Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor

GASTROENTEROLOGY 2004;126:796 – 808

Chemotherapy- and Radiotherapy-Induced Intestinal Damage Is Regulated by Intestinal Trefoil Factor P. L. BECK,* J. F. WONG,* Y. LI,* S. SWAMINATHAN,* R. J. XAVIER,‡ K. L. DEVANEY,‡ and DANIEL K. PODOLSKY‡ *University of Calgary, Gastrointestinal Research Group, Calgary, Alberta, Canada; and ‡Gastrointestinal Unit, Center for Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, Massachusetts

Background & Aims: Injury to the intestinal mucosa is frequently a dose-limiting complication of radiotherapy and chemotherapy. Approaches to limit the damage to the intestine during radiation and chemotherapy have been largely ineffective. Trefoil factors are produced throughout the gastrointestinal tract and regulate cell migration, restitution, and repair. Studies were undertaken to define the role of intestinal trefoil factor in modulating the intestinal response to chemotherapy and radiation. Methods: The effect of intestinal trefoil factor on migration and cell survival in intestinal epithelial monolayer exposed to methotrexate was studied in vitro. Chemotherapy and radiation damage was assessed in wild-type and intestinal trefoil factor–null mice in the presence or absence of supplemental intestinal trefoil factor administered in drinking water. Results: Radiation and chemotherapy induced a marked reduction in goblet cell number and intestinal trefoil factor messenger RNA, as well as intestinal trefoil factor promoter activity. Intestinal trefoil factor improved intestinal epithelial cell viability and wound repair after chemotherapy exposure in vitro. Intestinal trefoil factor– deficient mice (intestinal trefoil factorⴚ/ⴚ) were more susceptible to chemotherapy- and radiation-induced mucositis. Oral recombinant intestinal trefoil factor reduced the severity of both chemotherapy-induced and chemotherapy/radiotherapy-induced intestinal mucositis. Conclusions: These studies suggest that intestinal trefoil factor is involved in protection against and recovery from intestinal mucositis induced by radiation and chemotherapy.

ntestinal injury occurs commonly as a result of either chemotherapy or radiotherapy. This seems to reflect a special sensitivity of the rapidly-turning-over gastrointestinal tract epithelium to these cytotoxic therapies because of constitutive rapid cell turnover. Epithelial injury presumably results in disruption of the integrity of the surface mucosal barrier and enables bacteremia or translocation of bacterial products from the lumen, which contributes to morbidity and mortality.

I

At present there is no significantly effective treatment or preventive therapy for chemotherapy- and radiotherapy-induced intestinal damage. Efforts to develop strategies that modify chemotherapy regimens to accomplish better targeting of tumors have yielded limited success. Nonspecific agents such as antibodies and antacids are also essential.1– 4 There have been isolated reports suggesting that granulocyte colony-stimulating factor may reduce chemotherapy-induced intestinal damage by stimulating cell proliferation, but large controlled trials are lacking.5,6 Transforming growth factor-␤3 has been found to reduce the severity of chemotherapy-induced oral mucositis in a hamster model by inducing cell-cycle arrest before chemotherapy exposure,7–10 and interleukin (IL)-11 has been reported to reduce the severity of intestinal damage associated with radiotherapy and chemotherapy.9,11,12 In the same model, epidermal growth factor (EGF) was found to be protective against 5-fluorouracil (5-FU)–induced mucositis.5,13 However, IL-11 and EGF both have systemic actions, including stimulation of cell replication; this raises concerns that each could stimulate tumor cell growth and affect responses to chemotherapy. Recently, the cytokine IL-15 has also been found to provide selective protection against irinotecan-, 5-FU–, and leucovorin-induced and intestinal toxicity, perhaps acting through specific IL-15 receptors that are present in the intestinal crypt cells.14,15 Glucagon-like peptide-2 reduces chemotherapy-induced intestinal injury in animal models, in part by reducing chemotherapy-induced apoptosis.16,17 Finally, keratinocyte growth factor has Abbreviations used in this paper: BrdU, bromodeoxyuridine; BSA, bovine serum albumin; EGF, epidermal growth factor; 5-FU, 5-fluorouracil; IEC, intraepithelial carcinoma; IL, interleukin; ITF, intestinal trefoil factor; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt); MTX, methotrexate; RPA, RNA protection assay. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gastro.2003.12.004

March 2004

been reported to reduce the severity of radiation/chemotherapy-induced damage. It presumably acts through up-regulation of cell proliferation and possibly differentiation within the intestinal mucosa, as well as improvement of crypt stem cell survival.18 Mammalian trefoil factors are a family of 3 peptides that possess a common 3-loop structure and share overlapping functional properties. pS2 and spasmolytic polypeptide (also designated trefoil factor family 1 and trefoil factor family 2, respectively) are primarily produced by the mucin-secreting cells of the stomach. The third trefoil peptide, intestinal trefoil factor (ITF; also designated trefoil factor family 3), is secreted by goblet cells of the small and large intestine. These peptides promote epithelial cell migration, protect these cells from damage, and regulate restitution of the intestinal mucosa after damage both in vitro and in vivo.19 Although the actions of trefoil factors are not fully understood, the damage of intestinal mucosa results in coordinated up-regulation of trefoil gene expression and protein production.20 –23 Trefoil factors can effect upregulation of their own production.19,24 –26 These factors do not seem to have intrinsic activity in regulating cell proliferation, but they promote epithelial migration in vitro and crypt to villus migration in vivo. Thus, studies show that bromodeoxyuridine (BrdU)-labeled cells migrate more rapidly from the crypts to the villus tips in wild-type animals compared with ITF-null mice. Recent studies have shown that ITF increases the resistance of colonic epithelium to apoptosis induced by serum starvation, ceramide, or p53-dependent cell death induced by etoposide.27,28 Goblet cell ablation has been recognized as one of the most consistent hallmarks of chemotherapy- and radiotherapy-induced intestinal damage. Because this cell type is the main source of trefoil factors in the intestine, we hypothesized that radiation and chemotherapy induce a reduction in trefoil expression and that the loss of these factors impairs wound healing and restitution.

Materials and Methods Animals All mice used in these studies were either C57/B6 or Sv129/C57/B6. ITF⫺/⫺ mice have been previously described19; age- and sex-matched control mice were from Sv129/C57/B6 lines established with and bred alongside the ITF⫺/⫺ mice.

Regulation of Intestinal Trefoil Factor After Chemotherapy or Radiation Methotrexate (MTX) 150 mg/kg intraperitoneally (IP; single dose), 5-FU 75 mg 䡠 kg⫺1 䡠 day⫺1 IP for 3 days, or MTX

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

797

150 mg/kg IP (single dose) followed by 6 Gy of total-body irradiation (cesium source irradiator; Atomic Energy of Canada Inc., Ottawa, Ontario, Canada) was administered to mice. After mice were killed, total cellular RNA was extracted from the jejunum, ileum, and colon by using Trizol (Life Technologies Inc., Gaithersburg, MD). Northern blot analysis of ITF expression was performed.29,30 Transgenic mice producing ␤-galactosidase under regulation of the goblet cell–specific ITF promoter (ITF/␤-galactosidase mice)29 were exposed to 0 to 14 Gy of total-body irradiation and killed 24 hours later. Tissues from the jejunum, ileum, and colon were removed, and ITF promoter–regulated ␤-galactosidase was detected by Lac-Z staining as described previously.29

Effect of Intestinal Trefoil Factor on Methotrexate-Induced Inhibition of Wound Repair and Methotrexate-Induced Cell Death In Vitro An in vitro wounding model using intestinal epithelial cells (IEC) was used.31 To assess the role of ITF in wound repair after chemotherapy, confluent IEC-6 cells were incubated with either ITF (1 ␮g/␮L) or bovine serum albumin (BSA; 1 ␮g/␮L) for 48 hours before exposure to MTX 0.01 to 100 ␮g/␮L. After 3 hours, the cells were washed 3 times with media and reincubated with either ITF or BSA. Wound repair was determined at 24 hours as previously described.31 A further study was performed to address the concentrationdependent response characteristics of ITF in mediating wound repair after exposure to MTX. Confluent IEC-6 cells were incubated in ITF or BSA at doses of 2, 1, 0.5, 0.1, 0.01, or 0.001 ␮g/␮L for 48 hours before exposure to MTX 0.01 ␮g/␮L (a dose selected from the previous experiment). After 3 hours, the cells were washed 3 times with media and reincubated with either ITF or BSA at the previously described concentrations. Wound repair was determined at 24 hours, and the distance cell of migration across the wound edge was measured by an ocular micrometer. To determine whether ITF affected intestinal epithelial cell survival after MTX, IEC-6 cells were grown to 50% confluence in 24-well culture plates, and ITF 1 ␮g/␮L or BSA 1 ␮g/␮L was added for 24 hours before exposure to MTX (0 to 10 ␮g/mL). After 3 hours, the cells were rinsed with media 3 times, and cells were cultured for 24 hours in normal growth media with either ITF or BSA, as described previously. Trypan blue was used to quantify viable cells. As a further assessment of cell viability and proliferation, (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt) (MTS) assays were performed. The MTS assay is a modification of the 3-(4,5-dimethythiazol-2yl)-2,5-diphenyltetrazolium bromide assay and is based on the ability of viable cells to reduce a tetrazolium base compound to a blue formazan product as an indicator of both cell viability and proliferation.32 The Promega CellTiter 96 AQueous NonRadioactive Cell Proliferation Assay (catalog no. G5421; Madison, WI) was used in these studies. This assay has been well established as a reproducible means of assessing chemosensi-

798

BECK ET AL.

tivity.33,34 IEC-6 cells, plated at 60% confluence, were pretreated with either ITF or BSA before and after exposure to MTX (0.01 mg/mL) for 1 hour. The MTS assay was performed, and color change was assessed at 1, 2, 3, and 4 hours after the addition of the MTS reagents (per the manufacturer’s guidelines). The experiment was run 3 times with a minimum of 5 replicates for each condition per run.

In Vivo Assessment of Chemotherapy- and Radiotherapy-Induced Mucositis: Evaluation of Intestinal Trefoil Factorⴚ/ⴚ Mice— Susceptibility to Chemotherapy-Induced Mucositis ITF⫺/⫺ and control mice were given 5-FU (50 –75 mg 䡠 䡠 day⫺1 for 3 days IP).18 Mice were examined twice daily, and changes in body weight, fecal blood loss, and severity of diarrhea were determined as described previously.35 Diarrhea and fecal blood were assessed with a 0 –3 scale and a 0 – 4 scale, respectively, by using previously validated techniques.35 Susceptibility was also assessed in a second model of chemotherapy-induced mucositis. ITF⫺/⫺ mice or age- and sex-matched controls (Sv129/C57/B6) were given MTX 40 mg 䡠 kg⫺1 䡠 day⫺1 for 3 days (data adapted18). The animals were evaluated as described previously. In addition, intestinal permeability, which is proportional to mucosal damage, was determined by urinary chromium-51/ ethylenediaminetetraacetic acid (EDTA) excretion after the administration of MTX, as described previously.36,37 Urinary chromium-51/EDTA excretion is expressed as the percentage of the oral dose given. kg⫺1

Evaluation of the Ability of Recombinant Oral Intestinal Trefoil Factor to Protect Against Chemotherapy-Induced and Chemotherapy/Radiation-Induced Mucositis Recombinant human ITF or BSA was added to the drinking water of ITF⫺/⫺ and wild-type mice beginning 48 hours before the administration of 5-FU to yield a dose of 20 mg 䡠 kg⫺1 䡠 day⫺1 of either ITF or BSA. 5-FU was administered at a dose of 75 mg 䡠 kg⫺1 䡠 day⫺1 IP for 3 days. In separate experiments, ITF⫺/⫺ and matched control mice were given ITF or BSA orally (by gavage) at doses of 10 to 100 mg 䡠 kg⫺1 䡠 day⫺1 24 hours before MTX 150 mg/kg IP (single dose) followed by 6 Gy of total-body irradiation 1 hour after the administration of the MTX (data adapted18). The ITF and BSA were given by gavage daily, and mice were assessed as described previously. Mice were monitored and evaluated as described previously.

Assessment of Crypt Survival After Radiation We used a previously described murine model system to assess whether ITF influences crypt survival after irradiation.38 ITF⫺/⫺ and matched control animals were given ITF or

GASTROENTEROLOGY Vol. 126, No. 3

BSA by gavage at doses of 100 mg/kg 24 and 2 hours before 14 Gy of total-body irradiation, and mice were killed at 3.5 days. Crypt survival was determined by labeling cells in Sphase by administering BrdU 120 mg/kg (Sigma, St. Louis, MO) 2 hours before death.38

Evaluation of the Ability of Recombinant Oral Intestinal Trefoil Factor to Protect Against and Enhance Recovery After Chemotherapy/Radiation-Induced Mucositis These studies were designed to determine whether ITF could protect against injury when given before chemotherapy and radiotherapy and whether it could enhance recovery when given only after chemotherapy and radiotherapy. Four groups of wild-type animals (6 mice per group) were given ITF or BSA 10 mg 䡠 kg⫺1 䡠 day⫺1 by oral gavage 3 days before MTX 150 mg/kg IP (single dose), followed by 6 Gy of total-body irradiation 1 hour after administration of the MTX. Mice were then treated daily with ITF or BSA 10 mg 䡠 kg⫺1 䡠 day⫺1 by oral gavage. Thus, the groups were as follows: group 1, ITF before and after; group 2, ITF before and BSA after; group 3, BSA before and ITF after; and group 4, BSA before and BSA after. Animals were assessed as described previously. In addition, intestinal permeability was assessed at day 16 (48 hours after the last dose of either ITF or BSA) after radiotherapy/ chemotherapy by determination of lactulose/mannitol permeability. A total of 500 ␮L of a solution of lactulose 60 mg/mL and mannitol 40 mg/mL was given by gavage to mice with free access to water but no food; urine was collected over 12 hours and analyzed by high-performance liquid chromatography.39 To assess the frequency of apoptosis, Western blotting for activated caspase-3 was performed on mucosal scrapings from the jejunum. The jejunum tissue samples were placed in 2.5 mmol/L EDTA buffer and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY), and then aliquots were placed in sample buffer. The protein concentration was determined by Bradford assay. Protein samples were separated by 15% sodium dodecyl sulfate polyacrylamide gel (15%) electrophoresis and transferred onto pure nitrocellulose membranes (Bio-Rad, Hercules, CA). The resulting blot was blocked with 5% nonfat dry milk in PBS containing 0.05% Tween-20 and then incubated for 1 hour at 37°C in a 1:1000 dilution of anti–active caspase-3 antibody (rabbit; BD PharMingen, Mississauga, ON, Canada) and anti-actin antibody (rabbit; Sigma). The membrane was then washed and incubated with horseradish peroxidase– conjugated anti-rabbit immunoglobulin G 1:10,000 dilution for 30 minutes at 37°C, washed again, and detected with enhanced chemiluminescence Western blotting detection reagents (NEN Life Science Products Inc., Boston, MA) on X-OMAT AR film (Eastman Kodak Co., Rochester, NY). Band density was determined by using a calibrated imaging densitometer. A multitemplate RNA protection assay (RPA; BD PharMingen) was used to assess expression of specific molecules or receptors involved in apoptosis pathways, including Fas, Fas

March 2004

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

799

Figure 1. Effects of chemotherapy and radiation on goblet cells and ITF expression. (A) Goblet cell depletion in the jejunum and colon of wild-type mice 3.5 days after 14 Gy of total-body irradiation. Alcian blue periodic acid–Schiff staining of paraffin-embedded, formalin-fixed tissue. (B) Northern blot of ITF in wild-type mice 0 –10 days after MTX and 6 Gy of total-body irradiation. After the mice were killed, messenger RNA was extracted from colon tissue and run on a 1% formaldehyde agarose gel (10 ng of RNA per lane), transferred to a nylon membrane, and hybridized with a 32P-labeled recombinant ITF complementary DNA probe, as described previously.29,30 GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Reduction in ITF promoter activity after total-body irradiation in ITF/␤-galactosidase mice. Construction of ITF/␤-galactosidase reporter mice in which the ITF promoter is driving expression of ␤-galactosidase has been described previously.29 To further assess regulation of ITF expression, ITF/␤-galactosidase mice were exposed to 0, 8, or 14 Gy of total-body irradiation. Then, 3.5 days after irradiation, the animals were killed. Tissues were fixed and stained with Lac-Z to locate the expression of ␤-galactosidase and were counterstained with eosin Y as described previously.29 No specific ␤-galactosidase activity could be identified in any of the animals exposed to 8 or 14 Gy of irradiation.

ligand, Fas-associated death domain, tumor necrosis factor– associated apoptos–inducing ligand, tumor necrosis factor receptor 1–associated death domain protein, tumor necrosis factor receptor-p55, Bclw, Bfl-1, Bcl-xl, Bak, Bax, Bcl-2, and Bad (mAPO-2 and 3 templates). In brief, RNA was isolated from the jejunum or colon via Trizol (as described previously), and 10 ␮g of RNA per sample was used in the multitemplate RPA assay per the manufacturer’s guidelines.

Statistical Analysis Data are presented as the mean ⫾ SEM. Parametric data were analyzed with a 1-way analysis of variance followed by a Dunnett multiple comparisons posttest. Nonparametric data (scoring) were analyzed with a Kruskal–Wallis test (nonparametric analysis of variance) followed by a Dunn multiple comparisons posttest. Survival curves were created with the Kaplan–Meier method, and survival comparisons were performed with the logrank or Mantel–Haenszel test, which generate a 2-tailed P value.

Results Effect of Chemotherapy and Radiation on Intestinal Trefoil Factor Expression Both chemotherapy and radiotherapy resulted in a marked reduction in goblet cell numbers in vivo, followed by a gradual return to baseline numbers in surviving animals (Figure 1A). A decrease in ITF production as determined by Northern blotting of messenger RNA from the jejunum, ileum, and colon correlated with the reduction in apparent goblet cell numbers (Figure 1B). Steady-state ITF expression remained depressed 10 days after MTX 150 mg/kg and 6 Gy of total-body irradiation (Figure 1B). To further delineate the alterations in ITF expression, a transgenic mouse line was used that expresses ␤-galactosidase under regulation

800

BECK ET AL.

GASTROENTEROLOGY Vol. 126, No. 3

of the ITF promoter. When the ITF/␤-galactosidase mice were exposed to 6 to 14 Gy of total-body irradiation, there were no signs of ITF/␤-galactosidase–mediated Lac-Z staining in either the small intestine or colon in any of the mice examined at the standard 2.5-day time point (Figure 1C). Thus, radiation and chemotherapy deplete goblet cell numbers and ITF expression, and the reduction in ITF expression seems to result from direct effect on the ITF promoter. Effect of Chemotherapy on Intestinal Epithelial Cells In Vitro and Modulation by Intestinal Trefoil Factor To assess whether ITF can modulate the deleterious effects of chemotherapy on intestinal epithelial cells, 2 main responses were studied: the effects of ITF on cell viability and wound repair after exposure to chemotherapy. ITF enhanced IEC-6 wound repair in vitro after exposure to MTX 0.001 to 10 ␮g/mL (Figure 2A). A dose–response effect was noted with the ITF-enhanced wound repair after MTX exposure (Figure 2B). ITF also improved cell viability, as assessed by the trypan blue exclusion assay, after exposure to MTX 1 ␮g/mL (percentage of nonviable cells: ITF treated [1 ␮g/␮L], 8.0% ⫾ 1.35%; BSA treated [1 ␮g/␮L], 12.5% ⫾ 1.7%; P ⬍ 0.05). Cell viability and proliferation were further assessed via the MTS assay; ITF at concentrations from 0.01 to 1 mg/mL was more effective than BSA in protecting the cells against the deleterious effects of MTX (Figure 2C). Thus, it seems that ITF both protects cells from injury and promotes wound repair after exposure to chemotherapy. The Role of Intestinal Trefoil Factor in Modulating Susceptibility to ChemotherapyInduced Mucositis In Vivo ITF⫺/⫺ mice were studied to assess the role of ITF in modulating the response of the intestinal mucosa to chemotherapy. The ITF⫺/⫺ mice were markedly more susceptible to 5-FU–induced mucositis, as evidenced by more severe weight loss, diarrhea, fecal blood loss, and increased intestinal permeability (P ⫽ 0.03), resulting in reduced survival (P ⬍ 0.0085; Figure 3). A similar trend was seen in an MTX-induced model of mucositis; ITF⫺/⫺ mice had more severe diarrhea than wild-type mice (Figure 4). There was no premature death in either the ITF⫺/⫺ or control animals after MTX 40 mg 䡠 kg⫺1 䡠 day⫺1 for 3 days, and there were no differences in intestinal permeability when these groups were assessed at day 15. However, in a similar study, when MTX was given at 35 mg 䡠 kg⫺1 䡠 day⫺1 for 4 days, there was 50%

Figure 2. Effects of ITF on the response of IEC to methotrexate in vitro. (A) Impairment of epithelial wound repair by MTX in vitro is attenuated by ITF. BSA or ITF was added to wounded IEC-6 epithelial cell monolayers exposed to varying doses of MTX, and the number of cells migrating across the wound edge was counted at 24 hours (the mean of a minimum of 3 experiments involving a minimum of 4 individual wound preparations in 6-well plates: *P ⬍ 0.05; **P ⬍ 0.01). (B) Impairment of epithelial wound repair by MTX in vitro is attenuated by ITF in a concentration-dependent fashion. BSA or ITF was added to wounded IEC-6 epithelial cell monolayers at concentrations from 0.001 to 2 ␮g/␮L before and after exposure to MTX 0.01 ␮g/mL. Migration of cells across the wound edge was measured at 24 hours (the mean of a minimum of 2 experiments involving a minimum of 4 individual wound preparations in 6-well plates). (C) The deleterious effects of MTX on cell viability and proliferation were attenuated by ITF in vitro. Cell viability and proliferation were assessed via the Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation MTS Assay. IEC-6 cells, plated at 60% confluence, were pretreated with either ITF or BSA before and after exposure to MTX (0.01 mg/mL) for 1 hour. Color change was assessed at 1, 2, 3, and 4 hours after the addition of the MTS reagents (**P ⬍ 0.01, ITF treated vs. BSA treated). The experiment was run 3 times with a minimum of 5 replicates for each condition per run.

March 2004

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

801

Figure 3. The severity of 5-FU–induced mucositis was determined by endogenous ITF expression. (A) Age-, weight-, and sex-matched ITF⫺/⫺ mice and wild-type mice were given 5-FU 75 mg 䡠 kg⫺1 䡠 day⫺1 IP for 3 days, and changes in body weight were recorded and expressed as the percentage change in basal body weight (a minimum of 6 animals per group: *P ⬍ 0.05; **P ⬍ 0.01). (B) Fecal occult blood was determined by using Hemoccult SENSA paper (Beckman Coulter, Inc., Mississauga, Ontario, Canada) and was scored as described in the Materials and Methods section (a minimum of 6 animals per group: *P ⬍ 0.05; **P ⬍ 0.01). (C) Intestinal permeability was determined 4 days after the last dose of 5-FU by urinary excretion of an oral gavage of chromium-51 (51Cr)/EDTA (expressed as the percentage of the oral dose recovered in the urine over 24 hours; *P ⬍ 0.05; n ⫽ 5 animals per group). (D) Survival curves were created by using the Kaplan–Meier method, and survival comparisons were performed with the log-rank or Mantel–Haenszel test, which generated a 2-tailed P value.

mortality in the ITF⫺/⫺ group and no mortality in the wild-type controls. Effect of Exogenous Recombinant Intestinal Trefoil Factor on Intestinal Mucosal Susceptibility to Chemotherapy-Induced and Chemotherapy/Radiation-Induced Mucositis To assess the functional role of ITF in modulating intestinal mucosal injury implicit in the increased susceptibility of ITF-null mice to chemotherapy, recombinant human ITF (20 mg 䡠 kg⫺1 䡠 day⫺1 administered via drinking water) was given to ITF⫺/⫺ mice. Oral recombinant ITF reduced the severity of chemotherapy-induced weight loss and diarrhea during the initial study periods (days 1, 2, and 3; P ⬍ 0.5– 0.01; Figure 5). Oral ITF therapy was also effective at reducing the severity of mucositis induced by combined chemotherapy and radiotherapy (Figure 6). In these studies, ITF⫺/⫺

mice were susceptible to mucositis induced by combined chemotherapy/radiotherapy (6 Gy of total-body irradiation after a single 150 mg/kg IP dose of MTX). However, oral recombinant ITF (10 mg 䡠 kg⫺1 䡠 day⫺1) improved the mucositis in the ITF⫺/⫺ mice, resulting in reduced weight loss, less severe diarrhea, and reduced intestinal permeability (7.83% ⫾ 2.10% vs. 1.28% ⫾ 0.65% for ITF⫺/⫺ BSA-treated and ITF⫺/⫺ ITF-treated mice, respectively; P ⫽ 0.015; Figure 7). Mortality in the ITF⫺/⫺ mice treated with BSA was 50%, vs. no mortality in the ITF-treated group. It is important to note that wild-type control mice treated with ITF showed a similar reduction in mucositis-related weight loss (P ⬍ 0.05 at days 1, 3, and 21; Figure 6A) and diarrhea (P ⬍ 0.05– 0.01 at days 1 and 7; Figure 6B). Recombinant ITF reduced intestinal permeability 21 days after the single dose of MTX and 6 Gy of irradiation, as described previously. Significant reductions in intestinal permeability were also noted in both wild-type and ITF-null mice treated with ITF after combined

802

BECK ET AL.

GASTROENTEROLOGY Vol. 126, No. 3

either before or after the chemotherapy/radiotherapy had less weight loss than animals that did not receive ITF. The protective response of the ITF given only before injury was short lived, but both groups of animals that received ITF after injury did better than mice that did not receive ITF (Figure 9). There was no mortality in the 2 groups that received ITF after injury, but 1 of 6 mice died in the ITF before/BSA after group, and 2 of 6 mice died in the BSA before/BSA after group. There were no marked differences in diarrhea scores. However, at day 9 after injury, there was no detectable blood in the ITF before/ITF after group (fecal occult blood score, 0 ⫾ 0), whereas all other groups had detectable fecal blood loss (fecal occult blood score: ITF before/BSA after, 0.9 ⫾ 0.19; P ⬍ 0.05; BSA before/ITF after, 0.5 ⫾ 0.3, not significant; BSA before/BSA after, 1 ⫾ 0.27; P ⬍ 0.01; P values are vs. the ITF before/ITF after group). These differences in fecal blood loss resulted in a more profound reduction in hemoglobin concentration and hematocrit

Figure 4. The severity of MTX-induced mucositis was determined by endogenous ITF expression. (A) Age-, weight-, and sex-matched ITF⫺/⫺ mice and wild-type mice were given MTX 40 mg 䡠 kg⫺1 䡠 day⫺1 IP for 3 days, and changes in body weight were recorded and expressed as the percentage change in basal body weight (a minimum of 6 animals per group; *P ⬍ 0.05). (B) Assessment of the severity of diarrhea. MTX was given as described in Figure 3D, and stool consistency was assessed daily, in a blinded fashion, as described in the text (a minimum of 6 animals per group; **P ⬍ 0.01).

radiation/chemotherapy (Figure 7). Jejunal crypt survival and the number of BrdU-positive cells present in the colon were assessed at several time points in the previously mentioned studies. There was also improved jejunal crypt survival and there were more BrdU-positive cells in the colon in wild-type mice treated with BSA vs. similarly treated ITF⫺/⫺ mice after 14 Gy of total-body irradiation. Oral recombinant human ITF improved jejunal crypt survival and increased the number of BrdUpositive cells in the colon in ITF⫺/⫺ mice (Figure 8). To determine whether ITF was acting in the previous study in a protective fashion by reducing the initial injury induced by chemotherapy and radiotherapy or by acting to enhance recovery after the injury (or both), animals received either ITF or BSA for 3 days before chemotherapy and radiotherapy followed by either ITF or BSA. In this setting, ITF showed clear protective actions but showed even more marked enhancement of recovery (Figure 9). At day 2 after injury, mice that received ITF

Figure 5. Luminal recombinant human ITF reduces the severity of mucositis induced by combined chemotherapy. Age-, weight-, and sex-matched wild-type animals were treated with 3 daily doses of 5-FU 75 mg/kg IP. Animals were given recombinant human ITF or BSA (20 mg 䡠 kg⫺1 䡠 day⫺1) in drinking water throughout the experiment. (A) Body weights were recorded and expressed as percentage change in basal body weight (a minimum of 9 animals per group; *P ⬍ 0.05; **P ⬍ 0.01, ITF⫺/⫺ mice treated with BSA vs. ITF). (B) Assessment of the severity of diarrhea. Stool consistency was assessed daily, in a blinded fashion (a minimum of 9 animals per group; *P ⬍ 0.05; **P ⬍ 0.01, ITF⫺/⫺ mice treated with BSA vs. ITF).

March 2004

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

803

Fas-associated death domain, with smaller, more variable increases in Fas ligand, tumor necrosis factor–related apoptosis-inducing ligand, tumor necrosis factor receptor-p55, and tumor necrosis factor 1–associated death domain protein (not significant) in the jejunum in the animals that received chemotherapy/radiotherapy compared with untreated animals (Figure 9E). No significant differences were noted between the ITF and/or BSA treatment groups described previously. There were also increases in bcl-w, bcl-xl, bak, bax, and bad but no changes in bcl-2 or bfl-1 in the jejunum of mice that received chemotherapy/radiotherapy. However, again, there were no significant changes between the ITF and/or BSA treatment groups (Figure 9E). Similarly, there were no changes in any of the described molecules involved in apoptosis in colonic tissue (data not shown).

Discussion

Figure 6. Luminal recombinant human ITF reduces the severity of mucositis induced by combined radiation and chemotherapy. Age-, weight-, and sex-matched ITF⫺/⫺ and wild-type animals were treated with a single IP dose of MTX 150 mg/kg followed 1 hour later by 6 Gy of total-body irradiation. Animals were treated with oral ITF or BSA (10 mg 䡠 kg⫺1 䡠 day⫺1 administered via drinking water) throughout the experiment. (A) Body weights were recorded and expressed as percentage change in basal body weight (a minimum of 5 animals per group; **P ⬍ 0.01, ITF⫺/⫺ mice treated with BSA vs. ITF; ␣P ⬍ 0.05, wild-type mice treated with BSA vs. ITF). (B) Assessment of the severity of diarrhea. Stool consistency was assessed daily, in a blinded fashion (a minimum of 9 animals per group; *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001, ITF⫺/⫺ mice treated with BSA vs. ITF; ␣P ⬍ 0.05; ␣␣P ⬍ 0.01, wild-type mice treated with BSA vs. ITF).

in the animals that did not receive ITF after exposure to chemotherapy and radiotherapy. Small-intestinal permeability assessed via lactulose/mannitol permeability was lower in the animals that received ITF either before or after chemotherapy/radiotherapy, but these changes were not significant at P ⬍ 0.05 (data not shown). Western blotting of jejunal mucosal tissue showed increased rates of apoptosis in the animals that did not receive ITF after chemotherapy/radiotherapy. The lowest levels of activated caspase-3 were noted in the BSA/ITF and ITF/ITF treatment groups (Figure 9D). No differences were noted in activated caspase-3 levels in colonic mucosal tissues from any of the treatment groups compared with untreated animals (data not shown). When specific molecules or receptors involved in apoptosis pathways were assessed by multitemplate RPA, there were marked increases (P ⬍ 0.05) in caspase-8, Fas, and

Intestinal injury is a frequent complication of both radiation and chemotherapy. It is most frequently manifested by diarrhea, but hemorrhage and sepsis may result after disruption of mucosal surface integrity. Longterm sequelae include ischemia and stricture formation. The exquisite sensitivity of the intestine to these cytotoxic therapies likely results from the remarkable sustained proliferative activity in the intestinal tract epithelial cell compartment. Indeed, with the availability of growth factors to stimulate hematopoietic progenitor cells, gastrointestinal toxicity is now frequently rate limiting in the use of radiation and chemotherapy for the treatment of patients with a wide variety of malignancies. Accordingly, there is a compelling need to better

Figure 7. Luminal recombinant human ITF reduces impaired intestinal barrier function induced by combined radiation and chemotherapy in both wild-type (Wt) and ITF⫺/⫺ (ko) mice. Age-, weight-, and sexmatched wild-type animals were treated with a single IP dose of MTX 150 mg/kg followed 1 hour later by 6 Gy of total-body irradiation. Animals were treated with oral ITF or BSA (0, 20, or 100 mg 䡠 kg⫺1 䡠 day⫺1) throughout the experiment. Intestinal permeability was determined 21 days after the radiation and chemotherapy by urinary excretion of an oral gavage of chromium-51(Cr)/EDTA (expressed as the percentage of the oral dose recovered in the urine over 24 hours; *P ⬍ 0.05; n ⫽ 5 animals per group).

804

BECK ET AL.

Figure 8. Luminal recombinant human ITF improves jejunal crypt survival induced by combined radiation and chemotherapy in both wild-type and ITF⫺/⫺ mice. (A) ITF⫺/⫺ and matched control animals were given ITF or BSA by gavage at doses of 100 mg/kg 24 and 2 hours before 14 Gy of total-body irradiation. Animals were killed at 3.5 days, 2 hours after administration of BrdU. Tissues were processed and stained with anti-BrdU antibodies as described previously.38 BrdU staining was performed in the colon and jejunum in ITF⫺/⫺ mice treated with either oral ITF or BSA. Note the increased number of BrdU positive–staining cells both in the jejunal crypts and colonic tissue. (B) Assessment of jejunal crypt survival and number of BrdU positive– staining cells in colonic tissue. A minimum of 6 cross-sections of jejunum and colon were assessed per mouse, and the viability of each crypt was determined by histological appearance and the presence of 5 or more cells staining positively for BrdU within each crypt; the index used for assessment of colonic mucosa was the number of BrdUpositive cells per 1 mm of mucosa (measured along the muscularis mucosa), as described previously.38

understand endogenous mechanisms that can sustain mucosal integrity, modulate vulnerability to cytotoxic injury, and modulate reparative responses. ITF, a member of the trefoil peptide family, is expressed in large amounts by goblet cells throughout the small and large intestine. Trefoil peptide expression is up-regulated in immediate proximity to sites of injury in the gastrointestinal tract, irrespective of the nature of the insult.22,40 In vitro and in vivo studies have shown that

GASTROENTEROLOGY Vol. 126, No. 3

trefoil peptides promote cell migration and are potent mediators of intestinal mucosal homeostasis and wound repair. Thus, mice rendered ITF deficient by targeted gene deletion are exquisitely sensitive to intestinal injury and fail to mount an effective restitution response, the first critical phase of mucosal healing.19,40 ITF also blocks both p53-dependent and p53-independent apoptosis in vitro.27 The constellation of general enhanced restitution and protection from apoptotic stimuli might make ITF especially well suited to modulate intestinal vulnerability to these cytotoxic therapies. Prompt and sustained reduction of ITF content and expression followed exposure to either radiotherapy or chemotherapy. Transcription of ITF in intact goblet cells was suppressed after radiation exposure, as shown by a significant reduction in reporter ␤-galactosidase expression driven by the ITF promoter in transgenic mice.29 These findings lend circumstantial support to the notion that depletion of ITF may play a role in the susceptibility of intestinal mucosal damage by radiation and chemotherapy. These findings are also consistent with the earlier observation of an early decrease in ITF after chemotherapy in a rat model.40 Of interest, these studies also showed subsequent up-regulation of ITF. However, this induced expression occurs substantially later than the rapid up-regulation (hours to days) found in other forms of injury, perhaps accounting for the delayed recovery after chemotherapy and radiation. A putative role for ITF in the mucosal response to radiation and chemotherapeutic agents is corroborated by demonstration of the increased vulnerability of ITF-null mice to injury induced by either insult individually or the 2 administered in combination by using a variety of regimens. These mice were markedly more susceptible to both 5-FU–induced and MTX-induced intestinal injury, as assessed by a variety of parameters, including absolute survival. These findings are consistent with the inference that ITF both contributes to intrinsic intestinal mucosal defense and plays a requisite role in mucosal healing. The similarity of effect irrespective of the agent used suggests that these properties are generally relevant to cytotoxic therapy and are not a reflection of an idiosyncratic effect on a drug-specific mechanism of action. The protective effects of ITF have been shown. ITF⫺/⫺ mice are markedly more susceptible to both dextran sodium sulfate–induced and acetic acid–induced colonic injury.19 Furthermore, exogenous ITF can protect the epithelium against numerous deleterious agents, including ethanol, indomethacin, bacterial toxins, and various other chemicals and drugs.41,42 The exact mechanisms by which ITF exerts its protective effects are unclear. How-

March 2004

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

805

Figure 9. Luminal recombinant human ITF reduces the severity of mucositis when given before or after combined radiation and chemotherapy. Age-, weight-, and sex-matched wild-type animals were treated with a single IP dose of MTX 150 mg/kg followed 1 hour later by 6 Gy of total-body irradiation. Animals were treated with oral ITF or BSA (10 mg/kg by gavage daily) for 3 days before and/or after radiotherapy/chemotherapy throughout the experiment to day 14. ITF-ITF, ITF pretreatment and ITF treatment after radiotherapy/chemotherapy; ITF-BSA, ITF pretreatment and BSA treatment after radiotherapy/chemotherapy; BSA-ITF, BSA pretreatment and ITF treatment after radiotherapy/chemotherapy; BSA-BSA, BSA pretreatment and BSA treatment after radiotherapy/chemotherapy. A minimum of 6 animals per group were assessed. (A) Body weights were recorded and expressed as percentage change in basal body weight (a minimum of 6 animals per group. (B) Assessment of the severity of diarrhea and fecal blood loss on day 10 after radiotherapy/chemotherapy. Stool consistency was assessed daily, in a blinded fashion. Fecal occult blood was determined by using Hemoccult SENSA paper. (C) Complete blood counts at day 18, after radiotherapy/chemotherapy. Hgb, hemoglobin (g/L); Hct, hematocrit (⫻100); WBC, white blood cell count (⫻10); Plt, platelet count/10; Rx, radiotherapy. (D) Western blot of activated caspase-3 in the jejunum at day 18 after radiotherapy/chemotherapy. Animals that received ITF after radiotherapy/chemotherapy had lower levels of activated caspase-3 than those that did not receive ITF therapy. Representative Western blot. (E) Assessment of molecules associated with apoptosis. Determination by RNA protection assay on RNA from the jejunum by using 2 commercially available panels as described in the Materials and Methods section. GAPDH, GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ever, recent studies have shown that ITF can activate mitogen-activated protein kinase and lead to EGF receptor phosphorylation, suggesting a signaling role for this peptide.24 Other studies have shown that it can induce

redistribution of E-cadherin, which may be the mechanism of enhancement of cell migration.43 ITF also blocks the induction of both p53-dependent and p53-independent apoptosis.27,28 Further studies are required to deter-

806

BECK ET AL.

GASTROENTEROLOGY Vol. 126, No. 3

Figure 9 (Cont’d.)

mine the exact mechanisms by which ITF exerts its protective effects. The in vivo relevance of the initial in vitro findings is further validated by the observed ability of oral recombinant ITF to prevent chemotherapy-induced and/or radiation-induced injury and enhance healing. Although the effects of exogenous ITF administration were most pronounced in ITF-null mice, they were also apparent in wild-type mice competent to produce ITF endogenously. Oral recombinant ITF had the greatest effect when given after radiotherapy/chemotherapy, thus suggesting a prominent role for ITF in the recovery phase after radiotherapy/chemotherapy-induced mucositis. Remarkably, ITF therapy administered after radiotherapy/chemotherapy reduced ongoing apoptosis as assessed by activated caspase-3. ITF therapy did not markedly affect the expression of other molecules involved in apoptosis signaling pathways (including Fas, Fas ligand, tumor necrosis factor receptor, Fas-associated death domain, tumor necrosis factor 1-associated death domain protein, and tumor necrosis factor receptor 1–related apoptosis-inducing ligand) or the mitochondrial regulators of apoptosis (bcl-w, bfl-1, bcl-xl, bak, bax, bcl-2, or bad) as assessed via multitemplate RPA analysis. The precise mechanisms responsible for attenuation of injury from cytotoxic therapies generally remain to be

fully defined. However, it is noteworthy that in vitro studies suggest that the trefoil peptide ability to promote cellular restitution may be important given the ability of chemotherapy agents to inhibit this critical reparative process. The ability to block cell death may also be important. Previous studies have suggested that ITF has no primary effect on epithelial proliferation but could improve stem cell survival. Enhanced crypt survival and BrdU-positive cells in the colon of ITF-null mice pretreated with exogenous recombinant ITF suggest that the mechanism of action, in part, involves reducing epithelial cell death. In aggregate, these findings show the ability of the representative trefoil peptide ITF to inhibit the cellular effects of cytotoxic agents in vitro. These effects seem to be directly analogous to mucosa in vivo.

References 1. Khan SA, Wingard JR. Infection and mucosal injury in cancer treatment. J Natl Cancer Inst Monogr 2001;29:31–36. 2. Mori K, Tominaga K, Yokoyama K, Suga Y, Kishiro I, Tsurui M. Efficacy of famotidine in patients with acute gastric mucosal injury after continuous infusion of cisplatin plus vindesine. J Cancer Res Clin Oncol 1995;121:367–370. 3. Sartori S, Trevisani L, Nielsen I, Tassinari D, Panzini I, Abbasciano V. Randomized trial of omeprazole or ranitidine versus placebo in the prevention of chemotherapy-induced gastroduodenal injury. J Clin Oncol 2000;18:463– 467.

March 2004

4. Valls A, Pestchen I, Prats C, Pera J, Aragon G, Vidarte M, Algara M. Multicenter double-blind clinical trial comparing sucralfate vs placebo in the prevention of diarrhea secondary to pelvic irradiation. Med Clin (Barc) 1999;113:681– 684. 5. Melichar B, Kohout P, Bratova M, Solichova D, Kralickova P, Zadak Z. Intestinal permeability in patients with chemotherapyinduced stomatitis. J Cancer Res Clin Oncol 2001;127:314 – 318. 6. Kilic D, Sayan H, Gonul B, Egehan I. The effect of granulocyte macrophage-colony stimulating factor on glutathione and lipid peroxidation in a rat model. Eur J Surg Oncol 2000;26:701–704. 7. Sonis ST, Lindquist L, Van Vugt A, Stewart AA, Stam K, Qu GY, Iwata KK, Haley JD. Prevention of chemotherapy-induced ulcerative mucositis by transforming growth factor beta 3. Cancer Res 1994;54:1135–1138. 8. Potten CS, Booth D, Haley JD. Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo. Br J Cancer 1997;75:1454 –1459. 9. Potten CS. Interleukin-11 protects the clonogenic stem cells in murine small-intestinal crypts from impairment of their reproductive capacity by radiation. Int J Cancer 1995;62:356 –361. 10. Booth D, Haley JD, Bruskin AM, Potten CS. Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling. Int J Cancer 2000;86:53–59. 11. Du XX, Doerschuk CM, Orazi A, Williams DA. A bone marrow stromal-derived growth factor, interleukin-11, stimulates recovery of small intestinal mucosal cells after cytoablative therapy. Blood 1994;83:33–37. 12. Sonis S, Muska A, O’Brien J, Van Vugt A, Langer-Safer P, Keith J. Alteration in the frequency, severity and duration of chemotherapy-induced mucositis in hamsters by interleukin-11. Eur J Cancer B Oral Oncol 1995;31B:261–266. 13. Sonis ST, Costa JW Jr, Evitts SM, Lindquist LE, Nicolson M. Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy. Oral Surg Oral Med Oral Pathol 1992;74:749 –755. 14. Cao S, Black JD, Troutt AB, Rustum YM. Interleukin 15 offers selective protection from irinotecan-induced intestinal toxicity in a preclinical animal model. Cancer Res 1998;58:3270 –3274. 15. Reinecker HC, MacDermott RP, Mirau S, Dignass A, Podolsky DK. Intestinal epithelial cells both express and respond to interleukin 15. Gastroenterology 1996;111:1706 –1713. 16. Boushey RP, Yusta B, Drucker DJ. Glucagon-like peptide (GLP)-2 reduces chemotherapy-associated mortality and enhances cell survival in cells expressing a transfected GLP-2 receptor. Cancer Res 2001;61:687– 693. 17. Tavakkolizadeh A, Shen R, Abraham P, Kormi N, Seifert P, Edelman ER, Jacobs DO, Zinner MJ, Ashley SW, Whang EE. Glucagonlike peptide 2: a new treatment for chemotherapy-induced enteritis. J Surg Res 2000;91:77– 82. 18. Farrell CL, Bready JV, Rex KL, Chen JN, DiPalma CR, Whitcomb KL, Yin S, Hill DC, Wiemann B, Starnes CO, Havill AM, Lu ZN, Aukerman SL, Pierce GF, Thomason A, Potten CS, Ulich TR, Lacey DL. Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality. Cancer Res 1998;58:933–939. 19. Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 1996;274:262–265. 20. Babyatsky MW, Rossiter G, Podolsky DK. Expression of transforming growth factors alpha and beta in colonic mucosa in inflammatory bowel disease. Gastroenterology 1996;110:975– 984. 21. Wright NA, Poulsom R, Stamp GW, Hall PA, Jeffery RE, Longcroft JM, Rio MC, Tomasetto C, Chambon P. Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in dam-

INTESTINAL DAMAGE/INTESTINAL TREFOIL FACTOR

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

807

aged human gastrointestinal tissues. J Pathol 1990;162:279 – 284. Wright NA, Poulsom R, Stamp G, Van Noorden S, Sarraf C, Elia G, Ahnen D, Jeffery R, Longcroft J, Pike C, Rio M-C, Chambon P. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Gastroenterology 1993;104:12– 20. Itoh H, Tomita M, Uchino H, Kobayashi T, Kataoka H, Sekiya R, Nawa Y. cDNA cloning of rat pS2 peptide and expression of trefoil peptides in acetic acid-induced colitis. Biochem J 1996;318(Pt 3):939 –944. Taupin D, Wu DC, Jeon WK, Devaney K, Wang TC, Podolsky DK. The trefoil gene family are coordinately expressed immediateearly genes: EGF receptor- and MAP kinase-dependent interregulation. J Clin Invest 1999;103:R31–R38. Lefebvre O, Chenard MP, Masson R, Linares J, Dierich A, LeMeur M, Wendling C, Tomasetto C, Chambon P, Rio MC. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 1996;274:259 –262. Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, Wang TC. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest 2002;109:193–204. Taupin DR, Kinoshita K, Podolsky DK. Intestinal trefoil factor confers colonic epithelial resistance to apoptosis. Proc Natl Acad Sci U S A 2000;97:799 – 804. Kinoshita K, Taupin DR, Itoh H, Podolsky DK. Distinct pathways of cell migration and antiapoptotic response to epithelial injury: structure-function analysis of human intestinal trefoil factor. Mol Cell Biol 2000;20:4680 – 4690. Itoh H, Beck PL, Inoue N, Xavier R, Podolsky DK. A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells. J Clin Invest 1999;104:1539 – 1547. Suemori S, Lynch-Devaney K, Podolsky DK. Identification and characterization of rat intestinal trefoil factor: tissue- and cellspecific member of the trefoil protein family. Proc Natl Acad Sci U S A 1991;88:11017–11021. Dignass A, Lynch-Devaney K, Kindon H, Thim L, Podolsky DK. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. J Clin Invest 1994; 94:376 –383. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55– 63. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987;47:936 –942. Kawada K, Yonei T, Ueoka H, Kiura K, Tabata M, Takigawa N, Harada M, Tanimoto M. Comparison of chemosensitivity tests: clonogenic assay versus MTT assay. Acta Med Okayama 2002; 56:129 –134. Andres PG, Beck PL, Mizoguchi E, Mizoguchi A, Bhan AK, Dawson T, Kuziel WA, Maeda N, MacDermott RP, Podolsky DK, Reinecker HC. Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1⫹ lymphocyte-associated Th2-type immune response in the intestine. J Immunol 2000;164:6303– 6312. Reuter BK, Davies NM, Wallace JL. Nonsteroidal anti-inflammatory drug enteropathy in rats: role of permeability, bacteria, and enterohepatic circulation. Gastroenterology 1997;112: 109 –117. Beck PL, Xavier R, Lu N, Nanda NN, Dinauer M, Podolsky DK, Seed B. Mechanisms of NSAID-induced gastrointestinal injury

808

38.

39.

40.

41.

BECK ET AL.

defined using mutant mice. Gastroenterology 2000;119:699 – 705. Cohn SM, Schloemann S, Tessner T, Seibert K, Stenson WF. Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1. J Clin Invest 1997;99:1367–1379. Meddings JB, Swain MG. Environmental stress-induced gastrointestinal permeability is mediated by endogenous glucocorticoids in the rat. Gastroenterology 2000;119:1019 –1028. Xian CJ, Howarth GS, Mardell CE, Cool JC, Familari M, Read LC, Giraud AS. Temporal changes in TFF3 expression and jejunal morphology during methotrexate-induced damage and repair. Am J Physiol 1999;277:G785–G795. Kindon H, Pothoulakis C, Thim L, Lynch-Devaney K, Podolsky DK. Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology 1995;109:516 –523.

GASTROENTEROLOGY Vol. 126, No. 3

42. Babyatsky MW, deBeaumont M, Thim L, Podolsky DK. Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats. Gastroenterology 1996;110:489 – 497. 43. Podolsky DK. Mechanisms of regulatory peptide action in the gastrointestinal tract: trefoil peptides. J Gastroenterol 2000; 35(Suppl 12):69 –74.

Received July 24, 2002. Accepted December 4, 2003. Address requests for reprints to: Daniel K. Podolsky, M.D., Gastrointestinal Unit and Center for Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Boston, Massachusetts 02114. e-mail: [email protected]; fax: (617) 724-2136. Supported by Grants DK46906, DK07191, and DK43351 from the National Institutes of Health, as well as by grants from the Canadian Institute of Health Research and the Alberta Heritage Foundation for Medical Research.