Analía Trevani

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.

Desiccating stress‐induced disruption of ocular surface immune tolerance drives dry eye disease

Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surfaces mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF‐κB)‐ and time‐dependent disruption of the ocular surfaces immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF‐κB inhibitors reduced corneal epithelial damage and interleukin (IL)‐1β and IL‐6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF‐κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF‐κB activation could have therapeutic potential in dry eye.

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.

Immune complexes inhibit apoptosis of chronic lymphocytic leukaemia B cells

In the present study we examined the effect of immune complexes (IC) on the survival of chronic lymphocytic leukaemia (B‐CLL) B cells. Our results showed that either precipitating IC (pIC), Ab‐coated erythrocytes (E‐IgG) or heat‐aggregated IgG (aIgG) significantly inhibited spontaneous apoptosis of B‐CLL cells, as well as that induced by fludarabine, chlorambucil or dexamethasone. After depletion of T lymphocytes, monocytes and NK cells, incubation with IC was no longer able to delay B‐CLL cells apoptosis, suggesting that prevention of apoptosis depends on IC interaction with accessory leucocytes. The release of IFNγ by non‐malignant cells upon activation with IC was responsible, to some extent, for IC effects as shown by the fact that neutralizing anti‐IFNγ MoAb partially prevented their ability to inhibit B‐CLL cells apoptosis. The observation that treatment with IC resulted in increased expression of HLA‐DR on B‐CLL cells suggests that inhibition of apoptosis is associated with cellular activation.

Mucosal tolerance disruption favors disease progression in an extraorbital lacrimal gland excision model of murine dry eye

Dry eye is a highly prevalent immune disorder characterized by a dysfunctional tear film and a Th1/Th17xa0T cell response at the ocular surface. The specificity of these pathogenic effector T cells remains to be determined, but auto-reactivity is considered likely. However, we have previously shown that ocular mucosal tolerance to an exogenous antigen is disrupted in a scopolamine-induced murine dry eye model and that it is actually responsible for disease progression. Here we report comparable findings in an entirely different murine model of dry eye that involves resection of the extraorbital lacrimal glands but no systemic muscarinic receptor blockade. Upon ocular instillation of ovalbumin, a delayed breakdown in mucosal tolerance to this antigen was observed in excised but not in sham-operated mice, which was mediated by interferon γ- and interleukin 17-producing antigen-specific T cells. Consistently, antigen-specific regulatory T cells were detectable in sham-operated but not in excised mice. As for other models of ocular surface disorders, epithelial activation of the NF-κB pathway by desiccating stress was determinant in the mucosal immune outcome. Underscoring the role of mucosal tolerance disruption in dry eye pathogenesis, its prevention by a topical NF-κB inhibitor led to reduced corneal damage in excised mice. Altogether these results show that surgically originated desiccating stress also initiates an abnormal Th1/Th17xa0T cell response to harmless exogenous antigens that reach the ocular surface. This event might actually contribute to corneal damage and challenges the conception of dry eye as a strictly autoimmune disease.