RESUMEN
Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response.
Asunto(s)
Leishmania/metabolismo , Leishmaniasis/sangre , Leishmaniasis/fisiopatología , Neutrófilos/parasitología , Animales , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Sistema Inmunológico , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Psychodidae , Especificidad de la EspecieRESUMEN
Upon infection with the protozoan parasite Leishmania major, susceptible BALB/c mice develop unhealing lesions associated with the maturation of CD4(+)Th2 cells secreting IL-4. In contrast, resistant C57BL/6 mice heal their lesions, because of expansion and secretion of IFN-gamma of CD4(+) Th1 cells. The Fas-FasL pathway, although not involved in Th cell differentiation, was reported to be necessary for complete resolution of lesions. We investigate here the role of IFN-gamma and IL-4 on Fas-FasL nonapoptotic signaling events leading to the modulation of macrophage activation. We show that addition of FasL and IFN-gamma to BMMø led to their increased activation, as reflected by enhanced secretion of TNF, IL-6, NO, and the induction of their microbicidal activity, resulting in the killing of intracellular L. major. In contrast, the presence of IL-4 decreased the synergy of IFN-gamma/FasL significantly on macrophage activation and the killing of intracellular L. major. These results show that FasL synergizes with IFN-gamma to activate macrophages and that the tight regulation by IFN-gamma and/or IL-4 of the nonapoptotic signaling events triggered by the Fas-FasL pathway affects significantly the activation of macrophages to a microbicidal state and may thus contribute to the pathogenesis of L. major infection.
Asunto(s)
Proteína Ligando Fas/farmacología , Activación de Macrófagos , Transducción de Señal/inmunología , Receptor fas/metabolismo , Animales , Interferón gamma/farmacología , Interleucina-4/farmacología , Leishmania major/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB CRESUMEN
TNF is an essential player in infections with Leishmania major, contributing to the control of the inflammatory lesion and, to a lesser degree, to parasite killing. However, the relative contribution of the soluble and transmembrane forms of TNF in these processes is unknown. To investigate the role of transmembrane TNF (mTNF) in the control of L. major infections, mTNF-knock-in (mTNF(Delta/Delta)) mice, which express functional mTNF but do not release soluble TNF, were infected with L. major, and the development of the inflammatory lesion and the immune response was compared to that occurring in L. major-infected TNF(-/-) and wild-type mice. mTNF(Delta/Delta) mice controlled the infection and resolved their inflammatory lesion as well as wild-type mice, a process associated with the early clearance of neutrophils at the site of parasite infection. In contrast, L. major-infected TNF(-/-) mice developed non-healing lesions, characterized by an elevated presence of neutrophils at the site of infection and partial control of parasite number within the lesions. Altogether, the results presented here demonstrate that mTNF, in absence of soluble TNF, is sufficient to control infection due to L. major, enabling the regulation of inflammation, and the optimal killing of Leishmania parasites at the site of infection.
Asunto(s)
Inflamación/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas de la Membrana/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Formación de Anticuerpos/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inflamación/parasitología , Inflamación/patología , Interferón gamma/metabolismo , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/inmunología , Neutrófilos/parasitología , Neutrófilos/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Piel/metabolismo , Piel/parasitología , Piel/patología , Células TH1/inmunología , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.
Asunto(s)
Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Neutrófilos/inmunología , Animales , Médula Ósea/parasitología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Granuloma/patología , Interferón gamma/metabolismo , Interleucina-10/análisis , Interleucina-4/análisis , Procedimientos de Reducción del Leucocitos , Hígado/parasitología , Hígado/patología , Ratones , Óxido Nítrico Sintasa/biosíntesis , Bazo/química , Bazo/parasitología , Esplenomegalia/parasitología , Células TH1/inmunologíaRESUMEN
Interleukin (IL)-12p40, a subunit of IL-12p70 and IL-23, has previously been shown to inhibit IL-12p70 activity and interferon-gamma (IFN-gamma) production. However, recent evidence has suggested that the role of IL-12p40 is more complex. To study the contribution of IL-12p40 to immune responses against mycobacterial infections, we have used transgenic (tg) mice overexpressing IL-12p40 under the control of a major histocompatibility complex-II promoter. The IL-12p40 transgene was expressed during steady state at concentrations of 129 +/- 25 ng/ml of serum and 75 +/- 13 ng per spleen, while endogenous IL-12p40 was hardly detectable in control littermates. Bacille Calmette-Guérin (BCG) infection strongly induced the expression of IL-12p40 transgene in infected organs, and IL-12p40 monomeric and dimeric forms were identified in spleen of IL-12p40 tg mice. Excessive production of IL-12p40 resulted in a 14-fold increase in IL-12p70 serum levels in tg mice versus non-transgenic mice. IL-23 was also strongly elevated in the serum and spleens of IL-12p40 tg mice through BCG infection. While IFN-gamma and tumour necrosis factor protein levels were similar in IL-12p40 tg and non-transgenic mice, Th2 type immune responses were reduced in IL-12p40 tg mice. The number of BCG granulomas and macrophage expressing inducible nitric oxide synthase were similar in IL-12p40 tg and non-transgenic mice. IL-12p40 tg mice were as resistant as non-transgenic mice to BCG and Mycobacterium tuberculosis infections as they could efficiently control bacillary growth. These data show that high amounts of IL-12p40 promotes IL-12p70 and IL-23 formation, but that does not affect T helper 1 type immune responses and granuloma function, thus leading to normal mycobacterial clearance in infected organs.
Asunto(s)
Subunidad p40 de la Interleucina-12/inmunología , Interleucina-12/biosíntesis , Interleucina-23/biosíntesis , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/inmunología , Animales , Quimiocinas/biosíntesis , Granuloma/inmunología , Inmunidad Celular , Interferón gamma/biosíntesis , Subunidad p40 de la Interleucina-12/biosíntesis , Hepatopatías/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Bazo/inmunología , Células TH1/inmunología , Células Th2/inmunología , Tuberculosis/microbiología , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen-specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c-inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL-12p70 and IL-10. In contrast, L. major-infected BALB/c PMN transcribed and secreted high levels of IL-12p40 but did not secrete biologically active IL-12p70. Furthermore, IL-12p40 was shown not to associate with IL-23 p19 but formed IL-12p40 homodimers with inhibitory activity. No IL-10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL-12, and in BALB/c mice, secretion of IL-12p40 could contribute to impaired, early IL-12 signaling. These distinct PMN phenotypes may thus influence the development of L. major-specific immune response.
Asunto(s)
Leishmania major/inmunología , Neutrófilos/inmunología , Fenotipo , Animales , Células Cultivadas , Susceptibilidad a Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Innata , Interferón gamma/análisis , Interferón gamma/inmunología , Interleucina-10/análisis , Interleucina-10/inmunología , Subunidad p40 de la Interleucina-12/análisis , Subunidad p40 de la Interleucina-12/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/parasitología , Neutrófilos/patología , Nitritos/análisis , ARN Mensajero/metabolismo , Receptores Toll-Like/inmunología , Transcripción Genética , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.
Asunto(s)
Apoptosis/inmunología , Comunicación Celular/inmunología , Leishmania major/inmunología , Macrófagos Peritoneales/inmunología , Proteínas de la Membrana/fisiología , Neutrófilos/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Comunicación Celular/genética , Células Cultivadas , Técnicas de Cocultivo , Femenino , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Recuento de Leucocitos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/parasitología , Especificidad de la Especie , Factores de Tiempo , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Transcripción , Animales , Anticuerpos Antiprotozoarios/sangre , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Femenino , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Leishmania major/inmunología , Leishmaniasis/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN/química , ARN/genética , Receptor Notch1 , Receptores de Superficie Celular/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos Específicos , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.