RESUMO
Periodontitis is characterized by chronic inflammation and osteoclast-mediated bone loss regulated by the receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase-1 (mPGES-1) on RANKL- and lipopolysaccharide (LPS)-mediated osteoclastogenesis and prostaglandin E2 (PGE2 ) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) or 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644). Aminothiazoles significantly decreased the number of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells in cultures of RANKL- and LPS-stimulated RAW 264.7 cells, as well as reduced the production of PGE2 in culture supernatants. LPS-treatment induced mPGES-1 mRNA expression at 16 hrs and the subsequent PGE2 production at 72 hrs. Conversely, RANKL did not affect PGE2 secretion but markedly reduced mPGES-1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL-activated RAW 264.7 cells, TH-848 and TH-644 down-regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE2 production was also confirmed in LPS-stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS- and RANKL-mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.
Assuntos
Dinoprostona/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Tiazóis/farmacologia , Actinas/metabolismo , Animais , Catepsina K/genética , Catepsina K/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Prostaglandina-E Sintases/genética , Prostaglandina-E Sintases/metabolismo , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1+ and CD4+ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/imunologia , Interleucina-17/imunologia , Células T Matadoras Naturais/imunologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Quimiocinas/genética , Quimiocinas/imunologia , Modelos Animais de Doenças , Galactosilceramidas/farmacologia , Interleucina-8/genética , Interleucina-8/imunologia , Fígado/citologia , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Células T Matadoras Naturais/citologia , Neutrófilos/imunologiaRESUMO
Costimulatory and coinhibitory signals are important for the maintenance of immune homeostasis both in the steady state and during immune responses. In this study, we explore the relative contributions of these signals to the rapid production of large amounts of cytokines by activated invariant NKT cells (iNKT cells). We find that upon antigenic stimulation, iNKT cells rapidly up-regulate programmed death (PD)-1 and induce high levels of PD ligand 1 and costimulatory molecules on the surface of cognate Ag-presenting dendritic cells and that iNKT cells require a CD28 signal to secrete cytokines in the presence of a PD-1/PD ligand 1 interaction. CD28-deficient iNKT cells synthesized but failed to secrete cytokines during activation, and blockade of the PD-1 pathway restored the ability of CD28-deficient iNKT cells to secrete cytokines. The opposing functions of CD28 and PD-1 thus tightly regulate the unique effector function iNKT cells.
Assuntos
Antígenos de Diferenciação/imunologia , Antígenos CD28/metabolismo , Homeostase/imunologia , Células T Matadoras Naturais/imunologia , Animais , Células Apresentadoras de Antígenos , Antígenos CD28/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Sistema Imunitário/fisiologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Receptor de Morte Celular Programada 1 , Receptores Imunológicos/imunologia , Regulação para CimaRESUMO
Salvadora persica is commonly used chewing sticks in many parts of the world as an oral hygiene tool. This study measured the amount of benzyl isothiocyanate (BITC) released into the mouth and assessed its retention time in saliva. The study also tested if the released amount of BITC could potentially be antibacterial or cytotoxic. Twelve subjects brushed their teeth with fresh Miswak once, twice, and four times. The amount of BITC in the saliva and in the used brushes was quantified using gas chromatography-mass spectrometry. The antibacterial effect of BITC and Miswak essential oil (MEO) was tested against Haemophilus influenzae, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The cytotoxic effect on gingival fibroblasts and keratinocytes was tested using MTT. The highest amount of the active compounds was detected in saliva after using the Miswak tip for once and immediately. It significantly decreased when the Miswak tip was used more than once and thus after 10 min. The growth of the tested bacteria was inhibited by MEO and BITC in a dose dependent manner, P. gingivalis being the most sensitive. MTT assay showed that BITC and MEO were cytotoxic towards gingival fibroblasts while oral keratinocytes showed resistance. This study suggests that the Miswak tip should be cut before each use to ensure the maximum effect.
RESUMO
Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.
Assuntos
Laminina/genética , Placenta/fisiologia , Proteínas Recombinantes/química , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Laminina/isolamento & purificação , Camundongos , GravidezRESUMO
Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.
Assuntos
Laminina/metabolismo , Anticorpos Monoclonais , Especificidade de Anticorpos , Membrana Basal/embriologia , Membrana Basal/metabolismo , Eletroforese em Gel de Poliacrilamida , Endotélio/embriologia , Endotélio/metabolismo , Matriz Extracelular/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Laminina/imunologia , Especificidade de Órgãos , Testes de PrecipitinaRESUMO
α4-Laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by vascular and lymphatic endothelial cells, leukocytes and other normal cell types. These laminins are recognized by α6ß1 and α6ß4 integrins and MCAM (CD146), and promote adhesion and migration of the cells. α4-Laminins are also expressed and secreted by some tumor cells and strongly promote tumor cell migration. Moreover, the abluminal side of blood and/or lymphatic vessels and the nerve perineurium, common tracks of tumor cell dissemination, express α4-laminins, and these laminin isoforms, when expressed in the stroma, may contribute to tumor invasion. In the present study, we examined ten mAbs to human laminin α4 chain for their reactivity with the isolated laminin α4 globular domain, their ability to inhibit tumor cell adhesion and migration on laminins 411 and 421, and their effect on the binding of α6ß1 integrin and MCAM to both α4-laminins. Most of the mAbs reacted with the laminin α4 globular domain, but only two, mAbs FC10 and 084, significantly inhibited tumor cell adhesion and migration on laminin-411. When used in combination, these antibodies practically abolished the cell adhesion and migration on laminin-411 and significantly reduced the cellular responses on laminin-421. Accordingly, mAbs FC10 and 084 significantly inhibited the binding of purified α6ß1 integrin and MCAM to laminins 411 and 421. These results indicate that mAbs to the laminin α4 globular domain are able to inhibit tumor cell adhesion and migration on laminins 411 and 421, and that α6ß1 integrin and MCAM bind α4-laminins at very close sites on the globular domain. These reagents contribute to a better understanding of the biology of α4-laminins and may have a therapeutic potential in malignant and inflammatory diseases.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Neoplasias/genética , Antígeno CD146/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Laminina/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Estrutura Terciária de ProteínaRESUMO
α4-laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by blood and lymphatic vessels and some tumor cells. Laminin-411 promotes migration of leukocytes and endothelial cells, but the effect of this laminin and laminin-421 on tumor cells is poorly understood. In the present study, we demonstrate that laminin-411 and, to a greater extent, laminin-421 significantly promote migration of tumor cells originated from melanomas, gliomas and different carcinomas via α6ß1 integrin. In solid-phase binding assays, both laminins similarly bound α6ß1 integrin but only laminin-421, among several laminin isoforms, readily bound MCAM (CD146), a cell-surface adhesion molecule strongly associated with tumor progression. Accordingly, a function-blocking mAb to MCAM inhibited tumor cell migration on laminin-421 but not on laminins 411 or 521. In tumor tissues, melanoma cells co-expressed MCAM, laminin α4, ß1, ß2 and γ1 chains, and integrin α6 and ß1 chains. The present data highlight the novel role of α4-laminins in tumor cell migration and identify laminin-421 as a primary ligand for MCAM and a putative mediator of tumor invasion and metastasis.
Assuntos
Movimento Celular/fisiologia , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Neoplasias/fisiopatologia , Análise de Variância , Anticorpos Monoclonais/imunologia , Antígeno CD146/metabolismo , Linhagem Celular Tumoral , Humanos , Imuno-HistoquímicaRESUMO
Laminins, a large family of αßγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5ß1γ1) and 521 (α5ß2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3ß1/α6ß1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3ß1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMß1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
Assuntos
Anticorpos Monoclonais/farmacologia , Integrinas/antagonistas & inibidores , Laminina/antagonistas & inibidores , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Epitopos/imunologia , Feminino , Humanos , Hibridomas/imunologia , Integrinas/imunologia , Integrinas/metabolismo , Laminina/imunologia , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação ProteicaRESUMO
Natural killer (NK) cells are innate immune cells that are enriched in the liver, but the processes underlying NK cell trafficking to the liver and cellular activation within the liver of patients with T cell-mediated liver diseases remain poorly defined. Concanavalin A (Con A) hepatitis is a murine model mimicking many aspects of human T cell-mediated liver diseases. Here we demonstrate that severe hepatitis in CCR5-deficient (KO) mice is associated with increased hepatic NK cell recruitment driven by enhanced hepatic production of CCL5 acting via CCR1 and by enhanced hepatic NK cell activation relative to that observed in wild-type mice after Con A administration. Furthermore, NK cell depletion ameliorated severe hepatitis in CCR5 KO mice but did not alter hepatitis in wild-type mice after Con A treatment. We propose that in the setting of CCR5 deficiency NK cells assume a profound effector role in Con A hepatitis via enhanced CCL5-CCR1 driven hepatic recruitment in addition to augmented cytokine-driven NK cell activation to produce interferon-gamma. These results highlight the potential profound impact of altered chemokine receptor expression on the innate immune response in the setting of T cell-mediated hepatitis.
Assuntos
Concanavalina A/farmacologia , Hepatite/imunologia , Células Matadoras Naturais/imunologia , Fígado , Receptores CCR5/deficiência , Linfócitos T/imunologia , Animais , Antagonistas dos Receptores CCR5 , Células Cultivadas , Quimiocina CCL5/imunologia , Modelos Animais de Doenças , Humanos , Interferon gama/imunologia , Interleucina-4/imunologia , Células Matadoras Naturais/citologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Camundongos , Camundongos Knockout , Receptores CCR1 , Receptores CCR5/imunologia , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/imunologia , Baço/citologiaRESUMO
During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.