S.H. Murch

Tumour necrosis factor alpha in stool as a marker of intestinal inflammation

Measurement of disease activity in patients with inflammatory bowel disease is difficult. The best available methods are complex and time consuming, but it may be possible to use tumour necrosis factor alpha (TNF alpha) concentration in stool as a marker of disease activity. We measured TNF alpha concentrations in stool samples from normal children, infants with diarrhoea, and children with inflammatory bowel disease in active and inactive phases of the disease. In 10 normal children and 14 children with diarrhoea, median stool TNF alpha concentrations were 58 and 45 pg/g stool, respectively. Compared with diarrhoeal controls, stool TNF alpha concentrations were significantly increased in children with active Crohns disease (n = 13, median 994 pg/g, p less than 0.0002) and active ulcerative colitis (n = 4, range 276-5982 pg/g, p less than 0.003). In patients with inactive disease, either as a result of surgery or treatment with steroids, the concentration of stool TNF alpha fell to those of controls. Measurement of stool TNF alpha concentrations may provide a simple way to monitor disease activity in inflammatory bowel disease.

Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine

The spot‐ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor‐alpha (TNF‐α) and interferon‐γ (IFN‐γ), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF‐α production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF‐α‐secreting cells/106 isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohns disease (n= 9) all showed elevated frequencies of TNF‐á‐secreting cells (500–12 000 secreting cells/106 cells). In ulcerative colitis, four out of eight children had increased production of TNF‐α and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF‐α levels and the number of intestinal cells secreting TNF‐α. In controls and all groups of patients IFN‐γ‐secreting cells were uncommon. These results suggest that TNF‐α is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease.

High endothelin-1 immunoreactivity in Crohn's disease and ulcerative colitis

Both immunological hypersensitivity and vascular abnormalities have been implicated in the pathogenesis of inflammatory bowel disease. In an attempt to link the two hypotheses, we sought evidence of local production of endothelin-1, a potent vasoconstrictor, in patients with Crohns disease and ulcerative colitis. An immunohistochemical method was used to detect endothelin-1 in tissue samples from sixteen Crohns disease patients, nine ulcerative colitis patients, and thirteen controls. In the controls, positively staining cells were infrequent in both lamina propria (mean 0.9% of total cells, 95% confidence interval 0.1-1.7%) and submucosa (2.3%, 0.4-4.1%). The percentage of endothelin-immunoreactive cells was significantly higher in the two disease groups than in the controls. Among the Crohns disease patients, there were more immunoreactive cells in the submucosa than in the lamina propria (19.1%, 15.2-22.1% vs 12.3%, 8.1-16.5%; p less than 0.001), whereas the converse was true for the ulcerative colitis group (8.6%, 1.1-16.1% vs 24.4%, 14.1-34.6%; p less than 0.001). Immunoreactive macrophage aggregates around submucosal blood vessels were common in samples from Crohns disease patients. Endothelin concentrations, measured by radioimmunoassay, in supernatants of homogenised tissue samples were significantly higher in Crohns disease and ulcerative colitis than in controls. We suggest that local endothelin production by inflammatory cells may contribute to vasculitis in chronic inflammatory bowel disease by inducing intestinal ischaemia through vasoconstriction.

Disruption of sulphated glycosaminoglycans in intestinal inflammation

We have studied the distribution and nature of sulphated glycosaminoglycans (GAGs) within normal and inflamed intestine. There is increasing evidence that these negatively charged polysaccharides, which both regulate the ability of albumin to leave the vasculature and inhibit thrombosis, may be affected by inflammatory cells and their products. We obtained samples of freshly resected intestinal tissue from eight controls, eleven patients with Crohns disease, and six with ulcerative colitis. Sulphated GAGs were detected by means of a gold-conjugated poly-L-lysine probe, and the tissue density of anionic sites was assessed semiquantitatively by means of a Lennox graticule. In normal intestine there was staining in the vascular endothelium and the subepithelial basal lamina and throughout the extracellular matrix of the lamina propria and submucosa. Tissue from the patients with inflammatory bowel disease showed inflammation macroscopically and on histology. There were profound abnormalities of extracellular matrix GAGs, limited to the mucosa in ulcerative colitis and greatest in the submucosa in Crohns disease. There was also substantial loss of GAGs from the subepithelial basal lamina in both disorders and from the vascular endothelium in submucosa in Crohns disease. The extent of local GAG disruption was associated with the distribution of macrophages immunoreactive for tumour necrosis factor alpha and the activation marker RM 3/1. We suggest that inflammatory disruption of vascular and connective tissue GAGs may be an important pathogenetic mechanism, contributing to the leakage of protein and fluid, thrombosis, and tissue remodelling seen in inflammatory bowel disease.

3. Mucosal macrophages and cytokine production in the colon of children with Trichuris trichiura dysentery

Mucosal macrophages and accessory cells have been studied by immunohistochemistry in the lamina propria of the colon of children with Trichuris trichiura dysentery syndrome (TDS). No difference was found in the numbers of cells recognized by the monoclonal antibodies CD11c, CD68, or RFD7 between TDS children and local controls. However, large numbers of cells were recognized by an antibody against calprotectin (an anti-bacterial glycoprotein found in tissue infiltrating-monocytes) in TDS colonic mucosa, but few in control colon. Large numbers of cells containing tumour necrosis factor alpha (TNF alpha) were also seen in TDS mucosa; cells isolated from TDS mucosa secreted more TNF alpha than cells from control mucosa; and children with TDS had high levels of circulating TNF alpha. Non-specific macrophage-mediated inflammation and local cytokine production may therefore play a role in the pathogenesis of TDS.

Urinary growth hormone in growth-impaired children with chronic inflammatory bowel disease.

&NA; Growth impairment of undefined aetiology occurs in ˜30% of children with chronic inflammatory bowel disease. We measured urinary growth hormone concentrations in 36 children with chronic inflammatory bowel disease and 51 normal controls. The median urinary concentration of growth hormone in the stunted children with chronic inflammatory bowel disease was 15.8 ng/g creatinine (range 4.3‐32.6), compared with 11.7 ng/g creatinine (range 4.1‐35.9) in those with normal growth. The difference was statistically not significant (p = 0.15). Moreover, there was no significant difference between the patients and the control group. One stunted patient had a urinary growth hormone (UGH) concentration below the normal range, whereas four patients with normal height were also below the normal range. Four of these five patients (80%) were on corticosteroid treatment at the time of urinary collection, whereas only 26% of the patients with normal UGH were also on corticosteroid treatment. We conclude that growth retardation in children with chronic inflammatory bowel disease is probably not related to growth hormone deficiency but must involve other mechanisms.

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.