Lymphokines and the intestinal immune response. Role in inflammatory bowel disease

The study of production of and response to various soluble mediators by gut-derived mucosal mononuclear cells has contributed significantly to a better understanding of the regulation of the intestinal immune system. So far, a number of factors have been investigated in some detail or in a preliminary fashion: interleukin 1, interleukin 2, interleukin 4, interferon gamma and colony-stimulatory factors. Their level of activity, and their effect on the intestinal mucosal immune system are discussed, particularly in regard to their potential role in the pathogenesis of inflammatory bowel disease.     

Isotypic analysis of antibody response to a food antigen in inflammatory bowel disease

We studied the class-specific antibody response to the cow's milk antigen beta-lactoglobulin (beta-LG) in sera from patients with ulcerative colitis and Crohn's disease. IgG and IgM to beta-LG were significantly higher in patients when compared to healthy non-atopic controls, whereas IgA values were similar, and specific IgE absent in all groups. No correlation between IgG- or IgM-containing immune complexes was found with the corresponding isotype of antibody to beta-LG; however, IgM complexes correlated with serum total IgM in ulcerative colitis. In these patients, IgG antibodies were higher in active cases, whereas IgM increased in patients without signs of disease activity. Antibody titers did not correlate with disease duration or administration of antiinflammatory drugs. This pattern of anti-beta-LG reactivity suggests that the presence of intestinal lesions may be revealed by the selective increase of some antibody isotypes to orally administered antigens. Enhanced mucosal permeability may be studied by this type of serological analysis.     

Lymphocytotoxic antibody in inflammatory bowel disease. A family study.

The prevalence of lymphocytotoxic antibody in inflammatory bowel disease is 40 per cent. Twenty-seven of 90 relatives of 23 probands with the disease (30 per cent) demonstrated lymphocytotoxic antibody, as contrasted with only three of 69 control family members (4 per cent) (P less than 0.0001). Decreased lymphocytotoxicity against lymphocytes from patients with inflammatory bowel disease as compared to normal donor lymphocytes previously demonstrated in the serum of probands was also observed in the serums from family members of the probands. Nineteen of the 48 household contacts of probands (40 per cent) were positive for antibody, whereas eight of 42 nonhousehold contacts (19 per cent) demonstrated it (P less than 0.05). Eight of 16 spouses (50 per cent) of probands showed antibody. The increased prevalence of lymphocytotoxic antibody in family members of probands and its occurrence mainly in household contacts (consanguineous and non-consanguineous) may indicate the exposure of probands and their family members to a common environmental agent.     

Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in clinical specimens by immunofluorescence with a monoclonal antibody to B. fragilis lipopolysaccharide.

A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroides fragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group.     

Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease

The unfolded protein response as a consequence of endoplasmic reticulum (ER) stress has recently been implicated as a novel mechanism that may lead to inflammatory bowel disease (IBD). Impairment of proper ER stress resolution in highly secretory Paneth and, to a lesser extent, goblet cells within the epithelium can primarily lead to intestinal inflammation. An inability to manage ER stress may not only be a primary originator of intestinal inflammation as exemplified by genetic polymorphisms in XBP1 that are associated with IBD but also a perpetuator of inflammation when ER stress is induced secondarily to inflammatory mediators or microbial factors. Furthermore, ER stress pathways may interact with other processes that lead to IBD, notably autophagy.     

Carrageenan-induced intestinal injury in the rat--a model for inflammatory bowel disease

The cause of inflammatory bowel disease (IBD) remains unknown. In this report, an attempt is made to produce a suitable animal model for studying the pathobiology of IBD, especially its pathogenesis. Sprague-Dawley rats were divided into four groups of six (A, B, C, D). The experimental design involved prior parenteral sensitization of groups A and B by a 1.5 percent solution of lambda degraded carrageenan followed by oral administration of the same solution for 30 days to groups A and C. The animals were then sacrificed, and the small intestine was evaluated for injury. Oral carrageenan caused significant intestinal injury as evidenced by ulceration, abnormal villous pattern, degree and extent of inflammation [p = 0.0001 for groups (A + C) versus (B + D)]. Prior sensitization aggravated the effects of oral carrageenan. Overall, the inflammation produced was reminiscent of human IBD in that there was pin-point ulceration, focality of lesions and lymphoid hyperplasia with microgranulomas. It was concluded that this carrageenan model may prove to be particularly useful for studying the pathobiology of human IBD.     

Increase in podoplanin-expressing intestinal lymphatic vessels in inflammatory bowel disease

Ulcerative colitis (UC) and Crohn's disease (CD) are two presentations of inflammatory bowel diseases (IBDs). Whereas the inflammation in UC is confined to the mucosa/submucosa, CD is considered a transmural disease with characteristic lymphoid aggregates with or without epithelioid granulomas in the subserosa. Here we examined and quantified the distribution of lymphatic capillaries in small- and large-bowel resection specimens (non-IBD n=8; CD n=20 and UC n=13) using immunohistochemical staining with anti-human podoplanin antibody, an established marker for lymphatic endothelium. In normal small intestine, the lymphatic network originated in the capillaries beneath the surface epithelial cells, whereas it started in the lower third of the mucosa of the large intestine. Lymphatic microvessel counts revealed a statistically highly significant increase ( P<0.005 in the muscularis mucosae, P=0.012 in the tunica submucosa and P=0.012 in the tunica subserosa) in IBDs when compared with normal intestine. Numerical differences between CD and UC samples were not significant. Prominence of lymphatic capillaries could also be observed in areas where fibrosis replaced chronic inflammation. These findings suggested that lymph-vessel proliferation in IBDs may be triggered by chronic inflammation irrespective of its organization and is maintained in fibrotic end-stage disease.     

Assessment of systemic genetic damage in pediatric inflammatory bowel disease

The etiology of distal site cancers in inflammatory bowel disease (IBD) is not well understood and requires further study. We investigated whether pediatric IBD patients' blood cells exhibit elevated levels of genomic damage by measuring the frequency of mutant phenotype (CD59-/CD55-) reticulocytes (MUT RET) as a reporter of PIG-A mutation, and the frequency of micronucleated reticulocytes (MN-RET) as an indicator of chromosomal damage. IBD patients (n = 18 new-onset disease, 46 established disease) were compared to age-matched controls (constipation or irritable bowel syndrome patients from the same clinic, n = 30) and young healthy adults age 19–24 (n = 25). IBD patients showed no indication of elevated MUT RET relative to controls (mean ± SD = 3.1 ± 2.3 × 10⁻⁶ vs. 3.6 ± 5.6 x 10⁻⁶, respectively). In contrast, 59 IBD patients where %MN-RET measurements were obtained, 10 exceeded the upper bound 90% tolerance interval derived from control subjects (i.e., 0.42%). Furthermore, each of the 10 IBD patients with elevated MN-RET had established disease (10/42), none were new-onset (0/17) (p = .049). Interestingly, each of the subjects with increased chromosomal damage was receiving anti-TNF based monotherapy at the time blood was collected (10/10, 100%), whereas this therapy was less common (20/32, 63%) among patients that exhibited ≤0.42% MN-RET (p = .040). The results clearly indicate the need for further work to understand whether the results presented herein are reproducible and if so, to elucidate the causative factor(s) responsible for elevated MN-RET frequencies in some IBD patients.     

Antibodies detectable by counterimmunoelectrophoresis against Bacteroides antigens in serum of patients with inflammatory bowel disease.

Heat-extracted antigens from seven species of Bacteroides were used in passive hemagglutination and counterimmunoelectrophoretic tests. Sera from 87 normal persons (group I) and 15 patients with ulcerative colitis (group II) were of low and equal reactivity in passive hemagglutination tests; all positive tests were eliminated by 2-mercaptoethanol reduction of the sera. When these same sera were tested by counterimmunoelectrophoresis with six of the Bacteroides antigens, no significant difference in the percentage of positive reactions was noted. However, using the chi-square test, the seventh antigen, prepared from Bacteroides vulgatus, successfully distinguished the two populations at the 0.025 level. Counterimmunoelectrophoretic tests with the B. vulgatus antigen also provided a means to separate the patients in group II with active disease from those in remission at a P value of 0.01. All the sera from 12 patients with defined Crohn's disease activity indexes reacted with the B. vulgatus antigen in counterimmunoelectrophoretic tests. Reduction and alkylation of patient sera with 2-mercaptoethanol and iodoacetamide removed detectable antibody in 78% of the samples, which suggested a dominant role of immunoglobulin M in the response to Bacteroides antigens.     

Mouse models of intestinal inflammation as tools to understand the pathogenesis of inflammatory bowel disease

Mouse models of intestinal inflammation resemble aspects of inflammatory bowel disease in humans. These models have provided important insights into mechanisms that control intestinal homeostasis and regulation of intestinal inflammation. This viewpoint discusses themes that have emerged from mouse models of intestinal inflammation including bacterial recognition, autophagy, the IL‐23/Th‐17 axis of inflammation as well as the role of negative regulators. Many of the pathways highlighted by model systems have been identified in recent genome‐wide association studies in human validating the relevance of mouse models to human inflammatory bowel disease. Understanding of the complex biological mechanisms that lead to intestinal inflammation in mouse models may help to define targets for treatment of human diseases.     

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two-step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H(2)O(2)), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non-inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up-regulation of Mn-SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM-mediated damage, these changes should be coordinately counterbalanced by an increased H(2)O(2)-neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H(2)O(2)-related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H(2)O(2)-metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H(2)O(2)-mediated damage seemed to be restrained by the increasing numbers of CAT- and MPO-positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro-inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH(*)) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H(2)O(2) is hampered in these cells, thereby increasing not only the risk of detrimental effects of H(2)O(2) directly, but also those of its extremely reactive derivatives such as OH(*). Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes.     

Peripheral cell-mediated immune response to mycobacterial antigens in inflammatory bowel disease.

A mycobacterial etiology has been proposed in Crohn's disease (CD). We have sought evidence of increased or modified T lymphocyte immune responses to Mycobacterium tuberculosis and Myco, paratuberculosis in patients with CD (n = 13), compared with ulcerative colitis (UC; n = 17) and controls (n = 17). Peripheral blood cells were cultured with phytohaemagglutinin (positive mitogen control), mycobacterial purified protein derivative (PPD) preparations, lysates, column fractions and whole, heat-killed bacteria. Responses of T cells and T cell subsets were assessed by expression of activation markers (CD25, CD69), coupled with blastogenesis assays (3H-thymidine uptake) and estimates of proliferation. Virtually all patients responded to Myco. paratuberculosis and Myco. tuberculosis antigens. There were no significant differences between patient groups, although there was a very high overall correlation (r = 0.95; P < 0.0001) between responses to the two mycobacterial species. Most of the activation and proliferative responses resided in the CD4+ (T helper) subset. Although up to 15% of CD8+ (suppressor/cytotoxic) cells also became activated, the CD8+ cells did not proliferate subsequently. Cells expressing the alternate gamma delta form of the T cell receptor (TCR gamma delta+) did not activate or proliferate in response to mycobacterial antigens. There were no differences in any of these parameters between patient groups. We conclude that there is no specific increase or alteration in cell-mediated anti-mycobacterial immunity in inflammatory bowel disease (IBD). Thus our data do not support a mycobacterial etiopathology of Crohn's disease.     

The Mongolian gerbil as a model for inflammatory bowel disease

Mongolian gerbils are used as biomedical research models for a variety of diseases and are in some cases suited better than other rodents for basic research and therapeutic studies. The aim of this study was to establish and characterize a dextran sulphate sodium (DSS)‐induced model in gerbils for the human inflammatory bowel disease (IBD) and to utilize them for a therapeutic study in vivo. Four concentrations (0.5%, 1%, 2% and 4%) of DSS were administered via drinking water for 7 days; based on these results, a concentration of 3% DSS was given for 9 days in a second approach. Fluid uptake and general clinical condition were assessed daily using a clinical score. Caecum and colon were scored histologically. Fluid uptake was affected by addition of DSS to the drinking water. First clinical symptoms were observed at day 4 of DSS treatment with a considerable increase in clinical score parameters only in gerbils receiving 2% or 4% DSS. Histologically, ulceration and inflammation were observed predominantly in the caecum of gerbils treated with at least 1% DSS; reproducible inflammation in the colon required at least 2% DSS. Using 3% DSS for 9 days, considerably more inflammation was induced in the colon, comparable with lesions usually observed in the mouse model. Using an optimized protocol, DSS treatment induces reproducibly typhlocolitis in Mongolian gerbils, rendering them as a useful model for IBD.     

Entamoeba histolytica as a commensal intestinal parasite in homosexual men

Entamoeba histolytica is considered to be an uncommon, imported organism in the United Kingdom and in many parts of North America, but recent attention has been drawn to the possibility of sexual transmission of this parasite among homosexual men. To determine the prevalence and clinical importance of enteric parasitic infections in men attending a clinic in London for the treatment of sexually transmitted diseases, we studied 354 randomly selected patients who provided a single stool sample that was examined for E. histolytica and other intestinal parasites. Forty-five of the 225 homosexual patients (20 percent) were infected with E. histolytica, but no such infections were found among the 129 heterosexual subjects (P less than 0.0001). With the use of isoenzyme electrophoresis, 34 of the 45 E. histolytica isolates were classified according to zymodeme. All were Zymodeme I or III, which are considered to be nonpathogenic. There was no correlation between the presence of E. histolytica and gastrointestinal symptoms. These findings suggest that E. histolytica is a common commensal in the homosexual population and that, in the absence of evidence of invasive disease, treatment of persons passing cysts of the organism may have little practical benefit.     

Mucin depletion in inflammatory bowel disease.

The mucin and gland content of 26 rectal biopsy specimens--five normal specimens, 10 from patients with ulcerative colitis, and 11 from patients with Crohn's disease--were measured using a Quantimet image analyser. There was significantly less mucin in the groups with ulcerative colitis compared with either those with Crohn's disease or the normal controls. The difference in the gland content between the groups with ulcerative colitis and Crohn's disease and between the group with Crohn's disease and the normal controls did not reach significance. The results suggest that it is worth while assessing the mucin content of rectal biopsy specimens from patients with inflammatory bowel disease. In routine practice this assessment can be made by eye using a suitably stained section.     

Phage-display immunoprecipitation sequencing of the antibody epitope repertoire in inflammatory bowel disease reveals distinct antibody signatures

1. Download : Download high-res image (281KB)
2. Download : Download full-size image

     

Plasma viscosity in inflammatory bowel disease.

AIMS: To assess the relation of plasma viscosity to disease activity in patients with inflammatory bowel disease. METHODS: Crohn's disease (n = 60) and ulcerative colitis (n = 71) were diagnosed on the basis of typical histological or radiological features. Active Crohn's disease was defined as a Crohn's disease activity index of 150 or over. Active ulcerative colitis was defined as a liquid stool passed three times a day or more with blood. Blood samples were assessed for haemoglobin concentration, total white cell count, platelets, plasma viscosity, erythrocyte sedimentation rate, serum albumin, and C-reactive protein. RESULTS: Plasma viscosity was higher in those with active Crohn's disease compared with those with inactive Crohn's disease or active ulcerative colitis. Plasma viscosity correlated significantly with erythrocyte sedimentation rate, C-reactive protein, and platelet count in patients with Crohn's disease. In ulcerative colitis plasma viscosity correlated only with serum C-reactive protein. Plasma viscosity showed a low sensitivity for detecting active Crohn's disease, with 48% of those with active disease having a plasma viscosity within the laboratory reference range. CONCLUSIONS: Plasma viscosity is related to disease activity in Crohn's disease, but is insufficiently sensitive for it to replace erythrocyte sedimentation rate as a measure of the acute phase response in Crohn's disease.     

Interleukin-12 antagonists as new therapeutic agents in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs; Crohn's disease (CD) and ulcerative colitis) are chronic inflammatory diseases leading to destruction of gastrointestinal tissue. They are characterized by an exaggerated immune response. In CD, an increased expression of T-helper-1 (Th1) cytokines was observed in which interleukin-12 (IL-12) seems to play a pivotal role. Different immunosuppressive agents have been used to treat patients suffering from IBD, nevertheless remarkable side effects or treatment failure are limiting factors in this regard. Therefore, studies on more specific treatment of CD have recently been published, using recombinant anti-inflammatory cytokines or inhibitors of proinflammatory cytokines and their receptors. Beyond these principles anti-IL-12 strategies seem to play a promising role because of the central position of this Th1-inducing cytokine in the inflammatory cascade. Up to now anti-IL-12 antibodies, complement receptor-3 antibodies and IL-12p40 homodimers have been evaluated in their potential to suppress the mucosal inflammation. Based on our understanding of the pathogenesis of CD, the available data and experiences concerning these principles are presented in this review.