1 Department of Medicine, The pathology of Crohn's disease and ulcerative
colitis is characterized by chronic inflammation and destruction of the
gastrointestinal epithelium. Although suppression of inflammatory
mediators remains the principle component of current disease
therapeutics, strategies for enhancing repair and regeneration of the
compromised intestinal epithelium have not been widely explored. The
demonstration that a peptide hormone secreted by the intestinal
epithelium, glucagon-like peptide-2 (GLP-2), is a potent endogenous
stimulator of intestinal epithelial proliferation in the small bowel
prompted studies of the therapeutic efficacy of GLP-2 in CD1 and BALB/c
mice with dextran sulfate (DS)-induced colitis. We report here that a
human GLP-2 analog
(h[Gly2]GLP-2)
significantly reverses weight loss, reduces interleukin-1 expression,
and increases colon length, crypt depth, and both mucosal area and
integrity in the colon of mice with acute DS colitis. The effects of
h[Gly2]GLP-2 in the
colon are mediated in part via enhanced stimulation of mucosal
epithelial cell proliferation. These observations suggest that
exploitation of the normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing mucosal epithelium in
the presence of active intestinal inflammation.
intestine; inflammatory bowel disease; epithelium; growth factor; inflammation
INFLAMMATION OF THE intestinal epithelium, as
exemplified by Crohn's disease and ulcerative colitis, results in
considerable morbidity, and current therapeutic strategies, generally
directed at suppressing components of the inflammatory response, remain suboptimal (27). The identification of molecules important for maintaining the growth and integrity of the mucosal epithelium has
stimulated the development of novel approaches toward enhancement of
mucosal protection in the gut. For example, the observation that
trefoil peptides are abundantly expressed in the epithelium after
intestinal injury was followed by studies demonstrating that mice
deficient in intestinal trefoil factor are more susceptible to mucosal
injury and recombinant intestinal trefoil factor enhances epithelial
healing of the murine colon in vivo (35). Similarly, the
demonstration that the keratinocyte growth factor (KGF) stimulates epithelial cell proliferation in the gastrointestinal tract (30), taken
together with increased KGF expression in inflammatory bowel disease
(IBD) (51), suggests a possible link between KGF and intestinal
epithelial function in vivo. We have now examined the therapeutic
potential of a recently described intestinal growth factor,
glucagon-like peptide-2 (GLP-2), in mice with dextran sulfate
(DS)-induced colitis.
Despite ongoing advances in our understanding of the cell biology of
the gastrointestinal epithelium, principal strategies for treatment of
IBD remain focused on suppression of the cellular and humoral
inflammatory response. These approaches involve local or systemic
administration of corticosteroids, aminosalicylates, or
immunomodulatory agents such as azathioprine, mercaptopurine, cyclosporin, and methotrexate (27). Although these latter agents are
generally effective they do not specifically target the intestine and
their side effects may be considerable, precluding long-term use in
patients with chronic IBD. Newer targeted approaches to immunosuppressive and anti-inflammatory therapy, including use of
monoclonal antibodies against lymphocyte antigens (24, 48) or tumor
necrosis factor (TNF) (46, 50), interleukin-4 (IL-4) delivery via
adenoviral gene transfer (29), and antisense oligonucleotides for
suppression of intercellular adhesion molecule activity (ICAM), are
currently under evaluation.
The rapid turnover and renewal of differentiated cell types that
constitute the mucosal epithelium of the small and large bowel raise
the possibility that stimulation of epithelial proliferation may be
useful for enhancing repair of epithelial damage in vivo. Identification of growth factors produced locally in the bowel that
regulate crypt cell proliferation, such as epidermal growth factor,
transforming growth factor- GLP-2 administered to normal mice and rats increases growth of the
mucosal epithelium in small and large intestine (21, 53). The increase
in small bowel mass is attributable in part to activation of crypt cell
proliferation and inhibition of enterocyte apoptosis (53). GLP-2 also
promotes intestinal hexose transport via upregulation of
sodium-dependent glucose transporter 1 (SGLT-1) activity (11,
12). The importance of GLP-2 as a trophic factor for
intestinal epithelium is illustrated by studies demonstrating that
GLP-2 infusion prevents parenteral nutrition-associated mucosal hypoplasia in rats (10). To address the possibility that GLP-2 may be
therapeutically useful for enhancing the endogenous reparative response
to mucosal epithelial damage, we administered a degradation-resistant human GLP-2 analog,
h[Gly2]GLP-2 (6, 21),
to mice with experimental DS-induced colitis.
Animals and experimental protocol.
Groups of 6- to 8-wk-old female CD1 mice, 22-24 g or 8- to
9-wk-old female BALB/c mice, 18-21 g (Charles River), were housed in plastic bottom, wire-lid cages, maintained on a 12:12-h light-dark cycle, and allowed chow and water containing 0 or 5.0% DS ad libitum throughout the study. The experiments carried out with CD1 mice, designated experiments A and
B, were carried out with treatment groups containing four to five mice housed together. The experiments with BALB/c mice were carried out with five mice per control group for
saline- and
h[Gly2]GLP-2-treated
mice (not receiving DS) and 10 mice per treatment group for the DS arm
of the study, each BALB/c mouse being housed in a separate cage. CD1
mice were injected with either 0.5 ml saline or 750 ng
h[Gly2]GLP-2 in 0.5 ml
saline twice daily. BALB/c mice were injected with either 0.5 ml saline
or 350 ng h[Gly2]GLP-2
in 0.5 ml saline twice daily.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
(TGF-
), and insulin-like growth
factor I (18), provides an opportunity to manipulate mucosal epithelial
regeneration in experimental models of intestinal damage or resection.
The gastrointestinal tract also secretes regulatory peptides such as
gastrin and gastrin-releasing peptide, with intestinal growth-promoting
activity (28, 31, 56). The observation that injury of the intestinal
mucosa is frequently associated with increased secretion of the
proglucagon-derived peptides (PGDPs) (4), taken together with increased
intestinal growth in patients and rodents with glucagon-producing
tumors (20, 26, 47), resulted in the identification of GLP-2 as the
PGDP with intestinal growth factor-like activity (20).
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
days of oral DS. Two additional deaths in the BALB/c
group (1 in saline-treated DS group, 1 in
h[Gly2]GLP-2-treated
DS group) occurred on day 10 on the
morning when the mice were killed.
Experimental analyses.
Intestinal weights, morphology, enzymatic activity, and GLP-2 content
were assessed as described previously (6, 7, 20, 53). For analysis of
tissue PGDP content 2 cm of distal ileum (2 cm from cecum) and distal
colon (2 cm from the anus) were homogenized on ice in 5 ml of
extraction buffer (1 N HCl, 5% HCOOH, 1% trifluoroacetic acid, 1%
NaCl) and extracted as described previously (13). RNA was
prepared from homogenates of distal jejunum, ileum, and colon (13) and
analyzed as previously described (7). Blood for GLP-2 RIA was collected
in a final volume of 10% Trasylol, EDTA, Diprotin A (5,000 KIU/ml:32
mM:0.1 nM), and plasma was stored at 80°C before analysis by
RIA (6). Semiquantitative RT-PCR was carried out with aliquots analyzed
from a range (20-30) of cycle numbers to ensure linearity for
mouse TGF-
mRNA as previously described (8, 9, 34). The PCR
conditions were 94°C for 1 min and 68°C for 2 min for 30 cycles. Primers for TGF-
were 5'-TGCAGCACCCTGCGCTCGGAAGAT-3' and
5'-CCACCTGGCCAAATTCCTCCTCTG-3'. Occult blood testing
was carried out using Hematest reagent tablets (Bayer, Etobicoke,
Canada), as per the manufacturers' instructions. Myeloperoxidase (MPO)
activity was assayed spectrophotometrically as previously described
(5). Statistical differences between treatment groups were determined
by ANOVA using Tukey's studentized range test for multiple comparisons
at P = 0.05.
Histological analysis. Intestinal segments for histology were taken from proximal jejunum (8 cm distal to the pylorus), distal jejunum (18 cm distal to the pylorus), proximal ileum (10 cm before the cecum), and distal ileum (just proximal to cecum) and from the colon (1-3, 3-5, 5-7, and 7-9 cm distal to the cecum). Tissues were fixed in 10% buffered Formalin for 48 h and embedded in paraffin using standard techniques. Four- to six-micrometer cross sections were cut and stained with hematoxylin and eosin. Intestinal micrometry was performed using a Leica Q500MC image analysis system. Ten well-oriented villi and 25 well-oriented crypts from each small intestinal section were used to determine villus height and crypt depth. Disease severity was graded on a scale from 0-3 according to a standard scoring system (42): 0, normal bowel; 1, focal inflammatory cell infiltrate; 2, inflammatory cell infiltrate, gland drop out and crypt abscess; and 3, mucosal ulceration. Crypt cell proliferation index as assessed by proliferating cell nuclear antigen (PCNA) staining and colonic epithelial apoptosis index as assessed by percent TUNEL-positive cells was carried out as previously described (20, 53).
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RESULTS |
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Control mice not exposed to DS treated with either saline or
h[Gly2]GLP-2 gained
weight over the 9- to 10-day experimental period (Fig.
1). The slightly increased body weight gain
in the CD1
h[Gly2]GLP-2-treated
control mice is largely attributable to the relatively greater increase
in small bowel mass following treatment with the larger dose (750 ng
twice daily) of
h[Gly2]GLP-2 (Figs. 1
and 2).
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Mice receiving 5% DS in the drinking water developed loose blood-streaked stools after 4-5 days, became progressively more lethargic, and lost ~20-25% of their body weight at the end of the 9- to 10-day experiment (Fig. 1). In contrast, h[Gly2]GLP-2-treated mice receiving 5% DS appeared much healthier and lost significantly less weight over the 9- to 10-day experimental period (P < 0.05 for both CD1 and BALB/c experiments, DS-h[Gly2]GLP-2- vs. DS-saline-treated groups, Fig. 1).
GLP-1 has recently been shown to inhibit food and water intake (49, 55), whereas GLP-2 had no effect on food intake in mice over a 10-day experimental period (53). Nevertheless, one potential explanation for the different degree of illness and weight loss in our experiments might be due to theoretical effects of h[Gly2]GLP-2 on reduction of water intake and subsequent cumulative intestinal exposure to DS. Saline-injected control BALB/c mice not receiving 5% DS had a mean daily water intake of 6.5 ± 0.3 vs. 6.7 ± 0.4 ml for h[Gly2]GLP-2-treated control mice (P not significant). Furthermore, the cumulative intake of 5% DS water over the entire 9-day experiment, as well as the 5% DS water intake from experimental days 7-9, was significantly greater for h[Gly2]GLP-2-treated compared with saline-treated BALB/c mice receiving 5% DS (4.96 ± 0.5 vs. 5.9 ± 0.6 ml/day for saline- vs. h[Gly2]GLP-2-treated mice with DS colitis on days 7-9, P < 0.05). These observations demonstrate that the difference in disease severity between groups cannot be explained on the basis of any putative effects of GLP-2 on water intake and hence intestinal exposure to DS.
To determine the consequences of h[Gly2]GLP-2 administration in mice with DS-induced colitis, we examined the gastrointestinal tract from the stomach to the colon in control and DS colitis treatment groups. Although no visible or microscopic pathology was detected in the stomach of DS-treated CD1 mice, stomach weight was reduced in the DS-saline-treated group and was restored toward normal in the DS-h[Gly2]GLP-2-treated mice (Fig. 2A, P < 0.05). Control CD1 mice not exposed to DS but treated with h[Gly2]GLP-2, 750 ng twice daily for 10 days, had a significant increase in small bowel mass (Fig. 2B, 2.2 ± 0.04 vs. 1.3 ± 0.01 g h[Gly2]GLP-2 vs. control for experiment A, P < 0.05). CD1 mice with DS colitis treated with saline alone had a significant reduction in the mass of the small bowel (P < 0.05 for experiments A and B, Fig. 2B). In contrast, DS-h[Gly2]GLP-2-treated CD1 mice with colitis (experiments A and B) exhibited a significant increase in small bowel mass (Fig. 2B, 1.85 ± 0.2 vs. 0.86 ± 0.1 g, DS-h[Gly2]GLP-2- vs. DS-saline-treated mice for experiment A, P < 0.05) and a small but significant increase in small bowel length in experiment B (Fig. 2B, P < 0.05).
Treatment of healthy control CD1 mice with h[Gly2]GLP-2 produced a significant increase in large bowel weight (Fig. 2B, 0.27 ± 0.01 vs. 0.35 ± 0.01, P < 0.05 saline- vs. h[Gly2]GLP-2-treated animals), consistent with the results of previous studies (19). Similarly, mice with DS colitis (experiments A and B) treated with h[Gly2]GLP-2 had a significant increment in large bowel weight (0.24 ± 0.04 vs. 0.32 ± 0.02 g, DS-saline- vs. DS-h[Gly2]GLP-2-treated mice, P < 0.05, Fig. 2B, experiment A). Treatment of normal CD1 mice with h[Gly2]GLP-2, 750 ng twice daily, produced a small but significant increment in large bowel length (Fig. 2B, b and f). Mice with DS colitis also exhibited a significant decrease in large bowel length (P < 0.05, saline-treated controls vs. mice with DS colitis, Fig. 2B). Although large bowel length was greater in h[Gly2]GLP-2-treated mice with colitis (Fig. 2B) this difference was statistically significant for mice in experiment B (DS-saline- vs. DS-h[Gly2]GLP-2-treated mice, P < 0.05) but not in experiment A (Fig. 2B).
Similar results were observed for BALB/c mice with DS colitis treated with a lower dose of h[Gly2]GLP-2 (350 ng twice daily). The relative magnitude of increase in small bowel weight in wild-type BALB/c mice treated with 350 ng h[Gly2]GLP-2 was smaller than in CD1 mice but still significant (P < 0.05, saline- vs. h[Gly2]GLP-2-treated mice, Fig. 2B). The BALB/c mice receiving 5% DS appeared more ill than the CD1 mice, and a total of five BALB/c DS mice died during this experiment (3 in the saline-treated and 2 in the h[Gly2]GLP-2-treated group). The small bowel weight was significantly reduced in BALB/c DS-saline mice and increased significantly in mice treated with h[Gly2]GLP-2 (P < 0.05, saline- vs. h[Gly2]GLP-2-treated BALB/c mice, Fig. 2B). The large bowel weights of saline-treated BALB/c mice with DS colitis were significantly reduced compared with h[Gly2]GLP-2-treated mice with colitis, P < 0.05. Furthermore, the large bowel lengths were markedly reduced in both saline- and h[Gly2]GLP-2-treated BALB/c mice with colitis, but large bowel length was significantly greater in the h[Gly2]GLP-2-treated mice (P < 0.05, h[Gly2]GLP-2-treated vs. saline-treated BALB/c mice with DS colitis, Fig. 2B).
As small and large bowel wet weights in mice with intestinal inflammation potentially reflect cellular infiltration, hyperplasia, increased protein synthesis, and/or edema, we compared wet and dry small and large bowel weights in CD1 mice with and without colitis (Fig. 2C). The increase in small bowel wet and dry weights in control mice treated with h[Gly2]GLP-2 was most evident in the jejunum (P < 0.05, saline- vs. h[Gly2]GLP-2-treated control mice, Fig. 2C). Both saline- and h[Gly2]GLP-2-treated mice with DS colitis had increased wet colon weights (P < 0.05, control vs. DS colitis groups). In contrast, only the h[Gly2]GLP-2-treated mice with DS colitis had significantly increased dry colon weights (P < 0.05, saline- vs. h[Gly2]GLP-2-treated mice with DS colitis, Fig. 2C).
Control CD1 mice treated with
h[Gly2]GLP-2 exhibited
a significant increase in jejunal crypt and villus height that was most prominent in the proximal jejunum (P < 0.05, saline- vs.
h[Gly2]GLP-2-treated
mice, Fig. 3), consistent with previous
experiments (19, 54). Histological analysis of the intestine from DS
mice treated with saline injections demonstrated a reduction in small bowel villus and crypt height that was most marked in the jejunum (Fig.
3A). In contrast, mice with DS
colitis treated with
h[Gly2]GLP-2 exhibited
a significant increase in small bowel villus height and crypt depth in
both proximal and distal jejunum and ileum (Fig.
3A, P < 0.05 for
DS-h[Gly2]GLP-2- vs.
DS-saline-treated mice). In the colon, crypt depth was reduced in DS
colitis mice treated with saline alone; however, crypt depth was
significantly increased in mice with colitis treated with
h[Gly2]GLP-2
(P < 0.05, DS-h[Gly2]GLP-2- vs.
DS-saline-treated mice, Fig. 3B).
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Histological analysis of murine small bowel demonstrated an increase in both crypt cell proliferation and a decrease in epithelial cell apoptosis following treatment with GLP-2 (20, 53). Although increased numbers of PCNA-positive cells were observed in the colon of control CD1 mice treated with h[Gly2]GLP-2, the increase was not statistically significant (P = 0.1, Fig. 3C). In contrast, a highly significant increase in the number of PCNA-positive cells was detected in histological sections from h[Gly2]GLP-2-treated mice with DS colitis (Fig. 3C, DS-h[Gly2]GLP-2- vs. DS-saline-treated mice, P < 0.001). Although h[Gly2]GLP-2-treated control mice exhibited a significant decrease in apoptotic cells in the colon (P < 0.05), no significant change in the percentage of apoptotic cells was observed in DS-h[Gly2]GLP-2- vs. DS-saline-treated mice (Fig. 3D).
The colons from both CD1 and BALB/c mice with DS colitis contained
blood and exhibited varying degrees of mucosal infiltration with
leukocytes, loss of normal glandular architecture, and areas of both
crypt erosion and destruction. All BALB/c mice receiving DS were occult
blood positive when they were killed. Gross blood in the colon was
visible at necropsy in four saline-treated and two
h[Gly2]GLP-2-treated
BALB/c mice with colitis. The damage to the epithelial mucosa was
quantified by assessment of the total mucosal area in multiple
histological segments from proximal, middle, and distal colon from CD1
mice. An increase in mucosal surface area in all three regions of the
colon was observed in healthy control mice treated with
h[Gly2]GLP-2 (Fig.
4A,
P < 0.05 for
h[Gly2]GLP-2- vs.
saline-treated control mice). Mice treated with DS and saline
injections exhibited a significant decrease in mucosal surface area,
most prominent in the proximal colon (Fig.
4A). In contrast,
h[Gly2]GLP-2
administration to mice with DS colitis was associated with an increase
in mucosal area that was most significant in the proximal and middle
colon (Fig. 4A,
P < 0.05 for proximal and middle
colon, DS-h[Gly2]GLP-2- vs.
DS-saline-treated mice).
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To further quantify the extent of epithelial disruption in mice with
colitis, the presence or absence of intact epithelial mucosa was scored
in multiple sections. Mice treated with
h[Gly2]GLP-2
consistently exhibited a greater proportion of intact mucosal epithelium, and this difference was statistically significant for both
proximal and distal colon (Fig.
4B, P < 0.05 for proximal and distal colon,
DS-h[Gly2]GLP-2- vs.
DS-saline-treated mice). Furthermore, the percentage of histological
sections in the proximal colon exhibiting a pathological index (Fig.
4C) of two or three was greater in
saline-treated mice with DS colitis compared with mice treated with
h[Gly2]GLP-2 (Fig.
4C, P < 0.05 for grade II lesions,
DS-h[Gly2]GLP-2- vs.
DS-saline-treated mice). Analysis of MPO activity as an indirect
indicator of neutrophil infiltration demonstrated a marked induction of
MPO activity in the colon of BALB/c mice with DS colitis. No
significant differences in the relative levels of MPO activity were
detected in the colons from
h[Gly2]GLP-2-treated
control or DS mice (Fig. 5).
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To determine the effects of DS colitis and
h[Gly2]GLP-2 treatment
on gene expression in the small and large bowel, RNA prepared from CD1
mice in experiment A was analyzed by
Northern blotting and RT-PCR. Although the levels of SGLT-1 mRNA were
similar in jejunum and ileum from CD1 control mice and mice with DS
colitis, a slight decrease in SGLT-1 mRNA was detected in the colon of mice with DS colitis, consistent with destruction of epithelial mucosa
(Fig. 6, A and
B). In contrast, IL-1 mRNA transcripts, indirect markers of
intestinal inflammation, were markedly induced in the colon of mice
with DS colitis (Fig. 6, A and B), and the levels of IL-1 mRNA transcripts were clearly lower in the colons of
mice with colitis treated with
h[Gly2]GLP-2. The
reduction in IL-1 mRNA in
h[Gly2]GLP-2-treated
mice is unlikely due to a dilutional effect secondary to increased
bowel mass, as we did not observe corresponding reductions in the
colonic levels of proglucagon, SGLT-1, KGF, and 18S RNAs in RNA
analyses from the same mice.
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Because both KGF and TGF- have been shown to have therapeutic
activity in experimental models of intestinal inflammation (22, 58) we
examined whether the effects of
h[Gly2]GLP-2 treatment
might be mediated in part via local induction of these growth factors.
Northern blot analysis detected induction of colonic KGF gene
expression in mice with colitis (Fig.
6A). Nevertheless, there were no
significant differences in the levels of intestinal KGF mRNA in saline-
vs.
h[Gly2]GLP-2-treated
mice with DS colitis (Fig. 6A). The
levels of TGF-
mRNA transcripts were easily detectable and
comparable in control saline- and
h[Gly2]GLP-2-treated
healthy mice (Fig. 6C). In contrast,
a marked reduction of TGF-
mRNA was observed in colon RNA from both
saline- and h[Gly2]GLP-2-treated
mice with DS colitis, consistent with the presence of significant
destruction of the intestinal epithelium and/or toxicity from
DS on TGF-
-producing cell types. Thus no evidence for
h[Gly2]GLP-2-mediated
induction of intestinal KGF or TGF-
gene expression was observed in
these experiments.
Because previous studies have suggested that some forms of small
bowel injury are associated with increased levels of circulating enteroglucagons (4), we measured tissue levels of immunoreactive GLP-2
in the ileum and colon and levels of proglucagon mRNA transcripts in
jejunum, ileum, and colon of control and DS-treated mice. No significant change in the levels of proglucagon mRNA transcripts was
detected in the jejunum, ileum, or colon of mice with DS colitis (Fig.
6, A and
B). Immunoreactive GLP-2 levels were
not increased in the ileum; however, a significant decrease in the
levels of GLP-2 was observed in the colon of DS-treated mice, possibly
consistent with the colitis-associated destruction of the mucosal
epithelium and/or increased secretion and decreased storage of
tissue GLP-2 (Fig. 6D,
P < 0.05, control vs. DS-treated
mice). In contrast to the reduction in tissue levels of GLP-2, analysis
of the levels of circulating GLP-2 demonstrated increased levels in
mice with DS colitis (Fig. 7), with a
significant increase in plasma GLP-2 observed in the DS mice treated
with h[Gly2]GLP-2
(P < 0.05, h[Gly2]GLP-2-treated
mice without colitis vs.
h[Gly2]GLP-2-treated
mice with DS colitis).
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DISCUSSION |
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The mechanisms responsible for the development of ulcerative colitis in human patients remain incompletely understood and likely include an inappropriate immune response to dietary or microbial antigens, ultimately leading to cytokine activation and epithelial damage. The reproducible induction of experimental colitis in rodents following exposure to DS provides an opportunity to study the therapeutic efficacy of specific interventions on disease progression as assessed by histopathology of inflamed tissues in vivo. DS-induced rodent colitis pathologically resembles human ulcerative colitis, with development of mucosal edema, crypt erosions, and abscesses, leading to polyp formation and ultimately, progression to dysplasia and adenocarcinoma (14, 23, 40, 57). The precise mechanism(s) underlying the toxicity of DS for the colonic epithelium remains unclear but is not directly dependent on the presence of B, T, NK, or mast cells (1, 15, 37) and may include direct cytotoxicity, modulation of lymphocyte-epithelial cell interactions, and/or indirect cytotoxicity via liberation of multiple proinflammatory cytokines (39).
The majority of therapeutic maneuvers directed at suppressing the
development of DS-induced colitis have focused on interference with
immune-mediated mediators of cytotoxicity. Infusion of rat anti-neutrophil serum lowered the circulating neutrophil count, reduced
mucosal damage, and stabilized, but did not prevent weight loss (17)
and topical application of lidocaine, prednisolone, or sucralfate
reduced mucosal permeability, in rats with DS-induced colitis (3).
Neutralization of TNF by infusion of anti-TNF monoclonal or polyclonal
antibodies either had no effect (41) or exacerbated the development of
acute DS-induced colitis in mice (32), whereas infusion of homologous
IgG or a 5-lipoxygenase activating protein inhibitor improved
DS-induced colitis in rats and mice, respectively (38, 45). Although
fasting is associated with reduction of disease severity and decreased
IL-1 in mice with DS colitis (44), neutralization of IL-1 activity
had no effect on disease activity in mice (32); however, treatment with
IL-1 receptor antagonist attenuated the severity of granulomatous colitis in rats (36). The importance of ICAM expression in the pathophysiology of colitis was illustrated by increased ICAM-1 expression in inflamed colon and antisense oligonucleotides against ICAM-1 RNA inhibited ICAM-1 expression and ameliorated the severity of
DS-induced colitis in mice (2). Taken together, these observations illustrate that many of the therapeutic strategies employed for the
treatment of human IBD (27) generally give comparable results in
studies of rodents with experimental colitis.
Considerably less information is available on the feasibility of
therapeutic strategies designed to enhance epithelial repair following
resection of or damage to the intestine. The importance of intestinal
growth factors for regeneration of compromised epithelial mucosa is
illustrated by experiments in mice with genetic TGF- deficiency.
TGF-
-deficient intestine exhibits an increased susceptibility to
mucosal damage following exposure to DS, and treatment of
TGF-
-deficient mice with recombinant TGF-
reduced the severity of
intestinal injury (22). Similarly, KGF administration enhanced mucosal healing in rats with experimental colitis (58). Although growth hormone
and insulin-like growth factor I enhance intestinal adaptation in
rodents (18), the efficacy of growth hormone for stimulation of
intestinal adaptation in human studies of short bowel syndrome remains
unclear (52). DS has been shown to be cytotoxic for intestinal
epithelial cells (39), and DS directly inhibits proliferation of mouse
colonic epithelial cells in vitro (15); this inhibitory cytotoxic
effect may indirectly hinder endogenous mucosal regeneration and
recovery from DS-induced colitis in vivo. Accordingly, the observation
that GLP-2 exerts proliferative activity in normal colonic epithelium
(19) provides a potential explanation for the therapeutic efficacy of a
GLP-2 analog in mice with experimental colitis.
The data presented here clearly demonstrate that treatment of DS
colitis in mice with
h[Gly2]GLP-2 produced
significant reversal of weight loss. The increased body weight in
DS-h[Gly2]GLP-2-treated
mice is not simply due to direct GLP-2-mediated stimulation of food
intake, as we have previously demonstrated that GLP-2 treatment does
not modify eating behavior in mice (53). The observation that mice
treated with
h[Gly2]GLP-2 also
exhibit modestly decreased histological evidence of mucosal damage and
reduced expression of inflammatory cytokines such as IL-1 provides
additional evidence for the beneficial effects of
h[Gly2]GLP-2 treatment
in vivo. Nevertheless, it must be emphasized that
h[Gly2]GLP-2-treated
mice with colitis still exhibited significant histological evidence of
intestinal inflammation, mucosal epithelial destruction, and markedly
increased levels of tissue MPO activity. Similarly, comparable
therapeutic trials in mice with DS colitis have shown only a partial
reversal of weight loss using agents such as -melanocyte-stimulating hormone or TGF-
(22, 43).
Our current understanding of the mechanisms of GLP-2 action is focused
on the gastrointestinal tract, because no evidence for GLP-2-mediated
abnormalities or proliferation in other tissues was detected in mice
treated with daily GLP-2 for up to 3 mo (53). The beneficial effects of
h[Gly2]GLP-2 on weight
loss in our studies may be due in part to a combination of increased
nutrient absorption from the small bowel (7) and/or increased
healing and regeneration of the large bowel. The failure to detect
h[Gly2]GLP-2-induction
of KGF and TGF- expression in the normal or diseased colon is in
keeping with suggestions that the effects of GLP-2 on the intestinal
epithelium are direct, likely mediated via a separate and distinct
GLP-2 receptor.
Interestingly, although little is known about the factors regulating GLP-2 biological activity in the presence of intestinal disease, we observed a marked increase in small bowel dipeptidyl peptidase (DP) IV activity in saline-treated mice with DS colitis (data not shown). Induction of DP IV activity in the small bowel would be predicted to result in increased GLP-2 degradation and reduced levels of biologically active GLP-2 in vivo (21). These observations provide additional rationale for the use of DP IV-resistant GLP-2 analogs for treatment of intestinal diseases associated with inflammation and compromised epithelial repair and regeneration. In contrast to the reduction of DP IV activity, plasma levels of total immunoreactive GLP-2 were significantly higher in h[Gly2]GLP-2-treated mice with DS colitis compared with h[Gly2]GLP-2-treated controls. These observations suggest that GLP-2 secretion and/or clearance may be altered in the presence of intestinal inflammation, a possibility that merits further study. Taken together, these data emphasize the importance of understanding the physiological determinants of GLP-2 bioactivity in vivo.
Analysis of proglucagon gene expression in the colon of DS-treated mice demonstrated no significant change in the relative levels of proglucagon mRNA transcripts in mice with DS colitis. Nevertheless, given the considerable extent of mucosal damage and probable loss of GLP-2-producing enteroendocrine cells in the mucosal epithelium of the colon, the relatively normal (compared with control mice without colitis) level of proglucagon mRNA in the colon of mice with colitis is consistent with an upregulation of proglucagon gene expression in the remaining enteroendocrine cells of the colon. An increased number of proglucagon RNA transcripts per enteroendocrine cell has previously been observed in remnant rat intestine following small bowel resection (25). In contrast, the tissue levels of GLP-2 were significantly decreased in the colon of mice with DS colitis, likely due in part to decreased GLP-2 storage and increased GLP-2 secretion. Taken together, these observations suggest that epithelial damage is associated with signals that increase local GLP-2 synthesis and secretion leading to activation of intestinal repair mechanisms and epithelial restitution. Accordingly, significant intestinal resection or damage may be associated with relative local or circulating GLP-2 deficiency, and hence therapeutic administration of GLP-2 in these conditions may be viewed as a form of hormone replacement therapy that stimulates epithelial proliferation in both the small and large bowel. The significant improvement in body weight and multiple histological parameters of epithelial integrity and disease activity suggest that the therapeutic potential of epithelial growth factors, in conjunction with efforts directed at suppressing the inflammatory response, merits further study in models of disease associated with inflammation and destruction of the intestinal epithelial mucosa.
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ACKNOWLEDGEMENTS |
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We thank M. Hill and F. Wang for expert technical assistance and Dr. S. Asa for help with the histological analysis and immunocytochemistry.
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FOOTNOTES |
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This work was supported in part by a grant from the Medical Research Council of Canada (P. L. Brubaker and D. J. Drucker), and the National Science and Engineering Research Council of Canada and Allelix Biopharmaceuticals (D. J. Drucker). D. J. Drucker is a Scientist of the Medical Research Council of Canada and a consultant to Allelix Biopharmaceuticals.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Drucker, Toronto Hospital, 200 Elizabeth St. CCRW3-838, Toronto, Canada M5G 2C4.
Received 25 February 1998; accepted in final form 2 September 1998.
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