Expression of HLA-DR antigens by colonic epithelium in inflammatory bowel disease

The expression of HLA-DR and HLA-A,B,C antigens by human colonic epithelium has been examined in tissue sections of patients with inflammatory bowel disease using an immunohistological technique. Colonic epithelial cells from all 21 control subjects with histologically normal colonic mucosa were HLA-DR-. In contrast, in nine of 13 patients with active ulcerative colitis and 11 of 12 with active Crohn's disease the epithelium of involved colonic mucosa was HLA-DR+. HLA-DR antigens were found on the epithelium of only one of six patients with ulcerative colitis in remission and one of three with inactive Crohn's disease. Moreover, these antigens were not present on the epithelium of non-inflamed colonic mucosa in two patients with Crohn's disease in whom adjacent involved mucosa showed strong epithelial reactivity. This difference between patients with active and those with inactive disease is highly significant (P less than 0.005). These findings provide further evidence of the importance of cell-mediated immune mechanisms in the pathogenesis of inflammatory bowel disease.     

Expression of interleukin-8 gene in inflammatory bowel disease is related to the histological grade of active inflammation.

Interleukin-8 (IL-8) is a potent cytokine for recruitment and activation of neutrophils. To visualize its distribution in the intestinal mucosa and to understand better its possible role in the induction and promotion of inflammatory bowel disease, expression of the IL-8 gene was analyzed in resected bowel segments of 14 patients with active Crohn's disease or ulcerative colitis. In situ hybridization with IL-8 anti-sense RNA probes revealed strong and specific signals in the histologically affected mucosa. The number of cells expressing IL-8 gene correlated with the histological grade of active inflammation. In accordance with the characteristic histological signs of active disease, IL-8-expressing cells were diffusely distributed over the entire affected mucosa in patients with ulcerative colitis, whereas in patients with Crohn's disease, IL-8-expressing cells showed a focal distribution pattern. Cells expressing IL-8 were mainly located at the base of ulcers, in inflammatory exudates on mucosal surfaces, in crypt abscesses, and at the border of fistulae. Analysis of semi-serial sections pointed to macrophages, neutrophils, and epithelial cells as possible sources of this cytokine in active inflammatory bowel disease. We consistently failed to detect IL-8 messenger RNA in the mucosa of uninvolved bowel segments and in normal-appearing control mucosa of patients with colon cancer. In contrast, tissue specimens from two patients with acute appendicitis displayed IL-8-expressing cells in the mucosa. These results support the notion that IL-8 plays and important but nonspecific role in the pathogenesis of inflammatory bowel disease and that the production of IL-8 messenger RNA is restricted to areas with histological signs of inflammatory activity and mucosal destruction.     

Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease.

The perpetuation of inflammation in ulcerative colitis and Crohn's disease may be regulated in part by an increased secretion of proinflammatory cytokines due to either an appropriate response to initial stimulating agents, and/or due to an impaired down-regulation of cytokine secretion. The aim of this study was to determine the secretion patterns of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-1 beta, from isolated lamina propria mononuclear cells (LPMNC) isolated from colonic biopsies from patients with untreated ulcerative colitis or Crohn's disease. LPMNC isolated from involved inflammatory bowel disease (IBD) mucosa spontaneously produced increased amounts of TNF-alpha, and IL-6, and IL-1 beta. The TNF-alpha secretion from IBD LPMNC could be further enhanced by pokeweed mitogen stimulation. The secretion patterns of TNF-alpha and IL-1 beta by LPMNC from patients with either ulcerative colitis or Crohn's disease demonstrated a close correlation with the degree of tissue involvement and mucosal inflammation. LPMNC from non-involved ulcerative colitis mucosa secreted markedly increased levels of IL-6 compared with non-involved Crohn's disease mucosa or control mucosa. The heightened IL-6 secretion from LPMNC from non-involved ulcerative colitis mucosa without visible or microscopic signs of inflammation indicates that the pathophysiologic mechanisms involved in the initiation of inflammation may differ between ulcerative colitis and Crohn's disease. The determination of proinflammatory cytokine secretion by isolated LPMNC from colonoscopic biopsies may be a sensitive method for monitoring the severity of mucosal inflammation in IBD patients.     

Cyclooxygenase‐2 immunoreactivity in collagenous colitis

Collagenous colitis (CC) is an inflammatory bowel disease of unknown aetiology and pathogenesis. In ulcerative colitis and Crohn's disease, prostaglandins may be involved in the pathogenesis of inflammation, and increased expression of cyclo‐oxygenase‐2 (COX‐2) has been detected. The purpose of this study was to examine the presence and cellular localization of COX‐2 in colonic mucosa of patients with CC. Using immunohistochemistry, immunoflouresence and Western blot analysis, COX‐2 expression was evaluated in colonic mucosal biopsies from 10 patients with active untreated CC, and compared with samples from eight normal controls, and samples from eight patients with ulcerative colitis or Crohn's disease. Specimens from patients with CC expressed COX‐2 protein in increased amounts compared with controls, but similar to patients with ulcerative colitis and Crohn's disease. COX‐2 expression was localized to the mononuclear cells of the lamina propria. COX‐2 expression was most evident in macrophages. Co‐localization of COX‐2 and macrophages was increased in number in comparison with controls. In conclusion COX‐2 is expressed in increased amounts primarily in the macrophage subpopulation of the inflammatory infiltrate of lamina propria in CC. Increased recruitment of macrophages, increased expression of COX‐2 and increased prostaglandin synthesis may be involved in the pathogenesis of CC.     

Chemokines in the Inflammatory Bowel Diseases

Ulcerative colitis and Crohn's disease are characterized by chronic intestinal inflammation. Intestinal bacteria initiate the activation of intestinal inflammatory processes, which are mediated by proinflammatory cytokines and chemokines. In inflammatory bowel disease, intestinal inflammation is not downregulated, in part due to defective or absent inhibitory processes. Studies to date have demonstrated that IL-8, MCP-1, and ENA-78 are highly expressed in the intestinal mucosa in areas of active Crohn's disease and ulcerative colitis. Neutrophils and macrophages in the inflamed intestine synthesize and secrete large amounts of chemokines in patients with inflammatory bowel disease. Increased chemokine expression has also been observed in epithelial cells, endothelial cells, and smooth muscle cells. Future trials of specific agents capable of inhibiting chemokine synthesis and secretion or blocking chemokine–chemokine receptor interaction will be important to study in patients with ulcerative colitis and Crohn's disease.     

Granulomatous ulcerative colitis: a re-appraisal of the mucosal granuloma in the distinction of Crohn's disease from ulcerative colitis

AIMS: To determine whether the presence and location of giant cells or granulomas in relation to crypts distinguishes between ulcerative colitis and Crohn's disease. METHODS AND RESULTS: Twenty-nine large bowel mucosal biopsy specimens showing giant cells and/or granulomas in a background more typical of ulcerative colitis than Crohn's disease were collected between 1986 and 1996. Each was subject to detailed independent analysis by three histopathologists. Follow-up of the cases was by examination of all previous and subsequent gastrointestinal surgical or biopsy material and by scrutiny of the clinical notes by a gastroenterologist. On the basis of the accumulated histological data 10 of these 29 cases were accorded the diagnosis of ulcerative colitis. In nine of these 10 cases the clinical diagnosis, where known, was in keeping with this and all nine contained only crypt-associated giant cells and/or granulomas. The tenth case contained a solitary free-standing granuloma and clinically the patient had perianal disease, suggesting that the true diagnosis was Crohn's disease. CONCLUSIONS: Isolated giant cells and well-defined epithelioid granulomas distant from crypts do not, as a rule, occur in ulcerative colitis, and hence their presence in a colonoscopic biopsy showing features of chronic inflammatory bowel disease is a strong pointer towards the diagnosis of Crohn's disease. Crypt-associated giant cells and granulomas can occur in ulcerative colitis and in themselves are unreliable features for the discrimination between Crohn's disease and ulcerative colitis.     

Intraepithelial and lamina propria leucocyte subsets in inflammatory bowel disease: an immunohistochemical study of colon and rectal biopsy specimens.

AIMS--To gain new insights into the pathogenesis and differential diagnosis of ulcerative colitis and colonic Crohn's disease. METHODS--Immunohistochemistry for different leucocyte subsets was performed in biopsy specimens of the sigmoid colon and rectum from 55 patients with inflammatory bowel disease and 11 healthy controls. RESULTS--Colonic biopsy specimens from patients with active ulcerative colitis had significantly higher numbers of CD45+ and CD3+ leucocytes compared with those from patients with inactive disease, and higher numbers of total leucocytes and macrophages than those from patients with Crohn's disease. Rectal biopsy specimens from patients with Crohn's disease had greater numbers of intraepithelial leucocytes (CD45, CD3 and CD8 cells) than specimens from patients with active or inactive ulcerative colitis, or from healthy controls. CONCLUSIONS--Because of the phenotypic differences in the inflammatory infiltrate in the mucosa from the sigmoid colon and the rectum, the segment of the intestine to be biopsied should be specified. Assessment of the leucocytic component of the intraepithelial infiltrate in rectal biopsy specimens was more useful than examination of colonic biopsy specimens in the differential diagnosis of ulcerative colitis and Crohn's disease.     

Value of counting colonic mucosal Ig-containing cells in the differential diagnosis of chronic inflammatory bowel disease.

AIMS: To investigate whether counting cells containing immunoglobulin (Ig) subclass in colonic biopsy specimens of patients with chronic inflammatory bowel disease, in addition to conventional histological evaluation, can improve the differentiation of patients with Crohn's disease from those with ulcerative colitis. METHODS: The colonic and rectal biopsy specimens of 40 patients with chronic inflammatory bowel disease, comprising 20 patients with Crohn's disease and 20 with ulcerative colitis, were used and sections were stained specifically for IgA, IgM, and IgG heavy chains using an indirect immune peroxidase method. The immunoglobulin subclass containing cells were counted using an ocular grid counting method in a light microscope. A linear stepwise discriminant analysis was performed on Ig subclass containing cell counts in combination with 16 reproducible histological features. The results of this discriminant analysis were compared with the results of the discriminant analyses in which only the histological features were used. RESULTS: Applying stepwise discriminant analysis, two histological features (an excess of histiocytes in the lamina propria and the villous or irregular aspect of the mucosal surface) in combination with IgMax were selected as the most discriminatory parameters that distinguish Crohn's disease from ulcerative colitis. IgMmax was defined as the maximum value of the mean percentage of IgM containing cells over all the biopsy locations. The use of this combination resulted in a better classification in 20% of the patients with Crohn's disease and in 9% of the patients with ulcerative colitis compared with the use of histological features alone. CONCLUSIONS: Morphometric enumeration of Ig subclass containing cells in colonic mucosal biopsy specimens has diagnostic value as a means of differentiating individual patients with Crohn's disease from those with ulcerative colitis.     

Chemokine expression in IBD. Mucosal chemokine expression is unselectively increased in both ulcerative colitis and Crohn's disease

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by prominent cellular infiltrates in the bowel wall. Chemokines are chemotactic cytokines that are able to promote leukocyte migration to areas of inflammation and are also able to initiate cell activation events. They have recently been implicated in the pathophysiology of many disease states. The aim of this study was to detail the degree and distribution of specific chemokines, interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, -2, and -3, and macrophage inflammatory protein (MIP)-1alpha and -1beta, in IBD mucosa. Thirty-nine patients were included, ten controls, 20 ulcerative colitis (UC), and nine Crohn's disease (CD), with a range of disease activity. Colonic mucosal biopsies were collected from UC, CD, and control patients and embedded in glycol methacrylate. Two-micrometre-thick sections were cut and stained using immunohistochemistry for chemokine protein expression. Sections were analysed using a light microscope. Expression of all types of chemokine protein was detected in colonic mucosa from both control and IBD patients. Patterns of staining between IBD patients and controls differed significantly, but CD and UC patients demonstrated similar patterns of staining. Individual chemokine expression was found to be significantly up-regulated in IBD when patients were compared with the non-diseased group in all areas of the mucosal sections. Up-regulated chemokine expression correlated with increasing activity of the disease. It is concluded that human colonic chemokine expression is non-selectively up-regulated in IBD. The results supported the hypothesis that the degree of local inflammation and tissue damage in UC and CD is dependent on local expression of specific chemokines within IBD tissues.     

Immunohistological differential diagnosis of inflammatory colonic diseases

Immunohistological investigations were carried out on human colonic tissue from, I healthy mucosa, 2 slightly inflamed mucosa, 3 mucosa with ulcerative colitis, 4 mucosa with Crohn's colitis, using antibodies against immunoglobulins and complement components. All our antibodies, including F(ab')2 fragments, demonstrated a progressive increase of labelled cells from healthy mucosa through slightly inflamed mucosa to mucosa with ulcerative colitis, in contrast to a complete absence of labelled cells in cases of Crohn's disease. The results are discussed with regard to their pathogenesis and their clinical significance for the differentiation of ulcerative colitis and Crohn's colitis.     

Intramucosal ganglion cells are common in diverticular disease

AIMS: Ganglion cells were thought not to occur within the mucosa of the normal colon and found only in the setting of inflammatory bowel disease and neuronal intestinal dysplasia. The aim of this study was to firmly establish the incidence of intramucosal ganglion cells in diverticular disease, normal mucosa and in a spectrum of gastrointestinal diseases. METHODS: We retrospectively reviewed 50 resection specimens from cases of symptomatic diverticular disease and biopsies and/or resection specimens for several neoplastic and non-neoplastic gastrointestinal diseases (50 normal and 120 cases for a variety of gastrointestinal diseases). Normal cases were constituted by biopsies with no clinical history of large bowel disease and no pathology detected microscopically. RESULTS: All 50 cases of diverticular disease contained intramucosal ganglion cells, located within the muscularis mucosae (49/50 cases) as well as within the lamina propria in nine cases. Intramucosal ganglion cells occurred throughout the colorectum within the muscularis mucosae or lamina propria in normal mucosa in 11 cases and in a further 26 colorectal specimens with Crohn's disease (11/20), ulcerative colitis (11/20), adenocarcinoma (1/20), tubular adenoma (2/20), and mucosal prolapse (1/20). None of the 20 hyperplastic polyps contained intramucosal ganglion cells. CONCLUSIONS: We have firmly established the existence of the intramucosal ganglion cells in normal and diseased colorectum, especially in the mucosa of cases of diverticular disease (100% of cases), Crohn's disease and ulcerative colitis. These three conditions are linked by motility abnormalities which may underlie the reason for the presence of intramucosal ganglion cells.     

The rectal biopsy appearances in Salmonella colitis

Rectal biopsies were examined from 22 patients with Salmonella infection of food-poisoning type and from seven patients with inflammatory bowel disease and coincidental Salmonella infection. In the former group the changes observed were mucosal oedema with acute inflammation of varying severity but with preservation of the crypt architecture. Crypt abscesses were present in a few cases but were usually localized in the crypt and mucus depletion only occurred with severe inflammation. These features are not specific and are similar to those seen in other types of infective colitis such as Shigella dysentery, gonococcal proctitis and amoebic colitis. In the majority of cases of infective colitis the appearances are usually sufficiently distinctive, however, to distinguish them from those seen in ulcerative colitis and Crohn's disease. The changes in the biopsies from the seven patients with coincidental Salmonella infection were in general those of the underlying idiopathic inflammatory bowel disease.     

Enhanced formation of sulfidopeptide-leukotrienes in ulcerative colitis and Crohn's disease: inhibition by sulfasalazine and 5-aminosalicylic acid

Release of sulfidopeptide (SP)-leukotrienes (LT)in vitro from normal human colonic mucosa and from mucosal tissue obtained from patients with Crohn's disease (CD) and ulcerative colitis (UC) was investigated. It was found that inflamed mucosal tissue released significantly more SP-LT than normal colonic mucosa both under control conditions and after addition of calcium ionophore A23187. These results indicate the presence of endogenous stimuli as well as an increased responsiveness to an exogenous stimulus of LT formation in the inflamed mucosa. Sulfasalazine (SASP), a drug used in inflammatory bowel diseases, and its active metabolite 5-aminosalicylic acid (5-ASA) were found to inhibit colonic mucosal SP-LT formation, while only 5-ASA inhibited simultaneously synthesis of another arachidonic acid-derived inflammatory mediator, prostaglandin (PG) E₂. The results suggest that SP-LT might be important mediators of inflammation in CD and UC.     

Changes in neuropeptide-containing nerves in human colonic mucosa with inflammatory bowel disease

The distribution abnormality of vasoactive intestinal polypeptide-containing nerves (VIP-nerves) and substance P-containing nerves (SP-nerves) was immunohistochemically investigated in the colonic mucosa with inflammatory bowel disease (IBD) in relation to colonic glands and blood vessels In the lamina propria. In active ulcerative colitis (UC), VIP- and SP-nerves decreased in severe inflammatory lesions. VIP-nerves were almost absent particularly around crypt abscesses. Even In resolving and quiescent UC, VIP-nerves still decreased, depending on the decrease of glands and blood vessels. On the other hand, both nerves Increased in some hypervascular lesions. In the uninvolved mucosa of UC, they did not change their distribution. In Crohn's disease, the distribution abnormality of both nerves resembled that of UC. These results suggest that the changes in VIP-and SP-nerve distributions in the mucosa with IBD are subsequent to mucosal inflammation and damage. However, these peptides are known to be immunoregulators, and their distribution abnormalities may induce the disorder of immunoregulation in the IBD mucosa and cause the mucosal damage and/or chronicity.     

Type I Allergy to Normal Cellular Constituents in Chronic Inflammatory Bowel Disease?

The possibility of autoimmune type I reactions to cellular constituents was investigated in 22 patients with ulcerative colitis, 12 with Crohn's disease, and in 22 healthy volunteers. Nuclear components and colon mucosa fragments were tested as potential antigens by the basophil histamine release technique. One of 12 patients with ulcerative colitis responded to sonicated leukocyte nuclei and one of 12 patients with Crohn's disease responded to both nuclei and RNA. Increased serum levels of total IgE and antinuclear antibodies of IgE class were found in one and three of the 24 patients, respectively. Histamine release caused by colon mucosa fragments was not observed in a separate study consisting of 10 ulcerative colitis patients and 10 healthy volunteers. Autoimmune type I allergy to cellular constituents does not seem to be of significance for chronic inflammatory bowel disease and thus could not explain the involvement of tissue mast cells and eosinophils in this condition.     

Colitis in transgenic and knockout animals as models of human inflammatory bowel disease

Summary: Spontaneous colitis In knockout (KO) and transgenic rodents provides experimental models to study the development of mucosal inflammation and inflaminatory bowel disease (Crohn's disease and interactive colitis). Genetic and environmental factors, particularly the normal enteric flora, are important factors in the development of mucosal inflammation. The normal mucosal homeostasis is disrupted when there is either cytokine imbalance, abrogation of oral tolerance, alteration of epithelial barrier and function or loss of immunoregulatory cells. Some but not all immunodeficiencies, in the appropriate setting, lead to colitis. CD4-' T cells have been identified as the pathogenic T ceils in colitis, which mediate inflammation by either the Thl or the Th2 pathway. The Thi pathway dominates most colitis models and in Crohn's disease. In contrase. the colitis in TCRa KO mice shares many features of ulcerative colitis including the dominance of Th2 pathway in colonic inflammation. A major benefit of these models is in the development of therapeutic strategies for the treatment of inflammatory bowel 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.     

IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn's disease.

IL-8 is generating increasing interest as a powerful neutrophil chemoattractant and activator. To elucidate the mechanisms of neutrophil infiltration in inflammatory bowel disease, we examined 33 patients with ulcerative colitis (UC), 18 with Crohn's disease (CD), eight with some other type of colitis, and 18 normal control subjects for measurement of IL-8 in homogenates of colonic biopsy specimens. The affected colonic mucosa was found to contain significantly more IL-8 in patients with active inflammatory bowel disease than in patients with inactive disease (UC, P < 0.001; CD, P < 0.001), in patients with other types of colitis (UC, P < 0.05; CD, P < 0.01), or in normal control subjects (UC, P < 0.001; CD, P < 0.001). Colonic IL-8 levels correlated significantly with the macroscopic grade of local inflammation, especially in patients with UC (P < 0.001). Colonic IL-8 levels also correlated well with the neutrophil numbers in mucosal tissue (UC, r = 0.950, P < 0.001; CD, r = 0.940, P < 0.001), and with colonic IL-1 beta (r = 0.911, P < 0.001) and tumour necrosis factor-alpha (TNF-alpha) levels (r = 0.604, P < 0.001) in patients with these two conditions. These data suggest a potential role for IL-8 and its regulatory cytokines IL-1 and TNF-alpha in mediating neutrophil infiltration of the gut wall in inflammatory bowel disease.     

Differential diagnosis of Crohn's disease of the colon from ulcerative colitis: ultrastructure study with the scanning electron microscope

Mucosal biopsies from patients with Crohn's disease and with ulcerative colitis were studied by scanning electron microscopy. Important abnormalities of the mucosal surface were found in both diseases. For Crohn's disease, the characteristic abnormality was loss of the regularity of the polygonal units, but with preservation of the mucosal integrity and of the normal mucosal design. For ulcerative colitis, the abnormalities were disorganization of the cells, signs of sloughing, and superficial erosions. Patients with Crohn's disease always had a significantly increased number of muciparous cells, while those with ulcerative colitis had obvious signs of decreased mucus production. The lesions of ulcerative colitis could be seen under the scanning electron microscope in mucosal areas that appeared normal endoscopically. We feel therefore that scanning electron microscopy of biopsy specimens from patients with inflammatory bowel diseases can be of great help in differential diagnosis.