RESUMEN
BACKGROUND: A retroendocytotic pathway for high-density lipoprotein 3 (HDL(3)) in cultured intestinal epithelial cell lines has been described. In small intestinal crypt cells and Caco-2, HDL(3) is internalized, transported to lipid droplets and, after solubilization of these lipid droplets, resecreted. In the present study we examined the mechanisms of intracellular transport of HDL(3) in the Caco-2 cell line. METHODS: Apolipoprotein E free HDL(3 )was gold-labeled for transmission electron microscopy and 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide [DiI(3)] labeled for fluorescence and confocal laser scanning microscopy. For tubulin desintegration Caco-2 cells were incubated with taxol, colchicine and beta- and gamma-lumicolchicine. Tubulin staining was performed using a FITC labeled antibody. Uptake of HDL(3) was quantified by FACS analysis. RESULTS: HDL(3) was rapidly internalized and found to be in contact with lipid droplets in the perinuclear region after 10 min. By transmission electron microscopy a frequent colocalization of HDL(3)-containing vesicles and tubular structures was demonstrated. The close association of HDL(3)-containing vesicles with fluorescence stained tubulin could be confirmed by confocal laser scanning microscopy. Preincubation of the cells with taxol and colchicine did not completely prevent internalization but reduced it during a 2-hour incubation period to less than 50% of the control cells. The transport of DiI(3)-labeled HDL(3) to the lipid droplets in the perinuclear region was almost completely blocked by taxol and colchicine. CONCLUSION: Internalization and intracellular transport of HDL(3) in intestinal epithelial cells (Caco-2) is dependent on a tubulin-mediated mechanism.
Asunto(s)
Células CACO-2/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Lipoproteínas HDL/metabolismo , Tubulina (Proteína)/fisiología , Transporte Biológico/fisiología , Células CACO-2/ultraestructura , Carbocianinas , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Endocitosis/fisiología , Células Epiteliales/ultraestructura , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Mucosa Intestinal/ultraestructura , Lipoproteínas HDL3 , Microscopía Confocal , Tubulina (Proteína)/ultraestructuraRESUMEN
BACKGROUND: There is growing evidence that intestinal epithelial cells (IECs) are involved in the mucosal immune system. AIM: To assess the pattern of cytokines secreted by IECs and lamina propria mononuclear cells (LPMNCs). To achieve this, the expression and secretion of interleukin (IL)-1, IL-1 receptor antagonist (IL-1ra), IL-6, and IL-8 in human primary colonic and ileal IECs and LPMNCs from the same patient were studied. METHODS: IECs and LPMNCs were isolated from surgical specimens or endoscopic biopsy samples. mRNA expression was investigated by northern blot analysis. Secretion of IL-1beta, IL-6, IL-8, and IL-1ra was measured by enzyme linked immunosorbent assay. RESULTS: IL-1ra mRNA levels were higher in IECs than in LPMNCs in all probands. IL-8 mRNA was only present in low amounts in the IECs from two controls. In none of the specimens were IL-1beta and IL-6 mRNA present in IECs. Transcripts encoding IL-1beta, IL-1ra, IL-6, and IL-8 were identified in LPMNC preparations of all specimens. IECs from normal mucosa produced no detectable amounts of IL-1beta or IL-6, whereas LPMNCs did. IECs secreted some IL-8 (65 (9) pg/10(5) cells) but significantly more was generated by LPMNCs (408 (43) pg/10(5) cells, p<0.0001). However, IECs secreted more IL-1ra than did LPMNCs (120 (12) v 94 (11) pg/10(5) cells). In acute inflammation, IEC IL-1ra secretion was significantly increased. A correlation between secreted IL-1ra and the macroscopical degree of inflammation was found in Crohn's disease (r = 0.64, p<0.0001, n = 36) and ulcerative colitis (r = 0. 76, p<0.0001, n = 24). CONCLUSIONS: IECs from normal mucosa express and secrete IL-1ra and low amounts of IL-8, but no IL-1 or IL-6. In inflamed mucosa the secretion of IL-1ra by IECs is slightly increased but may be not sufficient to antagonise the greatly increased production of proinflammatory cytokines by LPMNCs and the IECs themselves.
Asunto(s)
Interleucina-1/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Células Epiteliales/metabolismo , Humanos , Interleucinas/metabolismo , Mucosa Intestinal/citología , Receptores de Interleucina-1/análisis , Receptores de Interleucina-1/antagonistas & inhibidoresRESUMEN
BACKGROUND AND AIMS: Macrophages play an important role during mucosal inflammation in inflammatory bowel disease (IBD). As the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) play an integral role in the activation of T cells by antigen-presenting cells (APC) we investigated the surface expression of B7-1 and B7-2 on colonic macrophages from normal and IBD mucosa. METHODS: Intestinal macrophages were isolated from biopsies of 13 control persons and 14 patients with IBD (seven with Crohn's disease (CD); and seven with ulcerative colitis (UC)). Cells were characterized by triple fluorescence flow cytometrical analysis using CD33 as macrophage marker. RESULTS: The expression of B7-1 (CD80) (9.2% +/- 4.2%) and B7-2 (CD86) (15.1% +/- 7.3%) was low on colonic macrophages from normal mucosa, indicating only a low antigen presenting potential. However, on macrophages from IBD colon there was a significant increase in the expression of co-stimulatory molecules (CD80, 33.8% +/- 8.9%, P = 0.00005 vs. control; CD86, 39.9% +/- 8.8%, P = 0.00002). There was no significant difference between CD and UC in the expression of CD80 (CD, 31.3% +/- 6.7%; UC, 34.4% +/- 13.3%) and CD86 (CD, 41.9% +/- 3.8%; UC, 35.6% +/- 13.8%). While in normal mucosa only 10.6% +/- 4.9% of the macrophages expressed CD14, more than 90% of the CD86/CD80 positive cells of the inflamed mucosa were positive for CD14. CONCLUSION: Colonic macrophages from normal mucosa rarely express the co-stimulatory molecules CD80 and CD86. In IBD a new macrophage population is found with high expression of co-stimulatory molecules presumably responsible for the perpetuated immune response.
Asunto(s)
Antígenos CD/biosíntesis , Antígeno B7-1/biosíntesis , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Glicoproteínas de Membrana/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Antígeno B7-2 , Recuento de Células , Diferenciación Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Citometría de Flujo , Humanos , Inflamación/inmunología , Receptores de Lipopolisacáridos/biosíntesis , Macrófagos/citología , Receptores de IgG/biosíntesis , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Células TH1/inmunología , Células Th2/inmunologíaAsunto(s)
Colon/citología , Íleon/citología , Mucosa Intestinal/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular , Células Cultivadas , Colon/patología , Colonoscopía , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Humanos , Íleon/patología , Mucosa Intestinal/patología , Mucosa Intestinal/ultraestructura , Microscopía Electrónica de Rastreo , Factores de TiempoRESUMEN
Macrophages play an important role in the intestinal mucosal immune system. However, they are a poorly defined cell population. We therefore determined their phenotype in normal colonic mucosa. Macrophages were isolated from colonic biopsies and surgical specimens by collagenase digestion. Colonic macrophages were positively sorted by anti-CD33 magnetic beads. Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, CD44, CD11b, CD11c, CD64, HLA-DR, CD80, CD86 and CD3/CD19 expression. CD33 was evaluated as a positive marker for intestinal macrophages. CD33+ cells isolated from normal colonic mucosa showed co-expression of the established intracellular macrophage marker CD68 in FACS analysis. CD33+ cells were capable of phagocytosis. Isolation of this cell population by magnetic anti-CD33 beads and culture resulted in a 4.2-40-fold increase in IL-1beta and 4.5-44-fold increase in tumour necrosis factor-alpha (TNF-alpha) secretion compared with unsorted lamina propria mononuclear cells (LPMC). Of the CD33+ cells, 90.9 +/- 6.9% (mean +/- s.d.) were CD44+. However, macrophages from colonic mucosa showed only a low expression of CD14 (10.5 +/- 3.8%), CD16 (10.1 +/- 3.9%), HLA-DR (27.3 +/- 9.2%), CD11b (17.4 +/- 6.8%), CD11c (17.8 +/- 10.4%). Furthermore, expression of CD80 (9.2 +/- 4.2%) and CD86 (15.1 +/- 7.3%) was low, suggesting a low ability of normal intestinal macrophages to activate T cells and T cell-mediated immune responses. We conclude that CD33 is useful for the isolation and flow cytometric characterization of colonic macrophages. These cells exhibit a single phenotype in normal mucosa (CD33++, CD44++, CD14-, CD16-, CD11b-, CD11c-, HLA-DRlow, CD80-, CD86-) lacking lipopolysaccharide (LPS) receptor and costimulatory molecules.
Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Colon/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Células Presentadoras de Antígenos/química , Células Presentadoras de Antígenos/inmunología , Antígeno B7-2 , Biomarcadores/análisis , Separación Celular , Colon/citología , Citometría de Flujo , Humanos , Inmunidad Mucosa , Interleucina-1/metabolismo , Mucosa Intestinal/citología , Macrófagos/citología , Macrófagos/metabolismo , Glicoproteínas de Membrana/análisis , Fenotipo , Receptores Inmunológicos/análisis , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Células TH1/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Intestinal macrophages play an important role in mucosal inflammation. In normal colonic mucosa we recently demonstrated a unique macrophage phenotype with attenuated immune functions. Here we present an analysis of the alterations of the phenotype of colonic macrophages in inflammatory bowel disease (IBD). METHODS: Intestinal macrophages were isolated from biopsies of patients with IBD (n =20). Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, HLA-DR, CD44, CD11b, CD11c and CD3/CD19 expression. RESULTS: In IBD there was an increase in expression not only of CD14 compared to control mucosa (36.0% +/- 13.2% vs. 10.5% +/- 3.8%, P< 0.0001) but also of CD16 (28.6% +/- 10.3% vs. 10.1% +/- 3.9%, P< 0.0001), HLA-DR (53.1% +/- 15.9% vs. 27.3% +/- 9.2%, P< 0.0005), CD11b (42.8% +/- 14.2% vs. 17.4% +/- 6.8%, P< 0.0001) and CD11c (35.1% +/- 15.9% vs. 17.8% +/- 10.4%, P< 0.005.). Furthermore, a hitherto undescribed new population of macrophages could be detected by flow cytometry only in patients with ulcerative colitis (CD16++, CD11b++, CD14(low), CD33(low), CD11c-) accounting for 5.8% of all cells isolated. CONCLUSION: In contrast to colonic macrophages from normal mucosa, there is a significantly higher expression of CD14, CD16, HLA-DR, CD11b and CD11c in IBD, indicating additional macrophage populations in the inflamed mucosa. This may reflect either a recruitment of new cells from the circulation or a change in phenotype of resident cells.
Asunto(s)
Antígenos CD/análisis , Colitis Ulcerosa/inmunología , Colon/inmunología , Colon/patología , Enfermedad de Crohn/inmunología , Mucosa Intestinal/inmunología , Macrófagos/patología , Antígenos de Diferenciación Mielomonocítica/análisis , Biopsia , Moléculas de Adhesión Celular/análisis , Colon/química , Citometría de Flujo/métodos , Antígenos HLA-DR/análisis , Humanos , Receptores de Hialuranos/análisis , Integrina alfaXbeta2/análisis , Mucosa Intestinal/química , Receptores de Lipopolisacáridos/análisis , Antígeno de Macrófago-1/análisis , Macrófagos/química , Receptores de IgG/análisis , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
BACKGROUND: Interleukin 1 (IL-1) alpha and beta are potent cytokines which play key roles in inflammation. They are controlled by IL-1 receptor antagonist (IL-1ra). AIMS: To investigate the influence of mucosal inflammation and IL-1ra genotype on the IL-1ra:IL-1 balance. PATIENTS AND METHODS: IL-1 alpha, IL-1 beta, and IL-1ra were measured by enzyme linked immunosorbent assay (ELISA) in biopsy specimens taken from inflamed and non-inflamed colon of 60 patients with Crohn's disease (CD), 34 with ulcerative colitis (UC), 15 inflammatory controls, and 103 non-inflamed controls. IL-1ra genotype was determined by polymerase chain reaction and gel electrophoresis. RESULTS: IL-1 alpha and IL-1 beta were significantly increased in inflamed mucosa in inflammatory bowel disease (IBD) (CD: 53.5 (22.4) and 409.9 (118.7) pg/mg protein, respectively; UC: 18.9 (6.8) and 214.5 (78.2) pg/mg, respectively) and non-IBD patients (19.2 (7.4) and 281.4 (121.0) pg/mg, respectively; p < 0.0001) compared with normal controls (2.8 (0.6) and 30.6 (5.6) pg/mg, respectively). In CD IL-1 alpha and beta were also significantly increased in non-inflamed mucosa (6.1 (1.3) pg/mg and 88.7 (17.4) pg/mg, respectively; p < 0.0012). IL-1ra:(IL-1 alpha+beta) ratios were significantly decreased in inflamed mucosa of patients with CD (182 (45); p < 0.0001), UC (425 (136); p = 0.0018) and without IBD (221 (76); p < 0.0001), and in non-inflamed mucosa in CD (369 (149); p < 0.0001) compared with normal controls (1307 (245); p < 0.0001). Patients with IL-1ra genotype 2 had slightly but significantly reduced mucosal IL-1ra concentrations (p = 0.003). The greatest difference was seen in colonic biopsy specimens from patients with inflamed Crohn's disease. CONCLUSION: Mucosal inflammation can modulate the balance of the IL-1:IL-1ra system in colonic mucosa.
Asunto(s)
Colon/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucina-1/análisis , Mucosa Intestinal/inmunología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/genética , Adulto , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Electroforesis en Gel de Agar , Ensayo de Inmunoadsorción Enzimática , Femenino , Genotipo , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Reacción en Cadena de la Polimerasa , Sialoglicoproteínas/análisisRESUMEN
To test whether there is a difference in the expression of interleukin 8 (IL8) between Crohn's disease and ulcerative colitis and to determine the main site of its synthesis this study analysed IL8 in mucosal biopsy specimens of patients with Crohn's disease and ulcerative colitis by enzyme linked immunosorbent assay (ELISA) and by in situ hybridisation. IL8 was measured by ELISA in 38 normal control patients, eight inflammatory control patients, 55 Crohn's disease biopsy specimens (26 patients), and 67 ulcerative colitis biopsy specimens (35 patients). IL8 mRNA was determined in samples by in situ hybridisation using a specific IL8 RNA probe. IL8 protein was significantly increased in macroscopically inflamed specimens of Crohn's disease (median 118 pg/specimen, p < 0.0001), ulcerative colitis (median 140 pg/specimen, p < 0.001), and inflammatory controls (median 30 pg/specimen, p = 0.010) compared with normal controls (median 4 pg/specimen). IL8 was also increased in uninflamed specimens of Crohn's disease (median 46 pg/specimen, p < 0.001) but not of ulcerative colitis patients (median 9 pg/specimen, p = 0.3). IL8 protein in the mucosa correlated significantly with macroscopic inflammation in Crohn's disease (r = 0.47, p < 0.001) and in ulcerative colitis (r = 0.60, p < 0.001). IL8 mRNA was detected by in situ hybridisation in 31 of 55 biopsy specimens (56%) of Crohn's disease patients, in 38 of 67 specimens of ulcerative colitis patients (57%), in five of eight inflammatory controls (63%) and in five of 38 normal controls (13%). Mucosal IL8 mRNA expression correlated with mucosal IL8 protein (r = 0.46, p < 0.001). IL8 mRNA was only detected in inflammatory cells of the interstitium but not in mucosal epithelial cells. IL8 is produced mainly in the lamina propria of the colon in inflammatory bowel disease and correlates with mucosal inflammation.
Asunto(s)
Colitis Ulcerosa/metabolismo , Colon/metabolismo , Enfermedad de Crohn/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Adolescente , Adulto , Anciano , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismoRESUMEN
BACKGROUND/AIMS: Interleukin (IL) 8 is a major neutrophil-activating cytokine synthesized by intestinal epithelial cell lines. The aim of this study was to improve the understanding of the regulation of IL-8 synthesis by investigating the roles of protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK) in the induction of IL-8. METHODS: HT-29 cells were stimulated with IL-1 beta or tumor necrosis factor alpha (TNF-alpha) together with activators or inhibitors of PKC and PKA or with inhibitors of PTK. The presence of IL-8 protein was detected by enzyme-linked immunosorbent assay and that of IL-8 messenger RNA by Northern blotting and in situ hybridization. RESULTS: TNF-alpha and IL-1 beta dose-dependently induced IL-8 production in HT-29 cells. Activation of PKC by phorbol myristate acetate also stimulated IL-8 production; however, the effects of IL-1 beta or TNF-alpha did not require PKC, as shown by the PKC inhibitor staurosporin or PKC depletion. Stimulation of PKA by forskolin or inhibition by H89 or H7 had no influence on the synthesis of IL-8. However, induction of IL-8 by IL-1 beta or TNF-alpha was reduced by the PTK inhibitors herbimycin (by 79% or 89%, respectively) and genistein (by > 95%). CONCLUSIONS: The synthesis of IL-8 is stimulated in HT-29 cells by IL-1 beta or TNF-alpha. This stimulation is independent from PKC or PKA but depends on protein tyrosine phosphorylation.