RESUMEN
The cross talk between NK cells and macrophages is emerging as a major line of defense against microbial infections and tumors. This study reveals a complex network of soluble mediators and cell-to-cell interactions allowing human classically activated (M1) macrophages, but not resting (M0) or alternatively activated (M2) macrophages, to prime resting autologous NK cells. In this article, we show that M1 increase NK cell cytotoxicity by IL-23 and IFN-ß-dependent upregulation of NKG2D, IL-1ß-dependent upregulation of NKp44, and trans-presentation of IL-15. Moreover, both IFN-ß-dependent cis-presentation of IL-15 on NK cells and engagement of the 2B4-CD48 pathway are used by M1 to trigger NK cell production of IFN-γ. The disclosure of these synergic cellular mechanisms regulating the M1-NK cell cross talk provides novel insights to better understand the role of innate immune responses in the physiopathology of tumor biology and microbial infections.
Asunto(s)
Interferón beta/inmunología , Interleucina-15/inmunología , Interleucina-1beta/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Antígenos CD/metabolismo , Comunicación Celular/inmunología , Línea Celular Tumoral , Proliferación Celular , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Interferón gamma/biosíntesis , Interleucina-1beta/biosíntesis , Subunidad p19 de la Interleucina-23 , Células Jurkat , Activación de Linfocitos/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Receptores Inmunológicos/metabolismo , Familia de Moléculas Señalizadoras de la Activación LinfocitariaRESUMEN
Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Bacteriólisis , Células Asesinas Naturales/inmunología , Mycobacterium kansasii , Mycobacterium tuberculosis , Perforina/metabolismo , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/farmacología , Bacteriólisis/efectos de los fármacos , Bacteriólisis/fisiología , Línea Celular Tumoral , Pared Celular/efectos de los fármacos , Humanos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/ultraestructura , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Subfamilia K de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Nanotubos , Receptor 2 Gatillante de la Citotoxidad Natural/antagonistas & inhibidores , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 2 Gatillante de la Citotoxidad Natural/genética , Receptor 3 Gatillante de la Citotoxidad Natural/antagonistas & inhibidores , Receptor 3 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 3 Gatillante de la Citotoxidad Natural/genética , Perforina/biosíntesis , Perforina/genética , Perforina/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
We have characterized the NK/NKT-like cells in patients with self-limiting hepatitis E infection. The distribution of peripheral NK/NKT-like cells, expressions of activation receptors, cytotoxic potential and effector function of NK/NKT-like cells from fresh peripheral blood mononuclear cells of 86 acute patients, 101 recovered and 54 control individuals were assessed. Activated NKT-like (CD16(+) CD56(+) CD3(+)) cells were high in the patient groups. On CD56(+) CD3(-) cells, NKp44 and NKp46 expressions were high in the acute patients, whereas NKp30, NKp44, NKp46 and NKG2D were high in the recovered individuals. On CD56(+) CD3(+) cells, NKp44, NKp46 and NKG2D expressions were high in the recovered but NKp30 was low in both the patient groups. Collectively, the current study elucidates the role of NK/NKT-like cells demonstrating phenotypic alterations of activated NKT-like cells and activation receptors, lack of CD107a expression and functional impairment of peripheral NK/NKT-like cells in self-limiting hepatitis E infection.
Asunto(s)
Expresión Génica , Hepatitis E/patología , Células Asesinas Naturales/fisiología , Receptor 1 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Células T Asesinas Naturales/fisiología , Adolescente , Adulto , Anciano , Antígenos CD/análisis , Femenino , Perfilación de la Expresión Génica , Hepatitis E/inmunología , Humanos , Células Asesinas Naturales/química , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK/análisis , Receptor 3 Gatillante de la Citotoxidad Natural/análisis , Células T Asesinas Naturales/química , Adulto JovenRESUMEN
Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.
Asunto(s)
Cartílago/inmunología , Condrocitos/metabolismo , Células Asesinas Naturales/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/inmunología , Cartílago/citología , Células Cultivadas , Niño , Preescolar , Pie Equinovaro , Citotoxicidad Inmunológica/inmunología , Humanos , Lactante , Leucocitos Mononucleares/inmunología , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismoRESUMEN
BACKGROUND: The human cytokine granulocyte-colony stimulatory factor (G-CSF) has found widespread application in the medical treatment of neutropenia and to mobilize hematopoietic stem cells used for transplantation. So far, the effect of G-CSF on natural killer (NK) cells has not been fully investigated. STUDY DESIGN AND METHODS: The effect of G-CSF on the phenotype, cytokine secretion profile, and cytotoxicity of NK cells was assessed. NK cells incubated in vitro in presence of G-CSF for 48 hours as well as NK cells isolated from peripheral blood of G-CSF-mobilized stem cell donors (in vivo) were used. RESULTS: In vitro, G-CSF caused a strongly altered phenotype in NK cells with 49% down regulation of NKp44 frequency. Furthermore, the expression of the activating receptors NKp46 and NKG2D decreased 40 and 64%, respectively. The expression of KIR2DL1 and KIR2DL2 decreased by 46% each. In cytotoxicity assays, the lytic capacity of G-CSF-exposed NK cells is reduced by up to 68% in vitro and up to 83% in vivo. Accordingly, granzyme B levels of in vivo G-CSF-exposed NK cells were reduced by up to 87% in comparison to nonstimulated NK cells. Cytokine production of in vitro and in vivo incubated NK cells was strongly decreased for interferon-γ, tumor necrosis factor-α, and granulocyte macrophage colony-stimulating factor as well as interleukin (IL)-6 and IL-8. Furthermore, we observed a reduction in proliferation and a positive feedback loop that increased the expression of the G-CSF receptor. CONCLUSION: G-CSF was demonstrated to be a strong inhibitor of NK cells activity and may prevent their graft-versus-leukemia effect after transplantation.
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Asesinas Naturales/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Citocinas/biosíntesis , Citocinas/genética , Depresión Química , Regulación de la Expresión Génica/efectos de los fármacos , Efecto Injerto vs Leucemia/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Granzimas/biosíntesis , Granzimas/genética , Humanos , Células Asesinas Naturales/metabolismo , Lenograstim , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Receptor 1 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 2 Gatillante de la Citotoxidad Natural/genética , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacologíaRESUMEN
CD4(+) T cell loss is central to HIV pathogenesis. In the initial weeks post-infection, the great majority of dying cells are uninfected CD4(+) T cells. We previously showed that the 3S motif of HIV-1 gp41 induces surface expression of NKp44L, a cellular ligand for an activating NK receptor, on uninfected bystander CD4(+) T cells, rendering them susceptible to autologous NK killing. However, the mechanism of the 3S mediated NKp44L surface expression on CD4(+) T cells remains unknown. Here, using immunoprecipitation, ELISA and blocking antibodies, we demonstrate that the 3S motif of HIV-1 gp41 binds to gC1qR on CD4(+) T cells. We also show that the 3S peptide and two endogenous gC1qR ligands, C1q and HK, each trigger the translocation of pre-existing NKp44L molecules through a signaling cascade that involves sequential activation of PI3K, NADPH oxidase and p190 RhoGAP, and TC10 inactivation. The involvement of PI3K and NADPH oxidase derives from 2D PAGE experiments and the use of PIP3 and H2O2 as well as small molecule inhibitors to respectively induce and inhibit NKp44L surface expression. Using plasmid encoding wild type or mutated form of p190 RhoGAP, we show that 3S mediated NKp44L surface expression on CD4(+) T cells is dependent on p190 RhoGAP. Finally, the role of TC10 in NKp44L surface induction was demonstrated by measuring Rho protein activity following 3S stimulation and using RNA interference. Thus, our results identify gC1qR as a new receptor of HIV-gp41 and demonstrate the signaling cascade it triggers. These findings identify potential mechanisms that new therapeutic strategies could use to prevent the CD4(+) T cell depletion during HIV infection and provide further evidence of a detrimental role played by NK cells in CD4(+) T cell depletion during HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteína gp41 de Envoltorio del VIH/metabolismo , Peróxido de Hidrógeno/metabolismo , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Fosfatidilinositol 3-Quinasas/fisiología , Secuencias de Aminoácidos/fisiología , Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , NADPH Oxidasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56(dim)CD16(bright) make up 90% circulating NK cells, whereas CD56(bright)CD16(-/dim) comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56(bright) NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-gamma and TNF-alpha and notably IL-12.
Asunto(s)
Antígeno CD56/biosíntesis , Células Dendríticas/metabolismo , Células Asesinas Naturales/metabolismo , Subgrupos Linfocitarios/metabolismo , Receptores de IgG/metabolismo , Antígeno CD56/genética , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/patología , Humanos , Inmunofenotipificación , Interleucina-12/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Activación de Linfocitos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Receptor 2 Gatillante de la Citotoxidad Natural/biosíntesis , Receptor 3 Gatillante de la Citotoxidad Natural/biosíntesis , Agregación de Receptores , Receptores de IgG/inmunologíaRESUMEN
Molecular and functional characterization of the natural cytotoxicity receptor (NCR) NKp44 in species other than Homo sapiens has been elusive, so far. Here, we provide complete phenotypic, molecular and functional characterization for NKp44 triggering receptor on Pan troglodytes NK cells, the closest human relative, and the analysis of NKp44-genomic locus and transcription in Macaca fascicularis. Similar to H. sapiens, NKp44 expression is detectable on chimpanzee NK cells only upon activation. However, basal NKp44 transcription is 5-fold higher in chimpanzees with lower differential increases upon cell activation compared with humans. Upon activation, an overall 12-fold lower NKp44 gene expression is observed in P. troglodytes compared with H. sapiens NK cells with only a slight reduction in NKp44 surface expression. Functional analysis of 'in vitro' activated purified NK cells confirms the NKp44 triggering potential compared with other major NCRs. These findings suggest the presence of a post-transcriptional regulation that evolved differently in H. sapiens. Analysis of cynomolgus NKp44-genomic sequence and transcription pattern showed very low levels of transcription with occurrence of out-of-frame transcripts and no surface expression. The present comparative analysis suggests that NKp44-genomic organization appears during macaque speciation, with considerable evolution of its transcriptional and post-transcriptional tuning. Thus, NKp44 may represent an NCR being only recently emerged during speciation, acquiring functional relevance only in non-human primates closest to H. sapiens.