RESUMO
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.
Assuntos
Podossomos , Humanos , Macrófagos/metabolismo , Fosforilação , Podossomos/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Quinases Associadas a rhoRESUMO
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.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-8/metabolismo , Leucotrieno D4/farmacologia , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Isoenzimas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptores de Leucotrienos/metabolismoRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Interleucina-33/metabolismo , Receptores de Leucotrienos/biossíntese , Linfócitos T CD4-Positivos/imunologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/imunologia , Receptores de Leucotrienos/imunologia , Regulação para CimaRESUMO
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.
Assuntos
Citoproteção/efeitos dos fármacos , Doença de Graves/tratamento farmacológico , Doença de Graves/radioterapia , Radioisótopos do Iodo/uso terapêutico , Metimazol/uso terapêutico , Lesões por Radiação/prevenção & controle , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/efeitos da radiação , Adulto , Idoso , Antitireóideos/uso terapêutico , Autoimunidade/efeitos dos fármacos , Autoimunidade/efeitos da radiação , Células Cultivadas , Citoproteção/imunologia , Feminino , Doença de Graves/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Células Matadoras Naturais/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologia , Adulto JovemRESUMO
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.
Assuntos
Movimento Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Leucotrieno B4/farmacologia , Receptores do Leucotrieno B4/fisiologia , Células Cultivadas , Humanos , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Células Matadoras Naturais/imunologia , RNA Mensageiro/análise , Receptores do Leucotrieno B4/análise , Receptores do Leucotrieno B4/genéticaRESUMO
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.
Assuntos
Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Cisteína Endopeptidases/imunologia , Regulação da Expressão Gênica , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Pyroglyphidae/imunologia , Receptores de Leucotrienos/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores , Células Cultivadas , Humanos , Hipersensibilidade/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptores de Leucotrienos/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Células Th17/citologia , Células Th17/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células Th17/metabolismoRESUMO
The constitutive commitment of neutrophils to apoptosis is a key process for the control and resolution of inflammation and it can be delayed by various inflammatory mediators including leukotriene B4 (LTB4). The mechanisms by which LTB4 contributes to neutrophil survival are still unclear and the present work aims at identifying intracellular pathways underlying this effect. Inhibition of human neutrophil apoptosis by LTB4 was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and by the specific MEK inhibitor PD98059. In contrast, inhibitors of p38 MAPK, Jak2/3 and Src did not hinder the anti-apoptotic effect of LTB4. We also investigated the effects of members of the Bcl-2 family as they play a crucial role in the regulation of programmed cell death. When neutrophils were incubated with LTB4 for 1 to 6 h, the mRNA levels of the anti-apoptotic protein Mcl-1 were upregulated approximately 2-fold, while those of the pro-apoptotic protein Bax were downregulated 3- to 4-fold, as determined by real-time PCR. Accordingly, Western blot analysis revealed that the expression of Mcl-1 was upregulated in presence of LTB4, while flow cytometric analysis revealed that Bax protein was downregulated. Furthermore, the modulatory effects of LTB4 on Mcl-1 and Bax proteins were abolished in the presence of either wortmannin or PD98059. Taken together, these results demonstrate the participation of PI3-K and MEK/ERK kinases, as well as regulatory apoptotic proteins such as Mcl-1 and Bax, in the anti-apoptotic effects of LTB4 in human neutrophils.
Assuntos
Apoptose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Leucotrieno B4/farmacologia , Proteínas de Neoplasias/fisiologia , Neutrófilos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Androstadienos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Neutrófilos/citologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , Valores de Referência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , Wortmanina , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
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 B(4) (LTB(4)) can also extend neutrophil survival, we proposed that glucocorticoids could prevent neutrophil apoptosis by up-regulating their expression of the high-affinity LTB(4) 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 LTB(4) antagonist LY255283. Moreover, LTB(4) 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 LTB(4)-treated cells showed enhanced responsiveness to LTB(4) in terms of intracellular Ca(2+) mobilization and chemotaxis. Whereas DEX and LTB(4) alone decreased neutrophil apoptosis by approximately 50%, neutrophils treated with both LTB(4) and DEX showed >90% survival at 24 h. Moreover, BLT1 antagonists prevented the increased neutrophil survival induced by DEX as well as by LTB(4). 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 LTB(4).