RESUMO
The increasing implication of lymphocytes in general physiology and immune surveillance outside of infection poses the question of how their antigen receptors might be involved. Here, we show that macromolecular aggregates of intraepidermal γδ T cell antigen receptors (TCRs) in the mouse skin aligned with and depended on Skint1, a butyrophilin-like (BTNL) protein expressed by differentiated keratinocytes (KCs) at steady state. Interruption of TCR-mediated 'normality sensing' had no impact on γδ T cell numbers but altered their signature phenotype, while the epidermal barrier function was compromised. In addition to the regulation of steady-state physiology, normality sensing licensed intraepidermal T cells to respond rapidly to subsequent tissue perturbation by using innate tumor necrosis factor (TNF) superfamily receptors. Thus, interfering with Skint1-dependent interactions between local γδ T cells and KCs at steady state increased the susceptibility to ultraviolet B radiation (UVR)-induced DNA damage and inflammation, two cancer-disposing factors.
Assuntos
Linfócitos Intraepiteliais , Receptores de Antígenos de Linfócitos T gama-delta , Animais , Butirofilinas , Epiderme , Linfócitos Intraepiteliais/metabolismo , Licenciamento , Camundongos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7(+) γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαß(+) repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7(+) cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4(+) cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.
Assuntos
Butirofilinas/imunologia , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Butirofilinas/genética , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Timo/imunologiaRESUMO
Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection.
Assuntos
Epiderme/imunologia , Células Epiteliais/imunologia , Imunoglobulinas/química , Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.
Assuntos
Linfócitos B/metabolismo , Movimento Celular/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Linfócitos B/imunologia , Sítios de Ligação , Adesão Celular/fisiologia , Linhagem Celular Transformada , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/imunologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/imunologia , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de ProteínaRESUMO
Chemerin is a potent macrophage chemoattractant protein. We used murine peritoneal exudate cells (PECs) in adhesion, flow cytometry, and confocal microscopy assays to test the hypothesis that chemerin can also contribute to inflammation by promoting macrophage adhesion. Chemerin stimulated the adhesion of PECs to the extracellular matrix protein fibronectin and to the adhesion molecule VCAM-1 within a minute, with an EC(50) of 322 and 196 pM, respectively. Experiments using pertussis toxin and PECs from ChemR23(-/-) mice demonstrated that chemerin stimulated the adhesion of macrophages via the Gi protein-coupled receptor ChemR23. Blocking Abs against integrin subunits revealed that 89% of chemerin-stimulated adhesion to fibronectin was dependent on increased avidity of the integrin VLA-5 (alpha(5)beta(1)) and that 88% of adhesion to VCAM-1 was dependent on increased avidity of VLA-4 (alpha(4)beta(1)). Although chemerin was unable to induce an increase in integrin affinity as judged by the binding of soluble ligand, experiments using confocal microscopy revealed an increase in valency resulting from integrin clustering as the mechanism responsible for chemerin-stimulated macrophage adhesion. PI3K, Akt, and p38 were identified as key signaling mediators in chemerin-stimulated adhesion. The finding that chemerin can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules, taken together with its ability to promote chemotaxis, suggests a novel role for chemerin in the recruitment and retention of macrophages at sites of inflammation.
Assuntos
Fatores Quimiotáticos/fisiologia , Fibronectinas/metabolismo , Mediadores da Inflamação/fisiologia , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Macrófagos Peritoneais/imunologia , Agregação de Receptores/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Adesão Celular/imunologia , Linhagem Celular , Quimiocinas , Quimiotaxia de Leucócito/imunologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Integrina alfa4beta1/fisiologia , Integrina alfa5beta1/fisiologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Chemerin is a chemotactic protein that binds to the G protein-coupled receptor, ChemR23. We demonstrate that murine chemerin possesses potent antiinflammatory properties that are absolutely dependent on proteolytic processing. A series of peptides was designed, and only those identical to specific C-terminal chemerin sequences exerted antiinflammatory effects at picomolar concentrations in vitro. One of these, chemerin15 (C15; A(140)-A(154)), inhibited macrophage (MPhi) activation to a similar extent as proteolyzed chemerin, but exhibited reduced activity as a MPhi chemoattractant. Intraperitoneal administration of C15 (0.32 ng/kg) to mice before zymosan challenge conferred significant protection against zymosan-induced peritonitis, suppressing neutrophil (63%) and monocyte (62%) recruitment with a concomitant reduction in proinflammatory mediator expression. Importantly, C15 was unable to ameliorate zymosan-induced peritonitis in ChemR23(-/-) mice, demonstrating that C15's antiinflammatory effects are entirely ChemR23 dependent. In addition, administration of neutralizing anti-chemerin antibody before zymosan challenge resulted in a significant exacerbation of peritoneal inflammation (up to 170%), suggesting an important endogenous antiinflammatory role for chemerin-derived species. Collectively, these results show that chemerin-derived peptides may represent a novel therapeutic strategy for the treatment of inflammatory diseases through ChemR23.