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.     

Ulcerative colitis--a disease characterised by the abnormal colonic epithelial cell?

The leakiness of the cell membranes of colonic epithelial cells isolated by the collagenase/Dispase technique from normal or diseased colons was assessed in a 4 h 51Cr release assay. Cells from normal, adenoma bearing or cancer bearing colons showed 51Cr release of 8% or less in almost all of 46 cell populations tested. In contrast, cells from mucosa affected by ulcerative colitis [11.9 (4.3%) n = 23] or Crohn's disease [8.4 (2.7%) n = 18] released significantly more 51Cr than the non-inflamed groups. Values are expressed as mean (SD). Overall, release values were greater in ulcerative colitis than Crohn's disease (p less than 0.01). In Crohn's disease, cells obtained from histologically inflamed mucosa released significantly more 51Cr [9.7 (2.5%) n = 11] than those from non-inflamed mucosa [6.4 (1.5%) n = 7, p less than 0.02] whereas, in ulcerative colitis, abnormal release values were found in 8 of 13 cell populations isolated from mucosa showing no histological evidence of active disease. In five patients with distal ulcerative colitis, cells from mucosa not apparently involved demonstrated normal 51Cr release in four of five studies despite abnormal release from cells from involved mucosa suggesting that a diffuse abnormality of the colonic epithelial cell is not usually present. These data indicate that chronic mucosal inflammation per se is associated with abnormalities of the colonic epithelial cell but that, in ulcerative colitis, the abnormality remains in many patients with quiescent disease. Identification of the local factors responsible for such an abnormality may contribute to an understanding of the pathogenesis of ulcerative colitis.     

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.     

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.     

Upregulation of Reg 1alpha and GW112 in the epithelium of inflamed colonic mucosa

BACKGROUND AND AIMS: Colonic epithelium is involved in the regulation of intestinal function and mucosal immune responses, and its function is altered in inflammatory bowel disease (IBD). However, a comprehensive analysis of the genetic alterations in inflamed colonic epithelium is not available at present. The aim of our study was to detect genes that are preferentially expressed in inflamed colonic epithelia and clarify the biochemical responses of epithelial cells in inflamed colonic mucosa. METHODS: cDNA representation difference analysis was used to identify candidate genes selectively expressed in inflamed colonic epithelia. Selective expression of these genes in the epithelium of inflamed colonic mucosa, including IBD and non-IBD tissues, was examined by real time polymerase chain reaction and in situ hybridisation. The effect of cell confluence and inflammatory mediators on Reg 1alpha gene expression was examined using a colon cancer cell line (HT29). RESULTS: We identified seven candidate genes that were presumed to be upregulated in the inflamed colonic epithelium. Of these, Reg 1alpha and GW112 were the dominant species and expression of these genes was confined to the crypt epithelium. In vitro studies using a colonic epithelial cell line suggested that cell confluence regulates Reg 1alpha gene expression. CONCLUSIONS: Selective expression of Reg 1alpha and GW112 genes in the crypt epithelium of inflamed colonic mucosa suggests the important regulatory functions of these genes.     

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.     

Immunohistochemical localization of vascular endothelial growth factor in colonic mucosa of patients with inflammatory bowel disease

BACKGROUND/AIMS: Significantly enhanced serum levels of VEGF (vascular endothelial growth factor) were found in patients with inflammatory bowel disease. Peripheral blood mononuclear cells have been identified as one of the origins of the circulating VEGF. The present investigation examines the localization of VEGF at the site of inflammation in colonic mucosa of patients with Crohn's disease and ulcerative colitis. METHODOLOGY: Immunohistochemical localization of VEGF and immunostaining for leukocytes were performed in colonic mucosal biopsies of 41 patients with Crohn's disease, 26 patients with ulcerative colitis and normal mucosal specimens of 5 patients with irritable bowel syndrome. Measurement of immunohistochemical staining for VEGF and for leukocytes within the epithelium and the lamina propria was performed separately by area morphometry using a computerized cell analysis system. RESULTS: In both patients with Crohn's disease and ulcerative colitis immunohistochemical staining for VEGF within the lamina propria of inflamed colonic mucosa was significantly higher compared with noninflamed mucosa (Crohn's disease: 4.26% vs. 0.07%, P < 0.001; ulcerative colitis: 3.68% vs. 0.32%, P = 0.001). There was a significant correlation between immunostaining for leukocytes and VEGF within the lamina propria in both patients with Crohn's disease (r = 0.73, P < 0.05)) and ulcerative colitis (r = 0.67, P < 0.05). In Crohn's disease immunostaining for VEGF within the epithelium was significantly higher in inflamed mucosa compared with noninflamed mucosa (9.85% vs. 0.63%, P < 0.001). In contrast, strong immunostaining for VEGF has been observed in the epithelium of noninflamed mucosa (7.60%, P < 0.003), as well as in inflamed mucosa of patients with active ulcerative colitis (9.68%, P < 0.002) compared with noninflamed mucosa of patients with inactive ulcerative colitis (1.39%). CONCLUSIONS: The present data indicate, that the increased VEGF expression within the epithelium and the interstitial accumulation of VEGF-producing leukocytes in inflamed mucosa may play an important role in the inflammatory mechanisms of Crohn's disease and ulcerative colitis.     

Prostanoid synthesis by cultured intestinal epithelial and mononuclear cells in inflammatory bowel disease.

Intestinal epithelial and mononuclear cells were isolated from normal colonic mucosa and from intestinal mucosa of inflammatory bowel disease patients. Prostanoid synthesis by primary cultures of intestinal mononuclear cells were four to six fold higher than its synthesis by primary cultures of epithelial cells. Prostaglandin E2, prostacyclin and thromboxane A2 synthesis by cultured mononuclear cells isolated from inflamed ileal mucosa of four Crohn's disease patients: 5.6 +/- 1.2; 3.2 +/- 1.9 and 2.4 +/- 1.4 (mean +/- SE) ng/1 X 10(6) cells were significantly higher than their respective synthesis by cultured mononuclear cells isolated from uninflamed ileal mucosa isolated from the same patients: 0.8 +/- 0.1; 0.3 +/- 0.1 and 0.2 +/- 0.03 ng/1 X 10(6) cells or from normal colonic mucosa: 1.5 +/- 0.3; 0.3 +/- 0.1 and 0.5 +/- 0.1 (N = 12) ng/1 X 10(6) cells. Prostanoid synthesis by primary cultures of intestinal mononuclear cells isolated from colonic mucosa of five ulcerative colitis patients was enhanced but not significantly different from its synthesis by cells isolated from normal subjects. These results suggest that the enhanced intestinal prostanoid synthesis in active Crohn's disease is derived from stimulated local mononuclear cells and may have an important role in the pathogenesis of the disease.     

Intestinal lymphokine-activated killer cells in inflammatory bowel disease

The role of non-specific cytotoxicity in the pathogenesis of inflammatory bowel disease (IBD) was investigated by assaying the natural killer (NK) and lymphokine-activated killer (LAK) cell activity of lamina propria mononuclear cells (LPMC) from 22 specimens of intestinal mucosa affected by IBD. Only minimal levels of NK activity were detected against K562 cells, as well as colon carcinoma cells, adenoma cells and fibroblasts freshly isolated from the intestinal mucosa. Culture of LPMC from IBD in the presence of interleukin-2 (IL-2) generated LAK cells that mediated high levels of activity against K562 cells and against neoplastic epithelial cells and fibroblasts derived from the intestinal mucosa. A group of 20 histologically normal specimens of intestinal mucosa showed similar levels of LAK activity against the K562 and intestinal cell targets. The minimal mucosal NK activity in IBD suggests that the cytotoxic properties of NK cells are not important in the pathogenesis of IBD. The presence of LAK precursor cells in the inflamed mucosa of IBD and their ability to lyse biologically relevant targets in vitro suggests that LAK cells have the potential to contribute to intestinal mucosal injury in IBD.     

Enhanced production of interleukin 1-beta by mononuclear cells isolated from mucosa with active ulcerative colitis of Crohn's disease.

IL1-beta production by mononuclear cells isolated from normal and active inflammatory bowel disease mucosa was studied. Significantly more IL1-beta was produced spontaneously by mononuclear cells from the inflamed mucosa compared with those from normal colonic mucosa (median 190 pg/ml (range 45-700) v 20 pg/ml (0-165)). Stimulation with lipopolysaccharide enhanced IL1-beta production by mononuclear cells from active inflammatory bowel disease mucosa but not those from normal mucosa. Depleting the mononuclear cells of macrophages, by panning with monoclonal antibody 3C10, reduced the amount of IL1-beta produced. Enhanced IL1-beta production from the inflamed mucosa may play an important role in the mediation of many inflammatory responses. The enhanced production appears to be the result of a recruited population of cells.     

Cellular basis for defective electrolyte transport in inflamed human colon

Electrolyte transport pathways in distal colonic mucosa from patients with noninflammatory and inflammatory (ulcerative colitis, Crohn's colitis) disease of the large bowel were studied in vitro with electrophysiological techniques. Noninflamed tissues exhibited substantial amiloride-sensitive electrogenic sodium transport. In contrast, inflamed but structurally intact tissues exhibited only a modest degree of electrogenic sodium transport, significant increases in total tissue conductance and apical membrane conductance, and a 100% increase in the arachidonic acid content of the cell membrane fraction of mucosal homogenates. Replacement of chloride with gluconate decreased total tissue conductance to a greater extent in inflamed than in noninflamed tissues, and total tissue conductance was higher in inflamed than in noninflamed tissues in the presence of transepithelial potassium and sodium gradients, suggesting enhanced mucosal "leakiness" to anions and cations in acute colitis. Apical addition of nystatin virtually abolished amiloride-sensitive apical sodium uptake in both groups, indicating that the ionophore formed channels in the apical membrane of noninflamed and diseased mucosa. Additional studies showed that mucosal inflammation decreased maximal activity of the basolateral sodium pump by 76%. Thus, defects in the biophysical properties of colonic epithelial cell membranes are likely to be important factors in the pathogenesis of diarrhea in ulcerative and Crohn's colitis.     

Epithelial expression of caveolin-2, but not caveolin-1, is enhanced in the inflamed mucosa of patients with ulcerative colitis

Caveolae are vesicular invaginations of the plasma membrane that act as a scaffold of the assembly of many classes of signaling molecules. Caveolins are the principal structural component of caveolae membranes, and three distinct forms of caveolins have been identified: caveolin-1, caveolin-2, and caveolin-3. In this study, we evaluated the changes in the caveolin-1 and caveolin-2 expression in the inflamed mucosa of patients with IBD. Tissue samples were obtained endoscopically from patients with ulcerative colitis (UC) (n = 18), Crohn's disease (n = 10) and ischemic colitis (n = 8). Normal colorectal tissues were also obtained (n = 15). The caveolin expression was evaluated by standard immunohistochemical procedure. In normal colonic mucosa, caveolin-1 expression was detected in the smooth-muscle cells of the muscularis mucosae and the endothelial cells, but caveolin-2 expression was not detected. In the inflamed mucosa of patients with active UC, caveolin-2 expression was clearly detectable as small scattered foci on the luminal surfaces of epithelial cells, but caveolin-1 expression was similar to that in normal mucosa. Caveolin-2 expression increased in accordance with the disease activity of UC. This enhanced caveolin-2 expression was not detected in active Crohn's disease or ischemic colitis. In conclusion, we demonstrated that the epithelial expression of caveolin-2 is markedly enhanced in the inflamed mucosa of patients with UC. It is likely that the enhanced caveolin-2 expression in patients with UC was associated with the altered signal transductions in the intestinal epithelial cells. Furthermore, our results suggest that there are differences in the phenotypic features of epithelial cells between UC and Crohn's disease.     

Elevation of interleukin-6 in inflammatory bowel disease is macrophage- and epithelial cell-dependent

Local interleukin-6 (IL-6) activity was studied using colonic mucosal tissues in inflammatory bowel disease (IBD) and inflammatory control patients. Active IBD specimens exhibited significantly higher IL-6 activity than control specimens in both cultures of isolated lamina propria mononuclear cells (LPMC) and mucosal tissues with an increased number of IL-6-producing cells. However, the activity in inactive IBD or inflammatory controls did not differ from controls. Northern blot analysis demonstrated IL-6 messenger RNA in LPMC and colonic epithelial cells isolated from active IBD specimens but not in control cells. Furthermore, immunofluorescent microscopic study of active IBD specimens showed more conspicuous staining of IL-6 in infiltrating LPMC (mostly CD68⁺ cells) and colonic epithelial cells. These results suggest that elevation of local IL-6 activity may be a characteristic feature of active IBD and both macrophages and colonic epithelial cells are the major cell types responsible for this phenomenon.     

Role of interleukin 1 in inflammatory bowel disease--enhanced production during active disease.

Interleukin 1 is a polypeptide cytokine produced by various cell types and has been shown to have a major role in inflammatory and immunological responses. In experimental colitis it proved to be a dominant mediator and a reliable marker of inflammation. The aim of the present study was to determine in vitro the extent of production and release of interleukin 1 from colonic mucosa of patients with active untreated inflammatory bowel disease. Colonic mucosal biopsy specimens were obtained during colonoscopy from 17 patients with ulcerative colitis, eight patients with Crohn's disease of the colon, and 16 normal control subjects. Interleukin 1 content was determined in fresh and 24 hour organ cultured mucosa as well as in cultured medium. Interleukin 1 content and release were significantly higher in the inflamed mucosa compared with that of control subjects. Prednisolone inhibited interleukin 1 release in a dose dependent fashion. We conclude that colonic mucosal interleukin 1 content and production is significantly raised in active inflammatory bowel disease and may have a role in the pathogenesis of the inflammatory response. Pharmacological suppression of tissue interleukin 1 production may have a beneficial therapeutic effect.     

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.     

Mucosal Expression of Interleukin-6 and Interleukin-8 Messenger RNA in Ulcerative Colitis and in Crohn's Disease

To elucidate the possible role ofproinflammatory cytokines in inflammatory bowel disease,the expression and localization of interleukin (IL)-6and IL-8 mRNAs were examined in colonic biopsy specimens obtained from 10 patients with activeulcerative colitis (UC), 5 with inactive UC, 6 withCrohn's disease (CD), and 5 normal controls. In situhybridization with digoxigenin-labeled probes andimmunohistochemistry for both cytokines were performed. The IL-6mRNA expression was enhanced in the inflamed mucosa in4 of 6 CD patients, while that of UC patients stayed atbaseline. In contrast, IL-8 mRNA expression was apparently augmented (P = 0.044) in 7 of 10active UC and 3 of 6 CD patients (NS). The cell countpositive for IL-8 mRNA per unit area was definitelyincreased in moderate/severe UC when compared to mild UC (53.1 ± 14.4/mm² vs 9.0± 5.1/mm², P = 0.028) according to thedegree of inflammation. IL-6 mRNA positive cells in CDwere preferentially located in deeper lamina propriathan IL-8 mRNA positive cells in UC. Interestingly, IL-8 mRNA wasexpressed in the mucosal epithelial cells in one UCpatient. The patients treated by corticosteroids tendedto show suppressed expression of each mRNA, except one patient with intractable UC. Our data suggestenhanced expression of mucosal IL-6 mRNA in CD and ofIL-8 mRNA in UC by infiltrating mononuclear cells,indicating the distinct participation of each cytokine in the pathogenesis of UC and CD. Moreover,intestinal epithelial cells in UC occasionally exhibitIL-8 mRNA.