RESUMEN
4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha 2A-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi from alpha 2A-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.
Asunto(s)
Plaquetas/enzimología , Proteínas de Unión al GTP/fisiología , Toxina del Pertussis , Fosfoproteínas , Proteína Quinasa C/sangre , Receptores Adrenérgicos alfa/fisiología , Factores de Virulencia de Bordetella/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Difosfato Ribosa/sangre , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Permeabilidad de la Membrana Celular , Activación Enzimática , Etilmaleimida/farmacología , Humanos , Fosforilación , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Virus-specific cytotoxic T lymphocytes (CTL) are thought to be responsible for the eradication of respiratory influenza virus infections by direct cytolysis of virus-infected epithelial cells. In this study, we provide evidence for a role for alveolar macrophages (AM) in the regulation of pulmonary virus-specific CTL responses. Prior to infection with influenza virus, AM were selectively eliminated in vivo with a liposome-mediated depletion technique, and virus-specific CTL activities of lung and mediastinal lymph node (MLN) cells were assayed ex vivo and compared with those for normal mice. AM depletion resulted in increased primary CTL responses and changed the kinetics of the CTL response. Flow cytometric analysis of lung and MLN cells showed that the percentage of CD8+ cells was not altered after AM depletion and that lung cells from AM-depleted mice had an increased capacity to lyse virus-infected cells. Upon restimulation in vitro, virus-specific CTL activity in lung cells of normal mice was similar to that in lung cells of AM-depleted mice. Furthermore, elimination of AM resulted in increased virus titers in the lung, but virus clearance as a function of time was not affected. Our results show that AM regulate virus-specific CTL responses during respiratory influenza virus infection by removing viral particles, by downregulating the priming and activity of CTL in MLN cells, and by inhibiting the expansion of virus-specific CTL in the lung.
Asunto(s)
Virus de la Influenza A/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Cinética , Recuento de Leucocitos , Liposomas , Pulmón/citología , Pulmón/inmunología , Pulmón/virología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , FenotipoRESUMEN
Owing to their scavenging and phagocytic functions, spleen macrophages are regarded to be important in the induction and maintenance of both innate and acquired immune defence mechanisms. In this study, we investigated the role of spleen macrophages in immunity against mouse hepatitis virus strain A59 (MHV-A59). Previous studies showed that spleen and liver macrophages are the first target cells for infection by MHV-A59 in vivo, suggesting that they could be involved in the induction of immune responses against MHV-A59. We used a macrophage depletion technique to deplete macrophages in vivo and studied the induction of virus-specific antibody and cytotoxic T-cell (CTL) responses and non-immune resistance against MHV-A59 in normal and macrophage-depleted mice. Virus titres in spleen and liver increased rapidly in macrophage-depleted mice, resulting in death of mice within 4 days after infection. Elimination of macrophages before immunization with MHV-A59 resulted in increased virus-specific humoral and T-cell proliferative responses. However, virus-specific CTL responses were not altered in macrophage-depleted mice. Our results show that spleen macrophages are of major importance as scavenger cells during MHV-A59 infection and are involved in clearance of virus from the host. In addition, macrophages may be involved in the regulation of acquired immune responses. In the absence of macrophages, increased virus-specific T-cell and antibody responses are detectable, suggesting that macrophages suppress MHV-A59-specific T- and B-cell responses and that other cells serve as antigen-presenting cells.
Asunto(s)
Infecciones por Coronavirus/inmunología , Hepatitis Viral Animal/inmunología , Macrófagos/inmunología , Virus de la Hepatitis Murina/inmunología , Bazo/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Presentación de Antígeno/inmunología , División Celular/inmunología , Inmunidad Celular , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Salmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mphi) after i. v. infection. It is generally assumed that Mphi restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mphi during experimental S. typhimurium infections, using a selective liposome-based Mphi elimination technique. Unexpectedly, elimination of Mphi prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mphi mediate the pathology caused by S. typhimurium. Removal of Mphi) during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mphi are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, Mphi appeared to be important effectors of protection against challenge infection since elimination of Mphi from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mphi play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.
Asunto(s)
Macrófagos/inmunología , Salmonelosis Animal/etiología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/farmacología , Ácido Clodrónico/administración & dosificación , Recuento de Colonia Microbiana , Femenino , Liposomas , Activación de Linfocitos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Salmonelosis Animal/prevención & control , Salmonella typhimurium/aislamiento & purificación , Linfocitos T/inmunología , VirulenciaRESUMEN
Macrophages and T lymphocytes play an important role in recovery from viral infections. During mouse hepatitis virus (MHV-A59) infection, a clear virus-specific class II-restricted cytotoxic T-cell response is generated. Transfer of these CD4+ cytotoxic T cells (CTL) into naive mice protects against a lethal challenge with MHV. However, their in vivo antiviral effector mechanism is not yet clear. To further investigate a possible effector mechanism, we studied the effect of adoptive transfer of CD4+ CTL on virus localization in spleen and liver. We showed that adoptive transfer of virus-specific T cells does not affect localization of MHV-A59 in different macrophage subsets. Interestingly, a rapid and large infiltrate of CD4+ T cells in and around MHV-A59-infected foci in the liver was observed early in infection, whereas no CD8+ T cells were detectable. Moreover, transfer of virus-specific T cells resulted in significantly decreased viral titres in the liver and spleen and a marginally increased anti-MHV-A59 IgM production. These results imply an important role for virus-specific CD4+ CTL in elimination of infectious MHV-A59 and induction of an effective immune response in the absence of CD8+ CTL.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Coronavirus/inmunología , Hepatitis Viral Animal/inmunología , Virus de la Hepatitis Murina , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Linfocitos T CD4-Positivos/trasplante , Técnicas de Cultivo de Célula , Infecciones por Coronavirus/terapia , Hepatitis Viral Animal/terapia , Inmunoterapia Adoptiva , Hígado/virología , Transfusión de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/aislamiento & purificación , Bazo/virología , Linfocitos T Citotóxicos/trasplanteRESUMEN
Different delivery vehicles may target to different antigen presenting cells (APC) because of their composition, size and/or physical properties. In this study, we examined the priming of cytotoxic T lymphocyte (CTL) responses to a soluble exogenous protein in vivo, using various delivery vehicles. In addition, we determined the role of macrophages as APC in vivo for each of these delivery vehicles by comparing the induction of antigen-specific CTL and serum antibodies in normal and macrophage-depleted mice. Influenza A virus-derived virosomes, liposomes and monophosphoryl lipid A/squalene (MPLSQ) efficiently induced antigen-specific CTL as well as antibody responses, of which virosomes proved to be the most efficient inducers. In mice that were immunized with cell-associated antigen, strong CTL responses but no antigen-specific antibodies were detectable, while aluminium hydroxide and aluminium phosphate elicited antigen-specific antibodies but no CTL responses. Elimination of macrophages in vivo before immunization abrogated CTL responses induced with liposomes and MPL/SQ, but did not affect induction of antigen-specific CTL with virosomes or cell-associated antigen. Importantly, serum antibody levels were not altered after macrophage depletion, regardless of the delivery vehicle used, suggesting that in the absence of macrophages, other APC may phagocytose the exogenous antigens for major histocompatibility complex (MHC) class II processing and presentation. These results suggest that soluble exogenous antigens delivered in different carrier systems may be processed differently by different APC in vivo for MHC class I- or class II-restricted presentation.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos/farmacología , Macrófagos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Formación de Anticuerpos , Citotoxicidad Inmunológica , Epítopos/farmacología , Terapia de Inmunosupresión , Virus de la Influenza A/inmunología , Inyecciones Intraperitoneales , Liposomas/administración & dosificación , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Vehículos Farmacéuticos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
CD4+ T cells play an important role in the aetiology of inflammatory bowel disease (IBD), but it is not clear which factor(s) cause activation of these cells. The aim of this study was to examine the effects of adoptive transfer of splenic (CD4+) T cells from TNBS/ethanol-sensitized donor rats to naive recipients and the migration pattern of transferred T cells. For the transfer experiments, colitis was induced in rats by colonic administration of TNBS/ethanol. Seventeen days later, either total splenic T cells or CD4+, or CD8+ T cells were transferred to naive recipients. At days 1, 2 and 3 after transfer, the recipients were killed and the migration pattern of the transferred T cells was studied, as well as inflammatory cells in several organs, including the colon. To determine cytokine profiles of the T cells, colitis was induced in mice. Therefore, different combinations of 2,4-dinitrobenzene sulfonic acid (DNBS) in ethanol or saline, or ethanol alone were intrarectally administered. At day 9 after induction of colitis, mice were killed and cytokine profiles in the colon were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The results show that CD4+ T cells from donor rats with TNBS/ethanol-induced colitis migrate in particular to the colon upon transfer to naive recipients, and that this process is down-regulated by CD8+ T cells. This migration is probably caused by T cell recognition of the colonic bacterial flora and initiates an inflammatory reaction in the recipient's colon, characterized by an increase of the recipient's own T cells, macrophages, and neutrophils. In the mice experiments we showed that a second administration of DNBS/ethanol or ethanol alone, which presumably causes bacterial translocation, results in increased numbers of T cells into the colon, accompanied by an increase in Th1 cytokines. These data suggest that Th1 cells recognize the colonic bacterial flora.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Colitis/inmunología , Regulación hacia Abajo , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colon/inmunología , Dinitrofluorobenceno/análogos & derivados , Inmunohistoquímica , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Ratas , Ácido TrinitrobencenosulfónicoRESUMEN
We previously reported the isolation of allergen-specific Th2 lines and clones from atopy patch test (APT) sites of atopic dermatitis (AD) patients. Upon stimulation with allergen or anti-CD3+ phorbol myristate acetate (PMA) IL-4 was released with or without IL-5, while no (or extremely low concentrations of) IL-2 and interferon-gamma (IFN-gamma) were detectable. A high IL-4/IFN-gamma ratio facilitates production of allergen-specific IgE, of which high levels are observed in AD patients. Here we show that the above mentioned Th2 cells are notably different from murine Th2 cells. Not IL-4, which is the autocrine acting growth factor for murine Th2 cells, but IL-2 was needed for proliferation of these human APT-derived Th2 lines and clones. Of significance, unless exogenous IL-2 was added, no proliferative response to allergen, presented by Epstein-Barr virus-transformed B (EBV-B) cells, non-T cells or IgE-bearing Langerhans cells (LC), occurred. Lack of proliferation and IL-2 production after full T cell receptor (TCR) triggering is a characteristic first described for in vitro anergized T cells. However, like the clones we describe in this study, anergic T cells may retain production of cytokines other than IL-2. A further resemblance between anergic T cells and the human Th2 clones reported here is that IL-4 can enhance IL-2-driven proliferation, but is not capable of inducing T cell growth by itself. The absence of IL-4-driven proliferation differentiates human Th2 cells from murine Th2 cells. Both produce IL-4 when stimulated in a cognate fashion, but only murine Th2 cells will proliferate. We conclude that the presently reported human Th2 cells are different from murine Th2 cells, in that they need other T cells to produce IL-2 required for their expansion. Moreover, the Th2 cells phenotypically resemble anergic T cells. As yet, however, we have no clue as to whether these features account for the current Th2 cells only or for human Th2 cells in general. We hypothesize that the Th2 phenotype of AD skin-derived, allergen-specific T cells may be induced in vivo by LC, which lack CD80, and therefore do not provide secondary signals through CD28-CD80 interaction.