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.

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.

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.

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.

Regulation of platelet-activating factor-mediated protein tyrosine phosphatase 1B activation by a Janus kinase 2/calpain pathway.

Atherosclerosis is a pro-inflammatory condition underlying many cardiovascular diseases. Platelet-activating factor (PAF) and interleukin 6 (IL-6) are actively involved in the onset and progression of atherosclerotic plaques. The involvement of monocyte-derived macrophages is well characterized in the installation of inflammatory conditions in the plaque, but less is known about the contribution of monocyte-derived dendritic cells (Mo-DCs). In the same way, the involvement of calcium, phospholipase C and A2 in PAF-induced IL-6 production, in different cells types, has been shown; however, the importance of the Jak/STAT pathway and its regulation by protein-tyrosine phosphatases in this response have not been addressed. In this study, we report that PAF stimulates PTP1B activity via Jak2, thereby modulating PAF-induced IL-6 production. Using HEK 293 cells stably transfected with the PAF receptor in order to discriminate the pathway components, our results suggest that Jak2 modulates PAF-induced IL-6 production via both positive and negative pathways. Jak2 kinase activity was necessary for maximal transactivation of the IL-6 promoter, as seen by luciferase assays, whereas the same kinase also downregulated this promoter transactivation through the activation of a calcium/calpain/PTP1B pathway. The same pathways were operational in monocyte-derived dendritic cells, since PAF-induced PTP1B activation negatively regulated PAF-induced IL-6 mRNA production and, in addition, Jak2 activated calpain, one of the components involved in PAF-induced PTP1B activation. Results obtained in this study indicate that Jak2 activation is important for maximal IL-6 promoter transactivation by PAF and that PTP1B is involved in the negative regulation of this transactivation. However, PTP1B does not directly regulate Jak2 activation, but rather Jak2 regulates PAF-induced PTP1B activation.

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.

Correction: Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0162995.].

Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues.

OBJECTIVE: IL-15 is an inflammatory cytokine secreted by many cell types. IL-15 is also produced during physical exercise by skeletal muscle and has been reported to reduce weight gain in mice. Contrarily, our findings on IL-15 knockout (KO) mice indicate that IL-15 promotes obesity. The aim of this study is to investigate the mechanisms underlying the pro-obesity role of IL-15 in adipose tissues. METHODS: Control and IL-15 KO mice were maintained on high fat diet (HFD) or normal control diet. After 16 weeks, body weight, adipose tissue and skeletal mass, serum lipid levels and gene/protein expression in the adipose tissues were evaluated. The effect of IL-15 on thermogenesis and oxygen consumption was also studied in primary cultures of adipocytes differentiated from mouse preadipocyte and human stem cells. RESULTS: Our results show that IL-15 deficiency prevents diet-induced weight gain and accumulation of lipids in visceral and subcutaneous white and brown adipose tissues. Gene expression analysis also revealed elevated expression of genes associated with adaptive thermogenesis in the brown and subcutaneous adipose tissues of IL-15 KO mice. Accordingly, oxygen consumption was increased in the brown adipocytes from IL-15 KO mice. In addition, IL-15 KO mice showed decreased expression of pro-inflammatory mediators in their adipose tissues. CONCLUSIONS: Absence of IL-15 results in decreased accumulation of fat in the white adipose tissues and increased lipid utilization via adaptive thermogenesis. IL-15 also promotes inflammation in adipose tissues that could sustain chronic inflammation leading to obesity-associated metabolic syndrome.

Cellular signalling of cysteinyl leukotriene type 1 receptor variants CysLT1-G300S and CysLT1-I206S.

Cysteinyl-leukotrienes are pro-inflammatory lipid mediators, involved in allergic asthma, that bind the G-protein-coupled receptors CysLT1, CysLT2 and GPR99. A polymorphism in one of these receptors, CysLT1-G300S was strongly associated with atopy, whereas the CysLT1-I206S polymorphism was not. In the present work, our aim was to characterize these two variants by studying their cellular signalling. Cell surface expression of mutant receptors in transfected HEK-293 cells was comparable to that of the wild-type receptor. Compared to CysLT1-WT, production of inositol phosphates as well as IL-8 and IL-13 promoter transactivation in response to either LTD4 or LTC4 was significantly increased in CysLT1-G300S-transfected cells. Moreover, LTD4-induced phosphorylation of the signalling effector Erk, but not p38, p65 or c-Jun was higher in CysLT1-G300S-transfected cells. On the other hand, the variant CysLT1-I206S did not show a significant difference in its signal transduction compared to the wild-type receptor. Taken together, our results indicate that the variant CysLT1-G300S can induce a greater signal than the CysLT1-WT receptor, a feature that may be relevant to its association with atopy.

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.

Impaired immune regulation after radioiodine therapy for Graves' disease and the protective effect of Methimazole.

Both therapies for Graves' disease (GD), radioactive iodine (RAI) and antithyroid drugs (ATD), were reported to have specific immune effects. We aimed at investigating the effects of RAI therapy on cellular subsets involved in immune regulation. We conducted a thirty day follow-up prospective cohort study of adult patients. Patients eligible for RAI therapy at our centre were approached. Twenty seven patients with GD were recruited, among whom 11 were treated with ATD. Twenty-two healthy subjects (HS) were also studied. Over time, frequency of regulatory T cells (Treg) and of invariant natural killer T cells (iNKT), along with Treg cell-mediated suppression and underlying mechanisms, were monitored in the peripheral blood. Variance in frequency of Treg and iNKT after RAI therapy was higher in GD patients than in HS over time (p < 0.0001). Reduced Treg suppressive function was observed after RAI therapy in GD patients (p = 0.002). ATD medication prior to RAI dampened these outcomes: less variation of Treg frequency (p = 0.0394), a trend toward less impaired Treg function, and prevention of reduced levels of suppressive cytokines (p < 0.05). Shortly after RAI therapy, alterations in immunoregulatory cells in patients with GD were observed and partially prevented by an ATD pretreatment. Worsening of autoimmunity after RAI was explained in previous studies by enhanced immune activity. This study adds new highlights on immune regulation deficiencies after therapeutic interventions in thyroid autoimmunity.

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.

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.

Rapid evaluation of the absence of inflammation after rupture of membranes.

OBJECTIVES: The aim of this study was to validate the results of an immunochromatographic bedside test to detect IL6 and IL8 in vaginal secretions after rupture of membranes (ROM) with results obtained by ELISA tests. METHODS: A prospective cohort of 60 women with ROM or preterm ROM (PROM) was recruited. An immunochromatographic bedside test was performed with vaginal secretions samplings at admission, every 48 hrs until labor and during labor. Remaining samples were frozen for ELISA analysis. The results of bedside tests were compared to those from ELISA analysis for 114 samples. RESULTS: With all samples combined, the positive predictive values were 50% for IL6 and 86.8% for IL8 and the negative predictive values were 97.4% for IL6 and 53.3% for IL8. Kappa coefficients were 0.54 for IL6 and 0.41 for IL8. CONCLUSION: Our findings show that a bedside test can detect the absence of IL6 in vaginal secretions. This result suggests that bedside test could be used for expectant management after premature PROM to inform the attending physician of the absence of inflammation in vaginal secretions.

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.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

Time-dependent effect of in utero inflammation: a longitudinal study in rats.

OBJECTIVE: To determine the impact of the duration of fetal exposure to inflammation on the neurological outcome of pups. METHOD: Time-pregnant Sprague-Dawley rats (n = 32) received intraperitoneal injection of lipopolysaccharides (LPS; 500 µg/kg), or an equivalent volume of vehicle 3, 6, 12 and 24 h before C-section. Maternal serum and amniotic fluid were tested for cytokines. Motor activity of resuscitated pups (n = 58) was analyzed using the open-field test (20 d). Brains were collected for histopathological examination. RESULTS: Perinatal mortality increased with the duration of fetal exposure to LPS. All parameters tested with the open-field test were lower in the LPS 12 h exposure group compared to the control group (p < 0.05). Tissue inhibitor of metalloproteinase 1 (TIMP-1) was statistically increased in maternal blood after 3, 6 and 12 h of LPS injection (p < 0.05 versus control). CONCLUSION: A threshold of duration of exposure to inflammation is demonstrated, before which delivery should be performed in order to prevent brain damage.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells.

INTRODUCTION: Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T(H)2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT(1) and CysLT(2) receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T(H)2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells. METHODS: A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD(4). The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively. RESULTS: IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD(4) weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD(4) had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT(1) but not CysLT(2). CysLT(1) expression was increased by IL-13 but not by IL-4 and/or LTD(4). Importantly, the upregulation of CysLT(1) by IL-13 preceded eotaxin-3 release. CONCLUSIONS: These results demonstrate a stepwise cooperation between IL-13 and LTD(4). IL-13 upregulates CysLT(1) expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

Rescue of internalization-defective platelet-activating factor receptor function by EBP50/NHERF1.

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in specific disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization and trafficking. EBP50/NHERF1 has been found to interact with a variety of proteins and these interactions are involved in a growing range of functions including the assembly of signalling complexes, receptor recycling and transport of proteins to the cell surface. Crucial roles of EBP50 in GPCR physiology include its involvement in internalization, recycling, and downregulation. We were interested in identifying the role of EBP50 in PAFR trafficking. Our results showed that EBP50 binds the PAFR in its basal state, while stimulation decreased the ratio of interaction between the two proteins. We also demonstrated that EBP50 could bind PAFR via its PDZ 2 domain. In addition, we studied the role of EBP50 in various functions of the PAFR such as PAF-induced inositol phosphate accumulation and receptor internalization: EBP50 decreased the WT PAFR response and rescued the function of internalization-deficient mutant receptors, as previously described for the arrestins and the GRKs. These results suggest new roles for EBP50, some of which could help understanding the complex formation after receptor activation.