RESUMEN
The essential oils of six endemic Malagasy Helichrysum species were investigated by GC (RI), GC-MS and 13C NMR spectrometry. In total, 153 compounds were identified accounting for 90.8% to 99.9% of the total composition. The main constituents were α-pinene for H. benthamii, 1,8-cineole for H. dubardii, (E)-ß-caryophyllene for H. indutum, and H. bojerianum. H. diotoides essential oil was characterized by the presence of two lilac alcohols and four lilac acetates whereas H. hirtum essential oil exhibited an atypical composition with 7ß-H-silphiperfol-5-ene, 7-epi-subergorgiol, and 7-epi-silphiperfol-5-en-13-oic acid as major components.
RESUMEN
The chemical composition of twenty-five essential oil samples from the aerial parts of two Malagasy endemic species Billburttia capensoides Sales & Hedge and B. vaginoides Sales & Hedge, were investigated for the first time. Based on chromatographic profiles, three selected samples were investigated using GC(RI), GC-MS and 13C NMR. The content of the main components varied drastically from sample to sample: p-mentha-1,3,8-triene (0.2-52.7%), terpinolene (2.8-40.7%) and dill apiole (0.0-22.2%). Statistical analysis of the 25 oil compositions allowed the distinction of two well-differentiated groups. Samples of group I contained mainly p-mentha-1,3,8-triene while the Group II was dominated by terpinolene and dill apiole.
Asunto(s)
Apiaceae/química , Aceites Volátiles/química , Componentes Aéreos de las Plantas/química , Anethum graveolens/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Monoterpenos Ciclohexánicos , Cromatografía de Gases y Espectrometría de Masas , Madagascar , Mentha/química , Aceites Volátiles/clasificación , Terpenos/químicaRESUMEN
Melicope belahe (Baill.) T.G. Hartley (Rutaceae) is an endemic species to Madagascar. The chemical composition of leaf essential oil is reported for the first time. A sample was extracted by hydrodistillation and analysis was carried out by combination of chromatographic (GC), spectroscopic and spectrometric (MS, 13C NMR) techniques. In total, 56 compounds have been identified. The chemical composition was dominated by α-pinene (42.6%) followed by linalool (6.2%) and (E)-ß-caryophyllene (5.2%).
Asunto(s)
Aceites Volátiles/química , Rutaceae/química , Monoterpenos Acíclicos , Monoterpenos Bicíclicos , Madagascar , Espectroscopía de Resonancia Magnética , Monoterpenos/análisis , Hojas de la Planta/química , Sesquiterpenos Policíclicos , Sesquiterpenos/análisisRESUMEN
Croton kimosorum Leandri is an endemic species to Madagascar. The chemical composition of aerial parts, leaf and stem oils is reported for the first time. Analysis was carried out by combination of chromatographic (CC, GC), spectroscopic and spectrometric (MS, 13C NMR) techniques. In total, 76 compounds have been identified. Essential oil isolated from aerial parts contained mainly linalool (21.6%), sabinene (10.4%), 1,8-cineole (6.3%), beta-pinene (6.2%), (E)-beta-caryophyllene (5.9%), terpinen-4-ol (4.8%), geraniol (4,5%) and germacrene D (2.3%). In comparison with the first sample, the composition of leaf and stem oils varied slightly, while essential oil isolated by vapor distillation from a semi-industrial still exhibited similar composition.
Asunto(s)
Croton/química , Aceites Volátiles/química , MadagascarRESUMEN
The volatile components isolated from leaves of Vepris madagascarica (Baillon) H. Perier (Rutaceae), an endemic species of north-eastern, sub-humid forests of Madagascar, were investigated by GC (Retention Indices), GC-MS and 13C NMR spectroscopy. Oil samples obtained on laboratory and industrial scales exhibited similar composition, dominated by phenylpropanoids. (E)-anethole (78.2% and 78.6%) was the major component followed by estragole (15.6% and 15.4%). In addition, trunk bark oil also contained (E)-anethole as its major component (84.6%), as well as various sesquiterpenes in low contents.
Asunto(s)
Anisoles/análisis , Aceites Volátiles/química , Rutaceae/química , Derivados de Alilbenceno , Madagascar , Corteza de la Planta/química , Hojas de la Planta/químicaRESUMEN
Ulcerative colitis (UC) and Crohn's disease (CD) are considered to be immunologically mediated disorders that share certain features with murine models of colitis. Whether any of these models are physiologically relevant to the human condition remains controversial. The hypothesis is that increased amounts of antibodies neutralizing transforming growth factor (TGF)-beta, interleukin (IL)-2 or IL-10 create a relative immunodeficient state in inflammatory bowel disease (IBD) that predisposes to disease. To evaluate this, serum samples from patients with UC or CD and from normal healthy individuals were studied by enzyme-linked immunosorbent assays. Antibodies recognizing TGF-beta were most prevalent in UC (P<0.01); anti-IL-10 antibodies were elevated in CD (P<0.05), while anti-IL-2 antibodies were the same for all three groups. Importantly, the percentage of IBD patients with at least one of the antibody levels greater than any control value was 30% for UC and 33% for CD. To verify the presence of these antibodies, immobilized TGF-beta was exposed to UC sera and the attached proteins identified by Western blot assay. The proteins proved to be exclusively immunoglobulin (Ig) G. To evaluate the neutralizing activity of these antibodies, cytokine-specific IgG from subjects in each group of patients was incubated with TGF-beta, IL-2 or IL-10 before addition to a bioassay with changes in viability determined by a colorimetric analysis. Antibodies from most individuals in all three groups neutralized the action of each cytokine. This study shows that about one-third of IBD patients may have a relative deficiency of TGF-beta, IL-2 or IL-10 due to an increase in neutralizing antibodies in their sera.
Asunto(s)
Autoanticuerpos/análisis , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Interleucina-10/deficiencia , Interleucina-2/deficiencia , Factor de Crecimiento Transformador beta/deficiencia , Adulto , Análisis de Varianza , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Estudios de Casos y Controles , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
Butyrate, a short-chain fatty acid released by colonic bacteria and administered therapeutically in inflammatory bowel diseases, exerts immunomodulatory properties. The aim of the study was to determine the functional consequences of butyrate exposure on the proinflammatory responsiveness of human intestinal epithelial cells (IEC). IL-8 promoter activity in IEC pretreated with butyrate then exposed to proinflammatory stimuli was assayed by transfection of luciferase constructs. IL-8 secretion was determined by ELISA and neutrophil migration by flow cytometry. Receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Butyrate modulated proinflammatory IL-8 secretion differentially in Caco-2 and HT-29 cells on the transcriptional level. Pointing to the potentially underlying mechanism of increased IL-1 beta-stimulated IL-8 secretion in HT-29 cells, butyrate up-regulated IL-1RI mRNA but not IL-1RII. Butyrate pretreatment of IEC lines stimulated by IL-1 beta modulated neutrophil migration significantly: reduction towards Caco-2 and enhancement towards HT-29/p cells. Pharmacological inhibition of protein tyrosine phosphatases or treatment with mesalamine or sulphasalazine diminished IL-1 beta-stimulated IL-8 secretion by butyrate-exposed HT-29 cells substantially. Immunomodulatory effects of butyrate on IEC are functionally relevant for neutrophil migration. Pharmacological inhibition of enhanced IL-1 beta-mediated IL-8 secretion in a subpopulation of IEC may improve the clinical efficacy of butyrate.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Butiratos/uso terapéutico , Interleucina-1/inmunología , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Antiinflamatorios no Esteroideos/uso terapéutico , Células CACO-2 , Quimiotaxis de Leucocito/efectos de los fármacos , Células HT29 , Humanos , Interleucina-8/genética , Mucosa Intestinal/efectos de los fármacos , Mesalamina/uso terapéutico , ARN Mensajero/análisis , Receptores de Interleucina-1/genética , Sulfasalazina/uso terapéutico , Transcripción GenéticaRESUMEN
A novel method for generating nontransformed human intestinal primary epithelial cell (HIPEC) lines in an in vitro culture system is reported here. Although several groups have reported the development of nontransformed intestinal epithelial cells (IEC) lines (Deveney et al, 1996; Latella et al, 1996; Pang et al, 1996; Perreault and Beaulieu, 1998), it still had been difficult to find an optimal condition to generate a pure population of nontransformed IEC in long-term cultures. It was hypothesized that an appropriate growth factor/cytokine milieu that would mimic the physiological microenvironment might favor the survival of the isolated cells and might play a critical role in epithelial cell growth. To test this hypothesis, isolated progenitor/crypt cells were cultured in collagen-coated petri dishes in the presence of mucosal tissue-derived growth factor containing culture supernatants (14-18 hours) and a combination of hormonal supplements. Cell attachment and growth was observed within 24 hours and confluent monolayers were seen between 7 and 12 days. Immunofluorescence staining and flow cytometric analysis of the cells demonstrated positive staining with anti-cytokeratin-18 antibody confirming their epithelial origin. The reproducibility of the method has been confirmed by establishing a number of HIPEC lines from various segments of the gastrointestinal tract. This novel method of HIPEC line generation, which maximizes the similarity of the ex vivo culture system to in vivo conditions, will serve as a valuable tool for the establishment of a large number of HIPEC lines (intestinal epithelial cell bank) and for subsequent use in studies of the immunological/physiological epithelial function in the intestine.
Asunto(s)
Mucosa Intestinal/citología , División Celular , Línea Celular , Supervivencia Celular , HumanosRESUMEN
BACKGROUND & AIMS: Regulatory cytokines mediate intestinal epithelial cell (IEC) participation in mucosal immune responses. The aim of this study was to investigate the expression of secretory and intracellular isoforms of interleukin 1 receptor antagonist (IL-1Ra) in human primary IECs and carcinoma-derived cell lines. METHODS: Primary IECs were isolated from patients with Crohn's disease or ulcerative colitis and from normal controls. Isoform-specific IL-1Ra messenger RNA (mRNA) and protein were assessed by reverse-transcription polymerase chain reaction and Western blot analysis. Expression during cellular differentiation was determined by in situ immunohistochemistry on sequentially released, native IECs and in vitro differentiated cell lines. Intracellular IL-1Ra I function was analyzed by permanent transfection of Caco-2 cells. RESULTS: Intracellular IL-1Ra I protein accumulated in surface IECs with extension to the crypts during inflammation. Secretory IL-1Ra and intracellular IL-1Ra II mRNA, but not the corresponding protein, was detected. Transcription of intracellular IL-1Ra I mRNA was significantly up-regulated with inflammation and in vitro by phorbol myristate acetate and interleukin 1beta. In vitro differentiated cells had higher constitutive intracellular IL-1Ra I protein content. Intracellular IL-1Ra I expression in Caco-2 cells decreased IL-1beta-stimulated interleukin 8 secretion. CONCLUSIONS: Native human IECs and certain cell lines constitutively express intracellular IL-1Ra type I, which is up-regulated by inflammation, inflammatory stimuli, and cellular differentiation. Constitutive expression of this anti-inflammatory cytokine may contribute to mucosal protection.
Asunto(s)
Mucosa Intestinal/metabolismo , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/biosíntesis , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Humanos , Inflamación , Proteína Antagonista del Receptor de Interleucina 1 , Mucosa Intestinal/citología , Isoformas de Proteínas , ARN Mensajero/biosíntesis , Sialoglicoproteínas/genéticaRESUMEN
Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1beta, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-alpha. Receptors for IL-1beta, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFalpha-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-gamma. Functionally, IL-1beta enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-alpha treatment enhanced the secretion of IL-8 and GRO-alpha in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.
Asunto(s)
Citocinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células CACO-2 , Línea Celular , Separación Celular , Citocinas/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Células HT29 , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-1/metabolismo , Interleucina-1/fisiología , Interleucina-6/metabolismo , Interleucina-6/fisiología , Mucosa Intestinal/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Receptores de Interleucina-1/biosíntesis , Receptores de Interleucina-6/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
NF-kappaB plays a major role in the transcriptional regulation of many proinflammatory genes in multiple cell lineages, including intestinal epithelial cells (IEC). Activation of NF-kappaB requires both phosphorylation and degradation of its natural cytoplasmic inhibitor, IkappaB. We tested whether a super-repressor of NF-kappaB activity, which is a mutated nondegradable IkappaB alpha resistant to phosphorylation and degradation, could be delivered into IEC using an adenoviral vector (Ad5 IkappaB) and determined the antiinflammatory potential of this inhibitor following different stimuli. We showed for the first time that recombinant adenovirus efficiently infected (>80%) transformed as well as primary IEC. Cytoplasmic levels of the NF-kappaB super-repressor protein were more than 50-fold higher than those of endogenous IkappaB, and this mutated IkappaB was resistant to IL-1beta-induced degradation. Immunofluorescent RelA nuclear staining was strongly inhibited in Ad5 IkappaB-infected IEC compared with control Ad5LacZ and NF-kappaB, but not AP-1 binding activity, was reduced by more than 70% as measured by electrophoretic mobility shift assay (EMSA). Induction of inducible nitric-oxide synthase (iNOS), IL-1beta, and IL-8 genes by IL-1beta, TNF-alpha, or PMA was blocked in Ad5 IkappaB-infected cells but not in Ad5 LacZ controls as assayed by RT-PCR and ELISA. In addition, IL-1beta-induced IL-8 secretion was totally inhibited by Ad5 IkappaB in primary colonic IEC. We conclude that an adenoviral vector efficiently transfers a nondegradable IkappaB in both transformed and native IEC. The strong inhibition of NF-kappaB activity and the resulting down-regulation of multiple proinflammatory molecules by Ad5 IkappaB suggests an exciting approach for in vivo intestinal gene therapy and illustrates the key role of NF-kappaB in transcriptional regulation of the inflammatory phenotype of IEC.
Asunto(s)
Inmunidad Mucosa , FN-kappa B/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/administración & dosificación , Factores de Transcripción , Adenovirus Humanos/genética , Células Cultivadas , Colon , Células Epiteliales/inmunología , Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Interleucina-1/genética , Interleucina-8/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Proteínas Recombinantes , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción ReIB , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND & AIMS: Intestinal epithelial cells constitute a barrier between host and external milieu and can play a role in signaling the influx of leukocytes during the acute mucosal inflammatory response. To further explore this role, the regulated expression of twelve C-X-C, C-C, and C-chemokines by human colon epithelial cells was characterized. METHODS: Chemokine production was assessed in HT-29 and Caco-2 human colon epithelial cells that were infected with Salmonella dublin or stimulated with interleukin 1 alpha or tumor necrosis factor alpha and in freshly isolated human colon epithelial cells by quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Expression of the neutrophil chemoattractants GRO-alpha, GRO-gamma, and interleukin 8 increased rapidly (2-3 hours) but transiently after infection or proinflammatory agonist stimulator. In contrast, expression of another neutrophil chemoattractant, epithelial cell-derived neutrophil activator 78, was delayed for 6-10 hours, and secretion continued to increase for 24 hours after infection. Among C-C chemokines known to chemoattract different leukocyte populations, monocyte chemotactic peptide 1 was rapidly expressed, whereas RANTES was up-regulated with delayed kinetics. Freshly isolated colon epithelial cells produced an array of chemokines similar to the cell lines, as well as macrophage inflammatory proteins 1 alpha and 1 beta. CONCLUSIONS: These data suggest that regulated chemokine production by epithelial cells results in temporal and spatial mucosal chemokine gradients that are important in both early and later phases of the mucosal inflammatory response.
Asunto(s)
Quimiocinas CXC , Quimiocinas/biosíntesis , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Mucosa Intestinal/inmunología , Adenocarcinoma , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CXCL1 , Factores Quimiotácticos/biosíntesis , Colon , Neoplasias del Colon , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/biosíntesis , Humanos , Inflamación , Interleucina-1/farmacología , Interleucina-8/biosíntesis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Cinética , Proteínas Inflamatorias de Macrófagos/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/farmacología , Salmonella , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The nuclear factor kappaB (NF-kappaB) regulates the transcription of genes bearing the kappaB consensus motif. Transmigration of NF-kappaB from the cytoplasm to the nucleus is regulated by the IkappaB family of inhibitory NF-kappaB-binding proteins. Dissociation of the NF-kappaB-IkappaB complex requires both phosphorylation and degradation of IkappaBs. We demonstrate that IL-1beta induces complete IkappaB alpha degradation in Caco-2 cell lines but not in HT-29, T84, SW-480 transformed cell lines, or native colonic epithelial cells. A similar lack of IkappaB alpha degradation in HT-29 cells followed TNF-alpha and bacterial polymer stimulation. IL-1beta stimulated NF-kappaB nuclear translocation and NF-kappaB-dependent IL-1beta and IL-8 expression in both Caco-2 and HT-29 cells as assayed by electrophoretic mobility shift assay, immunofluorescence, kappaB-luciferase transfection, reverse transcriptase-PCR analysis and ELISA. In HT-29 cells stimulated with IL-1beta, IkappaB alpha was phosphorylated and when cycloheximide blocked new protein synthesis, IkappaB alpha was partially degraded. NF-kappaB cytoplasmic to nuclear transmigration was incomplete and preceded IkappaB alpha degradation in 9T-29 cells, in contrast to complete coordinated NF-kappaB nuclear translocation and IkappaB alpha degradation in Caco-2 cells. Greater sensitivity of HT-29 cells to a calpain inhibitor, as measured by IL-8 secretion, suggested enhanced resistance to IkappaB alpha proteolysis. These data show that IL-1beta induces NF-kappaB activity and expression of NF-kappaB-dependent genes in colonic epithelial cells and suggest altered regulation of IkappaB alpha degradation compared with other cell lineages, which may result in their increased responsiveness to therapeutic blockade.
Asunto(s)
Colon/citología , Colon/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Línea Celular Transformada , Proteínas de Unión al ADN/biosíntesis , Epitelio/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-1/biosíntesis , Interleucina-1/farmacología , Interleucina-8/biosíntesis , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FosforilaciónRESUMEN
The intestinal mucosa is the largest surface area in the body which is continually exposed to an enormous amount of food antigens, viruses, bacteria, parasites or the by-products of these organisms. In such an antigen-loaded environment, specialized defence mechanisms must exist. There is clear evidence that the function of lymphocytes in the intestinal mucosa (IELs or LPLs) is different from that of lymphocytes of the peripheral blood, lymph node or spleen (these are antigen-free organs). The basic processes of these reactions are not completely understood. The role of differential antigen handling and presentation, and the non-random distribution of responsibilities between the professional and non-professional APC in this regard, have not been characterized. Thus, much remains to be learned about the basic mechanisms of antigen uptake, processing and presentation in the intestine which are necessary to induce an immune response. Diversity in APC function is a natural requirement for the maintenance of homeostasis in the intestine. Subpopulations of professional and non-professional APC may have been programmed to function in such a way that non-professional APCs may play a dominant role. It is anticipated that in vivo model systems will be developed and that eventually a clearer understanding will be gained in this rapidly evolving field.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Antígenos/fisiología , Intestinos/inmunología , Animales , Humanos , Intestinos/citologíaAsunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/fisiopatología , Activación de Linfocitos , Modelos BiológicosRESUMEN
Protooncogenes are cell cycle-related genes that are involved in cell growth of proliferation. Alterations in the level of expression of these genes, or expression of aberrant gene productions, have been observed in tumors and precancerous conditions. To determine if expression of these genes is altered in patients with inflammatory bowel disease (IBD) --who are at risk for development of colon cancer--we assayed transcripts of 15 protooncogenes in colonic epithelial cells of IBD patients and controls. Nine of these genes (H-ras, c-myc, c-fos, c-jun, junB, N-myc, c-abl, c-yes, and p53) were expressed in epithelial cells, whereas two (RB1 and N-ras) were not. expression of four other genes (c-src, K-ras, c-raf, and c-myb) was observed, but the intensity of these bands was too low for densitometric analysis. The steady-state levels of transcripts of H-ras and five nuclear protooncogenes (c-myc, c-fos, c-jun, junB, and N-myc) were lower in epithelial cells from involved or uninvolved IBD samples than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. The level of c-fos mRNA was two- to threefold higher in involved than in uninvolved areas of the colons of two ulcerative colitis (UC) patients, but not in one Crohn's disease (CD) patient. Message abundance of c-abl transcripts was two- to threefold lower in UC epithelial cells than in either the CD or control samples. The steady-state level of c-yes-encoded mRNA was considerably higher in IBD patients resected for colon cancer than in patients resected for active chronic IBD or in controls. The level of p53 message was constant in these samples. Increased levels of c-fos mRNA in involved UC relative to uninvolved UC may be related to the disease process. Decreased expression of c-abl transcript in UC may be a diagnostic marker for UC and may be related to the rate of cell turnover in these diseases. Enhanced expression of c-yes in IBD patients with tumors compared to active chronic IBD and controls suggests that expression of this gene may be a marker for development of colon cancer in IBD.
Asunto(s)
Colitis Ulcerosa/genética , Colon/química , Enfermedad de Crohn/genética , Proto-Oncogenes , ARN Mensajero/metabolismo , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/patología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Femenino , Expresión Génica , Humanos , Mucosa Intestinal/química , Mucosa Intestinal/patología , Masculino , Persona de Mediana EdadRESUMEN
Intestinal epithelial cells (IEC) have been shown to act as antigen-presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), IL-1 beta or TNF-alpha. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL-1) or bioassay (TNF and IL-6). Neither IL-1 beta nor TNF-alpha were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL-6 at levels comparable to those of macrophages. IEC IL-6 mRNA also increased approximately 200-fold during the first 24 h of culture. LPS, IFN-gamma or TNF-alpha had no effect on spontaneous IL-6 production, and neither resulted in the secretion of IL-1 beta or TNF-alpha. However, IL-1 beta up-regulated IL-6 synthesis by 6-7-fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL-6 expression but no detectable IL-1 beta, TGF-beta or TNF-alpha), adding to their uniqueness as APC.
Asunto(s)
Citocinas/metabolismo , Mucosa Intestinal/metabolismo , Epitelio/metabolismo , Expresión Génica , Humanos , Técnicas In Vitro , Interleucina-1/genética , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Mucosa Intestinal/inmunología , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A link between inflammation of the colon in inflammatory bowel disease (IBD) and the increased risk of colon cancer in ulcerative colitis (UC) may be provided by growth factor receptor genes. Their expression may be altered in response to growth factors present in the mucosa, and this, in turn, may induce further genetic changes, linked to carcinogenesis, in the cells of the colonic epithelium. To test this hypothesis, we assayed steady-state levels of eight growth factor receptor mRNAs in colonic epithelial cells of IBD patients and controls. Four of these genes (EGF-R, IGFI-R, CSF1-R, and PDGF-R-beta) were expressed in epithelial cells, whereas four (erbB-2, erbB-3, NGF-R, and met) were not. The level of the former in involved or uninvolved IBD was considerably lower than in normal epithelial cells from either sporadic colon cancer or diverticulitis patients. In contrast, expression was much higher in IBD patients with colon tumors than in active chronic IBD. The level of PDGF-R-beta mRNA was two- to fourfold higher in involved than in uninvolved areas of the colons of two UC patients, but not in one Crohn's disease patient. Message abundance of its ligand, PDGF-beta, however, was the same in paired UC samples. The pattern of expression of PDGF-beta and cripto was identical to that of EGF-R, whereas the level of mRNA of amphiregulin was the same in active chronic IBD and IBD patients with tumors. A fourth growth factor, Kfgf, was not expressed. Increased levels of PDGF-R-beta mRNA in involved UC relative to uninvolved UC may be related to the disease process in UC. Decreased expression of growth factor- and growth factor receptor-encoded mRNA in active chronic IBD may be related to the disease process, or it may be an effect of steroid therapy undergone by these patients. Enhanced expression of these genes in IBD patients with tumors compared to those without tumors suggests that this may be a marker for development of colon cancer in IBD.
Asunto(s)
Colitis Ulcerosa/genética , Colon/metabolismo , Neoplasias del Colon/genética , Enfermedad de Crohn/genética , ARN Mensajero/genética , Receptores de Factores de Crecimiento/genética , Adolescente , Adulto , Anciano , ADN Complementario , Femenino , Expresión Génica , Regulación de la Expresión Génica , Sustancias de Crecimiento/genética , Humanos , Immunoblotting , Masculino , Persona de Mediana EdadRESUMEN
Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.
Asunto(s)
Infecciones Bacterianas/inmunología , Enfermedades del Colon/inmunología , Citocinas/biosíntesis , Inflamación/metabolismo , Animales , Secuencia de Bases , Línea Celular , Quimiocina CCL2 , Factores Quimiotácticos/biosíntesis , Citocinas/genética , Células Epiteliales , Epitelio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Giardia lamblia , Giardiasis/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Interleucina-8/biosíntesis , Datos de Secuencia Molecular , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We have addressed the restriction elements involved in the interaction of lamina propria lymphocytes (LPL) and intestinal epithelial cells using the model of primary mixed cell culture reaction. Whereas peripheral blood T cells proliferate in response to both allogeneic non T cells and class II antigen-bearing intestinal epithelial cells (non T cells >> epithelial cells), LP T cells appear to proliferate preferentially in response to intestinal epithelial cells. The interaction between these cells does not appear to be restricted by conventional products of the major histocompatibility complex as neither monoclonal antibodies to class I nor to class II antigens inhibit the mixed cell cultures, whereas they are inhibitory in conventional mixed lymphocyte reactions. Furthermore, treatment of epithelial cells with interferon gamma fails to augment the cells' ability to induce proliferation of LPL while successfully enhancing proliferation of peripheral blood T cells in parallel cultures. Taken together, these data suggest that alternate restriction elements or mucosa-specific accessory molecules may exist on intestinal epithelial cells that are preferentially recognized by LPLs. Such a distinct regulatory network may be critical to the maintenance of immunologic homeostasis in the gut.