RESUMO
Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.
Assuntos
Fator B do Complemento/metabolismo , Edema/fisiopatologia , Hipersensibilidade Tardia/fisiopatologia , Mastócitos/efeitos da radiação , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Movimento Celular/fisiologia , Movimento Celular/efeitos da radiação , Fator B do Complemento/genética , Modelos Animais de Doenças , Edema/etiologia , Feminino , Hipersensibilidade Tardia/etiologia , Masculino , Mastócitos/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/fisiopatologia , Queimadura Solar/etiologia , Queimadura Solar/fisiopatologiaRESUMO
Natural killer T (NKT) cells have been implicated in diverse immune responses ranging from suppression of autoimmunity to tumor rejection. Thymus-dependent NKT cells are positively selected by the major histocompatibility complex class I-like molecule CD1d, but the molecular events downstream of CD1d are still poorly understood. Here, we show that distinct members of the Rel/nuclear factor (NF)-kappa B family of transcription factors were required in both hematopoietic and nonhematopoietic cells for normal development of thymic NKT cells. Activation of NF-kappa B via the classical I kappa B alpha-regulated pathway was required in a cell autonomous manner for the transition of NK-1.1-negative precursors that express the TCR V alpha 14-J alpha 18 chain to mature NK-1.1-positive NKT cells. The Rel/NF-kappa B family member RelB, on the other hand, had to be expressed in radiation resistant thymic stromal cells for the generation of early NK-1.1-negative NKT precursors. Moreover, NF-kappa B-inducing kinase (NIK) was required for both constitutive thymic DNA binding of RelB and the specific induction of RelB complexes in vitro. Thus, distinct Rel/NF-kappa B family members in hematopoietic and nonhematopoietic cells regulate NKT cell development with a unique requirement for NIK-mediated activation of RelB in thymic stroma.
Assuntos
Diferenciação Celular/fisiologia , Células Matadoras Naturais/fisiologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Subpopulações de Linfócitos T/fisiologia , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Receptor beta de Linfotoxina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Baço/citologia , Baço/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/alpha-galactosylceramide (CD1d/alphaGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/alphaGC tetramer-binding NKT cells, including NK1.1(+) and NK1.1(-) subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/alphaGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4(+)CD8(-)NK1.1(-)cells. NK1.1(+) NKT cells, including CD4(+) and CD4(-)CD8(-) subsets, appeared at days 7-8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4(+)NK1.1(-) NKT cells gave rise to NK1.1(+) NKT cells (including CD4(+) and CD4(-) subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1(-) NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1(-). Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells.
Assuntos
Antígenos CD1/imunologia , Antígenos CD4/imunologia , Células Matadoras Naturais/imunologia , Timo/imunologia , Animais , Animais Recém-Nascidos , Antígenos CD1/genética , Antígenos CD1d , Antígenos CD4/genética , Citocinas/análise , Feto , Citometria de Fluxo , Leucócitos/imunologia , Fígado/crescimento & desenvolvimento , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Precursores de Proteínas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Baço/embriologia , Baço/crescimento & desenvolvimento , Baço/imunologia , Timo/embriologia , Timo/crescimento & desenvolvimentoRESUMO
A defect in RelB, a member of the Rel/nuclear factor (NF)-kappa B family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-kappa B-inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/- x aly/+ compound heterozygous mice. After stimulation with alpha-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/- or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.
Assuntos
Diferenciação Celular/fisiologia , Células Matadoras Naturais/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/fisiologia , Fatores de Transcrição/metabolismo , Animais , Antígenos CD1/metabolismo , Antígenos CD1d , Células Cultivadas , Quimera , Fibroblastos/citologia , Fibroblastos/metabolismo , Receptores de Hialuronatos/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Receptor beta de Linfotoxina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Nódulos Linfáticos Agregados/anatomia & histologia , Nódulos Linfáticos Agregados/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/metabolismo , Fator de Transcrição RelB , Fatores de Transcrição/genética , Microglobulina beta-2/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Natural killer T (NKT) cells are a unique lymphocyte subtype implicated in the regulation of autoimmunity, particularly diabetes and experimental allergic encephalomyelitis in animal models. In some reports, NKT-cell regulation was revealed only following vigorous activation by a synthetic glycolipid, a process that might not occur naturally. Patients with diverse autoimmune diseases have reduced NKT-cell counts and, in diabetes and multiple sclerosis, effective NKT-cell regulation correlates with the secretion of Th2 cytokines. Although current controversy surrounds the importance of NKT cells and their modes of action, they represent a potentially important clinical target.
Assuntos
Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Animais , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Encefalomielite Autoimune Experimental/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Esclerose Múltipla/imunologiaRESUMO
Natural killer (NK) T cells using an invariant Valpha14 (Valpha14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1(-) Valpha14i NKT precursor cell differentiates and expands within the thymus before export to the peripheral tissues occurs. However, little is known about the signals that cause the emigration of Valpha14i NKT cells from the thymus to the periphery. Here we show that signaling of lymphotoxin (LT) alphabeta through the LTbeta receptor (LTbetaR) is indispensable for regulating peripheral but not thymic Valpha14i NKT cell numbers. Homing to and homeostatic proliferation of thymic Valpha14i NKT cells in peripheral organs, however, was not dependent on LTbetaR. Instead, our data indicate that a LTbetaR-expressing thymic stromal cell regulates the thymic emigration of Valpha14i NKT cells but not conventional T cell receptor alphabeta cells.
Assuntos
Movimento Celular/fisiologia , Células Matadoras Naturais/metabolismo , Linfotoxina-alfa/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Animais , Sobrevivência Celular , Feminino , Células Matadoras Naturais/citologia , Fígado/citologia , Fígado/fisiologia , Receptor beta de Linfotoxina , Linfotoxina-beta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Baço/citologia , Baço/fisiologia , Subpopulações de Linfócitos T/citologia , Timo/fisiologiaRESUMO
NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR alpha-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d-/- mice that are completely deficient for NKT cells. CD1d tetramer- CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer- CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.
Assuntos
Antígenos CD1/fisiologia , Integrina alfa2/biossíntese , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos CD1/biossíntese , Antígenos CD1d , Biomarcadores , Células Cultivadas , Citocinas/biossíntese , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Baço/imunologia , Timo/citologia , Timo/imunologiaRESUMO
NKT cells are enigmatic lymphocytes that respond to glycolipid Ags presented by CD1d. Although they are key immunoregulatory cells, with a critical role in immunity to cancer, infection, and autoimmune diseases, little is known about how they respond to antigenic challenge. Current theories suggest that NKT cells die within hours of stimulation, implying that their direct impact on the immune system derives from the initial cytokine burst released before their death. Here we show that NKT cell disappearance results from TCR down-regulation rather than apoptosis, and that they expand to many times their normal number in peripheral tissues within 2-3 days of stimulation, before contracting to normal numbers over subsequent days. This expansion is associated with ongoing cytokine production, biased toward a Th1 (IFN-gamma(+) IL-4(-)) phenotype, in contrast to their initial Th0 (IFN-gamma(+)IL-4(+)) phenotype. This study provides critical new insight into how NKT cells can have such a major impact on immune responses, lasting many days beyond the initial stimulation of these cells.
Assuntos
Antígenos/administração & dosagem , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos/farmacologia , Divisão Celular/imunologia , Separação Celular , Sobrevivência Celular/imunologia , Regulação para Baixo/imunologia , Galactosilceramidas/farmacologia , Injeções Intraperitoneais , Interferon gama/biossíntese , Interleucina-4/biossíntese , Células Matadoras Naturais/metabolismo , Fígado/citologia , Fígado/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/biossíntese , Subpopulações de Linfócitos T/metabolismo , Fatores de TempoRESUMO
Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.
Assuntos
Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos/química , Antígenos/metabolismo , Antígenos CD1/química , Antígenos CD1/metabolismo , Antígenos CD1d , Sítios de Ligação , Citocinas/biossíntese , Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Glicoesfingolipídeos/metabolismo , Técnicas In Vitro , Células Matadoras Naturais/metabolismo , Cinética , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Solubilidade , Subpopulações de Linfócitos T/metabolismoRESUMO
NKT cell development takes place in the thymus, beginning when these cells branch away from CD4+CD8+ mainstream thymocytes upon expression of the Valpha14Jalpha18 T cell receptor (TCR) and recognition of the CD1d molecule. Although NKT cells express an invariant TCR alpha chain, the diverse TCR beta expression leaves open the possibility that the development of these cells is shaped by glycolipid antigen recognition in the context of CD1d. Here, we show that the presence of an agonist glycolipid ligand, alpha-galactosylceramide, while NKT cells are developing in vitro or in vivo, specifically ablates their development. In contrast, the delayed introduction of this compound in vitro or in vivo, after NKT cells have developed, does not deplete these cells. These data indicate that NKT cells pass through a developmental window where they are susceptible to TCR-mediated negative selection, and suggest that NKT cells with a potentially high level of self reactivity can be removed from the NKT cell repertoire before they exit the thymus.