RESUMO
FcεRI engagement in mast cells (MCs) induces the activation of two distinct sphingosine kinase isoforms (SphK1 and SphK2) to produce sphingosine-1-phosphate, a mediator essential for MC responses. Whereas embryonic-derived SphK2-null MCs showed impaired responses to Ag, RNA silencing studies on other MC types indicated a dominant role for SphK1. Given the known functional heterogeneity of MCs, we explored whether the reported differences in SphK1 or SphK2 usage could be reflective of phenotypic differences between MC populations. Using lentiviral-based short hairpin RNA to silence SphK1 or SphK2, we found that SphK2 is required for murine MC degranulation, calcium mobilization, and cytokine and leukotriene production, irrespective of the tissue from which the MC progenitors were derived, the stage of MC granule maturity, or the conditions used for differentiation. This finding was consistent with the lack of a full allergic response in SphK2-null mice challenged to undergo passive cutaneous anaphylaxis. A redundant role for both SphKs was uncovered, however, in chemotaxis toward Ag in all MC types tested and in TNF-α production in certain MC types. In contrast, human MC responses were dependent only on SphK1, associating with a more robust expression of this isoform and a more varied representation of SphK variants relative to murine MCs. The findings show that the function of SphK1 and SphK2 can be interchangeable in MCs; however, an important determinant of SphK isoform usage is the species of origin and an influencing factor, the tissue from which MCs may be derived and/or their differentiation state.
Assuntos
Expressão Gênica , Mastócitos/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Cálcio/metabolismo , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Ativação Enzimática , Inativação Gênica , Vetores Genéticos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lentivirus , Leucotrienos/genética , Leucotrienos/imunologia , Lisofosfolipídeos/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Especificidade de Órgãos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Interferente Pequeno/genética , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transdução GenéticaRESUMO
Autocrine stimulation of S1PR2, a receptor for the lipid mediator sphingosine-1-phosphate (S1P), has been implicated in mast cell degranulation to IgE/antigen (Ag) although, paradoxically, its ligand cannot trigger substantial degranulation. Additionally, the in vivo role of S1PR2 in the overall allergic responses is unclear since S1PR2 was reported to be required for the onset of systemic anaphylaxis by IgE/Ag but, in apparent contradiction, also for the recovery from histamine-induced anaphylaxis in a mast cell independent manner. Here, we sought to clarify the role of S1PR2 in mast cell degranulation and in IgE and IgG-mediated anaphylaxis. Lack of S1PR2 reduced IgE/Ag-induced degranulation in in vitro experiments with mucosal mast cells, but had no effect on connective tissue type mast cells. This latter response correlated with a lack of involvement of S1PR2 in the onset of non-lethal IgE/Ag-mediated systemic and cutaneous anaphylaxis. However, S1pr2(-/-) mice were slow to recover (or did not recover) from FcÉRI-mediated anaphylaxis, an outcome that mirrored their known impairment in histamine clearance due to defective vascular tone. A minor role for S1PR2 in mast cell degranulation was uncovered upon engagement of low affinity receptors for IgG and in the onset of IgG-mediated anaphylaxis. Our findings show that S1PR2 is dispensable for initiating IgE/Ag-mediated connective tissue mast cell degranulation and anaphylaxis, but it is required for normal recovery from anaphylaxis.
Assuntos
Anafilaxia/imunologia , Anafilaxia/metabolismo , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Anafilaxia/genética , Animais , Degranulação Celular/imunologia , Imunização , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Receptores de Lisoesfingolipídeo/genética , Receptores de Esfingosina-1-FosfatoRESUMO
BACKGROUND: Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy. OBJECTIVE: We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy. METHODS AND RESULTS: Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1(-/-) and SphK2(-/-) mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4(+) effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals. CONCLUSION: Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Hipersensibilidade Alimentar/imunologia , Mastócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Administração Oral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células CACO-2 , Movimento Celular/genética , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Hipersensibilidade Alimentar/diagnóstico , Hipersensibilidade Alimentar/genética , Mucosa Gástrica/patologia , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina E/sangue , Imunoglobulina E/genética , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Ovalbumina/administração & dosagem , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Regulação para CimaRESUMO
Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.
Assuntos
Permeabilidade Capilar/imunologia , Hipersensibilidade/patologia , Mastócitos/patologia , Mastócitos/fisiologia , Mastocitose/patologia , PTEN Fosfo-Hidrolase/genética , Transferência Adotiva , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Degranulação Celular/imunologia , Divisão Celular/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Hipersensibilidade/imunologia , Hipersensibilidade/fisiopatologia , Mastocitose/imunologia , Mastocitose/fisiopatologia , Camundongos , Camundongos Mutantes , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/imunologiaRESUMO
Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.
Assuntos
Anafilaxia/enzimologia , Regulação Enzimológica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Transplante de Medula Óssea , Taxa de Filtração Glomerular , Células-Tronco Hematopoéticas/metabolismo , Histamina/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , PermeabilidadeRESUMO
The complement system provides the host with protection against pathogenic agents and in some cases can result in damage to host tissue. However, the exact mechanism of how complement kills gram-negative bacteria in lysozyme-neutralized and or lysozyme-depleted serum is still under active investigation. In previous studies, it has been demonstrated that inner membrane damage by the membrane attack complex contributes to depolarization and the subsequent collapse of the membrane potential. In these studies we have shown that the membrane attack complex and its precursors provide additional protective effect by the enhanced uptake of antibiotics in the death of E. coli J5. Specifically, the deposition of C5b fragments from C6 neutralized Pooled Normal Human Serum (PNHS) and C5b6 complexes from C7 neutralized PNHS on E. coli J5 contribute to antibiotic uptake and killing. Since C5b and C5b6 do not form pores, we suggest that disturbances and or cracks in the outer membrane by the deposited complexes accelerates uptake of the antibiotics and enhanced killing of E. coli J5 employed in these studies.