Lymphocyte subpopulations of intestinal mucosa in inflammatory bowel disease.

Lymphocyte subpopulations in peripheral blood (PBL) and intestinal mucosa (IML) of 10 patients with inflammatory bowel disease (IBD) were compared with those of 11 non-IBD controls. PBL were separated on Ficoll/hypaque gradients, and IML were isolated by incubation in dithiothreitol, EDTA, and collagenase. These methods yielded cells of good viability and with intact HLA A and B-antigens. T-cells, identified by neuraminidase-treated sheep RBC rosettes and non-specific esterase staining, comprised approximately 91% of the IML from normal mucosa of all groups. B-cells, identified by erythrocyte-antibody-complement rosettes and surface immunoglobulins, were only 7% of these IML populations. Cell yields were two-fold or more greater from abnormal IBD mucosa, with T-cells ranging from 55 to 95% and B-cells from 2 to 36%. The percentage of Fc receptor bearing cells was low in all specimens. By these methods, T-lymphocytes predominated in intestinal mucosa of both IBD and non-IBD patients, but there is marked increase in the percentage of B-cells isolated from abnormal mucosa in IBD.     

Transepithelial transport processes at the intestinal mucosa in inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) of unknown etiology. Oral absorption studies have shown an increased intestinal permeability for various sugar molecules in patients with IBD and their healthy relatives as a possible pathogenetic factor. However, the various transport pathways through the mucosal barrier have not yet been examined. This study therefore investigated whether antigens pass the epithelial barrier by a transcellular or a paracellular pathway. Mucosa of freshly resected specimens from CD (n = 10) or UC (n = 10) patients was investigated by immunoelectron microscopy and compared with healthy mucosa. Epithelial transport was studied with the antigens ovalbumin and horseradish peroxidase after defined incubation. Labeling density of subunit c of ATP synthetase was determined in mitochondria of enterocytes of all specimens. In all specimens epithelial transport of OVA and HRP was principally transcellular through enterocytes with normal ultrastructure, although some tight junctions in CD and UC were dilated. Antigens were transported within vesicles to the basolateral membrane 2.5 min after incubation. The level of enterocytes with electron-lucent cytoplasm containing a high amount of antigens was higher in CD and UC than in healthy mucosa, depending on the grade of inflammation. ATP synthetase was significantly decreased in electron-lucent cytoplasm of CD and UC to normal ultrastructure of healthy mucosa. Our study shows that ovalbumin and horseradish peroxidase taken up by the apical membrane reach the paracellular space by vesicular transport in healthy and IBD enterocytes within a few minutes. Transcellular pathway is affected in both CD and UC, which is indicated by a high level of antigens within the cytosol. We speculate that increased intestinal permeability in IBD results substantially from enhanced transcellular transport. Accepted: 4 January 1999     

Respiratory burst activity of intestinal macrophages in normal and inflammatory bowel disease.

Macrophages isolated from normal mucosa (greater than 5 cm from tumour) and inflamed mucosa (from patients with inflammatory bowel disease) of colon and ileum were studied for their ability to undergo a respiratory burst as assessed by reduction of nitroblue tetrazolium to formazan. Using phorbol myristate acetate (PMA) and opsonised zymosan as triggers, only a minority (median: 8% for zymosan and 9% for PMA) of macrophages isolated from normal colonic mucosa demonstrated release of oxygen radicals. In contrast, a significantly greater (median: 17% for zymosan and 45% for PMA) proportion of macrophages isolated from inflamed colonic mucosa were able to undergo respiratory burst. Studies with normal and inflamed ileum showed similar results. Stimulation of macrophages isolated from normal colon with interferon-gamma produced only a small increase in the proportion of cells showing release of oxygen radicals. We conclude that the respiratory burst capacity of majority of macrophages isolated from normal colon and ileum is downregulated and a greater proportion of macrophages isolated from inflamed colon and ileum are able to undergo a respiratory burst.     

Adhesion molecules in inflammatory bowel disease.

The ability of leucocytes to adhere to endothelium is essential for leucocyte migration into inflammatory sites. Some of these adhesion molecules are released from the cell surface and can be detected in serum. The soluble adhesion molecules intercellular adhesion molecule 1 (ICAM-1), E selectin, and vascular cell adhesion molecule 1 (VCAM-1) were studied in the serum of patients with Crohn's disease, ulcerative colitis, and healthy controls. A second blood sample was taken from patients with active disease after one month of treatment and a third two months after remission was achieved. Tissue expression of the same adhesion molecules was studied by immunohistology. Circulating VCAM-1 concentrations were significantly higher in patients with active ulcerative colitis (n = 11, median = 165 U/ml) compared with patients with inactive ulcerative colitis (n = 10, median = 117 U/ml, p < 0.005), active Crohn's disease (n = 12, median = 124 U/ml, p < 0.02), and controls (n = 90, median = 50 U/ml, p < 0.0001). Within each disease group there were no significant differences in E selectin or ICAM-1 concentrations between the active and inactive states, however, patients with active Crohn's disease had significantly higher ICAM-1 concentrations (n = 12, median = 273 ng/ml) than controls (n = 28, median = 168, p < 0.003). VCAM-1 concentrations fell significantly from pretreatment values to remission in active ulcerative colitis (p < 0.01). In Crohn's disease there was a significant fall in ICAM-1 both during treatment (p < 0.01) and two months after remission (p < 0.02). Vascular expression of ICAM-1 occurred more often and was more intense in inflamed tissue sections from patients with ulcerative colitis and Crohn's disease than from controls. Vascular labelling with antibody to E selectin also occurred more often in patients with active inflammatory bowel disease. In conclusion, increased circulating concentrations of selected adhesion molecules are associated with inflammatory bowel disease. There is also evidence of local upregulation, particularly of ICAM-1. Differential expression of adhesion molecules in tissue may play a part in the initiation of leucocyte migration and local inflammation; the function of circulating adhesion molecules is unknown, but may play a physiological part in blocking adhesion.     

炎症性肠病与肠黏膜免疫调节细胞

炎症性肠病(innammatory bowel disease,IBD)包括克罗恩痛(Crohn's disease,CD)和溃疡性结肠炎(ulcerative colitis,UC).其病因和发病机制尚未完全明确,免疫功能紊乱被认为是一重要因素,肠黏膜免疫调节细胞和多种细胞因子参与免疫反应和炎症过程.是当前关于IBD免疫发病机制的研究热点之一.本文仅此作一简要综述.     

Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria.

In contrast with normal subjects where IgA is the main immunoglobulin in the intestine, patients with active inflammatory bowel disease (IBD) produce high concentrations of IgG from intestinal lymphocytes, but the antigens at which these antibodies are directed are unknown. To investigate the specificities of these antibodies mucosal immunoglobulins were isolated from washings taken at endoscopy from 21 control patients with irritable bowel syndrome, 10 control patients with intestinal inflammation due to infection or ischaemia, and 51 patients with IBD: 24 Crohn's disease (CD, 15 active, nine quiescent), 27 ulcerative colitis (UC, 20 active, seven inactive). Total mucosal IgG was much higher (p < 0.001) in active UC (median 512 micrograms/ml) and active CD (256 micrograms/ml) than in irritable bowel syndrome controls (1.43 micrograms/ml), but not significantly different from controls with non-IBD intestinal inflammation (224 micrograms/ml). Mucosal IgG bound to proteins of a range of non-pathogenic commensal faecal bacteria in active CD; this was higher than in UC (p < 0.01); and both were significantly greater than controls with non-IBD intestinal inflammation (CD p < 0.001, UC p < 0.01) or IBS (p < 0.001 CD and UC). This mucosal IgG binding was shown on western blots and by enzyme linked immunosorbent assay (ELISA) to be principally directed against the bacterial cytoplasmic rather than the membrane proteins. Total mucosal IgA concentrations did not differ between IBD and controls, but the IgA titres against faecal bacteria were lower in UC than controls (p < 0.01). These experiments show that there is an exaggerated mucosal immune response particularly in active CD but also in UC directed against cytoplasmic proteins of bacteria within the intestinal lumen; this implies that in relapse of IBD there is a breakdown of tolerance to the normal commensal flora of the gut.     

Influence of inflammatory bowel disease on intestinal microflora.

The microflora of the jejunum, ileum, and colon has been studied from operative samples in Crohn's disease (n = 30), ulcerative colitis (n = 15), and controls (n = 40). There was no significant difference in the flora of patients with ulcerative colitis compared with controls. In Crohn's disease there was a significant increase in E. coli (P less than 0.001) and B. fragilis (P less than 0.001) in the ileum and of E. coli (P less than 0.001) and lactobacilli (P less than 0.01) in the colon. The abnormal ileal flora in Crohn's disease was unrelated to serological evidence of disease activity (indices: ESR, serum albumin, serum seromucoids), diameter of the ileum, or excision of the ileocaecal valve. The abnormal colonic flora in Crohn's disease was not related to presence of macroscopic colitis.     

T-cell co-stimulatory molecules: novel targets for the treatment of allergic airway disease.

The first two articles in this series discussed the fundamental concept of T-cell co-stimulation as a key event in the induction of any immune response, in addition to reviewing the current data on the role of co-stimulatory molecules for the induction and progression of allergic airway diseases. Based on these considerations, this final edition will delineate and discuss novel strategies for the prevention and/or therapy of allergic diseases based upon the modulation of co-stimulation.     

Effect of substance P on histamine secretion from gut mucosa in inflammatory bowel disease.

BACKGROUND: Mast cell hyperplasia in the gut is a feature of inflammatory bowel disease (IBD), but the role of mast cells in this disease is still unclear. Since mast cell-nerve interactions might have some impact on intestinal inflammation, the present study investigated whether the neuropeptide substance P causes histamine secretion from human gut mucosal mast cells. METHODS: Four hundred and eighteen colorectal endoscopic samples from 20 patients with IBD and 10 controls were studied. Colorectal biopsy samples were cultured in an oxygenated medium for spontaneous histamine release or were stimulated with substance P, anti-human immunoglobulin E, and a combination of substance P and anti-human immunoglobulin E. Histamine release was measured using a highly sensitive and specific radioimmunoassay. RESULTS: Substance P failed to induce mast cell activation in histologically normal mucosa from controls. In contrast, mucosal specimens taken from inflamed IBD tissue or from uninvolved Crohn disease tissue showed a considerably enhanced rate of histamine secretion towards substance P, alone or in combination with anti-IgE. Unaffected mucosa with ulcerative colitis appeared insensitive towards substance P. CONCLUSIONS: The neuropeptide substance P was shown to preferentially enhance mucosal mast cell mediator secretion in active IBD. Thus, it appears likely that mast cell-nerve interactions are involved in as yet uninvestigated neurovegetative histamine-releasing processes of the gut in IBD.     

Increased production of vascular endothelial growth factor by intestinal mucosa of patients with inflammatory bowel disease.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Recent studies have shown significantly increased VEGF serum levels in patients with active Crohn's disease and ulcerative colitis. The origin of the circulating VEGF is not yet completely described. The present investigation examines the VEGF production of colonic mucosa in consideration of mucosal disease activity in patients with inflammatory bowel disease. METHODOLOGY: Fifteen patients with inflammatory bowel disease were studied, 9 patients with Crohn's disease and 6 patients with ulcerative colitis. Biopsies were taken from endoscopically inflamed and non-inflamed colonic mucosa. Therefore, an analysis of the spontaneous VEGF production of cultured biopsies without stimulus and of the histological grade of inflammation scored on a scale of 0-3 (normal mucosa--severe chronic colitis) were performed. Eight patients with irritable bowel syndrome served as controls. VEGF levels in the supernatant of cultured mucosal biopsies were measured using an enzyme linked immunosorbent assay. RESULTS: VEGF production is expressed as pg/mg wet weight of the biopsies. Inflamed mucosa of patients with active ulcerative colitis (16.27 +/- 10.39, p = 0.003, n = 6) and active Crohn's disease (9.88 +/- 5.98, p < 0.012, n = 9) showed a significantly higher spontaneous production of VEGF by colonic mucosa than normal mucosa of controls (3.16 +/- 1.63, n = 8). In addition, there was an increased unstimulated VEGF production by cultured inflamed mucosa of patients with Crohn's disease compared with non-inflamed mucosa (3.88 +/- 3.66, p < 0.015, n = 9). In both Crohn's disease and ulcerative colitis, there was no significant difference between VEGF production by non-inflamed mucosa and normal mucosa of controls. CONCLUSIONS: The present study identifies the intestinal mucosa as one of the origins of the elevated VEGF serum levels in patients with active inflammatory bowel disease and verifies the findings of recent studies about the importance of VEGF in Crohn's disease and ulcerative colitis.     

Expression of vascular adhesion molecules in inflammatory bowel disease.

The expression of the vascular adhesion molecules ELAM-1 (endothelial leukocyte adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) was evaluated in colonic mucosa of patients with inflammatory bowel disease and normal controls by immunocytochemistry. VCAM-1 was found to be constitutively expressed in lymphoid aggregates in normal colonic mucosa and was not significantly enhanced or altered in distribution in mucosa of patients with inflammatory bowel disease regardless of the activity of the inflammatory process. In contrast, ELAM-1 was not detected by these techniques in normal colonic mucosa (n = 11) or in colonic mucosa of patients with inflammatory bowel disease which was either uninvolved or quiescent (n = 30). However, high levels of ELAM-1 were consistently found on endothelial surfaces in association with active inflammation in affected areas of colonic mucosa in patients with either ulcerative colitis (n = 27) or Crohn's colitis (n = 9). In addition, ELAM-1 appeared to be present within neutrophils which had migrated into crypt abscesses in affected mucosa. Similar analysis was carried out in the cotton-top tamarin (CTT), a primate that experiences an idiopathic chronic diffuse colitis resembling human ulcerative colitis. Although anti-human VCAM-1 antibodies did not react with the CTT, anti-human ELAM-1 stained endothelial surfaces in mucosal biopsies from CTT with active colitis. No ELAM-1 was identified in mucosa of CTT in which colitis activity was quiescent. Thus ELAM-1 is expressed on colonic endothelial surfaces in association with inflammation and may play an important role in facilitating leukocyte migration into sites of active IBD involvement.     

Targeting intestinal microflora in inflammatory bowel disease

TO THE EDITOR In their recent review article[1], Andoh and Fujiyama examined the various therapeutic approaches targeting intestinal microflora in patients with inflammatory bowel disease (IBD). I would like to provide some additional data to complete and update their comments. First of all, when considering the role of probiotics in 1BD treatment it must be emphasized that, in addition to Bifidobacteria, the Nissle 1917 E. coli strain and cocktails of microorganisms such as VSL # 3 mentioned in the article, other probiotic agents have been tested in the short- and long-term treatment of either ulcerative colitis and Crohn's disease, the results of those studies being reported in major international scientific journals.     

The intestinal microbiota and inflammatory bowel disease

The intestine hosts a complex ecosystem of microbial communities. Experimental data suggests that the microbiota has metabolic functions that contribute to nutrient and energy recovery from non-digestible substrates. Moreover, microbial colonization is essential for the normal development of the immune system and therefore seems to influence homeostasis between environmental antigen load and immune response. In genetically-susceptible individuals, an imbalance may give rise to diseases of immune dysregulation, including chronic inflammatory bowel diseases, in which there is an exaggerated immune response to harmless microbial antigens. Despite the availability of new molecular technologies, the normal composition of the human intestinal microbiota remains unknown. In the next few years, the results of international projects designed to determine the precise impact of the microbiota in various physiological and pathological processes will hopefully lead to major advances.     

T-cell co-stimulatory molecules: their role in allergic immune reactions.

The development of allergic diseases, such as allergic asthma, depends upon the initiation and maintenance of T-helper cell type-2-skewed allergen-specific immune reactions. Although it is clear that susceptibility to this process is under genetic and environmental control, the fine-tuning and regulation of the type-2 T-helper cell immune response is not yet fully understood. In this second article in the present series, current understanding regarding the involvement of T-cells and antigen-presenting cells is summarised, with emphasis on the interaction between these two types of immune regulatory cells by means of co-stimulatory molecules.     

IL-27在炎症性肠病炎症肠黏膜中的表达及意义

目的:检测克罗恩病(CD)和溃疡性结肠炎(UC)肠黏膜组织中IL-27 p28 mRNATL其蛋白、IL-27受体mRNA的表达,探讨其在CD和UC中的发病意义.方法:应用反转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹(Western blot)方法检测炎症性肠病患者炎症肠黏膜组织中IL-27 p28基因及蛋白、IL-27受体基因的表达,并与健康者作对照.结果:IL-27 p28 mRNA在CD患者中的阳性率和灰度分析表达较UC患者明显增高(X2=6.64,P<0.05;t=11.01,P<0.01),IL-27受体阳性率和灰度分析在CD患者较UC患者和健康对照者明显增高(阳性率:X2=10.91,P<0.016,X2=18.84,P<0.016).IL-27蛋白阳性率和灰度分析在CD患者中表达明显高于UC患者(X2=5.24,P<0.05;t=3.37,P<0.05),并且IL-27mRNA的表达与蛋白质表达密切相关.结论:IL-27 p28及其受体的上调,可能有助于CD患者炎症发展过程.     

肠黏膜非免疫细胞在炎症性肠病发病中的作用

肠黏膜非免疫细胞如上皮细胞、肥大细胞、血管内皮细胞和基质细胞等具有很多生物学功能,这些细胞与肠黏膜免疫细胞存在密切的相互作用,参与了肠黏膜的固有免疫和适应性免疫反应,维持肠黏膜结构和功能的稳定.炎症性肠病发病时,上述细胞间的相互作用失调,影响了肠黏膜功能的平衡,造成组织损伤和慢性炎症的发生.本文详细讨论了肠黏膜非免疫细胞的功能,及在炎症性肠病发病中的作用.     

Dendritic cells and scavenger macrophages in chronic inflammatory bowel disease.

We used enzyme (acid phosphatase [AP]) and immunohistochemical techniques and a set of monoclonal antibodies (CD11, CD5, CD4, CD19, CD8, OKIa), including two recently developed antibodies--for example, HECA-452 (specific for an adhesion molecule on high endothelial venules) and RFD1 (specific for 'active' human dendritic cells) to analyse the composition of the gut wall infiltrate of 10 well defined cases of chronic inflammatory bowel disease (CIBD) (six Crohn's disease (CD), four ulcerative colitis (UC]. Two polar forms in a spectrum of gut mononuclear phagocyte types (CD11+) were identified: at the one extreme scavenger macrophages with blunted projections (AP+, Heca-452-, RFD1-) and at the other extreme, dendritic cells with long dendritic cytoplasmic projections (AP-, Heca-452+, RFD1+). Dendritic cells were mainly found in highly organised lymphoid tissue present at the deeper layers in the gut wall (normal gut: underneath the muscularis mucosae and T-cell areas of lymph follicles [25-30 per follicle]; surrounding the broad zone of scavenger macrophages at the bottom of ulcers (CIBD) and fissures (CD) and in the lymphoid aggregates [25-30 dendritic cells per aggregate] adjacent to granulomas (CD]. These observations can be taken as evidence that exaggerated antigen handling and presentation and stimulation of the immune response takes place at these foci. The observation that scavenger macrophages were localised more superficial, as band like zones (normal gut: subepithelial; mainly surrounding ulcers (CIBD) and fissures (CD] can be taken as evidence that at these spots the ingestion and degradation of foreign material takes place.