RESUMO
We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover in Jurkat E6-1 CD4+ T-cells, to pursue previous reports that this viral coat protein: (a) inhibits receptor-activated inositol phosphate release; (b) stimulates basal inositol phosphate release; (c) inhibits inositol polyphosphate 5-phosphatase. Treatment of cells with up to 10 microg/ml gp120 from between 10 min and 24 h was without effect upon inositol phosphate turnover in both basal cells, and in C305 and OKT3 stimulated cells. This is the first report that biologically competent gp120 does not affect any aspect of inositol phosphate turnover in either basal or receptor-activated lymphocytes.
Assuntos
Proteína gp120 do Envelope de HIV/farmacologia , Fosfatos de Inositol/biossíntese , Cromatografia Líquida de Alta Pressão , Proteína gp120 do Envelope de HIV/isolamento & purificação , Humanos , Inositol Polifosfato 5-Fosfatases , Células Jurkat/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fosfolipases Tipo C/metabolismoRESUMO
Prostaglandin E2 is observed at elevated levels during human immunodeficiency virus (HIV) infection and thus may contribute to the HIV-dependent immunosuppression. The mechanisms responsible for this increase are not understood. Evidence indicates that the viral envelope proteins perturb membrane signaling mediated by the CD4 receptor, suggesting that the free envelope protein and/or the intact virus may be responsible for the increase in prostaglandin E2 levels. In this study, we have used THP-1 human monocytes and THP-1 cells differentiated by 12-O-tetradecanoylphorbol-13-acetate treatment into macrophages to determine if the HIV envelope protein, gp120, or an anti-CD4 receptor antibody stimulates prostaglandin formation by interacting with the CD4 receptor. Incubation of THP-1 cells with OKT4A antibody greatly stimulated the CD4-p56lck receptor complex as estimated by enhanced p56lck autophosphorylation, while the gp120 gave small but significant responses. Monocytic THP-1 cells poorly metabolized arachidonic acid to prostaglandin E2 and thromboxane B2 as measured by high-pressure liquid chromatography analysis. Western blot (immunoblot) and Northern (RNA) blot analyses revealed that unstimulated monocytes expressed little prostaglandin H synthase 1 and 2 (PGHS-1 and -2). Incubation of the monocytes with lipopolysaccharide, OKT4A, or gp120 did not increase the formation of prostaglandins. The expression of PGHS-1 or PGHS-2 was also not increased. Differentiation of the monocytes to macrophages by 12-O-tetradecanoylphorbol-13-acetate treatment resulted in increased expression of PGHS-1 and increased formation of prostaglandins compared with that for the monocytes. Lipopolysaccharide stimulation of the macrophages increased the formation of prostaglandins and increased the expression of PGHS-2 in the macrophages. However, OKT4A or gp120 preparation, at concentrations that stimulated p56lck autophosphorylation, did not enhance the formation of prostaglandins or the expression of PGHS-1 or PGHS-2. OKT4A and gp120 also did not stimulate the release of arachidonic acid, indicating that phospholipase A2 was not activated by the CD4 receptor in either the THP-1 monocytes or macrophages. These results indicate that activation of the CD4-p56lck receptor signal transduction pathway by the HIV envelope protein does not increase prostaglandin formation.
Assuntos
Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/fisiologia , Macrófagos/metabolismo , Monócitos/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandinas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD4/fisiologia , Células CHO , Diferenciação Celular , Linhagem Celular , Cricetinae , Dinoprostona/isolamento & purificação , Dinoprostona/metabolismo , Eicosanoides/metabolismo , Ativação Enzimática , Expressão Gênica , Proteína gp120 do Envelope de HIV/biossíntese , Proteína gp120 do Envelope de HIV/isolamento & purificação , Humanos , Isoenzimas/biossíntese , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Macrófagos/citologia , Monócitos/citologia , Acetato de Tetradecanoilforbol/farmacologia , Tromboxano B2/isolamento & purificação , Tromboxano B2/metabolismo , Quinases da Família src/metabolismoRESUMO
The binding of five different mouse monoclonal anti-dinitrophenyl (DNP) IgGl monomers, in the presence and absence of DNP-glycine, to Fc gamma RII on the mouse macrophage-like cell line J774 has been investigated. Membrane association constants were estimated using a competitive radioimmunoassay in which the ability of the IgG1 monomers to block the binding of 125I-labelled Fabs of the anti-Fc gamma RII monoclonal antibody 2.4G2 was monitored. The IgG1-Fc gamma RII association constants ranged from (8 +/- 1) x 10(4) M-1 to (6 +/- 2) x 10(5) M-1. No large differences in the IgG1-Fc gamma RII association constants were measured for different IgG1 antibodies or for any single IgG1 antibody in the presence and absence of DNP-glycine.
Assuntos
Anticorpos Monoclonais/imunologia , Haptenos/imunologia , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular , Hibridomas , Camundongos , RatosRESUMO
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
Assuntos
Antígenos de Diferenciação/metabolismo , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Animais , Antígenos de Diferenciação/isolamento & purificação , Ligação Competitiva , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Fragmentos Fab das Imunoglobulinas/metabolismo , Cinética , Membranas Artificiais , Camundongos , Microscopia de Fluorescência , Receptores Fc/isolamento & purificação , Receptores de IgG , Especificidade por Substrato , TermodinâmicaRESUMO
The association of a fluorescently labelled anti-dinitrophenyl monoclonal antibody (ANO2) with Langmuir-Blodgett monolayers composed of three different binary mixtures of phosphatidylcholine and dinitrophenyl-conjugated phosphatidylethanolamine has been characterized. Quantitative fluorescence microscopy measurements demonstrated that measurable amounts of antibodies bound to the monolayers only at high molar fractions of dinitrophenyl-conjugated lipid (greater than or equal to 5 mol%). Fluorescence pattern photobleaching recovery measurements showed that the apparent translational diffusion coefficients and mobile fractions of a fluorescent lipid were high for all monolayer compositions and that the antibody translational mobility was measurable but slow and depended on the two-dimensional antibody density. The results demonstrate that the ANO2-binding characteristics of Langmuir-Blodgett monolayers containing dinitrophenyl-conjugated phospholipids are substantially different from those of similar model systems but that the ANO2 antibodies, when bound, display similar diffusive behavior.