RESUMEN
The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11, or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2. Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.
Asunto(s)
Proteínas Munc18/genética , Proteínas Munc18/inmunología , Linfocitos T Citotóxicos/inmunología , Alelos , Línea Celular , Citotoxicidad Inmunológica , Femenino , Humanos , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Persona de Mediana Edad , MutaciónRESUMEN
Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.
Asunto(s)
Gastroenteritis/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Proteínas Munc18/genética , Proteínas Munc18/inmunología , Neutrófilos/inmunología , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/microbiología , Escherichia coli/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Femenino , Gastroenteritis/genética , Predisposición Genética a la Enfermedad , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/microbiología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/microbiología , Masculino , Neutrófilos/microbiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunologíaRESUMEN
Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins and SM (Sec1/Munc18) family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1), a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs) from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W(sh) mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.
Asunto(s)
Degranulación de la Célula/inmunología , Inmunoglobulina E/inmunología , Mastocitos/fisiología , Proteínas Munc18/metabolismo , Transducción de Señal/inmunología , Animales , Western Blotting , Cartilla de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Hígado/citología , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Munc18/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Syntaxin 11 (STX11) controls vesicular trafficking and is a key player in exocytosis. Since Stx11 mutations are causally associated with a familial hemophagocytic lymphohistio-cytosis, we wanted to clarify whether STX11 is functionally important for key immune cell populations. This was studied in primary cells obtained from newly generated Stx11(-/-) mice. Our data revealed that STX11 is not only widely expressed in different immune cells, but also induced upon LPS or IFN-γ treatment. However, Stx11 deficiency does not affect macrophage phagocytic function and cytokine secretion, mast cell activation, or antigen presentation by DCs. Instead, STX11 selectively controls lymphocyte cytotoxicity in NK and activated CD8(+) T cells and degranulation in neutrophils. Stx11(-/-) NK cells and CTLs show impaired degranulation, despite a comparable activation, maturation and expression of the complex-forming partners MUNC18-2 and VTI1B. In addition, Stx11(-/-) CTLs and NK cells produce abnormal levels of IFN-γ. Since functional reconstitution rescues the defective phenotype of Stx11(-/-) CTLs, we suggest a direct, specific and key role of STX11 in controlling lymphocyte cytotoxicity, cytokine production and secretion. Finally, we show that these mice are a very useful tool for dissecting the role of STX11 in vesicular trafficking and secretion.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Neutrófilos/inmunología , Proteínas Qa-SNARE/inmunología , Animales , Degranulación de la Célula/genética , Línea Celular , Citotoxicidad Inmunológica/genética , Humanos , Interferón gamma/inmunología , Lipopolisacáridos/inmunología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Munc18/inmunología , Mutación/genética , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/inmunologíaRESUMEN
Cytotoxic T lymphocytes, natural killer cells, and NKT cells are effector cells able to kill infected cells. In some inherited human disorders, a defect in selected proteins involved in the cellular cytotoxicity mechanism results in specific clinical syndromes, grouped under the name of familial hemophagocytic lymphohistiocytosis. Recent advances in genetic studies of these patients has allowed the identification of different genetic subsets. Additional genetic immune deficiencies may also induce a similar clinical picture. International cooperation and prospective trials resulted in refining the diagnostic and therapeutic approach to these rare diseases with improved outcome but also with improved knowledge of the mechanisms underlying granule-mediated cellular cytotoxicity in humans.
Asunto(s)
Citotoxicidad Inmunológica/fisiología , Células Asesinas Naturales/inmunología , Linfohistiocitosis Hemofagocítica , Células T Asesinas Naturales/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Síndrome de Chediak-Higashi/genética , Síndrome de Chediak-Higashi/inmunología , Gránulos Citoplasmáticos/metabolismo , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/inmunología , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Subgrupos Linfocitarios/inmunología , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/inmunología , Linfohistiocitosis Hemofagocítica/terapia , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , Síndrome de Activación Macrofágica/genética , Síndrome de Activación Macrofágica/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Proteínas Munc18/genética , Proteínas Munc18/inmunología , Perforina , Piebaldismo/genética , Piebaldismo/inmunología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Enfermedades de Inmunodeficiencia Primaria , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/inmunologíaRESUMEN
Rasmussen encephalitis (RE) is a rare form of severe unihemispheric epilepsy established to be an autoimmune disease. Here we demonstrate the presence of autoantibodies against Munc18-1 in 20% of patients collective with biopsy-proven RE. Intriguingly, brain biopsy specimens of these patients showed a striking perivascular accentuated infiltration of B-lymphocytes and plasma cells, suggesting a subgroup of RE patients harboring Munc18-1 antibodies and concomitant B- and plasma cell infiltration.
Asunto(s)
Autoanticuerpos/metabolismo , Linfocitos B/patología , Encefalitis , Proteínas Munc18/inmunología , Células Plasmáticas/patología , Adolescente , Adulto , Animales , Antígenos CD/metabolismo , Línea Celular Transformada , Niño , Encefalitis/inmunología , Encefalitis/metabolismo , Encefalitis/patología , Femenino , Humanos , Masculino , Ratones , TransfecciónRESUMEN
Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [(32)P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.