Inflammation and Immune Response

Altered Immune Response

While it is unlikely a specific microbial antigen will be established as a consistent causative factor in Crohn's Disease, an abnormal antibody response seems to be a factor. The inflammation appears to be, at least in part, a result of an overreaction to normal intestinal flora. Experts theorize either a microbial antigen may have precipitated the inflammation when elimination from the mucosa was unsuccessful, triggering ongoing inflammation, or an inherent dysregulation of the mucosal immunity exists, resulting in an overreaction to normal gut flora.⁷⁹ In either case, it is thought the antigen persists due to an inability of phagocytes to break down the cell walls of commensal microbes.

The presence of antibodies to microbial antigens in Crohn's Disease supports the theory that one aspect of Crohn's Disease pathology involves an abnormal immune response to otherwise normal intestinal flora. For example, Crohn’s disease is characterized by elevations in anti-Saccharomyces cerevisiae (brewer's yeast) antibodies in 49-60 percent of cases.⁸⁰ In addition, levels of protein-bound IgG (bound to proteins of non-pathogenic bacteria) have been found in the intestinal mucosa of patients with active Crohn's Disease to be significantly higher than patients with UC, irritable bowel syndrome, or non-specific IBD.⁸¹

Zareie et al found mononuclear cells from the lamina propria of the gut mucosal cells in Crohn's Disease patients were spontaneously activated, apparently by normal gram-negative, luminal bacteria. Activation resulted in secretion of tumor necrosis factor-alpha (TNF-alpha) and subsequent epithelial changes.⁸²

Cytokine Patterns

A T-cell mediated immune response has been identified in the mucosa of Crohn's Disease, in contrast to UC, and is postulated to be the primary precipitating event.⁸³ The ensuing production of inflammatory cytokines can cause ulceration and increased intestinal permeability.⁸³ Animal models confirm the generally held consideration that Crohn's Disease is primarily a T-helper 1- (Th1) dominant condition. In murine models, disease induced by a Th1 over-expression results in lesions histologically compatible with Crohn's Disease (including granulomas), while a T-helper 2- (Th2) mediated response results in lesions more closely resembling ulcerative colitis (including a lack of granulomas).^84,85

As mentioned, the characteristic granulomatous lesion seen in Crohn’s disease is evidence of a cell-mediated immune response. In human studies, while chronic Crohn's Disease appears to involve primarily an overactive Th1 response characteristic of a cell-mediated phenomenon,⁸⁶ some researchers have determined divergent cytokine patterns at different stages of the disease.⁸⁷ Chronic lesions are associated with high levels of interleukin-2 (IL- 2), interferon gamma (IFN-gamma),⁸⁷ TNF-alpha, and interleukin-12 and -18 (IL-12 and IL-18).⁸⁶ Desreumaux et al, however, found a distinctly different cytokine pattern in early CD. By examining ileal biopsy specimens of 17 patients compared to 11 controls, the researchers determined that early lesions were characterized by elevations in interleukin-4 (IL-4) and decreases in IFN-gamma, ⁸⁷ a pattern more consistent with an overactive Th2 immune response.

Other researchers have found, at least in animal models, the opposite may be true. In a mouse model of ileitis (a Crohn's Disease-like enteritis), researchers found elevated Th2 cytokines, including IL-4, during the chronic phase of the disease.⁸⁸

IL-18 is up-regulated in Crohn’s disease. Although typically considered to be an activator of Th1 responses, IL-18 has actually been shown to be a pleiotropic cytokine capable of mediating both Th1 and Th2 responses, providing more potential evidence for divergent cytokine patterns in the pathogenesis of Crohn's Disease.⁸⁸

Another study examined human colonic tissue samples and found IL-16 protein levels elevated in Crohn's Disease patients but not UC patients. The same study found an anti-human IL-16 antibody could suppress colonic injury in a murine model of Crohn’s-like experimental colitis.⁸⁹

Pro-inflammatory cytokines are normally kept in check by immunosuppressive cytokines such as transforming growth factor beta (TGFbeta). It is believed the transcription factor T-bet is integral to controlling the balance between proand anti-inflammatory cytokines.⁹⁰ T-bet is elaborated by Th1 cells, but not Th2 cells. IFN-gamma is enhanced by T-bet. Neurath et al examined Tbet activity in lamina propria T-cells of patients with Crohn's Disease as well as in animal models. The researchers discovered T-bet over-expression in the lamina propria T-cell nucleus in patients with Crohn's Disease, but not UC or controls. In the animal models, T-bet overexpression was consistent with Th1-mediated colitis (animal model of Crohn's Disease), while a T-bet deficiency was protective.

Tumor necrosis factor appears to play a significant role in the pathogenesis of Crohn's Disease. Mucosal biopsies of children with IBD compared to controls found a significantly greater number of TNF-alpha-secreting cells in patients with Crohn's Disease compared to those with UC or non-specific bowel inflammation.⁹¹ A significant difference between mild-to-moderate and severe disease was also noted for the Crohn's Disease subgroup, with severe disease demonstrating a significantly greater percentage of TNF-secreting cells. In animal models of Crohn’s ileitis, neutralization of TNF resulted in significant decrease in inflammation.⁹² Indeed, suppression of TNF-alpha is the primary mechanism of action of the monoclonal antibody category of Crohn's Disease medications (see Conventional Treatments below). TNF-alpha contributes to gut inflammation in several ways. It induces expression of adhesion factors that allow for inflammatory cells to infiltrate and activates macrophages to promote release of other pro-inflammatory mediators such as IFN-gamma.⁹³

TNF-alpha concentrations in the stool can be used to monitor disease activity in both Crohn's Disease and UC. Braegger et al compared 13 children with active Crohn's Disease to children with inactive Crohn's Disease, Ulcerative Colitis, diarrhea, and healthy controls. The average TNF concentrations in the stools of children with active Crohn's Disease ranged from 440-4,322 pg/g compared to a range of 40-84 pg/g in the children with diarrhea, inactive Crohn's Disease, and healthy controls.⁹⁴

A tumor necrosis factor-like cytokine, TL1A, has recently been identified as a co-contributor to IFN-gamma production. Tissue from IBD patients and controls was examined for the presence of TL1A, which was found to be up-regulated in patients with IBD, particularly those with active Crohn's Disease. It appears to be produced primarily in the macrophages and T lymphocytes from the lamina propria in Crohn's Disease patients, with the amount present correlating with disease severity. The result was a four-fold increase in IFN-gamma production. The authors concluded, ?Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as Crohn's Disease.⁹⁵

Oxidative Stress

Oxidative stress is thought to play a significant role in the pathogenesis of inflammatory bowel disease, including Crohn's Disease. Endogenous antioxidants such as superoxide dismutase (SOD), glutathione, and catalase are normally able to counteract oxidative stress in the intestinal mucosa. However, inflammation increases the demand for these important antioxidants and results in an imbalance between pro-oxidants and antioxidants, with subsequent mucosal damage.

In an Italian study, subjects (37 Crohn's Disease and 46 Ulcerative Colitis patients) were compared to 386 healthy controls. Oxidative DNA damage was measured examining the amount of 8-hydroxy-deoxy-guanosine (8-OhdG) present in blood. In addition, evaluation of plasma levels of vitamins A and E and carotenoids demonstrated significant decreases in patients with Crohn's Disease or Ulcerative Colitis compared to controls. The specific carotene found most significantly decreased was beta-carotene, with plasma levels only 50 percent of controls. No significant differences were noted between Crohn's Disease and Ulcerative Colitis patients. Levels of 8-OhdG were considerably higher in IBD patients than controls, illustrating increased oxidative DNA damage.⁹⁶

Researchers have studied the connection between oxidative stress and immune-regulated inflammatory factors. Reactive oxygen species (ROS) have been found to be involved in activation of nuclear factor-kappaB (NF-kappaB), which is necessary for encoding genes for TNF-alpha and some of the interleukins involved in inflammation. Antioxidant levels and inflammatory mediators were examined in 26 Crohn's Disease patients compared to 15 healthy controls. Selenium and glutathione peroxidase (GSHPx) activity were both decreased in Crohn's Disease patients, while TNF-alpha levels and ESR were increased and negatively correlated with selenium and GSHPx. Selenium levels decreased in accordance with disease activity, with the most severe disease manifestation exhibiting the lowest levels. These findings occurred in subjects who did not have evidence of malabsorption, indicating malabsorption is not the sole factor contributing to selenium deficiency. The researchers conclude, 'selenium supplementation in deficient patient groups [should be] regarded as a potential protecting factor against oxidative burst, NFkappaB activation and excessive inflammatory and immune response.'⁹⁷

A study examining indices of oxidative stress and plasma levels of vitamins A and E in 20 Crohn's Disease patients found higher peroxidative status and lower vitamin A and E levels compared to controls. Conservative surgery to remove bowel obstructions resulted in improvements in vitamin A status and oxidative stress measured by thiobarbituric acid reactive substances (TBARS).⁹⁸

A similar study found significantly higher levels of breath-pentane and -ethane and F2- isoprostane (measurements of oxidative stress) in 37 non-smoking Crohn's Disease patients compared to matched controls. At the same time, plasma levels of vitamin C and the carotenoids alpha- and beta-carotene, lycopene, and beta-cryptoxanthin were significantly lower in Crohn's Disease patients.⁹⁹

Pediatric patients also demonstrate signs of increased oxidative stress. In a study of 22 pediatric Crohn's Disease patients, malondialdehyde (MDA) levels were 70-percent higher than controls. Antioxidant levels were measured and only vitamin A was found significantly low, while alpha- and gammatocopherol and beta-carotene were no different than controls. Red blood cell levels of glutathione were higher in Crohn's Disease patients than controls. The researchers speculate these higher levels may be due to an attempt to compensate for increased oxidative stress.¹⁰⁰

Although oxidative stress is a factor in the pathogenesis of both UC and Crohn's Disease, the parameters may vary between the two. Both Ulcerative Colitis and Crohn's Disease are associated with increased levels of MDA, a sign of lipid peroxidation. However, one study found elevated levels of MDA in Crohn's Disease were associated with levels of the antioxidant metallothionein (a hydroxyl radical scavenger) and manganese-dependent SOD (active in the mitochondria); whereas, in Ulcerative Colitis the MDA levels were associated with catalase, GSHPx, and myeloperoxidase. Based on this data, the researchers suggest the likelihood of the ROS hydroxyl radicals and superoxide anions in the pathogenesis of Crohn's Disease, while hydrogen peroxide and hypochlorous acid may be more associated with Ulcerative Colitis.¹⁰¹



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