William F. Doe

Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease.

Evidence of in vivo oxidant-induced injury in inflammatory bowel disease (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol redox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific probe [14C]-iodoacetamide (IAM), there was decreased labeling of a number of proteins indicating oxidation of thiol groups in CEC from inflamed mucosa compared to paired normal mucosa, especially the loss of thiol labeling of a 37-kD protein which was almost completely lost. The loss of reduced protein thiol status for the 37-kD band was paralleled by loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essential reduced cysteine (Cys149) at the active site. The identity of the 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inflammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OCl- >> ChT > H2O2 > NO (52 +/- 3, 250 +/- 17, 420 +/- 12, 779 +/- 120 microM oxidant) and OCl- >> ChT > NO > H2O2 (89 +/- 17, 256 +/- 11, 407 +/- 105, 457 +/- 75 microM oxidant), respectively, for GAPDH enzyme activity. This study provides direct evidence of in vivo oxidant injury in CEC from inflamed mucosa of IBD patients. Oxidation and inhibition of essential protein function by inflammatory cells is a potential mechanism of tissue injury that may contribute to the pathogenesis of the disease and supports the exploration of compounds with antioxidant activity as new therapies for IBD.

Depleted mucosal antioxidant defences in inflammatory bowel disease

Experimental approaches designed to define the role of reactive oxygen and nitrogen species generated by inflammatory cells in the tissue injury seen in inflammatory bowel disease rarely consider the chemical antioxidant defences against such increased oxidant stress in the mucosa. In this investigation, we have analysed components of the aqueous and lipid phase antioxidant mucosal defences by measuring the total peroxyl radical scavenging capacity and the levels of urate, glutathione, alpha-tocopherol, and ubiquinol-10 in paired noninflamed and inflamed mucosal biopsies from inflammatory bowel disease patients. Compared to paired noninflamed mucosa, decreases were observed in inflamed mucosa for total peroxyl radical scavenging capacity (55%, p = 0.0031), urate [Crohns disease (CD), 62.2%, p = 0.066; ulcerative colitis (UC), 47.3%, p = 0.031], glutathione (UC, 59%, 7/8 patients, ns), total glutathione (UC 65.2%, 6/8 patients, ns), ubiquinol-10 (CD, 75.7%, p = 0.03; UC, 90.5%, p = 0.005). The mean alpha-tocopherol content was unchanged. These observations support our earlier findings of decreased reduced and total ascorbic acid in inflamed IBD mucosa and demonstrate that the loss of chemical antioxidant defences affects almost all the major components. The decreased antioxidant defences may severely compromise the inflamed mucosa, rendering it more susceptible to oxidative tissue damage, hindering recovery of the mucosa and return of epithelial cell layer integrity. The loss of chemical antioxidant components provides a strong rational for developing novel antioxidant therapies for the treatment of inflammatory bowel disease.

Enhanced expression and production of monocyte chemoattractant protein‐1 in inflammatory bowel disease mucosa

Peripheral blood monocytes are recruited to the inflamed mucosa of inflammatory bowel disease but the specific chemotactic signals responsible for their attraction are not known. Monocyte chemoattractant protein‐1 (MCP‐1) is a chemokine with potent monocyte attracting and activating properties and the aim of this study was to examine its expression and production in inflammatory bowel disease. In situ hybridisation demonstrated mRNA for MCP‐1 in macrophages, some of which had been recently recruited from the circulation as demonstrated by their co‐expression of the monocyte marker CD14, as well as in smooth muscle and endothelial cells in inflamed mucosa. Immuno‐histochemical studies showed a corresponding distribution of MCP‐1 protein production by macrophages, smooth muscle, and endothelial cells. By contrast minimal MCP‐1 mRNA expression and protein were found in histologically normal mucosa. Macrophages isolated from surgically resected inflammatory bowel disease colons and examined by Northern analysis expressed MCP‐1 mRNA significantly more frequently (15/24 vs. 0/19, P< 0.0001) than macrophages from histologically normal mucosa from colon cancer resections. Blood monocytes stimulated by lipopolysaccharide and treated with hydrocortisone, 5‐aminosalicylic acid, or cyclosporin A showed reduced MCP‐1 expression and production in the presence of these agents. This study demonstrates increased expression of MCP‐1 mRNA and protein and cells of origin of MCP‐1 in inflamed intestinal mucosa. Together with the demonstrated influence of therapeutic agents on monocyte MCP‐1 production the findings suggest a role for MCP‐1 in monocyte attraction to the mucosal lesion of inflammatory bowel disease.

Interleukin 8: cells of origin in inflammatory bowel disease.

Neutrophils are important cellular mediators in inflammatory bowel disease (IBD). Interleukin (IL)8, a powerful neutrophil chemoattractant, is found in increased quantities in inflamed mucosa, but the cells of origin are uncertain. IL8 gene expression was studied by in situ hybridisation in uninflamed intestinal tissue resected for colon carcinoma (n = 7) and in inflamed colonic tissue resected for IBD (n = 11). Immunohistochemistry was used to assess the phenotype of IL8 expressing macrophages and the production of IL8 protein. Macrophages isolated from intestinal resections and lipopolysaccharide stimulated peripheral blood monocytes treated with 5-aminosalicylic acid, hydrocortisone, and cyclosporin A were examined for IL8 mRNA by northern blotting and IL8 secretion by enzyme linked immunosorbent assay (ELISA). In all cases IL8 mRNA was detected by in situ hybridisation in macrophages and neutrophils adjacent to ulceration in inflamed bowel, but not detected in uninflamed mucosa from carcinoma resections. Recently recruited CD14 positive macrophages were responsible for some of this IL8 expression. IL8 protein was present in the same distribution as mRNA. Epithelial cells in normal and inflamed tissue showed neither mRNA nor protein. IL8 mRNA was expressed significantly more commonly by macrophages from IBD affected than from normal mucosa, and IL8 secretion by IBD but not normal colon macrophages was augmented significantly by lipopolysaccharide treatment. IL8 expression and production by lipopolysaccharide treated blood monocytes was inhibited by the therapeutic agents tested. These results show that neutrophils and recently recruited macrophages are responsible for production of IL8 in IBD, suggesting a mechanism for a continuing cycle of neutrophil attraction. Agents used therapeutically in these diseases may be effective in part by disrupting this cycle.

Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa

Alterations in phenotype and function of intestinal macrophages occur in inflammatory bowel disease (IBD) but it is unclear whether these changes result from the recruitment of circulating monocytes to the intestine or from proliferation of resident intestinal macrophages. We sought to demonstrate the arrival of blood monocytes, the precursors of macrophages, in IBD mucosa. Peripheral blood mononuclear cells were isolated from 23 patients with clinically active intestinal inflammation (13 Crohns disease, eight ulcerative colitis, two infective colitis), then radiolabelled with 99mtechnetium (Tc)‐stannous colloid (n=13) or 111indium (In)‐oxine (n=10) before re‐injection and abdominal scanning. Four patients had demonstrable intestinal monocyte uptake using [99mTc]‐stannous colloid, while six [111In]‐oxine‐labelled monocyte scans were positive. Uptake sites correlated with actively inflamed regions. Patients demonstrating monocyte uptake had been treated with corticosteroids for a significantly (P < 0.02) shorter duration (median 3 vs 20 days) than those with negative scans. There was no significant difference between positive and negative scans for disease category, clinical or histological disease activity, or radioisotope used. Biopsies of inflamed mucosa from two patients suffering ulcerative colitis who had positive scans showed a high proportion of CD14‐positive macrophages, 4–9% of which contained autoradiographic grains. These results demonstrate that blood monocytes are recruited to the mucosa of actively inflamed bowel, and suggest that this process may be inhibited by corticosteroids. Moreover, the phenotype of the recently‐arrived monocytes indicates their susceptibility to stimulation by lipopolysaccharide, and suggests a mechanism for the continuing inflammation in the bacterial product‐rich milieu of IBD.

Enhanced Mucosal Cytokine Production in Inflammatory Bowel Disease

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohns disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.

Evidence for a CD14+ population of monocytes in inflammatory bowel disease mucosa—implications for pathogenesis

Lipopolysaccharide (LPS) is abundant in the intestinal lumen. CD14 is the receptor for the LPS‐LPS binding protein complex, and its presence on mononuclear phagocytes allows cell activation by pg/ml concentrations of LPS. We have shown that the recently recruited blood monocyte in inflammatory bowel disease mucosa is CD14+. This study examined the expression of CD14 on macrophages in inflamed (n= 13) and uninflamed (n= 7) intestine by immunohistochemistry, and on disaggregated lamina propria mononuclear cells (12 from inflamed, 17 from uninflamed intestine) and peripheral blood mononuclear cells (n= 26) by flow cytometry, using a panel of three MoAbs directed against CD14. Immunohistochemistry revealed that 3·7% of macrophages in uninflamed intestine were CD14+, while 25·1 % of macrophages in active inflammatory bowel disease expressed CD14 (P < 0·02). Flow cytometry demonstrated that CD14 expression by macrophages from Crohns disease and ulcerative colitis was augmented significantly (P= 0·02 and P= 0·01, respectively) compared with uninflamed intestine, with a discrete population of macrophages in inflammatory bowel disease, not present in normal intestine, which strongly expressed CD14. The characteristically high levels of CD14 on blood monocytes were unaffected by the presence of intestinal inflammation. Given the exposure of lamina propria cells to LPS present in the lúmen of the terminal ileum and colon, the increased numbers of CD14+ macrophages in inflammatory bowel disease may result in greatly increased production of inflammatory mediators, thereby suggesting a mechanism for the perpetuation of mucosal inflammation.

Inflammation location, but not type, determines the increase in TGF-β1 and IGF-1 expression and collagen deposition in IBD intestine

Background and AimsInflammatory bowel disease (IBD) is frequently complicated by extracellular matrix (ECM) changes that may result in fibrosis. Transforming growth factor (TGF)-&bgr;1 and insulin-like growth factor (IGF)-1 mediate numerous ECM changes. Our aim was to determine whether TGF-&bgr;1 and IGF-1 are involved in intestinal ECM collagen regulation and what impact the inflammatory infiltrate has on their expression. MethodsTGF-&bgr;1 and IGF-1 mRNA and protein were assessed in fibrosed Crohns disease (CD), inflamed CD, inflamed ulcerative colitis (UC), and control intestine using in situ hybridization and immunohistochemistry. Collagen types I and III were quantified by electron immunohistochemistry. ResultsIn CD, increased TGF-&bgr;1 and IGF-1 mRNA expression was transmural. In UC, the increase was confined to the lamina propria and submucosa. In both, distribution of TGF-&bgr;1 and IGF-1 protein matched mRNA expression and coincided with the distribution of the inflammatory infiltrate. An increase in the collagen type III:I ratio in both CD and UC also coincided with the inflammatory infiltrate. ConclusionsThese findings suggest that TGF-&bgr;1 and IGF-1 are involved in intestinal ECM remodeling in IBD, and their enhanced expression depends on the presence and location of inflammatory infiltrates rather than the type of IBD.

Altered Response of Intestinal Mucosal Fibroblasts to Profibrogenic Cytokines in Inflammatory Bowel Disease

Background and AimsFibrosis is a major complication of inflammatory bowel disease (IBD), which may be mediated by the intestinal fibroblast. Our aim was to isolate and characterize mucosal fibroblasts from histologically normal intestine (control), ulcerative colitis (UC), inflamed Crohns disease (CD), and fibrosed CD intestine. MethodsFibroblasts were characterized by light and electron microscopy and immunohistochemistry. Fibroblast collagen secretion and proliferation were determined by 3H-proline and 3H-thymidine incorporation, and the effects of exposure to interleukin (IL)-1&bgr;, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-&bgr;1, insulin-like growth factor (IGF)-1, and macrophage colony stimulating factor (M-CSF) were determined. ResultsNo difference in doubling time was observed between the fibroblast populations from UC and CD intestine. All proliferated faster than fibroblasts from control intestine. Collagen secretion from IBD fibroblasts, independent of type, was increased compared with control fibroblasts and PDGF, bFGF, and TGF-&bgr;1-induced collagen secretion from IBD fibroblasts. ConclusionsThese results suggest the presence of an activated subpopulation of fibroblasts in both UC and CD tissue irrespective of the presence of tissue fibrosis or disease type.

Isolation of Colonic Crypts That Maintain Structural and Metabolic Viability In Vitro

The aim of this study was to develop a method by which colonic epithelial cells can be isolated from resected mucosa or colonoscopic biopsy specimens and viability maintained in the short term. The principles of the technique are to digest the lamina propria from the epithelium with Dispase and collagenase, to disrupt the epithelium by trituration, and to purify the epithelial cells by seiving and differential sedimentation. Whole and partial crypts were isolated with consistently high purity of 93.5% +/- 1.2% (excluding red cells). Structural integrity was confirmed by light and electron microscopy, exclusion of trypan blue, minimal leakage of lactic dehydrogenase over 5 h (4.1% +/- 1.7%), and 51Cr leakage of less than 2% per hour over 16 h. Functional integrity was supported by continued deoxyribonucleic acid synthesis [( 3H]thymidine uptake) over 16 h and the formation of epithelial monolayer cultures on plastic. Thus, this simple method yields a highly enriched cell population that maintains high viability in vitro for at least 16 h. Such cells may be useful for the study of the biology of colonic epithelial cells.