2Department of Gastroenterology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Trinitrobenzene sulfonic acid (TNBS) and oxazolone are used to induce colitis for the investigation of inflammatory reactions in the colon. Although these chemicals are presumed to bind proteins in the colonic mucosa and then induce colitis as haptens, hapten-protein formation has not yet been confirmed in the colonic mucosa. We developed a mouse model of colitis characterized by hapten-protein visualization, using 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl), which emits fluorescence after binding to proteins. The enema of 1 mg/mL NBD-Cl induced severe diarrhea, rectal bleeding, and body weight reductions in BALB/c mice. Mucosal signs indicative of colitis, such as redness and swelling observed under stereomicroscopy or inflammatory cell infiltration and crypt-epithelium destruction under microscopy, were manifested around NBD-proteins visualized with fluorescence. Fluorescence microscopy showed the infiltration of F4/80+ cells around areas of NBD-proteins, and flow cytometry indicated the uptake of NBD-proteins by CD11b+ cells. We also found critical roles for T cells and interleukin-6 in colitis induction with NBD-proteins. NBD-Cl-induced colitis presents a unique model to study the relevance between hapten-protein formation and inflammatory reactions and offers a method to assess experimental interventions on colitis induction in the mucosa, where hapten-protein formation is confirmed.
Ulcerative colitis and Crohn's disease are chronic inflammatory disorders of the gastrointestinal tract and are broadly categorized as inflammatory bowel disease (IBD) (1). The precise pathogenesis of IBD still remains unclear, and a cure for IBD is not yet available. Although glucocorticoids, 5-aminosalicylic acid, immunosuppressive agents, anti-tumor necrosis factor-α antibodies, and leukocyte/granulocyte apheresis are useful to induce remission and prevent disease recurrence, IBD can be a refractory disease (2,3). Therefore, there is a need for the development of novel anti-inflammatory treatments for IBD patients.
Experimental colitis in mice is a useful model for the investigation of inflammatory reactions in the colon and also for the evaluation of novel anti-inflammatory treatments. Intrarectal administration of trinitrobenzene sulfonic acid (TNBS) or oxazolone is a wellknown method to induce colitis in mice (4-7). TNBS and oxazolone are considered to be haptens, because these chemicals are presumed to bind to endogenous proteins in the colonic mucosa and induce a local immunologic response through macrophage and T cell activation. However, we cannot observe hapten-protein formation in either TNBS- or oxazolone-induced colitis, because TNBS- and oxazolone-protein conjugates are not distinguishable from TNBS or oxazolone alone, respectively, in the colonic mucosa. Hapten-protein visualization allows us to investigate the relevance between hapten-protein formation and inflammatory reactions. Furthermore, the visualization of hapten-protein conjugates can enable the assessment of experimental interventions on colitis induction in the mucosa, where hapten-protein formation is confirmed.
4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) is a chemical that fluoresces upon binding to proteins through the substitution reaction of 4-Cl with amino or thiol groups of amino acid residues (8). The fluorescence of NBD-proteins (excitation 460 nm, emission 535 nm) is similar to those of fluorescein and green fluorescence proteins, so NBD-proteins can be detected using experimental settings for the observation of fluorescein and green fluorescence proteins. NBD-Cl resembles dinitrochlorobenzene in chemical structure and reaction patterns, thereby inducing allergic contact dermatitis in a manner similar to dinitrochlorobenzene (9). Thus, we expected that the intrarectal administration of NBD-Cl might induce colitis in mice through the formation of NBD-proteins and that these could be visualized in the colonic mucosa under fluorescence observation.
In the present study, we examined colitis induction by NBD-Cl in mice and the fluorescence observation of NBD-proteins in the colonic mucosa.Materials and methods Reagents
We obtained NBD-Cl from Tokyo Chemical Industry (Tokyo, Japan or TCI-America, Portland, OR, USA). NBD-Cl was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) to a concentration of 200 mg/mL for the stock solution, which was stored at -80°C. NBD-Cl stock solution was diluted with ethanol (Wako Chemicals, Osaka, Japan) and then with distilled water to prepare the enema solution. Rat anti-mouse interleukin-6 receptor (IL6R) monoclonal IgG antibody MR16-1 (10,11) was kindly provided by Chugai Pharmaceutical Company (Shizuoka, Japan). Control rat IgG was purchased from Sigma-Aldrich. Diethyl ether, 4% paraformaldehyde solution, ammonium chloride, acetone, and olive oil were purchased from Wako Chemicals. RPMI1640, HEPES, gentamicin, penicillin-G, streptomycin, amphotericin B, fetal bovine serum, and phosphate-buffered saline were purchased from Invitrogen (Carlsbad, CA, USA).Colitis induction
Eight-week-old female BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan) and kept in a 12-h light/dark cycle with controlled humidity (60–80%) and temperature (22° ± 1°C) under specific pathogen-free conditions. Food and water were freely available. All animal experiments were performed according to the guidelines of the Institute for Laboratory Animal Research and with the approval of the ethics committee of the Nagoya University School of Medicine.
To induce colitis, NBD-Cl was intrarectally administered to mice in a similar manner to TNBS and oxazolone (7, 12-14). Briefly, we lightly anesthetized mice with diethyl ether and inserted a rubber catheter (2-mm outer diameter) fitted onto a 1-mL syringe via the anus. The tip was positioned 2 cm proximal to the anus, and then 100 µL 40% ethanol containing 0–2 mg/mL NBD-Cl (0 mg/mL NBD-Cl enemas contained only 40% ethanol) was slowly administered to the mice through the catheter. Mice were kept in a head-down position for 30 s and then returned to their cages. Ethanol (40%) is used to help haptens go through the intestinal epithelial barrier (7, 12-14).