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Published online before print November 3, 2003
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* Department of Gastroenterology and
Liver Unit, Institut de Malalties Digestives, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Spain;
Department of Pathology, School of Clinical and Laboratory Sciences, University of Newcastle-upon-Tyne, Royal Victoria Infirmary, United Kingdom;
Department of Pathology, Hospital Mutua of Terrassa, Barcelona, Spain;
¶ Histopathology Unit, Cancer Research UK, London Research Institute, United Kingdom; and
|| Novo Nordisk A/S, Bagsvaerd, Denmark
1 Correspondence: Gastroenterology Department, Hospital Clínic, IDIBAPS, University of Barcelona, Villarroel 170, 08036 Barcelona, Spain. E-mail: panes{at}medicina.ub.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: trefoil peptides • TFF2 • intravital microscopy • cell adhesion molecule • MAdCAM-1 • VCAM-1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The potential reparative properties of these peptides have been demonstrated in recent studies using transgenic animals in which expression of individual trefoil protein genes has been ablated or augmented [4 5 6 ]. Studies using TFF3 knockout mice revealed a normal phenotype but increased sensitivity to damaging agents such as dextran sulfate sodium (DSS) [6 ]. In addition, intracolonic (ic) administration of TFF2 has been shown to enhance mucosal repair in a model of dinitrobenzenesulphonic acid (DNBS)-induced colitis in rats [7 ]. However, the potential, preventive effect and the relative therapeutic value of trefoil proteins by diverse routes of administration (topical vs. systemic) have not been assessed directly in experimental colitis.
Central to the pathology of IBD is the role of molecules that regulate the recruitment of leukocytes such as cell adhesion molecules (CAMs) [8 ]. Leukocyte and endothelial CAMs participate in transmigration of leukocytes from the vascular compartment to sites of inflammation or immune reaction. This process results from a complex series of events involving rolling, activation, firm adhesion, and subsequent migration of leukocytes across the vascular endothelium. Vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) are endothelial CAMs of the immunoglobulin (Ig) superfamily, with a critical role in mediating the firm adhesion of leukocytes to endothelial cells in IBD [9 ].
Trefoil peptides are thought to have a major role in the maintenance of epithelial integrity, but potential interactions between these proteins and nonepithelial glycoprotein-secreting tissues or cells have not been investigated fully. CAMs expressed by endothelial cells, such as VCAM-1 and MAdCAM-1, and leukocyte integrins, are glycoproteins that may be classed as mucins on the basis of their O-glycosylation pattern and proline-, serine-, and threonine-rich domains [10
]. TFF3 has been shown to reduce the expression of several families of adhesion molecules such as E-cadherin, 
- and ß-catenin, and associated proteins in colorectal carcinoma cell lines to modulate epithelial cell adhesion and to facilitate cell migration [11
]. It is possible that trefoil proteins may also influence migration of inflammatory cells through modulation of adhesion molecule function or expression in endothelial cells. A recent study, showing that TFF2 and TFF3 are expressed in rat lymphoid tissues and participate in the immune response induced by the bacterial lipopolysaccharide in which monocyte migration is stimulated, supports this hypothesis [12
].
In the present investigation, we have used a well-established model of experimental colitis induced by oral administration of DSS in mice. This model is characterized by morphological changes that mimic human ulcerative colitis [13 ]. The overall aims of the present work were to study the preventive and therapeutic efficacy of TFF2 in experimental colitis, to compare the efficacy of different routes of administration of TFF2, and to examine the possible effects of trefoil peptide administration on the expression of endothelial CAMs and leukocyte recruitment in the context of the intestinal inflammatory response.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study groups
Study 1. ic TFF2 pretreatment
Colitic mice were pretreated with ic administration of recombinant, glycosylated, human TFF2, a 14-kDa protein, at a dose of 2.0 mg/kg in 1% carboxymethylcellulose (MC; n=10), once daily for 10 days, starting 2 h before induction of colitis and until sacrifice at Day 10. Recombinant TFF2 was expressed in yeast (Saccharomyces cerevisiae) and purified, as described previously [15
]. The control group consisted of mice (n=10) treated with vehicle (0.1 ml MC). For treatment administration, a flexible, plastic cannula (PE-10, Portex, Hythe, UK) with an outside diameter of 1 mm was inserted into the colon so that the tip was 5 cm proximal to the anus.
Study 2. Subcutaneous (sc) TFF2 pretreatment
To evaluate the effects of systemic administration of trefoil peptide, mice were treated once daily, starting 2 h before induction of colitis and during 10 days with sc recombinant, glycosylated TFF2 at doses 2.0 mg/kg in vehicle, 0.1 ml 1% MC (n=10). These doses were based on previous in vivo studies showing therapeutic effectiveness in models of gastric injury [16
]. A colitic control group (n=10) was treated with sc vehicle.
Study 3. ic TFF2 treatment of established colitis
Twenty colitic mice were treated with ic recombinant, glycosylated TFF2 after removing toxic DSS, which is from Day 4 to Day 9 after induction of colitis. The daily dose of TFF2 was 2.0 mg/kg.
Assessment of inflammatory damage
Clinical parameters including body weight, stool consistency (score: 0, normal stools; 1, soft stools; 2, liquid stools), and rectal bleeding (score: 0, negative fecal occult blood test; 1, positive fecal occult blood test; 2, visible rectal bleeding) were assessed daily. For assessment of rectal bleeding, HemoFEC (Boehringer Mannheim, Barcelona, Spain) was used. The disease activity index (DAI) was determined at Day 10 and combines the aforementioned parameters as described by Murthy et al. [17
]. Mice were killed by cervical dislocation at Day 10 after the induction of colitis. The colon was excised and opened by a longitudinal incision, rinsed with saline, and weighed, and its length was measured after exclusion of the cecum. To evaluate histological damage, colonic samples were fixed in 4% formalin and embedded in paraffin, and sections (5–7 µm) were stained with hematoxylin and eosin following standard procedures. A single pathologist, unaware of the treatment group, assessed sections. Colitis was graded quantitatively according to previously defined criteria, which take into account the lesion depth and the percentage of colon with mucosal injury over the total colon length [18
]. This histological score grades the degree of colon inflammation, and higher values indicate more severe lesions.
In vivo assessment of leukocyte–endothelial cell interactions
Leukocyte–endothelial cell interactions in colonic submucosal and lamina propria venules were characterized using intravital microscopy in control and colitic animals and in mice pretreated or treated with ic TFF2. Studies were performed at Day 10 after induction of colitis (n=6 animals per group).
Fluorescence intravital microscopy
Mice were anesthetized with sc ketamine (150 mg/kg body weight; Ketolar, Parke-Davies, Morris Klein, NJ) and xylazine (7.5 mg/kg body weight; Sigma Chemical Co., St. Louis, MO), and a tail vein was cannulated. Throughout the experiments, rectal temperature was monitored using an electrothermometer and was maintained between 36.5°C and 37.5°C with an infrared heat lamp. The abdomen was opened via a midline incision, and a segment of the distal colon was chosen for microscopy examination, exteriorized, and covered with cotton gauze soaked with bicarbonate buffer. Mice were then placed on an adjustable microscope stage, and the colon was extended over a nonautofluorescent plastic coverslip that allowed observation of a 2-cm2 segment of tissue. An inverted microscope (Diaphot 300, Nikon, Tokyo, Japan) with a CF Fluor 40x objective lens (Nikon) was used. A charge-coupled device (CCD) camera (Model XC-77, Hamamatsu Photonics, Hamamatsu, Japan) with a C2400 CCD camera control unit and a C2400-68 intensifier head (Hamamatsu Photonics) mounted on the microscope projected the image onto a monitor (Trinitron KX-14CP1, Sony, Tokyo, Japan), and the images were recorded using a videocassette recorder (SR-S368E, JVC, Tokyo, Japan) for off-line analysis. A video date–time generator (Panasonic Digital AV Mixer WJ-AVE55, Matsushita Communication Industrial, Tokyo, Japan) displayed these parameters on recorded and live images. Leukocytes were in vivo-labeled by intravenous (iv) injection of rhodamine-6G (Molecular Probes, Leiden, The Netherlands) as described previously [19
]. Rhodamine-6G-associated fluorescence was visualized by epi-illumination at 510–560 nm using a 590-nm emission filter. Single, unbranched submucosal and lamina propria venules with internal diameters (ID) of 25–40 µm were selected for observation. Venular ID was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station). The flux of rolling leukocytes, leukocyte rolling velocity, number of adherent leukocytes, venular blood flow (Vbf), and venular wall shear rate (
) were determined off-line after playback of the videotapes. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of free-flowing leukocytes in the same vessel. The flux of rolling leukocytes was measured as the number of rolling leukocytes that passed a fixed point within a small (10 µm) viewing area of the vessel in a 1-min period. Leukocyte rolling velocity was calculated as the mean of 10 rolling leukocyte velocities and expressed in µm/s. Leukocytes were considered adherent to venular endothelium when stationary for 30 s or longer and expressed as the number per 100 µm length of venule. Vbf was estimated from the mean of the velocity of three free-flowing leukocytes (ffv), using the empirical relationship of Vbf = ffv/1.6. was calculated, assuming cylindrical geometry, using the Newtonian definition = 8 (Vbf/ID). In each animal, three to six random venules were examined, and results were calculated as the mean of each parameter in all venules examined.
Effects of TFF2 on adhesion molecule expression in colitis
Thirty mice were used to characterize endothelial expression of intercellular adhesion molecule 2 (ICAM-2; see below), VCAM-1, and MAdCAM-1 in the colon under baseline conditions (n=15) and after induction of colitis (n=15) at Day 10. Additional groups of animals were studied to assess the effect of topical TFF2 pretreatment (n=10) or treatment (n=10) on endothelial expression of VCAM-1 and MAdCAM-1.
Dual radiolabeled monoclonal antibody (mAb) technique
Expression of CAMs was characterized using this technique [20
]. ICAM-2, which is constitutively expressed on endothelial cells and is not increased in response to cellular activation [21
], was measured to provide an estimation of endothelial surface area relative to tissue weight with which to correct CAM expression.
The mAb used to quantify endothelial expression of CAMs were 3C4, a rat IgG2a
against mouse ICAM-2 [22
]; MK1.91, a rat IgG1 against mouse VCAM-1 [23
]; MECA-367, a rat IgG2a against mouse MAdCAM-1 [24
]; and UPC-10, a nonbinding IgG [25
]. MK1.91 was purified by protein G chromatography at Pharmacia Upjohn Laboratories (Kalamazoo, MI). 3C4 and MECA367 were purchased from PharMingen (San Diego, CA). UPC-10 was purchased from Sigma Chemical Co. and was dialyzed to remove sodium azide. The binding mAb directed against ICAM-2, VCAM-1, and MAdCAM-1 were labeled with 125I, whereas the nonbinding mAb UPC-10 was labeled with 131I (Amersham Ibérica, Madrid, Spain). Radioiodination of the mAb was performed by the iodogen method as described previously [26
]. Labeled mAb were stored at 4°C and used within 3 weeks after the labeling procedure. The specific activity of labeled mAb was 
0.5 mCi/mg.
Animals were anesthetized with sc ketamine (150 mg/kg) and xylazine (7.5 mg/kg), and the left carotid artery and a tail vein were cannulated with PE-10 tubing. For assessment of endothelial expression of ICAM-2, VCAM-1, and MAdCAM-1, a mixture of 10 µg 125I-3C4 and 60 µg unlabeled 3C4, 10 µg 125I-MK1.91 and 20 µg unlabeled MK1.91, and 10 µg 125I-MECA-367 without additional unlabeled MECA-367 was administered, respectively. In all cases, 10 µg 131I-UPC-10 was added to the injection mixture. Doses of anti-ICAM-2, anti-VCAM-1, and anti-MAdCAM-1 mAb proved to be saturating in previous assays [27
, 28
]. The mixture of binding and nonbinding mAb was administered through the tail vein catheter. Blood samples were obtained through the carotid artery catheter 5 min after injection of the mAb mixture. Thereafter, animals were heparinized (1 mg/kg sodium heparin iv) and rapidly exsanguinated. Entire organs were then harvested and weighed. 125I (binding mAb) and 131I (nonbinding mAb) activities in each organ and 100 µl aliquots of cell-free plasma were counted in a Cobra II -counter (Packard, Canberra, Australia) with automatic correction for background activity and spillover. The injected activity in each experiment was calculated by counting a 3-µl sample of the 300-µl injection mixture containing the radiolabeled mAb. The accumulated activity of each mAb in an organ was expressed as nanograms of binding mAb per gram of tissue. The formula used to calculate ICAM-2, VCAM-1, or MAdCAM-1 expression was as follows: Endothelial expression = [(cpm 125I organxg-1xcpm 125I injected-1)–(cpm 131I organxg-1xcpm 131I injected-1)x(cpm 125I in plasma)/(cpm 131I in plasma)] x ng injected mAb. This formula was modified from the original method [20
] to correct the tissue accumulation of nonbinding mAb for the relative plasma levels of binding and nonbinding mAb [29
].
Effects of TFF2 on endothelial cell adhesion molecule expression in vitro
To determine whether changes in adhesion molecule expression in response to TFF2 treatment were a direct effect of the peptide, primary cultures of human umbilical vein endothelial cells (HUVEC) under basal or stimulated conditions were exposed to different concentrations of TFF2. HUVEC were cultured in Endothelial Cell Basal Medium-2 (Bio-Whittaker, Verviers, Belgium) in 96-well plates. When endothelial monolayers were confluent, they were incubated for 20 h at 37°C and 5% CO2 with different doses of TFF2 (10 nM, 100 nM, and 1000 nM) with or without tumor necrosis factor (TNF-; 10 ng/ml; Sigma Chemical Co.). This cytokine was chosen because of the central role it has in the pathogenesis of IBD. The concentration of TFF2 used was based on previous studies showing efficacy as proangiogenic factors [30
]. Thereafter, cells were washed and incubated for 45 min at room temperature (RT) with a mouse anti-VCAM-1 antibody (Hae2d) or a mouse anti-E-selectin antibody (E83), as a positive control for endothelial activation. Dr. Pablo Engel (Liver Unit, Institut de Malalties Digestives, Hospital Clínic, Barcelona, Spain) provided antibodies. After several washes, cells were incubated with a 1/1000 dilution of rabbit anti-mouse peroxidase-conjugated antibody for 30 min at RT and added a developing solution of O-phenylenediamine dihydrochloride (Sigma Chemical Co.). After 30 min, absorbance was read at a wavelength of 450 nm. Results were expressed as optical density units. Given that MAdCAM-1 is not expressed by HUVEC, this CAM could not be studied.
Measurement of cytokine production
Levels of murine TNF- and interleukin-6 (IL-6) were quantified in protein extracts from colon homogenates by using commercially available enzyme-linked immunosorbent assay (ELISA) kits supplied by R&D Systems (Abingdon, UK) and Diaclone (Besançon, France), respectively. Results were expressed in pg/mg protein. Moreover, plasma samples were taken after centrifugation at 1000 g during 10 min at 4°C of 1 ml blood, obtained from each mouse by cardiac puncture with a heparinized syringe for measurements of circulating TNF- (R&D Systems) and IL-6 (Diaclone) by ELISA. Results were expressed in pg/ml serum. Assessment of cytokine production was performed in controls, colitic-untreated animals, and colitic animals pretreated with ic TFF2, which induced significant amelioration of colitis (n=7–8 per group).
Statistical analyses
Statistical analysis was performed by using the nonparametric tests Kruskal-Wallis and the Mann-Whitney tests, ANOVA with the Bonferroni (post-hoc) test, and Student’s unpaired t-test when appropriate. All values are expressed as mean ± SEM. Statistical significance was set at P< 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study 2. Preventive effect of sc TFF2
Pretreatment with sc-administered TFF2 also reduced the severity of colitis induced in mice by DSS. Compared with sc vehicle-treated mice, daily pretreatment with sc TFF2 significantly diminished rectal hemorrhage score (1.1±0.1 vs. 1.4±0.1; P<0.05) and colon weight (257±18 vs. 354±8 mg; P<0.0001) as well as the colon weight-to-length ratio (3.6±0 vs. 4.7±0 mg/mm; P<0.0001). By the end of the study (Day 10), sc administration of TFF2 had no significant effects on body weight loss (TFF2 -1.7±0.8, vehicle -1.4±0.7 g), stool consistency (TFF2 1.3±0.4, vehicle 1.1±0.1), DAI (TFF2 2±0.5, vehicle 1.9±0.1), colon length (TFF2 74±6, vehicle 72±2 mm), or the histological damage score (TFF2 71±17, vehicle 71±11).
Colitis was less severe in animals treated with ic TFF2 than in those treated by the sc route. Compared with the latter group, topical administration of TFF2 significantly decreased the DAI score and improved stool consistency at Day 10 (Table 1) . Although there also was a trend for reduction of other parameters of disease severity examined in animals receiving TFF2 by ic route, no significant differences were observed between this group and animals pretreated sc in body weight loss, rectal bleeding, colon weight, colon length, ratio of colon weight to length, or histological damage score (Table 1) .
Study 3. Curative effect of ic TFF2
Treatment with ic TFF2 from Day 4 to Day 9 after induction of colitis did not have any beneficial effect for any of the parameters analyzed. Comparison of TFF2-treated colitic mice with vehicle-treated colitic mice showed that body weight loss (-1.7±1.0 vs. -1.0±0.5 g), stool consistency (1.1±0.1 vs. 1.0±0.1), rectal bleeding (1.1±0.1 vs. 1.2±0.1), DAI (1.7±0.2 vs. 2.0±0.3), colon weight (339.4±14.8 vs. 359.5±9.5 mg), colon length (65.4±1.7 vs. 73.9±2.0 mm), colon weight-to-length ratio (5.2±0.2 vs. 4.9±0.1 mg/mm), and the histological damage score (97.2±24.6 vs. 57.2±10.9) was not significantly different in the two treatment groups.
Leukocyte–endothelial cell interactions in colonic venules
When compared with control animals, colitic mice showed a pronounced increase in leukocyte–endothelial cell interactions. There was a 2.5-fold increase in the flux of rolling leukocytes at Day 10 after induction of colitis (Figs. 1A
and 2A
and 2B
). Few adherent leukocytes were present in venules of control mice, and a 19-fold increase in leukocyte adhesion was observed in colitic animals (Figs. 1B
and 2A
and 2B
). No differences in leukocyte rolling velocity, venular blood flow, or venular wall shear rate were observed between control and colitic mice (not shown).
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Effects of TFF2 on adhesion molecule expression in colitis
The endothelial surface area for the colon per gram of tissue was estimated from endothelial binding of the anti-ICAM-2 mAb 3C4. Values were 350.0 ± 92.0 ng mAb/g tissue in colitic animals and 277.2 ± 27.8 ng mAb/g tissue in control animals; this difference was not significant (P=0.47). Binding of the anti-ICAM-2 mAb was also similar in control and colitic mice in other organs including liver, pancreas, mesentery, stomach, small intestine, and cecum (data not shown). Values for endothelial anti-VCAM-1 and anti-MAdCAM-1 binding were not, therefore, corrected for changes in endothelial surface area relative to organ weight, which might result from edema or capillary recruitment.
Endothelial VCAM-1 and MAdCAM-1 expression in the colons of colitic mice increased significantly (threefold; P=0.01) compared with noncolitic mice (Fig. 3 ). VCAM-1 expression was also increased significantly in the mesentery, ileum, and cecum, and MAdCAM-1 expression was higher in the mesentery, stomach, and cecum (Table 2 ). No accumulation of the anti-MAdCAM-1 antibody was observed in liver, spleen, or other extra-abdominal organs such as heart or lung, which is consistent with the restricted expression of this adhesion molecule in gastrointestinal organs (data not shown). The increase in endothelial expression of VCAM-1 and MAdCAM-1 in colonic vessels correlated significantly with the severity of DSS-induced colitis measured using DAI score and pathological alterations such as the colon weight-to-length ratio (Fig. 4 ).
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Treatment of established colitis with TFF2, which did not cure intestinal inflammation, did not reduce significantly endothelial VCAM-1 expression in the colon (Fig. 3) or in other gastrointestinal organs (Table 2) . An increase in MAdCAM-1 expression in response to TFF2 administration was again observed in the colon (Fig. 3) , jejunum, and ileum (Table 2) as compared with vehicle-treated colitic animals.
Effects of TFF2 on endothelial cell adhesion molecule expression in vitro
Resting HUVEC expressed low levels of E-selectin and VCAM-1 (Fig. 5
). Following activation with TNF-, there was a dramatic increase in the cell-surface expression of these adhesion molecules. Coincubation with TFF2 at concentrations from 10 to 1000 nM did not modify TNF-induced VCAM-1 expression (Fig. 5A)
. As for E-selectin expression, a minor, although significant, reduction of TNF--induced expression was observed when cells were incubated with 10 nM TFF2 (Fig. 5B)
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and IL-6 quantified in protein extracts of colon homogenates were increased in colitic animals with respect to control mice, fivefold and 18-fold, respectively (Fig. 6
). Treatment with ic TFF2 significantly reduced the levels of both cytokines in colonic tissue (Fig. 6)
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levels remained under the detection limits in control and colitic mice. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There have been only two previous therapeutic studies using trefoil peptides in experimental colitis. Mashimo et al. [6 ] demonstrated that mice lacking TFF3 had impaired mucosal healing and died from extensive colitis after oral administration of DSS. The failure in healing was shown to be a result of the absence of TFF3. After rectal application of acetic acid, TFF3-deficient mice treated by luminal instillation of rat TFF3 showed normal healing with enhanced epithelial migration and marked attenuation of gross injury [6 ]. A recent study showed that ic TFF2 enhances the rate of colonic epithelial repair in the DNBS/ethanol-induced colitis model in the rat [7 ], which resembles human Crohn’s disease. There are no published data on the potential therapeutic value of TFF2 in DSS-induced colitis, which resembles human ulcerative colitis, or on the relative efficacy of different routes of administration of TFF2. Further, the possibility that trefoil peptides might act by modulation of inflammatory cell migration pathways has not been investigated previously.
In the current study, we demonstrate that ic TFF2 ameliorates DSS-induced colitis when administered in a preventative mode. The clinical course and colonic, pathological changes such as colon weight-to-length ratio were reduced in response to TFF2 treatment. In contrast to the study by Tran et al. [7 ], we did not observe any beneficial effect of TFF2 when this treatment was administered after induction of colitis. Although the doses of TFF2 used in both studies are very similar, a number of factors may explain this apparent discrepancy, including the different experimental models of colitis (DNBS/ethanol vs. DSS) and different animal species studied (rat vs. mouse).
Studies comparing the efficacy of different routes of administration of TFF2 in the prevention of gastric mucosal damage induced by ethanol or indomethacin have produced conflicting results. Some authors found greater protection with topical administration [31 ], whereas others found greater protection with systemic administration [3 ], and others reported that both routes of administration were equally effective [32 , 33 ]. A possible endocrine-mediated effect, which may imply the presence of a receptor for the peptides, has been proposed. A putative receptor for TFF3 has been reported [34 ], and there is evidence that parenterally administered porcine TFF2 is taken up by mucus-secreting cells in the stomach and secreted into the mucus layer in the same way as endogenous TFF2 [35 ]. To determine whether the protective effects of the TFF2 are dependent on the route of administration, we compared systemic (sc) and luminal TFF2 administration. The results show that both routes of administration can afford amelioration of various parameters of colitis severity, but the luminal route was superior for reduction of the DAI, a very important clinical index. This difference may be related to the ability of trefoil peptides to bind mucins and hence, to improve organization of the mucus layer that protects the apical side of the mucosa from deleterious luminal agents.
We used fluorescence intravital microscopy to characterize changes in leukocyte–endothelial cell interactions in colonic submucosal and lamina propria venules. We have shown that pretreatment with ic TFF2, which improves clinical and pathological scores in DSS-induced colitis, decreased leukocyte adhesion to levels observed in control animals. Administration of TFF2 after onset of colitis, which was clinically ineffective, did not modify leukocyte adhesion in intestinal venules.
To explore whether the reduced adhesion of leukocytes to venular endothelium in the inflamed intestine was related to differences of the known determinants of leukocyte recruitment in this pathological condition, we measured VCAM-1 and MAdCAM-1 expression using the dual radiolabeled mAb technique. We corroborated previous findings that expression of VCAM-1 and MAdCAM-1 is increased significantly in colitic animals; studies in the trinitrobenzene sulfonic acid [29 ] and DSS models of colitis [9 ] have shown a marked increase of VCAM-1 in the colonic endothelium. For MAdCAM-1, previous studies have shown that IL-10-deficient mice with active colitis have much higher levels of MAdCAM-1 in the colon [36 ], and tissue sections from patients with active ulcerative colitis or Crohn’s disease have increased endothelial MAdCAM-1 expression as compared with those from normal subjects [37 ]. Although DSS-induced colitis affects predominantly the distal colon [13 ], we also found increased VCAM-1 expression in the ileum and cecum and increased endothelial MAdCAM-1 in the stomach and cecum. Higher levels of CAMs in these vascular beds probably result from DSS-induced mucosal lesions in these upper segments of the gut.
It is interesting that we have found a strong positive correlation between endothelial expression of VCAM-1 and MAdCAM-1 in colonic vessels and severity of DSS-induced colitis, evaluated by DAI and the colon weight-to-length ratio. Measurement of the expression of these CAMs may prove, therefore, a useful parameter with which to evaluate the severity of colitis and the effects of therapeutic interventions in experimental models. It could also provide a rational method for monitoring the severity of IBD in patients.
VCAM-1-mediated leukocyte recruitment seems to be a key factor in the pathogenesis of DSS-induced colitis, as immunoneutralization of VCAM-1 significantly ameliorates the severity of the experimental colitis [9 ]. TFF2 pretreatment decreased endothelial VCAM-1 expression in the colon in association with an amelioration of colitis and a decrease in leukocyte adhesion. This is consistent with the notion that pretreatment with TFF2 significantly improves colonic inflammation. To our surprise, MAdCAM-1 expression was increased in the colon, jejunum, and ileum in colitic animals receiving TFF2. The explanation for the paradoxical increase in MAdCAM-1 expression after pretreatment or treatment with TFF2 is not readily apparent. It is possible that TFF2 may stabilize MAdCAM-1 molecules on the surface of endothelial cells through interactions with their mucin-like regions.
Data derived from our in vitro experiments assessing the effects of TFF2 on TNF--stimulated endothelial cells indicate that the observed reduction of VCAM-1 expression in colitic animals in response to ic TFF2 was probably an indirect effect resulting from modulation of other factors involved in the inflammatory cascade, as incubation of HUVEC in the presence or absence of different concentrations of TFF2 did not modify TNF-induced VCAM-1 up-regulation. Among the other factors that may be affected by treatment with TFF2, activation of the immune cells and the resulting cytokine production are probably relevant, as concentrations of TNF- and IL-6 in colonic tissue as well as circulating levels of IL-6 were significantly reduced in response to TFF2 treatment. This may be a consequence of "improved" barrier function of the intestinal epithelium afforded by TFF2, as has been shown previously [1
].
In conclusion, our work demonstrates clearly an effect of TFF2 in establishing protection against colonic inflammatory damage, especially after topical application. However, in the model of colitis used in the current study, TFF2 did not show efficacy in the treatment of established colitis. These observations suggest that luminal application of TFF2 might be useful for prevention of disease flares in ulcerative colitis. Further studies assessing the value of these peptides in milder forms of colitis as the sole treatment or as coadjuvant therapy are warranted.
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ACKNOWLEDGEMENTS |
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Received August 22, 2003; revised October 6, 2003; accepted October 7, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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