Maryse Thivierge

IL-13 and IL-4 Up-Regulate Cysteinyl Leukotriene 1 Receptor Expression in Human Monocytes and Macrophages

The cysteinyl (Cys) leukotrienes (LT)C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of asthma. The human LTD4 receptor (CysLT1R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT1R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT1R mRNA levels, with consequently enhanced CysLT1R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT1R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, but not IFN-γ, induced a similar pattern of CysLT1R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT1R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed enhanced responsiveness to LTD4, but not to LTB4, in terms of Ca2+ mobilization, as well as augmented chemotactic activity. Our findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT1R expression on monocytes and macrophages, and consequently their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases.

IL-5 Up-Regulates Cysteinyl Leukotriene 1 Receptor Expression in HL-60 Cells Differentiated into Eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD4 receptor, CysLT1R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD4 receptors. The present work was undertaken to study the potential modulation of CysLT1R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT1R mRNA expression, with consequently enhanced CysLT1R protein expression and function in HL-60/eos. CysLT1R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1–20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT1R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT1R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed enhanced responsiveness to LTD4, but not to LTB4 or platelet-activating factor, in terms of Ca2+ mobilization, and augmented the chemotactic response to LTD4. Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases.

Platelet-activating factor enhances interleukin-6 production by alveolar macrophages.

The production of the cytokine interleukin-6 (IL-6) by rat alveolar macrophages (AMs) was analyzed after their stimulation with muramyl dipeptide (1 microgram/ml), in the presence of graded concentrations of platelet-activating factor (PAF). Significantly enhanced production of IL-6 was observed at 10(-10) to 10(-8) mol/L PAF, with peak effect at 10(-10) mol/L. This enhancement was blocked by three structurally unrelated specific PAF receptor antagonists BN 52021, WEB 2170, and CV 3988. The biologically inactive PAF precursor/metabolite, lyso-PAF, and the enantiomer enantio-PAF failed to induce significant enhancement in IL-6 production. In parallel, addition of PAF to AM triggered leukotriene B4 (LTB4) release. Inhibition of 5-lipoxygenase pathway by AA-861 or MK 886 inhibited the PAF-induced augmentation of both IL-6 and LTB4 production, suggesting an implication of endogenous leukotrienes in this mechanism. Furthermore, addition of exogenous LTB4 to AMs could augment their IL-6 production, with peak activity at 10(-12) mol/L LTB4, and reverse the inhibitory effects of 5-lipoxygenase inhibitors. Taken together, these observations suggest that PAF can modulate lung immune and inflammatory responses by enhancing IL-6 production and that this activity may be dependent on secondary 5-lipoxygenase metabolites. This may have clinical relevance in PAF-mediated events in the lung, such as the cellular components of late-phase asthma.

Augmented expression of platelet-activating factor receptor gene by TNF-alpha through transcriptional activation in human monocytes.

Tumor necrosis factor α (TNF‐α) is a cytokine produced by activated monocytes and often associated with platelet‐activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G‐protein‐coupled receptor constitutively expressed on monocytes. TNF‐α (100–400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half‐life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24‐h‐treated cells without change in receptor affinity. Increased interleukin‐6 production in response to PAF was also found in 24‐h TNF‐α‐pretreated monocytes. These observations provide new evidence for TNF‐α and PAF interactions in human monocytes during inflammatory processes through up‐regulation of PAFR expression by TNF‐α. J. Leukoc. Biol. 61: 106–112; 1997.

Cysteinyl-Leukotriene Receptor Type 1 Expression and Function Is Down-Regulated during Monocyte-Derived Dendritic Cell Maturation with Zymosan: Involvement of IL-10 and Prostaglandins

TLRs sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). DCs have been shown to produce leukotrienes and, conversely, leukotrienes are known to modulate several DC functions. In this study, we examined the modulation of expression and function of cysteinyl-leukotriene receptor type 1 (CysLT1) on human monocyte-derived DCs during their differentiation and subsequent maturation with zymosan, a TLR2 agonist. Maturation of DCs with zymosan reduced CysLT1 mRNA levels and protein expression in a time-dependent fashion and was associated with a diminution of functional responsiveness to leukotriene D4 as assessed by intracellular calcium mobilization, CCL2 and CCL3 production, and chemotaxis. The effect of zymosan was mediated by both TLR2 and dectin-1 activation. Zymosan also induced a rapid expression of cyclooxygenase-2 and the production of PGE2 and IL-10. Addition of an anti-IL-10 neutralizing Ab or inhibitors of cyclooxygenase greatly reduced the ability of zymosan to down-regulate CysLT1 expression. Down-regulation of CysLT1 expression by zymosan could be reproduced by a combination of IL-10 and PGE2, and was dependent on MAPK activation. Taken together, our findings indicate that zymosan down-regulates CysLT1 expression in DCs with consequently reduced functional responsiveness of the cells to leukotriene D4 stimulation. This effect is partially dependent on an endogenous production of PGs and IL-10 by DCs.

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.

Modulation of platelet-activating factor receptor (PAFR) gene expression via NFκB in MonoMac-1 cells

The inflammatory phospholipid platelet-activating factor (PAF) exerts its actions through the activation of a specific G-protein-coupled receptor (PAFR) that is constitutively expressed on the cell surface of human monocytes and monocytic cell lines. The expression of PAFR can be upregulated by various inflammatory mediators that may amplify and perpetuate the pathogenic actions of PAF. We previously reported that PAFR expression could be upregulated by the inflammatory cytokines interferon g [1] and TNFa [2]. Phorbol-12-myristate 13-acetate (PMA) [3] and lipopolysaccharides (LPS) (unpublished data) could also modulate PAFR gene expression. The human leukocytic PAFR has been cloned by Nakamura et al. [4] and other groups in 1991. The promoter of this gene contains many sites for the Rel nuclear factor kB (NFkB). NFkB activation is observed in response to various stimuli among which TNFa and LPS (reviewed in 5). As these compounds can upregulate PAFR gene expression in monocytes, we hypothesized that NFkB activation could be a central element of this transcriptional activation. We decided to determine the role of NFkB activation in the increase of PAFR mRNA expression seen in the human monocytic cell line MonoMac-1 in response to TNF a.

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.

Upregulation of Cysteinyl-Leukotriene 1 Receptor Expression by Interleukin-5 in Human Leukocytes

The cysteinyl-leukotrienes (cysLTs), LTC4, LTD4, and LTE4 are potent lipid mediators implicated mainly in acute bronchoconstriction and chronic airway inflammation in asthma [1]. Blood eosinophils from asthmatic patients synthesize increased amounts of cys-LTs. Cys-LTs, in turn, have in vitro chemoattractant activity for human eosinophils. LTD4-induced eosinophil infiltration of the airway could be blocked by the cysLT antagonist MK-571 [2].