RESUMO
BACKGROUND: Histologically nonresponsive coeliac disease (NRCD) is a potentially serious condition diagnosed during the follow-up of coeliac disease (CD) when patients have persistent villous atrophy despite following a gluten-free diet (GFD). AIM: As current assessments of recovery are limited to invasive and costly serial duodenal biopsies, we sought to identify antibody biomarkers for CD patients that do not respond to traditional therapy. METHODS: Bacterial display peptide libraries were screened by flow cytometry to identify epitopes specifically recognised by antibodies from patients with NRCD, but not by antibodies from responsive CD patients. Deamidated gliadin was confirmed to be the antigen mimicked by library peptides using ELISA with sera from NRCD (n = 15) and responsive CD (n = 45) patients on a strict GFD for at least 1 year. RESULTS: The dominant consensus epitope sequence identified by unbiased library screening QPxx(A/P)FP(E/D) was highly similar to reported deamidated gliadin peptide (dGP) B-cell epitopes. Measurement of anti-dGP IgG titre by ELISA discriminated between NRCD and responsive CD patients with 87% sensitivity and 89% specificity. Importantly, dGP antibody titre correlated with the severity of mucosal damage indicating that IgG dGP titres may be useful to monitor small intestinal mucosal recovery on a GFD. CONCLUSIONS: The finding of increased levels of anti-dGP IgG antibodies in CD patients on strict GFDs effectively identifies patients with NRCD. Finally, anti-dGP IgG assays may be useful to monitor mucosal damage and histological improvement in CD patients on a strict GFD.
Assuntos
Doença Celíaca/dietoterapia , Dieta Livre de Glúten , Gliadina/imunologia , Adolescente , Adulto , Idoso , Anticorpos/sangue , Biomarcadores/metabolismo , Biópsia , Doença Celíaca/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Sensibilidade e EspecificidadeRESUMO
The mucosa that lines the human colon and small intestine is a site of chronic regulated "physiologic" inflammation. This contrasts markedly with other mucosal sites in that if the numbers of T and B cells, eosinophils, mast cells, macrophages, and dendritic cells that are present in the human intestinal tract were to be present in other sites, those sites would be considered to be sites of chronic pathological inflammation. This review examines the role of the intestinal epithelium in the development of "physiologic" intestinal mucosal inflammation and focuses on its role in signalling and mediating host innate and adaptive mucosal immune responses.
Assuntos
Células Epiteliais/imunologia , Imunidade Celular/imunologia , Mucosa Intestinal/imunologia , Bactérias/imunologia , Humanos , Imunidade Ativa/imunologia , Imunidade Inata/imunologiaRESUMO
The intestinal mucosa contains a subset of lymphocytes that produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine (MDC/CCL22) is a recently described CC chemokine known to chemoattract the Th2 cytokine producing cells that express the receptor CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion was upregulated in colon epithelial cell lines in response to proinflammatory cytokines or infection with enteroinvasive bacteria. Inhibition of nuclear factor (NF)-kappaB activation abolished MDC/CCL22 expression in response to proinflammatory stimuli, demonstrating that MDC/CCL22 is a NF-kappaB target gene. In addition, tumor necrosis factor-alpha-induced MDC/CCL22 secretion was differentially modulated by Th1 and Th2 cytokines. Supernatants from the basal, but not apical, side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the constitutive and regulated production by intestinal epithelial cells of a chemokine known to function in the trafficking of T cells that produce anti-inflammatory cytokines.
Assuntos
Quimiocinas CC/biossíntese , Mucosa Intestinal/metabolismo , Animais , Polaridade Celular/fisiologia , Quimiocina CCL22 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Colo/metabolismo , Citocinas/metabolismo , Citocinas/farmacologia , Infecções por Escherichia coli/metabolismo , Transplante de Tecido Fetal , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Intestinos/embriologia , Intestinos/transplante , Camundongos , Camundongos SCID , NF-kappa B/fisiologia , RNA Mensageiro/metabolismo , Infecções por Salmonella/metabolismo , Linfócitos T/fisiologia , Células Th2/metabolismo , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Gene expression profiling offers new opportunities for understanding host-cell responses to microbial pathogens and their products. Current strategies involve either first identifying mRNAs that differ in their expression status under different experimental conditions and later defining the identity of the respective genes (for example, differential display or serial analysis of gene expression), or alternatively assessing changes in the expression of already defined genes (for example, cDNA or oligonucleotide microarrays). Early studies indicate the power of gene expression profiling for providing new insights into groups of genes whose expression is altered during the course of host-microbe interactions, and for the discovery of cellular genes that were not previously recognized to be regulated by infection.
Assuntos
Perfilação da Expressão Gênica , Sistema Imunitário/imunologia , Infecções/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Sistema Imunitário/metabolismo , Infecções/imunologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The chemokine TARC is a ligand for the chemokine receptor CCR4 expressed on T helper (Th)2-type CD4 T cells. Allergic airway inflammation is characterized by a local increase in cells secreting Th2-type cytokines. We hypothesized that bronchial epithelial cells may be a source of chemokines known to chemoattract Th2 cells. Regulated TARC expression was studied using normal human bronchial epithelial cells and a human lung xenograft model. TARC expression was increased in normal human bronchial epithelial cells in response to tumor necrosis factor-alpha stimulation, and further upregulation of TARC was observed with interferon (IFN)-gamma but not interleukin (IL)-4 costimulation. TARC functions as a nuclear factor (NF)-kappa B target gene, as shown by the abrogation of TARC expression in response to proinflammatory stimuli when NF-kappa B activation is inhibited. In an in vivo model, minimal constitutive TARC expression was observed in human lung xenografts. Consistent with our findings in vitro, TARC messenger RNA (mRNA) expression was upregulated in the xenografts in response to IL-1, and costimulation with IFN-gamma but not IL-4 further increased TARC mRNA and protein expression. In addition, bronchoalveolar lavage fluid from asthmatic subjects after allergen challenge contained significantly increased levels of TARC, suggesting that TARC production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma.
Assuntos
Brônquios/citologia , Quimiocinas CC/genética , Células Epiteliais/imunologia , Transplante de Pulmão/imunologia , Células Th2/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular Transformada , Quimiocina CCL17 , Quimiocinas CC/análise , Células Epiteliais/citologia , Transplante de Tecido Fetal , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Neoplasias Pulmonares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , NF-kappa B/genética , RNA Mensageiro/análise , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.
Assuntos
Quimiocinas CC , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Proteínas Inflamatórias de Macrófagos/biossíntese , Adenoviridae/imunologia , Animais , Quimiocina CCL20 , Ensaio de Imunoadsorção Enzimática , Epitélio/imunologia , Epitélio/metabolismo , Transplante de Tecido Fetal , Humanos , Indicadores e Reagentes , Interleucina-1/farmacologia , Proteínas Inflamatórias de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR6 , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Proteínas Repressoras/fisiologia , Infecções por Salmonella/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas , Regulação para Cima/genéticaRESUMO
BACKGROUND & AIMS: Human intestinal epithelial cells inducibly express neutrophil and monocyte chemoattractants, yet little is known about the regulated production of T-cell chemoattractants by the intestinal epithelium. IP-10, Mig, and I-TAC are 3 CXC chemokines that are known to act as CD4(+) T-cell chemoattractants. METHODS: We studied constitutive chemokine expression in human colon, and defined the regulated expression of these chemokines by reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistology using cultured human intestinal epithelial cell lines and a novel adaptation of an in vivo human intestinal xenograft model. RESULTS: IP-10 and Mig were constitutively expressed by normal human colon epithelium, and their cognate receptor, CXCR3, was expressed by mucosal mononuclear cells. Interferon (IFN)-gamma stimulation increased mRNA expression and the polarized basolateral secretion of these chemokines by human colon epithelial cell lines; infection with enteroinvasive bacteria, or stimulation with the proinflammatory cytokines tumor necrosis factor alpha and interleukin 1alpha, strongly potentiated IFN-gamma-induced epithelial cell IP-10, Mig, and I-TAC production. Epithelial cell mRNA and protein expression of IP-10, Mig, and I-TAC were rapidly up-regulated in human intestinal xenografts in response to stimulation with IFN-gamma alone or in combination with IL-1. CONCLUSIONS: The constitutive and regulated production of the IFN-gamma-inducible chemokines IP-10, Mig, and I-TAC by human intestinal epithelium, and the expression of their cognate receptor, CXCR3, by mucosal mononuclear cells, suggest that the intestinal epithelium can play a role in modulating physiologic and pathologic T cell-mediated mucosal inflammation.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/citologia , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/farmacologia , Mucosa Intestinal/imunologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Quimiotaxia de Leucócito/efeitos dos fármacos , Enterite/imunologia , Enterite/metabolismo , Transplante de Tecido Fetal , Feto/citologia , Expressão Gênica/fisiologia , Células HT29 , Humanos , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Transplante de Neoplasias , RNA Mensageiro/análise , Receptores CXCR3 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transplante HeterólogoRESUMO
Cytokines are key communication molecules between host cells in the defense against the enteric pathogen, Salmonella. Infection with Salmonella induces expression of multiple chemokines and proinflammatory cytokines in cultured intestinal epithelial cells and macrophages. In animal models, protective roles have been shown for IL-1alpha, TNFalpha, IFN-gamma, IL-12, IL-18 and IL-15, whereas IL-4 and IL-10 inhibit host defenses against Salmonella.
Assuntos
Citocinas/biossíntese , Imunidade Inata , Infecções por Salmonella/imunologia , Salmonella/patogenicidade , Animais , Células Cultivadas , Modelos Animais de Doenças , Epitélio/microbiologia , Humanos , Intestinos/citologia , Intestinos/microbiologia , Macrófagos/microbiologia , Camundongos , Salmonella/fisiologia , Infecções por Salmonella/microbiologiaRESUMO
Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human beta-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection with Helicobacter pylori or stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response to H. pylori infection and proinflammatory mediators.
Assuntos
Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica , Helicobacter pylori/fisiologia , Interleucina-1/farmacologia , Proteínas/genética , beta-Defensinas , Defensinas , Humanos , Lipopolissacarídeos/farmacologia , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a process accompanied by increased epithelial expression of proinflammatory cytokines. To further define the role intestinal epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection on high density cDNA arrays was used to characterize the mRNA expression profile of approximately 4,300 genes in human intestinal epithelial cells after infection with the prototypic invasive bacteria, Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and protein assays. Epithelial infection with Salmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines (granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory protein-2alpha), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most prominent. Furthermore, the transcription factor NF-kappaB is shown to be important for inducible mRNA expression for a broad group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach to characterizing and understanding host-pathogen interactions.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Salmonella/patogenicidade , Sequência de Bases , Northern Blotting , Citocinas/genética , Primers do DNA , DNA Complementar , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HT29 , Humanos , NF-kappa B/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cryptosporidium parvum induces moderate levels of apoptosis of cultured human intestinal epithelial cells, which are maximal at 24 h after infection. Apoptosis is further increased in C. parvum-infected cells by inhibition of NF-kappaB. C. parvum infection also attenuates epithelial apoptosis induced by strongly proapoptotic agents. The data suggest C. parvum has developed strategies to limit apoptosis in order to facilitate its growth and maturation in the early period after epithelial cell infection.
Assuntos
Apoptose , Cryptosporidium parvum/fisiologia , Mucosa Intestinal/parasitologia , Animais , Humanos , Mucosa Intestinal/patologia , NF-kappa B/fisiologia , Células Tumorais CultivadasRESUMO
Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.
Assuntos
Antiprotozoários/farmacologia , Arginina/metabolismo , Giardia lamblia/patogenicidade , Giardíase/prevenção & controle , Inibidores do Crescimento/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Óxido Nítrico/biossíntese , Animais , Ligação Competitiva , Células CACO-2/enzimologia , Células CACO-2/metabolismo , Células CACO-2/parasitologia , Comunicação Celular/imunologia , Polaridade Celular/imunologia , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/imunologia , Giardíase/imunologia , Giardíase/parasitologia , Glutationa/análogos & derivados , Glutationa/farmacologia , Inibidores do Crescimento/biossíntese , Células HT29/enzimologia , Células HT29/metabolismo , Células HT29/parasitologia , Humanos , Mucosa Intestinal/enzimologia , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Compostos Nitrosos/farmacologia , S-NitrosoglutationaRESUMO
Cryptosporidium parvum can be regarded as a minimally invasive mucosal pathogen, since it invades surface epithelial cells that line the intestinal tract but does not invade deeper layers of the intestinal mucosa. Nonetheless, infection can be associated with diarrhea and marked mucosal inflammation. This article briefly reviews in vitro and in vivo models useful for studying the pathogenesis of C. parvum infection and explores the role of innate and acquired immune responses in host defense against this protozoan parasite.
Assuntos
Quimiocinas CXC , Criptosporidiose/parasitologia , Cryptosporidium parvum/patogenicidade , Peptídeos e Proteínas de Sinalização Intercelular , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Animais , Formação de Anticorpos , Células Cultivadas/parasitologia , Quimiocina CXCL1 , Fatores Quimiotáticos/análise , Criptosporidiose/imunologia , Criptosporidiose/patologia , Cryptosporidium parvum/citologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/parasitologia , Células Epiteliais/patologia , Substâncias de Crescimento/análise , Humanos , Imunidade Celular , Imunidade Inata , Interferon gama/imunologia , Interleucina-8/análise , Intestinos/parasitologia , Intestinos/patologia , Regulação para CimaRESUMO
The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.
Assuntos
Antibacterianos/metabolismo , Mucosa Intestinal/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , beta-Defensinas , Células CACO-2/metabolismo , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Defensinas , Regulação da Expressão Gênica/imunologia , Células HT29 , Humanos , Interleucina-1/farmacologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Biossíntese Peptídica/imunologia , Proteínas/genética , RNA Mensageiro/biossíntese , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Transplante Heterólogo/imunologiaRESUMO
The intestinal epithelium produces and responds to cytokines and lipid mediators that play a key role in the induction and regulation of mucosal inflammation. The lipid mediator platelet-activating factor (PAF) can be produced and degraded by the human intestinal epithelium and is known to mediate a range of proinflammatory and other biological effects in the intestinal mucosa. In the studies herein, we assessed whether or not human intestinal epithelial cells express cell surface or intracellular PAF receptors (PAF-R), whether expression of these receptors can be regulated, and whether human intestinal epithelial cells respond to PAF. Several human colon epithelial cell lines (HT-29, Caco-2, T84, HCT-8, HCA-7, I407, and LS-174T) were shown by RT-PCR to constitutively express mRNA for PAF-R. In addition, PAF-R expression was demonstrated by immunoblot analysis and PAF-R was shown to be constitutively expressed on the cell surface of several of these cell lines, as assessed by flow cytometry. PAF-R expression by human colon epithelial cells was upregulated by stimulation with retinoic acid but not by stimulation with PAF, proinflammatory agonists (tumor necrosis factor-alpha, interleukin-1, interferon-gamma), or transforming growth factor-alpha. PAF-R on intestinal epithelial cells were functional, as PAF stimulation of the cells increased tyrosine phosphorylation of several cellular proteins, including proteins of 75 and 125 kDa, and this response was blocked by a PAF-R antagonist. Consistent with the findings using cell lines, PAF-R were also constitutively expressed by normal human colon and small intestinal epithelium in vivo, as shown by immunohistology. The constitutive and regulated expression of functional PAF-R by human intestinal epithelium suggests PAF produced by the intestinal epithelial cells or cells underlying the epithelium has autocrine or paracrine effects on intestinal epithelial cells.
Assuntos
Mucosa Intestinal/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Linhagem Celular , Membrana Celular/metabolismo , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Humanos , Immunoblotting , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/genética , RNA Mensageiro/metabolismo , Tirosina/metabolismoRESUMO
Celiac disease (CD) patients usually express a DQ2 heterodimer, whose chains DQalpha1*0501/DQbeta1*0201, are encoded by the genes HLA-DQA1*0501 and DQB1*0201, respectively. Among the DQ2 carriers, the risk of developing disease was shown to correlate with the number of DQbeta1*0201 chains encoded. Studying two separate cohorts of Italian and Tunisian patients, we now show a significant association of celiac disease with expression of either the DQ2 or DR53 heterodimers. The risk is maximal for individuals that carry both DQ2 and DR53 heterodimers. When twenty synthetic peptides overlapping most of A-gliadin sequence were tested for the binding to various purified DR molecules, it was found that DR53 molecules bind selectively and with high affinity (IC50<1 microM) to A-gliadin-derived peptides. These data suggest that both HLA DQ2 and DR53 molecules are associated with increased genetic risk for CD, and provide a possible biochemical basis for this complex association.
Assuntos
Doença Celíaca/genética , Gliadina/metabolismo , Antígenos HLA-DR/genética , Ligação Competitiva , Estudos de Coortes , Dimerização , Predisposição Genética para Doença , Genótipo , Antígenos HLA-DQ/genética , Antígenos HLA-DR/química , Antígenos HLA-DR/isolamento & purificação , Cadeias HLA-DRB4 , Humanos , Itália , Ligação Proteica , Fatores de Risco , TunísiaRESUMO
BACKGROUND & AIMS: Intestinal epithelial cells produce an array of proinflammatory chemokines that can provide signals to mucosal immune and inflammatory cells. To determine if chemokines can also signal epithelial cells, we characterized the expression of chemokine receptors on human colon epithelial cells in vitro and in vivo. METHODS: Expression of chemokine receptor messenger RNAs (mRNAs) by the human colon epithelial cell lines HT-29, HT-29.18.C1, Caco-2, T84, HCA-7, and LS174T was assessed by reverse-transcription polymerase chain reaction. Chemokine receptors on intestinal epithelial cells in vitro were determined by flow cytometry, and expression in vivo was determined by immunostaining of human colon. Interleukin (IL)-8 and growth-related (GRO) alpha secretion were assayed by enzyme-linked immunosorbent assay. RESULTS: Human colon epithelial cells constitutively expressed mRNAs for an array of CC and CXC chemokine receptors, including CCR1-8 and CXCR4, but little if any CXCR1 or CXCR2. Further studies focused on CXCR4 and CCR5 because mRNA for those chemokine receptors was abundantly expressed by each of the colon epithelial cell lines, and these receptors were present on the cell surface. Analogous to their localization on polarized cell lines, CXCR4 and CCR5 had a predominant apical and, to a lesser extent, basolateral distribution on human enterocytes, as demonstrated by immunostaining of human colon. Human colon epithelial cells stimulated with stromal cell-derived factor 1alpha and macrophage inflammatory protein (MIP)- 1alpha or MIP-1beta, which are the chemokine ligands for CXCR4 or CCR5, up-regulated production of the CXC chemokines IL-8 and GROalpha. CONCLUSIONS: Human colon epithelial cells express chemokine receptors. Human colonocytes have the potential to serve as targets for chemokine signaling.
Assuntos
Colo/química , Mucosa Intestinal/química , Receptores de Quimiocinas/análise , Animais , Humanos , Camundongos , RNA Mensageiro/análise , Receptores CCR5/análise , Receptores CXCR4/análise , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Células Tumorais CultivadasRESUMO
Human intestinal epithelial cells up-regulate the expression of an inflammatory gene program in response to infection with a spectrum of different strains of enteroinvasive bacteria. The conserved nature of this program suggested that diverse signals, which are activated by enteroinvasive bacteria, can be integrated into a common signaling pathway that activates a set of proinflammatory genes in infected host cells. Human intestinal epithelial cell lines, HT-29, Caco-2, and T84, were infected with invasive bacteria that use different strategies to induce their uptake and have different intracellular localizations (i.e., Salmonella dublin, enteroinvasive Escherichia coli, or Yersinia enterocolitica). Infection with each of these bacteria resulted in the activation of TNF receptor associated factors, two recently described serine kinases, I kappa B kinase (IKK) alpha and IKK beta, and increased NF-kappa B DNA binding activity. This was paralleled by partial degradation of I kappa B alpha and I kappa B epsilon in bacteria-infected Caco-2 cells. Mutant proteins that act as superrepressors of IKK beta and I kappa B alpha inhibited the up-regulated transcription and expression of downstream targets genes of NF-kappa B that are key components of the epithelial inflammatory gene program (i.e., IL-8, growth-related oncogene-alpha, monocyte chemoattractant protein-1, TNF-alpha, cyclooxygenase-2, nitric oxide synthase-2, ICAM-1) activated by those enteroinvasive bacteria. These studies position NF-kappa B as a central regulator of the epithelial cell innate immune response to infection with enteroinvasive bacteria.
Assuntos
Quimiocinas CXC , Infecções por Enterobacteriaceae/imunologia , Proteínas I-kappa B , Peptídeos e Proteínas de Sinalização Intercelular , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , NF-kappa B/fisiologia , Células CACO-2 , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CXCL1 , Fatores Quimiotáticos/biossíntese , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Infecções por Enterobacteriaceae/genética , Ativação Enzimática/imunologia , Regulação da Expressão Gênica/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Genes Reporter/imunologia , Substâncias de Crescimento/biossíntese , Células HT29 , Humanos , Quinase I-kappa B , Imunidade Inata , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Mucosa Intestinal/enzimologia , Mucosa Intestinal/microbiologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Isoenzimas/biossíntese , Proteínas de Membrana , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Human immunodeficiency virus (HIV) can infect human colon epithelial cell lines by both CD4-dependent and -independent mechanisms. The present studies assessed cellular factors that are important for HIV-1 transcription in human colon epithelial cells. The HIV-1 long terminal repeat (LTR) was shown to contain functional DNA cis-regulatory elements downstream of the viral transactivator-responsive element in the transcribed noncoding 5' leader sequence. These downstream regulatory elements, termed DSE, can bind c-Fos and JunD and transmit protein kinase C activation signals to the HIV LTR. Moreover, specific Jun and Fos transcription factors can transactivate HIV-1 provirus in human colon epithelial cells. The DSE also bind related proteins of the CREB/ATF family. In this regard, the DSE behave as 12-0-tetradecanoylphorbol 13-acetate responder element-like cAMP-responsive elements because they bind both AP-1 and CREB/ATF transcription factors, thereby permitting induction of the HIV-1 LTR by both protein kinase C and A activation signals.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/virologia , Infecções por HIV/metabolismo , HIV-1/genética , Mucosa Intestinal/virologia , Proteína Quinase C/metabolismo , Transcrição Gênica , Linhagem Celular , Colo/citologia , Colo/efeitos dos fármacos , Colo/virologia , Infecções por HIV/genética , Repetição Terminal Longa de HIV/genética , Humanos , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
The properties of two candidate Salmonella typhi-based live oral typhoid vaccine strains, BRD691 (S. typhi Ty2 harboring mutations in aroA and aroC) and BRD1116 (S. typhi Ty2 harboring mutations in aroA, aroC, and htrA), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in S. typhi does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.