RESUMO
New approaches for the treatment of inflammatory disorders such as rheumatic arthritis (RA) and inflammatory lung disease (asthma) are needed because a significant population of patients do not experience sustained relief with currently available therapies. The tyrosine kinase Syk plays a crucial role in inflammatory signaling pathways and has gained much attention as a potential target for treatment of inflammatory disorders. We have shown that our Syk siRNA injected directly into limb joints of arthritic mice, diminishes joint swelling and reduces levels of Syk kinase and inflammatory cytokines in joint tissue. Further, our Syk siRNA, administered via nasal instillation, inhibits recruitment of inflammatory cells to the bronchoalveolar fluid of allergen-sensitized mice. We propose that targeting Syk via localized application of Syk siRNA provides an opportunity for specific knockdown of Syk kinase with minimal potential for systemic effects.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Asma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Interferente Pequeno/uso terapêutico , Alérgenos/imunologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Western Blotting , Feminino , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Quinase Syk , TransfecçãoRESUMO
In addition to their primary role in hemostasis and wound healing, platelets play important roles in a multitude of physiological functions including immune and inflammatory responses. We present data that platelets, by virtue of their expression of the human specific FcγR, FcγRIIA, bind IgG complexes in vivo and that circulating phagocytes from healthy individuals internalize platelets in vivo. Human platelets, as a consequence of their expression of FcγRIIA, may thus, contribute to the clearance of IgG-containing complexes from the circulation.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Plaquetas/imunologia , Fagócitos/imunologia , Receptores de IgG/imunologia , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Movimento Celular , Endocitose/imunologia , Citometria de Fluxo , Saúde , Humanos , Imunoglobulina G/imunologia , Camundongos , Monócitos/citologia , Monócitos/imunologia , Monócitos/ultraestrutura , Fagócitos/citologia , Fagocitose/imunologia , Ligação Proteica , Doadores de TecidosRESUMO
Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.
Assuntos
Antígeno de Macrófago 1/imunologia , Fagocitose , Receptores de IgG/imunologia , Animais , Células COS , Chlorocebus aethiops , Feminino , Humanos , Antígeno de Macrófago 1/metabolismo , Masculino , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/imunologia , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de IgG/química , Receptores de IgG/metabolismoRESUMO
Fc gamma receptors (FcgammaRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcgammaR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src-related tyrosine kinases (SRTKs) and phosphatidyl inositol 3-kinases (PI3K) play important roles in FcgammaR phagocytosis. Herein, we demonstrate that these kinases are not required for FcgammaR endocytosis. Endocytosis of heat-aggregated IgG (HA-IgG) by COS-1 cells stably transfected with FcgammaRIIA or chimeric FcgammaRI-gamma-gamma (EC-TM-CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG-sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow-derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcgammaRIIA was cross-linked using HA-IgG or EA. However, inhibition of FcgammaR phosphorylation prevented only FcgammaR phagocytosis. Finally, biochemical analyses of PI3K(p85)-Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcgammaR phagocytosis and endocytosis. Overall, our results indicate that FcgammaR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition-based model in which PI3K and c-Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de IgG/imunologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/imunologia , Células COS , Chlorocebus aethiops , Eritrócitos/imunologia , Humanos , Imunoglobulina G/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Modelos Imunológicos , Fagocitose/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-cbl/imunologia , Receptores de IgG/genética , Ovinos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinase SykRESUMO
The protein tyrosine kinase Syk is critically involved in immunoreceptor signaling in hematopoietic cells. Recent studies demonstrate Syk expression in nonhematopoietic cells, including fibroblasts, endothelial cells, hepatocytes, and breast epithelium. However, the role of Syk in these cells is uncertain. We hypothesized that Syk is expressed in respiratory epithelial cells (EC) and that it functions as a signaling molecule involved in inflammatory responses in the epithelium. With the use of immunohistochemistry, Western blot, PCR, and laser scanning confocal microscopy, Syk was detected in human, rat, and mouse bronchial epithelium in situ and in cultured human bronchial EC in primary cells and the cell lines HS-24 and BEAS-2B. Syk-dependent signaling pathways in EC were initiated by engagement of beta1-integrin receptors. Stimulation of beta1-integrin receptors by fibronectin or antibody cross-linking caused redistribution of Syk from a cytoplasmic to plasma membrane localization. In stimulated cells, Syk and beta1-integrin colocalized. In addition, following beta1-integrin receptor engagement, tyrosine phosphorylation of Syk was observed. Expression of the intercellular adhesion molecule-1 (ICAM-1) and production of IL-6, both important molecules in lung inflammation, was downregulated in EC treated with Syk small interfering RNA or Syk inhibitor piceatannol. We propose that Syk is involved in signaling pathways induced by integrin engagement in airway EC. Syk-mediated signaling regulates IL-6 and ICAM-1 expression and may be important in the pathophysiology of lung inflammation.
Assuntos
Brônquios/metabolismo , Mediadores da Inflamação/metabolismo , Integrina beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , Regulação para Baixo , Células Epiteliais/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Endogâmicos BN , Estilbenos/farmacologia , Distribuição Tecidual/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Proteína-Tirosina Quinase ZAP-70RESUMO
There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcgammaRIIA, which uses the Ig tyrosine-activating motif (ITAM) within its own cytoplasmic domain, and FcgammaRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated gamma-chain. For example, in transfected epithelial cells and COS-1 cells, FcgammaRIIA mediates phagocytosis of IgG-coated red blood cells more efficiently than does FcgammaRI/gamma, and enhancement of phagocytosis by Syk kinase is more pronounced for FcgammaRI/gamma than for FcgammaRIIA. In addition, structure/function studies indicate that the gamma-chain ITAM and the FcgammaRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcgammaRIIA and FcgammaRI/gamma, we examined the interaction of FcgammaRIIA and the FcgammaRI/gamma chimera FcgammaRI-gamma-gamma (extracellular domain-transmembrane domain-cytoplasmic domain) with Syk kinase and with the Src-related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS-1 cells. Our data indicate that FcgammaRIIA interacts more readily with Syk than does FcgammaRI-gamma-gamma and suggest that one consequence may be the greater phagocytic efficiency of FcgammaRIIA compared with FcgammaRI/gamma. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcgammaRI-gamma-gamma and FcgammaRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcgammaRIIA and FcgammaRI-gamma-gamma are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcgammaR-mediated phagocytosis.
Assuntos
Antígenos CD/metabolismo , Precursores Enzimáticos/metabolismo , Monócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/metabolismo , Quinases da Família src/metabolismo , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/genética , Células COS , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Monócitos/efeitos dos fármacos , Mutação , Fagocitose/efeitos dos fármacos , Receptores de IgG/efeitos dos fármacos , Receptores de IgG/genética , Transdução de Sinais/fisiologia , Estilbenos/farmacologia , Quinase Syk , Transfecção , Quinases da Família src/antagonistas & inibidoresRESUMO
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD31), a 130-kD transmembrane glycoprotein that functions in adhesion and signaling, is thought to play a role in some forms of leukocyte transmigration. In the lung, PECAM-1 is highly expressed, yet there have been few studies examining its role in pulmonary pathology. We therefore examined the inflammatory response (measured by bronchoalveolar lavage cell counts and protein content) after several types of lung injury in wild-type and PECAM-1 knockout mice. Consistent with studies in other organs, instillation of an endothelial stimulant (interleukin-1) was PECAM-1-dependent. In contrast, we noted that three other forms of acute lung injury (acid aspiration, adenoviral instillation, and tumor necrosis factor instillation) were completely PECAM-1-independent. Interestingly, in situ immune complex deposition injury, another complex lung disease, was also PECAM-1-dependent. This surprising finding was investigated in more detail and found to be due to a defect in macrophage activation, and not to a blockade of leukocyte transmigration. Experiments in bone marrow chimeric mice as well as ex vivo data demonstrated that Fcgamma receptor-dependent phagocytosis and tumor necrosis factor release were significantly reduced in macrophages derived from PECAM-1 knockout mice. Although PECAM-1 may not be required for transmigration of leukocytes into the alveolar space in many forms of complex lung inflammation, it is important in the function of Fcgamma receptors on alveolar macrophages.
Assuntos
Macrófagos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Receptores de IgG/fisiologia , Animais , Sequência de Bases , Primers do DNA , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Pneumonia/fisiopatologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcγRIIA, which uses the Ig tyrosine-activating motif (ITAM) within its own cytoplasmic domain, and FcγRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated γ-chain. For example, in transfected epithelial cells and COS-1 cells, FcγRIIA mediates phagocytosis of IgG-coated red blood cells more efficiently than does FcγRI/γ, and enhancement of phagocytosis by Syk kinase is more pronounced for FcγRI/γ than for FcγRIIA. In addition, structure/function studies indicate that the γ-chain ITAM and the FcγRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcγRIIA and FcγRI/γ, we examined the interaction of FcγRIIA and the FcγRI/γ chimera FcγRI-γ-γ (extracellular domain-transmembrane domain-cytoplasmic domain) with Syk kinase and with the Src-related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS-1 cells. Our data indicate that FcγRIIA interacts more readily with Syk than does FcγRI-γ-γ and suggest that one consequence may be the greater phagocytic efficiency of FcγRIIA compared with FcγRI/γ. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcγRI-γ-γ and FcγRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcγRIIA and FcγRI-γ-γ are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcγR-mediated phagocytosis.
RESUMO
We constructed chimeric receptors to dissect the role of the transmembrane (TM) domain in cell surface expression of and phagocytosis by the gamma chain-dependent Fcgamma receptors FcgammaRIIIA and FcgammaRI. FcgammaR chimeras containing the TM and cytoplasmic (CY) domains of the gamma chain were expressed on the cell surface and mediated an efficient phagocytic signal. In contrast, chimeras containing the FcgammaRIIIA TM were poorly expressed. Receptors containing the FcgammaRI TM and the gamma chain CY but lacking the gamma chain TM also were expressed efficiently and mediated phagocytosis, suggesting that a gamma chain dimer induced by the gamma chain TM is not required for efficient phagocytosis. Cotransfection of FcgammaRI or FcgammaRIIIA with the chimera CD8-gamma-gamma (EC-TM-CY) resulted in FcgammaR cell surface expression and phagocytosis, whereas CD8-CD8-gamma, whose TM does not associate with FcgammaR, allowed cell surface expression of (but not phagocytosis by) FcgammaRI. CD8-CD8-gamma also did not allow surface expression of FcgammaRIIIA. Exchanging FcgammaRI and CD8 TMs indicated that the C-terminal 11 amino acids of the FcgammaRI TM are essential for association of FcgammaRI with the gamma chain and phagocytosis. The data indicate that specific sequences in the FcgammaRIIIA and FcgammaRI TMs govern their different interactions with the gamma chain in cell surface expression and phagocytosis and that gamma chain TM sequences are not required for gamma chain-mediated phagocytosis. The data identify a specific region of the FcgammaRI TM and its asparagine as important for FcgammaRI cell surface expression in the absence of the gamma chain and for distinguishing the FcgammaRI and FcgammaRIIIA phenotypes.
Assuntos
Fagocitose , Receptores de IgG/química , Sequência de Aminoácidos , Animais , Células COS , Eritrócitos , Mutação , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Receptores de IgG/biossíntese , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ovinos , TransfecçãoRESUMO
Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcgammaRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) in FcgammaRIIB-mediated inhibition of FcgammaR signaling. Coexpression of SHP-1 enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA phagocytosis in COS-1 cells. SHP-1 also enhances the reduction in FcgammaRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP-1. Furthermore, the activation of SHP-1 tyrosine phosphorylation is observed following stimulation of FcgammaRII in COS-1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP-1 also enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA, indicating that FcgammaRIIB can use more than one pathway for its inhibitory action. In addition, SHP-1 and SHIP-1 can inhibit FcgammaRIIA phagocytosis and signal transduction in the absence of FcgammaRIIB. The data support emerging evidence that SH2-containing phosphatases, such as SHP-1 and SHIP-1, can modulate signaling by "activating" receptors.
Assuntos
Antígenos CD/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de IgG/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Antígenos CD/química , Células COS , Células Cultivadas , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Monócitos/enzimologia , Fagocitose , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/química , Fosforilação , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Quinases/metabolismo , Receptores de IgG/química , Quinase Syk , Tirosina/metabolismoRESUMO
Granulocyte colony-stimulating factor (G-CSF) induces proliferation, maturation, and functional activities of myeloid progenitors and mature neutrophils through a specific receptor, the G-CSF-R. Different signals are mediated by distinct regions of the cytoplasmic domain of G-CSF-R, but the precise role of each region has not yet been fully clarified. We evaluated the involvement of Syk kinase, essential in mediating phagocytic signals by Fcgamma receptors, in G-CSF-induced phagocytosis, using murine myeloid 32D cells transfected with wild-type (WT) human G-CSF-R (hG-CSF-R) or with a G-CSF-R mutant truncated at cytoplasmic amino acid 715. The G-CSF-R mutant lacks the immunoreceptor tyrosine-based activation motif (ITAM), putative binding site for Syk. Following treatment of WT hG-CSF-R transfectants with IgG-coated particles, there was a significant increase in phagocytosis in G-CSF-stimulated cells, in which Syk tyrosine phosphorylation occurred, paralleled by enhancement of its tyrosine kinase activity. In the mutant transfectants, no significant increase in phagocytosis or Syk tyrosine phosphorylation occurred after stimulation with G-CSF. We also demonstrated that tyrosine phosphorylation of the Src kinases Hck and Lyn occurs following G-CSF stimulation of cells expressing WT G-CSF-R, but that Hck is not phosphorylated in mutant G-CSF-R transfectants. The increase in phagocytosis following G-CSF stimulation cannot be attributed to a rapid de novo increase in expression of Fcgamma receptors. G-CSF induced expression of Fcgamma receptors only after prolonged stimulation. Our data provide evidence that the carboxy-terminal region of G-CSF-R plays a role in the phagocytosis of IgG-coated particles and that Syk and Hck kinase tyrosine phosphorylation is involved.
Assuntos
Fagocitose , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de IgG/biossíntese , Transdução de Sinais , Quinase Syk , Transfecção , Quinases da Família src/metabolismoRESUMO
Although the cytoplasmic domain of the human FcgammaRIa alpha-chain lacks tyrosine-based phosphorylation motifs, it modulates receptor cycling and receptor-specific cytokine production. The cytoplasmic domain of FcgammaRIa is constitutively phosphorylated, and the inhibition of dephosphorylation with okadaic acid, an inhibitor of type 1 and type 2A protein serine/threonine phosphatase, inhibits both receptor-induced activation of the early tyrosine phosphorylation cascade and receptor-specific phagocytosis. To explore the basis for these effects of the cytoplasmic domain of FcgammaRIa, we developed a series of human FcgammaRIa molecular variants, expressed in the murine macrophage cell line P388D1, and demonstrate that serine phosphorylation of the cytoplasmic domain is an important regulatory mechanism. Truncation of the cytoplasmic domain and mutation of the cytoplasmic domain serine residues to alanine abolish the okadaic acid inhibition of phagocytic function. In contrast, the serine mutants did not recapitulate the selective effects of cytoplasmic domain truncation on cytokine production. These results demonstrate for the first time a direct functional role for serine phosphorylation in the alpha-chain of FcgammaRIa and suggest that the cytoplasmic domain of FcgammaRI regulates the different functional capacities of the FcgammaRIa-receptor complex.