Essential role for mast cell tryptase in acute experimental colitis

Abstract

Patients with inflammatory bowel disease (IBD) have increased numbers of human tryptase-β (hTryptase-β)-positive mast cells (MCs) in the gastrointestinal tract. The amino acid sequence of mouse mast cell protease (mMCP)-6 is most similar to that of hTryptase-β. We therefore hypothesized that this mMCP, or the related tryptase mMCP-7, might have a prominent proinflammatory role in experimental colitis. The dextran sodium sulfate (DSS) and trinitrobenzene sulfonic acid (TNBS) colitis models were used to evaluate the differences between C57BL/6 (B6) mouse lines that differ in their expression of mMCP-6 and mMCP-7 with regard to weight loss, colon histopathology, and endoscopy scores. Microarray analyses were performed, and confirmatory real-time PCR, ELISA, and/or immunohistochemical analyses were carried out on a number of differentially expressed cytokines, chemokines, and matrix metalloproteinases (MMPs). The mMCP-6-null mice that had been exposed to DSS had significantly less weight loss as well as significantly lower pathology and endoscopy scores than similarly treated mMCP-6-expressing mice. This difference in colitis severity was confirmed endoscopically in the TNBS-treated mice. Evaluation of the distal colon segments revealed that numerous proinflammatory cytokines, chemokines that preferentially attract neutrophils, and MMPs that participate in the remodeling of the ECM were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6-null mice. Collectively, our data show that mMCP-6 (but not mMCP-7) is an essential MC-restricted mediator in chemically induced colitis and that this tryptase acts upstream of many of the factors implicated in IBD.

Fig. 1.

mMCP-6⁺ MCs are present in the normal adult mouse colon. (A and B) Chloroacetate esterase histochemistry was used to identify MCs in colonic tissue sections from normal adult B6 mouse colon. Positive cells (red, marked with black arrows) were detected in the mucosal (A) and submucosal (B) layers. Dashed lines indicate layer boundaries. (C and D) Fluorescent immunohistochemistry was used to visualize mMCP-6⁺ MCs in the colon. mMCP-6⁺ MCs (red cells indicated by white arrows) were observed in the mucosa (C), submucosa (D), and serosa (not shown). (Scale bars: 50 μm.)

Fig. 2.

The 6⁻/7⁻ B6 mice are protected from DSS-induced colitis. (A) DSS-treated 6⁻/7⁻ mice lost significantly less of their initial body weight than similarly treated 6⁺/7⁻ mice. Weight loss scores are reported as mean ± SEM. *P = 0.0014; **P < 0.0001. (B) Using a 20-point scoring system, we found DSS-treated 6⁻/7⁻ B6 mice also had significantly lower histopathology scores than the DSS-treated 6⁺/7⁻ mice. Data are presented in a scatter plot.

Fig. 3.

DSS-induced colitis histopathology is less severe in 6⁻/7⁻ mice than in 6⁺/7⁻ mice. (A–E) The colons of the DSS-treated and untreated 6⁺/7⁻ and 6⁻/7⁻ mice were harvested, fixed, and stained. (A) Severe inflammation, in particular ulceration (black arrows), loss of crypts (large black arrowheads), and increased inflammatory infiltrate can be seen in the representative colon from a DSS-treated 6⁺/7⁻ mouse. The majority of the infiltrating inflammatory cells are neutrophils, as assessed at higher power by H&E staining (black arrows in B) and by chloroacetate esterase histochemistry (pink-staining cells indicated by black arrows) (C). In contrast, the histopathology of the colon of the DSS-treated 6⁻/7⁻ mouse in D resembles that of the untreated 6⁺/7⁻ mouse in E. (Scale bars: 50 μm.) (F) There are significant differences in the numbers of neutrophils per high-power field (mean ± SEM) in the colon segments of DSS-treated 6⁺/7⁻ and 6⁻/7⁻ mice. *P = 0.007.

Fig. 4.

DSS-treated 6⁻/7⁻ mice have a more benign endoscopic appearance than DSS-treated 6⁺/7⁻ mice. (A) Representative images from the endoscopy grading scale for the scores 0, 1, 2, and 3. Note the apparent loss of vascularity with a score of 1, the appearance of contact bleeding with a score of 2, and the mucosal erosions in a mouse with a score of 3. (B) Inflammation was quantitated using the endoscopy scale at days 7, 9, and 14. In agreement with the histopathology data, DSS-treated 6⁻/7⁻ mice had significantly lower endoscopy scores at days 9 and 14 than DSS-treated 6⁺/7⁻ mice. Endoscopy scores are reported as mean ± SEM. *P < 0.0001.

Fig. 5.

mMCP-6–null 6⁻/7⁺ B6 mice resemble mMCP-6–null 6⁻/7⁻ B6 mice in the DSS-induced colitis model, whereas mMCP-5–null B6 mice resemble WT B6 mice. (A and B) In two experiments, DSS-treated 6⁻/7⁺ B6 mice displayed significantly less weight loss (A) and histopathology scores (B) than similarly treated 6⁺/7⁻ B6 mice. Weight loss scores are reported as mean ± SEM. *P < 0.0005. Histopathology data are presented in a scatter plot. (C and D) DSS-treated mMCP-5–null B6 mice displayed weight loss (C) and histopathology scores (D) similar to those of DSS-treated 6⁺/7⁻ B6 mice. Weight loss scores are reported as mean ± SEM. Histopathology data are presented in a scatter plot.

Fig. 6.

Levels of the transcripts that encode numerous chemokines, cytokines, and MMPs are reduced in the colons of DSS-treated 6⁻/7⁻ B6 mice. Microarray and real-time PCR assays were performed on RNA isolated from the distal colons of untreated and DSS-treated 6⁺/7⁻ and 6⁻/7⁻ mice to identify transcripts that are expressed differentially in this colitis model at day 9. The resulting GeneChip data (Dataset S1) revealed that many chemokines, cytokines, MMPs, and other proteins that have been implicated by others in patients with IBD were differentially expressed at the RNA level. To confirm and extend these microarray data, real-time PCR approaches were used to compare the levels of the transcripts that encode CXCL1, IL-1β, MMP3, MMP13, IL-6, and SLC26A3 in RNA samples from larger numbers of untreated and DSS-treated 6⁺/7⁻ and 6⁻/7⁻ mice. Data are expressed as the fold change (mean ± SEM) in these groups of mice relative to the untreated 6⁺/7⁻ B6 mice.

Fig. 7.

MMP3 and SLC26A3 protein levels in DSS-treated 6⁻/7⁻ mice relative to DSS-treated 6⁺/7⁻ mice. (A–C) MMP3 protein levels were assessed by immunohistochemistry. As shown in A, there is minimal MMP3 staining in the colonic mucosa and submucosa of untreated WT mice. However, the inflamed mucosal and submucosal layers of DSS-treated 6⁺/7⁻ mice (black arrows in B) have increased staining, corresponding to MMP3, in contrast to the DSS-treated 6⁻/7⁻ mice (C) that resemble the untreated WT mice. (D–F) The same representative mouse groups demonstrating staining for SLC26A3. In agreement with the RNA data, there is decreased expression of SLC26A3 in the DSS-treated 6⁺/7⁻ mice (E) compared with WT untreated mice (black arrows in D) and DSS-treated 6⁻/7⁻ mice (black arrows in F). (Scale bars: 50 μm.)