Sylvie Turcotte

Modulation of Leukotriene B4 Receptor-1 Expression by Dexamethasone: Potential Mechanism for Enhanced Neutrophil Survival

Glucocorticoids can down-regulate many inflammatory and immune responses and constitute a powerful therapeutic tool in a number of diseases. However, they have a somewhat paradoxical effect on neutrophils, in that they prolong their survival. Because leukotriene B4 (LTB4) can also extend neutrophil survival, we proposed that glucocorticoids could prevent neutrophil apoptosis by up-regulating their expression of the high-affinity LTB4 receptor (BLT1). Here we show that, indeed, dexamethasone (DEX) up-regulates the steady-state levels of BLT1 mRNA in human neutrophils. The effect was time and concentration dependent, being maximal at 4 h and at 10–100 nM DEX. The effect was also dependent on transcriptional activity, whereas BLT1 mRNA stability was not affected. DEX-induced up-regulation of BLT1 expression was prevented by pretreatment with the LTB4 antagonist LY255283. Moreover, LTB4 itself up-regulated the expression of BLT1 mRNA. BLT1 protein expression on neutrophils exposed to DEX for 24 h was also up-regulated 2- to 3-fold, and DEX-treated as well as LTB4-treated cells showed enhanced responsiveness to LTB4 in terms of intracellular Ca2+ mobilization and chemotaxis. Whereas DEX and LTB4 alone decreased neutrophil apoptosis by ∼50%, neutrophils treated with both LTB4 and DEX showed >90% survival at 24 h. Moreover, BLT1 antagonists prevented the increased neutrophil survival induced by DEX as well as by LTB4. Taken together, our results suggest that DEX-induced up-regulation of BLT1 expression in neutrophils may be one mechanism through which glucocorticoids can prolong neutrophil survival, namely by enhancing cell responses to the antiapoptotic effect of LTB4.

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10−10 M PAF. Sequential incubation of EC with PAF and TNFα resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNFα was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNFα, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Platelet-Activating Factor Induces Th17 Cell Differentiation

Th17 cells have been implicated in a number of inflammatory and autoimmune diseases. The phospholipid mediator platelet-activating factor (PAF) is found in increased concentrations in inflammatory lesions and has been shown to induce IL-6 production. We investigated whether PAF could affect the development of Th17 cells. Picomolar concentrations of PAF induced IL-23, IL-6, and IL-1β expression in monocyte-derived Langerhans cells (LCs) and in keratinocytes. Moreover, when LC were pretreated with PAF and then cocultured with anti-CD3- and anti-CD28-activated T cells, the latter developed a Th17 phenotype, with a significant increase in the expression of the transcriptional regulator RORγt and enhanced expression of IL-17, IL-21, and IL-22. PAF-induced Th17 development was prevented by the PAF receptor antagonist WEB2086 and by neutralizing antibodies to IL-23 and IL-6R. This may constitute a previously unknown stimulus for the development and persistence of inflammatory processes that could be amenable to pharmacologic intervention.

Enhanced cysteinyl-leukotriene type 1 receptor expression in T cells from house dust mite-allergic individuals following stimulation with Der p.

In order to determine the potential for allergen to modulate T cell expression of the CysLT1 receptor and responsiveness to leukotrienes, peripheral blood mononuclear cells from house dust mite-allergic or nonallergic individuals were incubated with D. pteronyssinus allergen (Der p). Baseline CysLT1 expression was similar in both groups of donors, but Der p significantly enhanced CysLT1 expression in CD4+ and CD8+ T cells of only allergic individuals and induced enhanced responsiveness of CD4+ T cells to LTD4 in terms of calcium mobilisation. This effect was prevented by the CysLT1 antagonist MK571. Der p also induced IL-4 and IL-10 production, and neutralizing antibody to IL-4 prevented both the enhanced CysLT1 expression and the enhanced responsiveness of T cells to LTD4 induced by Der p. In allergic individuals, Der p also induced T cell proliferation and a Th2-biased phenotype. Our data suggest that, in allergen-sensitized individuals, exposure to allergen can enhance T cell expression of CysLT1 receptors through a mechanism involving IL-4 production. This, in turn, would induce CD4+ T cell responsiveness to cysteinyl-leukotrienes and Th2 cell activation.

Differential Contribution of BLT1 and BLT2 to Leukotriene B4-Induced Human NK Cell Cytotoxicity and Migration

Accumulating evidence indicates that leukotriene B4 (LTB4) via its receptors BLT1 and/or BLT2 (BLTRs) could have an important role in regulating infection, tumour progression, inflammation, and autoimmune diseases. In the present study, we showed that LTB4 not only augments cytotoxicity by NK cells but also induces their migration. We found that approximately 30% of fresh NK cells express BLT1, 36% express BLT2, and 15% coexpress both receptors. The use of selective BLTR antagonists indicated that BLT1 was involved in both LTB4-induced migration and cytotoxicity, whereas BLT2 was involved exclusively in NK cell migration, but only in response to higher concentrations of LTB4. BLT1 and BLT2 expression increased after activation of NK cells with IL-2 and IL-15. These changes of BLTR expression by cytokines were reflected in enhanced NK cell responses to LTB4. Our findings suggest that BLT1 and BLT2 play differential roles in LTB4-induced modulation of NK cell activity.

Adhesion-independent synergy of monocytes and endothelial cells in cytokine production: regulation of IL-6 and GM–CSF production by PAF

Co-Cultures of monocytes (MO) and endothelial cells (EC) were studied for their capacity to synergize in the production of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM–CSF), two cytokines potentially important in vascular physiopathology. Resting monocytes produced detectable amounts of IL-6 but no GM–CSF, whereas confluent EC produced significant quantities of GM–CSF, but minimal IL-6. In co-cultures without stimuli, additive synthesis of both cytokines was observed. When EC were pretreated, however, with either PAF, TNF or both stimuli, before addition of MO, synergistic production of IL-6 was observed. In contrast, GM–CSF production was not enhanced by coculture of monocytes with activated EC. When either cell population was fixed with paraformaldehyde or killed by freeze-thawing before addition to the co-culture, cytokine levels reverted to those produced by the unaffected population alone. On the other hand, separating the two cell populations by a cell-impermeable membrane in transwell cultures did not affect the synergistic production of the cytokines. Taken together, our data suggest that EC and MO can synergize in response to stimuli by producing IL-6 and that this synergy is dependent on the integrity of both cell populations, but independent of cell-cell contact.

IL-33 Upregulates Cysteinyl Leukotriene Receptor Type 1 Expression in Human Peripheral Blood CD4+ T Lymphocytes

IL-33 and cysteinyl leukotrienes (cysLTs) are key components of asthma pathogenesis, and both contribute to the initiation and maintenance of the type 2 inflammatory environment. However, little is known about the potential interactions between the two mediators. In this work, we aimed at studying the regulation of expression of the cysLT receptors CysLT1 and CysLT2 by IL-33 in human PBLs. Our results show that the IL-33/ST2L axis increases CysLT1 but not CysLT2 expression in a concentration- and time-dependent manner in PBLs. IL-33–induced CysLT1 upregulation was observed at the protein but not at the mRNA level and was accompanied by an increase in LTD4-induced calcium mobilization and migration of CD4+ T lymphocytes. We also show that purified naive CD4+ T lymphocytes expressed ST2L and responded to IL-33 in the absence of Ag or TCR stimulation, suggesting a mechanism independent of Ag presentation. These results contribute to expanding our knowledge in the field of IL-33 by proposing a new mode of action of the cytokine on T cells and by extending its role to the regulation of naive T cell trafficking, therefore reinforcing its interest as a potential therapeutic target for the treatment of asthma.