RESUMO
Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.
Assuntos
Fatores Imunológicos/farmacologia , Imunomodulação , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Receptores de Interleucina-8A/biossíntese , Receptores de Interleucina-8B/biossíntese , Receptores do Leucotrieno B4/imunologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Ilhas de CpG/genética , Regulação para Baixo/imunologia , Feminino , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Interleucina-8/imunologia , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Ovalbumina , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/biossíntese , Receptor Toll-Like 9/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The 5-HT(3) and 5-HT(4) receptor antagonists alosetron and piboserod, and the muscarinic receptor antagonists PNU-171990A (2-(diisopropylamino)ethyl 1-phenylcyclopentanecarboxylate, hydrochloride) and PNU-174708A (2-(diisopropylamino)ethyl 1-phenylcyclohexanecarboxylate) were studied by electromyography, defining the migrating myoelectric complex (MMC) after i.v. administration in conscious rats. Alosetron prolonged the MMC cycle length from 16.6 to maximally 30.4 min at the dose 0.5 mg kg(-1). Piboserod promptly abolished MMC pattern and prolonged cycle length from 16.5 to >60 min at 0.5 mg kg(-1). PNU-171990A and PNU-174708A had no effect on basal cycle length up to a dose of 20 mg kg(-1). In controls, saline did not change the MMC pattern, while L-hyoscyamine at the same dose, 20 mg kg(-1), prolonged cycle length from 17.6 to 29.0 min. None of the drugs affected duration or propagation velocity of phase III of MMC. Blockade of 5-HT(4) receptors seems to exert a powerful inhibitory effect on motility, 5-HT(3) receptor blockade is less efficient and muscarinic receptor blockade has low efficacy.
Assuntos
Antagonistas Muscarínicos/farmacologia , Complexo Mioelétrico Migratório/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Animais , Atropina/farmacologia , Carbolinas/farmacologia , Ácidos Carboxílicos , Ciclopropanos , Relação Dose-Resposta a Droga , Eletromiografia , Ésteres/farmacologia , Indóis/farmacologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Complexo Mioelétrico Migratório/fisiologia , Oxazinas/farmacologia , Ratos , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/fisiologia , Receptores 5-HT4 de Serotonina/efeitos dos fármacos , Receptores 5-HT4 de Serotonina/fisiologiaRESUMO
It has been shown that Bacteroides thetaiotaomicron, a representative member of the gut microflora, signals intestinal epithelial cells both in vivo and in vitro and modulate specific glycosylation processes that may mediate intestinal functions. However it is not known whether these modulations depend on the presence of live bacteria or may be elicited by soluble factors produced in vitro by this bacterium. We used lectins and an histochemical approach to survey tissue sections prepared from various cellular compartments of the small and large intestine of NRMI/KI mice grown under gnotobiotic conditions. We compared the results obtained with bacterial culture supernatant and live B. thetaiotaomicron to those obtained from germ-free mice or mice having a conventional microflora. This approach allowed us to conclude that (1) a small but specific number of glycan patterns were restored after treatment with bacterial culture supernatant and (2) the B. thetaiotaomicron associated mice restored a larger number of patterns, however, the complete conventional mice pattern must be a function of the whole microflora in the gut. The possibility to modulate this complex glycosylation pattern by introducing exogenous bacteria and bacterial products should be considered as a promising approach towards understanding the molecular basis of microbial-host interactions.
Assuntos
Bacteroides/metabolismo , Glicosilação , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Lectinas de Plantas/metabolismo , Animais , Sítios de Ligação , Enterócitos/citologia , Enterócitos/metabolismo , Enterócitos/microbiologia , Feminino , Vida Livre de Germes , Processamento de Imagem Assistida por Computador , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestino Grosso/citologia , Intestino Grosso/microbiologia , Intestino Delgado/citologia , Intestino Delgado/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Transdução de SinaisRESUMO
Glutathione transferases (GSTs) play a central role in the cellular defense against harmful endogenous compounds and xenobiotics in mouse and man. The gastrointestinal channel is constantly exposed to bacteria, bacterial products, and xenobiotics. In the present study the distribution of alpha, mu, and pi class GSTs was examined immunohistologically in the colon of conventional and germ-free (GF) mice subjected to experimental colitis. The tissues samples were from conventional mice with and without colitis induced by dextran sulfate sodium (DSS); GF mice treated with DSS or carrageenan; and GF mice inoculated with normal mouse bacterial flora as well as with Lactobacillus GG. In conventional as well as in GF mice the mu and pi class GSTs showed reduced intestinal expression when colitis was induced. In con-rast, the level of GSTs reacting with antibodies directed against the alpha class, in particular mGST A4-4, was elevated after induction of inflammation. Of special interest is mGST A4-4 because of its high catalytic activity with toxic products of lipid peroxidation. In the colon of conventionalized GF mice that were given mouse intestinal flora, the mGST A4-4 expression was increased with time for several weeks, but then showed a decrease to a normal level. Additionally, the inoculation of GF mice with Lactobacillus GG induced all the intestinal GSTs studied.
Assuntos
Colite/enzimologia , Colite/microbiologia , Colo/enzimologia , Colo/microbiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa Transferase/biossíntese , Animais , Colite/induzido quimicamente , Sulfato de Dextrana , Vida Livre de Germes , Glutationa Transferase/metabolismo , Histocitoquímica , Inativação Metabólica , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Lactobacillus/metabolismo , CamundongosRESUMO
BACKGROUND AND AIMS: Dextran sulfate sodium (DSS) induced colitis exhibits a predominantly NF-kappaB dependent proinflammatory cytokine profile and shares similarities with human inflammatory bowel disease (IBD). Lamina propria macrophages of IBD patients display elevated levels of NF-kappaB p65. Knowing the role of NF-kappaB in IBD, we investigated the beneficial cellular mechanisms underlying the lasting effect of a single p65 antisense treatment in DSS-colitis mice. METHODS: One local dose of p65 antisense oligonucleotides was administered in DSS colitis mice. Ten days later the mice were killed and examined at cellular and biochemical levels. The level of p65 in lamina propria cells was determined by electrophoretic mobility shift assay and by intracellular immunofluorescent staining of nuclear p65 levels, using laser scanning cytometer. RESULTS: FACS analysis demonstrated a considerable drop in infiltrating lymphocytes and a drastic reduction in CD14+ cells in mice treated with p65 antisense oligonucleotides. Moreover, abrogation of inflammation extended all the way to the cecum in treated mice. Treatment was correlated with decreased DNA binding activity of NF-kappaB. CONCLUSION: Our data strongly support a model in which p65 antisense treatment possesses the capacity to disrupt the pathogenic autocrine loop propagated by NF-kappaB at the chronic phase of IBD.
Assuntos
Colite/terapia , Receptores de Lipopolissacarídeos/imunologia , Linfócitos/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Colite/induzido quimicamente , Colite/imunologia , Sulfato de Dextrana , Feminino , Humanos , Masculino , Camundongos , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , Fatores de TempoRESUMO
The glycosylation of the intestinal cell layer is thought to control several key functions of the gut such as vectorial transports, defence against microbial agents or immunological processes. It has been assumed that the gut microflora may modulate the glycosylation pattern of the intestinal cell layer. However, there is no direct evidence for this regulatory process. The first goal of this work was to establish the germ-free mice intestinal glycosylation baseline using a histochemical approach and a panel of ten lectins with defined glycan specificities to tissue sections prepared from various cellular compartments of the small and large intestine. Using this baseline, we have studied the contribution of the gut microflora on the carbohydrate composition of glycoconjugates of intestinal cells by comparing the germ-free and conventional mice glycosylation patterns. Analysis of the germ-free mice intestinal glycosylation baseline revealed that the expression of glycans depends on the proximodistal gradient (small to large intestine) and on the cell lineage (absorptive, goblet, crypt, and Paneth cells), indicating that mice are able to create and maintain a strict topological and cell lineage-specific regulation of glycosyltransferase expression. By comparing germ-free and conventional mice, we find that the gut microflora specifically modulates the gut glycosylation pattern, quantitatively as well as qualitatively by changing the cellular and subcellular distribution of glycans. This is the first report in mice to directly demonstrate the critical contribution of microflora to intestinal glycosylation, a key characteristic of the gut.