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A Role of a Lymphocyte Tryptase, Granzyme A, in Experimental Ulcerative Colitis

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A Role of a Lymphocyte Tryptase, Granzyme A, in Experimental Ulcerative Colitis Biosci. Biotechnol. Biochem., 71 (1), 234–237, 2007 Note A Role of a Lymphocyte Tryptase, Granzyme A, in Experimental Ulcerative Colitis y Hirofumi HIRAYASU,* Yumiko YOSHIKAWA,* Satoshi TSUZUKI, and Tohru FUSHIKI Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received June 23, 2006; Accepted October 4, 2006; Online Publication, January 7, 2007 [doi:10.1271/bbb.60354] In the large-intestinal mucosae of rats orally admin- site, and the mucosae from at least two rats (about 2 ng istered dextran sulfate sodium, which induces an enter- of GzmA) were required for detection by binding assay. itis resembling ulcerative colitis (UC), the activity for On the other hand, the activity of GzmA can be granzyme A, a lymphocyte tryptase, increased at an determined sensitively by hydrolysis of N -benzyloxy- earlier stage than that at which UC markers (growth- L-lysine thiobenzyl ester (BLT), a synthetic substrate for regulated gene product/cytokine-induced neutrophil the enzyme,6,8) which is completely inhibited in the chemoattractant-1 and caspase-3) increased. This sug- presence of a recombinant rat PSTI6) (more than 2 mM) gests involvement of the enzyme in the exacerbation and (Fig. 1A). Therefore, of the hydrolysis of BLT by perpetuation of enteritis. samples from large-intestinal mucosae, hydrolysis in which hydrolysis in the absence of PSTI is subtracted Key words: granzyme A; cytotoxic T-lymphocytes; ul- from hydrolysis in the presence of PSTI should cor- cerative colitis; dextran sulfate sodium; respond to that catalyzed by GzmA. In addition, neither pancreatic secretory trypsin inhibitor the binding between GzmA and 125I-labeled PSTI7) nor BLT hydrolysis by a recombinant rat GzmA was A family of serine proteases termed granzymes are inhibited by soybean trypsin inhibitor (SBTI, type I-S, stored in granules of cytotoxic T-lymphocytes (CTLs) Sigma, St. Louis, MO) or leupeptin (Peptide Institute, and natural killer (NK) cells located in peripheral blood Osaka, Japan) (Fig. 1A). Therefore, these protease and lymphoid tissues including the intestines.1) Of these, inhibitors can be included to reduce the consumption granzyme A (GzmA), a tryptase, has been found to of the substrate by proteases other than GzmA (such as participate in the exclusion of abnormal cells such as kallikrein). In this study, mucosal extracts from Spra- virus-infected cells via induction of apoptosis in target gue-Dawley (SD) rats (Japan SLC, Hamamatsu, Japan) cells and/or the stimulation of cytokine (chemokine) were prepared as follows: Mucosa obtained from the production in immune and non-immune cells.1) On the whole large intestine of each rat (about 0.2 g) was other hand, there have been reports suggesting the solubilized on ice for 1 h with 2 ml of a buffer (20 mM involvement of this tryptase in the pathogenesis of HEPES, pH 8.0, and 145 mM NaCl, containing 2% v/v intestinal diseases with severe inflammation and epithe- Triton X-100), followed by gel filtration using a PD-10 lial loss, such as ulcerative colitis (UC).2,3) In this study, column (GE Healthcare, Uppsala, Sweden) equilibrated we investigated the role of GzmA in UC using rats with buffer containing 0.1% (v/v) Triton X-100. Protein orally administered dextran sulfate sodium (DSS), which concentrations of the gel-filtrated sample (mucosal is known to induce an enteritis that closely resembles extract) were determined by a standard method. The UC.4,5) handling of animals was in accordance with the guide- Using pancreatic secretory trypsin inhibitor (PSTI), a lines of Kyoto University for the care and use of candidate physiological inhibitor of GzmA,6) first we laboratory animals. Also, this study was approved by the developed a method of measuring the level of the Kyoto University Animal Committee of the Graduate enzyme in the large-intestinal mucosae of rats. We have School of Agriculture. Representative BLT hydrolysis in found that 125I-labeled purified rat PSTI binds only to a the presence of the extracts is shown in Fig. 1B. Total protein, identical with GzmA, in large-intestinal muco- BLT hydrolysis caused by the addition of the extract sa.7) However, the content of GzmA was very low at the was reduced by about 72% of the original when SBTI * The two authors contributed equally to this work. y To whom correspondence should be addressed. Fax: +81-75-753-6264; E-mail: [email protected] Abbreviations: BLT, N -benzyloxy-L-lysine thiobenzyl ester; CTLs, cytotoxic T-lymphocytes; DSS, dextran sulfate sodium; GzmA, granzyme A; GRO/CINC-1, growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1; IL, interleukin; NK cells, natural killer cells; PSTI, pancreatic secretory trypsin inhibitor; SBTI, soybean trypsin inhibitor; UC, ulcerative colitis Role of Granzyme A in Experimental Colitis 235 (20 mM) and leupeptin (2 mM) were included, which was further reduced by 91% by concomitant inclusion of a recombinant rat PSTI (2 mM). The BLT hydrolysis catalyzed by GzmA increased in proportion to the extracts added in a range of protein concentrations from 0 to 600 mg/ml (r ¼ 0:945, P ¼ 0:0013, as determined by linear correlation analysis) (Fig. 1C). In this method, up to about 50 pg of GzmA in 200 ml of reaction volume could be determined, allowing determination not only from a number of rats and but for other biochemical parameters in the same samples. Male 6-week-old SD rats were given distilled water including 30 g/l of DSS (M.W. ¼ 5;000, Wako Pure Chemicals, Tokyo) ad libitum for the induction of UC- like enteritis.5) The rats were sacrificed before DSS administration (day 0) and at 2, 4, 6, 8, and 10 d after administration. Large-intestinal mucosal extracts from the rats were prepared as described above, except that 20 mM of SBTI was included in the HEPES buffer during extraction and gel-filtration. GzmA activity was deter- mined using 60 ml of the extracts (approximately 100 mg protein included) in 200 ml of reaction volume. In this determination of GzmA activity, SBTI, again, and leupeptin were added such that the protease inhibitors come to final concentrations of 20 mM and 2 mM re- spectively in the reaction volume. The activities of other mucosal proteases, dipeptidyl peptidase IV and leucine aminopeptidase, were determined by the same method, as described previously.9) The level of growth-regulated gene product/cytokine-induced neutrophil chemoattrac- tant-1 (GRO/CINC-1)10) and caspase-3 activity11) in the mucosal extracts were determined with the Rat GRO/ CINC-1 Assay Kit (Immuno-Biological Laboratories, Fujioka, Japan) and with the Caspase-3 Colorimetric Activity Assay Kit (Chemicon International, Temecula, CA), respectively. Under our experimental conditions, the days needed Fig. 1. Development of a Method of Measuring GzmA Activity in for appearance of hematochezia, a representative UC- Large-Intestinal Mucosal Extracts of Rats. like symptom, were 7:3 Æ 0:7 d(mean Æ S.D.). This A, PSTI but not SBTI or leupeptin inhibits the BLT hydrolysis catalyzed by GzmA. A recombinant GzmA (about 5 ng)6,8) was result and weight gain rates of the DSS-treated rats incubated with 200 mM BLT and 500 mM dithiobis(2-nitrobenzoic during the whole study period (data not shown) were 4) acid), in a buffer (20 mM HEPES, pH 8.0, 145 mM NaCl, and 0.1% similar to those reported previously. In the present v/v Triton X-100) in the presence or absence of the indicated study, we determined the mucosal level of GRO/CINC- protease inhibitors at 25 C in a final volume of 200 ml: recombinant 1 as a marker for UC-like enteritis. GRO/CINC-1 is re- rat PSTI (open circles); SBTI (closed circles); leupeptin (open triangles). Absorbance at 405 nm was measured with a microplate presentative of a complex family of pro-inflammatory reader. The activity of recombinant GzmA in the absence of each cytokines known as chemotactic cytokine, which be- inhibitor was taken as 100%. Values are means Æ SEM of three longs to the IL-8 family.10) In rats, since the IL-8 gene separate experiments performed in duplicate. B, Representative BLT has not been identified yet, GRO/CINC-1 is used as an hydrolysis by large-intestinal mucosal extract. One hundred ml of the IL-8-like cytokine. GRO/CINC-1 is known to increase extract was added to the same volume of the HEPES buffer with or without 2 mM of PSTI, 20 mM of SBTI, and 2 mM of leupeptin (Leup). in the intestinal mucosae of rats administered DSS and BLT hydrolysis without any inhibitors was taken as 100%. BLT to correlate with the macroscopic grade and infiltrated hydrolysis catalyzed by GzmA in the extract, which was inhibited neutrophil numbers of local inflammation.5) As shown in in the presence of PSTI, is indicated as GzmA activity. C, Dose- Fig. 2A, the mucosal level gradually increased. Statisti- dependent increases in BLT hydrolysis catalyzed by GzmA in the cally significant increases compared to the value at day 0 extracts. The extract was added at the indicated protein concen- trations in a final reaction volume of 200 ml. GzmA activity was were found at day 8 and day 10. All these results calculated as shown in panel B, and expressed as the amounts of indicate the induction of UC-like enteritis under our BLT hydrolyzed in 1 min. Each value was expressed as the mean of experimental conditions. Caspase-3 is one of the key four separate assays performed in duplicate. enzymes for the induction of apoptosis, and enhanced 236 H. HIRAYASU et al.
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