炎症性肠病患者CD27-CD70共刺激途径的激活研究

目的 探讨CD27-CD70共刺激途径在炎症性肠病患者外周循环和肠黏膜中的表达,比较炎症性肠病患者CD27-CD70表达与正常对照者间的差异.方法 共纳入62例克罗恩病(CD)、64例溃疡性结肠炎(UC)患者和56名正常对照者.应用酶联免疫吸附试验分析炎症性肠病患者和正常对照者血浆中CD27-CD70蛋白的表达;SYBR-green实时PCR法分析炎症性肠病患者和正常对照者外周血单个核细胞中CD27-CD70 mRNA的表达;免疫组化法分析炎症性肠病患者和正常对照者肠黏膜组织CD27-CD70蛋白的表达.结果 CD和UC患者血浆中CD27-CD70的表达均显著高于正常对照者(P值均<0.05).ROC曲线的分析表明,CD27-CD70对区分CD患者和正常对照者以及区分UC患者和正常对照者具有显著的诊断价值(P值均<0.05).CD患者血浆中CD27和CD70水平与内镜下疾病活动性(EDAI)无关(r值分别=0.055和0.024,P值分别为0.673和0.852),且UC患者血浆中CD27和CD70水平与EDAI无关(r值分别=0.077和0.021,P值分别为0.547和0.869).CD患者和UC患者外周血单个核细胞CD27和CD70 mRNA表达均显著高于正常对照者外周血单个核细胞中mRNA的表达(P值均=0.000).免疫组化实验表明CD患者和UC患者肠黏膜组织CD27和CD70的表达均显著高于正常对照者(P值均=0.000).结论 炎症性肠病患者血浆、外周血单个核细胞和肠黏膜中均存在CD27-CD70途径的激活,但血浆中CD27-CD70的水平不能反映内镜下疾病活动程度.选择性干预CD27-CD70共刺激通路可能成为缓解或减轻炎症性肠病进程的有益尝试.     

Loss of interleukin-2-producing intestinal CD4+ T cells in inflammatory bowel disease.

Interleukin-2 activity of intestinal lamina propria mononuclear cells is decreased in Crohn's disease and ulcerative colitis patients compared with control patients with noninflammatory bowel disease. Factors that might be responsible for this phenomenon were investigated. Most interleukin-2 activity was produced by helper (CD4+) T cells. These were present in comparable numbers in both inflammatory bowel disease and control cultures, but the frequency of interleukin-2-producing cells was significantly (3-4 times) lower among Crohn's disease and ulcerative colitis than control cells. In agreement with this finding, levels of interleukin-2 messenger RNA were substantially decreased in both forms of inflammatory bowel disease compared with controls. Mucosal CD8+ T cells and plastic-adherent cells were unable to suppress interleukin-2 activity by autologous or allogeneic CD4+ T cells. The rate of interleukin-2 absorption was similar for inflammatory bowel disease and control cells. Induction of interleukin-2 by different stimuli (phorbol ester, phytohemagglutinin, or anti-CD3 monoclonal antibody) before or after incubation under basal conditions ("resting") failed to normalize the capacity to generate interleukin-2 by Crohn's disease and ulcerative colitis cells. Prostanoids (prostaglandin E2 and 6-keto-prostaglandin F1 alpha) were produced in large amounts in cultures of inflammatory bowel disease cells, but inhibition by indomethacin failed to restore interleukin-2 activity to control levels. Finally, supernatants from Crohn's disease and ulcerative colitis cell cultures failed to suppress interleukin-2 production by control CD4+ T cells. Our results show that the low interleukin-2 activity detected in inflammatory bowel disease mucosa is not caused by activated suppressor cells, excessive lymphokine utilization or immune stimulation, a defective response to activation signals, or production of inhibitory substances. Rather, the low interleukin-2 activity appears to be related to a loss of interleukin-2-producing mucosal CD4+ T cells. It is concluded that abnormalities of intestinal CD4+ T-cell function are associated with the immunopathogenesis of Crohn's disease and ulcerative colitis.     

Increased concentrations of interleukin 1 beta, interleukin-2, and soluble interleukin-2 receptors in endoscopical mucosal biopsy specimens with active inflammatory bowel disease.

Concentrations of interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2), and soluble IL-2 receptors (sIL-2R) were determined by enzyme linked immunosorbent assays (ELISA) in supernatants of sonicated endoscopical mucosal biopsy specimens from 31 patients with inflammatory bowel disease and 19 controls. IL-1 beta was detected in 53% of the patient supernatants (p = 0.0001), IL-2 in 35% (p = 0.0031), compared with none of the controls. Soluble IL-2R was present in 55% and 26% of the specimens, respectively (p = 0.07). The concentrations of IL-1 beta (p = 0.00015), IL-2 (p = 0.0019), and sIL-2R (p = 0.0073) were highest in the most inflamed biopsy specimens, compared with less inflamed specimens and controls. There were no significant differences in IL-1 beta, IL-2, and sIL-2R concentrations between ulcerative colitis (16) and Crohn's disease patients (15). The results suggest that enhanced cellular immunity operates in vivo at the mucosal level in active inflammatory bowel disease.     

Coexpression of CD4 and CD8 on peripheral blood T cells and lamina propria T cells in inflammatory bowel disease by two colour immunofluorescence and flow cytometric analysis.

Using two colour immunofluorescence with fluorescein isothiocyanate and phycoerythrin labelled monoclonal antibodies and multiparameter flow cytometry, we investigated the coexpression of CD4 and CD8 antigens on peripheral blood lymphocytes and lamina propria lymphocytes of patients with ulcerative colitis and Crohn's disease and normal control subjects. Both the absolute number and the proportion of peripheral blood CD4+, CD8+ cells in inflammatory bowel disease were small but significantly increased compared with those in normal control subjects. Peripheral blood lymphocytes activated with phytohaemagglutinin showed appreciably increased coexpression of CD4+, CD8+. These CD4, CD8 positive cells were large and granular. Thus the increased number of peripheral blood CD4+, CD8+ cells in inflammatory bowel disease suggests that chronic immune activation occurs not only in the active state of the disease but also in remission. The proportion of CD4+, CD8+ cells in the lamina propria was greater than in peripheral blood in normal subjects, suggesting chronic immune stimulation of the local immune system. This was also seen in patients with Crohn's disease or inactive ulcerative colitis. The proportion of CD4+, CD8+ cells was, however, significantly less in the lamina propria of patients with active ulcerative colitis. Whether this implies a possible defect in mucosal immunoregulation in active ulcerative colitis cannot be determined from these results.     

Soluble interleukin-6 receptors in inflammatory bowel disease: relation to circulating interleukin-6.

The in vivo appearance of soluble interleukin (IL)-6 receptor (sIL-6R) in serum from patients with inflammatory bowel disease was examined using an enzyme linked immunosorbent assay (ELISA). The serum sIL-6R concentrations in patients with active disease (ulcerative colitis, 148.4 (5.1); Crohn's disease, 142.3 (9.3) ng/ml; mean (SEM)) were significantly raised compared with those in patients with inactive disease (ulcerative colitis, 116.2 (7.2); Crohn's disease, 114.3 (7.1) ng/ml), some other type of colitis (104.8 (11.6) ng/ml), or in normal subjects (107.3 (2.4) ng/ml). These differences were also seen in paired samples examined during both active and inactive phases. Additionally, serum sIL-6R and IL-6 concentrations correlated significantly with C-reactive protein levels in both ulcerative colitis and Crohn's disease patients (r = 0.23 and 0.56, respectively; p < 0.05 for both). Furthermore, gel filtration analysis of serum from these patients showed two major peaks of immunoreactive IL-6-one peak corresponding to free IL-6 and another peak to sIL-6R-bound IL-6-this was further confirmed by a luminescence sandwich ELISA. These results, together with its in vitro effects, indicate that natural sIL-6R may function as a powerful enhancer of the IL-6-dependent immune processes observed in inflammatory bowel disease.     

炎症性肠病患者CD40-CD154共刺激途径的激活及其与内镜下疾病活动程度的相关性研究

目的探讨CD40-CD154共刺激途径在炎症性肠病患者外周循环和肠黏膜中的表达,比较炎症性肠病患者CD40-CD154表达和正常对照者的差异,分析CD40-CD154表达与内镜下疾病活动性的相关性。方法研究对象为克罗恩病患者62例、溃疡性结肠炎患者64例和正常对照者56例。对炎症性肠病患者和正常对照者,分别应用酶联免疫吸附试验（ELISA）、SYBR—green real time PCR方法、免疫组化法,分析血浆中、外周血单个核细胞中、肠黏膜组织中CD40-CD154的表达情况。结果克罗恩病、溃疡性结肠炎患者血浆、外周血单个核细胞及肠黏膜组织中,CD40和CD154的表达均显著高于正常对照者（P均〈0．05）,但外周循环和肠黏膜中CD40及CD154的表达和内镜下疾病活动性无关（P均〉0．05）。结论炎症性肠病患者血浆、外周血单个核细胞和肠黏膜中均存在CD40-CD154途径的激活,但CD40-CD154的高表达不能反映内镜下疾病活动程度。     

Selective resistance of mucosal T-cell activation to immunosuppression in Crohn''s disease

BACKGROUND & AIMS: The inappropriately high state of T-cell activation found in Crohn's disease could be due to failure to respond to inhibitory signals. We tested the hypothesis that Crohn's disease mucosal T-cells are resistant to the immunosuppressive action of interleukin4. PATIENTS: Patients with Crohn's disease, ulcerative colitis, and other malignant and non-malignant conditions undergoing bowel resection. METHODS: The effect of interleukin-4 on lamina propria mononuclear cells from Crohn's disease, ulcerative colitis and control mucosa was assessed on various T-cell functions: interleukin-2-induced cytotoxicity, soluble interleukin-2 receptor and interleukin-2 production, and expression of mRNA for interleukin-2R and interferon-gamma. RESULTS: Cytotoxicity of control and ulcerative colitis cells was markedly decreased by interleukin-4, whereas Crohn's disease cells failed to be inhibited. Addition of interleukin-4 to interleukin-2-stimulated cultures decreased soluble interleukin-2R production significantly less in Crohn's disease and ulcerative colitis than control cells. In the same cultures, residual levels of interleukin-2 were significantly increased in control and ulcerative colitis, but not Crohn's disease cultures. Finally, Crohn's disease cells were significantly more resistant to interleukin-4-mediated inhibition of spontaneous and interleukin-2-induced expression of interleukin-2Ralpha and interferon-gamma mRNA compared to control cells. CONCLUSIONS: The effector function, receptor expression and cytokine production of Crohn's disease mucosal T-cells are resistant to interleukin4-mediated inhibition. Failure to respond to down-regulatory signals may contribute to persistent T-cell activation and chronicity of inflammation in Crohn's disease.     

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.     

Activated platelets are the source of elevated levels of soluble CD40 ligand in the circulation of inflammatory bowel disease patients

BACKGROUND: The CD40/CD40L system, a key regulator and amplifier of immune reactivity, is activated in inflammatory bowel disease (IBD) mucosa. AIMS: To determine whether plasma levels of sCD40L are elevated in Crohn's disease (CD) and ulcerative colitis (UC) patients compared with normal controls, to investigate the cellular source of sCD40L, and to explore CD40L induction mechanisms. PATIENTS: CD, UC, and normal control subjects were studied. METHODS: The concentration of sCD40L in plasma and supernatants of freshly isolated platelets and autologous peripheral blood T cells (PBT) was measured by ELISA. Surface CD40L expression level was measured by flow cytometry in resting and thrombin activated platelets, and unstimulated and CD3/CD28 stimulated PBT before and after coculture with human intestinal microvascular endothelial cells (HIMEC). RESULTS: Compared with normal controls, plasma sCD40L levels were significantly higher in both CD and UC patients and proportional to the extent of mucosal inflammation. Platelets from IBD patients displayed a significantly higher surface CD40L expression than those from control subjects, and released greater amounts of sCD40L than autologous PBT. Contact with IL-1beta activated HIMEC induced significant upregulation of CD40L surface expression and release by platelets. CONCLUSIONS: Elevated levels of sCD40L in the circulation of IBD patients reflect enhanced surface expression and release of CD40L by platelets. This phenomenon translates to an increased platelet activation state apparently induced by passage through an inflamed mucosal microvascular bed, a conclusion supported by the positive correlation of plasma sCD40L levels with the extent of anatomical involvement by IBD. These results suggest that platelet-endothelial interactions critically contribute to activation of the CD40 pathway in IBD.     

Association between enhanced soluble CD40 ligand and prothrombotic state in inflammatory bowel disease

BACKGROUND: Inflammatory bowel disease is associated with an increased incidence of thromboembolic complications. The aim of this study was to investigate the role of the soluble CD40 ligand (sCD40L), which displays prothrombotic properties, in patients with ulcerative colitis (UC) and Crohn's disease (CD) in comparison with inflammatory and healthy controls. METHODS: Plasma levels of sCD40L, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin (TAT) complex and soluble P-selectin were measured in 104 inflammatory bowel disease patients (54 ulcerative colitis and 50 Crohn's disease), in 18 cases with other causes of intestinal inflammation and in 80 healthy controls using commercially available enzyme-linked immunosorbent assays. Plasma levels of sCD40L were correlated with disease activity, type, localization and treatment as well as with the measured thrombophilic parameters. RESULTS: CD patients had significantly higher sCD40L levels than both groups of controls (CD vs HC P < 0.001; CD vs non-IBD P < 0.05). UC patients had higher but not significantly different sCD40L levels compared with the controls. Both UC and CD patients with active disease had significantly higher sCD40L levels in comparison with patients with inactive disease. Plasma levels of sCD40L were correlated with platelet count (r = 0.27, P = 0.001). They also showed a correlation with prothrombin F1+2 (r = 0.16, r = 0.03) and TAT (r = 0.15, r = 0.04) as well as with P-selectin (r = 0.19, P = 0.01). CONCLUSIONS: The increased sCD40L plasma levels may represent, at least in some degree, a molecular link between inflammatory bowel disease and the procoagualant state.     

The importance of peripheral immune cells in inflammatory bowel disease.

Background/aims: Inappropriate down regulation of an activated immune system is considered as the main pathogenetic mechanism in inflammatory bowel disease. Migration of circulating cells to a diseased intestine is considered as an important factor in the pathogenesis of inflammatory bowel disease. We aimed to evaluate some features of circulating immune cells in inflammatory bowel disease. Methods: Twenty-two control, 29 Crohn's disease and 17 ulcerative colitis patients were studied. CD2, CD3, CD4, CD8, CD11b, CD11c, CD25, CD45RA, CD45RO, CD54 and HLA DR on the surface of peripheral blood lymphocytes and CD11b, CD11c, CD45RA and CD45RO on the phagocytes were researched with two-color immunofluorescence flow cytometry. Results: The percentages of CD2+ and CD4+ lymphocytes were found significantly reduced in ulcerative colitis. CD3+ and CD8+ lymphocytes in inflammatory bowel disease were higher than in controls. CD45RA+ lymphocytes were found significantly decreased in ulcerative colitis and active Crohn's disease. CD45RO+ lymphocytes and CD45RO+, CD11b+ and CD11c+ phagocytes were significantly increased in Crohn's disease. Conclusions: We demonstrated that there were significant differences between ulcerative colitis and Crohn's disease in the expression of some important surface markers on the peripheral blood immune cells. It seems that circulating CD11b-CD11c and CD45RA-CD45RO expressing phagocytes are important in inflammatory bowel disease and may be useful in distinguishing Crohn's disease from ulcerative colitis. These findings may give us some clues about the immunopathogenesis of inflammatory bowel disease.     

Normal inflammatory bowel disease mucosa conceals alterations in natural killer cell activity.

Non-major histocompatibility complex-restricted cytotoxicity or natural killer (NK) activity could be detected in all intestinal lamina propria mononuclear cell preparations of histologically normal mucosa from 57 patients with gastrointestinal disease. Similar levels of NK activity were detected among the different disease groups. Within the inflammatory bowel disease patient group, however, Crohn's disease patients showed a threefold higher level of NK activity than detected in ulcerative colitis patients. Cytotoxicity levels in Crohn's disease patients were also higher than in the control carcinoma patients, whereas ulcerative colitis patients had considerably lower cytotoxicity levels than the carcinoma patients. Thus, unaffected normal inflammatory bowel disease mucosa conceals alterations in NK activity which might occur before the inflammation. The colon adenocarcinoma cell line Caco-2 was found to be a representative target for detecting individual differences in NK activity of lamina propria mononuclear cells compared with standard K-562 targets. The latter can be of relevance when studying mucosal immunoregulatory mechanisms in intestinal disease.     

Depletion of immunoglobulin M memory B cells is associated with splenic hypofunction in inflammatory bowel disease

OBJECTIVES: IgM memory B cells that are responsible for the protection against infections by encapsulated bacteria, require the spleen for their generation and/or survival. Since the association between inflammatory bowel disease and functional hyposplenism is well described, our aim was to verify whether IgM memory B cells mirror the reduced splenic function in Crohn's disease and ulcerative colitis patients. METHODS: Peripheral blood samples were obtained from 32 Crohn's disease and 29 ulcerative colitis patients, 33 healthy controls, and 27 splenectomized patients. Perendoscopic intestinal biopsies were also collected from 15 of 32 Crohn's disease patients, 14 of 29 ulcerative colitis patients and 13 of 33 control subjects. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function and flow cytometry was performed to analyze both peripheral and mucosal B cells. RESULTS: Twelve of 32 Crohn's disease patients and 13 of 29 ulcerative colitis patients had pitted red cell values >4% and were considered to be hyposplenic. In inflammatory bowel disease patients circulating IgM memory B cells were significantly lower than in control subjects. We observed a significant inverse correlation between the frequency of circulating IgM memory B cell and the pitted red cell values in inflammatory bowel disease patients with hyposplenism. To exclude the possibility that the reduction of circulating IgM memory B cells reflected their recruitment in the inflamed bowel mucosa, lamina propria B-cell populations were also characterized. We found that the frequency of IgM memory B cells was similar in the blood and in the lamina propria of the same patient. CONCLUSIONS: Our findings show that peripheral IgM memory B cells are reduced in inflammatory bowel disease patients and this defect seems to be related to the impairment of splenic function.     

In vitro effects of oxpentifylline on inflammatory cytokine release in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease. AIM: To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn's disease and patients with ulcerative colitis. METHODS: PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs). RESULTS: PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn's disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants. CONCLUSIONS: PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.     

Local immune mechanisms in inflammatory bowel disease and colorectal carcinoma. Natural killer cells and their activity

Mononuclear cell (MNC) populations isolated from intestinal mucosa, mesenteric lymph nodes, and peripheral blood have been assessed for their natural killer (NK) (Leu-7+) cell proportions and NK cell activity against K-562 erythroleukemic target cells. In peripheral blood, normal proportions of Leu-7+ cells were found in patients with Crohn's disease or ulcerative colitis, whereas increased proportions in colorectal carcinoma may have been related to the higher mean age of these patients. Low proportions of Leu-7+ cells (less than 3%) were present in intestinal MNCs in Crohn's disease, ulcerative colitis, colon cancer, and miscellaneous intestinal diseases. All groups of patients had diminished NK activity of peripheral blood MNCs compared with a group of healthy controls. Intestinal NK cell activity from histologically normal mucosa correlated with autologous peripheral blood NK cell activity (p less than 0.001) but no such correlation was seen for patients with inflammatory bowel disease. Mucosal or nodal NK cell activity showed a wide range of activity but did not relate to the underlying disease, mucosal histopathology, drug therapy, or, in patients with cancer, Dukes' grading. Intestinal MNCs from all patient groups responded to stimulation with lymphoblastoid interferon, except in a small number of patients whose unstimulated activity was not detectable. In conclusion, the NK cell on intestinal mucosa behaves similarly in various intestinal diseases. However, the disparity between NK activity of autologous peripheral blood and intestinal MNCs in inflammatory bowel disease highlights the difficulty in extrapolating peripheral blood findings to mucosal immune events.     

Flow cytometric analysis of peripheral blood lymphocytes in ulcerative colitis and Crohn''s disease.

Using two colour immunofluorescence with fluorescein isothiocyanate and phycoerythrin labelled monoclonal antibodies, multi-parameter flow cytometry was used to examine the antigenic characteristics of peripheral blood lymphocytes in whole blood of patients with ulcerative colitis and Crohn's disease who were not taking immunosuppressive drugs. The numbers of CD4+ and CD8+ lymphocytes in patients with ulcerative colitis and Crohn's disease remained unchanged so that the CD4/CD8 ratio was the same as that of normal control subjects. In Crohn's disease there were many activated T cells (CD3+, CD25+). Although natural killer cells in active Crohn's disease were lower than in normal control subjects, cytotoxic T lymphocytes, as defined by CD3+, CD16+, did not differ in patients with inflammatory bowel disease compared with normal control subjects. For B cell subsets, there were differences in Leu-1+ B cells, Leu-8+ B cells, Fc epsilon R+B cells (Leu-16+, Leu-20+), and activated B cells (Leu-12+, Leu-21+) between patients with inflammatory bowel disease and normal control subjects. These differences are compatible with local activation of B cells in the inflamed colon.     

Intestinal immune reactivity to interleukin 2 differs among Crohn's disease, ulcerative colitis, and controls

When stimulated by the lymphokine interleukin 2, human intestinal mucosal mononuclear cells mediate lymphokine-activated killer cell activity. When supplied with optimal doses of exogenous interleukin 2, lamina propria mononuclear cells isolated from inflammatory bowel disease and control tissue display comparable levels of cytotoxicity in vitro. However, cultures of Crohn's disease- and ulcerative colitis-derived cells contain significantly decreased interleukin 2 activity, suggesting that in vivo the availability of interleukin 2 may be limited, perhaps resulting in impaired cytotoxic function. To test this hypothesis, lamina propria mononuclear cells from inflammatory bowel disease and control patients were stimulated to produce endogenous interleukin 2, which was then used to induce autologous lymphokine-activated killer cells. When tested against K562 and Daudi target cells, Crohn's disease cells, despite producing only one-third of the amount of interleukin 2 generated by control cells, exhibited comparable levels of cytotoxicity. In contrast, ulcerative colitis cells produced substantially less interleukin 2 and exhibited remarkably low lymphokine-activated killer cell activity. When the same cells were supplied with an amount of human recombinant interleukin 2 equivalent to the average titer found in control cultures, similar results were obtained, and Crohn's disease cells even showed a significantly greater cytolytic activity than controls. These results suggest that the observed differences in lymphokine-activated killer cell activity cannot be attributed to the level of interleukin 2 alone, and that response to this lymphokine is different among Crohn's disease, ulcerative colitis, and control intestine. In Crohn's disease, there is either an increased number of interleukin 2-responsive cells or an exacerbated reactivity to interleukin 2. In ulcerative colitis, a loss of interleukin 2-responsive cells, a hyporesponsiveness to interleukin 2, or both might be present. In conclusion, this study demonstrates that reactivity to interleukin 2 distinguishes inflammatory bowel disease from control intestinal mononuclear cells, and, under appropriate experimental conditions, it can be used to uncover abnormalities of intestinal immunity.     

Increased group II phospholipase A2 in colonic mucosa of patients with Crohn's disease and ulcerative colitis.

The immunochemical protein content of group II phospholipase A2 (PLA2) and PLA2 enzymatic activity were measured for colonic mucosal biopsy samples obtained from patients with either Crohn's disease of the colon or ulcerative colitis, and control patients without inflammatory bowel disease. Immunoreactive group II PLA2 (IR-PLA2 II) content and PLA2 activity in actively inflamed colonic mucosa of Crohn's disease patients were significantly higher than those in inactively inflamed mucosa of Crohn's disease patients and the colonic mucosa of controls. IR-PLA2 II content and PLA2 activity in severely inflamed mucosa of ulcerative colitis patients were significantly higher than those in the colonic mucosa of the controls. Mucosal PLA2 enzymatic activity was closely correlated with mucosal IR-PLA2 II content in patients with Crohn's disease and ulcerative colitis. These results suggest that an increase in PLA2 enzymatic activity in inflamed colonic mucosa of Crohn's disease and ulcerative colitis was mainly attributed to increased protein content of group II PLA2, and that an increase in mucosal group II PLA2 may be involved in the pathogenesis of intestinal inflammation of Crohn's disease and ulcerative colitis.     

Circulating Soluble Interleukin-2 Receptor α and β Chain in Inflammatory Bowel Disease

Objectives : Inflammatory bowel disease is characterized by T cell activation. Activated T cells shed interleu-kin-2 receptors (IL-2R) in a soluble form. A positive correlation between sIL-2Rα (CD25) and disease activity in inflammatory bowel disease has been shown previously, whereas IL-2Rβ (CD122) has never before been investigated in this respect. Serum from 27 patients with ulcerative colitis (UC), 31 with Crohn's disease (CD), and 29 healthy volunteers was obtained. Methods : Disease activity was scored according to a semiquantitative score for UC and by Crohn's disease activity index for CD. sIL-2Rα and -β chains were assessed by a sandwich ELISA technique using monoclonal antibodies specific for CD25 and CD122, respectively. Results : The median concentration of sIL-2Rα was 4424 pg/ml in healthy controls, 6460 in UC ( p < 0.004), and 6371 in CD ( p < 0.01). The corresponding value of sIL-2Rβ in healthy volunteers was 605 pg/ml; in active UC, significantly lower levels were found at 233 pg/ml ( p < 0.01), whereas in inactive UC, no such difference was observed at 725 pg/ml ( p > 0.05). In CD, the levels were 839 pg/ml in inactive and 920 pg/ml in active disease stages ( p > 0.05 vs controls). A positive and significant correlation existed between sIL-2R levels of α and β chains in CD ( r = 0.64; p < 0.01) but not in UC ( r = -0.32; p > 0.05) or in healthy volunteers ( r = 0.16; p > 0.05). Conclusion : Future longitudinal studies will be necessary to learn whether this newly assessed sIL-2Rβ (CD122), which may interfere with IL-15R, could be used to predict disease exacerbation and to monitor anti-inflammatory therapy in UC.     

Bacterial L-form isolation from inflammatory bowel disease patients

This study was designed to investigate a possible relationship between bacterial L forms and inflammatory bowel disease. Homogenates of intestinal mucosal biopsies from Crohn's disease, ulcerative colitis, and control patients underwent bacterial culture on hypertonic media designed for the recovery of L-form bacteria and parental organisms. L forms were recovered from 24 of 71 Crohn's disease, 51 of 121 ulcerative colitis, and 2 of 140 control biopsy specimens. These isolation rates are significantly different when Crohn's disease biopsy specimens (p less than 0.001) and ulcerative colitis biopsy specimens (p less than 0.001) are compared with controls. Six different L-form types were recovered, of which the most common were Escherichia coli and Streptococcus fecalis. No marked differences were observed in L-form recovery rates or L-form types recovered between Crohn's disease and ulcerative colitis patients. Drug treatment of inflammatory bowel disease patients did not affect L-form recovery rates or the type of L forms recovered. The results suggest either that L forms are involved in the causation of inflammatory bowel disease or that their presence in mucosal biopsy tissues is a result of the disease process.