RESUMEN
BACKGROUND: In normal mucosa, intestinal lamina propria macrophages (IMACs) maintain tolerance against food antigens and the commensal bacterial flora. Several mechanisms have been identified that mediate tolerance. The ubiquitin-proteasome system (UPS) is a large multiprotein complex that degrades cellular proteins. As the UPS may modulate immune functions of IMACs, we performed a detailed investigation of UPS expression and function under normal conditions and in cells derived from patients suffering from inflammatory bowel disease (IBD). METHODS: IMACs were isolated from intestinal mucosa. mRNA expression of macrophages differentiated in vitro (i.v. MACs) and IMACs was compared by Affymetrix® oligonucleotide arrays. Quantitative Taqman-PCR was performed on five exemplary proteasomal and five ubiquitinylation genes each. Proteins were analyzed by immunohistochemistry and Western blotting. Proteasome function was assessed by a fluorimetric test. RESULTS: Affymetrix analysis showed downregulation of mRNA expression of almost all represented proteasomal and of 22 ubiquitination-associated genes in IMACs as compared to i.v. MACs and monocytes. By quantitative PCR, up to tenfold higher mRNA expression of 10 exemplary genes of the UPS (UBE2A, UBE2D2, UBE2L6, USP14, UBB and ATPase2, ß2, ß5, ß2i/MECL-1, ß5i/LMP7) was demonstrated in i.v. MACs as compared to IMACs. Immunohistochemistry and Western blots confirmed these findings in intestinal mucosa of controls and patients suffering from diverticulitis. In contrast, a significant increase in protein amounts was found in mucosa of patients with IBD. CONCLUSION: Reduced expression of subunits of the UPS in IMACs of normal mucosa supports the concept of the presence of a nonreactive, anergic macrophage phenotype in the gut under normal conditions. Reinduction in IMACs of IBD mucosa reflects activated IMACs which can present antigenic peptides and thus support inflammation.
Asunto(s)
Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Regulación hacia Abajo , Mucosa Intestinal/enzimología , Macrófagos/enzimología , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Adenosina Trifosfatasas/genética , Diferenciación Celular , Células Cultivadas , Colitis Ulcerosa/enzimología , Colon/enzimología , Enfermedad de Crohn/enzimología , Diverticulitis/enzimología , Diverticulitis/genética , Humanos , Análisis por Micromatrices , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/sangreRESUMEN
BACKGROUND: The first identified susceptibility gene for Crohn's disease, NOD2, acts as a sensor for the bacterial-wall peptidoglycan fragment muramyl dipeptide (MDP) and activates the transcription factor nuclear factor-κB (NF-κB). Upon NF-κB activation, intestinal macrophages (IMACs) induce expression of macrophage inflammatory protein (MIP)-3α to attract memory T lymphocytes. We therefore investigated the influence of NOD2 ligation of IMAC differentiation and functional MIP-3α induction. METHODS: Human embryonal kidney HEK293 cells were transfected with NOD2 wild-type (NOD2(WT)) and the NOD2 SNP13 variant (NOD2(L1007fsinsC)) and stimulated with MDP. Recruitment of CD45R0+ and Th17 cells was determined by immunohistochemistry. RESULTS: Endogenous NOD2 stimulation was followed by a dose-dependent increase in MIP-3α secretion in MONO-MAC-6 (MM6) cells. MIP-3α mRNA was also significantly (*p < 0.05) induced in HEK293 transfected with NOD2(WT) via MDP ligation. In vivo cell-cell contacts between IMACs and CD45R0+ memory T cells as well as recruitment of Th17 cells in patients of NOD2 variants were unchanged as compared to wild-type patients. CONCLUSION: Our data demonstrate a dose-dependent increase in MIP-3α secretion in the human myeloid cell line MM6 upon MDP. However, MIP-3α-driven recruitment of Th17 cells or CD45R0+ memory T lymphocytes is not affected in patients carrying heterozygous NOD2 variants.
Asunto(s)
Quimiocina CCL20/metabolismo , Enfermedad de Crohn/metabolismo , Macrófagos/metabolismo , Proteína Adaptadora de Señalización NOD2/fisiología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adolescente , Adulto , Anciano , Enfermedad de Crohn/patología , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293/efectos de los fármacos , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Macrófagos/patología , Persona de Mediana Edad , FN-kappa B/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación Transcripcional , Transfección , Adulto JovenRESUMEN
BACKGROUND: Environmental factors are thought to play an important role in the development of Crohn's disease (CD). Immune responses against auto-antigens or food antigens may be a reason for the perpetuation of inflammation. METHODS: In a pilot study, 79 CD patients and 20 healthy controls were examined for food immunoglobulin G (IgG). Thereafter, the clinical relevance of these food IgG antibodies was assessed in a double-blind cross-over study with 40 patients. Based on the IgG antibodies, a nutritional intervention was planned. The interferon (IFN)gamma secretion of T cells was measured. Eosinophil-derived neurotoxin was quantified in stool. RESULTS: The pilot study resulted in a significant difference of IgG antibodies in serum between CD patients and healthy controls. In 84 and 83% of the patients, respectively, IgG antibodies against processed cheese and yeast were detected. The daily stool frequency significantly decreased by 11% during a specific diet compared with a sham diet. Abdominal pain reduced and general well-being improved. IFNgamma secretion of T cells increased. No difference for eosinophil-derived neurotoxin in stool was detected. CONCLUSION: A nutritional intervention based on circulating IgG antibodies against food antigens showed effects with respect to stool frequency. The mechanisms by which IgG antibodies might contribute to disease activity remain to be elucidated.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Enfermedad de Crohn/dietoterapia , Enfermedad de Crohn/inmunología , Hipersensibilidad a los Alimentos/inmunología , Inmunoglobulina G/inmunología , Dolor Abdominal/fisiopatología , Adolescente , Adulto , Análisis de Varianza , Enfermedad de Crohn/sangre , Estudios Cruzados , Defecación/fisiología , Progresión de la Enfermedad , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Neurotoxina Derivada del Eosinófilo/análisis , Neurotoxina Derivada del Eosinófilo/inmunología , Heces , Femenino , Alimentos , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Probabilidad , Pronóstico , Recurrencia , Valores de Referencia , Medición de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
Haem oxygenase-1 (HO-1) up-regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up-regulation of HO-1 expression in patients with Crohn's disease (CD) and ulcerative colitis (UC) was demonstrated. A HO-1 gene promoter microsatellite (GT)(n) dinucleotide repeat polymorphism was associated with regulation of HO-1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO-1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5'-flanking region of the HO-1 gene containing the (GT)(n) dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)(n) repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short-, middle- and long-sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)(n) dinucleotide repeats of the HO-1 promotor region have no significance for the pathophysiology and disease course of IBD.