1 Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, North Carolina 27599; and 2 Human Genome Sciences, Incorporated, Rockville, Maryland 20850
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ABSTRACT |
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Keratinocyte growth
factor-2 (KGF-2, repifermin) is a homolog of KGF-1 with epithelial
mitogenic activities. We investigated the therapeutic role of KGF-2
in intestinal ulceration and its mechanisms of protection. KGF-2
(0.3-5 mg/kg) was administered before or after induction of small
intestinal ulceration by indomethacin (Indo) in prevention and
treatment protocols. In acute studies, KGF-2 was injected for up to 7 days before or daily for 5 days after Indo. In a 15-day chronic study,
KGF-2 was injected intravenously daily beginning before or 7 days after
Indo. Injury was evaluated by blinded macroscopic and microscopic
inflammatory scores, epithelial BrdU staining, tissue IL-1,
PGE2, and hydroxyproline concentrations, and collagen type
I RNA expression. In vitro effects of KGF-2 were evaluated by
epithelial cellular proliferation, restitution of wounded monolayers,
PGE2 secretion, and expression of COX-2 and collagen mRNA.
Intravenous KGF-2 significantly decreased acute intestinal injury by
all parameters and significantly decreased chronic ulceration.
Pretreatment, daily infusion, and delayed treatment were effective.
KGF-2 promoted in vitro epithelial restitution with only modest effects
on epithelial cell proliferation, stimulated COX-2 expression in
cultured epithelial cells, and upregulated in vitro and in vivo
PGE2 production. KGF-2 did not affect in vivo fibrosis,
although it induced collagen expression in cultured intestinal
myofibroblasts. These results suggest that KGF-2 inhibits intestinal
inflammation by stimulating epithelial restitution and protective PGs.
immunoregulation; inflammatory bowel disease; cytokines; prostaglandins
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INTRODUCTION |
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THE INTESTINAL EPITHELIUM is composed of a dynamic single layer of epithelial cells that separates the highly concentrated bacterial antigens and toxins in the lumen of the distal intestinal tract from the gut-associated lymphoid tissue (21). Defective mucosal barrier function either caused by an intrinsic defect or the result of a secondary consequence of inflammation can lead to unrestrained luminal antigen uptake, which overwhelms the innate host defense mechanisms of the mucosal immune response (12, 14, 31). Therefore, efficient epithelial repair is an essential component in the resolution of intestinal inflammation.
Growth factors, which are a class of soluble mediators that
promote mitogenicity (17), chemotaxis (42),
cytokine release (28), and fibrogenesis (4)
in a number of cells, are important contributors to tissue repair,
remodeling, and fibrosis (4). Keratinocyte growth factor-1
(KGF-1) is the seventh member of the fibroblast growth factor family
and is a polypeptide mitogen secreted by fibroblasts and endothelial
cells that acts in a paracrine fashion primarily on epithelial cells,
which do not express this molecule but bear its receptor
(11). KGF-1 stimulates mitogenic and motogenic activity in
epithelial cells in vitro. In vivo administration of recombinant KGF-1,
which was first isolated from human lung fibroblasts, was shown to
induce proliferation of type II pneumocytes (38) and to
stimulate proliferation of epithelial cells throughout the
gastrointestinal tract and liver (15). The role of KGF-1 in intestinal inflammation is unclear, but increased expression of
KGF-1 mRNA has been reported in the mucosa of patients with inflammatory bowel diseases (IBD) (2, 10) and in a T
lymphocyte-mediated model of in vitro crypt hyperplasia
(3). In this in vitro model, KGF-1 expression by
mesenchymal cells was upregulated by tumor necrosis factor-
(TNF-
), and neutralization of endogenous KGF-1 partially inhibited T
cell-mediated crypt hyperplasia (3). Exogenous injection
of recombinant KGF-1 into rats significantly reduced the severity and
extent of mucosal injury induced by trinitrobenzene sulfonic acid
(TNBS) (45). Although intraperitoneal injection of KGF-1
promoted epithelial proliferation in normal rats and potentiated
healing of TNBS-induced colitis, it is uncertain whether the protective
effect of KGF-1 is a result of its mitogenic properties or is due to an
anti-inflammatory effect.
The recently cloned KGF-2 (fibroblast growth factor-10, repifermin) (5) was found to have homology with KGF-1 and to bind to the same fibroblast growth factor receptor 2IIIb on epithelial cells (16). However, KGF-2 also binds to an additional receptor, fibroblast growth factor receptor 1IIIb (23). Fibroblast growth factor-10 knockout mice demonstrate that this molecule functions as a regulator of embryonic brain, lung, and limb development (34). The role of KGF-2 in the gastrointestinal tract is not well studied, although it attenuated colitis induced by dextran sulfate sodium in mice in a recent study (25). In the present study, we investigated the protective and therapeutic effects of exogenous KGF-2 on indomethacin-induced small intestinal ulceration in rats. Furthermore, we explored the effects of KGF-2 on in vivo and in vitro wound healing, epithelial proliferation, and prostaglandin production to determine its mechanisms of protection and quantified fibrosis to explore potential detrimental effects of this molecule.
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MATERIALS AND METHODS |
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Animals
Specific pathogen-free, inbred female Lewis rats (180-200 g) were obtained from Charles River Laboratories (Raleigh, NC). Rats were fed standard rat chow ad libitum and were weighed daily. All rat experiments were conducted according to the highest standards of humane animal care as outlined in the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892] and approved by the University of North Carolina Institutional Animal Care and Use Committee.Experimental Design
Prevention of acute indomethacin-induced small intestinal ulceration by KGF-2. Indomethacin (Sigma Chemical, St. Louis, MO) was dissolved in absolute ethanol (50 mg/ml) by intermittent vortexing for 1 h at room temperature, then diluted 1:4 (vol/vol) in 5% sodium bicarbonate to produce a stock solution of 10 mg/ml. A final dilution of 7.5 mg indomethacin/kg body wt in 0.3 ml of sodium bicarbonate was administered subcutaneously once daily for 2 days. Negative control rats received 0.3 ml of vehicle containing a mixture of 80% sodium bicarbonate and 20% ethanol. Recombinant KGF-2 was supplied by Human Genome Sciences (Rockville, MD). KGF-2 (1 or 5 mg/kg) was administered intravenously or subcutaneously for 6 days beginning 1 day before indomethacin injection. Animals were divided into five groups: 1) 1 mg/kg iv KGF-2 (n = 7); 2) 1 mg/kg sc KGF-2 (n = 7); 3) 5 mg/kg sc KGF-2 (n = 7); 4) 5 mg/kg iv human serum albumin (HSA) (n = 7) as a positive control (all rats receiving indomethacin subcutaneously); and a negative control group receiving vehicle subcutaneously and HSA (5 mg/kg) intravenously (n = 5).
In a separate dose-ranging study, rats received intravenous KGF-2 (0.3, 1.0, or 3.0 mg/kg) for 6 days beginning 1 day before subcutaneous indomethacin (n = 12/group). Positive control rats received indomethacin subcutaneously and HSA (1 mg/kg) intravenously, and negative controls received vehicle subcutaneously and HSA intravenously. To explore the ability of KGF-2 to prevent subsequent intestinal injury, KGF-2 (0.3, 1.0, or 3.0 mg/kg) was administered intravenously on daysPrevention and treatment of chronic indomethacin-induced intestinal ulceration by KGF-2. Indomethacin (7.5 mg/kg) was delivered by the same schedule as described in Prevention of acute indomethacin-induced small intestinal ulceration by KGF-2, and KGF-2 (0.3 or 1 mg/kg) was delivered once daily by intravenous injection in the tail vein beginning 1 day before indomethacin for 14 days after the first indomethacin injection (prevention protocol). In a treatment protocol, KGF-2 (1 mg/kg) was delivered intravenously daily beginning 7 days (7 rats) after initiation of indomethacin treatment and continued for 14 days. Positive control rats received HSA (1 mg/kg iv) for 15 days, beginning 1 day before indomethacin (n = 7); negative controls received HSA (1 mg/kg) beginning 1 day before buffer injection (n = 5).
Clinical Assessment of Inflammation
Clinical manifestations of inflammation were assessed by monitoring the body weight of animals daily. Rats were euthanized by inhalation of 100% CO2. Cardiac blood was obtained for cell counts, which were processed using an automated cell counter (CDC Technology, Oxford, CT). Liver, spleen, and intestine weights were recorded and normalized for the body weight of each rat. Gross intestinal inflammatory scores were quantified by blinded observers at the time of necropsy using a previously validated scale (32). Values of 0-4 (4 as the most severe) were assigned based on the severity of mesenteric contraction, the severity of adhesions, and the extent of intestinal wall thickening. The resulting gross gut score is the sum of these values, the maximum possible being 12. Also, the percentage of the surface area covered by ulceration and the number of ulcers in the most involved 10-cm length of small intestine were determined; this segment of involved intestine was weighed after gently removing the luminal contents. Samples of intestine, liver, and spleen were fixed in 10% formalin, embedded in paraffin, and sectioned for histochemical staining with hematoxylin and eosin, Masson trichrome, and Alcian blue stains. A histological inflammatory score was evaluated for each animal by a blinded observer as previously described and validated (24). In brief, values from 0 to 4 (4 as the most severe) were assigned for both acute and chronic inflammation in coded multiple cross sections of the mid small intestine. The acute and chronic scores of at least three histological segments were averaged, a score of 8 (4 acute, 4 chronic) representing the maximum possible combined total histological inflammatory score. The percentage of surface area covered by microscopic ulceration was also measured in at least three sections of small intestine from each rat. Immunologic assessment of inflammation was performed by determining the intestinal concentration of immunoreactive rat interleukin-15-Bromo-2'-Deoxyuridine Staining for Assessment of Proliferating Cells
Rats in the chronic indomethacin-induced colitis experiment were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg; Sigma Chemical) 1 h before necropsy. Paraffin-embedded sections of 8-µm thickness were deparaffinized and pretreated with 2 N hydroxychloride and pepsin (Sigma Chemical). Endogenous peroxidase was quenched with 1% H2O2. Slides were blocked with 1% normal horse serum in PBS with 0.1% BSA and then incubated with anti-BrdU (DAKO, Carpinteria, CA) in a 1:100 dilution. After being washed with PBS, sections were incubated with biotinylated antimouse IgG (Vector Laboratories, Burlingame, CA) and then with a peroxidase-linked avidin-biotin complex (Vector Laboratories) diluted 1:100 in PBS with 0.1% BSA. Slides were exposed to 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical) substrate for 3 min. Sections were counterstained with hematoxylin. The total number of proliferating cells per crypt was counted at a magnification of ×100 in 10 crypts by two independent investigators blinded to treatment.Cell Culture
Nontransformed rat intestinal epithelial cells IEC-6 (ATCC CRL 1592) used between passages 8 and 18 were cultured with DMEM (GIBCO BRL, Long Island, NY) with 5% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine, antibiotics, and insulin (4 U/ml). Caco-2 epithelial cells (ATCC HTB-37) were also used. Caco-2 cells were cultured in MEM (GIBCO BRL) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1% nonessential amino acids, and antibiotics. HT-29 epithelial cells (ATCC HTB-38) were cultured in DMEM (GIBCO BRL) with 10% FBS and antibiotics. Rat subepithelial intestinal myofibroblasts, passages 3-10, isolated from the colon of neonatal Lewis rats (19, 41) were cultured in Dulbecco's MEM/F-12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics.RNA Preparation and RT-PCR
Intestinal tissues were snap-frozen in isopentane and kept atNorthern Blot Analysis
Total RNA (5 µg/lane) was electrophoresed through 1% agarose/formaldehyde gels. The size-fractioned RNA was transferred to a nylon membrane (Hybond-N, Amersham Life Sciences, Arlington Heights, IL) and hybridized with a [32P]dCTP-labeled (3,000 Ci/mmol, ICN, Costa Mesa, CA) 1300-bp cDNA probe encoding rat collagen type 1 (13). Northern blot hybridization of rat mRNA revealed an established pattern of two different molecular mass transcripts of 4.7 and 5.7 kb, which is characteristic for rat collagen type 1 (13, 18, 41). Hybridizations were performed in Rapid-Hyb buffer (Amersham) for 2-3 h at 65°C followed by washing under high-stringency conditions to reduce background using variable (0.1-2×) concentrations of saline-sodium phosphate-EDTA buffer with 0.1% SDS. The membranes were exposed to Kodak-X-OMAT film (Rochester, NY) atHydroxyproline Content of Rat Small Intestine
To determine whether administration of KGF-2 (1 mg/kg iv, days 0-7) altered the collagen content of intestines from normal or indomethacin-treated rats, tissue hydroxyproline content was measured using modifications of the procedure of Deterding et al. (8). Briefly, 10 cm of normal or ulcerated rat mid small intestine were lyophilized, and the dry weight was recorded. The dry samples were minced and hydrolyzed with 6 N HCl for 18 h at 120°C. Oxidation of the samples at room temperature was made with chloramine T solution (44). Elrlich's reagent was used to develop the color reaction of 30 min at 65°C (44). The reaction was read at 560 nm, and the amount of hydroxyproline was determined from the hydroxyproline standards (Sigma Chemical).Western Blot Analysis for COX-2 Concentrations
For analysis of COX-2 expression, Caco-2 cells (5 × 105/well) were stimulated with various concentrations of KGF-2 (1.0-100 ng/ml), medium alone, TNF-Cell Proliferation Assay
Cells (IEC-6 or Caco-2) were seeded into 24-well plates (5 × 104 cells/well) in the presence of DMEM containing 5% FBS. When ~50% confluent, cells were washed three times and then cultured for an additional 24 h in DMEM containing 0.1% FBS. Cultures were then supplemented with KGF-2 in concentrations ranging from 0.1 to 100 ng/ml. After 20 h at 37°C, 1 µCi/well of [3H]thymidine was added; after 4 h, cells were washed with cold PBS three times and precipitated with 10% TCA; acid-insoluble materials were lysed with 0.1 N NaOH. Incorporation of radiolabeled thymidine was determined by a liquid scintillation counter. [3H]thymidine incorporation in KGF-2-related cultures was expressed as a percentage of [3H]thymidine incorporation in control cultures.Monolayer Wound Repair
An in vitro wound healing assay was performed using a modified method (30). Briefly, reference lines were drawn horizontally across the outer bottom of 24-well plates. Caco-2 cells were seeded and grown to confluence, then incubated with media containing 0.1% FBS for 24 h. Linear wounds were made with a sterile plastic pipette tip perpendicular to the lines on the bottom of the well. Various concentrations of KGF-2 (1-100 ng/ml), transforming growth factor-Statistical Analysis
Results are expressed as means ± SE. Significance of differences was assessed by univariate analysis and ANOVA or the unpaired Student's t-test (hydroxyproline measurements only). Correlation of tissue injury with PGE2-to-IL-1 ![]() |
RESULTS |
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Continuous KGF-2 Treatment Reduces Acute Indomethacin-Induced Injury
In an experiment investigating activities of KGF-2 administered intravenously or subcutaneously, indomethacin-injected rats developed progressive weight loss after 2 days that was almost completely prevented by daily intravenous injection of KGF-2 beginning 1 day before indomethacin administration and continuing for 5 days (Fig. 1). There were significant differences in weight loss between the indomethacin plus HSA group and the groups treated with intravenous KGF-2 or high-dose subcutaneous KGF-2 (5 mg/kg) from day 3 to day 5. Low-dose subcutaneous KGF-2 (1 mg/kg) did not prevent weight loss. The extent of mucosal ulceration in the most affected 10-cm segment of mid small bowel, as determined by the percentage of surface area ulcerated, was markedly reduced in rats treated with intravenous KGF-2, compared with indomethacin-injected rats treated with HSA or subcutaneous KGF-2 (Table 1). The number of ulcers was also decreased in rats receiving intravenous KGF-2 compared with those receiving indomethacin plus HSA (3.7 ± 1.1 vs. 8.7 ± 1.7, respectively; P < 0.01). There were highly significant differences in intestinal adhesions (P = 0.019), mesenteric contractions (P = 0.0003), intestinal thickness (P = 0.00002), and the total gross gut score (P = 0.0003) between the indomethacin plus HSA-treated group and rats treated with 1 mg/kg iv KGF-2. Subcutaneous injection of KGF-2 was less effective in preventing grossly evident intestinal injury, although the average wet weights and percent ulceration of surface area of the most affected 10-cm length of small intestine in all treated groups were significantly decreased compared with positive controls. IL-1
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Microscopic interpretation by a blinded observer showed marked
reduction of acute and chronic histological inflammatory scores in the
intravenous KGF-2-treated group compared with the indomethacin plus HSA
group (Fig. 2). Subcutaneous KGF-2 (1 mg/kg) reduced acute and total inflammatory scores but did not
significantly inhibit chronic inflammation, and the 5 mg/kg sc dose had
no beneficial effect. Similar to the macroscopic observations, KGF-2 at
the 1 mg/kg iv (1.0% of surface area) and 1 mg/kg sc doses (1.6% of surface area) decreased the extent of microscopic mucosal ulceration compared with the indomethacin plus HSA positive control group (8.9%)
(P = 0.001 and 0.002, respectively). Mucosal ulcers in indomethacin plus HSA-treated rats (Fig.
3A) were active with exudate
and necrosis. In some KGF-2-treated rats, ulcers were beginning to heal
by the time of necropsy (5 days after beginning indomethacin) as shown
by reepithelialization of the ulcer base and margins (Fig. 3,
B and C).
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Dose-Dependent Effects of Continuous KGF-2 in Reducing Acute Intestinal Ulceration
In a separate experiment to determine the optimal dose of intravenous KGF-2, rats were pretreated intravenously with KGF-2 at 0.3, 1, or 3 mg · kg
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Pretreatment with KGF-2 Prevents Acute Intestinal Injury
Because daily administration of KGF-2 in the protocol discussed in Dose-Dependent Effects of Continuous KGF-2 in Reducing Acute Intestinal Ulceration could manifest its protective effects by treating the early phase of indomethacin-induced intestinal injury as well as preventing the onset of intestinal ulceration, we next determined whether pretreatment alone with KGF-2 could prevent acute intestinal ulceration and weight loss. In a purely prophylactic study, we demonstrated that KGF-2 injected intravenously daily for 3 days before indomethacin decreased all parameters of intestinal injury and systemic weight loss in a dose-dependent fashion (Table 3). Of interest, 3 mg/kg KGF-2 was more effective than 1 mg/kg in this prevention protocol, in contrast to the continuous treatment study depicted in Table 2.
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In a second purely prophylactic experiment, we sought to determine
whether a single injection of KGF-2 could prevent indomethacin-induced injury and, if so, to identify the duration of this protective effect.
A single injection of KGF-2 (1 mg/kg iv) significantly attenuated acute
mid small bowel ulceration and inflammation 1, 3, or 5 days, but not 7 days, before indomethacin administration (Table
4). No significant improvement in weight
loss was noted after pretreatment for longer than 3 days. Together,
these studies demonstrate that short-term prophylactic administration
of KGF-2 has a protracted ability to prevent acute intestinal injury.
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Prevention and Treatment of Chronic Indomethacin-Induced Small Intestinal Ulceration by KGF-2
We next investigated whether an optimal intravenous dose of KGF-2 could prevent chronic small bowel ulceration and reverse established inflammation. Within the 14 days of observation, control animals did not regain their preinjury weight, whereas high-dose KGF-2 (1 mg · kg
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KGF Modestly Accelerates Epithelial Growth
The number of BrdU-staining epithelial cells in the small intestine of rats 14 days after indomethacin was decreased in positive controls (8.2 ± 1.9 vs. 9.6 ± 0.8 in vehicle plus HSA, P = 0.03; Fig. 4), showing that chronic inflammation decreased mucosal cellular proliferation in this model. Long-term KGF-2 administration increased in vivo epithelial proliferation (9.4 ± 3.4 for continuous daily 1 mg/kg KGF-2 and 10.8 ± 1.2 for delayed treatment, P = 0.02 and 0.001 vs. indomethacin plus HSA, respectively). Interestingly, proliferation was consistently higher on the nonmesenteric side of the small intestine (Fig. 4), away from the ulceration, which typically occurs on the mesenteric border (Fig. 3). Moreover, the single layer of cells reepithelializing ulcers (Fig. 3C) did not exhibit BrdU staining.
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We demonstrated little or no evidence of KGF-2- induced
gastrointestinal mucosal proliferation in normal (noninflamed) rats. A
single injection of KGF-2 (1 mg/kg iv) followed by BrdU injection 24 h later failed to show enhanced proliferation of enterocytes. However, periodic acid-Schiff staining did show an increase in goblet
cell number. In a second, long-term study KGF-2 (0.01, 0.1, and
1 mg/kg) was injected intravenously daily into normal rats for 28 days.
Histopathological analysis demonstrated no microscopic changes in the
intestinal mucosa with no demonstrable epithelial hyperplasia. To
quantitate the ability of KGF-2 to induce goblet cell hyperplasia, six
normal rats were injected with KGF-2 (1 mg · kg1 · day
1 iv) for 6 days. Image analysis demonstrated a significant (P < 0.05) increase in the percentage of the epithelial surface area occupied by goblet cells in the ileum (17.7 ± 2.0 vs. 7.0 ± 0.5 for control) and colon (27.4 ± 2.7 vs. 18.9 ± 0.5 for control).
In vitro studies provided evidence of a modest proliferative effect of
KGF-2 in some, but not all, colonic cell lines. Addition of KGF-2
(1-100 ng/ml) to serum-deprived medium slightly enhanced growth of
Caco-2 cells in a dose-dependent manner as determined by
[3H]thymidine incorporation (Fig.
5). KGF-2 (100 ng/ml) increased cellular
proliferation in Caco-2 cells by twofold. A slight proliferation was
noted in human HT-29 epithelial cells in response to KGF-2, with no
effect on rat IEC-6 cells (Fig. 5).
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KGF-2 Promotes Caco-2 Monolayer Restitution
To investigate the effect of KGF-2 on migration of cells after in vitro "wounding," Caco-2 cells were grown to confluence and then changed to serum-deprived conditions. The width of the acellular region was measured at various time intervals after creation of linear wounds, and data were calculated as the percent change over baseline values. As demonstrated in Fig. 6, KGF-2 significantly enhanced wound healing by cellular migration in a dose- (%change of width at 24 h with 1 vs. 100 ng/ml KGF-2 is 36% vs. 57%, respectively) and time- (%change of width at 6 vs. 24 h with 1 ng/ml is 13% vs. 36%, respectively) dependent fashion. The effect of KGF-2 (10 ng/ml) was equivalent to that of TGF-
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KGF-2 Increases Expression and Activity of COX
KGF-2 increased expression of COX-2 mRNA in Caco-2 cells in a dose-dependent fashion (Fig. 7A). Also, Western blotting showed a dose-dependent increase of COX-2 protein after incubation with KGF-2 (Fig. 7B). PGE2 levels in supernatants of cultured epithelial cells were also increased by KGF-2 (100 ng/ml) to levels comparable with TNF-
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Consistent with the ability of KGF-2 to upregulate prostaglandins in
vitro, tissue PGE2 levels in the small intestine of rats treated with KGF-2 (1 mg/kg iv) were twofold higher compared with the
indomethacin plus HSA injection group (Fig.
8A, 1,338 ± 281 vs.
696 ± 145 pg/mg tissue wt; P = 0.04) in the acute
continuous treatment study, despite significantly lower inflammatory
scores (Fig. 2) and IL-1 tissue concentrations (Fig. 8B)
in the KGF-2-treated rats. The ratio of tissue PGE2 to
IL-1
was >10-fold higher in the KGF-2 (1 mg/kg iv)-treated group
compared with the indomethacin plus HSA control group (0.27 ± 0.17 vs. 0.02 ± 0.003, P = 0.08) (Fig.
8C). The PGE2-to-IL-1
ratio in the vehicle
plus HSA control group was quite high (2.76 ± 1.27) because of
the very low tissue IL-1
concentrations. This ratio of protective to
proinflammatory molecules correlated well with tissue injury as
manifested by the gross gut score (r = 0.94, P < 0.03). In keeping with these results, the ratio of
COX-2 to IL-1
mRNA expression in mid small intestinal tissues was
slightly higher in KGF-2-treated rats (3.00 ± 0.95 in
indomethacin plus KGF-2-injected rats vs. 2.39 ± 0.47 in the
indomethacin plus HSA controls).
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KGF-2 Promotes Collagen Type I Synthesis in Cultured Intestinal Myofibroblasts Without Stimulating In Vivo Fibrosis
To investigate whether KGF-2 promotes collagen matrix synthesis, a rat intestinal myofibroblast cell line (41) was stimulated with various concentrations of KGF-2 for 24 h. There was a dose-dependent increase in collagen type I mRNA expression in KGF-2-treated cells compared with control cells and TGF-
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DISCUSSION |
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This study shows that exogenous KGF-2 administration improved
wound healing in acute and chronic small intestinal ulceration in rats
and suggests that these effects are mediated by enhanced epithelial
migration as well as upregulation of protective prostaglandins. KGF-2
had beneficial effects on restoring weight loss, improving macroscopic
and microscopic small intestinal inflammation and ulceration,
decreasing anemia, and lowering tissue IL-1 levels in an acute,
continuous administration protocol. Continuous treatment with KGF-2 (1 mg/kg) was also effective in attenuating chronic intestinal ulceration
and, in contrast to KGF-1 in the TNBS model (45), KGF-2
exhibited therapeutic activity by reversing established small
intestinal ulceration in a 7-day delayed-treatment protocol. Of
interest, prophylactic administration of KGF-2 for up to 5 days before
indomethacin prevented intestinal injury. Optimal doses of KGF-2 were 1 mg · kg
1 · day
1, with
higher doses being less effective, consistent with the bell-shaped
dose-response curve of several biological molecules, including
recombinant IL-10 (33) and anti-TNF-
monoclonal
antibody (36). Intravenous administration was more
effective than subcutaneous delivery, possibly because of differences
in pharmacokinetics between the two routes of administration.
The role of endogenous KGF-2 in intestinal inflammation is still unknown, but our results suggest a potential therapeutic activity of this growth factor with several observed mechanisms of action that appear to be largely independent of cellular proliferation. In our experiments, high concentrations of KGF-2 (100 ng/ml) had only a modest effect on in vitro epithelial proliferation. Chronic in vivo administration of KGF-2 normalized epithelial proliferation in the antimesenteric region of the small intestine but had almost no proliferative effect in areas of active ulceration, as measured by BrdU incorporation. Furthermore, the regions of reepithelialization of ulcer bases showed no cell division. Previous studies (39) have shown that indomethacin induces transmural ulceration on the mesenteric border of the mid small intestine and decreases epithelial cell proliferation with certain dose ranges in rats. These findings suggest that KGF-2 stimulates epithelial healing by a mechanism independent of cellular proliferation.
Mucosal restitution occurs by rapid migration of viable epithelial
cells from adjacent areas to cover the damaged area without proliferation (4, 6, 43). Subsequent restoration of the normal epithelial architecture is mediated by stem cell proliferation, forming regenerative crypts, with crypt-to-surface cell migration and
differentiation (4, 6, 43). Our in vitro results
demonstrate that KGF-2 promotes cellular migration in a dose-dependent
fashion. Many growth factors, including TGF-, TGF-
, acidic or
basic fibroblast growth factor, hepatocyte growth factor, and
intestinal trefoil peptides, promote epithelial restitution
(9), with TGF-
playing a central role in all but
trefoil peptide-stimulated repair (4). A dose of 1 ng/ml
of KGF-2 was equivalent to 2 ng/ml of TGF-
in the epithelial cell
migration in vitro assay. The lack of BrdU staining in the single layer
of cells reepithelializing ulcer bases further supports epithelial
migration as the major mechanism of initial ulcer healing and provides
in vivo confirmation of the observation that growth factor-induced cell
migration is independent of cell proliferation (1).
Promotion of epithelial restitution by KGF-2 with subsequent ulcer
healing would prevent further influx of bacteria, toxins, and antigens
from the gut lumen. A number of rodent models demonstrate that normal
luminal bacteria and bacterial products are essential for chronic
intestinal inflammation (31). Decreased stimulation of
lamina propria macrophages by luminal bacterial cell wall polymers due
to ulcer healing may account for the observed decrease in IL-1
tissue concentrations, despite the lack of a direct effect of KGF-2 on
cytokine-stimulated epithelial cell nuclear factor-
B (NF-
B)
activation (D. S. Han and R. B. Sartor, unpublished results).
In addition to promotion of cellular migration, KGF-2 upregulated COX-2
expression, thereby stimulating the production of protective
prostaglandins that could inhibit the onset of intestinal injury and
enhance mucosal healing. Prostaglandin synthesis is an important
mediator of mucosal integrity and homeostasis in the gastrointestinal
tract (37). Mucosal prostaglandin production is regulated
by constitutive COX-1 and inducible COX-2, which is upregulated in
intestinal epithelial cells in active IBD (35) through the
activation of NF-B by proinflammatory cytokines (18). We demonstrated that KGF-2 induces the expression of COX-2 in cultured
intestinal epithelial cells and stimulates PGE2 production in vitro and in vivo, consistent with upregulation of COX-2 expression in endothelial cells by basic fibroblast growth factor
(20). Stenson et al. (7, 27, 37) showed the
key role of epithelial PGE2 in mucosal protection and
healing in radiation-induced enteritis and dextran sulfate
sodium-induced colitis and the importance of this molecule in oral
tolerance. In the dextran sulfate sodium model, another model of
experimental intestinal inflammation attenuated by KGF-2
(25), PGE2 reversed injury-induced defective
epithelial cell proliferation (37), which is a feature of
indomethacin-induced mucosal injury (39). The importance
of prostaglandins, especially those induced by upregulation of COX-2,
in mucosal protection is further illustrated by more aggressive dextran
sulfate sodium-induced colitis in COX-2
/
mice (26).
In addition to effects on mucosal cytoprotection and epithelial
proliferation, PGE2 inhibits macrophage activation and
proinflammatory cytokine production (22), consistent with
our observation of decreased tissue IL-1
concentrations and enhanced
tissue PGE2-to-IL-1
ratios after KGF-2 administration. Furthermore, the correlation of tissue PGE2-to-IL-1
ratios with intestinal injury supports the key role of balanced
inflammatory mediators and cytokines in mucosal protection. Although
these results concentrate on COX-2 and PGE2, it is quite
probable that KGF-2 induces expression of additional protective
molecules that diminish tissue injury by additive or synergistic
interactions with PGE2. For example, in preliminary
studies, we have documented upregulation of the intracellular isotype
form of IL-1 receptor antagonist in cultured colonic epithelial cells
with KGF-2 (Han and Sartor, unpublished data). In addition, long-term
KGF-2 administration stimulated goblet cell hyperplasia, similar to the
effects of its homologue KGF-1 (15), presumably resulting
in enhanced mucin secretion, which could provide additional mucosal protection.
Growth factors are integrally involved in mucosal homeostasis and
tissue repair. For example, TGF- regulates epithelial proliferation, differentiation, and restitution; inhibits T lymphocyte proliferation while promoting oral tolerance; and stimulates extracellular matrix deposition (4, 42). In contrast, KGF-2 has no known
activities on T cell regulation. However, one potential detrimental
consequence of treatment with growth factors is stimulation of
fibrogenesis, because TGF-
, insulin-like growth factor, and KGF-1
have been implicated in collagen deposition in the intestine through
direct effects on intestinal mesenchymal cells (40, 41, 45,
46). Excessive collagen deposition can lead to clinically
significant fibrosis, as demonstrated by colonic obstruction after
mucosal administration of a TGF-
1 plasmid expressed in
an adenoviral vector (40). Our in vitro results using
cultured rat intestinal myofibroblasts show increased expression of
collagen type I RNA by KGF-2. However, extracts from the small
intestines of rats with indomethacin-induced ulceration exhibited no
significant upregulation of collagen type I RNA or hydroxyproline
concentrations by a therapeutically active dose of KGF-2, and serum
hydroxyproline measurements were not increased after KGF-2
administration. The absence of detectable in vivo fibrogenesis offers a
conceptual advantage of KGF-2 over other growth factors as a
therapeutic candidate for IBD.
In summary, in vivo beneficial effects of KGF-2 in preventive, continuous treatment and therapeutic protocols with no apparent toxicity or induction of fibrosis suggest potential therapeutic application of this molecule to human IBD. Possible mechanisms of protection include accelerated wound healing by increased migration of epithelial cells, with or without enhanced proliferation, and increased mucosal PGE2 production. The latter mechanism is more likely to be involved in the ability of KGF-2 pretreatment to attenuate intestinal injury up to 5 days before indomethacin injection. It is likely that optimal therapeutic effects of protective growth factors may be achieved in conjunction with simultaneous use of immunosuppressive agents and broad-spectrum antibiotics to synergistically restore mucosal barrier function while decreasing antigenic stimulation and blocking activation of regulatory macrophages and TH1 lymphocytes.
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ACKNOWLEDGEMENTS |
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We thank Julie Vorobiov of the Immunoassay Core of the Center for Gastrointestinal Biology and Disease for performing immunoassays and Susie May for secretarial support.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-40249 and DK-34987 (R. B. Sartor). D. S. Han was supported by the Korean Science and Engineering Foundation and Human Genome Sciences, Inc.
Present address of D. S. Han: Hanyang University, Kuri Hospital, Kuri, Republic of Korea 470-701.
Address for reprint requests and other correspondence: R. Balfour Sartor, Div. of Digestive Disease, CB# 7038, 032 Glaxo Bldg., Univ. of North Carolina, Chapel Hill, NC 27599 (E-mail: rbs{at}med.unc.edu).
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. Section 1734 solely to indicate this fact.
Received 2 September 1999; accepted in final form 9 May 2000.
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