Dextran sulfate sodium-induced murine colitis activates NF-kappa B and increases galanin-1 receptor expression

Jorge A. Marrero1, Kristina A. Matkowskyj1, Kenny Yung1, Gail Hecht1, and Richard V. Benya1,2

Departments of 1 Medicine and 2 Pharmacology, University of Illinois at Chicago and Chicago Veterans Affairs Medical Center, West Side Division, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Galanin is widely distributed in enteric nerve terminals and acts to modulate intestinal motility by altering smooth muscle contraction. This ligand causes Cl- secretion when colonic epithelial cells express the galanin-1 receptor (Gal1-R) subtype. Because Gal1-R expression by colonic epithelia is upregulated by the transcription factor nuclear factor-kappa B (NF-kappa B), increasingly appreciated as critical in the pathophysiology of inflammatory bowel disease, we wondered whether the diarrhea associated with this condition could be due to NF-kappa B-mediated increases in Gal1-R expression. To test this hypothesis, we provided oral dextran sulfate sodium (DSS) to C57BL/6J mice. Although Gal1-R are not normally expressed by epithelial cells lining the mouse colon, DSS treatment resulted in increased NF-kappa B activation and Gal1-R expression. Whereas galanin had no effect on murine colonic tissues studied ex vivo, it progressively increased short-circuit current and colonic fluid secretion in DSS-treated mice. Concomitant parenteral administration of the NF-kappa B inhibitor dexamethasone attenuated the activation of this transcription factor by DSS, inhibiting the increase in Gal1-R expression. Although Gal1-R-specific antagonists do not exist, intracolonic administration of commercially available galanin antibody diminished the DSS-induced increase in colonic fluid accumulation. Overall, these data demonstrate that a significant component of the excessive fluid secretion observed in DSS-treated mice is due to increased Gal1-R expression.

galanin; secretion; inflammatory bowel disease


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GALANIN IS A NEUROPEPTIDE originally isolated from porcine small intestine (37) that is 30 amino acids long in humans (7) and 29 amino acids long in all other species (for review, see Ref. 21). Galanin is widely distributed along the length of the gastrointestinal (GI) tract, with immunoreactivity to this peptide found in the enteric nerve terminals of all species studied (4, 19, 42). Recent molecular studies indicate that galanin acts by binding to one of three different receptor subtypes now identified as the galanin-1 (Gal1-R), galanin-2, and galanin-3 receptors. All three subtypes have been cloned and sequenced, including the human (16) and murine Gal1-R (41).

Although pharmacological studies indicate that all three galanin receptor subtypes are expressed by smooth muscle cells lining the GI tract (14, 15), we have previously shown (5, 6, 23) that only Gal1-R mRNA is found in epithelial cells lining the human colon. When galanin binds to receptors on smooth muscle cells, it causes both contraction and relaxation, thereby acting to modulate intestinal motility (for review, see Ref. 30). In contrast, we have recently demonstrated (5) that galanin activation of Gal1-R expressed by the human colon epithelial cell line T84 results in Cl- secretion. This finding therefore suggested the possibility that galanin activation of Gal1-R expressed by epithelial cells lining the GI tract could be important in regulating intestinal fluid secretion.

Our cloning of the human GAL1R gene (6, 24) revealed the presence of multiple recognition sites for the inflammation-associated transcription factor nuclear factor-kappa B (NF-kappa B) in the 5'-flanking region, which we have shown to be functional using reporter gene studies (24). This transcription factor is activated in many inflammatory disorders of the GI tract, including idiopathic inflammatory bowel disease (IBD) (26, 27, 36), and subsequent to infection with many different enteric pathogens (13, 34, 46). We have therefore hypothesized that the excessive colonic fluid secretion observed in diarrheal diseases associated with inflammation could at least partly be due to NF-kappa B-induced upregulation of Gal1-R. In support of this hypothesis, we recently have shown (17) that infection of human T84 cells in vitro, or murine colonocytes in vivo, with pathogenic Escherichia coli results in NF-kappa B activation and Gal1-R upregulation. Increased Cl- secretion consequently occurs (17), thereby supporting a role for galanin and the Gal1-R in mediating the excessive fluid secretion observed in infectious diarrhea. In contrast, nothing is known about whether Gal1-R expression is increased in noninfectious inflammatory disorders affecting the colon. The aim of this study, therefore, was to determine whether enhanced Cl- secretion induced by noninfectious inflammatory conditions could also be attributable to increased expression and subsequent activation of Gal1-R.

In this study, we used the dextran sulfate sodium (DSS) model of murine colitis (9, 28) to determine whether Gal1-R expression was upregulated in association with colonic inflammation and to evaluate the ability of galanin to increase colonic fluid secretion. We show here that DSS-induced colonic inflammation is associated with increased NF-kappa B activation and Gal1-R expression. The increase in Gal1-R expression allows galanin to increase the amount of colonic fluid generated. These data therefore support a role for Gal1-R expression in mediating the diarrhea associated with colonic inflammation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Specific pathogen-free C57BL/6J male mice, at least 8 wk old, were obtained from Jackson Laboratory (Bar Harbor, ME) and used after reaching weights of 25-30 g. DSS (mol wt = 40,000) was obtained from ICN Biomedicals (Aurora, OH), and galanin was from Bachem (Torrance, CA). The antibody specific for the p65 subunit (active conformation) of NF-kappa B was from Boehringer Mannheim (Mannheim, Germany), and the monoclonal antibody to human galanin, which has 100% cross-reactivity with murine galanin, was from Peninsula Labs (San Francisco, CA). Pentobarbital was from Abbott (North Chicago, IL), and 2-0 silk was from Johnson and Johnson, (Somerville, NJ). All other supplies were from Sigma Chemical (St. Louis, MO).

Animal care and induction of colitis. All animals were studied at 8 wk of age. Mice were kept in microisolator cages and provided free access to food and water. Colitis was induced by allowing animals access to 2% (wt/vol) DSS in their drinking water ad libitum as previously described (9, 28). Animals were fasted for 24 h before euthanasia, while retaining free access to water with DSS. This study was approved by the University of Illinois at Chicago Animal Care and Use Committee.

Histological analysis. Freshly resected specimens were placed in 10% formalin and fixed in paraffin-embedded blocks. Blocks were sectioned at a thickness of 5 µm and stained with hematoxylin and eosin using standard techniques (1). The severity of colitis was graded on a scale from 0 to 3, as previously described (28), by three separate investigators blinded as to the conditions of the animal from which the colon was obtained. Briefly, 0 indicated normal mucosa; 1, focal inflammatory cell infiltration including neutrophils; 2, inflammatory cell infiltration, gland dropout, and cryptitis; and 3, frank mucosal ulceration.

Immunohistochemistry. A standard three-stage indirect immunoperoxidase technique was used for all immunohistochemistry (20). We utilized an anti-peptide antibody specific for the Gal1-R at a dilution of 1:500 as previously described for 60 min at 22°C (17). The commercially available antibody for the p65 subunit of NF-kappa B, which only detects this molecule when biologically active, was used as previously described at a dilution of 1:200 (27).

In all cases, fixed tissue sections were rehydrated in graded alcohols and then rinsed in a running water bath. Endogenous peroxidase activity was quenched by preincubating slides in 3% hydrogen peroxide in a light-impermeable chamber. After being washed in PBS, slides were incubated in blocking solution [5% skim milk (vol/vol) and 0.15% H2O2 (vol/vol) in deionized water]. Primary antibody was then applied after a PBS wash, and the tissues were incubated for 1 h in a humidity chamber. Control tissues were processed similarly, except that primary antibody was not applied. After being washed again in PBS, the tissues were incubated with biotinylated anti-rabbit IgG for 15 min. The slides were then incubated with streptavidin conjugated to horseradish peroxidase for 15 min and washed again in PBS buffer. Incubating slides with liquid DAB substrate-chromogen system for 2 min identified bound antibody. After a final wash in PBS and distilled water, the slides were counterstained with a 50% dilution of Gill's modified hematoxylin for 4 min, dehydrated in graded alcohols, and mounted with a coverslip using Permount.

Chromogen quantification. We have developed a novel algorithm for quantitative immunohistochemistry that is based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image (25). This algorithm allows us to determine the absolute amount of antibody-specific chromogen per pixel for any cellular region or structure. In brief, digital images are acquired at ×1,000 using a Microlumina ultraresolution scanning digital camera (3,380 × 2,700 pixels; Leaf Systems, Fort Washington, PA) attached to a Nikon E600 microscope system. Chromogen abundance is quantified by calculating the cumulative signal strength within three randomly selected areas. For any image file of dimensions N1 by N2 pixels, and where n1 and n2 identify the specific position of the data within the digital image file, the cumulative signal strength (or mathematical energy, E) is defined as
<IT>E</IT> = <FENCE><LIM><OP>∑</OP><LL><IT>n</IT><SUB>1</SUB> = 1</LL><UL><IT>N</IT><SUB>1</SUB></UL></LIM> <LIM><OP>∑</OP><LL><IT>n</IT><SUB>2</SUB> = 1</LL><UL><IT>N</IT><SUB>2</SUB></UL></LIM> [<IT>f</IT><SUB>red</SUB>(<IT>n</IT><SUB>1</SUB>,<IT>n</IT><SUB>2</SUB>)]<SUP>2</SUP></FENCE><SUP>1/2</SUP> + <FENCE><LIM><OP>∑</OP><LL><IT>n</IT><SUB>1</SUB> = 1</LL><UL><IT>N</IT><SUB>1</SUB></UL></LIM> <LIM><OP>∑</OP><LL><IT>n</IT><SUB>2</SUB> = 1</LL><UL><IT>N</IT><SUB>2</SUB></UL></LIM> [<IT>f</IT><SUB>green</SUB>(<IT>n</IT><SUB>1</SUB>,<IT>n</IT><SUB>2</SUB>)]<SUP>2</SUP></FENCE><SUP>1/2</SUP>

 + <FENCE><LIM><OP>∑</OP><LL><IT>n</IT><SUB>1</SUB> = 1</LL><UL><IT>N</IT><SUB>1</SUB></UL></LIM> <LIM><OP>∑</OP><LL><IT>n</IT><SUB>2</SUB> = 1</LL><UL><IT>N</IT><SUB>2</SUB></UL></LIM> [<IT>f</IT><SUB>blue</SUB>(<IT>n</IT><SUB>1</SUB>,<IT>n</IT><SUB>2</SUB>)]<SUP>2</SUP></FENCE><SUP>1/2</SUP>
The amount of chromogen per pixel is then determined by subtracting the energy of the control slide (i.e., not exposed to primary antibody) from that in the homologous region of the experimental slide (i.e., exposed to primary antibody). Chromogen quantity is expressed in the valueless units of "energy units per pixel" (eu/pix).

Electrophysiology. Mice were euthanized by CO2 asphyxiation. The cecum and rectum were identified, and a full-thickness piece of proximal colon was resected and immediately mounted in an Ussing chamber. The colon was mounted as a sheet on a 4-mm-internal diameter ring and incubated at 37°C in Ringer solution that was continually gassed with 95% O2-5%CO2. Transepithelial resistance was determined as described previously (17). Briefly, electrical current (100 µA) was applied as a 2-s pulse under current-clamped conditions across the tissue using Ag-AgCl electrodes connected to the reservoirs via salt bridges, and the resultant potential difference was recorded every 5 min for 30-40 min. The short-circuit current (Isc) was then measured and recorded every minute after the addition of secretagogue under voltage-clamped conditions. Tissues were equilibrated for 15-30 min to allow for the development of a stable baseline. Mucosal and serosal sides were then exposed to 1 µM galanin, a dose previously shown to maximally yet specifically activate Gal1-R-induced Cl- secretion (5). After return to stable baseline, the tissues were then stimulated by adding 100 µM carbachol to the basal reservoir.

Evaluation of colonic fluid secretion. Animals were fasted for 24 h before surgery while continuing to have free access to water containing 2% DSS. Anesthesia was achieved using pentobarbital sodium (50 µg/mg ip). The adequacy of anesthesia was assessed by interdigital toe pinch, as previously described (43). Under sterile conditions using a dissecting microscope, a small laparotomy incision was made, the cecum identified, and the most distal segment of colon exposed. Using 2-0 silk, a ligature was first placed on the most distal aspect of this segment of colon, followed by placement of a second ligature at the most proximal portion of the colon to generate a loop that was ~5 cm in length. Before the proximal suture was completely tied off, a 26-gauge TB needle was inserted through the area defined by the proximally placed yet loosely tied suture. The knot was closed around the needle now inside the colonic lumen. Loops were injected with 200 µl of 1 µM galanin or saline (control), the needle was withdrawn, and the suture was tightened. The bowel was placed back into the peritoneal cavity, and the laparotomy incision was closed using a running (mattress) suture. The mice were placed back in their cage with free access to water without DSS but not food. After 4 h, the mice were killed by CO2 asphyxiation and the colonic loops were removed and their lengths and weights recorded. Fluid secretion was calculated by determining the ratio of loop weight (mg) to length (cm), as previously described (8).

Statistical evaluations. All comparisons between multiple groups of animals (i.e., Table 1) were performed by ANOVA, whereas comparisons between paired groups of animals (i.e., Table 2) were performed using the Student's paired t-test. In all cases, data were analyzed using StatView (Abacus Concepts, Berkeley, CA), with P < 0.05 being considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DSS causes a progressive and severe colitis. Mice given 2% DSS ad libitum in their drinking water developed a progressively worsening colitis (Table 1). Overall, animals lost nearly one-third of their body weight after 6 days, with losses seen as early as 24 h after 2% DSS was started. In contrast, histological evidence of inflammation was not observed until 3 days after DSS administration, with significant shortening in overall colonic length not observed for another 2 days (Table 1). The development of gross bleeding and loose stools paralleled the development of the histological colitis, which was not detected until mice had been exposed to DSS for 4 or more days (Table 1). We found that DSS-induced inflammation was so rapidly progressive that animals were unable to reliably survive beyond 7 days of DSS exposure. For ethical reasons, therefore, all mice were killed after 6 days of exposure to DSS.

                              
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Table 1.   DSS causes progressive colonic inflammation associated with increased NF-kappa B and Gal1-R expression

The worsening colitis was associated with increased activation of NF-kappa B that in turn paralleled the increases observed in Gal1-R expression (Fig. 1; Table 1). Epithelial cells lining the normal uninflamed mouse colon do not normally express active NF-kappa B or Gal1-R protein (Fig. 1A and Ref. 17). Increased amounts of activated NF-kappa B were observed as soon as 1 day after starting DSS, preceding significant increases in Gal1-R expression (Table 1). Immunohistochemically, the initial increases in activated NF-kappa B were most prominently observed in the nuclei (Fig. 1D). Yet the sensitivity of quantitative immunohistochemistry is such that increases in cytoplasmic chromogen due to NF-kappa B were detected as soon as 1 day after mice were exposed to DSS (Table 1). After 3 days of exposure to DSS, levels of activated NF-kappa B as determined by quantitative immunohistochemistry reached a steady state (Table 1). Although significant increases in Gal1-R chromogen quantity were not seen until 2 days after DSS was started, levels continued to rise thereafter (Table 1; Fig. 1). This increase in Gal1-R expression was confirmed by Western blot analysis of proteins isolated from colonic epithelial cells. Although no specific protein could be identified in control animals, a ~40-kDa protein was prominently identified in the colonic epithelia of mice given DSS for 6 days (Fig. 1B).


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Fig. 1.   Immunohistochemistry performed on murine colonic epithelial cells using antibodies against galanin-1 receptor (Gal1-R) (A, C, E, G) or activated subunit of nuclear factor-kappa B (NF-kappa B) p65 (D, F, H). Whole sections of murine colon were resected, placed in 10% formalin, and processed as described in METHODS 2 (C, D), 4 (E, F), or 6 (G, H) days after initial exposure to 2% dextran sulfate sodium (DSS). D: arrowhead, greater amounts of immunostaining in nucleus for NF-kappa B at outset of DSS treatment. B: Western blot analysis performed against proteins isolated from epithelial cells lining colon of control animals and mice exposed to DSS for 6 days. LM, lane marker; D0, day 0 of DSS exposure (control); D6, day 6 of DSS exposure. Molecular mass shown on left in kDa × 10-3; arrowhead, ~40-kDa protein present only in intestinal epithelial cells exposed to DSS. Values are representative of 5 separately treated animals per condition. A and C-H: magnification, ×400.

Galanin increases Isc only in inflamed colonic tissues. Consistent with the absence of Gal1-R on colonic epithelial cells by immunohistochemistry in control mice, normal colons mounted in Ussing chambers failed to show any increase in Isc when exposed to ligand. Specifically, basal Isc in control animals was 3.4 ± 0.2 µA/cm2 and remained unchanged at 3.6 ± 0.2 µA/cm2 after exposure to 1 µM galanin. We used carbachol as a control secretagogue because, similar to galanin, this drug causes Cl- secretion via a Ca2+-dependent mechanism after binding to a specific heptaspanning receptor. In contrast to what was observed with galanin, these tissues rapidly responded to 100 µM carbachol so that Isc rapidly rose from 3.5 ± 0.7 to 11.2 ± 0.5 µA/cm2 (n = 4). For the first 2 days of DSS exposure, galanin continued to have no effect on altering Isc in mouse colon (Fig. 2A). However, with the onset of detectable inflammation after 3 days of exposure to DSS and associated with the immunohistochemical evidence of extranuclear Gal1-R expression, galanin increased Isc. Indeed, the galanin-induced increase in Isc (Fig. 2A) paralleled the degree of colonic inflammation and the amount of Gal1-R detectable by immunohistochemistry (Fig. 1). After 6 days of DSS, basal Isc was unchanged (3.3 ± 0.5 µA/cm2) from that observed in mice not exposed to DSS (3.4 ± 0.2 µA/cm2). However, 1 µM galanin markedly increased Isc (10.7 ± 0.8 µA/cm2) to an extent similar to that observed with stimulation with carbachol (basal, 3.4 ± 0.4 µA/cm2; 100 µM carbachol, 9.1 ± 0.7 µA/cm2).


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Fig. 2.   Ability of galanin to increase short-circuit current (Isc) in murine colonocytes as a function of time of exposure to 2% DSS. A: mouse colon was mounted in an Ussing chamber after exposure to DSS for indicated amount of time. After development of stable baseline, colon was treated with 1 µM galanin. Maximal increases in Isc were recorded and expressed as %increase observed with 100 µM carbachol. Overall, 100 µM carbachol increased Isc 3.2 ± 0.3-fold. B: time course of galanin-induced increases in Isc after 6-day exposure to DSS. As described in METHODS, Isc was recorded each minute after exposure to 1 µM galanin. Values are means ± SE of a minimum of 5 separate experiments.

The kinetics of galanin-induced increases in Isc were similar to what we previously reported (5). This peptide caused a rapid yet transient increase in Isc so that maximal increases were observed between 2 and 3 min after simulation with galanin and return to basal levels was noted ~8 min later (Fig. 2B).

DSS-induced inflammation decreases transepithelial resistance. Although basal Isc was not changed in DSS-treated mice compared with control mice, resistance was markedly diminished in a manner consistent with that recently reported for human colonic epithelium obtained from patients with IBD (35). Although tissue resistance was 530 ± 40 Omega  · cm2 for untreated control animals, that of mice treated with DSS for 6 days was 310 ± 30 Omega  · cm2. Although galanin acted to increase Isc, it had no effect on resistance (330 ± 30 Omega  · cm2 in mice treated with 2% DSS for 6 days and exposed to 1 µM galanin). Thus these data indicate that galanin acts primarily by increasing Isc.

Galanin potentiates fluid collection in colonic closed loops. The ability of galanin to increase Isc, which we have previously shown is due to increased Cl- secretion in T84 cells (5), does not necessarily mean that this peptide causes increased fluid secretion into the colonic lumen. To determine the ability of galanin to induce fluid secretion, we evaluated the effect of this peptide using closed colonic loops. After 4 h, closed loops in untreated control animals weighed 29 ± 4 mg/cm (Fig. 3). As expected given the absence of detectable Gal1-R in colonic epithelial cells of control animals, instilling 1 µM galanin into the colonic loop did not significantly alter the amount of fluid secretion (Fig. 3). After 6 days of exposure to DSS, however, closed colonic loop weights increased by 2.3 ± 0.2-fold compared with that observed in untreated control animals. This increase in closed loop weight was markedly potentiated by galanin so that after 6 days of DSS, this peptide increased colonic loop weights by an additional 2.2 ± 0.4-fold compared with loops injected only with saline (Fig. 3, inset). Therefore, overall galanin increased fluid secretion in animals exposed to DSS for 6 days by 5.1 ± 0.4-fold compared with control animals not given DSS.


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Fig. 3.   Effect of 2% DSS exposure on colonic fluid secretion as determined by closed-loop experiments. Saline or 1 µM galanin was injected into closed colonic loop formed by suturing closed proximal and distal aspects of the organ. Animals were allowed to recover, and 4 h after injection colonic loop was excised (inset), measured for length, and weighed. Values are expressed in weight per length and represent means ± SE of a minimum of 5 separate experiments.

Effect of dexamethasone-induced inhibition of NF-kappa B activation and Gal1-R expression on colonic fluid secretion. Gal1-R-specific antagonists do not currently exist, as all previously described galanin antagonists have been shown to act as partial agonists at this receptor subtype (5, 18, 29, 40, 45). To explore the contribution of the Gal1-R to the excessive fluid secretion observed in DSS murine colitis, we attempted to inhibit the expression of this receptor by treating mice with a NF-kappa B inhibitor. Steroids are known to have multiple different effects, including inhibiting the activation of NF-kappa B present in the colonic epithelia of patients with IBD (3). We also have demonstrated (17) that dexamethasone inhibits NF-kappa B activation, and associated Gal1-R expression, in the colons of C57BL/6J mice subsequent to infection with enteric pathogens. Because this agent can be readily given to animals without reservation, we investigated whether parenterally administered dexamethasone had any effect on the colitis caused by 2% DSS in mice. Mice were given 0.15 µg/mg dexamethasone in 0.9% saline intraperitoneally every 12 h, a concentration similar to that used in the treatment of acute idiopathic colitis in humans, with the first dose administered 2 h before their initial exposure to DSS. In the absence of dexamethasone, and as described above, 6 days of DSS caused a marked colitis (Table 1) that resulted in significant increases in NF-kappa B activation and Gal1-R expression (Fig. 1). In contrast, dexamethasone markedly attenuated the amount of inflammation, NF-kappa B activation, and Gal1-R expression observed (Fig. 4, Table 2). Finally, although increased fluid secretion was seen in DSS-treated animals not given dexamethasone (Fig. 3), concomitant administration of this anti-inflammatory agent decreased fluid secretion to levels similar to those observed for untreated control animals (Table 2). Not unexpectedly given the absence of Gal1-R in DSS-treated animals also given dexamethasone, galanin had no effect on colonic fluid secretion in mice given DSS as well as this steroid (Table 2).


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Fig. 4.   Effect of parenteral dexamethasone administration on NF-kappa B activation and Gal1-R expression in colonic epithelial cells of mice treated with 2% DSS. Mice given DSS alone had marked increases in epithelial cell NF-kappa B activation (A) and Gal1-R expression (C). In contrast, mice concomitantly receiving dexamethasone (0.15 µg/mg ip every 12 h) showed no evidence of NF-kappa B activation (B) or Gal1-R expression (D). Values are representative of 3 separately treated animals per condition. Magnification: ×400.


                              
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Table 2.   Effect of dexamethasone and galanin antibody on development of DSS-induced colitis, NF-kappa B activation, Gal1-R expression, and fluid secretion

Intraluminal administration of antibody to galanin inhibits fluid secretion. To confirm the contribution of Gal1-R activation to the excessive fluid secretion observed, we intraluminally instilled galanin antibody in DSS-treated mice. Although colonic fluid secretion in mice exposed to DSS for 5 days was approximately twofold greater than observed in control mice, intraluminal injection of galanin antibody completely eliminated the DSS-induced increase in colonic fluid secretion (Table 2). We instilled sufficient antibody in 200 µl of saline to achieve a 1:500 final dilution based on the amount of colonic fluid secretion normally observed over a 4-h period. The amount of colonic fluid generated over 4 h from the colonic loops of DSS-treated mice in the presence of galanin antibody (27 ± 3 mg/cm) was not significantly different from that observed in untreated control mice processed in parallel (29 ± 4 mg/cm).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we show that in the DSS model of murine colitis, a significant proportion of the increase in colonic fluid secretion is due to Gal1-R upregulation. We previously showed (5) that Gal1-R are expressed by the human colonic epithelial cell line T84 and that when activated cause Cl- secretion by a Ca2+-dependent mechanism. We also have shown (17) that although murine colonic epithelial cells do not normally express Gal1-R, infection with pathogenic E. coli results in upregulation of this receptor via an NF-kappa B-mediated process. This physiological observation therefore is in accordance with our prior biochemical finding that GAL1R gene expression is regulated by this inflammation-associated transcription factor (24).

Recent studies have indicated that NF-kappa B is activated in many inflammatory disorders of the human colon in addition to infection (13, 34, 46), including Crohn's disease and ulcerative colitis (26, 36). This has led to an awareness that many therapeutic agents used to treat patients with IBD, including sulfasalazine (39) and steroids (3, 31, 32, 44), mediate their effects by acting as NF-kappa B inhibitors. In this study, we show for the first time that DSS activates NF-kappa B and that the increased activation of this transcription factor is associated with increased Gal1-R expression. Although we do not provide definitive proof in this study that NF-kappa B is directly responsible for the DSS-induced increase in Gal1-R expression, we do show that the increase in the expression of this receptor permits galanin to increase Isc (Fig. 2) and net colonic fluid secretion (Fig. 3). At the very least, these data support the possibility that increased Gal1-R expression may be an important component of the excessive fluid secretion observed in IBD.

Our electrophysiological findings are in agreement with the few such studies performed on inflamed human tissues. In patients with IBD it has come to be appreciated that significant colonic fluid accumulation occurs via increases in paracellular permeability (35). We likewise show that the colonic resistance decreased by >40% in mice given 2% DSS for 6 days, similar to the ~50% decrease reported in patients with active colitis (35). It also has been reported that basal Isc is not increased in humans with active IBD, supporting the argument that the associated diarrhea is largely a problem of increased paracellular permeability. Similarly, we did not find any significant change in basal Isc in DSS-treated vs. control mice. However, we did find that whereas galanin had no effect on control murine colonic epithelium, this peptide hormone markedly increased Isc in DSS-treated mice (Fig. 2). The failure to see elevations in basal Isc, along with our showing that galanin increases Isc in DSS-treated mice, is possibly due to the fact that this ligand is present in enteric nerve terminals lining the GI tract of all species studied, including humans (4, 19). Removal of colonic epithelial tissues for study ex vivo either displaces them from the source of stimulatory ligand or dilutes the ligand available when they are placed in an Ussing chamber. That galanin is important in mediating fluid secretion is nonetheless demonstrated by its ability to potentiate colonic fluid secretion in vivo (Fig. 3), with galanin antibody markedly diminishing the amount of fluid secretion observed (Table 2), in DSS-treated mice.

It should be emphasized that the basic mechanism of action of DSS in causing colitis has yet to be established. Studies suggest that DSS mediates its effects by altering mucosal prostanoid concentrations (38) and host immunologic responsiveness (2, 11, 12) and by being directly toxic to the gut epithelial cells (9, 10). However, and particularly in light of our recent finding that enteric pathogens increase Gal1-R expression in gut epithelia (17), it is the ability of DSS to alter the composition of the colonic microflora that may be of greatest significance (28). DSS has been shown to alter the composition of the intestinal flora by two different mechanisms. First, DSS can be phagocytosed by gut macrophages, causing them to have reduced phagocytic ability of normal commensal organisms, allowing expansion of the Gram-negative anaerobic population (22). Second, DSS also has been shown, by virtue of it directly injuring intestinal epithelia, to permit increased uptake of bacterial endotoxin and peptidoglycan-polysaccharide polymers (33). Thus DSS may cause colonic injury, as well as increase Gal1-R expression, by altering the composition of the host bacterial flora.

This study does not attempt to validate the use of DSS-treated mice as a model for the study of IBD. Rather, this study is the first to show that DSS activates NF-kappa B and increases Gal1-R expression in the colonic epithelium and that the increase in Gal1-R expression is directly responsible for a significant proportion of the colonic fluid secretion observed. Overall, these findings support a possible role for galanin and the Gal1-R in the pathophysiology of IBD-associated diarrhea.


    ACKNOWLEDGEMENTS

This study was supported by an American Digestive Health Foundation/Astra Merck Advanced Research Training Award (J. A. Marrero), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50694 (G. Hecht) and DK-51168 (R. V. Benya), Veterans Administration Merit Reviews (G. Hecht and R. V. Benya), and the Veterans Administration Research Enhancement Awards Program (G. Hecht and R. V. Benya).


    FOOTNOTES

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 and other correspondence: R. V. Benya, Dept. of Medicine, Univ. of Illinois at Chicago, 840 South Wood St. (M/C 787), Chicago, IL 60612 (E-mail: rvbenya{at}uic.edu).

Received 11 August 1999; accepted in final form 14 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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