic pH increases the avidity of both, FcγRII and FcγRIII. Additional experiments were performed to analyse whether the binding of IgG to FcγRI also showed pH dependence. To this aim, we employed interferon‐γ‐treated human neutrophils and mouse inflammatory macrophages, previously incubated with blocking antibodies directed to FcγRII and FcγRIII. Acidic pH did not enhance the binding of AIgG nor monomeric IgG under these experimental conditions. Further studies are required to determine whether the enhancement of FcγR avidity for IC could be attributed to titration of histidine(s) residues on the Fc fragment of IgG.