Relationship between endogenous colony stimulating factors and apoptosis in human colon cancer cells: role of cyclo-oxygenase inhibitors.

1. Nonsteroidal anti-inflammatory drug (NSAID) usage is associated with gastrointestinal inflammatory damage and aggravation of gut inflammatory conditions. NSAIDs also exert a preventive effect against colon cancer that seems to be due to increased colon cell apoptosis. NSAIDs have been shown to modulate the release of colony stimulating factors (CSFs) in some cells. In the present study we analysed the effect of these drugs on secretion of CSFs and apoptosis in human colon epithelial cells (HT-29). 2. HT-29 cells secreted bioactive levels of GM-CSF, G-CSF and M-CSF when stimulated with IL-1ss and TNF-alpha, and diclofenac (10(-7)-10(-4) M), indomethacin (10(-7)-10(-4) M) and sodium salicylate (10(-5)-10(-2) M) induced concentration-dependent increases in GM-CSF secretion. 3. Reduced secretion of G-CSF and M-CSF and increased cell apoptosis were observed with the highest concentrations of these non-selective NSAIDs. 4. No changes in any CSF release or HT-29 cell apoptosis were detected in the presence of the COX-2 selective inhibitor DFP (10(-7)-10(-4) M). 5. Neither the exogenous addition of CSFs nor the blockade of secreted CSFs modified apoptosis in HT-29 cells stimulated with cytokines and/or NSAIDs. 6. These results suggest that colon epithelial cells can contribute to local inflammatory responses by releasing CSFs and thus extend the life span of local leukocytes. Modulation of CSF levels by non-selective NSAIDs may be involved in the pro-inflammatory effects of these agents in the gut.

The effect of treatment on lymphokine-secreting cells in the intestinal mucosa of children with Crohn's disease.

BACKGROUND: Recent studies have shown both interleukin 2 (IL-2) and interferon gamma (IFN) to be elevated in patients with active Crohn's disease compared to ulcerative colitis or non-inflammatory bowel disease controls. However the effect of treatment on these lymphokines has not been studied. PATIENTS AND METHODS: Using a reverse haemolytic plaque assay the percentage of lymphokine-secreting cells was determined in the intestinal mucosa of children with Crohn's disease before and after 8 weeks of treatment with either enteral nutrition, cyclosporin or steroids. RESULTS: Before treatment, a high percentage of cells isolated from mucosal biopsies secreted IL-2 or interferon-gamma. Eight weeks' treatment with the immunosuppressive agents cyclosporin, or with corticosteroids, produced a significant reduction in the percentage of IL-2 secreting cells, although only for the former was there also a reduction in interferon-gamma secreting cells. Enteral nutrition however, produced a reduction in lymphokine-secreting cells equivalent to cyclosporin and produced the best histological and clinical improvement. CONCLUSION: Enteral nutrition and cyclosporin can down-regulate lymphokine secretion in the gut in Crohn's disease.

Decreased mucosal interleukin-4 (IL-4) production in gut inflammation.

AIMS: To determine the prevalence of cells secreting interleukin-4 (IL-4) in the gut mucosa of children with chronic inflammatory bowel disease. METHODS: Mononuclear cells were isolated from intestinal biopsy specimens from control children (n = 10) and children with active inflammatory bowel disease (Crohn's disease n = 15, ulcerative colitis n = 9, indeterminate colitis n = 3). Spontaneous IL-4 production was then measured by SPOT-enzyme linked immunosorbant assay (ELISA) using a pair of non-competing anti-IL-4 monoclonal antibodies. The percentage of T cells in the isolated cells were also determined and the prevalence of IL-4 secreting cells calculated per 10,000 T cells. RESULTS: In control children the mean number of IL-4 secreting cells was 15.1 per 10,000 T cells. In Crohn's disease and ulcerative colitis the means were 5.3 and 5.2, respectively. In two children with indeterminate colitis numbers were also low. There was no difference in the percentage of T cells in the cell preparations isolated from each patient group. The reduction of IL-4 secreting cells in patients with Crohn's disease was not caused by steroids. CONCLUSIONS: In idiopathic inflammatory bowel disease IL-4 secreting cells are reduced in diseased mucosa.

Diarrhoeal disease: current concepts and future challenges. Intestinal cytokines in inflammatory bowel disease and invasive diarrhoea.

In the intestine large numbers of bacteria and their products are separated by a single epithelial layer from resident inflammatory cells (macrophages and lymphocytes). Many of these bacterial products, such as lipopolysaccharides and peptidoglycans, are potent stimulators of free radical and inflammatory cytokine production by macrophages. This can occur in vivo in response to mucosal invasion by enteropathogenic bacteria or because of inappropriate activation of these cells, as in chronic inflammatory bowel disease. In this review we present evidence for production of cytokines in normal intestine and in intestinal inflammatory conditions. The adverse effects of cytokine production upon intestinal homeostasis, in particular disruption of epithelial integrity and prothrombotic changes in the vascular endothelium, are also discussed.

The frequency of cells secreting interferon-gamma and interleukin-4, -5, and -10 in the blood and duodenal mucosa of children with cow's milk hypersensitivity.

Enzyme-linked immunoabsorbant spots (ELISPOTs) have been used to analyze the frequency of cells spontaneously secreting interferon-gamma (INF-gamma), IL-4, IL-5, or IL-10 in mononuclear cells isolated from the blood of children with cow's milk-sensitive enteropathy (CMSE), cow's milk allergy (CMA), and age-matched controls. In addition, cytokine profiles of duodenal lamina propria lymphocytes were compared in patients with CMSE and control subjects. In blood, spontaneous cytokine-secreting cells were uncommon, but there was significantly increased IFN-gamma, IL-4, IL-5, and IL-10 ELISPOTs in children with CMSE and CMA compared with control subjects. IL-4 ELISPOTs were significantly greater in the blood of children with CMA compared with those with CMSE. In the lamina propria the frequencies of spontaneous cytokine-secreting cells were high compared with that in blood. Significantly increased ELISPOTs for IFN-gamma and IL-4 were found in CMSE compared with controls. IL-5 ELISPOTs were unchanged, and IL-10 ELISPOTs were reduced in CMSE compared with controls. These results show a general enhancement of Th1 and Th2-type cytokine-secreting cells in the blood of children with cow's milk hypersensitivity, although the increased IL-4-secreting cells in blood in CMA may be of relevance in view of the fact that this disease is IgE-mediated. In the lamina propria, there is also enhancement of IFN-gamma- and IL-4-secreting cells in CMSE compared with control subjects; however, cells secreting IFN-gamma are 10 times more numerous than cells secreting IL-4, showing a dominance of Th1-type responses in both controls and CMSE patients.

Activation of mucosal V beta 3+ T cells and tissue damage in human small intestine by the bacterial superantigen, Staphylococcus aureus enterotoxin B.

Staphylococcus aureus enterotoxin B (SEB) was added to explants of fetal human intestine in organ culture or administered into the lumen of fetal small intestine prior to culture. Both routes produced a massive increase in lamina propria T cells expressing V beta 3, and to a lesser extent, those expressing V beta 5 and V beta 12. SEB-activated lamina propria T cells produced interleukin-2 and interferon-gamma and T cell activation was accompanied by tissue damage, which was inhibited by FK506.

Suppression of T cell-mediated injury in human gut by interleukin 10: role of matrix metalloproteinases.

BACKGROUND & AIMS: Activation of lamina propria T cells with pokeweed mitogen in human fetal gut explant cultures results in severe mucosal injury. In the same system, Staphylococcus aureus enterotoxin B produces villus atrophy and crypt cell hyperplasia. The aim of this study was to examine the ability of interleukin (IL)-10 to modify these changes. METHODS: Mucosal pathology and lamina propria glycosaminoglycans were assessed histologically, and CD3(+), CD25(+) and alpha-actin smooth muscle-positive cells were determined by immunohistochemistry. Reverse plaque assays and quantitative reverse-transcriptase polymerase chain reaction were used to analyze the cytokine response. Matrix metalloproteinase production was analyzed by Western blotting, in situ hybridization, and zymography. RESULTS: Both experimental enteropathies produced mucosal damage, although injury was greater after pokeweed mitogen activation than S. aureus enterotoxin B. In both cases, the increase in cytokine-secreting cells and transcripts observed after T-cell activation was inhibited by IL-10. IL-10 also inhibited the increase in collagenase and stromelysin-1 in the explant culture supernatants and the loss of glycosaminoglycans. IL-10 decreased metalloproteinase production in control explants and increased matrix. In mesenchymal cells, IL-10 had a minor effect on metalloproteinase production. CONCLUSIONS: IL-10 down-regulates mucosal T-cell activation, metalloproteinase production, and loss of extracellular matrix and prevents mucosal damage in the gut.

Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease.

BACKGROUND/AIMS: Cytokines are thought to be important in mediating tissue damage in inflammatory bowel disease (IBD). Many of the in vivo activities of tumor necrosis factor alpha (TNF-alpha) match the changes found in IBD, but its importance is controversial. METHODS: A sensitive, reverse hemolytic plaque assay was used to determine the frequency of TNF-alpha secreting cells isolated from mucosal biopsy specimens of children with Crohn's disease or ulcerative colitis (UC) and non-IBD controls before and after medical treatment. RESULTS: Frequency of TNF-alpha secreting cells was significantly increased in biopsy specimens from children with mild, nonspecific inflammation compared with those with histologically normal intestine. Frequency did not increase in UC compared with children with nonspecific inflammation but was significantly greater in Crohn's disease than in UC. After treatment, the frequency of TNF-alpha secreting cells was reduced in patients receiving cyclosporin A, not reduced in patients with steroids or enteral nutrition, and not changed with treatment in UC. CONCLUSIONS: TNF-alpha secreting cells are increased in the mucosa of inflamed intestine, regardless of pathogenesis. In patients with IBD, higher levels are seen in Crohn's disease than in UC, probably reflecting the extensive T-cell activation in Crohn's disease. No relation existed between histological healing and the frequency of TNF-alpha-secreting cells.