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Department of Pediatrics, Division of Endocrinology (L.S., L.E.U.), and Departments of Physiology (P.K.L., K.M.H.) and Pediatrics (M.H.U.), University of North Carolina, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: Dr. P. K. Lund, Department of Physiology, CB#7545 University of North Carolina, Chapel Hill, North Carolina 27599-7545. E-mail: empk{at}med.unc.edu
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Abstract |
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messenger RNAs (mRNAs) were quantified by Northern blot hybridization
and expressed as a percentage of mRNA abundance in fed controls. In DSS
mice, IL-1ß mRNA was elevated in the fed group (954 ± 155%;
P < 0.001), but was suppressed in fasted animals
(71.1 ± 11%). IGF-I mRNA also was elevated in fed DSS mice
(421 ± 71%; P < 0.01). This increase was
attenuated in fasted DSS mice (202 ± 17%; P
< 0.01 compared to fed DSS mice). Tumor necrosis factor- mRNA was
increased in fed DSS mice (162 ± 15%; P <
0.01), but was not significantly lower in fasted animals. By in
situ hybridization, IL-1ß mRNA was localized to the lamina
propria of colonic mucosa in fed DSS animals, but was not detectable in
other groups. We conclude that fasting has a protective effect on the
progression of acute DSS-induced colitis. This is associated with
decreased expression of IL-1ß and IGF-I mRNAs in the colon. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I has unique properties that are potentially relevant to IBD; it is a potent mitogen for fibroblasts (12), an inducer of collagen synthesis (13), and an important participant in tissue remodeling and repair (14). Expression of IGF-I is ubiquitous, occurring in monocytes (15), macrophages (16), fibroblasts (17), and cellular elements throughout the gastrointestinal tract (18, 19). IGF-I receptor types I and II also are distributed widely in the alimentary tract, and the bowel is an established target organ for IGF-I (8, 20, 21).
Interleukin-1 (IL-1) is synthesized in the intestinal wall in both the presence and absence of inflammation, being produced by intestinal mononuclear cells of the lamina propria (22). IL-1 receptors are present on intestinal cells and resemble the type I IL-1 receptor characterized in other cell types (23). Proinflammatory cytokines such as IL-1 appear to induce IGF-I in vitro, linking IGF-I to key mediators of the inflammatory response in IBD (15). A causative role of IL-1 in experimental enterocolitis has been confirmed by studies showing that treatment with IL-1 receptor antagonists attenuates acute and chronic enterocolitis (5, 7), whereas exposure to neutralizing antibodies against IL-1 receptor antagonists prolongs intestinal inflammatory responses (6).
Fasting decreases circulating concentrations of IGF-I (24, 25) and
expression of IGF-I in nonhepatic tissues (26), including the jejunum
(19). However, little is known about the effects of fasting or nutrient
status on the expression of IGF-I in the colon. The present study was
undertaken to obtain insight into nutritional effects on IGF-I,
IL-1ß, and tumor necrosis factor- (TNF) gene expression in
colitis. We used an experimental mouse model in which acute colitis is
produced by adding dextran sulfate sodium (DSS) to the drinking water
(27, 28, 29, 30, 31, 32).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animal experiments were approved by the institutional animal care and use committee of the University of North Carolina-Chapel Hill. Study protocols were in compliance with the Guide for the Care and Use of Laboratory Animals published by the NIH.
Experimental design
One animal from each of 15 pairs was given no food (fasted) for
48 h before death, whereas the other animal was fed ad
libitum (fed) throughout the study. In 10 pairs of mice, acute
colitis was produced by adding 5% (wt/vol) DSS (mol wt,
34,000–45,000) to the drinking water for 5 days before death. The
control group of 5 pairs of mice was given normal drinking water
(without DSS) throughout the study. After 5 days, the mice were
anesthetized by an im injection of 900 µg/g BW ketamine (Parke Davis,
Morris Plains, NJ) and 20 µg/g BW xylazine (Mobay Corp., Shawnee,
KS). The animals were killed after the colon and cecum had been rapidly
removed and irrigated with ice-cold saline, and the colon was subjected
to uniform tension with a 5-g weight for measurement of length.
RNA extraction
Colon and cecum from each animal were pooled to provide
sufficient tissue for suitable yields of RNA. Freshly dissected tissues
were homogenized immediately in guanidine thiocyanate (33) before
storing at -80 C. This appears essential for extraction of intact RNA
from this bowel segment (Lund, P. K., unpublished observations). Total
RNA was separated from DNA and protein by centrifugation of homogenates
over 5.7 M cesium chloride (34). The concentration of total
RNA was determined by absorbance at 260 nm. RNA integrity was verified
by electrophoresis of total RNAs on 1% agarose gels containing
ethidium bromide.
Northern blot analysis and quantification of hybridization
signals
Aliquots of total RNA (40 µg) were denatured with glyoxal and
dimethylsulfoxide, size-fractionated on 1% agarose gels, and
transferred to GeneScreen (New England Nuclear, Boston, MA).
Transferred RNA was immobilized, and blots were stained and then
photographed (7). The abundance of 28S ribosomal RNA was quantified by
densitometry as a control for the amount of total RNA loaded per sample
(19). Blots were prehybridized and hybridized in buffer containing 50%
deionized formamide, 3 x SSC (standard saline citrate), 10
x Denhardt’s solution (1 x Denhardt’s solution = 0.02% each BSA,
Ficoll, and polyvinylpyrrolidone), 50 mM Tris (pH 7.0),
0.1% SDS, 0.2 mg/ml sonicated denatured salmon sperm DNA (Sigma
Chemical Co., St. Louis, MO), and 2 µg/ml nonpolyadenylated
[poly(A)-] RNA. Prehybridization was performed at 55 C
for 6–24 h, and hybridization was conducted at 55 C for 24 h with
1 x 106 cpm/ml (5–10 ng/ml)
[32P]UTP-labeled antisense RNA probes added. Blots were
washed at 60 C (IL-1ß or IGF-I probes) or 65 C (TNF probe) in
1 x SSC-0.1% SDS three times for 15 min each time and in
0.1 x SSC twice for 15 min each time. Blots were then exposed to
Kodak Biomax MR film (Eastman Kodak, Rochester, NY) with intensifying
screens at -80 C. Positive controls were run on each gel.
Autoradiograms of Northern blots were analyzed by densitometric
scanning, and the abundance of messenger RNA (mRNA) was normalized to
28S ribosomal RNA (19).
Probes used in Northern and in situ hybridizations
Complementary DNA clones for mouse IL-1ß (DuPont Merck
Pharmaceutical Co., Glenolden, PA), mouse TNF (kindly provided by
Dr. Bruce Beutler, University of Texas, Southwestern Howard Hughes
Medical Institute, Houston, TX), and rat IGF-I (P2; 786-bp insert) (35)
inserted into PSP64 (mIL-1ß), pGEM3 (mTNF), or pGEM3Z (rIGF-I)
vectors (Promega Biotech, Madison, WI) were linearized and used as a
template for the SP6 (IL-1ß) or T7 (TNF and IGF-I)
polymerase-directed synthesis of 32P-labeled (Northern) or
35S-labeled (in situ) complementary (antisense)
RNA probes (36, 37).
In situ hybridization and histology
A 1.0-cm segment of distal colon from each mouse was embedded in
OCT compound (Miles, Elkhart, IN), frozen in isopentane at -40 to -50
C, and stored at -80 C until used. In situ hybridization
histochemistry was performed on 10-µm thick sections using a
modification (7) of methods previously described by Watson et
al. (38). The hybridization mix contained 75% formamide, 10%
dextran sulfate, 3 x SSC, 50 mM Na phosphate buffer
(pH 7.4), 1 x Denhardt’s solution, 0.1 mg/ml yeast transfer RNA,
10 mM dithiothreitol, and 1–2 x 106 cpm
radiolabeled probe/slide. Sections were observed and photographed under
light- and darkfield illumination (Olympus BH-2 microscope, Olympus
Corp., Lake Success, NY). The specificity of hybridization signals
using the IL-1ß antisense probe was verified by two negative controls
performed on adjacent sections: 1) sections pretreated with
ribonuclease A (200 µg/ml; Sigma) before hybridization with the
antisense probe, and 2) hybridization with sense strand IL-1ß probe.
Such controls uniformly yielded negative results (hybridization
indistinguishable from area of slide with no section). Sections were
also stained with hematoxylin and eosin to assess morphology or
Masson’s Trichrome stain to visualize collagen (39). A colitis
activity score was used to score each section in a blind fashion based
by the method of Dieleman et al. (29) and as detailed in
Table 2
. Four different sections from each animal were used to derive a
mean score for inflammation and damage. The total activity score ranged
from 0–6, which represented the sum of scores from 0–3 for damage and
inflammation (Table 2).
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, and IGF-I mRNA in fed DSS-treated animals
(fed-DSS group), fasted DSS-treated animals (fasted-DSS group), and
fasted water-treated animals (fasted-H2O group) are
expressed as a percentage of the abundance in control mice, which were
fed and received normal drinking water throughout the study
(control-fed). Statistical comparisons between pairs of fed and fasted
animals were performed using Student’s paired t test. When
comparing DSS-treated and water controls, Student’s t test
for unpaired groups was used. P < 0.05 was considered
statistically significant. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The average daily intakes of DSS-water or normal water were similar in
fed and fasted animals [4.6 ± 0.2 g (DSS-fed)
vs. 4.8 ± 0.5 g (DSS-fasted) and 7.0 ±
0.8 g (control-fed) vs. 7.3 ± 1.2 g
(control-fasted)]. The average daily intake of chow did not differ
between the fed DSS-treated and control animals [3.6 ± 0.7
g (DSS-fed) vs. 4.4 ± 0.4 g (control-fed)].
Weight loss during the 2 days of fasting was not significantly
different in control-fasted (4.4 ± 0.4 g) and DSS-fasted
animals (3.4 ± 0.4 g). Fasting caused the colon wet weight
to decrease in DSS-treated animals, but not in control mice (Table 1).
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). In DSS-fasted
animals, inflammation and tissue damage were greatly reduced (Fig. 1).
The quantitative microscopic colitis activity score (see
Materials and Methods and Table 2
) was
5.3 ± 0.2 in DSS-fed and 2.4 ± 0.1 in DSS-fasted animals
(P < 0.0001). In both fed and fasted control mice, no
inflammation or tissue damage was noted (colitis activity score of 0.0
in both groups).
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). The
DSS-fed animals showed markedly increased abundance of IL-1ß mRNA in
colon (954 ± 155% of that observed in control-fed animals;
P < 0.001; Fig. 2). The abundance of IL-1ß mRNA in
the DSS-treated group that was fasted for 2 days was markedly lower
than that in DSS-fed animals and was even lower than that in the fed,
water controls (Fig. 2; 71.1 ± 11% of control-fed). Fasting of
water control animals also decreased IL-1ß mRNA (Fig. 2; 34 ±
6% of control-fed), although this difference was not as great as in
DSS-treated animals.
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). IL-1ß mRNA was
not detected in colon of DSS-fasted animals (Fig. 3), probably
reflecting the lower mRNA abundance and the lower sensitivity of the
in situ hybridization procedure compared with those of the
Northern blot hybridization. In control mice receiving no DSS, in
situ hybridization similarly revealed no detectable IL-1ß mRNA
(not shown).
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mRNA expression was detectable in the colon of all
animals by Northern blot hybridization (Fig. 4).
Treatment of fed animals with 5% DSS for 5 days increased the
abundance of TNF mRNA in the colon (Fig. 4A; 162 ± 15% of
control-fed group; P < 0.01). Fasting for 2 days
before death modestly reduced the abundance of TNF mRNA in some, but
not all, DSS-treated animals, and this variable reduction did not
achieve statistical significance. TNF mRNA also did not differ
significantly between fed and fasted water controls.
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).
DSS-fed animals had a greater abundance of both size classes of IGF-I
mRNA in colon than the control-fed group [Fig. 5A; 421 ± 71%
(1.2 kb; P < 0.01) and 236 ± 65% (7.5 kb;
P < 0.05)]. The abundance of both size classes of
IGF-I was lower in the fasted DSS-animals than in the DSS-fed group
[Fig. 5A; 202 ± 17% (1.2 kb; P < 0.01) and
127 ± 14% (7.5 kb; P < 0.05) relative to fed,
water controls], but was not significantly different in the fed and
fasted water control groups (see Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with our prior observations, fed DSS-treated mice developed bloody diarrhea within 3 days, and histology at 5 days confirmed severe acute colitis with crypt loss. The mechanism by which DSS causes acute ulcerative colitis is not clear. It seems, however not to require activation of T or B cells, because it occurs in immunodeficient mice lacking these cells (29). Therefore, it is not likely that inflammation is the primary reason for the epithelial loss and erosion that occur in fed DSS-treated animals. Instead, it is more likely that the inflammation is secondary to tissue damage caused by the DSS treatment per se. This is supported by a report of decreased [3H]thymidine uptake by mouse colonic epithelial cells exposed to DSS in vitro (29). Macrophages that have phagocytized DSS show a subsequent decrease in bacterial phagocytosis (32), heralding a decline in the mucosal defense system to bacteria. This is consistent with the observation that the population of intestinal microflora in DSS-treated animals is increased (27).
We show that fasting impedes the progression of inflammation and tissue damage in the DSS model of experimental colitis. Similarly, patients with Crohn’s disease or ulcerative colitis show remission after bowel rest with total parental nutrition (41). As in our animal model, the mechanisms underlying this effect are unknown. Retrospective, case-controlled studies of humans have focused on correlations between the intake of certain foods and the prevalence of inflammatory bowel disease (42, 43). Although not conclusive, positive correlations have been made between IBD and the frequency of intake of "fast food" (42) or of fat-rich, "western" foods (43). A study in which the diet of animals with experimental colitis was manipulated show that feeding a diet rich in fish oil had a protective effect on the development of colitis (30). Our finding that fasting impedes the progression of DSS-induced colitis indicates that additional studies of the effects of specific nutrients in this model may help determine enteral diets that might alter the progression of colitis.
A possible mechanism for the beneficial effect of fasting is that lack of substrate may lead to a reduction in colonic microflora, and this may reduce the load of bacterial antigens that elicit an inflammatory response after DSS causes mucosal damage. Although, it is not known whether fasting affects the gastrointestinal microflora, the question is amenable to testing. Alternatively, fasting may reduce rates of mucosal cell proliferation, and this may reduce susceptibility to DSS-induced mucosal damage. Finally, metabolic or hormonal changes associated with fasting may alter the profile of proinflammatory cytokines. Our data on IL-1 and IGF mRNAs in fasted and fed DSS-treated animals indicate that this may be the case.
Using Northern blotting and in situ hybridization, we observed markedly increased expression of IL-1ß mRNA in acute DSS-induced colitis. These results are similar to those of Cominelli et al. (5), who showed that increased IL-1ß gene expression and synthesis occur early in the coarse of immune complex-induced colitis in rabbits. In DSS-treated animals, we show a localization of IL-1ß mRNA to the lamina propria layer of colon, which is consistent with the observation that IL-1 is produced by intestinal mononuclear cells of the lamina propria (22). We have attempted to localize TNF and IGF-I mRNAs in colon. Unfortunately, the levels of these mRNAs are too low for this to be accomplished with in situ hybridization.
Fasting of DSS-treated animals markedly reduced the abundance of IL-1ß mRNA in colon to a level even lower than that in fed controls. In addition, IL-1ß mRNA was undetectable by in situ hybridization in sections of colon from fasted animals. The lower IL-1ß gene expression in fasted DSS-treated animals is associated with a minor degree of inflammation and tissue damage in the colon of the same animals. This positive correlation between expression of the IL-1ß gene and acute experimental colitis supports the concept that IL-1 is an important mediator of colitis in humans (1, 2) and animals (5, 6, 7, 9).
The increased expression of TNF mRNA in DSS-treated animals is
consistent with a report of increased levels of TNF in colon tissue
of rats with DSS-induced colitis (31). We found a great difference in
the magnitude of the increase in mRNA expression, favoring IL-1ß over
TNF, suggesting differential regulation and function of these
cytokines in the pathogenesis of acute colitis. This is supported by
the observation that expression of IL-1ß mRNA was greatly suppressed
by fasting, whereas TNF mRNA was unaffected. This may reflect
changes in the cellular composition of the inflammatory infiltrate.
Further, in patients with ulcerative colitis, expression of IL-1ß
mRNA is localized to subepithelial macrophages, whereas TNF mRNA is
found in the deeper lamina propria of colon (1).
The increased expression of IGF-I mRNA in colon from animals with DSS-induced colitis is consistent with reports showing up-regulation of IGF-I mRNA in colon of rat models of chronic experimental enterocolitis (9, 44). Interestingly, IGF-I-binding sites are also increased in colon tissue from rats with experimental colitis (10). Although the role of locally expressed IGF-I in colitis is not clear, IGF-I may be induced as part of a tissue repair mechanism during chronic inflammation (8). On the other hand, excess local expression of IGF-I may promote excessive wound healing and fibrogenic complications of chronic enterocolitis (8, 9). The present findings of increased IGF-I expression during acute DSS-induced colitis indicate that up-regulated colonic synthesis of IGF-I is an early event during bowel inflammation, possibly promoting tissue repair. Supporting this possibility is the observation that systemic IGF-I administration reduces the severity of DSS-induced colitis in rats and promotes colonic tissue repair (21).
Using a well established model for acute colitis, we show that inflammation and damage in the colon are associated with increased expression of mRNAs encoding IL-1, TNF, and IGF-I. We found a preventive effect of fasting on the development of acute colitis, associated with a decrease in the expression of IL-1 and IGF-I.
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Acknowledgments |
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Footnotes |
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2 Recipient of a research fellowship from the European Society for
Pediatric Endocrinology.
Received July 15, 1996.
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References |
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