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Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997), pp. 1969 ± 1980

Protease Activity in a Hapten-Induced Model of Ulcerative Colitis in Rats JAMES V . HAWKINS, DVM, MS, EV A L. EMMEL, BS, JENNIFER J. FEUER, BA, MARK A. NEDELMAN, MS, CATHERINE J. HARVEY, BV Sc, HILTON J. KLEIN, V MD, MS, HARRY ROZ MIAREK, DVM, PhD, ANN R. KENNEDY, DSc, GARY R. LICHTENSTEIN, MD, and PAUL C. BILLINGS, PhD

In¯ ammatory bowe l dise ase (IBD) is a painful and de bilitating condition affe cting the mucosal lining of the colon and othe r areas of the gastrointe stinal tract. IBD gene rally falls into two major cate gorie s: ulce rative colitis (UC) and Crohn’ s disease. We have utilize d dinitrobe nzene sulfonic acid (DNBS) to induce e xpe rime ntal UC in rats. Histopathologic analysis indicate s that DNBS induce s a condition in anim als similar to human UC. Bioche mical results reve aled 6- to 10-fold e le vate d le vels of se rine prote ase activity in colon tissue from animals with UC as compare d with matche d controls. We also obse rve d e le vate d le vels of prote ase activity in tissue samples obtaine d from human patie nts with UC. Hence , our re sults de monstrate that prote ase activity is increased in rode nt and human UC. The se prote ase s may play a signi® cant role in de struction of colonic tissue in IBD. Protease inhibitors that targe t se rine prote ases may be use ful pharmacologic al age nts to limit tissue de struction in IBD. KEY WORDS: protease activity; ulce rative colitis.

In¯ ammatory bowel dise ase s were de scribed as early as the 19th century and subse que ntly divide d into two major cate gorie s, ulcerative colitis and Crohn’ s disease (re vie wed in 1 and 2). There is a small subse t of IBD cases that doe s not fall into eithe r group, as well as some othe r rare dise ase s that are classi® e d as type s of IBD. In the Unite d State s, it is estimated that 1± 2 Manuscript received October 9, 1996; accepte d May 2, 1997. From Laboratory Animal Medicine, De partme nts of Radiation Oncology and Pathology, Gastroe nterology Division, De partme nt of Medicine, School of Me dicine, De partment of Pathology, School of Dental Medicine , Unive rsity of Pennsylvania, Philade lphia, Pennsylvania 19104; Centocor, Inc. Toxicology & Preclinical Pharmacology, 200 Gre at V alle y Parkway, Malve rn, Pennsylvan ia 19355; and Laboratory Animal Re sources, Merck Re se arch Laboratories, Building 44-201, We st Point, Pennsylvania 19486. This project was supported in part by NIH grant CA46496 and a Fe llowship from Merck Re search Laboratories, West Point, Pennsylvania. Jame s V . Hawkins’ pre se nt addre ss: Bioresources, He nry Ford Hospital, 2799 W. Grand Blvd., Detroit, Michigan 48202. Address for reprint re quests: Paul C. Billings, De partment of Pathology, School of De ntal Me dicine, Unive rsity of Pennsylvania, 4010 Locust St., Philadelphia, Pennsylvania 19104.

million pe ople are affe cted, with 20,000 ± 25,000 ne w case s annually with incide nce e stimates of 1± 10 pe r 100,000 (3). Advance s in the clinical manage ment of IBD will re quire new insights into disease pathoge nesis. UC is a disease that affects the mucosal lining of the colon (1, 2, 4). Endoscopic and histopathologic evaluation of UC patie nts show a symme trical, continuous in¯ ammation of the colonic mucosa (4). The disease usually starts in the re ctum and progre sse s towards the cecum. The condition is continuous, although it may appe ar to be patchy. The re is a cyclical patte rn of erosion and in¯ ammation, followe d by he aling in some patie nts, and there is ofte n an in¯ ammatory exudate on the mucosal surface (1, 2, 4). The acute phase of UC is characte rize d by crypt absce sse s and ulce rations exte nding to the muscularis laye r with a promine nt in® ltration of in¯ ammatory cells. The chronic phase has additional ® brosing and thicke ning

1969

Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997) 0163-2116/97/0900-1969$12.50/0 Ñ

1997 Plenum Publishing Corporation

HAWKINS E T AL

of the gut wall with lymphoplasm acytic in® ltration and distortion of the mucosal archite cture (4). While the cause of IBD is not known, a number of factors have bee n implicate d. These include a varie ty of bacte ria and viruse s, a ge ne tic pre dispositio n (the re is a familial tende ncy) , metabolic de fe cts, mucosal defe cts, immunologic al disturbance s, T-ce ll activation, autoimm unity, intrace llular signaling, alte red leve ls of spe ci® c in¯ ammatory mediators and cytokine s, and dietary factors (1, 2, 5± 8). Whate ve r the pre cipitating factor, the pathoge ne sis of IBD is gre atly in¯ ue nce d by the in¯ ammatory re sponse mounte d by the host. Se veral studie s have establishe d a pote ntial link betwe en prote ase and prote ase inhibitor leve ls and progre ssion of IBD. Incre ase d fe cal prote ase activity has bee n obse rve d in UC patie nts and was corre late d with dise ase se verity (9). Most of the activity was de rive d from se rine prote ase s of pancre atic and granulocytic cell origin and was inhibite d by DFP, a pote nt serine prote ase inhibitor. The le ve l of prote ase activity was also correlate d with dise ase se verity (9). Decrease d le vels of the endoge nous a 2-macroglobulin have bee n observe d in patie nts with active IBD (10) . 5-Aminosalic ylic acid has bee n shown to block de struction of e ndoge nous a 1 -prote inase inhibitor, a spe ci® c antagonist of ne utrophil elastase (11) . Inhibition of elastase may he lp explain the the rape utic bene ® t of this drug. Furthe r, the amount of pancre atic secretory trypsin inhibitor (PSTI) , a 56-amino acid se rine prote ase inhibitor prese nt in the intestinal lume n, is lowe r in colonic tissue from UC and Crohn’ s disease patie nts (12) . A reduction in PSTI leve ls could rende r the intestinal mucosa le ss re sistant to prote olytic attack and more susceptible to in¯ ammation. Inte restingly, camostat mesilate , a se rine prote ase inhibitor, induce d long-te rm re mission in two patie nts with re fractory UC (13). Camostat mesilate could act by inhibiting spe ci® c in¯ ammatory prote ase s, the re by de creasing tissue damage . Animal mode ls have be en utilize d to acquire insights into IBD pathoge ne sis. A hapte n-induce d animal mode l has bee n de velope d base d on the the ory that an antige n hype rsensitivity compone nt plays a role in the etiology of in¯ ammatory bowe l disease. This model uses e thanol to remove the prote ctive mucus coating and the hapte n 2,4,6-trinitrobe nze nesulfonic acid (TNBS), give n as an ene ma, to induce a chronic colitis in rats (14, 15) . The TNBS model produce s acute and chronic in¯ ammation of the colon with in® ltration of neutrophils, macrophage s, lymphocyte s, and mast cells and produce s acute and

1970

chronic pathology more close ly re sembling Crohn’ s disease than UC. Unfortunate ly, TNBS is an e xplosive and hazardous mate rial, no longe r available in the United State s. An alte rnative mode l, using dinitrobe nzene sulfonic acid ( DNBS) has be e n de ve lope d by Ce ntocor (Malve rn, Pe nnsylvania) . DNBS is less hazardous than TNBS and can be used safe ly in a well-ve ntilate d room with personne l wearing prote ctive glove s, clothing, and goggle s. The DNBS model produce s acute and chronic in¯ ammation and ulceration in the colon similar to TNBS. Both compounds bind to prote ins, but TNBS has an additional active nitro group and binds more re adily at lowe r conce ntrations. Howe ver, DNBS is more se le ctive and binds only to the e -amino groups of lysine (16). In the current inve stigation, DNBS was use d to produce IBD in rats. Histopathologic al analysis indicate s that DNBS produce s a condition close ly rese mbling human UC. We used this model to analyze prote ase activity in diseased colonic tissue . O ur results demonstrate that se rine prote ases are signi® cantly e le vate d in UC tissue vs normal colon. Hence, our re sults indicate that prote ase le vels are e le vate d in UC and thus may play a role in the e xtensive tissue damage in IBD. MATERIALS AND METHODS Ch em icals. DNBS was purchased from Aldrich Chemical Co (St. Louis, Missouri). Ge latin was purchased from DIFCO (Detroit, Michigan). EDTA, E TO H, DFP, NEM, odianisidine dihydrochloride, trypsin and pepstatin we re obtained from Sigma. Methoxy¯ urane was supplied by Pitman-Moore (Washington Crossing, New Jersey) . Anim als. These studies we re approved by the Institutional Re view Board of the University of Pe nnsylvania and the Animal Care and Use Committe es of the Unive rsity of Pe nnsylvania and Ce ntocor. Sprague-Dawley rats (250 ± 300 g; virus-antibody-fre e) were obtained from Charles Rive r Laboratories, housed in microisolator cages on aspen chip bedding, and maintaine d in accordance with the Guide for the Care and Use of Laboratory Animals. Rats were fed Laboratory Rodent Diet # 5001 from Purina Mills (Richmond, Indiana). The rats we re divided into four groups: (1) procedural or negative control group, (2) control group give n saline, (3) ve hicle control group (0.25 ml of 50% EtOH), and (4) DNBS group (30 mg DNBS dissolved in 0.25 ml of 50% EtO H) . The compounds we re administered by enema with a me tal gavage tube inserte d rectally until the tip was 8 cm from the anus, which is approximate ly the location of the splenic ¯ e xure. The animals were lightly anesthetized with me thoxy¯ urane for e nema installation and killed with CO 2 at the e nd of the study. Body we ights we re compared for the saline (N 5 16), EtOH (N 5 16) , and DNBS (N 5 20) treatme nt groups. A baseline body weight was taken at day 0 (range: 250 ± 300 g) Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

PRO TEASE ACTIVITY IN ULCERATIVE CO LITIS T ABLE 1. B REAKDOWN

OF

A NIMALS U SED

Day killed*

Treatm ent group

Anim als per group (N)

1

3

7

14

Analysis

Untreated Saline EtO H² DNBS Untreated Saline EtO H DNBS

3 6 17 17 3 5 15 15

1 1 5 5 1 1 5 5

1 1 2 2 1 0 0 0

1 2 5 5 1 2 5 5

0 2 5 5 0 2 5 5

Histopathology Histopathology Histopathology Histopathology Protease Protease Protease Protease

* Day afte r the e nema when animals were sacri® ced. ² EtO H is use d as the solvent vehicle to administe r DNBS.

and subsequently on days 3 and 7. Data we re analyze d using a two-way ANOV A with repeate d me asures and Duncan’s multiple range te st. Histop ath ology of DNBS Model. Animals in each tre atme nt group were killed on day 1± 14, afte r the e nema (Table 1) . The colon was dissecte d fre e with approximately 8 cm of colon remove d (with the lesion approximately in the center) and opened longitudinally. Feces we re removed, and the tissue was rinsed in saline. The colon was cut lengthwise and half was placed into neutral-buffered 10% formalin and the remaining tissue was frozen in Dry Ice and stored ( 2 80 °C). The formalin-® xe d colon tissue was paraf® n e mbe dded and stained with hematoxylin and eosin (17). A sample from each e nd and two samples from the center of the colon we re examined. Histological scoring was done on coded samples by a pathologist who was unaware of the time point or treatme nt status of the tissue. This scoring system is an adaptation from an e arlier report (18) . Data we re analyzed with a two-way ANOV A with a randomized block design. Determ in ation of Myelop eroxidase Activity. To ascertain neutrophil in® ltration, mye loperoxidase (MPO ) assays (19) we re performed on colon tissue from animals in each tre atme nt group at days 3 and 7 afte r the enema. Tissues we re homogenized in 50 mM phosphate (pH 6.0), 0.5% hexadecyltrimethylammonium bromide (HTAB) buffe r (50 mg tissue/ml), and centrifuged (5000g, 7 min). The supernatant fraction was analyze d for MPO activity. E nzyme s assays contained 350 m g of tissue e xtract, 0.11 M o-dianisidine dihydrochloride, and 0.0005% H 2 O 2 . The reactions we re monitored at 460 nm. One unit of activity e quals 1 m M peroxide/min at 25 °C. Data were analyzed using a two-way ANO VA. Colon Sam pling an d An alysis for Proteas e Activity. Rats in each tre atme nt groups we re killed on days 1± 14, afte r the enema (Table 1) . Samples from animals with normal colonic tissue we re taken from the middle of the colon segme nt. For animals with UC, tissue samples we re taken from the center of the ulcer (C), 1 cm from the cente r (I), and 2 cm from the cente r (E). For analysis, a small piece of tissue was collected from a section of colon, weighed, and placed in a 2-ml microfuge tube with cold PBS. The tissue was homogenized (10 sec, on ice) with a Tekmar Ultra-Turrax homogenizer (Cincinnati, Ohio) and subsequently sonicated for 10 sec on ice. The homogenate was centrifuged (14,000g, 5 min at 5°C) and the supernatant fraction was analyze d for protease activity. Protein concentration in Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

tissue extracts was dete rmined with the Bio-Rad Protein Assay. Protease activity present in colonic tissue was dete rmined on gelatin zymograms. Brie¯ y, 12% SDS polyacrylamide gels of 0.75 mm thickness (20) we re prepared containing 0.1% gelatin (21). Stock solutions of ge latin (2% ) we re prepared in distilled wate r. An e qual amount of protein from e ach sample (10 ± 25 m g) was applied to the gel in standard SDS-ge l loading buffer containing 0.1% SDS but lacking b -me rcaptoethanol and samples we re not boiled prior to loading. The ge ls we re run (200 V for 1 hr) in a Bio-Rad Mini Protean II apparatus and then soaked in 200 ml of 2% Triton X-100 in distilled wate r on a gyratory shaker (15 min, 20°C). Next, the ge ls were incubated in reaction buffe r [50 mM Tris (pH 8.0), 1 mM CaCl2 ], unless otherwise indicated, for 12 hr at 37 °C and the n stained in amido black and subsequently destained. Protease activity shows up as clear bands (indicative of cleavage of the gelatin substrate) on a blue background. For dete rmination of pH optima, the zymograms were incubated in: 50 mM acetate buffer (pH 5.2), 50 mM Tris (pH 8 and 9), or 50 mM glycine± NaO H (pH 10.2) (22), containing 1 mM CaCl2 . Protease activity was quantitated by densitometry using an NIH image graphics program (ve rsion 1.54) on a Macintosh computer. Statistical analysis for protease activity was performed using one-way ANO VA. Hu m an Sam p les. IRB approval was obtained for studies involving human tissue. Human colonic biopsy samples of normal and involved regions of the colon were obtained, afte r informed consent was obtained, from patie nts with ulcerative colitis (see n in the Departme nt of Me dicine, Hospital of the University of Pennsylvania). The samples we re immediately placed on ice, brought to the laboratory and stored at 2 80°C. For analysis, specimens were prepared in the same manner as rat colon tissue. Data Analysis. Statistical analysis of data was carried out using statistics programs from SAS (Statistical Analysis Syste m, Cary, North Carolina) or SPSS (v. 6.1.2 for Windows; SPSS Inc., Chicago, Illinois).

RESULTS To assess the e ffe ct of e ach treatment on the animals, body weights were de te rmine d at de ® ne d time pe riods afte r the e nema. In the saline control group, TABLE 2. C HANGE

IN

A NIMAL B ODY W EIGHT T IME A FTER T REATMENT

Day weighed after treatm ent

Treatm ent

3 3 3 7 7 7

Saline ETO H DNBS Saline ETO H DNBS

AS A

FUNCTION

OF

Chan ge from baseline* 15 6

2 5.6 6

2 28.4 6 39.5 6 16.9 6 2 1.5 6

1.3 2.4 1.8 3.0 2.8 2.7

* Baseline body weight (g) is de® ne d as animal we ight at day 0. Change from base line indicates the change in body weight (g) from day 0. There we re 16 ± 20 rats/group on day 3 and 13± 16 rats/group on day 7.

1971

HAWKINS E T AL T ABLE 3. H ISTOPATHOLOGY Time

Mucosa

OF

C OLONIC T ISSUE

Lam ina propria

FROM

E T OH-T REATED R ATS

Subm ucosa

Muscularis layers/serosa

Day 1

ulcerate d

ede ma and neutrophils

ne utrophils, plasma cells, macrophage s, e osinophils, edema, he morrhage and acute vasculitis with mural ne crosis

neutrophils and me sothelial ce ll hype rtrophy

Day 7

epithelial rege ne ration with cuboidal to columnar ce lls ove r areas of previous ulceration; crypt and goble t cell hype rplasia with branching glands

neutrophils, plasma ce lls, lymphocyte s, ® broblasts, edema, and lymphangiectasia

ne utrophils, plasma cells, macrophage s, e osinophils, mature granulation tissue, and lymphangie ctasia

neutrophils and me sothelial ce ll hype rtrophy over the se rosa

Day 14

he aling; dilation and branching of glands, crypts and goble t ce ll hype rplasia

lymphocytes, plasma ce lls, scattered neutrophils and ® brosis

plasma ce lls, lymphocyte s, e osinophils, ® brosis, he mosiderosis and lymphangie ctasia

me sothelial ce ll hype rtrophy

the anim als had gaine d 15 g on day 3, while the EtO H and DNBS groups lost 5.56 g and 28.4 g, respe ctively. There were signi® cant diffe rences among the three groups and among the diffe re nt time points (Table 2).

Colon tissue from anim als in e ach group was e xamine d histologically. As expe cted, tissue from animals in the negative control group had no abnormal histopathological ® ndings. The saline control colons

T ABLE 4. H ISTOPATHOLOGY

OF

Mucosa

Lam ina propria

Subm ucosa

Day 1

ulceration, coagulation necrosis, ® brin, hemorrhage , neutrophils, and minimal epithelial ce ll regene ration by ¯ attened cuboidal cells

neutrophils, edema and hemorrhage

neutrophils, eosinophils, macrophage s, plasma cells, e de ma, hemorrhage , acute vasculitis with mural necrosis, ® brin thrombi and prominent endothelial cells

Day 7

® brin and neutrophils if the e ntire se ction was ulcerate d, or e pithelial re ge neration by cuboidal to columnar cells and adjace nt crypt and goblet ce ll hype rplasia with branching and dilation of glands

lymphocytes, macrophages , e osinophils, mature granulation tissue, hemosiderosis and lymphangie ctasia

Day 14

® brin if the e ntire section was ulce rated, or epithelial rege ne ration by cuboidal to columnar ce lls and adjace nt crypt and goblet cell hype rplasia with branching and dilation of glands

lymphocytes, plasma cells, few neutrophils, and ® brosis

1972

C OLONIC T ISSUE

FROM

DNBS-T REATED R ATS Muscularis/serosa neutrophils, macrophage s, ® brin thrombi, coagulation necrosis, and promine nt endothelial cells

neutrophils, macrophage s, lymphocytes, mature granulation tissue and mesothe lial ce ll hype rtrophy over the serosa

few neutrophils, plasma cells, lymphocyte s, macrophage s, eosinophils, mature granulation tissue, ® brosis, and hemoside rosis

few neutrophils, macrophage s, lymphocytes, plasma cells, mature granulation tissue, and mesothe lial ce ll hype rtrophy over the serosa

Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

PRO TEASE ACTIVITY IN ULCERATIVE CO LITIS

Fig 1. UC Lesion. Gross appe arance of a re prese ntative tissue section of colon seve n days afte r DNBS administration. Note the ulce ration in the ce nter with diminishing se verity toward the periphery.

had a fe w scatte red ne utrophils in the lamina propria. The day 1 ethanol control mucosa was ulce rated, but by day 7, the ethanol control mucosa was de veloping epithe lial regene ration with cuboidal to colum nar cells over are as of previous ulceration. Afte r 14 days, the EtO H colonic mucosa was he aling and e xhibite d dilation and branching of glands, crypts and goble t cell hype rplasia (Table 3).

Tissue from DNBS-tre ate d animals had more se vere le sions than e ither saline or EtO H controls (Table 4). The day 1 DNBS mucosa had ulceration, coagulation ne crosis, ® brin, hemorrhage , ne utrophils, and minimal epithe lial cell re ge ne ration by ¯ atte ne d cuboidal cells. By day 7, the colonic mucosa containe d ® brin and ne utrophils whe re the e ntire section was ulce rated, or e pithe lial re ge neration by cuboidal to

Fig 2. Tissue section of colon afte r DNBS administration. This is a re presentative section of colon obtained from a rat seve n days afte r treatmen t with DNBS. The mucosa was ulcerate d and containe d ® brin, ne utrophils, epithelial re ge neration, and adjace nt crypt and goblet ce ll hype rplasia with branching and dilation of glands. Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

1973

HAWKINS E T AL T ABLE 5. COMPARISON

OF

H ISTOPATHOLOGY OF U LCERATIVE C OLITIS WITH DNBS MODEL

Mucosa/lamina propria Multifocal ulceration/ne crosis Hemorrhage Granulation tissue E pithelial re generation Crypt and goble t ce ll hype rplasia Gland branching Goble t ce ll mucin de pletion Crypt absce sse s Ne utrophils Macrophage s E osinophils Lymphoplasmac ytic in® ltrate Granulomas Submucosa Hemorrhage Granulation tissue Fibrosis E dema V asculitis Ne utrophils Macrophage s E osinophils Lymphoplasmac ytic in® ltrate Granulomas Muscularis laye rs Granulation tissue Ne utrophils Macrophage s E osinophils Lymphoplasmac ytic in® ltrate Se rosa In¯ ammation

IN

H UMANS

Ulcerative colitis*

DNBS model

X X X X X X X X X X X X none

X X X X X X X X X X X X none

X X X X X X X X X none

X X X X X X X X X none

rare rare rare rare rare

X X X X X

none

X

* X indicates that a characteristic is present. Information on human ulce rative colitis was compiled from re fere nce 4.

columnar cells. Tissue obtaine d on day 14 containe d ® brin whe re the entire section was ulcerate d, or e pithe lial re ge ne ration. Examples of the gross appe arance and histopathology of DNBS-tre ated tissue s are prese nte d in Figure s 1 and 2. Except for the lack of granulomas and mast T ABLE 6. MPO A CTIVITY IN C OLON T ISSUE AS T IME P OST TREATMENT * G roup

Posttreatm ent analysis (day)

Treatm en t group

1 2 3 4 5 6

3 3 3 7 7 7

Saline EtO H DNBS Saline EtO H DNBS

A

FUNCTION

OF

MPO activity (units/g tissue)² 3.2 28.9 23.3 2.7 21.4 24.2

6

6

6

6

6

6

1.2 4.5 0.8 0.6 3.5 2.2

* Animals were tre ated as indicated and afte r three or se ven days, animals we re killed and MPO activity was dete rmine d in colon tissue. ² Statistical analysis (two-way ANO VA): group 1 vs 2 or 3, P , 0.0001; group 4 vs 5 or 6, P , 0.0001.

1974

cells, this model is similar to the pre viously de scribe d TNBS mode l (14) . In the TNBS model, a granuloma was de ® ne d as a colle ction of ® ve epithe lioid macro-

Fig 3. Assay sensitivity. Known amounts of trypsin were run on a 12% polyacrylamide gel containing 0.1% ge latin as substrate. The gel was incubated in 50 mM Tris HCl (pH 8.0) , 1 mM CaCl2 , and staine d. Lane s 1± 8 contain 0 ± 100 ng trypsin, re spectively. Note that 1 ng of trypsin is re adily dete ctable in this system. Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)


Fig 4. Prote ase activity in normal tissue. Colon tissue was obtained from an untreated rat and the fe cal pe llets were re move d. The colon was cut into two pieces and one piece was rinsed three times in PBS (washed) . The samples were homogenize d and analyze d for protease activity on gelatin zymograms. 2 , untreate d sample ; 1 , sample was incubate d with trypsin prior to analysis. Note that normal colon tissue has little protease activity de tectable in this assay. Furthe r, rinsing the tissue with PBS, prior to analysis, has little e ffect on protease levels. Numbe rs on right, molecular mass in kilodaltons.

Fig 5. Prote ase activity in DNBS-treate d animals. Colon tissue sample s were obtained from animals on days 1, 7, and 14 after DNBS treatme nt and analyzed on ge latin zymograms. Protease activity increase d at day 1, was maximal at day 7, and declined by day 14. Colon tissue from an untreate d animal (C) is presented in lane 1. The ticks to the right of each column are the mole cular mass markers for that sample (112, 53, and 20 kDa, respe ctive ly); numbe rs on the right indicate molecular mass in kilodaltons.

phage s with or without Langhan’ s-type giant cells, ne crosis, or foreign bodie s (14) . The histopathologic fe ature s of the DNBS-induce d UC in rats are similar to the disease in humans (Table 5). The histopathologica l analysis reve aled that colon tissue from DNBS animals is signi® cantly more damage d than that in EtOH tre ated animals (P , 0.0066) but the differe nce starts to diminish by day 7. With the cente r sections grade d over time , there is a signi® cant incre ase in seve rity (P , 0.0001) from day 1 vs days 3, 7, and 14. There is not, howeve r, a signi® cant difference in le sion scoring among days 3, 7, and 14. With the e dge sections there is a signi® cant differe nce (P , 0.017) betwe en days 1 and 3 and day 14, but no diffe re nce be twee n the othe r time points. To asse ss leukocyte in® ltration into the tissue , mye lope roxidase (MPO ) activity was de te rmine d. MPO is pre sent in the azurophilic granule s of neutrophils and has be en use d as a quantitative measure of in¯ ammation in intestinal tissue (19) . MPO assays were pe rforme d on colon tissue from animals obtaine d on day 3 or 7 after the e nema. There was an approximate ly seve n fold increase in MPO activity in the EtO H or DNBS groups over the saline group, but

little differe nce betwe en the EtO H and DNBS groups on days 3 and 7 (Table 6). In the next series of expe riments, prote ase activity in rat colon tissue was analyze d. To e stablish assay sensitivity, de® ne d amounts of trypsin were run on a zymogram. Under the conditions utilize d, we can readily detect 1 ng of trypsin (Figure 3). For analysis of rat colon, tissue e xtracts were prepare d from control and tre ated anim als and analyze d for prote ase activity on gelatin zymograms. Colon tissue from normal (untre ate d) rats has little inhe re nt prote ase activity (Figure 4). To de te rmine if the normal bacte rial ¯ ora of the colon has a signi® cant in¯ ue nce on the assay, a rat colon was disse cted out and the fecal pellets were manually re moved. The colon was cut into two pie ces, and one piece was directly homoge nized (unwashe d) while the re maining piece was rinsed three time s in PBS (washe d). The le ve l of prote ase activity was similar in the washe d and unwashe d sample s (Figure 4). We also asse sse d whether colon tissue contains late nt activity, as some prote ases are synthe size d in a pro-form and require prote olytic processing for activity. For the se e xpe riments, the sample s were incubate d with trypsin, prior


1975

HAWKINS E T AL

Fig 6. Prote ase activity as a function of time and location. Colon sample s were obtained from animals at 1, 7, and 14 days after DNBS tre atme nt and analyzed on gelatin zymograms for protease activity. C, tissue from ce nter of the lesion or ulcer; I, interme diate piece 1 cm from the center; E, end piece, 2 cm from the cente r of the lesion. Prote ase activity incre ases at day 7 and drops by day 14. NE G, colon tissue from untreate d animal. Numbers on the right indicate molecular mass in kilodaltons.

to being run on the zymogram . Trypsin pretreatment had no e ffe ct on prote ase activity. Tissue from animals tre ated with DNBS was e xamined next. Tissue sample s were obtaine d from control and DNBS-treate d animals at 1, 7, and 14 days afte r tre atme nt and assaye d for prote ase activity (Figure 5). The amount of prote ase activity increases at day 1, pe aks at day 7, and is still e le vate d at day 14, when the le sion is starting to heal. The se results de monstrate that prote ase activity is signi® cantly e le vate d in animals with UC. High leve ls of prote ase activity were also obse rve d in tissue obtaine d from the center of the lesion and in adjace nt tissue 1± 2 cm from the cente r (Figure 6). We ne xt evaluate d the e ffect of mechanical trauma resulting from the e nema and EtO H, which is the

1976

Fig 7. Prote ase activity in differe nt treatme nt groups. Colon tissue was obtained from animals that were untreated or receive d an ene ma consisting of saline, EtO H, or DNBS. Low levels of protease activity were prese nt in tissue obtained from control animals (lanes 1± 3). Howe ver, the DNBS-treate d rats (lane 4) show a ® ve- to six-fold increase in protease activity over the controls as we ll as having the most seve re lesions on histopathology. The se re sults also indicate that e thanol or the mechanical trauma of giving the enema (saline) doe s not cause a major increase in protease levels. Numbe rs on the right indicate molecular mass in kilodaltons.

solve nt ve hicle used to administe r DNBS. Low leve ls of prote ase activity were obse rved in colon tissue obtaine d from the se anim als (Figure 7). The se results sugge st that ele vate d prote ase activity in DNBStre ated animals is a conse que nce of the in¯ ammatory reaction in the colon, induce d by the DNBS, rathe r than mechanical trauma or e thanol. The prote ase activity pre se nt in colon tissue obtaine d from DNBS-tre ate d animals was characte rized furthe r. Tissue e xtracts were run on gelatin zymogram s; the zymograms were incubate d in 2% Triton X-100 and subse que ntly incubate d in buffers of increasing pH. Maximal prote ase activity was obse rved at pH 8 ± 10 (Figure 8). To de te rmine the type (s) of prote ases prese nt in UC tissue , the e ffect of se ve ral spe ci® c prote ase inhibitors on e nzyme activity was de termined. BBI and DFP (inhibitors of se rine prote ase s) had the most pronounce d effect, inhibiting . 80% of prote ase activity. In contrast, EDTA (an inhibitor of metalloprote ases), pe pstatin (an inhibitor of aspartic prote ases), and NEM (thiol prote ase inhibitor) showe d le ss effect (Figure 9). These results Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)


DISCUSSION

Fig 8. Prote ase activity as a function of pH. Colon tissue obtained from an animal seve n days afte r DNBS treatment was analyze d for protease activity. After running, the zymograms were incubated in buffer at the indicated pH. Note that maximal protease activity is obse rved at pH 8 ± 10. Numbe rs on the right indicate mole cular mass in kilodaltons.

indicate that the majority of prote ase activity observe d on the zymograms is due to se rine prote ases. The e xpe rime nts with rat colon de monstrate d gre atly elevate d le ve ls of prote ase activity in UC tissue . To determine whe the r a similar situation also occurs in humans, we e xamine d human colonic mucosa for prote ase activity. For these studie s, biopsy spe cimens were obtaine d during routine endoscopic examination of patie nts with ulce rative colitis and consiste d of small pie ces of tissue take n from the colonic mucosa from involve d and normal-appe aring are as. Signi® cantly e levate d le vels of prote ase activity were pre sent in tissue obtaine d from involve d are as of the colon (Figure 10) . We also obse rved signi® cantly elevate d le vels of a spe ci® c prote ase (mass of ; 44 kDa) in involve d are as. Tre atme nt of sample s with trypsin (prior to running on the zymograms) reve ale d that this prote ase is pre sent in all samples. These results sugge st that this prote ase is prese nt in normal colonic mucosa, but be come s activate d in UC le sions. Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

In this re port, we de scribe the use of DNBS to induce IBD in rats. The DNBS model has an in¯ ammatory response in rats similar to the TNBS model use d pre viously. Both mode ls have crypt abscesse s and about half of the TNBS rats have granulomas (14) , whe reas no granulom as are see n with DNBS. Our observations indicate the DNBS mode l produce s a colitis more like UC than Crohn’ s dise ase (Table 5). The histopathologica l scoring of tissue damage also follows a logical patte rn. Afte r the initial damage , the re is a pe riod of acute , the n chronic in¯ ammation, followe d by tissue rege neration be ginning at about day 7. As in the TNBS mode l, DNBS induce s a strong in¯ ammatory response and a signi® cant incre ase in MPO activity compare d to controls. The re is also weight loss during the ® rst wee k (up to 10% ) that is similar to that reporte d afte r TNBS adm inistration (14) . Since TNBS is no longe r available in the Unite d State s, DNBS should be an alte rnative compound for IBD studie s. Furthe r, DNBS is safe r than TNBS and the refore, e asier to handle . Prote ase activity was marke dly incre ased in DNBStre ated animals and was maximal in colon tissue seve n days after DNBS administration. Protease activity was highe st at pH 8 ± 10 and the inhibitor studie s de monstrate that the majority of the activity visualize d on the zymograms re sults from se rine prote ase s. The elevation of se rine prote ase activity in rat UC correlate s well with re ports of e le vate d fe cal serine prote ase activity in human UC patie nts (9, 10) and elevate d prote ase le vels in human biopsy sample s (Figure 10) , re porte d in this study. An intriguing ® nding was the ef® cie nt inhibition of prote ase activity by the BBI, a soybe an-de rive d prote ase that inhibits trypsin and chymotrypsin. BBI has bee n shown to block tumor de velopme nt in se ve ral anim al mode l systems including dimethyhydrazine induce d colon carcinoge ne sis in mice (23) . Although the mechanisms of anticance r activity by BBI are not known, we have hypothe sized that it inhibits speci® c prote ases involve d in the tumor cell deve lopme nt (21) . We have identi® e d a prote ase of approximate ly 45 kDa that is inhibite d by BBI. This prote ase has bee n observe d in a numbe r of differe nt cell type s including human and murine ® broblasts (21, 24) and a human bre ast cance r cell line (25) . An intere sting prope rty of this prote ase is that it require s trypsin tre atme nt to be observe d on ge latin zymograms, sugge sting that it is synthe size d as a proe nzyme and require s prote olytic processing to become function-

1977

HAWKINS E T AL

Fig 9. E ffects of inhibitors on protease activity. UC sample s were obtained from animals se ven days after DNBS tre atment and analyzed on ge latin zymograms. After e lectrophoresis, the zymograms were incubated in re action buffer containing the indicated protease inhibitor. BBI and DFP (inhibitors of serine proteases; lanes 2 and 3) inhibited most protease activity in the samples. EDTA (inhibitor of metalloproteases; lane 3) , pepstatin (inhibitor of aspartic protease s; lane 5) , and NEM (inhibitor of thiol proteases; lane 6) had less effect. The lane de signated ``None’’ (lane 1) was incubate d in the absence of protease inhibitor. Numbe rs on right, mole cular mass in kilodaltons.

ally active . Surprisingly, we obse rve d a similar prote ase that is active in dise ase d human colon tissue , but not active in normal tissue . Howe ver, this prote ase is pre sent in normal colon tissue that has bee n treate d with trypsin. The se results sugge st that this prote ase is activate d in human UC. While the e xact source of prote ase s observe d in UC colonic tissue sample s has not bee n ide nti® e d, they are like ly de rived from in¯ ammatory cells (lymphocyte s, macrophage s, mast cells, and ne utrophils ) pre sent in areas of active dise ase . These prote ase s could also come from mese nchymal cells responding to the re le ase of cytokine s from stimulate d in¯ ammatory cells. Pote ntial candidate serine prote ases in DNBS colon tissue are e lastase (Mr 30 kDa) and cathepsin G (Mr 30 kDa), pre sent in the azurophilic granule s of neutrophils (26) . The se prote ases have bee n associate d with a number of dise ase processe s, and they are e ffective ly inhibite d by BBI (27, 28) . Howeve r, we obse rved little prote ase activity in this size range in our tissue sample s, sugge sting that enzyme s obse rve d in our study arise from anothe r source . Anothe r conce rn was the possibility that bacte rial prote ases are be ing visualize d on the zymograms. Se veral factors argue against this. We observe d little

1978

diffe re nce in prote ase activity whe n colon tissue was unwashe d or washe d with PBS prior to analysis (Figure 4). Furthe r, we have found that extracts and conditione d medium prepare d from E. coli and othe r bacte ria have little activity on the zymogram s. Take n toge the r, the se re sults indicate that the prote ase s obse rved on the zymograms are host de rive d and not from bacteria. Bacte ria may well play a role in the proce ss of prote ase induction, howe ve r. Bacte rialde rive d toxins, or othe r compounds, may induce spe ci® c in¯ ammatory prote ase s in the host. Bacteria may also have a role in the pathology of ulcerative colitis since antibiotics have be en shown, in some individuals, to have a bene ® cial e ffe ct (1, 2). It should also be note d that normal e nte ric bacteria are part of an accurate UC mode l. Work by othe r inve stigators also sugge sts that prote ase s play a key role in IBD. Camostat mesilate , a serine prote ase inhibitor, induce d re mission in two UC patie nts with mild to mode rate UC. In these patie nts, re mission was maintaine d for 20 and 24 months (13) . The be ne® cial e ffe ct of camostat mesilate could re sult from its ability to inhibit se rine prote ase activity pre sent in UC. Incre ase d fe cal serine prote ase activity was observe d in UC patie nts while the le ve ls of endoge nous prote ase inhibitors such as Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)


ACKNOWLEDGMENTS We thank Beatrice E nge lsberg and Jonathan Cryer for the ir technical assistance, Jeffre y R. Parno for statistical advice , and Dr. Gary Wu, Dr. Sheri Motze l, and Dr. Norton Taichman for helpful suggestions.

REFERENCES

Fig 10. Analysis of human biopsy sample s for protease activity. Human colonic biopsy sample s of normal and involve d regions of the colon were obtained from patients with ulce rative colitis. The sample s were homogenize d and analyze d for protease activity on gelatin zymograms. Twe nty-® ve micrograms of protein was loade d into each lane of the ge l. Sample s were untreate d or incubated with trypsin (trypsin activate d) prior to be ing run on the zymogram. Involve d, tissue biopsy from involve d re gion of the colon; Uninvolve d, mate rial obtained from normal are as. Note that the involve d regions have highe r leve ls of protease activity compare d with the uninvolve d re gions. Also note the absence of the 44 kDa protease in the uninvolved sample which was not trypsin activate d (last lane on right). Numbe rs on right, Mr in kilodaltons. Arrow on left de signate s position of 44 kDa protease activity.

pa nc re atic se cre tory tryp sin inh ibit or an d a 1 prote inase inhibitor, are de crease d in UC patie nts (9, 10) . Lower prote ase inhibitor le vels could e nhance tissue de struction by in¯ ammatory prote ase s in the vicinity of active colonic in¯ ammation. In conclusion, DNBS is an effective re age nt for inducing a UC-like disease in rats. Using this model, we have demonstrate d that prote ase activity is signi® cantly e le vate d in UC tissue . The data from this proje ct corre late well with re sults of human studie s. It se e ms like ly that elevate d prote ase activity in UC cause s e xtensive tissue destruction to the epithe lial cells lining the gastrointe stinal tract and also to supporting smooth muscle and conne ctive tissue . Inhibition of prote ase activity should signi® cantly diminish tissue damage in IBD, reducing dise ase se verity, and improving the quality of life for the se individuals. Conse que ntly, prote ase inhibitors may be valuable pharmacologic al age nts, which could be use d with curre ntly pre scribe d antiin¯ ammatory drugs and othe r medications, to tre at IBD patie nts. Digestive Diseases and Sciences, Vol. 42, No. 9 (Septem ber 1997)

1. Glickman RM: In¯ ammatory bowel disease: Ulce rative colitis and crohn’s disease. In Harrison’ s Principles of Internal Me dicine. KJ Isselbache r, et al (eds). New York, McGraw-Hill, 1994, pp 1403± 1417 2. Podolsky DK: In¯ ammatory bowel disease. N Engl J Me d 325:928 ± 937, 1008 ± 1016, 1991 3. Calkins BM, Mendeloff AI: The epide miology of idiopathic in¯ ammatory bowel disease . In In¯ ammatory Bowel Dise ase , 4th ed. JB Kirsner, RG Shorter (e ds). Philade lphia, Williams and Wilkins, 1995, pp 31± 68 4. Crawford JM: The gastrointestinal tract. In Robbins Pathologic Basis of Dise ase , 5th ed. RS Cotran, V Kumar, SL Robbins, FJ Schoe n (eds). Philadelphia, WB Saunders, 1994, pp 755± 829 5. Brynskov J, Nielse n OH, Ahnfelt-Ronne L, Be ndtzen K: Cytokines and their natural re gulation in in¯ ammatory bowel disease. Dig Dis 12:290 ± 304, 1994 6. Grisham MB: Oxidants and fre e radicals in in¯ ammatory bowel disease. Lance t 344:859 ± 861, 1994 7. Hodgson HJ: Immunological aspects of in¯ ammatory bowel disease of the human gut. Age nts Actions Special Confere nce Issue : C27± C31, 1992 8. Rudolph U, Finegold MJ, Rich SS, Harriman GR, Srinivasan Y, Brabet P, Boulay G, Bradle y A, Birnbaume r L: Ulcerative colitis and adenocarcin oma of the colon in G alpha i2-de ® cie nt mice . Nature Gene t 10:143± 150, 1995 9. Bohe M: Pancre atic and granulocytic e ndoproteases in fae cal extracts from patients with active ulce rative colitis. Scand J Gastroente rol 22:59 ± 64, 1987 10. Bohe M, Ge nell S, Ohlsson K: Prote ase inhibitors in plasma and faecal extracts from patie nts with active in¯ ammatory bowel disease. Scand J Gastroe nterol 21:598 ± 604, 1986 11. Ottonello L, Dapino P, Pastorino G, V itale E , Dallegri F: The drug 5-aminosalicylic acid rescues alpha 1-prote inase inhibitor from the neutrophil oxidative inactivation. A possible contribution to its therape utic action in ulcerative colitis. Dige stion 51:140 ± 145, 1992 12. Playford RJ, Hanby AM, Patel K, Calam J: In¯ uence of in¯ ammatory bowel disease on the distribution and conce ntration of pancreatic se cre tory trypsin inhibitor within the colon. Am J Pathol 146:310 ± 316, 1995 13. Se nda SY, Fujiyama Y, Bamba T, Hosoda S: Treatmen t of ulcerative colitis with camostat mesilate , a se rine protease inhibitor. Intern Med 32:350 ± 354, 1993 14. Morris GP, Beck PL, He rridge MS, Depew WT, Sze wczuk MR, Wallace JL: Hapte n-induced mode l of chronic in¯ ammation and ulce ration in the rat colon. Gastroe nterology 96:795± 803, 1989 15. Elson CO, Sartor RB, Tennyson GS, Riddell RH: Expe rime ntal mode ls of in¯ ammatory bowel disease. Gastroente rology 109:1344 ± 1367, 1995 16. Nahas F, Le skowitz S: The ability of hapten-conjugate d cells to induce cell-mediated cytotoxicity is affe cte d by the mode of

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HAWKINS E T AL hapten linkage . Cell Immunol 54:241± 247, 1980 17. Prophet EB, Mills B, Arrington JB, Sobin LH (eds): Arme d Force s Institute of Pathology, Laboratory Methods in Histotechnology. Washington, DC, Ame rican Re gistry of Pathology, 1992, p 55 18. Wallace JL, Kee nan CM: An orally active inhibitor of leukotriene synthe sis accele rates healing in a rat mode l of colitis. Am J Physiol 258:527± 534, 1990 19. Krawisz JE, Sharon P, Stenson WF: Quantitative assay for acute intestinal in¯ ammation based on myeloperoxidase activity. Assessme nt of in¯ ammation in rat and hamster mode ls. Gastroente rology 87:1344 ± 1350, 1984 20. Laemmli UK: Cleavage of structural proteins during the assembly of bacte riophage T4, Nature 227:680 ± 685, 1970 21. Billings PC, Habres JM: A growth regulate d protease activity which is inhibited by the anticarcinoge nic Bowman-Birk inhibitor. Proc Natl Acad Sci USA 89:3120 ± 3124, 1992 22. Gomori G: Pre paration of buffers for use in e nzyme studies. Methods E nzymol 1:138 ± 146, 1955 23. Billings PC, Ne wberne P, Kennedy AR: Protease inhibitor

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