RESUMO
BACKGROUND & AIMS: Intraepithelial T lymphocyte cells (IEL) are the first immune cells to respond to pathogens; they help maintain the integrity of the epithelial barrier. We studied the function of the mouse glycoprotein Signaling Lymphocyte Activation Molecule Family receptor (SLAMF) 4 (encoded by Slamf4) on the surface of CD8αß αß T-cell receptor (TCR)(+) IELs, and the roles of these cells in homeostasis of the small intestine in mice. METHODS: SLAMF4(-) CD8(+) αßTCR(+) cells isolated from spleens of OT-I Rag1(-/-) mice were induced to express gut-homing receptors and transferred to C57BL/6J mice; levels of SLAMF4(+) cells were measured in small intestine tissues. After administration of anti-CD3 or antigen, with or without anti-SLAM4, to C57BL/6J and Slamf4(-/-) mice, CD8αß αßTCR(+) IELs were collected; cytokine production and cytotoxicity were measured. Depletion of CX3CR1(+) phagocytes was assessed in mice by live-cell confocal imaging or by cytofluorometry; small intestine tissues were analyzed by histology and inflammation was quantified. RESULTS: Splenic CD8(+) αßTCR(+) cells began to express SLAMF4 only after migrating to the small intestine. Injection of C57BL/6J mice with anti-SLAMF4 and anti-CD3 increased levels of interleukin 10 and interferon gamma secretion by IEL, compared with injection of anti-CD3 only. Similarly, the number of granzyme B(+) cytotoxic CD8(+) αßTCR(+) IELs increased in Slamf4(-/-) mice after injection of anti-CD3 and anti-SLAMF4, administration of antigen, or injection of anti-CD3. Surprisingly, in vivo activation of CD8αß(+) IELs with anti-CD3 or antigen caused transient depletion of CX3CR1(+) phagocytes, which was prolonged by co-injection with anti-SLAMF4 or in Slamf4(-/-) mice. Anti-CD3 aggravated inflammation in the small intestines of Slamf4(-/-) mice and Eat2a(-/-)Eat2b(-/-) mice, indicated by flattened villi and crypt hyperplasia. CONCLUSIONS: In mice, the intestinal environment induces SLAMF4 expression and localization to the surface of CD8(+) αßTCR(+) IELs. Signaling via SLAMF4 controls expansion of cytotoxic CD8αß(+) IELs, which regulate the reversible depletion of lamina propria phagocytes and inflammation in the small intestine.
Assuntos
Antígenos CD/metabolismo , Proliferação de Células , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Ativação Linfocitária , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD/genética , Receptor 1 de Quimiocina CX3C , Movimento Celular , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Hiperplasia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagócitos/imunologia , Fagócitos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária , Baço/imunologia , Baço/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
The homophilic cell surface receptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6(-/-) C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram(-) bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag(-/-) mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6(-/-) Rag(-/-) mice were markedly reduced as compared with those of Rag(-/-) mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6(-/-) Rag(-/-) mice than in Rag(-/-) animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag(-/-) mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram(-) bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag(-/-) mice.
Assuntos
Antígenos CD/imunologia , Citrobacter rodentium/imunologia , Colite/imunologia , Imunidade Inata/imunologia , Receptores de Superfície Celular/imunologia , Animais , Colite/microbiologia , Colo/imunologia , Colo/microbiologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Células Th17/imunologia , Interleucina 22RESUMO
Glucocorticoid-induced TNF receptor family-related protein (GITR) regulates the function of both T cells and antigen-presenting cells (APCs), while the function of GITR ligand (GITR-L) is largely unknown. Here we evaluate the role of GITR-L, whose expression is restricted to APCs, in the development of enterocolitis. On injecting naive CD4(+) T cells, GITR-L(-/-)Rag(-/-) mice develop a markedly milder colitis than Rag(-/-) mice, which correlates with a 50% reduction of Ly6C(+)CD11b(+)MHCII(+) macrophages in the lamina propria and mesenteric lymph nodes. The same result was observed in αCD40-induced acute colitis and during peritonitis, suggesting an altered monocyte migration. In line with these observations, the number of nondifferentiated monocytes was approximately 3-fold higher in the spleen of GITR-L(-/-)Rag(-/-) mice than in Rag(-/-) mice after αCD40 induction. Consistent with the dynamic change in the formation of an active angiotensin II type 1 receptor (AT1) dimer in GITR-L(-/-) splenic monocytes during intestinal inflammation, the migratory capability of splenic monocytes from GITR-L-deficient mice was impaired in an in vitro transwell migration assay. Conversely, αGITR-L reduces the number of splenic Ly6C(hi) monocytes, concomitantly with an increase in AT1 dimers. We conclude that GITR-L regulates the number of proinflammatory macrophages in sites of inflammation by controlling the egress of monocytes from the splenic reservoir.
Assuntos
Glucocorticoides/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Monócitos/citologia , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Antígenos CD40 , Camundongos , Camundongos Knockout , Camundongos Mutantes , Monócitos/efeitos dos fármacosRESUMO
Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages (MΦs) by IFN-γ or bacteria. In this article, we report that a very high NADPH oxidase (Nox2) enzyme activity was found in Slamf8(-/-) MΦs in response to Escherichia coli or Staphylococcus aureus, as well as to PMA. The elevated Nox2 activity in Slamf8(-/-) MΦs was also demonstrated in E. coli or S. aureus phagosomes by using a pH indicator system and was further confirmed by a reduction in the enzyme activity after transfection of the receptor into Slamf8-deficient primary MΦs or RAW 264.7 cells. Upon exposure to bacteria or PMA, protein kinase C activity in Slamf8(-/-) MΦs is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which, in turn, leads to greater Nox2 activity. Taken together, the data show that, in response to inflammation-associated stimuli, the inducible receptor Slamf8 negatively regulates inflammatory responses.
Assuntos
Antígenos CD/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD/imunologia , Western Blotting , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/imunologia , Fagossomos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Regulação para CimaRESUMO
BACKGROUND & AIMS: Signaling lymphocyte activation molecule (Slamf)1 is a co-stimulatory receptor on T cells and regulates cytokine production by macrophages and dendritic cells. Slamf1 regulates microbicidal mechanisms in macrophages, therefore we investigated whether the receptor affects development of colitis in mice. METHODS: We transferred CD45RB(hi) CD4(+) T cells into Rag(-/-) or Slamf1(-/-)Rag(-/-) mice to induce colitis. We also induced colitis by injecting mice with an antibody that activates CD40. We determined the severity of enterocolitis based on disease activity index, histology scores, and levels of cytokine production, and assessed the effects of antibodies against Slamf1 on colitis induction. We quantified migration of monocytes and macrophage to inflamed tissues upon induction of colitis or thioglycollate-induced peritonitis and in response to tumor necrosis factor-α in an air-pouch model of leukocyte migration. RESULTS: Colitis was reduced in Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after transfer of CD45RB(hi) CD4(+) T cells or administration of the CD40 agonist. The numbers of monocytes and macrophages were reduced in inflamed tissues of Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after induction of colitis and other inflammatory disorders. An antibody that inhibited Slamf1 reduced the level of enterocolitis in Rag(-/-) mice. CONCLUSIONS: Slamf1 contributes to the development of colitis in mice. It appears to indirectly regulate the appearance of monocytes and macrophages in inflamed intestinal tissues. Antibodies that inhibit Slamf1 reduce colitis in mice, so human SLAMF1 might be a therapeutic target for inflammatory bowel disease.
Assuntos
Antígenos CD/fisiologia , Colite/fisiopatologia , Receptores de Superfície Celular/fisiologia , Animais , Antígenos CD/genética , Antígenos CD40/efeitos adversos , Movimento Celular , Quimiocina CCL2/sangue , Quimiocina CCL7/sangue , Colite/sangue , Colite/induzido quimicamente , Modelos Animais de Doenças , Intestinos/patologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Família de Moléculas de Sinalização da Ativação Linfocitária/antagonistas & inibidores , Adenina/análogos & derivados , Animais , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Camundongos SCID , Piperidinas , Pirazóis/farmacologia , Pirimidinas/farmacologiaRESUMO
Whereas the SLAMF-associated protein (SAP) is involved in differentiation of T follicular helper (Tfh) cells and antibody responses, the precise requirements of SLAMF receptors in humoral immune responses are incompletely understood. By analyzing mice with targeted disruptions of the Slamf1, Slamf5, and Slamf6 genes, we found that both T-dependent and T-independent antibody responses were twofold higher compared to those in single knockout mice. These data suggest a suppressive synergy of SLAMF1, SLAMF5, and SLAMF6 in humoral immunity, which contrasts the decreased antibody responses resulting from a defective GC reaction in the absence of the adapter SAP. In adoptive co-transfer assays, both [Slamf1 + 5 + 6] (-/-) B and T cells were capable of inducing enhanced antibody responses, but more pronounced enhancement was observed after adoptive transfer of [Slamf1 + 5 + 6] (-/-) B cells compared to that of [Slamf1 + 5 + 6] (-/-) T cells. In support of [Slamf1 + 5 + 6] (-/-) B cell intrinsic activity, [Slamf1 + 5 + 6] (-/-) mice also mounted significantly higher antibody responses to T-independent type 2 antigen. Furthermore, treatment of mice with anti-SLAMF6 monoclonal antibody results in severe inhibition of the development of Tfh cells and GC B cells, confirming a suppressive effect of SLAMF6. Taken together, these results establish SLAMF1, SLAMF5, and SLAMF6 as important negative regulators of humoral immune response, consistent with the notion that SLAM family receptors have dual functions in immune responses.
RESUMO
Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.
Assuntos
Antígenos CD/fisiologia , Células Mieloides/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Quimiotaxia/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Peritonite/metabolismo , Peritonite/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
Glucocorticoid-induced tumor necrosis factor receptor family-related protein (TNFRSF18, CD357) is constitutively expressed on regulatory T cells (Tregs) and is inducible on effector T cells. In this report, we examine the role of glucocorticoid-induced TNF receptor family-related protein ligand (GITR-L), which is expressed by antigen presenting cells, on the development and expansion of Tregs. We found that GITR-L is dispensable for the development of naturally occurring FoxP3(+) Treg cells in the thymus. However, the expansion of Treg in GITR-L (-/-) mice is impaired after injection of the dendritic cells (DCs) inducing factor Flt3 ligand. Furthermore, DCs from the liver of GITR-L (-/-) mice were less efficient in inducing proliferation of antigen-specific Treg cells in vitro than the same cells from WT littermates. Upon gene transfer of ovalbumin into hepatocytes of GITR-L (-/-)FoxP3(GFP) reporter mice using adeno-associated virus (AAV8-OVA) the number of antigen-specific Treg in liver and spleen is reduced. The reduced number of Tregs resulted in an increase in the number of ovalbumin specific CD8(+) T effector cells. This is highly significant because proliferation of antigen-specific CD8(+) cells itself is dependent on the presence of GITR-L, as shown by in vitro experiments and by adoptive transfers into GITR-L (-/-) Rag (-/-) and Rag (-/-) mice that had received AAV8-OVA. Surprisingly, administering αCD3 significantly reduced the numbers of FoxP3(+) Treg cells in the liver and spleen of GITR-L (-/-) but not WT mice. Because soluble Fc-GITR-L partially rescues αCD3 induced in vitro depletion of the CD103(+) subset of FoxP3(+)CD4(+) Treg cells, we conclude that expression of GITR-L by antigen presenting cells is requisite for optimal Treg-mediated regulation of immune responses including those in response during gene transfer.