RESUMO
Recurrent Crohn's disease originates with small erosions in the follicle-associated epithelium overlying the Peyer's patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohn's disease. Ileal tissues were obtained after surgery performed on Crohn's patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohn's samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN(+) and CD83(+), in the subepithelial dome. Moreover, the CD83(+) cells in Crohn's tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohn's disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN(+) cells in Crohn's tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-alpha. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohn's disease.
Assuntos
Doença de Crohn/imunologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Escherichia coli/fisiologia , Nódulos Linfáticos Agregados/imunologia , Adulto , Idoso , Antígenos CD/biossíntese , Translocação Bacteriana , Moléculas de Adesão Celular/biossíntese , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Íleo/imunologia , Íleo/microbiologia , Íleo/patologia , Imunoglobulinas/biossíntese , Imuno-Histoquímica , Lectinas Tipo C/biossíntese , Masculino , Glicoproteínas de Membrana/biossíntese , Microscopia Confocal , Pessoa de Meia-Idade , Nódulos Linfáticos Agregados/microbiologia , Nódulos Linfáticos Agregados/patologia , Receptores CCR7/biossíntese , Receptores de Superfície Celular/biossíntese , Receptor 4 Toll-Like/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Antígeno CD83RESUMO
Stress has been shown to have both central and peripheral effects, promoting psychological illness (such as anxiety and depression), as well influencing peripheral disease in the intestine. Stress in humans can exacerbate symptoms of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), lowering visceral pain thresholds and decreasing mucosal barrier function. Studies in rodents have revealed that both acute and chronic exposure to stressors can lead to pathophysiology of the small and large intestine, including altered ion secretion and increased epithelial permeability (by both transcellular and paracellular pathways). Prolonged exposure to stress can induce low-grade inflammation, cause ultrastructural epithelial abnormalities, and alter bacterial-host interactions allowing greater microbial translocation. In this review, we discuss the stress response and the effects of both acute and chronic stress to induce pathophysiological damage to the gut. We present the potential pathways involved, and the proposed mechanisms of action mediating the effects. Furthermore, we explore the impact of early life stress on colonic physiology in neonatal rodents and the implications for gut dysfunction in adulthood.
Assuntos
Mucosa Intestinal/fisiopatologia , Estresse Fisiológico/complicações , Estresse Psicológico/complicações , Doença Aguda , Animais , Animais Recém-Nascidos , Doença Crônica , Modelos Animais de Doenças , Humanos , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/fisiopatologia , Mucosa Intestinal/metabolismo , Intestino Grosso/metabolismo , Intestino Grosso/fisiopatologia , Intestino Delgado/metabolismo , Intestino Delgado/fisiopatologia , Síndrome do Intestino Irritável/etiologia , Síndrome do Intestino Irritável/metabolismo , Síndrome do Intestino Irritável/fisiopatologia , Privação Materna , Permeabilidade , RoedoresRESUMO
BACKGROUND: Dendritic cell (DC) redistribution during early stages of enteritis may be related to ileal barrier dysfunction. We used a rat model of ileitis to examine this hypothesis. METHODS: Sprague-Dawley rats were injected with indomethacin or saline and euthanized 2, 6, 12, or 24 hours later. Ileal segments and mesenteric lymph nodes were obtained for morphological, bacterial, or functional studies. To determine the role of Toll-like receptor (TLR)-2 and -4 blockages, rats were pretreated with normal IgG, anti-TLR-2, or anti-TLR-4 antibodies prior to indomethacin or saline, and ileal segments were collected 24 hours later. RESULTS: In control rats, CD103+DC were mainly located in the lamina propria (LP) and some expressed TLR-2. TLR-4+ cells with different morphology and distribution from CD103+DC were also detected. In indomethacin-treated rats at 6-24 hours, inflammation was evident as was redistribution of CD103+DC from LP to Peyer's patches. We also observed TLR-2+ monocyte depletion and changes in TLR-4 distribution. At 2-6 hours we detected opened tight junctions as well as abnormal trans- and para-epithelial enteric bacterial infiltration, while macromolecular permeability was not significantly enhanced until 24 hours. In the absence of indomethacin, anti-TLR-2 blockage induced a significant increase of LP CD103+DC, while in the presence of indomethacin, anti-TLR-2 or -4 blockages significantly inhibited (P < 0.05) the reduction of LP CD103+DC. CONCLUSIONS: During the early stages of indomethacin-induced ileitis, epithelial barrier damage and abnormal bacterial infiltration into the mucosa occurred in conjunction with initial redistribution of CD103+DC. Furthermore, we showed that TLR-2 and -4 blockade regulates CD103+DC distribution during early phases in this experimental model.
Assuntos
Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Ileíte/metabolismo , Íleo/metabolismo , Mucosa Intestinal/metabolismo , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Antígenos CD/metabolismo , Western Blotting , Células Dendríticas/ultraestrutura , Modelos Animais de Doenças , Progressão da Doença , Enterócitos/ultraestrutura , Células Epiteliais/ultraestrutura , Peroxidase do Rábano Silvestre/farmacocinética , Ileíte/induzido quimicamente , Ileíte/patologia , Íleo/efeitos dos fármacos , Íleo/patologia , Imuno-Histoquímica , Indometacina/toxicidade , Cadeias alfa de Integrinas/metabolismo , Mucosa Intestinal/patologia , Masculino , Microscopia Eletrônica , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Previously, we showed that corticotropin-releasing factor (CRF) injected i.p. mimicked epithelial responses to stress, both stimulating ion secretion and enhancing permeability in the rat colon, and mast cells were involved. However, the ability of CRF-sensitive mucosal/submucosal loops to regulate intestinal barrier and the participation of resident mast cells are unclear. METHODS: We examined colonic epithelial responses to stress-like peptides in Wistar-Kyoto (WKY), and mast cell-deficient (Ws/Ws) and their +/+ littermate control rats in distal segments mounted in Ussing chambers. Short-circuit current (ion secretion), flux of horseradish peroxidase (macromolecular permeability), and the release of rat mast cell protease II were measured in response to CRF [10(-6) to 10(-8)M] or sauvagine [10(-8) to 10(-10)M] in tissues pretreated with astressin, doxantrazole, or vehicle. RESULTS: Stress-like peptides (sauvagine > CRF) induced a dose-dependent increase in short-circuit current (maximal at 30 min), and significantly enhanced horseradish peroxidase flux and protease II release in WKY. Epithelial responses were inhibited by both astressin and doxantrazole, and significantly reduced in tissues from Ws/Ws rats. CONCLUSION: The stress mediators CRF and sauvagine modulate barrier function in the rat colon acting on mucosal/submucosal CRF receptor-bearing cells, through mast cell-dependent pathways.
Assuntos
Colo/citologia , Transporte de Íons/fisiologia , Mastócitos/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Estresse Fisiológico/imunologia , Proteínas de Anfíbios/administração & dosagem , Proteínas de Anfíbios/metabolismo , Animais , Colo/metabolismo , Colo/patologia , Hormônio Liberador da Corticotropina/administração & dosagem , Hormônio Liberador da Corticotropina/fisiologia , Relação Dose-Resposta a Droga , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Epitélio/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Mastócitos/patologia , Neuropeptídeos/administração & dosagem , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/metabolismo , Ratos , Ratos Endogâmicos WKY , Ratos Mutantes , Receptores de Hormônio Liberador da Corticotropina/agonistas , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia , Estatísticas não Paramétricas , Tioxantenos/administração & dosagem , Xantonas/administração & dosagemRESUMO
BACKGROUND: We previously showed that neonatal maternal separation (MS) of rat pups causes immediate and long-term changes in intestinal physiology. AIM: To examine if administration of probiotics affects MS-induced gut dysfunction. METHODS: MS pups were separated from the dam for 3 h/day from days 4 to 19; non-separated (NS) pups served as controls. Twice per day during the separation period, 10(8) probiotic organisms (two strains of Lactobacillus species) were administered to MS and NS pups; vehicle-treated pups received saline. Studies were conducted on day 20, when blood was collected for corticosterone measurement as an indication of hypothalamus-pituitary-adrenal (HPA) axis activity, and colonic function was studied in tissues mounted in Ussing chambers. Ion transport was indicated by baseline and stimulated short-circuit current (Isc); macromolecular permeability was measured by flux of horseradish peroxidase (HRP) across colonic tissues; and bacterial adherence/penetration into the mucosa was quantified by culturing tissues in selective media. Colonic function and host defence were also evaluated at day 60. RESULTS: Isc and HRP flux were significantly higher in the colon of MS versus NS pups. There was increased adhesion/penetration of total bacteria in MS pups, but a significant reduction in Lactobacillus species. Probiotic administration ameliorated the MS-induced gut functional abnormalities and bacterial adhesion/penetration at both day 20 and 60, and reduced the elevated corticosterone levels at day 20. CONCLUSIONS: The results indicate that altered enteric flora are responsible for colonic pathophysiology. Probiotics improve gut dysfunction induced by MS, at least in part by normalisation of HPA axis activity.
Assuntos
Corticosterona/metabolismo , Gastroenteropatias/psicologia , Privação Materna , Probióticos/uso terapêutico , Estresse Psicológico/complicações , Animais , Animais Recém-Nascidos , Feminino , Gastroenteropatias/prevenção & controle , Lactobacillus , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Estresse Psicológico/metabolismo , Resultado do TratamentoRESUMO
We examined ileal dendritic cell (DC) subpopulations in a rat model of indomethacin-induced enteritis to determine changes in phenotype and distribution associated with increased mucosal permeability during acute and chronic stages of inflammation. Sprague-Dawley rats were treated with indomethacin (7.5 mg/kg subcutaneously, 2 injections 48 h apart). Animals were killed at day 4 (acute stage) or at day 15 or 30 (chronic stages); control rats were injected with saline. DC distribution was evaluated by immunohistochemistry for CD103, CD11b, CD83, and CD163; inflammation was assessed by light microscopy; and permeability was determined by flux of horseradish peroxidase in Ussing chambers. In controls, both immature DC subpopulations, CD103+CD11b+CD163-CD83- and CD103+CD11b-CD163-CD83-, were observed in the lamina propria, and the CD11b- population also was present in Peyer's patches. In acute inflammation, permeability was increased (P<0.01), and inflamed areas with or without ulcers were observed. CD103+ and CD11b+ (CD83-) DCs were absent from inflamed areas, reduced in noninflamed tissues, but present in Peyer's patches. In the chronic stage at day 15, CD103+ and CD11b+ cells were located in inflamed and noninflamed areas and in Peyer's patches. In addition, CD83+ DCs were detected in inflamed areas. At day 30, when we observed a complete microscopic resolution of inflammation, numbers of CD103+ and CD11b+ DCs were increased, and there were CD83+ DCs beneath the epithelial cell layer. We conclude that antigen uptake in acute inflammation may activate resident immature DCs, inducing their migration to lymphoid tissue where they mature and then return to the intestine to play a role in the local inflammatory response.
Assuntos
Doença de Crohn/imunologia , Células Dendríticas/imunologia , Ileíte/imunologia , Íleo/imunologia , Doença Aguda , Animais , Antígenos CD/análise , Doença Crônica , Doença de Crohn/patologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Ileíte/patologia , Íleo/metabolismo , Íleo/patologia , Imunoglobulinas/análise , Imuno-Histoquímica , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Glicoproteínas de Membrana/análise , Permeabilidade , Ratos , Ratos Sprague-Dawley , Antígeno CD83RESUMO
BACKGROUND: Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. METHODS: Indomethacin administration to rats induced a long-lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short-circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. RESULTS: Rats with induced enteritis in both phases demonstrated a long-lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. CONCLUSIONS: The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.
Assuntos
Translocação Bacteriana/fisiologia , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Intestino Delgado/fisiopatologia , Animais , Transporte Biológico , Eletrofisiologia/métodos , Enterócitos/imunologia , Enterócitos/metabolismo , Enterócitos/microbiologia , Enterócitos/patologia , Epitélio/fisiologia , Epitélio/ultraestrutura , Imunidade nas Mucosas , Indometacina/farmacologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Peroxidase/biossíntese , Ratos , Ratos Sprague-Dawley , RecidivaRESUMO
Although Helicobacter spp. have been viewed as bacteria with low pathogenicity, many investigators have shown that these low-grade pathogens have the potential to become a severe threat in immunocompromised, inbred, and transgenic animals. Therefore the presence of Helicobacter spp. in experimental animals is considered to be an unacceptable variable. In this study a formulation of medicated feed was designed and tested in an attempt to eradicate Helicobacter spp. from an infected rat breeding colony. Two feeding protocols were used: 1) treating Helicobacter-infected pregnant dams to produce clean offspring and 2) treating infected adult animals long enough to eliminate the organisms. Bacterial DNA was extracted from feces and amplified using primers that recognized the Helicobacter spp.-specific region of the 16S rRNA gene. Fecal samples from the weanlings from protocol 1 tested negative for Helicobacter spp. at 1 week before and 2 and 12 weeks after weaning. Infected adult rats from protocol 2 tested negative after three cycles of 2 weeks on and 2 weeks off the medicated feed. Animals from both protocols have remained Helicobacter-free for 8 months.
Assuntos
Animais de Laboratório/microbiologia , Infecções por Helicobacter/veterinária , Helicobacter/genética , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/microbiologia , Amoxicilina/uso terapêutico , Ração Animal , Animais , Claritromicina/uso terapêutico , Primers do DNA , Eletroforese em Gel de Ágar , Fezes/microbiologia , Infecções por Helicobacter/tratamento farmacológico , Metronidazol/uso terapêutico , Omeprazol/uso terapêutico , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , RatosAssuntos
Técnicas de Cocultura/métodos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Separação Celular , Meios de Cultivo Condicionados , Humanos , Sistema Imunitário/citologia , Transporte de Íons , Leucócitos Mononucleares/imunologiaAssuntos
Bactérias/patogenicidade , Técnicas de Cocultura/métodos , Epitélio/microbiologia , Actinas/metabolismo , Apoptose , Fenômenos Fisiológicos Bacterianos , Técnicas Bacteriológicas , Cálcio/metabolismo , Linhagem Celular , Epitélio/metabolismo , Epitélio/patologia , Helicobacter pylori/patogenicidade , Helicobacter pylori/fisiologia , Humanos , Junções Íntimas/metabolismoRESUMO
BACKGROUND: Food allergy and chronic intestinal inflammation are common in western countries. The complex of antigen/IgE is taken up into the body from the gut lumen with the aid of epithelial cell-derived CD23 (low affinity IgE receptor II) that plays an important role in the pathogenesis of intestinal allergy. This study aimed to elucidate the role of mast cell on modulation of antigen/IgE complex transport across intestinal epithelial barrier. METHODS: Human intestinal epithelial cell line HT29 cell monolayer was used as a study platform. Transepithelial electric resistance (TER) and permeability to ovalbumin (OVA) were used as the markers of intestinal epithelial barrier function that were recorded in response to the stimulation of mast cell-derived chemical mediators. RESULTS: Conditioned media from naïve mast cell line HMC-1 cells or monocyte cell line THP-1 cells significantly upregulated the expression of CD23 and increased the antigen transport across the epithelium. Treatment with stem cell factor (SCF), nerve growth factor (NGF), retinoic acid (RA) or dimethyl sulphoxide (DMSO) enhanced CD23 expression in HT29 cells. Conditioned media from SCF, NGF or RA-treated HMC-1 cells, and SCF, NGF, DMSO or RA-treated THP-1 cells enhanced immune complex transport via enhancing the expression of the CD23 in HT29 cells and the release of inflammatory mediator TNF-α. Nuclear factor kappa B inhibitor, tryptase and TNF-α inhibited the increase in CD23 in HT29 cells and prevents the enhancement of epithelial barrier permeability. CONCLUSIONS: Mast cells play an important role in modulating the intestinal CD23 expression and the transport of antigen/IgE/CD23 complex across epithelial barrier. (Tu YH, Oluwole C, Struiksma S, Perdue MH, Yang PC. Mast cells modulate transport of CD23/IgE/antigen complex across human intestinal epithelial barrier.
RESUMO
BACKGROUND: Psychological stress is one of the factors associated with many human diseases; the mechanisms need to be further understood. METHODS: Rats were subjected to chronic water avoid stress. Intestinal epithelial heat shock protein (HSP) 70 was evaluated. The intestinal epithelial permeability was examined with Ussing chamber technique. RESULTS: HSP70 was detected in normal intestinal epithelial cells. Psychological stress decreased HSP70 in the intestinal epithelial cells that correlated with the stress-induced intestinal epithelial hyperpermeability. Pretreatment with HSP70 abrogated stress-induced intestinal barrier dysfunction. CONCLUSIONS: Chronic stress inhibits HSP70 activity in rat intestinal epithelial layer that is associated with intestinal epithelial barrier dysfunction, which can be prevented by pretreatment with HSP70 protein. (Yang PC, Tu YH, Perdue MH, Oluwole C, Struiksma S. Regulatory effect of heat shock protein 70 in stress-induced rat intestinal epithelial barrier dysfunction.
RESUMO
We investigated myeloid-dendritic cell (DC) marker and Toll-like receptor (TLR)-2 and 4 distributions in ileal samples from Crohn's disease (CD) patients (n = 14) and controls (n = 13). In controls, no TLR-2+ cells were observed, and higher numbers of TLR-4+ and DC-SIGN+ cells (P < 0.01) were detected in ileal samples when compared versus colonic tissues. In non-inflamed CD ileum, TLR-4+ and DC-SGN+ cells were depleted from superficial areas of the villus, and a significant CD1a+ cell infiltration (P < 0.01) was observed when compared to ileal controls and non-inflamed colonic CD samples. In inflamed CD ileum, DC-SIGN+, CD1a+, TLR-4+and few TLR-4+DC-SIGN+ cells were detected as well as CD83 depletion. No correlation between TLR-2 and DC markers was detected in CD samples. A unique distribution of myeloid-DC markers characterized the CD ileum. Also, the presence of significant amounts of ileal CD1a+ cells may provide a relevant DC-mediated mechanism for antigen recognition in the pathogenesis of CD.
Assuntos
Doença de Crohn/imunologia , Células Dendríticas/imunologia , Íleo/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Adulto , Análise de Variância , Feminino , Humanos , Técnicas Imunoenzimáticas , MasculinoRESUMO
Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.
Assuntos
Colo/metabolismo , Doenças do Colo/etiologia , Secreções Intestinais/metabolismo , Mastócitos/patologia , Estresse Psicológico/complicações , Animais , Aderência Bacteriana , Colo/patologia , Doenças do Colo/metabolismo , Doenças do Colo/microbiologia , Doenças do Colo/patologia , Hormônio Liberador da Corticotropina/administração & dosagem , Modelos Animais de Doenças , Esquema de Medicação , Condutividade Elétrica , Hiperplasia , Bombas de Infusão Implantáveis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Transporte de Íons , Masculino , Mastócitos/microbiologia , Permeabilidade , Ratos , Ratos Endogâmicos , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Estresse Psicológico/induzido quimicamente , Estresse Psicológico/metabolismo , Estresse Psicológico/patologia , Fatores de TempoRESUMO
A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.
Assuntos
Dinitrofenóis/toxicidade , Epitélio/fisiologia , Infecções por Escherichia coli/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Desacopladores/toxicidade , Trifosfato de Adenosina/biossíntese , Western Blotting , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Impedância Elétrica , Epitélio/efeitos dos fármacos , Infecções por Escherichia coli/metabolismo , Retroalimentação Fisiológica/fisiologia , Humanos , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Leupeptinas/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , NF-kappa B/fisiologia , Nitrilas/farmacologia , Permeabilidade/efeitos dos fármacos , Estresse Fisiológico/induzido quimicamente , Estresse Fisiológico/metabolismo , Sulfonas/farmacologia , Sais de Tetrazólio , TiazóisRESUMO
Dysregulated epithelial cell kinetics associated with mucosal barrier dysfunction may be involved in certain intestinal disorders. We previously showed that chronic psychological stress, in the form of repetitive sessions of water avoidance stress (WAS), has a major detrimental impact on ileal barrier function. We hypothesized that these changes were related to a disturbance in enterocyte kinetics. Rats were submitted to WAS (1 h/day) for 5 or 10 days. As previously shown, permeability to macromolecules was enhanced in rats stressed for 5 and 10 days compared with controls. WAS induced a decrease in crypt depth at day 5 associated with an increased number of apoptotic cells. Cell proliferation was significantly increased at days 5 and 10. Villus height and the specific activity of sucrase were significantly reduced at day 10. We concluded that WAS induces a disturbance of epithelial cell kinetics, with the pattern depending on the duration of the stress period. These findings help to explain the mechanism underlying altered epithelial barrier function resulting from exposure to chronic psychological stress.
Assuntos
Células Epiteliais/citologia , Íleo/citologia , Estresse Psicológico/fisiopatologia , Animais , Apoptose/fisiologia , Aprendizagem da Esquiva/fisiologia , Proliferação de Células , Íleo/enzimologia , Íleo/patologia , Permeabilidade , Ratos , Sacarase/metabolismo , ÁguaRESUMO
Chronic psychological stress impacts many functions of the gastrointestinal tract. However, the effect of stress on nutrient absorption is poorly documented. This study was designed to investigate glucose transporters in rats submitted to different periods of water-avoidance stress (WAS). Rats were subjected to WAS (1 h/day) for 1, 5, or 10 consecutive days. Four hours after the last WAS session, rats were killed and segments of jejunum were mounted in Ussing chambers to study electrophysiological properties of the jejunum and Na+-dependent glucose absorption kinetics. Mucosa was obtained to prepare brush-border membrane vesicles (BBMV) used to measure [14C]fructose uptake as well as sodium-glucose transporter 1 (SGLT-1) and GLUT2 expression by Western blot analysis. Exposure of animals to WAS induced a decrease in Na+-dependent glucose absorption Vmax after 1, 5, and 10 days without any change in SGLT-1 expression. Potential difference across the jejunum was decreased for all stressed groups. Furthermore, we observed an increase in phloretin-sensitive uptake of [14C]fructose by BBMV after 1, 5, or 10 days of WAS, which was not present in control animals. This suggested the abnormal appearance of GLUT2 in the brush border, which was confirmed by Western blot analysis. We concluded that psychological stress induces major changes in glucose transport with a decrease in Na+-dependent glucose absorption and an increase in GLUT2 expression at the brush-border membrane level.
Assuntos
Transportador de Glucose Tipo 2/biossíntese , Glucose/metabolismo , Absorção Intestinal/fisiologia , Jejuno/metabolismo , Microvilosidades/metabolismo , Sódio/fisiologia , Estresse Psicológico/metabolismo , Animais , Western Blotting , Cultura em Câmaras de Difusão , Ingestão de Alimentos , Eletrofisiologia , Manobra Psicológica , Cinética , Membranas/metabolismo , Floretina/farmacologia , Ratos , Ratos Endogâmicos BN , Transportador 1 de Glucose-Sódio/metabolismo , Natação/psicologia , Aumento de Peso/fisiologiaRESUMO
Neonatal maternal separation (MS) predisposes adult rats to develop stress-induced mucosal barrier dysfunction/visceral hypersensitivity and rat pups to develop colonic epithelial dysfunction. Our aim was to examine if enhanced epithelial permeability in such pups resulted from abnormal regulation by enteric nerves. Pups were separated from the dam for 3 h/day (days 4-20); nonseparated (NS) pups served as controls. On day 20, colonic tissues were removed and mounted in Ussing chambers. Horseradish peroxidase (HRP) flux was used to measure macromolecular permeability. HRP flux was increased in MS versus NS pups. The enhanced flux was inhibited by the cholinergic muscarinic antagonist atropine and the nicotinic antagonist hexamethonium. The cholinergic component was greater in tissues from MS versus NS pups, suggesting that increased cholinergic activity was responsible for the MS elevated permeability. Western blots and immunohistochemistry of colonic tissues demonstrated increased expression of choline acetyltransferase (ChAT) in MS pups, indicating greater synthesis of acetylcholine. Since a previous study indicated that corticotrophin-releasing factor (CRF) mediates barrier dysfunction in MS pups, we examined if the two pathways were linked. In MS tissues, nonselective CRF receptor antagonism inhibited the enhanced flux, and the addition of atropine did not produce further inhibition. Using selective receptor antagonists, we identified that CRF receptor 2 was involved in mediating this effect. These findings suggest that CRF, via CRF receptor 2, acts on cholinergic nerves to induce epithelial barrier dysfunction. Our study provides evidence that MS stimulates synthesis of acetylcholine, which, together with released CRF, creates a condition conducive to the development of epithelial barrier defects.
Assuntos
Fibras Colinérgicas/fisiologia , Colo/inervação , Colo/fisiologia , Mucosa Intestinal/fisiologia , Privação Materna , Animais , Animais Recém-Nascidos , Atropina/farmacologia , Betanecol/farmacologia , Colina O-Acetiltransferase/biossíntese , Hormônio Liberador da Corticotropina/antagonistas & inibidores , Feminino , Hexametônio/farmacologia , Peroxidase do Rábano Silvestre/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
We previously reported that CD23/FcepsilonRII (low-affinity IgE receptor) is expressed on human intestinal epithelial cells and is responsible for transepithelial transport of IgE. In this study, we compared the transport of IgE with that of immune complexes in both the apical-to-serosal and the serosal-to-apical directions across HT29 epithelial cell layers and examined the effects of two p38 MAPK inhibitors, SKF86002 and SB203580, on the expression and function of CD23. Our study showed that both p38 MAPK inhibitors at 10 microM significantly inhibited constitutive and IL-4-upregulated CD23 protein expression in epithelial cells. Both inhibitors, in a concentration-dependent manner, also significantly reduced IgE binding and uptake into cells. Transepithelial transport of IgE and immune complexes across the epithelial barrier were similarly inhibited. IL-4 upregulated the phosphorylation and activity of p38 MAPK and the phosphorylation of the downstream substrate MAPKAPK-2 (MK-2). The inhibitors exerted effects in the pathway post the p38 MAPK; SB203580 significantly inhibited the phosphorylation of MK-2. Our results indicate that CD23 expression in these human intestinal epithelial cells is mediated through the p38 MAPK pathway and that inhibition of p38 MAPK consequently interferes with the transport of IgE and immune complexes across the intestinal epithelial barrier.
Assuntos
Imunoglobulina E/metabolismo , Absorção Intestinal/fisiologia , Mucosa Intestinal/metabolismo , Receptores de IgE/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Humanos , Transdução de Sinais/fisiologiaRESUMO
Previous studies have shown that early life stress in the form of intermittent maternal separation (MS) predisposes adult rats to develop stress-induced intestinal mucosal dysfunction and visceral hypersensitivity. However, the mechanism involved in the functional abnormalities is unclear. Our aim was to study immature animals during or shortly after exposure to MS to determine whether there are early pathophysiological changes in the gut. Sprague-Dawley rat pups were individually separated from the dam for 3 h/d from 4 to 21 d of age; nonseparated (NS) control pups remained in the home cage with the dam. On d 19-20, d 24-25, and d 29-30, blood was collected for corticosterone measurement, and colonic tissues were removed for functional and morphologic assessment. Corticosteroid levels were elevated in MS pups compared with NS, indicating that MS was indeed stressful. The distal colon demonstrated significantly enhanced ion secretion and macromolecular permeability at d 19-20 and d 24-25, returning to normal by d 29-30. Electron microscopy and bacterial culture studies indicated bacteria adhering to and penetrating into the colonic epithelium of the MS pups at all time points, while such events were rare in NS pups. The pathophysiological changes were inhibited by injecting pups sc with a corticotropin-releasing hormone (CRH) receptor antagonist daily during MS. Our studies indicate that early psychological trauma predisposes neonatal rats to develop persistent mucosal barrier dysfunction, including impaired host defense to luminal bacteria, by a mechanism involving peripheral CRH receptors.