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The Journal of Immunology, 2006, 177: 3355-3361.
Copyright © 2006 by The American Association of Immunologists, Inc.

Corticotropin-Releasing Hormone Receptor 2-Deficient Mice Have Reduced Intestinal Inflammatory Responses1

Efi Kokkotou*,{dagger}, Daniel Torres*, Alan C. Moss*, Michael O’Brien{ddagger}, Dimitri E. Grigoriadis§, Katia Karalis{dagger},¶ and Charalabos Pothoulakis2,*,{dagger}

* Gastrointestinal Neuropeptide Center, Gastroenterology Division, Beth Israel Deaconess Medical Center, Boston, MA 02215; {dagger} Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, {ddagger} Department of Pathology, Boston University Medical Center, Boston, MA; § Department of Molecular Biology, Neurocrine Biosciences Inc., San Diego, CA; and Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston MA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Corticotropin-releasing hormone (CRH) and urocortins (Ucn) bind with various affinities to two G-protein-coupled receptors, CRHR1 and CRHR2, which are expressed in brain and in peripheral tissues, including immune cells. CRHR2-deficient mice display anxiety-like behavior, hypersensitivity to stress, altered feeding behavior and metabolism, and cardiovascular abnormalities. However, the phenotype of these mice in inflammatory responses has not been determined. In the present study we found that compared with wild-type CRHR2-null mice developed substantially reduced intestinal inflammation and had lower intestinal mRNA expression of the potent chemoattractants keratinocyte chemokine and monocyte chemoattractant protein 1 following intraluminal exposure to Clostridium difficile toxin A, a potent enterotoxin that mediates antibiotic-associated diarrhea and colitis in humans. This effect was recapitulated by administration of astressin 2B, a selective CRHR2 antagonist, before toxin A exposure. Moreover, Ab array analysis revealed reduced expression of several inflammatory chemokines, including keratinocyte chemokine and monocyte chemoattractant protein 1 in toxin A-exposed mice pretreated with astressin 2B. Real-time RT-PCR of wild-type mouse intestine showed that only UcnII, but not other Ucn, was significantly up-regulated by ileal administration of toxin A at 4 h compared with buffer exposure. We also found that human colonic epithelial HT-29 cells express CRHR2{alpha} mRNA, whereas expression of beta and {gamma} spliced variants was minimal. Moreover, treatment of HT-29 cells with UcnII, which binds exclusively to CRHR2, stimulated expression of IL-8 and monocyte chemoattractant protein 1. Taken together, these results provide direct evidence that CRHR2 mediates intestinal inflammatory responses via release of proinflammatory mediators at the colonocyte level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Corticotropin-releasing hormone receptor 2 (CRHR2)3 is a seven transmembrane G-protein-coupled receptor that binds CRH and urocortins (Ucn) with various affinities. UcnII and UcnIII are exclusive ligands for CRHR2, whereas CRH and Ucn bind also to CRHR1 (1). Both CRHR1 and CRHR2 are present in the brain but have distinct anatomical patterns, implicating different functional roles (2). For example, CRHR1 mediates the effects of CRH on the hypothalamic-pituitary-adrenal axis, including the acute stress response, and on cortical, cerebellar, and sensory pathways, whereas CRHR2 participates in the integration of neuroendocrine, autonomic, and behavioral functions (3). In humans, three CRHR2 isoforms have been described: CRHR2{alpha}, beta, and {gamma}. Outside the CNS, CRHR2{alpha} is the most commonly found isoform, CRHR2{gamma} appears to be restricted to the brain, whereas CRHR2beta has been described only in muscle (4, 5). Peripheral CRHR2 expression is more abundant in lung, heart, vascular system, myometrium, gastrointestinal tract, skin, and immune cells, playing a role in a variety of physiological functions (4, 6). The simultaneous generation of CRHR2-deficient mice by three independent groups enhanced our understanding of several CRHR2-related functions (7, 8, 9). Upon restrain stress, CRHR2-deficient mice had higher adrenocorticotropin and corticosterone levels indicating hypersensitivity to stress (7, 8). These mice had also increased anxiety-like behavior in the elevated plus maze, open-field, the dark-light emergence, and the acute locomotor activity tests, as well as difficulties in coping with novelty (7, 8, 9). CRHR2-null mice also displayed altered metabolic responses, including a substrate preference for fatty acids, and hyperphagia not associated with extensive body weight gain, most likely due to increased thermogenesis (10, 11). Furthermore these mice showed cardiovascular abnormalities, such as elevated blood pressure and decreased cardiac contractility (8, 12).

Compared with the proinflammatory role of CRH in peripheral tissues, the role of Ucn appears to be less clear (13, 14). In vitro, Ucn and UcnII mediate anti-inflammatory effects by causing macrophage apoptosis (15) and stimulate the production of IL-6, which has immunomodulatory effects, in aortic smooth muscle cells (16), via CRHR2 activation. In vivo, Ucn suppresses experimental autoimmune encephalomyelitis in rats (17), reduces LPS-induced serum TNF{alpha} and IL-1beta levels in mice (18), and decreases mortality after cecal ligation and puncture or following injection with bacterial endotoxin (19). However, the interpretation of those studies might be complicated by the fact that Ucn can directly up-regulate glucocortocoid production, which has anti-inflammatory effects (17, 18). On the other hand, UcnII treatment worsens Listeria monocytogenes infection in mice via up-regulation of IL-10 (20). Moreover, Ucn is up-regulated in the stomach of patients with Helicobacter pylori gastritis (21) and the colonic lamina propria cells of patients with ulcerative colitis (22). Treatment with Ucn also protects mice against 2,4,6-trinitrobenzene sulfonic acid-induced colitis and reduces the levels of Th1 cytokines associated with this response (23). Using an RNA interference approach La Fleur et al. showed that UcnII silencing in the ileum of rats did not significantly affect the inflammatory response to Clostridium difficile toxin A (24). However, the role of CRHR2 and its specific ligand UcnII in intestinal inflammation has not been fully elucidated.

The presence of CRHR2 on resident and circulating immune cells such as granulocytes, monocytes, plasma, and mast cells (14, 25, 26) and its up-regulation on intestinal plasma cells and macrophages in patients with ulcerative colitis (22), along with the altered susceptibility of CRH-deficient mice to inflammatory (27, 28, 29) and autoimmune conditions (30), prompted us to examine intestinal inflammatory responses in CRHR2-null mice. For that purpose, we applied the C. difficile toxin A-mediated mouse model of acute intestinal inflammation, which has been previously used by us to demonstrate a peripheral proinflammatory role for CRH (29, 31) mediated, at least in part, by CRHR1 (31). Toxin A is a potent enterotoxin that causes antibiotic-associated inflammatory diarrhea in hospitalized patients. This disease affects millions of patients per year in the United States alone and accounts for more than $1 billion in hospitalization costs (32). In the present study, we found significantly reduced toxin A-associated inflammatory responses in CRHR2-deficient mice and in wild-type (WT) mice treated with astressin 2B, a highly selective CRHR2 antagonist (33). Furthermore, we found a link between CRHR2 signaling in colonic epithelial cells and chemokine production, an important component of inflammatory responses against microbial pathogens and their products.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Induction of intestinal inflammation

CRHR2-deficient mice were provided by Dr. W. Vale (Salk Institute, La Jolla, CA) (7). Male CRHR2 knockout (KO) mice and their WT littermates (n = 17–18/group) were derived from heterozygous breedings. Mouse ileal loops were ligated in anesthetized animals via laparotomy and injected with 0.15 ml of purified C. difficile toxin A (10 µg) or buffer, as previously described (29). The purification of toxin A from toxigenic C. difficile strain 10465 (American Type Culture Collection) cultures has been previously described by us (34). In the experiments involving treatment with astressin 2B (Neurocrine Biosciences), CD1 male mice (n = 7–8/group) were injected i.p. with 200 µl of an astressin 2B solution (300 µg/kg, 30 µg/kg, or 3 µg/kg in saline supplemented with 0.75% DMSO and 1% BSA) or vehicle, 30 min before the administration of toxin A or buffer. Previous studies indicated the specificity of this peptide antagonist for CRHR2 and its ability to alter CRH or Ucn-associated responses in vivo (33, 35). In separate experiments, CD1 mice were treated with toxin A or buffer for 4 h for evaluation of mRNA expression of Ucns (n = 6/group). Animal studies were approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center.

Analysis of inflammatory responses

Four hours after ileal toxin A injection, mice were sacrificed, and fluid secretion was determined as the loop weight (mg) -to-length (cm) ratio. Full-thickness loop sections were processed for histology or frozen immediately at –80°C for further analysis. Histological severity of enteritis was graded blindly by an experienced pathologist (M.O.), using previously established parameters (36). Expression of the various cytokines and Ucns was measured by real-time RT-PCR in a 5700 Sequence Detection System (gene expression assays; Applied Biosystems) or by ELISA (DuoSet; R&D Systems). Myeloperoxidase (MPO) activity in tissue lysates was determined by an enzymatic method using human MPO (0.1 U/100 µl; Sigma-Aldrich) as a standard, as previously described (29). Total protein concentration of each sample was determined by the DC protein assay (Bio-Rad).

Antibody array analysis

Clarified ileal loop lysates (100 µg in 1 ml of buffer) pooled from four animals per group, were incubated with a mouse Ab array spotted with Abs against 40 different cytokines (mouse inflammation assay 3.1; Ray Biotech) according to the manufacturer’s instructions and as previously described by us (37).

Cell stimulations

HT-29 human colonic adenocarcinoma cells growing at 80–90% confluence in McCoy’s 5A medium supplemented with 10% FBS and 1% antibiotic-antimycotic (Invitrogen) were serum-deprived for 16 h, and then stimulated with 10–7 M UcnII (Bachem) for 3 h. RNA was purified using the RNeasy kit followed by DNase treatment (Qiagen) and IL-8, monocyte chemoattractant protein 1 (MCP-1), and GAPDH mRNA levels were analyzed by real-time RT-PCR (gene expression assay; Applied Biosystems) as described above. Expression of the various CRHR2 subtypes in HT-29 cells was assessed likewise, using the isoform-specific primers described by Kostich et al. (5).

Statistical analysis

Results are expressed as mean ± SEM and data have been analyzed by t test or by ANOVA with Bonferroni/Dunn post hoc analysis using the StatView statistical software program (SAS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CRHR2-deficient mice have reduced intestinal secretion, histological damage, and inflammation in response to toxin A

We injected closed ileal loops of anesthetized CRHR2-deficient mice and their WT littermates with purified toxin A or vehicle and measured toxin A-associated inflammatory responses after 4 h. As shown in Fig. 1A, histologic analysis of the intestinal tissue revealed that WT mice developed severe epithelial cell damage associated with neutrophil tissue transmigration and edema of the intestinal mucosa as a result of toxin A treatment (bottom left panel). In contrast, CRHR2-deficient mice had significantly reduced ileal tissue pathology (bottom right panel), also reflected by histological scoring of their inflammation (p = 0.03, Fig. 1B). Another marker of inflammation in this model is fluid accumulation in the loop lumen. As shown in Fig. 1C, toxin A-induced intestinal fluid secretion, measured as the loop weight-to-length ratio, was ~30% less in CRHR2-deficient compared with WT mice (68.8 ± 7.5 vs 100 ± 8, respectively, p = 0.008), whereas basal fluid secretion in buffer-injected loops was similar between the two genotypes.


Figure 1
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  		FIGURE 1. CRHR2-deficient mice have reduced intestinal inflammatory responses. Closed ileal loops were prepared in male CRHR2-deficient mice (n = 17–18/group) (filled bars) and their WT littermates (open bars) and injected with 10 µg of purified C. difficile toxin A, a potent enterotoxin, or buffer. At 4 h posttreatment, inflammatory responses were evaluated in the intestinal tissue. A, Representative H & E-stained transverse sections of ileal loops from WT (left) or CRHR2 KO mice (right), treated with buffer (top) or toxin A (bottom) (magnification, x200). B, Intestinal inflammation was graded blindly by an experienced pathologist in H & E-stained histological sections of ileal loops by a score of 0–3 for epithelial damage, vascular congestion and edema of the mucosa, and neutrophil margination and infiltration. The sum of the three scores for each group is presented. C, Intestinal fluid secretion in the lumen of ileal loops of toxin A- or buffer-treated WT and CRHR2-deficient mice is expressed as the weight-to-length ratio of each loop (percentage of control). *, p < 0.05; **, p < 0.01.

 
Astressin 2B, a selective CRHR2 antagonist, mimics the outcome of CRHR2 genetic ablation in the toxin A mouse model of intestinal inflammation

We next used a pharmacological approach to confirm the reduced toxin A-associated inflammatory responses found in CRHR2-deficient mice. Astressin 2B is a highly selective CRHR2 antagonist (33) that does not cross the blood-brain barrier (35). Mice were pretreated with three different doses of astressin 2B, 30 min before injection of toxin A into ileal loops and toxin A-associated responses were determined at 4 h. Astressin 2B, itself, had no effect on tissue histology in saline-treated mice (Fig. 2A, top panel). However, in toxin A-treated mice, i.p administration of astressin 2B inhibited toxin A-induced inflammatory responses. At the microscopic level, astressin 2B-treatment resulted in considerably less intestinal epithelial damage, congestion and edema and lower levels of immune cell infiltration (Fig. 2A, bottom right panel), compared with vehicle-treated mice (Fig. 2A, bottom left panel). Neutrophil infiltration score was 1.15 ± 0.18 in toxin A-treated mice receiving vehicle injections compared with 0.14 ± 0.14 in mice receiving the lower dose of astressin 2B (3 µg/kg) (p = 0.0017) (Fig. 2B). However, a dose-responsive effect of astressin 2B was not evident in this assay. Reduced neutrophil infiltration in the presence of astressin 2B was also confirmed by tissue MPO activity, a specific marker for activated neutrophils. Baseline MPO levels were 54.0 ± 22.1 U/g protein and were increased to 189.9 ± 32.3 U/g upon toxin A treatment. Toxin A-treated mice that received astressin 2B (30 µg/kg) had MPO levels similar to baseline 61.6 ± 21.5 U/g, p < 0.003). Along these lines, toxin A-injected mice pretreated with vehicle showed a dramatic increase in intraluminal fluid secretion, whereas mice receiving astressin 2B had a substantially reduced toxin A-associated secretory response (51.8 ± 3.7 vs 145.2 ± 12.9 weight-to-length ratio, 300 µg/kg astressin 2B vs vehicle treatments, p < 0.0001) (Fig. 2C), consistent with the astressin 2B-mediated reduced inflammatory responses in this model. This response was dose-dependent.


Figure 2
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  		FIGURE 2. Pharmacological antagonism of CRHR2 by astressin 2B reduces intestinal inflammation. WT mice (n = 8/group) were pretreated i.p. with 3, 30, and 300 µg/kg astressin 2B or vehicle, and after 30 min, ileal loops were formed and injected with either buffer (PBS) or purified toxin A (10 µg). After 4 h, ileal loops were harvested and the severity of inflammation was assessed at the microscopic level. A, Representative H & E-stained intestinal sections from buffer- (top) or toxin A-treated (bottom) mice, in the absence (left) or presence (right) of astressin 2B treatment (magnification, x200). B, Neutrophil infiltration of the ileal tissue due to toxin A exposure was blindly scored in astressin 2B- or vehicle-treated mice from 0 to 3 by an experienced pathologist. C, Intestinal secretion in closed ileal loops was measured as the loop weight (mg) to length (cm) ratio and expressed as percentage of control. **, p < 0.01 for comparisons between toxin A- or buffer-treated loops; +, p < 0.05 and ++, p < 0.01 for comparisons among toxin A-treated and toxin A + astressin 2B-treated groups.

 
Role of CRHR2 in toxin A-induced chemokine release

As an unbiased approach to investigate potential links between CRHR2 signaling and proinflammatory cytokine expression in the toxin A enteritis model, we used an inflammatory Ab array to screen tissue lysates obtained from toxin A-exposed ileal loops of animals with or without astressin 2B administration. We found that, among 40 cytokines and chemokines tested, toxin A administration was associated with increased expression of IL-6, and the potent chemoattractants eotaxin, MCP-1, and keratinocyte chemokine (KC). Moreover, expression of these inflammatory mediators was significantly reduced by astressin 2B administered before toxin A exposure (Fig. 3A). We next confirmed the results of the Ab array by ELISA. In buffer-treated loops, KC expression was similar between vehicle and astressin 2B-exposed mice (52.0 ± 6.2 and 33.8 ± 81.1 pg/mg of protein, respectively). However, ileal toxin A exposure stimulated increased KC expression in vehicle-treated mice, whereas astressin 2B administration resulted in ~50% reduced KC intestinal levels (261.2 ± 85.5 vs 109.4 ± 11.6 pg/mg, respectively, p = 0.001) (Fig. 3B), consistent with lower neutrophil infiltration in this group (Fig. 2, A and B). Similarly, toxin A stimulated an increase in MCP-1 protein levels that was reduced by astressin 2B pretreatment (246.2 ± 34 vs 165 ± 28 arbitrary units (AU) in vehicle vs 30 µg/kg astressin 2B-treated toxin A-exposed mice, respectively, p = 0.046) (Fig. 3C). Lower KC mRNA levels were also observed in toxin A-treated loops of CRHR2 KO mice (48.4 ± 14.1 AU) compared with WT (165.8 ± 28.2 AU, p = 0.003) (Fig. 3D), in line with reduced toxin A-associated inflammatory responses in these mice (Fig. 1). A similar pattern was observed in MCP-1 expression in WT compared with CRHR2-deficient mice (142 ± 33 vs 36 ± 10 AU in WT vs CRHR2 KO mice, respectively, p = 0.01) (Fig. 3E).


Figure 3
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  		FIGURE 3. CRHR2 has an effect on chemokine release. A, CD1 mice were treated i.p. with buffer or astressin 2B (30 µg/kg) 30 min before C. difficile toxin A administration in closed ileal loops. Tissue lysates were prepared 4 h later from 4 loops/group and applied to an Ab array membrane detecting 40 inflammatory cytokines and chemokines. Visual inspection of the blot revealed several differentially regulated spots, as marked with rectangles, corresponding to KC (1), eotaxin (2), IL-6 (3), and MCP-1 (4). B, CD1 mice (n = 7–8/group) were treated as above and tissue KC and MCP-1 levels were evaluated by ELISA, normalized by protein concentration and expressed as picograms per milligram of total protein. C, CRHR2 KO mice (filled bars) and their WT littermates (open bars) (n = 7–8/group) were treated with 10 µg of toxin A in closed ileal loops, and at 4 h posttreatment, RNA was prepared from the intestinal tissue and analyzed for KC and MCP-1 expression by real-time RT-PCR. mRNA expression is presented in arbitrary units and has been normalized by GAPDH expression. **, p < 0.01 for comparisons among WT and CRHR2 KO mice and between toxin A- or buffer-treated groups; ++, p < 0.01 for comparisons among toxin A-treated and toxin A + astressin 2B-treated groups.

 
UcnII expression is up-regulated in response to toxin A-mediated inflammation

In a previous study, we have shown that exposure of mouse ileal loops to C. difficile toxin A resulted in the local up-regulation of CRH expression (31). However, expression of the other members of the CRH family of peptides has not been explored. Mouse ileal loops were exposed to toxin A, and at 4 h, ileal loops were processed for mRNA measurements of Ucn, UcnII, and UcnIII. As shown in Fig. 4, only UcnII, but not the other Ucns, was significantly up-regulated by ileal administration of toxin A at 4 h compared with buffer exposure (178 ± 18 vs 100 ± 13 AU, respectively, p < 0.001).


Figure 4
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  		FIGURE 4. UcnII expression is increased during toxin A-mediated intestinal inflammation. Closed ileal loops of anesthetized CD-1 mice were injected with buffer or toxin A (10 µg) (n = 6/group), and after 4 h, tissues were harvested. Ucn, UcnII, UcnIII, and GAPDH mRNA expression was evaluated by real-time RT-PCR. Results are expressed as arbitrary units and have been normalized by GAPDH expression. **, p < 0.01 between 0 and 4 h time points.

 
Chemokine induction in HT-29 human colonic epithelial cells in response to UcnII treatment

Based on previous studies showing profound up-regulation of CRHR2 expression in intestinal epithelium of mice treated with C. difficile toxin A (31), we examined whether CRHR2 ligand coupling is associated with chemokine release in colonic epithelial cells in vitro. We first examined by real-time RT-PCR the expression of the different isoforms of CRHR2 receptor in human colonic adenocarcinoma HT-29 cells, and found that these cells express mRNA for CRHR2{alpha}, the most common isoform, whereas expression of mRNAs for the beta and {gamma} CRHR2 isoforms was almost undetectable (Fig. 5A). Treatment of HT-29 cells for 3 h with UcnII (10–7 M), which binds exclusively to CRHR2, stimulated a 2- to 3-fold increase in the expression of IL-8 (18.5 ± 2.3 vs 8.5 ± 1.9 AU, UcnII vs vehicle, p = 0.011) (Fig. 5B) and of MCP-1 (15 ± 0.9 vs 8.1 ± 2 AU, UcnII vs vehicle, respectively, p = 0.013) (Fig. 5C). These findings are consistent with our in vivo results showing reduced expression of KC (the mouse equivalent of human IL-8) and MCP-1 in CRHR2-deficient (Fig. 3, D and E) and astressin 2B-treated WT mice (Fig. 3, B and C) in response to toxin A.


Figure 5
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  		FIGURE 5. UcnII treatment stimulates chemokine expression in HT-29 human colonic epithelial cells. A, HT-29 cells were analyzed by real-time RT-PCR for the expression of the various CRHR2 isoforms ({alpha}, beta, {gamma}). B and C, HT-29 cells (six independent cultures) were treated with UcnII (10–7 M) for 3 h and IL-8 (B) and MCP-1 (C) mRNA expression was analyzed by real-time RT-PCR. Results are expressed as arbitrary mRNA units and have been normalized by GAPDH expression. *, p < 0.05, **, p < 0.01 for control vs UcnII treatments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we show that inhibition of CRHR2 signaling, either by genetic ablation or by pharmacological antagonism results in reduced inflammatory diarrhea, and diminished mucosal chemokine levels in a mouse model of C. difficile toxin A-mediated enteritis. UcnII, a CRH-like peptide that binds selectively to CRHR2 (38), was also up-regulated by toxin A in mouse ileal mucosa, indicating that it may represent an important CRHR2 ligand involved in intestinal inflammation. In support of this notion, exposure to UcnII of human colonocytes leads to increased expression of the potent chemoattractants IL-8 and MCP-1. To our knowledge, this is the first direct demonstration that the CRHR2 is linked to inflammatory responses in the intestine, or any other organ, suggesting involvement of this receptor in innate immunity. Moreover, the presence of CRHR2 receptors in human colonocytes has not been previously recognized.

Our Ab array analysis and subsequent ELISA and real-time RT-PCR studies indicate that CRHR2 is linked to the expression of several chemokines during intestinal inflammation. Although our in vitro results suggest that this effect is most likely mediated via CRHR2-dependent chemokine release from colonic epithelial cells, activation of CRHR2-bearing immune cells cannot be excluded. In support of this notion, Mousa et al. suggested that CRHR2 expression is evident in circulating or locally recruited at the sites of inflammation granulocytes, monocytes, and lymphocytes (26). Related to the CRHR2-mediated chemotactic effects, in another study CRH treatment stimulated migration of human monocytes in vitro, although the specific CRHR mediating this effect has not been determined (39). Moreover, treatment of murine macrophages with CRH and UcnII resulted in CRHR2-mediated up-regulation of TLR4 expression, an important pathogen recognition receptor and a potent inducer of IL-6 (40). Similarly, CRH can also stimulate activation of NF-{kappa}B, a transcription factor associated with inflammatory responses (41). Taken together, these results suggest that CRHR2 may be an important component of the innate immunity.

Results of the present study, indicating that colonic epithelial HT-29 cells express functional CRHR2, are consistent with findings of prior in situ and immunohistochemical studies suggesting the presence of CRHR2 in intestinal epithelial cells and its up-regulation during toxin A-induced inflammation (31). Along these lines, the presence of CRHR2 mRNA at the base of mouse duodenal villus epithelium (42) and in the luminal surface of the crypts in rat colon (43) has been previously demonstrated. Interestingly, increased CRHR2 expression is noted in lamina propria plasma cells, and macrophages in colonic tissues from patients with ulcerative colitis (22), suggesting participation of this receptor in inflammatory bowel disease. Saruta et al. also demonstrated increased colonic Ucn immunoreactivity in lamina propria plasma cells and enterochromaffin cells in these patients (22).

Studies so far have established that CRHR2 activation results in delayed gastric transit in mice, which is most likely mediated by Ucn and UcnII (2, 44). Our findings in the present study revealed that among the Ucns that we examined, only UcnII mRNA expression was increased in the ileum of toxin A-injected mice. Such a finding, together with the ability of UcnII to directly stimulate IL-8 and MCP-1 expression in human colonocytes in vitro, suggest that UcnII might be an important mediator for inflammation. These results may be related to the results of Brar et al. (45), who showed that UcnII stimulated Erk1/2 phosphorylation in CRHR2-expressing cells, because IL-8 expression depends on Erk1/2 activation (46). Interestingly, patients with C. difficile colitis have increased fecal expression of IL-8 (47), and IL-8 has been associated with the pathophysiology of inflammatory bowel disease (48). UcnII, however, also enhances IL-10 and decreases TNF-{alpha} release in a macrophage cell line (20), indicating that UcnII may not only exert proinflammatory responses. In another study, La Fleur et al. used RNA interference technology to generate ileal-specific CRH and UcnII phenotypic KO rats and examined their inflammatory responses in the toxin A model of intestinal inflammation (24). Consistent with our previous reports (29, 31), they found that CRH inhibition resulted in reduced ileal inflammation, whereas UcnII inhibition had no protective effect in toxin-associated responses (24). Different experimental approaches or different species (rat vs mouse) may account for these different results.

Our results demonstrate that astressin 2B treatment reduces toxin A-mediated secretion and inflammation. Although the effect of astressin 2B in toxin A-mediated ileal fluid secretion was dose-dependent (Fig. 2C), the inhibitory effect of this CRHR2 antagonist in toxin A-induced neutrophil infiltration was similar among the three different doses tested (Fig. 2B). These differences may reflect the sensitivity of the two different techniques used in these responses, the one being quantitative (fluid secretion), whereas the other more subjective and semiquantitative (neutrophil infiltration score). Based on the minimal ability of astressin 2B to cross the blood-brain barrier (35), it is likely that the site of action of this antagonist is the ileal mucosa. Moreover, it is unlikely that the hypothalamic-pituitary-adrenal axis is involved in astressin 2B-mediated anti-inflammatory responses, because this drug is unable to significantly modify CRH-induced adrenocorticotropin release (35).

In summary, results of the present study provide direct evidence for a significant proinflammatory role of intestinal CRHR2 and its specific ligand UcnII in human colonic epithelial cells in vitro and in acute, enterotoxin-mediated intestinal secretion and inflammation in mice. Because a local CRH peptides/receptor circuit seems to be involved in human and experimental inflammatory bowel disease (22, 49, 50), our results suggest the possibility for a novel therapeutic target for intestinal inflammatory conditions, or possibly other forms of inflammation, namely CRHR2 antagonists.


   Acknowledgments
 
We are grateful to Dr. Wylie Vale (Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California) for providing the CRHR2 KO mice. We also thank Nashima Mustafa for excellent technical assistance with the in vivo experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
D.E.G. is an employee at Neurocrine Biosciences Incorporated.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research Grants P0-1 DK 33506, DK 38458, and DK 47977 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Charalabos Pothoulakis, Beth Israel Deaconess Medical Center, Harvard Medical School, Division of Gastroenterology, Dana 601, 330 Brookline Avenue, Boston, MA 02215. E-mail address: cpothoul{at}bidmc.harvard.edu Back

3 Abbreviations used in this paper: CRH, corticotropin-releasing hormone; Ucn, urocortin; CRHR, CRH receptor; KC, keratinocyte chemokine; MPO, myeloperoxidase; MCP-1, monocyte chemoattractant protein 1; AU, arbitrary units; WT, wild type; KO, knockout. Back

Received for publication May 3, 2006. Accepted for publication June 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hoare, S. R., S. K. Sullivan, J. Fan, K. Khongsaly, D. E. Grigoriadis. 2005. Peptide ligand binding properties of the corticotropin-releasing factor (CRF) type 2 receptor: pharmacology of endogenously expressed receptors, G-protein-coupling sensitivity and determinants of CRF2 receptor selectivity. Peptides 26: 457-470. [Medline]
  2. Martinez, V., L. Wang, M. Million, J. Rivier, Y. Tache. 2004. Urocortins and the regulation of gastrointestinal motor function and visceral pain. Peptides 25: 1733-1744. [Medline]
  3. Chalmers, D. T., T. W. Lovenberg, E. B. De Souza. 1995. Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression. J. Neurosci. 15: 6340-6350. [Abstract/Free Full Text]
  4. Lovenberg, T. W., D. T. Chalmers, C. Liu, E. B. De Souza. 1995. CRF2 {alpha} and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology 136: 4139-4142. [Abstract]
  5. Kostich, W. A., A. Chen, K. Sperle, B. L. Largent. 1998. Molecular identification and analysis of a novel human corticotropin-releasing factor (CRF) receptor: the CRF2{gamma} receptor. Mol. Endocrinol. 12: 1077-1085. [Abstract/Free Full Text]
  6. Kishimoto, T., R. V. Pearse, 2nd, C. R. Lin, M. G. Rosenfeld. 1995. A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle. Proc. Natl. Acad. Sci. USA 92: 1108-1112. [Abstract/Free Full Text]
  7. Bale, T. L., A. Contarino, G. W. Smith, R. Chan, L. H. Gold, P. E. Sawchenko, G. F. Koob, W. W. Vale, K. F. Lee. 2000. Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat. Genet. 24: 410-414. [Medline]
  8. Coste, S. C., R. A. Kesterson, K. A. Heldwein, S. L. Stevens, A. D. Heard, J. H. Hollis, S. E. Murray, J. K. Hill, G. A. Pantely, A. R. Hohimer, et al 2000. Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2. Nat. Genet. 24: 403-409. [Medline]
  9. Kishimoto, T., J. Radulovic, M. Radulovic, C. R. Lin, C. Schrick, F. Hooshmand, O. Hermanson, M. G. Rosenfeld, J. Spiess. 2000. Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nat. Genet. 24: 415-419. [Medline]
  10. Bale, T. L., K. R. Anderson, A. J. Roberts, K. F. Lee, T. R. Nagy, W. W. Vale. 2003. Corticotropin-releasing factor receptor-2-deficient mice display abnormal homeostatic responses to challenges of increased dietary fat and cold. Endocrinology 144: 2580-2587. [Abstract/Free Full Text]
  11. Carlin, K. M., W. W. Vale, T. L. Bale. 2006. Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis. Proc. Natl. Acad. Sci. USA 103: 3462-3467. [Abstract/Free Full Text]
  12. Bale, T. L., M. Hoshijima, Y. Gu, N. Dalton, K. R. Anderson, K. F. Lee, J. Rivier, K. R. Chien, W. W. Vale, K. L. Peterson. 2004. The cardiovascular physiologic actions of urocortin II: acute effects in murine heart failure. Proc. Natl. Acad. Sci. USA 101: 3697-3702. [Abstract/Free Full Text]
  13. Bamberger, C. M., A. M. Bamberger. 2000. The peripheral CRH/urocortin system. Ann. NY Acad. Sci. 917: 290-296. [Abstract/Free Full Text]
  14. Baigent, S. M.. 2001. Peripheral corticotropin-releasing hormone and urocortin in the control of the immune response. Peptides 22: 809-820. [Medline]
  15. Tsatsanis, C., A. Androulidaki, E. Dermitzaki, I. Charalampopoulos, J. Spiess, A. Gravanis, A. N. Margioris. 2005. Urocortin 1 and urocortin 2 induce macrophage apoptosis via CRFR2. FEBS Lett. 579: 4259-4264. [Medline]
  16. Kageyama, K., T. Suda. 2003. Urocortin-related peptides increase interleukin-6 output via cyclic adenosine 5'-monophosphate-dependent pathways in A7r5 aortic smooth muscle cells. Endocrinology 144: 2234-2241. [Abstract/Free Full Text]
  17. Poliak, S., F. Mor, P. Conlon, T. Wong, N. Ling, J. Rivier, W. Vale, L. Steinman. 1997. Stress and autoimmunity: the neuropeptides corticotropin-releasing factor and urocortin suppress encephalomyelitis via effects on both the hypothalamic-pituitary-adrenal axis and the immune system. J. Immunol. 158: 5751-5756. [Abstract]
  18. Agnello, D., R. Bertini, S. Sacco, C. Meazza, P. Villa, P. Ghezzi. 1998. Corticosteroid-independent inhibition of tumor necrosis factor production by the neuropeptide urocortin. Am. J. Physiol. 275: E757-E762. [Medline]
  19. Gonzalez-Rey, E., A. Chorny, N. Varela, G. Robledo, M. Delgado. 2006. Urocortin and adrenomedullin prevent lethal endotoxemia by down-regulating the inflammatory response. Am. J. Pathol. 168: 1921-1930. [Abstract/Free Full Text]
  20. Sashinami, H., K. Kageyama, T. Suda, A. Nakane. 2005. Urocortin 2 suppresses host resistance to Listeria monocytogenes infection via up-regulation of interleukin-10. Endocrinology 146: 5003-5011. [Abstract/Free Full Text]
  21. Chatzaki, E., I. Charalampopoulos, C. Leontidis, I. A. Mouzas, M. Tzardi, C. Tsatsanis, A. N. Margioris, A. Gravanis. 2003. Urocortin in human gastric mucosa: relationship to inflammatory activity. J. Clin. Endocrinol. Metab. 88: 478-483. [Abstract/Free Full Text]
  22. Saruta, M., K. Takahashi, T. Suzuki, A. Torii, M. Kawakami, H. Sasano. 2004. Urocortin 1 in colonic mucosa in patients with ulcerative colitis. J. Clin. Endocrinol. Metab. 89: 5352-5361. [Abstract/Free Full Text]
  23. Gonzalez-Rey, E., A. Fernandez-Martin, A. Chorny, M. Delgado. 2006. Therapeutic effect of urocortin and adrenomedullin in a murine model of Crohn’s disease. Gut 55: 824-832. [Abstract/Free Full Text]
  24. la Fleur, S. E., E. C. Wick, P. S. Idumalla, E. F. Grady, A. Bhargava. 2005. Role of peripheral corticotropin-releasing factor and urocortin II in intestinal inflammation and motility in terminal ileum. Proc. Natl. Acad. Sci. USA 102: 7647-7652. [Abstract/Free Full Text]
  25. Papadopoulou, N. G., L. Oleson, D. Kempuraj, J. Donelan, C. L. Cetrulo, T. C. Theoharides. 2005. Regulation of corticotropin-releasing hormone receptor-2 expression in human cord blood-derived cultured mast cells. J. Mol. Endocrinol. 35: R1-R8. [Abstract/Free Full Text]
  26. Mousa, S. A., C. P. Bopaiah, C. Stein, M. Schafer. 2003. Involvement of corticotropin-releasing hormone receptor subtypes 1 and 2 in peripheral opioid-mediated inhibition of inflammatory pain. Pain 106: 297-307. [Medline]
  27. Venihaki, M., P. Dikkes, A. Carrigan, K. P. Karalis. 2001. Corticotropin-releasing hormone regulates IL-6 expression during inflammation. J. Clin. Invest. 108: 1159-1166. [Medline]
  28. Venihaki, M., J. Zhao, K. P. Karalis. 2003. Corticotropin-releasing hormone deficiency results in impaired splenocyte response to lipopolysaccharide. J Neuroimmunol. 141: 3-9. [Medline]
  29. Anton, P. M., J. Gay, A. Mykoniatis, A. Pan, M. O’Brien, D. Brown, K. Karalis, C. Pothoulakis. 2004. Corticotropin-releasing hormone (CRH) requirement in Clostridium difficile toxin A-mediated intestinal inflammation. Proc. Natl. Acad. Sci. USA 101: 8503-8508. [Abstract/Free Full Text]
  30. Benou, C., Y. Wang, J. Imitola, L. VanVlerken, C. Chandras, K. P. Karalis, S. J. Khoury. 2005. Corticotropin-releasing hormone contributes to the peripheral inflammatory response in excperimental autoimmune encephalomyelitis. J. Immunol. 174: 5407-5413. [Abstract/Free Full Text]
  31. Wlk, M., C. C. Wang, M. Venihaki, J. Liu, D. Zhao, P. M. Anton, A. Mykoniatis, A. Pan, J. Zacks, K. Karalis, C. Pothoulakis. 2002. Corticotropin-releasing hormone antagonists possess anti-inflammatory effects in the mouse ileum. Gastroenterology 123: 505-515.
  32. Kyne, L., R. J. Farrell, C. P. Kelly. 2001. Clostridium difficile. Gastroenterol. Clin. North Am. 30: 753-777. [Medline]
  33. Rivier, J., J. Gulyas, D. Kirby, W. Low, M. H. Perrin, K. Kunitake, M. DiGruccio, J. Vaughan, J. C. Reubi, B. Waser, et al 2002. Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists. J. Med. Chem. 45: 4737-4747. [Medline]
  34. Pothoulakis, C., J. T. LaMont, R. Eglow, N. Gao, J. B. Rubins, T. C. Theoharides, B. F. Dickey. 1991. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein. J. Clin. Invest. 88: 119-125. [Medline]
  35. Rivier, C. L., D. E. Grigoriadis, J. E. Rivier. 2003. Role of corticotropin-releasing factor receptors type 1 and 2 in modulating the rat adrenocorticotropin response to stressors. Endocrinology 144: 2396-2403. [Abstract/Free Full Text]
  36. Pothoulakis, C., I. Castagliuolo, T. LaMont, A. Jaffer, J. C. O’Keane, R. M. Snider, S. E. Leeman. 1994. CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin. Proc. Natl. Acad. Sci. USA 91: 947-951. [Abstract/Free Full Text]
  37. Rhee, S. H., E. Im, M. Riegler, E. Kokkotou, M. O’Brien, C. Pothoulakis. 2005. Pathophysiological role of Toll-like receptor 5 engagement by bacterial flagellin in colonic inflammation. Proc. Natl. Acad. Sci. USA 102: 13610-13615. [Abstract/Free Full Text]
  38. Reyes, T. M., K. Lewis, M. H. Perrin, K. S. Kunitake, J. Vaughan, C. A. Arias, J. B. Hogenesch, J. Gulyas, J. Rivier, W. W. Vale, P. E. Sawchenko. 2001. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl. Acad. Sci. USA 98: 2843-2848. [Abstract/Free Full Text]
  39. Genedani, S., M. Bernardi, M. G. Baldini, A. Bertolini. 1992. Influence of CRF and {alpha}-MSH on the migration of human monocytes in vitro. Neuropeptides 23: 99-102. [Medline]
  40. Tsatsanis, C., A. Androulidaki, T. Alissafi, I. Charalampopoulos, E. Dermitzaki, T. Roger, A. Gravanis, A. N. Margioris. 2006. Corticotropin-releasing factor and the urocortins induce the expression of TLR4 in macrophages via activation of the transcription factors PU.1 and AP-1. J. Immunol. 176: 1869-1877. [Abstract/Free Full Text]
  41. Zhao, J., K. P. Karalis. 2002. Regulation of nuclear factor-{kappa}B by corticotropin-releasing hormone in mouse thymocytes. Mol. Endocrinol. 16: 2561-2570. [Abstract/Free Full Text]
  42. Perrin, M., C. Donaldson, R. Chen, A. Blount, T. Berggren, L. Bilezikjian, P. Sawchenko, W. Vale. 1995. Identification of a second corticotropin-releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc. Natl. Acad. Sci. USA 92: 2969-2973. [Abstract/Free Full Text]
  43. Chatzaki, E., P. D. Crowe, L. Wang, M. Million, Y. Tache, D. E. Grigoriadis. 2004. CRF receptor type 1 and 2 expression and anatomical distribution in the rat colon. J. Neurochem. 90: 309-316. [Medline]
  44. Million, M., C. Maillot, P. Saunders, J. Rivier, W. Vale, Y. Tache. 2002. Human urocortin II, a new CRF-related peptide, displays selective CRF(2)-mediated action on gastric transit in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 282: G34-G40. [Abstract/Free Full Text]
  45. Brar, B. K., A. Chen, M. H. Perrin, W. Vale. 2004. Specificity and regulation of extracellularly regulated kinase1/2 phosphorylation through corticotropin-releasing factor (CRF) receptors 1 and 2beta by the CRF/urocortin family of peptides. Endocrinology 145: 1718-1729. [Abstract/Free Full Text]
  46. Rhee, S. H., A. C. Keates, M. P. Moyer, C. Pothoulakis. 2004. MEK is a key modulator for TLR5-induced interleukin-8 and MIP3{alpha} gene expression in non-transformed human colonic epithelial cells. J. Biol. Chem. 279: 25179-25188. [Abstract/Free Full Text]
  47. Steiner, T. S., C. A. Flores, T. T. Pizarro, R. L. Guerrant. 1997. Fecal lactoferrin, interleukin-1beta, and interleukin-8 are elevated in patients with severe Clostridium difficile colitis. Clin. Diagn. Lab. Immunol. 4: 719-722. [Medline]
  48. MacDermott, R. P., I. R. Sanderson, H. C. Reinecker. 1998. The central role of chemokines (chemotactic cytokines) in the immunopathogenesis of ulcerative colitis and Crohn’s disease. Inflamm. Bowel Dis. 4: 54-67. [Medline]
  49. Kawahito, Y., H. Sano, S. Mukai, K. Asai, S. Kimura, Y. Yamamura, H. Kato, G. P. Chrousos, R. L. Wilder, M. Kondo. 1995. Corticotropin releasing hormone in colonic mucosa in patients with ulcerative colitis. Gut 37: 544-551. [Abstract/Free Full Text]
  50. van Tol, E. A., P. Petrusz, P. K. Lund, M. Yamauchi, R. B. Sartor. 1996. Local production of corticotropin releasing hormone is increased in experimental intestinal inflammation in rats. Gut 39: 385-392. [Abstract/Free Full Text]



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