RESUMO
We have previously shown that interspecies incompatibility of CD47 plays an important role in triggering rejection of xenogeneic hematopoietic cells by macrophages. However, whether CD47 incompatibility also induces rejection of nonhematopoietic cellular xenografts remains unknown. Herein, we have addressed this question in a mouse model of hepatocyte transplantation in which CD47(-/-) hepatocytes were used to resemble xenografts for CD47 incompatibility. We show that intrasplenic transplantation of CD47(-/-), but not wild-type (WT) hepatocytes, into partially hepatectomized syngeneic WT mice resulted in a rapid increase in Mac-1(+) cells with an activation phenotype (i.e., Mac-1(+)CD14(+) and Mac-1(+)CD16/32(high)), compared to nontransplant controls. In addition, CD47(-/-) hepatocytes were more severely damaged than WT hepatocytes as indicated by the greater AST and ALT serum levels in these mice. Furthermore, long-term donor hepatocyte survival and liver repopulation were observed in mice receiving WT hepatocytes, whereas CD47(-/-) hepatocytes were completely rejected within 2 weeks. These results suggest that CD47 on donor hepatocytes prevents recipient myeloid innate immune cell activation, hence aiding in graft survival after hepatocyte transplantation. Thus, CD47 incompatibility is likely to present an additional barrier to hepatocyte xenotransplantation.
Assuntos
Antígeno CD47/genética , Deleção de Genes , Rejeição de Enxerto/genética , Hepatócitos/transplante , Animais , Antígeno CD47/imunologia , Células Cultivadas , Rejeição de Enxerto/imunologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Imunidade Inata , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/imunologiaRESUMO
TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica.
Assuntos
Técnicas de Inativação de Genes , Intestinos/microbiologia , Mucinas/genética , Proteínas Musculares/genética , Peptídeos/genética , Nódulos Linfáticos Agregados/microbiologia , Yersiniose/genética , Yersiniose/transmissão , Yersinia enterocolitica/patogenicidade , Animais , Feminino , Imunidade Inata , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/patologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Knockout , Mucinas/imunologia , Proteínas Musculares/imunologia , Peptídeos/imunologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Nódulos Linfáticos Agregados/patologia , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Baço/patologia , Fator Trefoil-2 , Yersiniose/imunologia , Yersiniose/patologia , Yersinia enterocolitica/imunologiaRESUMO
Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.
Assuntos
Anticorpos Neutralizantes/farmacologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Células Matadoras Naturais , Vírus da Leucemia Murina/imunologia , Vírus Reordenados/imunologia , Animais , Anticorpos Neutralizantes/efeitos adversos , Citotoxicidade Imunológica/efeitos dos fármacos , Citometria de Fluxo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Humanos , Injeções Intraperitoneais , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/metabolismo , Ativação Linfocitária , Depleção Linfocítica , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/biossíntese , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Transgênicos , Monócitos/citologia , Monócitos/imunologia , Monócitos/virologia , Vírus Reordenados/genética , Vírus Reordenados/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1(+) CD27(-) NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1(+) CD27(+) NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Imunização/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Vacinas de DNA/imunologia , Animais , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/genética , Células Matadoras Naturais/química , Fígado/imunologia , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Plasmídeos , Baço/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Vacinas de DNA/genéticaRESUMO
BACKGROUND: Carotid artery lesions from symptomatic patients are characterized by inflammation and neovascularization. The adipokine leptin promotes angiogenesis and activates inflammatory cells, and the leptin receptor (ob gene-encoded receptor), ObR, is expressed in advanced atherosclerotic lesions. The present study quantitatively analyzed ObR messenger RNA (mRNA) expression and immunoreactivity in carotid artery plaques from symptomatic and asymptomatic persons. Plaque angiogenesis, gene expression of vascular endothelial growth factor (VEGF), and macrophage density were also analyzed. METHODS: Carotid endarterectomy specimens were collected from 26 patients undergoing surgery for hemispheric cerebrovascular symptoms (n = 13) or progressive asymptomatic internal carotid stenosis (n = 13). A representative sample, including part of the most active site, was collected from each lesion and evaluated by real-time polymerase chain reaction analysis for ObR(long) and ObR(common) isoforms, VEGF(165), and macrophage adhesion molecule-1 (Mac-1) mRNA, and by immunohistochemistry for ObR, von Willebrand factor (vWF), and CD68 antigen expression. RESULTS: All plaques exhibited advanced atherosclerosis (American Heart Association class IV through VI). Transcript levels were preferentially elevated in symptomatic plaques for ObR(long) (P = .0006) and ObR(common) (P = .033), with a simultaneous upregulation of VEGF(165) (P = .001) and Mac-1 mRNA expression (P = .003). Immunohistochemical analysis confirmed a significant increase of ObR antigen levels (P = .011) and CD68-positive inflammatory cells (P = .049) in symptomatic plaques, whereas neovascularization, evident in all plaques, was similar in both groups (P = .7). CONCLUSION: The ObR(long) and ObR(common) genes are upregulated and their protein preferentially synthesized in clinically symptomatic carotid plaques. Moreover, ObR expression is positively correlated with augmentation of gene transcripts related to macrophage density and neovascularization. These data suggest that ObR(long) and ObR(common) may be linked with histologic features of carotid plaque instability, which are associated with cerebral ischemic symptoms.
Assuntos
Estenose das Carótidas/metabolismo , Transtornos Cerebrovasculares/etiologia , Macrófagos/patologia , Receptores para Leptina/análise , Idoso , Biomarcadores/análise , Biomarcadores/sangue , Proteína C-Reativa/análise , Estenose das Carótidas/complicações , Estenose das Carótidas/patologia , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/patologia , Feminino , Humanos , Imuno-Histoquímica , Interleucina-6/sangue , Leptina/sangue , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/genética , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas , RNA Mensageiro/análise , Receptores para Leptina/sangue , Receptores para Leptina/genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND/AIMS: The aim of the present study was to investigate the participation of the adhesion molecules and their ligands in the inflammatory process in secondary cholangitis. METHODOLOGY: Biopsy specimens were collected from the common bile duct from 29 patients with extrahepatic bile obstruction. Immunohistochemistry was used to study the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin, LFA-1, PECAM-1, Mac-1, and VLA-4). The patients were categorized into 2 groups - chronic exacerbated cholangitis and chronic sclerotic cholangitis. RESULTS: An increased ICAM-1 expression was demonstrated and also de novo VCAM-1 and E-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis. The inflammatory cells were strongly LFA-1-, Mac-1- positive. In chronic sclerotic cholangitis the E-selectin expression persisted on vascular endothelium in the fibrous tissue and reduced inflammatory infiltrate showed LFA-1 and VLA-4 positivity. The newly formed vessels in the fibrous connective tissue were PECAM-1-positive. CONCLUSIONS: From these results, it could be concluded that in chronic exacerbated cholangitis with complete bile obstruction the firm adhesion is mediated by ICAM-1/LFA-1 and ICAM-1/Mac-1 and less by VCAM-1/VLA-4 pathways. In chronic sclerotic cholangitis, caused by incomplete obstruction, the firm adhesion was maintained by ICAM-1/LFA-1 and less by VCAM-1/VLA-4 pathways. It seems likely that PECAM-1 and VCAM-1/VLA-4 play additional roles in neutrophil recruitment.
Assuntos
Moléculas de Adesão Celular/análise , Colangite/patologia , Colestase Extra-Hepática/patologia , Doenças do Ducto Colédoco/patologia , Adulto , Idoso , Ducto Colédoco/patologia , Selectina E/análise , Feminino , Humanos , Técnicas Imunoenzimáticas , Integrina alfa4beta1 , Molécula 1 de Adesão Intercelular/análise , Testes de Função Hepática , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno de Macrófago 1/análise , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
Earlier work has shown that the transcription factor C/EBPalpha induced a transdifferentiation of committed lymphoid precursors into macrophages in a process requiring endogenous PU.1. Here we have examined the effects of PU.1 and C/EBPalpha on fibroblasts, a cell type distantly related to blood cells and akin to myoblasts, adipocytes, osteoblasts, and chondroblasts. The combination of the two factors, as well as PU.1 and C/EBPbeta, induced the up-regulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells. They also up-regulated these markers in mouse embryo- and adult skin-derived fibroblasts. Based on cell morphology, activation of macrophage-associated genes, and extinction of fibroblast-associated genes, cell lines containing an attenuated form of PU.1 and C/EBPalpha acquired a macrophage-like phenotype. The lines also display macrophage functions: They phagocytose small particles and bacteria, mount a partial inflammatory response, and exhibit strict CSF-1 dependence for growth. The myeloid conversion is primarily induced by PU.1, with C/EBPalpha acting as a modulator of macrophage-specific gene expression. Our data suggest that it might become possible to induce the transdifferentiation of skin-derived fibroblasts into cell types desirable for tissue regeneration.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Transdiferenciação Celular , Fibroblastos/citologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Transdiferenciação Celular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células NIH 3T3 , Fagocitose/genética , Proteínas Proto-Oncogênicas/genética , Retroviridae/genética , Transativadores/genética , Transfecção , Regulação para CimaRESUMO
OBJECTIVE: To assess the importance of the interaction between leukocyte integrin Mac-1 (a Mb 2) and platelet glycoprotein (GP) Ib-a for leukocyte recruitment after vascular injury and the effect of the neutralization of the Mac-1-GPIba interaction on cell proliferation and the neointimal hyperplasia triggered by the vascular injury. METHODS: A peptide called M2 or anti-M2 antibody was developed to block the Mac-1-GPIba interaction. This peptide was injected and compared to a control-peptide in C57B1/6J mice submitted to vascular injury of the femoral artery with a guide wire. One, five or 28 days after the vascular injury, the femoral arteries were removed for morphometric and immunohistochemical analyses. RESULTS: The blocking of the Mac-1-GPIba interaction promoted a statistically significant reduction in the number of leukocytes in the neointimal layer on the first day after the vascular injury (control: 7.9+/-5.0% of the cell total versus anti-M2: 2.0+/-1.6%, p=0.021), as well as determined a statistically significant decrease in leukocyte accumulation in the neointimal layer on days 5 and 28 (control: 42.3+/-12.9% versus anti-M2: 24.6+/-10.8%, p=0.047 and control: 7.9+/-3.0% versus anti-M2: 3.3+/-1.3%, p=0.012; respectively). Cell proliferation in the neointimal layer of the vessel five days post-injury was reduced with the blocking of the Mac-1-GPIba interaction (control: 5.0+/-2.9% of the cell total versus anti-M2: 1.8+/-0.5%; p=0.043), along with a significant decrease in cell proliferation in the vessel neointimal layer 28 days post-injury (control: 3.8+/-1.7% versus anti-M2: 2.0+/-1.2%; p=0.047). The blocking of the Mac-1-GPIba interaction also determined a statistically significant decrease of the intimal thickening 28 days post-injury (control: 10,395+/-3,549 microm(2) versus anti-M2: 4,561+/-4,915 microm(2); p=0.012). CONCLUSION: Leukocyte recruitment after a vascular injury depends on the Mac-1-GPIba interaction and the neutralization of this interaction inhibits cell proliferation and neointimal formation.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Artéria Femoral/lesões , Leucócitos/fisiologia , Antígeno de Macrófago 1/fisiologia , Peptídeos/administração & dosagem , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/metabolismo , Proliferação de Células , Artéria Femoral/metabolismo , Imunoglobulina G/administração & dosagem , Inflamação/metabolismo , Antígeno de Macrófago 1/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Peptídeos/imunologia , Adesividade Plaquetária/fisiologia , Coelhos , Estatísticas não Paramétricas , Túnica Íntima/imunologia , Túnica Íntima/patologiaRESUMO
OBJETIVO: Avaliar a importância da interação entre a integrina Mac-1 dos leucócitos (a Mb 2) e a glicoproteína (GP) Iba das plaquetas para o recrutamento de leucócitos após a lesão vascular e o efeito da neutralização da interação Mac-1-GPIba sobre a proliferação celular e a hiperplasia neointimal desencadeadas por lesão vascular. MÉTODOS: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação Mac-1-GPIba . Esse peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos a lesão vascular da artéria femoral com corda-guia. Um, cinco ou 28 dias após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imuno-histoquímica. RESULTADOS: O bloqueio da interação Mac-1-GPIba promoveu uma redução estatisticamente significativa do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9±5,0 por cento do total de células versus anti-M2: 2,0±1,6 por cento, p=0,021), bem como determinou uma diminuição estatisticamente significativa do acúmulo de leucócitos na neoíntima em cinco e 28 dias (controle: 42,3±12,9 por cento versus anti-M2: 24,6±10,8 por cento, p=0,047 e controle: 7,9±3,0 por cento versus anti-M2: 3,3±1,3 por cento, p=0,012; respectivamente). A proliferação celular na camada média do vaso em cinco dias pós-lesão foi reduzida com o bloqueio da interação Mac-1-GPIba (controle: 5,0±2,9 por cento do total de células versus anti-M2: 1,8±0,5 por cento; p=0,043), assim como houve diminuição significativa da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8±1,7 por cento versus anti-M2: 2,0±1,2 por cento; p=0,047). O bloqueio da interação Mac-1-GPIba também determinou uma redução estatisticamente significativa do espessamento intimal em 28 dias pós-lesão (controle: 10.395±3.549 µm² versus anti-M2: 4.561±4.915 ...
OBJECTIVE: To assess the importance of the interaction between leukocyte integrin Mac-1 (a Mb 2) and platelet glycoprotein (GP) Ib-a for leukocyte recruitment after vascular injury and the effect of the neutralization of the Mac-1-GPIba interaction on cell proliferation and the neointimal hyperplasia triggered by the vascular injury. METHODS: A peptide called M2 or anti-M2 antibody was developed to block the Mac-1-GPIba interaction. This peptide was injected and compared to a control-peptide in C57B1/6J mice submitted to vascular injury of the femoral artery with a guide wire. One, five or 28 days after the vascular injury, the femoral arteries were removed for morphometric and immunohistochemical analyses. RESULTS: The blocking of the Mac-1-GPIba interaction promoted a statistically significant reduction in the number of leukocytes in the neointimal layer on the first day after the vascular injury (control: 7.9±5.0 percent of the cell total versus anti-M2: 2.0±1.6 percent, p=0.021), as well as determined a statistically significant decrease in leukocyte accumulation in the neointimal layer on days 5 and 28 (control: 42.3±12.9 percent versus anti-M2: 24.6±10.8 percent, p=0.047 and control: 7.9±3.0 percent versus anti-M2: 3.3±1.3 percent, p=0.012; respectively). Cell proliferation in the neointimal layer of the vessel five days post-injury was reduced with the blocking of the Mac-1-GPIba interaction (control: 5.0±2.9 percent of the cell total versus anti-M2: 1.8±0.5 percent; p=0.043), along with a significant decrease in cell proliferation in the vessel neointimal layer 28 days post-injury (control: 3.8±1.7 percent versus anti-M2: 2.0±1.2 percent; p=0.047). The blocking of the Mac-1-GPIba interaction also determined a statistically significant decrease of the intimal thickening 28 days post-injury (control: 10,395±3,549 µm² versus anti-M2: 4,561±4,915 µm²; ...
Assuntos
Animais , Masculino , Camundongos , Coelhos , Anticorpos Monoclonais/administração & dosagem , Artéria Femoral/lesões , Leucócitos/fisiologia , Antígeno de Macrófago 1/fisiologia , Peptídeos/administração & dosagem , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Anticorpos Monoclonais/imunologia , Plaquetas/metabolismo , Proliferação de Células , Artéria Femoral/metabolismo , Imunoglobulina G/administração & dosagem , Inflamação/metabolismo , Modelos Animais , Antígeno de Macrófago 1/análise , Peptídeos/imunologia , Adesividade Plaquetária/fisiologia , Estatísticas não Paramétricas , Túnica Íntima/imunologia , Túnica Íntima/patologiaRESUMO
This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.
Assuntos
4-Butirolactona/análogos & derivados , Lignanas/farmacologia , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , Piper/química , Receptores de Formil Peptídeo/antagonistas & inibidores , Superóxidos/metabolismo , 4-Butirolactona/farmacologia , Adulto , Cálcio/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Antígeno de Macrófago 1/análise , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosforilação , Receptores de Formil Peptídeo/metabolismoRESUMO
Leukocyte adhesion deficiency type I (LAD I) is characterized by recurrent and fatal bacterial infections, and caused by the mutation of the CD18 gene. A 9-month-old infant whose umbilical cord separated at day 10 of life had sepsis, complicated otitis media and neutrophilia. Molecular analysis showed homozygous intron 7 (+1) g > a in the CD18 gene, resulting in three splicing transcriptions that inserted 64, 298 (5' end of intron 7), and 1157 (whole intron 7) nucleotides into the 300th amino acid of Ile and stopped at the 326th (inserted 64 and 1157 nucleotides) and the 344th (inserted 64 nucleotides), respectively. The two truncated mutations lost cysteine-rich, transmembrane, and cytoplasma domains. Increased susceptibility to infections correlated to polymorphonuclear cell dysfunction, including absent expression of adhesion molecule (CD11b/CD18), impaired chemotaxis, and decreased phagocytosis. Both his heterozygous parents revealed non-random skewing only to the wild type. The skewing pattern and severe phenotype make stem cell transplantation an optimal option.
Assuntos
Antígenos CD18/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Síndrome da Aderência Leucocítica Deficitária/imunologia , Neutrófilos/fisiologia , Antígenos CD18/análise , Antígenos CD18/química , Quimiotaxia de Leucócito , Humanos , Peróxido de Hidrogênio/metabolismo , Lactente , Antígenos CD15/análise , Antígeno de Macrófago 1/análise , Masculino , Mutação , Fagocitose , Cordão UmbilicalRESUMO
NK cells are important for the clearance of tumors, parasites, and virus-infected cells. Thus, factors that control NK cell numbers and function are critical for the innate immune response. A subset of NK cells express the inhibitory killer cell lectin-like receptor G1 (KLRG1). In this study, we identify that KLRG1 expression is acquired during periods of NK cell division such as development and homeostatic proliferation. KLRG1(+) NK cells are mature in phenotype, and we show for the first time that these cells have a slower in vivo turnover rate, reduced proliferative response to IL-15, and poorer homeostatic expansion potential compared with mature NK cells lacking KLRG1. Transfer into lymphopenic recipients indicate that KLRG1(-) NK cells are precursors of KLRG1(+) NK cells and KLRG1 expression accumulates following cell division. Furthermore, KLRG1(+) NK cells represent a significantly greater proportion of NK cells in mice with enhanced NK cell numbers such as Cd45(-/-) mice. These data indicate that NK cells acquire KLRG1 on their surface during development, and this expression correlates with functional distinctions from other peripheral NK cells in vivo.
Assuntos
Células Matadoras Naturais/fisiologia , Receptores Imunológicos/biossíntese , Animais , Homeostase , Interleucina-12/farmacologia , Interleucina-18/farmacologia , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/análise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Regulação para CimaRESUMO
The cell composition of early hepatic lesions of experimental murine tularemia has not been characterized with specific markers. The appearance of multiple granulomatous-necrotic lesions in the liver correlates with a marked increase in the levels of serum alanine transferase and lactate dehydrogenase. Francisella tularensis, detected by specific antibodies, can be first noted by day 1 and becomes associated with the lesions by 5 days postinoculation. These lesions become necrotic, with some evidence of in situ apoptosis. The lesions do not contain B, T, or NK cells. Rather, the lesions are largely composed of two subpopulations of Mac-1(+) cells that are associated with the bacteria. Gr-1(+) Mac-1(+) immature myeloid cells and major histocompatibility complex class II-positive (MHC-II(+)) Mac-1(+) macrophages were the most abundant cell phenotypes found in the granuloma and are likely major contributors in controlling the infection in its early stages. Our findings have shown that there is an early development of hepatic lesions where F. tularensis colocalizes with both Gr-1(+) Mac-1(+) and MHC-II(+) Mac-1(+) cells.
Assuntos
Francisella tularensis , Granuloma/patologia , Fígado/patologia , Antígeno de Macrófago 1/análise , Macrófagos/química , Tularemia/patologia , Animais , Apoptose , Biomarcadores/análise , Feminino , Granuloma/microbiologia , Antígenos de Histocompatibilidade Classe II/análise , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos , Necrose/patologia , Receptores de Quimiocinas/análise , Tularemia/imunologiaRESUMO
Integrins are type I heterodimeric (alpha/beta) cell adhesion molecules. They trigger cell-signaling by recruiting cytosolic molecules to their cytoplasmic tails. Integrin alpha cytoplasmic tail contributes towards integrin function specificity, an important feature of integrins having different alpha subunits but sharing the same beta subunit. Herein, we show that the src family kinase Hck co-capped selectively with leukocyte integrin alpha(M)beta(2) but not alpha(L)beta(2) or alpha(X)beta(2). This was disrupted when the alpha(M) cytoplasmic tail was substituted with that of alpha(L) or alpha(X). Co-capping was recovered by alpha(L) or alpha(X) cytoplasmic tail truncation or forced separation of the alpha and beta cytoplasmic tails via salt-bridge disruption.
Assuntos
Antígeno de Macrófago 1/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Humanos , Integrina alfaXbeta2/análise , Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fagossomos/enzimologia , Proteínas Proto-Oncogênicas c-hck/análise , Deleção de SequênciaRESUMO
Migration of activated macrophages is essential for resolution of acute inflammation and the initiation of adaptive immunity. Here, we show that efficient macrophage migration in inflammatory environment depends on Mac-1 recognition of a binary complex consisting of fibrin within the provisional matrix and the protease tPA (tissue-type plasminogen activator). Subsequent neutralization of tPA by its inhibitor PAI-1 enhances binding of the integrin-protease-inhibitor complex to the endocytic receptor LRP (lipoprotein receptor-related protein), triggering a switch from cell adhesion to cell detachment. Genetic inactivation of Mac-1, tPA, PAI-1 or LRP but not the protease uPA abrogates macrophage migration. The defective macrophage migration in PAI-1-deficient mice can be restored by wild-type but not by a mutant PAI-1 that does not interact with LRP. In vitro analysis shows that tPA promotes Mac-1-mediated adhesion, whereas PAI-1 and LRP facilitate its transition to cell retraction. Our results emphasize the importance of ordered transitions both temporally and spatially between individual steps of cell migration, and support a model where efficient migration of inflammatory macrophages depends on cooperation of three physiologically prominent systems (integrins, coagulation and fibrinolysis, and endocytosis).
Assuntos
Movimento Celular/imunologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Movimento Celular/genética , Endocitose , Fibrinogênio/metabolismo , Humanos , Imunoprecipitação , Integrases/metabolismo , Proteínas Relacionadas a Receptor de LDL/análise , Proteínas Relacionadas a Receptor de LDL/genética , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/genética , Macrófagos/química , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Inibidor 1 de Ativador de Plasminogênio/genética , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Chelation therapy with sodium edetate (EDTA) improved renal function and slowed the progression of renal insufficiency in patients subjected to lead intoxication. This study was performed to identify the underlying mechanism of the ability of EDTA treatment to protect kidneys from damage. METHODS: The effects of EDTA administration were studied in a rat model of acute renal failure induced by 60 minutes ischemia followed or not by 60 minutes reperfusion. Renal ischemic damage was evaluated by histological studies and by functional studies, namely serum creatinine and blood urea nitrogen levels. Treatment with EDTA was performed 30 minutes before the induction of ischemia. Polymorphonuclear cell (PMN) adhesion capability, plasmatic nitric oxide (NO) levels and endothelial NO synthase (eNOS) renal expression were studied as well as the EDTA protection from the TNFalpha-induced vascular leakage in the kidneys. Data was compared by two-way analysis of variance followed by a post hoc test. RESULTS: EDTA administration resulted in the preservation of both functional and histological parameters of rat kidneys. PMN obtained from peripheral blood of EDTA-treated ischemized rats, displayed a significant reduction in the expression of the adhesion molecule Mac-1 with respect to controls. NO was significantly increased by EDTA administration and eNOS expression was higher and more diffuse in kidneys of rats treated with EDTA than in the controls. Finally, EDTA administration was able to prevent in vivo the TNFalpha-induced vascular leakage in the kidneys. CONCLUSION: This data provides evidence that EDTA treatment is able to protect rat kidneys from ischemic damage possibly through the stimulation of NO production.
Assuntos
Quelantes/farmacologia , Ácido Edético/farmacologia , Isquemia/prevenção & controle , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/fisiopatologia , Injúria Renal Aguda/prevenção & controle , Animais , Nitrogênio da Ureia Sanguínea , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Adesão Celular/fisiologia , Quelantes/uso terapêutico , Creatinina/sangue , Modelos Animais de Doenças , Progressão da Doença , Ácido Edético/uso terapêutico , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Isquemia/tratamento farmacológico , Isquemia/fisiopatologia , Rim/química , Rim/patologia , Antígeno de Macrófago 1/análise , Masculino , Neutrófilos/química , Neutrófilos/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/análise , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controle , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Conjugated linoleic acid (CLA) refers to a group of naturally occurring positional and geometrical conjugated dienoic isomers of linoleic acid (C18:2), of which the cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) isomers predominate. Accumulating evidence has demonstrated that CLA isomers are capable of inhibiting the growth of a variety of cancer cell lines in vitro; however, their modulatory effects on the proliferation and differentiation of myeloid leukemia cells remain poorly understood. In the present study, CLA was shown to inhibit the proliferation of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. Morphological, flow cytometric and functional analyses revealed that CLA induced the differentiation of WEHI-3B JCS cells into matured macrophage-like cells, as indicated by increases in the cytoplasm:nucleus ratio and vacuolation, the expression of macrophage differentiation antigens (Mac-1 and F4/80) and the enhanced monocytic serine esterase activity of CLA-treated WEHI-3B JCS cells. RT-PCR analysis showed that CLA up-regulated the expression of TNF-alpha, IL-1beta and IFN-gamma genes in WEHI-3B JCS cells, which had previously been shown to play an important role in triggering the differentiation of myeloid leukemia cells. Moreover, CLA-treated WEHI-3B JCS cells had also shown reduced tumorigenicity in vivo. Collectively, our results indicate that CLA might exert its growth-inhibitory effects on myeloid leukemia cells by triggering their terminal differentiation, which is mediated, at least in part, by modulation of the cytokine gene expression in the leukemia cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Animais , Antígenos de Diferenciação/análise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Antígeno de Macrófago 1/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin alphaMbeta2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. METHODS: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90 degrees light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. RESULTS: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. CONCLUSIONS: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.
Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Separação Celular , Citometria de Fluxo , Monócitos/metabolismo , Neutrófilos/metabolismo , Difosfato de Adenosina/farmacologia , Adulto , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Antígeno CD11b/análise , Agregação Celular/fisiologia , Comunicação Celular/fisiologia , Colágeno/farmacologia , Fator Xa/metabolismo , Feminino , Fibrinogênio/metabolismo , Humanos , Antígeno de Macrófago 1/análise , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Tromboplastina/metabolismoRESUMO
BACKGROUND: Fibroblasts can be misidentified as macrophages because both cell types share antigens that are associated with popular antibodies targeting the monocyte/macrophage lineage. With the recent description of fibroblast-specific protein 1 (FSP1), we revisited the specificity of antibodies directed against macrophages to determine systematically which antibodies best distinguish both cell types in fibrotic tissues. METHODS: Tissue fibrosis was produced in mice carrying the GFP transgene encoding green fluorescent protein under the control of the FSP1 promoter. Single cell suspensions from these marked tissues were submitted for flow cytometry using antibodies against Mac-1, Mac-2, Mac-3, F4/80, CD68, major histocompatibility complex (MHC) class II, and CD45, and cDNA amplification of mRNA encoding the above target antigens was performed using specific primer sets in sorted pools of cells. Fibrotic tissues were also stained by immunohistochemistry with the same antibodies and examined under confocal microscopy. RESULTS: Comparison overlap between FSP1(+) fibroblasts with each of the macrophage markers demonstrated that all antimacrophage antibodies (Mac-1, Mac-2, Mac-3, CD68, MHC class II, and CD45) except one (F4/80) recognize both cell types. CONCLUSION: Antibodies directed against F4/80 clearly distinguish macrophages from FSP1(+) fibroblasts in fibrotic tissues and is the preferred antibody in mice.
Assuntos
Especificidade de Anticorpos , Fibroblastos/imunologia , Macrófagos/imunologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação/análise , Antígenos de Diferenciação Mielomonocítica/análise , Proteínas de Ligação ao Cálcio/análise , Fibrose , Galectina 3/análise , Rim/patologia , Antígeno de Macrófago 1/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100RESUMO
Children treated by peritoneal dialysis (PD) are at increased risk of infections. IgG receptors (FcgammaRs) and complement receptors (CRs) on white blood cells (WBCs) are important for the phagocytic process. We have investigated FcgammaR and CR expression on monocytes, macrophages and neutrophils in blood and in peritoneal dialysis effluent (PDE) of 39 PD children. WBCs were isolated from blood and PDE, labelled with FITC-conjugated CD16 (FcgammaRIII), CD32 (FcgammaRII), CD64 (FcgammaRI), CD11b (CR3) and CD35 (CR1) monoclonal antibodies, and analysed by flow cytometry. Peritoneal cells had lower percentages of FcgammaR-positive or CR-positive cells than blood. On the other hand, the receptor number per cell [mean fluorescence intensity (MFI)] was higher on peritoneal macrophages and neutrophils than blood, except for CD16. The FcgammaR and CR expression in blood and dialysate did not change significantly during the first year of PD treatment. During a peritonitis episode the MFI of all receptors in blood increased only on monocytes, with the exception of CD32. The percentages of FcgammaR-positive and CR-positive macrophages and neutrophils in the PDE increased, whereas the MFI did not increase consistently. Peritoneal cells of PD children showed a lower percentage of FcgammaR-positive and CR-positive neutrophils and macrophages, combined with an increased MFI, indicating a state of activation. Blood and peritoneal cells are capable of up-regulating the receptor expression during peritonitis but probably not to a maximum level.