RESUMO
CD38 is a 45,000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B-cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B-cell development. Pre-pro-B, pro-B, pre-B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B-cell maturation. To assess the role of CD38 during B-cell maturation, CD38-deficient mice were analysed. CD38(-/-) mice showed a significant increase in both the frequency of B-lineage cells and the absolute numbers of pre-pro-B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross-linking in Ba/F3 cells promoted apoptosis and marked extracellular signal-regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B-cell precursors from bone marrow. These data demonstrate that B-cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK-associated regulation of the B-lineage differentiation pathway.
Assuntos
ADP-Ribosil Ciclase 1/genética , Células da Medula Óssea/citologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Células Precursoras de Linfócitos B/citologia , ADP-Ribosil Ciclase 1/biossíntese , Animais , Apoptose/imunologia , Células da Medula Óssea/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Linhagem da Célula/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Flavonoides/farmacologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/imunologiaRESUMO
Bacterial clearance is one of the most important beneficial consequences of the innate immune response. Chemokines are important mediators controlling leukocyte trafficking and activation, whereas reactive oxygen and nitrogen species are effectors in bacterial killing. In the present work, we used in vivo and in vitro models of infections to study the role of monocyte chemoattractant protein 1 (MCP-1)/CCL2 and nitric oxide (NO) in the bacterial clearance in sepsis. Our results show that MCP-1/CCL2 and NO levels are increased in the peritoneal cavity of mice 6 h after sepsis induced by cecal ligation and puncture. Pretreatment with anti-MCP-1/CCL2 monoclonal antibodies increased the number of colony-forming units (CFUs) recovered in the peritoneal lavage fluid. Moreover, CFU counts were increased in the peritoneal fluid of CCR2 mice subjected to cecal ligation and puncture. In vitro stimulation of peritoneal macrophages with recombinant MCP-1/CCL2 reduced CFU counts in the supernatant after challenge with Escherichia coli. Conversely, treatment with anti-MCP-1/CCL2 increased CFU counts under the same experimental condition. Stimulation of cultured macrophages with MCP-1/CCL2 and interferon had a synergistic effect on NO production. Macrophages from CCL2 mice showed a consistent decrease in NO production when compared with wild-type controls after stimulation with LPS + interferon. Finally, we showed incubation of macrophages with E. coli, and the ERK inhibitor U0126 increased CFU numbers and decreased intracellular levels of NO. In conclusion, we demonstrated for the first time that MCP-1/CCL2 has a crucial role in the clearance of bacteria by mechanisms involving increased expression of inducible NO synthase and production of NO by ERK signaling pathways.
Assuntos
Infecções Bacterianas/imunologia , Quimiocina CCL2/imunologia , Óxido Nítrico/imunologia , Sepse/imunologia , Animais , Líquido Ascítico/microbiologia , Infecções Bacterianas/microbiologia , Butadienos/farmacologia , Células Cultivadas , Quimiocina CCL2/deficiência , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Nitrilas/farmacologia , Fagocitose/imunologia , Proteínas Recombinantes/imunologia , Sepse/microbiologiaRESUMO
OBJECTIVE: To analyze the effect of corticosteroid administration on the concentration of hyaluronan (HA) in bronchoalveolar lavage (BAL) in a murine model of eosinophilic airway inflammation and to study the mechanisms involved. MATERIALS AND METHODS: Untreated-mice or mice treated with 1 µg/g/day betamethasone (Bm) or 0.25 µg/g/day(-1) budesonide (Bd) were sensitized and challenged with Dermatophagoides pteronyssinus (Dp) or saline (control group). The concentration of HA in BAL was determined by ELISA. In vitro migration assays were performed using a Boyden chamber and the expression of HA synthases (HAS) was analyzed by RT-PCR. RESULTS: We found a significant increase (P < 0.01) in the levels of HA in BAL from Dp-treated mice that was prevented by Bm or Bd. Corticosteroids also inhibited the increase in HAS expression, and the phosphorylation of Akt and ERK in the lungs of challenged mice. Finally, we found that low molecular weight HA induces the chemotaxis of BAL cells in vitro through a mechanism mediated by CD44. CONCLUSION: We conclude that corticosteroids prevent the increase in HA in BAL from Dp-challenged mice. This effect is associated with reduced expression of HAS and reduced phosphorylation of Akt and ERK in the lungs of challenged mice.
Assuntos
Betametasona/farmacologia , Budesonida/farmacologia , Eosinofilia/imunologia , Glucocorticoides/farmacologia , Ácido Hialurônico/imunologia , Pneumonia/imunologia , Alérgenos , Animais , Anti-Inflamatórios/farmacologia , Antígenos de Dermatophagoides , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Glucuronosiltransferase/genética , Hialuronan Sintases , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/imunologiaRESUMO
Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.
Assuntos
Actinas/metabolismo , Candida albicans/metabolismo , Candidíase/metabolismo , Cofilina 1/metabolismo , Imunidade Inata/fisiologia , Leucotrieno B4/metabolismo , Macrófagos Alveolares/metabolismo , Actinas/genética , Actinas/imunologia , Animais , Candida albicans/imunologia , Candidíase/genética , Candidíase/imunologia , Cofilina 1/genética , Cofilina 1/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Lectinas Tipo C , Leucotrieno B4/genética , Leucotrieno B4/imunologia , Quinases Lim/genética , Quinases Lim/imunologia , Quinases Lim/metabolismo , Macrófagos Alveolares/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-delta , Ratos , Ratos WistarRESUMO
The effects of Leishmania mexicana metacyclic promastigotes upon MAP kinase signalling in mouse bone marrow macrophages and subsequent expression of the disease regulatory proteins iNOS and COX-2 were studied. At a ratio of 5:1, promastigotes caused a marked increase in phosphorylation of the three major MAP kinases, ERK, p38 and JNK. MAP kinase signalling was substantially reduced in TLR-4(-/-) but not TLR-2(-/-) deficient macrophages and completely abolished in double TLR-2/4(-/-) macrophages. A similar outcome was observed using cysteine peptidase B deficient amastigotes. Furthermore, whilst promastigotes had no independent effect on iNOS or COX-2 expression, they prolonged the induction of these proteins stimulated by LPS and enhanced PGE(2) and NO production. Induction of COX-2 and iNOS was also TLR-4 dependent. Blockade of either PGE(2) or NO production with indomethacin or l-NAME reversed promastigote inhibition of LPS induced IL-12 production. Promastigotes also increased macrophage arginase-1 expression and enhanced arginase activity, both of which were substantially reduced in TLR-4 but not TLR-2 deficient macrophages. Surprisingly, arginase inhibition by Nor-NOHA also caused a reversal of promastigote mediated inhibition of macrophage IL-12 production. These data demonstrate for the first time the role of TLR-4 in mediating the effects of L. mexicana promastigotes on MAP kinase activation, up-regulation of COX-2, iNOS as well as arginase-1 expression in macrophages and further shows that PGE(2), NO and arginase activity all contribute substantially to the inhibition of host cell IL-12 production.
Assuntos
Ativação Enzimática/imunologia , Interleucina-12/biossíntese , Leishmaniose/imunologia , Macrófagos/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Arginase/biossíntese , Arginase/imunologia , Western Blotting , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-12/imunologia , Leishmania mexicana/imunologia , Leishmaniose/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologiaRESUMO
Lymphocytes of human T-lymphotropic virus type-I (HTLV-I) infected patients were previously found tolerant to mitogenic stimuli as well as glucocorticoid treatment. These data suggest that common signaling events are impaired during this infection. The underlying mechanisms of these phenomena may include changes in cellular composition, cytokine milieu and the differential activation of mitogen-activated protein kinases (MAPKs). We investigated the role of (i) p38 and ERK MAPKs, (ii) lymphocyte subpopulations, (iii) and cytokines implicated in antigen or glucocorticoid-induced immunomodulation. Twenty-one asymptomatic carriers (AC), 19 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 21 healthy subjects took part in this study. Lymphocytes were isolated and cultured in vitro to assess lymphocyte proliferation and sensitivity to dexamethasone. The expression of phospho-MAPKs, lymphocyte subsets and cytokines were assessed by flow cytometry. Patients with HAM/TSP had a higher p38/ERK ratio (p<0.05) associated with a reduced response to mitogens (phytohaemagglutinin or PMA+ionomycin) (p<0.001) and higher sensitivity to dexamethasone (p<0.05). HAM/TSP patients presented increased frequency of activated T cells and CD8(+)CD28(-) regulatory T cells, being negatively related to the mitogenic response. These data suggest that multiple underlying mechanisms could be involved with HTLV-related changes in cellular response to mitogens and glucocorticoids.
Assuntos
Apresentação de Antígeno/imunologia , Infecções por HTLV-I/enzimologia , Infecções por HTLV-I/imunologia , Tolerância Imunológica/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Dexametasona/farmacologia , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Feminino , Citometria de Fluxo , Glucocorticoides/farmacologia , Infecções por HTLV-I/fisiopatologia , Humanos , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Linfócitos T/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Recognition of microbial products by macrophages (Mphi) stimulates an inflammatory response and plays a critical role in directing the host immune response against infection. In the present work, we showed for the first time that synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG) are able to stimulate, in the presence of interferon-gamma (IFN-gamma), both arginase and inducible nitric oxide synthase (iNOS) in murine Mphi. Unexpectedly, IFN-gamma, a cytokine believed to be an inhibitor of arginase activity, intervened in the activation of this enzyme. A significant increase in arginase activity was observed upon a short pre-incubation (1 hr) with IFN-gamma and subsequent CpG stimulation. Therefore, a very interesting observation of this study was that the CpG-mediated arginase activity is dependent on IFN-gamma priming. The increase in arginase activity as a result of stimulation with CpG plus IFN-gamma was correlated with augmented expression of the arginase II isoform. The use of pharmacological specific inhibitors revealed that arginase activity was dependent on p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinase (ERK), but independent of c-Jun N-terminal kinase (JNK) activation. This report reveals a singular effect of the combination of CpG and IFN-gamma, one of the mayor cytokines produced in response to CpG administration in vivo.
Assuntos
Arginase/biossíntese , Sequência Rica em GC/imunologia , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Animais , Antracenos/farmacologia , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Flavonoides/farmacologia , Imidazóis/farmacologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Piridinas/farmacologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CD38 is a surface receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed on the surface of both mature and immature B cells. In this work, the function of CD38 in the maturation of murine B lymphocytes in the spleen was analyzed. The results showed that CD38 is highly expressed on Transitional 2 (T2) B lymphocytes with an intermediate expression on Transitional 1 (T1) and mature follicular B cells (M). Correlating with a high expression of CD38, T2 cells are also larger and more granular than T1 or M B cells. T2 cells also showed high levels of other molecules, which indicate an activated phenotype. CD38 crosslinking induced proliferation and maturation of T2 B lymphocytes; in contrast, T1 subset died by apoptosis. Finally, CD38 stimulation of T2 B lymphocytes obtained from Btk-, Lyn-, or Fyn-deficient mice showed a defective differentiation; similarly, drugs interfering with PI3K or ERK decreased the proliferation or differentiation of this subset. This suggests that these molecules participate in the CD38 signaling pathway. As a whole, the results indicate that CD38 plays an important role in the regulation of B-cell maturation in the spleen.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , ADP-Ribosil Ciclase 1/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica/genética , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , Transdução de Sinais/genética , Baço/citologia , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/imunologia , Hemócitos/imunologia , Mytilus/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteína Quinase C/imunologia , Animais , Separação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Hemócitos/citologia , Hemócitos/enzimologia , Imunidade Inata , Mytilus/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/imunologia , Transdução de Sinais/imunologia , Zimosan/metabolismoRESUMO
The strong inflammatory response observed in neurodegenerative diseases can depend on the impairment of the endogenous control of microglial activation, triggering the release of potentially detrimental factors such as cytokines, nitric oxide (NO) and superoxide anion (O(2)(-)). Our aim was to study the activation of microglial cells and the transduction pathways involved in their modulation by IL-1beta and TNF-alpha. Microglial and mixed glial cell cultures from neonatal rats were exposed to IFN-gamma and/or IL-1beta and TNF-alpha. We analyzed NO secretion and the activation of ERK and STAT1. We found that astrocytes modulated microglial cell activation, decreasing production of NO. IFN-gamma induced an 18- to 25-fold increase in NO, associated to a 3- to 5-fold increase in ERK phosphorylation in microglial cultures. IL-1beta, but not TNF-alpha, inhibited IFN-gamma-induced production of NO in microglia by 87%. It also reduced IFN-gamma-induced phosphoERK (pERK) by 40%, without affecting phosphoSTAT1 (pSTAT1). In contrast, in microglial cultures exposed to media conditioned by astrocytes, IL-1beta did not inhibit pERK, whereas it reduced activation of STAT1. Inducible NO synthase expression induced by IFN-gamma in microglial cultures was reduced when the activation of ERK was prevented. We propose that IL-1beta modulates IFN-gamma-induced production of oxidative molecules through cross talk between STAT1 and MAPK pathways, regulating the amplitude and duration of microglial activation. Modulation of ERK was observed at 30 min, whereas inhibition of pSTAT was observed later (at 4 h), indicating that it was an early and transient phenomenon.
Assuntos
Astrócitos/imunologia , Encefalite/imunologia , Gliose/imunologia , Interleucina-1beta/imunologia , Microglia/imunologia , Transdução de Sinais/imunologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Células Cultivadas , Encefalite/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gliose/fisiopatologia , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Microglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/efeitos dos fármacos , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcgammaRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcgammaRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcgammaRIIA and mutants that associated with lipid rafts efficiently activated NF-kappaB, in an ERK-dependent manner. In contrast, WT FcgammaRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcgammaRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcgammaRIIA-mediated responses, like NF-kappaB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.