RESUMO
Generation of an effective cellular immune response is key to the successful development of both humoral and cellular immune defences against most pathogens. However, while the type of cellular immune response elicited by any given pathogen is dictated by the entire array of antigens and molecules which comprise that pathogen, most studies of human immune responses to bacterial pathogens tend to focus on selected antigens. This is a result, in part, of a desire to find those antigens that will generate a desired immune response, as well as limited technology for monitoring the complex array of responses generated by an intact organism. Utilizing Streptococcus mutans as a model Gram-positive organism, a novel flow cytometric assay that permits the identification of individual cells within a responding population, and highly sensitive cytokine assays, we show for the first time that CD8 T cells and natural killer (NK) cells comprise a significant component of the response to this organism in humans. This is despite the fact that CD8 T cells are traditionally thought to respond to endogenously derived antigens only. In addition, we provide the first evidence that a Gram-positive organism can actively inhibit interleukin-2 (IL-2), an important autocrine growth factor for T cells. The latter observation could represent an additional mechanism by which Gram-positive organisms evade host defences.
Assuntos
Interleucina-2/metabolismo , Monócitos/imunologia , Streptococcus mutans/imunologia , Linfócitos T/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Divisão Celular , Células Cultivadas , Depressão Química , Citometria de Fluxo , Humanos , Interleucina-2/imunologia , Células Matadoras Naturais/imunologiaRESUMO
The primary function of polymorphonuclear neutrophils (PMN) in the immune response appears to be acute phagocytic clearance of foreign pathogens and release of inflammatory mediators. Consistent with their assumed lack of major histocompatibility complex (MHC) class II expression, PMN have not been considered to play a role in antigen presentation and T-cell activation. However, recent reports have shown that human PMN can express MHC class II molecules both in vitro and in vivo after stimulation with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). Thus, under appropriate conditions, PMN could play a significant role in immune regulation, including T-cell activation. In this report, we demonstrate that human class II-expressing PMN can serve as accessory cells in superantigen (SAg)-mediated T-cell activation. This accessory activity for SAg presentation was present only after induction of MHC class II expression, and was especially pronounced following culture of PMN with GM-CSF plus IFN-gamma, which acted synergistically to induce MHC class II molecules on PMN. Moreover, the level of MHC class II expression and the magnitude of SAg-induced T-cell responses were found to be highly correlated and distinctly donor dependent, with PMN from some donors repeatedly showing fivefold higher responses than PMN from other donors. On the other hand, culture of PMN with GM-CSF plus IFN-gamma under conditions that resulted in optimal MHC class II expression did not enable them to function as antigen-presenting cells for either intact tetanus toxoid (TT) or for a TT peptide. These results delineate a new pathway for T-cell activation by SAg that may play an important role in the severity of SAg-induced inflammatory responses. They also identify a donor-specific polymorphism for induction of PMN MHC class II expression which may be of significance for therapies involving GM-CSF and IFN-gamma.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Neutrófilos/imunologia , Superantígenos/imunologia , Linfócitos T/imunologia , Toxoide Tetânico/imunologia , Divisão Celular , Células Cultivadas , Humanos , Neutrófilos/citologia , Linfócitos T/citologiaRESUMO
Hydroquinone (HQ) is a major metabolite of benzene and is present in large quantities in cigarette tar as a result of the combustion of tobacco leaf pigments. We hypothesize that the immunosuppressive effects of cigarette smoking are due, in part, to the deposition of large quantities of HQ in the lungs. Exposure of primary human T lymphoblasts (HTL) in vitro to 50 microM HQ blocked IL-2-dependent proliferation by > 90% with no loss in viability. Inhibition of DNA synthesis was observed immediately after the addition of HQ to the cells. However, this effect could be reversed up to 6 hr later by simply washing the cells and reculturing them in the absence of HQ. HQ did not significantly alter intracellular glutathione levels up to 24 hr later, and the presence of 50 microM 2-mercaptoethanol or 500 microM dithiothreitol during the treatment did not prevent inhibition of DNA synthesis. HQ did not block binding of 125I-IL-2 to the cells, but inhibited the IL-2-dependent progression of HTL through S phase of the cell cycle. These observations demonstrate that HQ, in concentrations comparable to those found in cigarette tar, is a potent inhibitor of IL-2-dependent T cell proliferation and may therefore help to explain the potent immunosuppressive effects of cigarette smoke on lung T lymphocytes.
Assuntos
Hidroquinonas/toxicidade , Tolerância Imunológica/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Hidroquinonas/metabolismo , Interleucina-2/imunologia , Interleucina-2/farmacologia , Receptores de Interleucina-2/efeitos dos fármacos , Receptores de Interleucina-2/fisiologia , Fase S/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
It has been reported that the expression of major histocompatibility complex (MHC) class II can be regulated by lead (Pb) in murine cells. In both human and mouse, the expression of MHC class II and invariant chain (Ii) can be regulated by cytokines, including interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). Herein we report that in humans, as with IL-4, Pb enhanced MHC class II antigen DR (HLA-DR) surface expression by monocytes and B cells; Ii surface expression by monocytes and B cells was not affected by Pb while it was enhanced by IL-4. IFN-gamma increased HLA-DR and Ii surface expression by monocytes but it decreased HLA-DR and Ii surface expression by B cells. Total cellular HLA-DR expression by peripheral blood mononuclear cells (PBMC) was increased by Pb, IFN-gamma, or IL-4. Total cellular Ii (p33 and p35) expression by PRMC was not affected by Pb or IFN-gamma while it was increased by IL-4. In PBMC, the steady-state mRNA levels of HLA-DR alpha and Ii were not affected by Pb; IFN-gamma increased HLA-DR alpha mRNA expression but not Ii; IL-4 increased both mRNA levels of HLA-DR alpha and Ii. Furthermore, Pb, IFN-gamma, or IL-4 significantly increased the total cellular level of HLA-DR:Ii complexes in PBMC while they had no effect on cell surface HLA-DR:Ii complex expression. Overall, these results suggest that, in vitro, Pb, IFN-gamma, and IL-4 differentially modulate HLA-DR and Ii expression by human PBMC.
Assuntos
Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos HLA-DR/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Interferon gama/farmacologia , Interleucina-4/farmacologia , Chumbo/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Adolescente , Antígenos de Diferenciação de Linfócitos B/efeitos dos fármacos , Sinergismo Farmacológico , Antígenos HLA-DR/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-4/metabolismo , RNA Mensageiro/biossínteseRESUMO
It has been shown that lead (Pb) potentiates lipopolysaccharide (LPS) lethality in animals by increasing the secretion and uptake or reactivity of tumor necrosis factor-alpha (TNF-alpha). Herein we report that PbCl2 increased TNF-alpha secretion from LPS-treated human peripheral blood mononuclear cells (PBMC) in a concentration- and time-dependent manner. PbCl2 also increased total cellular TNF-alpha levels but had no effect on the steady-state levels of TNF-alpha mRNA. PbCl2 decreased membrane-associated TNF-alpha (mTNF-alpha) on LPS-treated monocytes, whereas PbCl2 increased TNF-alpha receptor (TNF-R) p55 surface expression, and had no effect on TNF-R p75 surface expression by LPS-treated monocytes. Overall, the results suggest that PbCl2 increases TNF-alpha expression by posttranscriptional mechanisms in human PBMC, and enhances the reactivity and uptake of TNF-alpha by increasing the surface expression of TNF-R p55.
Assuntos
Chumbo/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Sequência de Bases , Humanos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transcrição GênicaAssuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Antígenos HLA-DR/biossíntese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neutrófilos/química , Diferenciação Celular , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
The immunotoxic effects of the environmental carcinogen benzo[a]pyrene (BaP) have been evaluated using mouse, but not human, lymphocytes. Since susceptibility to immunomodulation by BaP may be species-related, the effects of BaP on human peripheral blood mononuclear cells (HPBMC) from several individuals were investigated in vitro. HPBMC were stimulated by the T-cell mitogen concanavalin A (con A) in the presence of BaP (10(-9) to 10(-6) M) or vehicle control (acetone) and evaluated after 3 days for [3H]TdR incorporation, cell numbers, cell-cycle distribution, viability, and expression of cell surface or intracellular activation antigens. IL-2 production was quantified at 24 hr. Incorporation of [3H]TdR was the parameter most sensitive to BaP with an IC50 relative to controls of 4.5 +/- 3.1 x 10(-8) M with a range for individuals of 1.8 x 10(-8) to 6.6 x 10(-8) M (n = 15). The P450 inhibitor 7,8-benzoflavone (10(-6) M) partially prevented BaP inhibition of [3H]TdR incorporation. Maximum suppression of this parameter required that BaP be present in culture > 24 hr. The number of cells recovered from culture was decreased by 10(-8) to 10(-6) M BaP, but viability was only slightly diminished by 10(-7) to 10(-6) M BaP. BaP had little or no effect on the percentages of cells with induced IL-2 (CD25) or transferrin (CD71) receptors or on the amount of IL-2 activity present in 24-hr culture supernatants. Despite the BaP-mediated decrease in [3H]TdR incorporation, cell-cycle analysis indicated that BaP at 10(-7) and 10(-6) M increased the percentages of cells in S phase, and correspondingly decreased the percentages of cells in the G0/G1 phase. Also, BaP (10(-7) to 10(-6) M) decreased the percentages of G0/G1 cells that were positive for the intracellular activation antigen PCNA (or Ki-67), but had no affect on this percentage if cells were prevented from traversing S phase by mimosine or aphidicolin. These results suggest that BaP inhibits DNA synthesis and proliferation of con A-stimulated, human peripheral blood T-cells at culture concentrations as low as 10(-8) M by a P450-dependent process, but has little or no effect on measured G1-associated events at culture concentrations up to 10(-6) M.
Assuntos
Benzo(a)pireno/farmacologia , Divisão Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Antígenos de Superfície/análise , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Humanos , Interleucina-2/metabolismo , Antígeno Ki-67 , Leucócitos Mononucleares/imunologia , Proteínas de Neoplasias/análise , Proteínas Nucleares/análise , Antígeno Nuclear de Célula em Proliferação , Estimulação QuímicaRESUMO
The effect of administration of lead acetate (10 mM in the drinking water) for 8 weeks on the in vivo sheep red blood cell (SRBC) specific plaque-forming cell (PFC) responses of inbred A, BALB/c, C57Bl/6, DBA/1, SJL, and NZW/NZB F1 mice and outbred CFW mice was examined to determine if lead was immunomodulatory in a genetically related manner. Lead did not suppress the SRBC-specific PFC/10(6) splenocytes or PFC/spleen response in any mouse strain when compared to the responses of strain-matched control mice. In addition, 10 mM lead-treated BALB/c mice manifested augmented PFC/10(6) splenocytes (17%; p less than .05) but unchanged PFC/spleen responses. Correspondingly, serum concentrations of SRBC-specific antibody (measured by radioimmunoassay) and serum immunoglobulin G, M, or A isotypes were also unchanged by lead acetate treatment in all tested mouse strains. There were no observable lead-related histopathological changes or deposition of immune complexes or antibasement membrane antibody in the kidneys of treated mice. Further, splenocytes from lead-treated, SRBC-immunized mice cultured with T-independent antigens (TNP-LPS, TNP-Ficoll) or with a T-dependent antigen (SRBC) exhibited direct and indirect specific PFC responses that were unchanged from those of control mice. The H-2K/D haplotypes of the outbred CFW mice were determined by microcytotoxicity to include r, q, u, and s. These results suggest that lead acetate (10 mM) administered po for 8 weeks does not suppress the primary direct humoral immune response to SRBC in inbred and outbred mice of several H-2 haplotypes (k/d; d; b; q; d,z; s; r; and u).
Assuntos
Antígenos T-Independentes/imunologia , Chumbo/imunologia , Compostos Organometálicos , Baço/imunologia , Administração Oral , Animais , Formação de Anticorpos , Peso Corporal/efeitos dos fármacos , Feminino , Haploidia , Técnica de Placa Hemolítica , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Radioimunoensaio , Especificidade da EspécieRESUMO
The interaction of highly purified Pseudomonas aeruginosa cytotoxin (PAC) with murine splenocytes was examined. Added at culture initiation, PAC (0.1 to 0.5 microgram/ml) inhibited subsequent [3H]deoxythymidine incorporation measured between 42 to 48 h. Incorporation of [3H]deoxythymidine was inhibited 50% in lipopolysaccharide-, phytohemagglutinin-, and concanavalin A-stimulated cultures by 0.20, 0.32, and 0.39 microgram of PAC per ml, respectively. It is concluded that PAC exhibits a narrow inhibitory concentration response range of 0.1 to 0.5 microgram/ml which, secondarily, is affected by the presence of mitogens. Antitoxin added at splenocyte culture initiation, directly after PAC, yielded greater than or equal to 86% protection against PAC inhibition of [3H]deoxythymidine incorporation. Addition of antitoxin to cultures at different times after PAC demonstrated a time-dependent loss of antitoxin protective effect over a 12-h period, indicating that PAC became cell associated and refractory to antitoxin within this time period. PAC preincubated with splenocytes at 4 degrees C for less than or equal to 1 h could not be removed by washing of cells and was fully inhibitory to [3H]deoxythymidine incorporation when these cells were cultured at 37 degrees C. This finding was confirmed by demonstrating that 125I-labeled PAC bound immediately to cells. It is concluded that PAC action on splenocytes is dose- and time-dependent and consists of a two-phase process: (i) a very rapid binding of PAC to the cell surface available to antitoxin, and (ii) a slower toxicity development phase of ca. 12 h, during which PAC becomes refractory to antitoxin.