RPTPε promotes M2-polarized macrophage migration through ROCK2 signaling and podosome formation.

Cysteinyl-leukotrienes (cys-LTs) have well-characterized physiopathological roles in the development of inflammatory diseases. We have previously found that protein tyrosine phosphatase ε (PTPε) is a signaling partner of CysLT1R, a high affinity receptor for leukotriene D4 (LTD4). There are two major isoforms of PTPε, receptor-like (RPTPε) and cytoplasmic (cyt-)PTPε, both of which are encoded by the PTPRE gene but from different promoters. In most cells, their expression is mutually exclusive, except in human primary monocytes, which express both isoforms. Here, we show differential PTPε isoform expression patterns between monocytes, M1 and M2 human monocyte-derived macrophages (hMDMs), with the expression of glycosylated forms of RPTPε predominantly in M2-polarized hMDMs. Using PTPε-specific siRNAs and expression of RPTPε and cyt-PTPε, we found that RPTPε is involved in monocyte adhesion and migration of M2-polarized hMDMs in response to LTD4 Altered organization of podosomes and higher phosphorylation of the inhibitory Y-722 residue of ROCK2 was also found in PTPε-siRNA-transfected cells. In conclusion, we show that differentiation and polarization of monocytes into M2-polarized hMDMs modulates the expression of PTPε isoforms and RPTPε is involved in podosome distribution, ROCK2 activation and migration in response to LTD4.

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.

Role of Protein Tyrosine Phosphatase Epsilon (PTPε) in Leukotriene D4-Induced CXCL8 Expression.

Phosphorylation on tyrosine residues is recognized as an important mechanism for connecting extracellular stimuli to cellular events and defines a variety of physiologic responses downstream of G protein-coupled receptor (GPCR) activation. To date, few protein tyrosine phosphatases (PTPs) have been shown to associate with GPCRs, and little is known about their role in GPCR signaling. To discover potential cysteinyl-leukotriene receptor (CysLT1R)-interacting proteins, we identified protein tyrosine phosphatase ε (PTPε) in a yeast two-hybrid assay. Since both proteins are closely linked to asthma, we further investigated their association. Using a human embryonic kidney cell line 293 (HEK-293) cell line stably transfected with the receptor (HEK-LT1), as well as human primary monocytes, we found that PTPε colocalized with CysLT1R in both resting and leukotriene D4 (LTD4)-stimulated cells. Cotransfection of HEK-LT1 with PTPε had no effect on CysLT1R expression or LTD4-induced internalization, but it inhibited LTD4-induced CXC chemokine 8 (CXCL8) promoter transactivation, protein expression, and secretion. Moreover, reduced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), but not of p38 or c-Jun-N-terminal kinase 1 or 2 mitogen-activated protein kinases (MAPKs), was observed upon LTD4 stimulation of HEK-LT1 coexpressing cytosolic (cyt-) PTPε, but not receptor (R) PTPε The increased interaction of cyt-PTPε and ERK1/2 after LTD4 stimulation was shown by coimmunoprecipitation. In addition, enhanced ERK1/2 phosphorylation and CXCL8 secretion were found in LTD4-stimulated human monocytes transfected with PTPε-specific siRNAs, adding support to a regulatory/inhibitory role of PTPε in CysLT1R signaling. Given that the prevalence of severe asthma is increasing, the identification of PTPε as a new potential therapeutic target may be of interest.

Regulation of platelet-activating factor-induced interleukin-8 expression by protein tyrosine phosphatase 1B.

BACKGROUND: Platelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation. METHODS: We used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting. RESULTS: Here, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression. CONCLUSION: The negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.

Constitutively active Stat5b signaling confers tolerogenic functions to dendritic cells of NOD mice and halts diabetes progression.

Defects in dendritic cells (DCs) development and function lead to autoimmune disorders. Autoimmune diabetes in humans and NOD mice results from a breakdown of self-tolerance, ending in T cell-mediated ß-cell destruction. DCs dysfunction in NOD mice results in part from a defect in the JAK-STAT5 signaling pathway associated with the idd4 susceptibility locus. The involvement of Stat5b in DCs tolerogenic functions remains unknown. We have generated transgenic mice (NOD.CD11cStat5b-CA) expressing a constitutively active form of the Stat5b gene (Stat5b-CA) under control of CD11c promoter. All NOD.CD11cStat5b-CA mice were protected against diabetes. Protection was associated with an increased in the pool and suppressive function of Tregs, a promotion of Th2 and Tc2 immune response and a decreased percentage of CD8+ T cells. Splenic DCs of NOD.CD11cStat5b-CA mice acquired a mature phenotype, promoted and induced better conversion of CD4+CD25-Foxp3- T cells into Tregs (CD4+CD25+Foxp3+ T cells) than DCs of NOD mice. Stat5b-CA.DC-educated CD4+CD25- T cells delayed diabetes onset whereas Stat5b-CA.DC-educated Tregs blocked ongoing diabetes in 8-10 weeks old NOD recipient mice. Importantly, injection of Stat5b.CA.DC to 8-10-week old NOD mice halted diabetes progression and educated their splenocytes to loose their diabetogenic potential when transferred to NOD.SCID mice. Our work is the first to report that an active form of Stat5b restored DCs tolerogenic functions that re-educated Tregs to re-establish and to sustain long-term protective immune response against diabetes in NOD mice.

Interleukin-15-mediated inflammation promotes non-alcoholic fatty liver disease.

Interleukin-15 (IL-15) is essential for the homeostasis of lymphoid cells particularly memory CD8(+) T cells and NK cells. These cells are abundant in the liver, and are implicated in obesity-associated pathogenic processes. Here we characterized obesity-associated metabolic and cellular changes in the liver of mice lacking IL-15 or IL-15Rα. High fat diet-induced accumulation of lipids was diminished in the livers of mice deficient for IL-15 or IL-15Rα. Expression of enzymes involved in the transport of lipids in the liver showed modest differences. More strikingly, the liver tissues of IL15-KO and IL15Rα-KO mice showed decreased expression of chemokines CCl2, CCL5 and CXCL10 and reduced infiltration of mononuclear cells. In vitro, IL-15 stimulation induced chemokine gene expression in wildtype hepatocytes, but not in IL15Rα-deficient hepatocytes. Our results show that IL-15 is implicated in the high fat diet-induced lipid accumulation and inflammation in the liver, leading to fatty liver disease.

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.

Effect of interleukin-6 receptor blockade on feto-maternal outcomes in a rat model of intrauterine inflammation.

AIM: To study the effect of blocking the inflammatory cascade with interleukin-6 receptor antibody (anti-IL-6R) on feto-maternal outcomes in a rat model. METHODS: Pregnant Sprague-Dawley rats (n = 38) were injected intraperitoneally (day 22) (control, anti-IL-6R 30 µg/kg, lipopolysaccharide [LPS] 250 µg/kg or 500 µg/kg alone or combined with anti-IL-6R) followed by preterm caesarian performed 12 h later. Resuscitated pups (n = 179) were given to surrogate mothers. Primary outcomes were maternal and pup mortality. RESULTS: Fifty percent of pregnant rats died after LPS 500 µg/kg + anti-IL-6R injection but none in other groups. Neonatal mortality at 24 h was 63% and 86% in LPS 500 µg/kg and LPS 500 µg/kg + anti-IL-6R groups, respectively (P < 0.05). Surviving pups in the latter group presented a severe growth deficit compared to the LPS 500 µg/kg group (P < 0.01) and showed no difference with controls for open field testing. Maternal cytokine analysis after LPS 500 µg/kg + anti-IL-6R injection showed a tendency for increased IL-1 production (P = 0.06). CONCLUSION: Paradoxically, the association of pregnancy, inflammation and anti-IL-6R increases the inflammatory effects of LPS.

Differential signaling defects associated with the M201V polymorphism in the cysteinyl leukotriene type 2 receptor.

The cysteinyl-leukotrienes (cysLTs) LTC(4), LTD(4), and LTE(4), are involved in a variety of inflammatory diseases, including asthma, and act on at least two distinct receptors, CysLT(1) and CysLT(2). Specific antagonists of CysLT(1) are currently used to control bronchoconstriction and inflammation in asthmatic patients. The potential role of CysLT(2) in asthma remains poorly understood. A polymorphism in the CysLT(2) gene, resulting in a single amino acid substitution (M201V), was found to be associated with asthma in three separate population studies. Here, we investigated whether the M201V mutation affected the affinity of CysLT(2) for its natural ligands and its signaling efficiency. Human embryonic kidney 293 cells were stably transfected with either wild-type (wt) or mutant (M201V) CysLT(2). Affinity of the M201V receptor for LTC(4) was reduced by 50%, whereas affinity for LTD(4) was essentially lost. LTC(4)-induced production of inositol phosphates (IPs) in M201V-expressing cells was significantly decreased at suboptimal concentrations of the ligand, but no difference was observed at high concentrations. In contrast, LTD(4)-induced IP production was 10- to 100-fold less in M201V- than in wt-expressing cells. Similar results were also observed with the transactivation of the interleukin-8 promoter induced by LTC(4) or LTD(4). Moreover, in contrast to wt-expressing cells, phosphorylation of nuclear factor κB p65 was absent in LTD(4)-stimulated M201V-expressing cells. Likewise, phosphorylation of c-Jun N-terminal kinase was not induced in LTD(4)-stimulated M201V cells, whereas activation of extracellular response kinase and p38 was maintained, at least at higher LTD(4) concentrations. Our results indicate that the M201V polymorphism drastically affects CysLT(2) responses to LTD(4) and less to LTC(4).

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 D(4) 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 PGE(2) 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 PGE(2), 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 D(4) stimulation. This effect is partially dependent on an endogenous production of PGs and IL-10 by DCs.

Caveolae facilitate but are not essential for platelet-activating factor-mediated calcium mobilization and extracellular signal-regulated kinase activation.

Certain proteins, including receptors and signaling molecules, are known to be enriched in caveolae and lipid rafts. Caveolin-1, the major structural protein of caveolae, specifically interacts with many signaling molecules and, thus, caveolae and lipid rafts are often seen as preassembled signaling platforms. A potential binding site for caveolin-1 is present in the platelet-activating factor receptor (PAFR) sequence, and many downstream signaling components of PAFR activation preferentially localize in caveolae. The aim of this study was to investigate whether the PAFR was localized in caveolae/lipid raft domains and, if so, what would be the significance of such localization for PAFR signaling. In this study, we demonstrate that PAFR localizes within membrane microdomains, in close proximity to caveolin-1 in living cells, with potential interaction through a caveolin-1-binding sequence in the PAFR C terminus. Caveolin-1, however, is not essential for PAFR localization in lipid rafts. Disruption of caveolae/lipid rafts with methyl-beta-cyclodextrin markedly reduced PAF-triggered inositol phosphate production and cytosolic calcium flux, suggesting that PAFR signaling through the Galphaq protein was critically dependent on integrity of lipid rafts and/or caveolae. Interestingly, whereas in caveolin-1-expressing cells lipid raft disruption markedly decreased PAFR-mediated activation of the ERK/MAPK pathway, in cells lacking caveolae, such as leukocytes, lipid raft disruption had either the same inhibitory effect (Ramos B cells) or no effect (monocytes) on PAFR capacity to signal through the ERK/MAPK pathway. In conclusion, PAFR appears to localize within caveolae or lipid rafts in different cell types, and this location may be important for specific signaling events.

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-1ß 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.

FGF2 in asthmatic airway-smooth-muscle-cell hyperplasia.

Airway smooth muscle (ASM)-cell hyperplasia is a cardinal feature of the remodeled airways in asthma and contributes to airway hyper-responsiveness. Several upregulated mediators are potentially involved in this architectural change. Recently, many investigators have turned their interest toward fibroblast growth factor (FGF)2. This opinion article describes the current knowledge on the biology of this growth factor, reviews the papers that have measured its baseline or allergen-induced expression in human asthmatics and summarizes observations supporting its role as an ASM cell mitogen. The possibility that FGF2 is involved in ASM-cell hyperplasia is raised, not only because it induces ASM-cell proliferation by itself but because of recent findings showing that FGF2 confers to ASM cells the ability to proliferate in response to different asthma mediators.

Regulation of platelet-activating factor-mediated interleukin-6 promoter activation by the 48 kDa but not the 45 kDa isoform of protein tyrosine phosphatase non-receptor type 2.

BACKGROUND: An underlying state of inflammation is thought to be an important cause of cardiovascular disease. Among cells involved in the early steps of atherosclerosis, monocyte-derived dendritic cells (Mo-DCs) respond to inflammatory stimuli, including platelet-activating factor (PAF), by the induction of various cytokines, such as interleukin 6 (IL-6). PAF is a potent phospholipid mediator involved in both the onset and progression of atherosclerosis. It mediates its effects by binding to its cognate G-protein coupled receptor, PAFR. Activation of PAFR-induced signaling pathways is tightly coordinated to ensure specific cell responses. RESULTS: Here, we report that PAF stimulated the phosphatase activity of both the 45 and 48 kDa isoforms of the protein tyrosine phosphatase non-receptor type 2 (PTPN2). However, we found that only the 48 kDa PTPN2 isoform has a role in PAFR-induced signal transduction, leading to activation of the IL-6 promoter. In luciferase reporter assays, expression of the 48 kDa, but not the 45 kDa, PTPN2 isoform increased human IL-6 (hIL-6) promoter activity by 40% after PAF stimulation of HEK-293 cells, stably transfected with PAFR (HEK-PAFR). Our results suggest that the differential localization of the PTPN2 isoforms and the differences in PAF-induced phosphatase activation may contribute to the divergent modulation of PAF-induced IL-6 promoter activation. The involvement of PTPN2 in PAF-induced IL-6 expression was confirmed in immature Mo-DCs (iMo-DCs), using siRNAs targeting the two isoforms of PTPN2, where siRNAs against the 48 kDa PTPN2 significantly inhibited PAF-stimulated IL-6 mRNA expression. Pharmacological inhibition of several signaling pathways suggested a role for PTPN2 in early signaling events. Results obtained by Western blot confirmed that PTPN2 increased the activation of the PI3K/Akt pathway via the modulation of protein kinase D (PKD) activity. WT PKD expression counteracted the effect of PTPN2 on PAF-induced IL-6 promoter transactivation and phosphorylation of Akt. Using siRNAs targeting the individual isoforms of PTPN2, we confirmed that these pathways were also active in iMo-DCs. CONCLUSION: Taken together, our data suggest that PTPN2, in an isoform-specific manner, could be involved in the positive regulation of PI3K/Akt activation, via the modulation of PKD activity, allowing for the maximal induction of PAF-stimulated IL-6 mRNA expression.

Leukotriene D4 up-regulates furin expression through CysLT1 receptor signaling.

Leukotriene (LT)D(4) is suggested to play a role in airway remodeling, which is characterized by fibrogenesis and airway smooth muscle cell hyperplasia. In this study, we investigated the effects of LTD(4) on the expression of furin, a proprotein convertase involved in the maturation/activation of several substrates implicated in the remodeling processes. HEK293 cells stably transfected with the CysLT1 receptor were used to study the transcriptional regulation of furin by LTD(4). Stimulation of the cells with LTD(4) resulted in a time- and concentration-dependent induction of furin mRNA and protein expression. The study of furin gene (fur) promoters P1, P1A, and P1B revealed a selective transactivation of the P1 promoter by LTD(4). Mutations in the activator protein (AP)-1-binding element of the P1 promoter resulted in the partial loss of transactivation by LTD(4). Binding of AP-1 transcription factor to fur P1 promoter after stimulation with LTD(4) was demonstrated by electrophoretic mobility shift assay, and supershift assays indicated the formation of c-Jun/c-Fos complexes. LTD(4) induced the maturation of the furin substrates membrane-type 1 matrix metalloproteinase and transforming growth factor-beta1, which was inhibited by the furin inhibitor alpha1-PDX. Finally, LTD(4) induced furin gene expression in monocytic THP-1 cells, which was abrogated using a selective CysLT1 receptor antagonist and inhibitors of the mitogen-activated protein kinases MEK-1, p38, and JunK. Our data show for the first time that LTD(4), via the CysLT1 receptor, can transcriptionally activate furin production with consequent maturation of furin substrates relevant to airway remodeling. These findings suggest that CysLT1 is involved in remodeling processes through modulation of furin transcription.

Interleukin-4 and interleukin-13 enhance human bronchial smooth muscle cell proliferation.

BACKGROUND: T(H)2 inflammation and bronchial smooth muscle cell (BSMC) hyperplasia are characteristic features of asthma, but whether these phenomena are linked remains unknown. This study aims to define the effect of the T(H)2 cytokines IL-4 and IL-13 on human BSMC proliferation when administered alone or in combination with the fibroblast growth factor 2 (FGF2) growth factor. In addition, the effects of the proinflammatory mediators TNFalpha and IL-1 beta and the involvement of members of the well-known family of platelet-derived growth factor (PDGF) mitogens were tested. METHODS: BSMC proliferation was measured by crystal violet staining and PDGF and PDGF receptor (PDGFR) expression were determined by RT-PCR, immunocytochemistry, ELISA, flow cytometry and dot plot analysis. RESULTS: Neither IL-4 nor IL-13 alone induced BSMC proliferation, despite both being potent inducers of PDGF-CC. However, following a pretreatment with FGF2, which increased PDGFR alpha chain expression, both IL-4 and IL-13 increased FGF2-induced BSMC proliferation in a time- and concentration-dependent manner. TNFalpha and IL-1 beta did not affect basal or FGF2-induced BSMC proliferation, but both proinflammatory mediators enhanced the proliferative synergism between FGF2 and the T(H)2 cytokines. CONCLUSIONS: IL-4 and IL-13 potently induce FGF2-primed BSMC proliferation via an autocrine loop involving PDGFRalpha and PDGF-CC, and this proliferative synergism is amplified by proinflammatory cytokines.

Lipopolysaccharide and hypoxia/ischemia induced IL-2 expression by microglia in neonatal brain.

Using a model of perinatal brain lesions induced by lipopolysaccharide and hypoxia/ischemia, we hypothesized that interleukin-2 (IL-2), a neurotoxic cytokine, was enhanced within injured brains. We showed that lipopolysaccharide and hypoxia/ischemia enhanced both intracerebral IL-2 mRNA and protein levels, with a maximum increase upon lipopolysaccharide and hypoxia/ischemia. The lack of detectable T lymphocytes suggested the synthesis of IL-2 by neural cells. Lipopolysaccharide and hypoxia triggered IL-2 synthesis by cultured microglia with a peak after exposure to lipopolysaccharide and hypoxia. Double-labeling showed, in vivo and in vitro, that IL-2 immunoreactivity was colocalized with a microglia/macrophage marker. These results disclosed the ability of microglia to produce IL-2 and also suggest the implication of IL-2 in neural cell death triggered by perinatal lipopolysaccharide and hypoxia/ischemia exposures.

Inverse agonist-induced signaling and down-regulation of the platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in several diseases such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G-protein-coupled receptor family. Following stimulation, PAFR becomes rapidly desensitized; this refractory state is dependent on PAFR phosphorylation, internalization and down-regulation. In this report, we show that the PAFR inverse agonist, WEB2086, can induce phosphorylation and down-regulation of PAFR. Using selective inhibitors, we determined that the agonist, PAF, and WEB2086 could induce phosphorylation of PAFR by PKC. Moreover, dominant-negative (DN) mutant of PKC isoforms beta inhibited WEB2086-stimulated PAFR phosphorylation, whereas PAF-stimulated phosphorylation was inhibited by DN PKCalpha and delta. WEB2086 also induced PAFR down-regulation which could be blocked by PKC inhibitors and by DN PKCbeta. WEB2086-induced down-regulation was dynamin-dependent but arrestin-independent. Unlike PAF, WEB2086-stimulated intracellular trafficking of PAFR was independent of Rab5. Specific inhibitors of lysosomal proteases and of proteasomes were both effective in reducing WEB2086-induced PAFR down-regulation, indicating the importance of receptor targeting to both lysosomes and proteasomes in long-term cell desensitization to WEB2086. These results indicate that although both agonists and inverse agonists induce receptor PAFR down-regulation, this may be accomplished through different signal transduction and trafficking pathways.

RNA-binding protein HuR is required for stabilization of SLC11A1 mRNA and SLC11A1 protein expression.

The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3' untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization plays an important role in the regulation of SLC11A1 expression. We also observe that down-regulation of HuR expression by RNA interference (RNAi) results in a decrease in SLC11A1 expression which can be restored by the addition of recombinant HuR protein to the RNAi-treated cells. Finally, we show that HuR overexpression in HL-60 cells significantly increases the SLC11A1 mRNA stability. Taken together, our data demonstrate that HuR is a key mediator of posttranscriptional regulation and expression of the SLC11A1 gene.

Controversy surrounding the increased expression of TGF beta 1 in asthma.

Asthma is a waxing and waning disease that leads to structural changes in the airways, such as subepithelial fibrosis, increased mass of airway smooth muscle and epithelial metaplasia. Such a remodeling of the airways further amplifies asthma symptoms, but its etiology is unknown. Transforming growth factor beta1 is a pleiotropic cytokine involved in many fibrotic, oncologic and immunologic diseases and is believed to play an essential role in airway remodeling that occurs in asthmatic patients. Since it is secreted in an inactive form, the overall activity of this cytokine is not exclusively determined by its level of expression, but also by extensive and complex post-translational mechanisms, which are all important in modulating the magnitude of the TGFbeta1 response. Even if TGFbeta1 upregulation in asthma is considered as a dogma by certain investigators in the field, the overall picture of the published literature is not that clear and the cellular origin of this cytokine in the airways of asthmatics is still a contemporaneous debate. On the other hand, it is becoming clear that TGFbeta1 signaling is increased in the lungs of asthmatics, which testifies the increased activity of this cytokine in asthma pathogenesis. The current work is an impartial and exhaustive compilation of the reported papers regarding the expression of TGFbeta1 in human asthmatics. For the sake of comparison, several studies performed in animal models of the disease are also included. Inconsistencies observed in human studies are discussed and conclusions as well as trends from the current state of the litterature on the matter are proposed. Finally, the different points of regulation that can affect the amplitude of the TGFbeta1 response are briefly revised and the possibility that TGFbeta1 is dysregulated at another level in asthma, rather than simply in its expression, is highlighted.