- Top of page
- Materials and Methods
Treatment with the nonsteroidal anti-inflammatory drugs piroxicam or sulindac was recently shown to accelerate the development of colitis in interleukin (IL)-10-deficient (IL-10−/−) mice. Although NSAIDs have been hypothesized to decrease the barrier function of the intestinal epithelium, the mechanism by which this accelerates colitis in IL-10−/− mice is not well understood. In this study, the effects of piroxicam on the colonic mucosa of IL-10−/− C57BL/6 mice were evaluated histologically. The effect of piroxicam on intestinal epithelial cells in vitro was assessed using colorimetric and fluorescent assays for cell viability and apoptotic cell death. Interactions of intestinal bacteria with the colonic mucosa were evaluated by rRNA-directed fluorescence in situ hybridization. In vivo treatment of C57BL/6 IL-10−/− mice with oral piroxicam markedly enhanced apoptosis of colonic epithelium and resulted in focal erosion of the mucosal surface, enhanced bacterial adhesion and invasion, and accelerated the development of colitis. In vitro, piroxicam induced apoptosis of CT26 murine intestinal epithelial cells in a dose-dependent fashion. Piroxicam-induced apoptosis of CT26 cells could not be prevented by addition of exogenous IL-10; however, IL-10 did significantly enhance their rate of proliferation. Thus, exposure to piroxicam enhances intestinal epithelial apoptosis both in vitro and in vivo and facilitates adhesion and invasion of intestinal bacteria into mucosal tissues in vivo. The role of IL-10 in this process requires further study. These studies support the hypothesis that increased exposure of mucosal cells to intestinal bacteria may lead to development of intestinal inflammation in IL-10−/− or other genetically susceptible individuals.
A number of experimental models for inflammatory bowel disease (IBD) have been established in rodents.1–4 These models vary in the anatomic portion of the gut affected, the depth of inflammation, and the involvement of TH1 versus TH2 cytokines; however, together they have provided important evidence that abnormal immune activation and cytokine production are important in the development and perpetuation of IBD. Colitis develops spontaneously when mice deficient in the TH2 regulatory cytokine IL-10 are colonized with intestinal bacteria but not when they are kept under germ-free conditions. Marked variations in the age of onset and severity of inflammation occur because of both environmental and genetic (strain) effects.5–7 Most of the reported studies used interleukin (IL)-10-deficient (IL-10−/−) mice on a 129/SvEv or Balb/C background, because these strains of mice develop colitis more rapidly than do mice on the C57BL/6 background. However, Berg et al8 recently showed that the relatively slow development of colitis in IL-10−/− mice on the C57BL/6 background can be accelerated by administration of the nonsteroidal anti-inflammatory drugs (NSAIDs) piroxicam or sulindac. Exposure of IL-10−/− mice to NSAIDs results in rapid (<2 wk) and uniform development of colitis that persisted long-term after the NSAIDs are discontinued. The histology of colitis and cytokine production in NSAID-treated animals were reported to be similar to what is observed when these animals spontaneously develop colitis. Colitis did not develop when wild-type mice were treated with NSAIDs. Coadministration of a prostaglandin agonist with the NSAID prevented the development of colitis, indicating that development of colitis was related to inhibition of prostaglandin synthesis.8 However, the precise mechanisms by which NSAIDs rapidly accelerate the development of colitis in IL-10−/− mice are not yet well understood.
Conventional NSAIDs such as piroxicam and sulindac inhibit cyclooxygenase enzymes (COX-1 and COX-2) that are important for prostaglandin synthesis. COX-1 is constitutively expressed within the gastrointestinal tract, where it regulates the production of prostaglandins that are essential for maintaining mucosal barrier function. NSAID inhibition of COX-1 may compromise the gastrointestinal defense mechanisms by decreasing production of prostaglandins that maintain mucosal integrity. Development of colitis is currently thought to involve an aberrant immune response to bacterial antigens. Thus, decreases in the integrity of the intestinal epithelial barrier resulting from NSAID exposure may allow antigens from intestinal bacteria to contact immune cells in the lamina propria, eventually leading to an excessive inflammatory response in genetically susceptible IL-10−/− animals.
We used piroxicam as described by Berg et al8 to study the mechanism by which NSAIDs accelerate the development of colitis in the relatively colitis-resistant C57BL/6 strain of IL-10−/− mice. We found that piroxicam enhances colonic epithelial apoptosis and increases intestinal epithelial exposure to bacteria. Increased exposure to bacteria may potentially trigger immune responses that lead to the development of colitis in these animals.
- Top of page
- Materials and Methods
Colitis is known to be a potential side effect of NSAID treatment in both in humans and rodents. NSAIDs inhibit the cyclooxygenase enzymes that are responsible for synthesis of the prostaglandins that regulate mucosal integrity and the proinflammatory prostaglandins. However, the precise mechanisms by which NSAID treatment induce colitis have not previously been established. Our data show that exposure to piroxicam induces apoptosis of colon epithelial cells, as evidenced by decreased cell survival, increased annexin V positivity, and the appearance of histone-associated oliconucleosomes within the cytoplasm of cells treated with piroxicam in vitro. Mice treated with piroxicam in vivo showed classic morphological evidence of colon epithelial apoptosis, confirmed by immunodetection of enhanced activation of caspase-3 in colon epithelial cells. The data presented further show a link between NSAID (piroxicam)-induced apoptosis, enhanced bacterial adhesion and invasion, and accelerated development of colitis in genetically susceptible IL-10−/− mice.
IL-10 is a regulatory cytokine that is primarily considered to have immunosuppressive properties. IL-10 normally decreases the production of TH1 cytokines by T cells and can also suppress macrophage activity. Loss of the regulatory functions of IL-10 with regard to immune responses against colonic bacteria has been suggested to be responsible for the development of spontaneous colitis in IL-10−/− mice. Genetic lack of IL-10 or its inhibition by neutralizing antibodies to the IL-10 receptor (IL-10R) have been shown to enhance autoreactive proliferative responses of T cells in response to staphylococcal enterotoxin B (SEB).15 IL-10 expression was shown to protect syngeneic intestinal epithelial cells from apoptosis induced by SEB-primed spleen or colonic T cells, although this was attributed to the regulatory effects of IL-10 on activated T cells.15 However, it is possible that IL-10 may also affect epithelial cells directly. Intestinal epithelial cells have been shown to express mRNA for both subunits of the IL-10 receptor-signaling complex.16 Furthermore, both epithelial cell lines and freshly isolated murine intestinal epithelial cells derived from small or large intestine demonstrate specific saturable binding of IL-10.16 IL-10 has previously been shown to protect thyroid epithelial cells against apoptosis by up-regulating expression of the antiapoptotic cellular FLICE inhibitory protein (cFLIP) and Bcl-xL.17,18 Autocrine production or exogenous addition of IL-10 protected normal and malignant thyroid epithelial cells against apoptosis induced by chemotherapeutic agents. Exposure of these cells to neutralizing antibodies against IL-10 down-modulated Bcl-2 and Bcl-xL, induced apoptotic cell death, and enhanced susceptibility of the remaining population to chemotherapeutic agents.19 However, in contrast to what is seen in the thyroid, in our studies, addition of exogenous IL-10 to isolated intestinal epithelial cells in vitro failed to increase their resistance to piroxicam-induced apoptosis. We hypothesize that, by increasing the proliferation of colonic epithelial cells (Fig. 4), IL-10 may also enhance epithelial regeneration and repair and thus potentially mitigate negative effects of piroxicam-induced apoptosis in the colon in vivo. This can be tested in future studies designed to clarify the role of IL-10 in colonocyte survival, including the possibility of indirect effects through other cell types that are present in the colonic microenvironment.
The marked changes in distribution of bacteria within the colon after piroxicam treatment are of considerable interest. Evidence that colonic bacteria, particularly Bacteroides species, may participate in the pathogenesis of IBD has been presented for both rodents and humans.20–24 Increased numbers of mucosa-associated Bacteroides organisms in human patients with IBD have also been detected by culture and by FISH.25 The presence of bacteria adherent to and within colon epithelial cells in piroxicam-exposed but not control mice clearly implicates these organisms in the pathogenesis of the colitis that develops after piroxicam exposure. We hypothesize that increased immune system exposure to antigens from these bacteria results in development of chronic bowel inflammation in susceptible individuals. However, the data presented here do not allow direct effects of piroxicam on mucosal integrity or bacterial growth to be distinguished from those that arise as a result of inflammation. Further studies including time-courses that relate changes in intestinal bacteria-mucosal interactions to the onset of inflammation will be required to address these issues.
NSAIDs have been previously documented to increase the incidence of apoptosis in colonic cells. Administration of sulindac and the selective COX-2 inhibitor nimesulide to mice enhanced apoptosis in colonic epithelium of mice with azoxymethane and dextran sulfate sodium-induced colitis. NSAID-treated mice had decreased numbers of aberrant crypt foci and tumors, which was attributed to the increased level of apoptosis.26 Nonselective NSAIDs such as sulindac and piroxicam inhibit both the COX-1 and/or COX-2 cyclooxygenase enzymes. COX-2 expression is stimulated by inflammatory mediators and results in preferential synthesis of the prostanoids (PG) PGI2 and PGE2.27 Treatment with PGE2 or with agents that induce its synthesis inhibits apoptosis induced by camptothecin in T24 bladder epithelial cells.28 Overexpression of COX-2 has been proposed to enhance neoplastic transformation by increasing cellular proliferation and decreasing apoptosis.29,30 Although these studies seem to implicate COX-2 in regulating apoptosis, simultaneous inhibition of both COX-1 and COX-2 seems to be required for acceleration of colitis in IL-10−/− mice, because use of either COX-1- or COX-2-selective inhibitors alone does not induce colonic inflammation.8
Given these data, it is of interest to examine the potential role of conventional and COX-selective NSAIDs in the pathogenesis of human colitis. A recent study of 105 cases of newly diagnosed colitis and 105 age- and sex-matched controls showed increased incidence of NSAIDs or salicylate exposure in colitis patients before disease onset (odds ratio, 9.1; 95% confidence limits, 4.5-21.9; P < 0.001).31 Results of retrospective studies of the effects of NSAIDs on IBD activity have been mixed.32 A relapse rate of 25% was recorded in a cohort of patients with quiescent IBD with arthritis or arthralgias who were prospectively exposed to NSAIDs.33 In a group of 33 patients with IBD who were prescribed the COX-2-selective NSAIDs celecoxib or rofecoxib, 13 (39%) had a flare of IBD within 6 weeks of initiating COX-2 therapy.34 However, a prospective open-label monocentric trial of 32 patients with clinically inactive or mild IBD using the COX-2-selective NSAID rofecoxib showed no significant exacerbation of IBD.32
Regular intake of NSAIDs has been shown to be beneficial in reducing the risk of colorectal cancer in the general population and in patients with inherited mutations that place them at increased risk for colorectal cancer.35 Use of the NSAID-like drugs sulfasalazine and its metabolite mesalamine (5-aminosalicylic acid) for therapy for colitis has been shown to protect against development of colorectal carcinoma in patients with IBD. Treatment with 5-aminosalicylic acid was associated with increased apoptosis in tumors or abnormal mucosa, without long-term effects on normal mucosa in human, mouse, and in vitro studies.35,36 Thus treatments that moderately increase apoptosis of (possibly mutant) colon epithelial cells may be beneficial for chemoprevention of colorectal cancer, as long as such treatment does not affect mucosal integrity.
In summary, the NSAID piroxicam enhances apoptosis of intestinal epithelial cells both in vitro and in vivo, leading to the development of epithelial erosions, direct adherence of bacteria to the mucosal surface, and bacterial invasion of epithelial cells. We hypothesize that the increased prevalence and accessibility of intestinal bacterial antigens to immune cells that results from piroxicam-induced apoptosis of the colonic epithelium facilitates the development of acute and chronic colonic inflammation in IL-10−/− mice. Published data suggest that NSAIDs may similarly increase apoptosis in the human colon. Further studies should be undertaken to determine whether NSAID use is harmful for individuals whose genetic makeup makes them particularly susceptible to NSAID-induced colonic apoptosis or who may be at increased risk for developing prolonged or inappropriate immune responses in response to mucosal contact with intestinal bacteria.