RESUMO
Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule.
Assuntos
Artrite Experimental/etiologia , Ativação de Neutrófilo , Receptores de IgG/fisiologia , Motivos de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgG/química , Relação Estrutura-Atividade , Tirosina/fisiologiaRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that is released by platelets and endothelial cells and has been implicated in diverse biological functions. We hypothesized that S1P may influence immune complex-mediated polymorphonuclear neutrophil activation. Using flow cytometry and fluorescence spectrometry, we found that exogenous addition of S1P led to an enhanced polymorphonuclear neutrophil Fcgamma receptor-mediated rise in intracellular Ca(2+) and reactive oxygen species generation in a pertussis toxin-independent manner, while having only a small effect by itself. Thus, S1P amplifies a positive feedback loop where Fcgamma receptor-mediated rises in Ca(2+) and reactive oxygen species are interdependent, with reactive oxygen species acting to increase tyrosine phosphorylation and activity of upstream signaling intermediates. S1P augmentation of Fcgamma receptor signaling translates to downstream functional consequences, including shape change and recruitment to endothelial surfaces coated with suboptimal levels of immune complexes. Taken together, S1P from activated platelets or endothelial cells may serve to amplify leukocyte recruitment and tissue injury at sites of immune complex deposition in vasculitis.
Assuntos
Adjuvantes Imunológicos/sangue , Quimiotaxia de Leucócito/imunologia , Lisofosfolipídeos/sangue , Ativação de Neutrófilo/imunologia , Infiltração de Neutrófilos/imunologia , Receptores de IgG/sangue , Esfingosina/análogos & derivados , Regulação para Cima/imunologia , Adjuvantes Imunológicos/metabolismo , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/fisiologia , Sinalização do Cálcio/imunologia , Adesão Celular/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Células HL-60 , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/fisiologia , Lisofosfolipídeos/metabolismo , Microcirculação/imunologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de IgG/fisiologia , Esfingosina/sangue , Esfingosina/metabolismoRESUMO
The immunoreceptor tyrosine-based inhibitory motif-containing FcgammaRIIb modulates immune function on multiple cell types including B cells, monocytes/macrophages, and dendritic cells. The promoter for the human FCGR2B is polymorphic, and the less frequent 2B.4 promoter haplotype is associated with the autoimmune phenotype of systemic lupus erythematosus. In the present study, we demonstrate that the 2B.4 promoter haplotype of FCGR2B has increased binding capacity for GATA4 and Yin-Yang1 (YY1) transcription factors in both B lymphocytes and monocytes, and that overexpression of GATA4 or YY1 enhances the FCGR2B promoter activity. The 2B.4 haplotype leads to elevated expression of the endogenous receptor in heterozygous donors by approximately 1.5-fold as assessed on EBV-transformed cells, primary B lymphocytes, and CD14(+) monocytes. This increased expression accentuates the inhibitory effect of FcgammaRIIb on B cell Ag receptor signaling, measured by Ca(2+) influx and cell viability in B cells. Our results indicate that transcription factors GATA4 and YY1 are involved in the regulation of FcgammaRIIb expression, and that the expression variants of FcgammaRIIb lead to altered cell signaling, which may contribute to autoimmune pathogenesis in humans.
Assuntos
Autoimunidade , Proteínas de Ligação a DNA/metabolismo , Haplótipos , Regiões Promotoras Genéticas , Receptores de IgG/genética , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Células COS , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA4 , Regulação da Expressão Gênica , Humanos , Receptores de Lipopolissacarídeos/análise , Monócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores de IgG/fisiologia , Fator de Transcrição YY1RESUMO
Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI-evoked responses but still allows the formation of loose platelet aggregates. By using mice deficient in G(alpha)q or specific thromboxane A2 and ADP antagonists, we show that these autocrine agents mediated aggregation but not collagen-induced Ca2+ mobilization or PS exposure. Collectively, these data indicate that integrin alpha2beta1 facilitates the central function of GPVI in the platelet activation processes that lead to thrombus formation, whereas the autocrine thromboxane A2 and ADP serve mainly to trigger aggregate formation.
Assuntos
Plaquetas/metabolismo , Integrina alfa2beta1/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Trombose/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Colágeno/administração & dosagem , Colágeno/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Integrina alfa2beta1/sangue , Integrina alfa2beta1/genética , Camundongos , Camundongos Knockout , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/genética , Receptores de IgG/genética , Receptores de IgG/fisiologia , Tromboxano A2/metabolismoRESUMO
In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.
Assuntos
Células Dendríticas/metabolismo , Granulócitos/metabolismo , Imunoglobulinas/química , Glicoproteínas de Membrana/biossíntese , Monócitos/metabolismo , Receptores Imunológicos/biossíntese , Homologia de Sequência de Aminoácidos , Adulto , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sinalização do Cálcio/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Cromossomos Humanos Par 7 , Clonagem Molecular , DNA Complementar/isolamento & purificação , Células Dendríticas/imunologia , Granulócitos/imunologia , Humanos , Imunoglobulinas/genética , Integrina alfaXbeta2/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Receptores de Lipopolissacarídeos/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/imunologia , Família Multigênica/imunologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , RNA Mensageiro/biossíntese , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/fisiologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Solubilidade , Células U937 , Domínios de Homologia de src/imunologiaRESUMO
Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (=177-fold of controls administered Ag alone), whereas it was abrogated in FcR gamma-/- mice and augmented in Fc gamma RII-/- mice (=5147-fold of controls). The response to IgE/Ag complexes in FcR gamma-/- and Fc gamma RII-/- mice was similar to that se for wild-type mice, demonstrating that non-Fc gamma R-dependent responses were normal. Our observations suggest that IgG/Ag complexes enhance Ab responses via Fc gamma Rs. Moreover, they reveal a strong negative regulation of Ab responses to IgG/Ag exerted by Fc gamma RII.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunoglobulina G/fisiologia , Imunoglobulinas/biossíntese , Receptores de IgG/deficiência , Receptores de IgG/genética , Animais , Complexo Antígeno-Anticorpo/fisiologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Mutantes , Fragmentos de Peptídeos/imunologia , Receptores de IgG/fisiologia , Tirosina/imunologiaRESUMO
Receptors for the Fc region of IgG (Fc gamma R) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of Fc gamma R leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc gamma R subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct Fc gamma R, Fc gamma RIIa and Fc gamma RIIIb, and can be induced to express Fc gamma RI by IFN-gamma. We have previously shown that stimulation of PMN through Fc gamma RIIIb results in enhanced Fc gamma RIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate Fc gamma R responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by Fc gamma RIIa and Fc gamma RI in PMN and amplified receptor-triggered tyrosine phosphorylation of Fc gamma R-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking Fc gamma RIIIb similarly enhanced phosphorylation of Fc gamma RIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in Fc gamma RIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of Fc gamma R signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.
Assuntos
Fagocitose/imunologia , Espécies Reativas de Oxigênio/fisiologia , Receptores de IgG/fisiologia , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/fisiologia , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Monócitos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxidantes/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/metabolismo , Quinase Syk , Tirosina/metabolismo , Células U937RESUMO
Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.
Assuntos
Complexo Antígeno-Anticorpo/farmacologia , Cálcio/sangue , Ativação de Neutrófilo/imunologia , Neutrófilos/fisiologia , Receptores de IgG/fisiologia , Animais , Células Cultivadas , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hidroquinonas/farmacologia , Imunoglobulina G/farmacologia , Inositol 1,4,5-Trifosfato/sangue , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Coelhos , Espécies Reativas de Oxigênio , Albumina Sérica/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The initial stages of an immune response are regulated at the level of the cell-surface antigen and Fc receptors. The extracellular portions of these receptors provide immune specificity and determine the nature of the responding effector cells, whereas the intracellular portion transduces signals into the cell and determines the intensity and duration of the immune response. Recent studies led to the identification of two types of modules within the cytoplasmic region of receptor subunits that are critical for the activation and termination of signal transduction pathways. Phosphorylation of the conserved tyrosine residues within the two modules, the immunoreceptor tyrosine-based activation motif (ITAM) and the immunoreceptor tyrosine-based inhibition motif (ITIM), is followed by the recruitment of different sets of SH2-containing molecules to the receptor site. These proteins regulate the receptor-linked signal transduction pathways in a positive or a negative fashion, which is a reminiscent of the ancestral Yin-Yang principle.
Assuntos
Antígenos CD/fisiologia , Ativação Linfocitária/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de IgG/fisiologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Humanos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/fisiologia , Dados de Sequência Molecular , Receptores Imunológicos/fisiologia , Receptores KIR , Transdução de Sinais/fisiologiaRESUMO
In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.
Assuntos
Neutrófilos/fisiologia , Fagocitose/fisiologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/fisiologia , Receptores de Complemento/fisiologia , Receptores de IgG/fisiologia , Transdução de Sinais/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Amilorida/farmacologia , Sequência de Bases , Antígenos CD18/fisiologia , Cálcio/metabolismo , DNA Complementar/genética , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Capeamento Imunológico , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Neutrófilos/ultraestrutura , Proteínas Opsonizantes , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Explosão Respiratória , Trocadores de Sódio-Hidrogênio/biossíntese , Trocadores de Sódio-Hidrogênio/genética , ZimosanRESUMO
We have investigated the mechanisms underlying calcium signalling evoked by cross-linking of the high affinity IgG receptor (Fc gamma RI) in populations of the human monocyte-like cell line, U937, following activation of the cells by cytokine treatment, or differentiation to a more macrophage-like state by treatment with dibutyryl cAMP (Bt2cAMP). We have shown previously that a larger and more prolonged entry of external calcium occurs in Bt2cAMP-differentiated cells, although there is a smaller initial release from internal stores in these cells than in those activated by IFN-gamma treatment. In this paper we demonstrate, by use of the endomembrane Ca(2+)-ATPase inhibitor, thapsigargin, that this effect is explained (at least in part) by an enhanced capacity for store regulated entry of calcium in Bt2cAMP-differentiated cells.
Assuntos
Bucladesina/farmacologia , Cálcio/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Anticorpos , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ácido Egtázico/análogos & derivados , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fura-2 , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/fisiologia , Interferon gama/farmacologia , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Monócitos/efeitos dos fármacos , Organelas/fisiologia , Receptores de IgG/fisiologia , Terpenos/farmacologia , Tapsigargina , Fatores de TempoRESUMO
A high dose intravenous immunoglobulin (IVIG) therapy is used in the treatment of a wide range of autoimmune disorders. However, the mechanisms of the action of IVIGs remain poorly understood. To analyse the mechanisms of effects of IVIGs on immunoglobulin (Ig) production of B cells, the effects of IVIGs on B lymphoblastoid cell lines transformed by Epstein-Barr virus (LCLs) were investigated. The productions of IgG or IgM of LCLs were dose-dependently suppressed by polyethylene glycol (PEG)-treated IVIG or pH 4-treated IVIG though the productions were not or only slightly suppressed by pepsin-treated IVIG. The suppression by IVIGs was blocked by anti-human IgG Fc or anti-Fc gamma RII. C mu gene expression and mu s C terminal gene expression of LCLs were suppressed by PEG-treated IVIG, whereas neither C mu gene expression nor mu s C terminal gene expression of LCLs were suppressed by pepsin-treated IVIG. Although the increase in intracellular calcium concentration in LCLs was not suppressed by pepsin-treated IVIG, the increase was suppressed by PEG-treated IVIG. This suppressing effect of PEG-treated IVIG on intracellular calcium concentration of LCLs was blocked by anti-human IgG Fc or anti- Fc gamma RII. Our results suggest that IVIGs suppressed the Ca(2+)-dependent signal transduction through Fc gamma R on B-cell membrane, consequently, the transcription of C mu mRNA, especially secreted mu mRNA was suppressed in the B cells.