| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Article |
Departments of Pharmacology (E.C., I.C., A.G.), Gastroenterology (C.L., I.A.M.), Pathology (M.T.), and Clinical Chemistry (C.T., A.N.M.), University of Crete School of Medicine, Heraklion 71110, Greece
Address all correspondence and requests for reprints to: Dr. A. Gravanis, Department of Pharmacology, University of Crete School of Medicine, Heraklion GR-71110, Crete, Greece. E-mail: gravanis{at}med.uoc.gr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and CRH-R2ß receptors and the CRH-binding protein, in contrast to CRH, which has low affinity toward the CRH-R2 subtypes (2). The CRH family of peptides is expressed along the whole length of the gastrointestinal (GI) tract. Indeed, CRH is produced by enterochromaffin cells in human colon (3), whereas Ucn is detectable in both rat stomach and colon (4, 5, 6). Recently published reports suggest that the CRH family of peptides and their receptors participate in the regulation of GI motility as well as in the GI response to inflammatory processes. Indeed, it is now well established that CRH is present in the colonic mucosa of patients with ulcerative colitis, playing a local proinflammatory role (7). In addition, Ucn has been identified in macrophages in the lamina propria of human colonic mucosa, participating in the regulation of the local inflammatory response (8). In general, it appears that the effect of the CRH family of peptides in the GI tract is receptor type specific and that the CRH-R1 and CRH-R2 receptors have more or less opposing effects. Indeed, activation of the CRH-R1 receptor results in amplification of colonic propulsive activity, whereas activation of the CRH-R2 receptor results in inhibition of gastric emptying rate in mice (9) and rats (10).
Based on these and other data, we hypothesized that most effects of the CRH family of peptides in the stomach are probably mediated by CRH-R2. As a result, the aim of the present study was to 1) examine the presence of CRH vs. Ucn transcripts and peptides in human gastric mucosa, and 2) examine the association between CRH and Ucn and the inflammatory response of gastric mucosa to a specific gastric mucosa noxious agent. The model of gastritis chosen in this prospective study was that caused by Helicobacter pylori because of its localized and well circumscribed nature and its complete reversibility after appropriate eradication treatment. Our fresh tissue samples were obtained from gastroscopic biopsies. The design of our study was based on our pilot data showing that the CRH transcript and peptide may not be detectable in normal human gastric mucosa, whereas Ucn may be present and localized to gastric epithelial cells. Our data confirmed our initial observations and indicated an interesting association between Ucn and gastric inflammatory response to H. pylori infection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Patients with a history of epigastric pain and/or dyspeptic symptoms of more than 1-month duration underwent gastroscopy according to existing diagnostic protocols in effect at University Hospital (Heraklion, Crete, Greece). Patients with a history of duodenal or gastric ulcer, esophagitis either as a result of gastroesophageal reflux or infection, motility disorders of the esophagus and upper GI tract, gallbladder or bile duct lithiasis, pancreatitis, cirrhosis, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), diabetes mellitus, or cancer were excluded. Excluded were also patients taking any medicine except antacids during the previous month. After careful exclusion of all of the above-mentioned cases, patients who underwent gastroscopy were divided into two groups: controls (n = 8) and patients with diagnosed H. pylori gastritis (n = 15). Between the two groups there were no differences regarding age, gender, dietary habits, or smoking. Controls were patients with normal endoscopic findings and the absence of inflammatory elements in antral biopsies. Patients with chronic gastritis due to H. pylori were individuals with compatible history and diagnostic findings, including flat or raised erosions of gastric mucosa and exudative elements. The lesions were usually more prominent in gastric antrum. The presence of H. pylori was detected histologically and by placement of antral biopsies in a urea-containing medium, allowing simple detection from the pH change that occurs as ammonia is produced by the organism (CLO-test, 
West, Bentley, Australia). A second gastroscopy was performed 2 months after eradication treatment, consisting of a double antibiotic 10-d scheme (amoxicillin, 1 g, orally, twice daily; clarithromycin, 500 mg, orally, twice daily) and omeprazole (20 mg, orally, twice daily for 10 d and then 20 mg daily for 1 month). To attain a more representative measurement of immunoreactive (ir-) Ucn levels in stomach antrum, samples were collected from antrum (lesser and greater curves, front and back wall) by endoscopic biopsy forceps. Samples from each patient were pooled and frozen immediately at –70 C.
The histological grading of gastritis was based on the Sydney classification and was performed by the same person, who was not aware of the different groups of patients. Chronic inflammation was graded as mild, moderate, or severe, noted by +, 2+, and 2++, respectively. The absence of any inflammation was indicated as zero. The presence of H. pylori was graded as +, 2+, or 2++ depending on the density of its presence on the superficial epithelium of antrum mucosa. Normal biopsies were considered those with absent inflammation and negative for H. pylori. Human term placenta was obtained from women undergoing labor at the Obstetrics and Gynecology Department, Heraklion University Hospital. Informed consent and full ethics committee approval had been obtained before the collection of the specimens.
RIA for Ucn
Pooled gastroscopic antral biopsy samples from each patient were homogenized in ice-cold 0.1 N HCl and centrifuged at 10,000 x g for 20 min at 4 C. Peptides were extracted as previously described (11). Briefly, the supernatants were acidified by 10 vol 0.1 N HCl and centrifuged at 10,000 x g for 10 min, and the new supernatants were extracted by activated Sep-Pak C18 cartridge column (Sep-Pak, Waters Corp., Milford, MA), washed with 20 ml 0.1 N HCl, eluted with 3 ml 80% acetonitrile/20% 0.01 N HCl, and dried under vacuum (Speed-Vac). The ir-Ucn was assayed using a Ucn RIA kit (Peninsula Laboratories, Inc., Belmont, CA) following the manufacturer’s instructions. The rabbit antiserum used (RIK 8034) exhibits 100% cross-reactivity to human Ucn and no cross-reactivity to human CRH, Ucn II and III, sauvagine, or urotensin I and II. In our hands the sensitivity of the assay was 10 pg, and the 50% inhibitory concentration was 109 pg/tube. Results were expressed as picograms of ir-CRH per mircogram of total cellular protein determined on whole cellular homogenates by the Bradford method (12).
Immunohistochemistry
Immunostaining was performed on formalin-fixed, paraffin-embedded tissue sections. Paraffin sections from biopsies of stomach antrum were cut and stained by the standard alkaline phosphatase-antialkaline phosphatase method (DAKO Corp., Glostrup, Denmark). Briefly, paraffin sections were heated in a microwave in a solution of sodium citrate before incubation with the antibodies. Xylene-dewaxed and alcohol-rehydrated sections were placed in coplin jars filled with a 0.1 M trisodium citrate solution and heated three times in a conventional microwave oven for 5 min each time at 700 watts. Slides were then allowed to cool for 15 min and then were washed in Tris-buffered saline. After blocking nonspecific binding sites by incubation with normal rabbit serum (1:20, 30 min, room temperature) in a humid chamber, first antibody incubation was performed (1 h, room temperature) using the rabbit anti-Ucn polyclonal serum described above (IHC 8034, Peninsula Laboratories, Inc.; dilution, 1:1000). After washing with Tris-buffered saline, sections were incubated with antirabbit IgG and the alkaline phosphatase-antialkaline phosphatase complex (DAKO Corp.). Fast Red TR (DAKO Corp.) was used as a chromogen with a light hematoxylin counterstain, and sections were mounted with warm glycergel (DAKO Corp.). Negative control sections were included in each experiment by incubation with nonimmune IgG instead of the primary antibody or using antiserum inactivated by 1 µM Ucn peptide (Sigma-Aldrich, St. Louis, MO) overnight at room temperature. Photographs were taken in a standard light microscope using Kodak Elite Chrome film 100 ASA (Eastman Kodak Co., Rochester, NY).
RT-PCR
Total RNA was extracted from frozen biopsies of stomach antrum using the TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Contaminant genomic DNA was removed by the addition of deoxyribonuclease (Life Technologies, Inc.). RT was performed using the Superscript Preamplification System (Life Technologies, Inc.) and random hexamers in a total volume of 20 µl. Two microliters of the RT product were used as template, amplified by PCR using 2 mM MgCl2, one strength PCR buffer, 0.2 mM of sense and antisense primers, 0.2 mM deoxy-NTPs, and 2.5 U Taq polymerase (Life Technologies, Inc.) in a final reaction volume of 50 µl. PCR was performed in a DNA Thermal Cycler (Perkin-Elmer Corp., Norwalk, CT) with the following cycling parameters: a preamplification cycle (denaturation for 5 min at 98 C, annealing for 1 min at 65 C, and extension for 1 min at 72 C), 2 cycles with annealing for 1 min at 63 C, 35 cycles of amplification (denaturation for 1 min at 95 C, annealing for 1 min at 60 C, and extension for 1 min at 72 C), and a final extension step for 7 min at 72 C. The oligonucleotides were designed according to the published human sequences for Ucn (sense, 5'-CAGGCGAGCGGCCGCG-3'; antisense, 5'-CTTGCCCACCGAGTCGAAT-3') (13) and CRH (sense, 5'-CAACTTTTTCCGCGTGTTGCT-3'; antisense, 5'-ATGGCATAAGAGCAGCGCTAT-3') (14). The size of the amplified product was expected to be 145 bp for Ucn and 360 bp for CRH. Oligonucleotides were custom-synthesized by MWG-Biotech, AG (Munich, Germany). Negative control samples where no RT enzyme was added in a positive for Ucn RNA sample or without DNA template were included in every assay to exclude the possibility of genomic or other DNA contamination. PCR for actin was also performed using a standard procedure (15) to assure good quality of RNA and cDNA preparations. The amplified PCR products were fractionated by 1% agarose gel electrophoresis, detected by ethidium bromide staining under UV.
Statistical analysis
The levels of immunoreactive Ucn in gastric biopsies from normal controls and from patients with H. pylori gastritis and after eradication of infection were measured by RIA and were expressed as the mean ± SEM picograms of Ucn per microgram of protein of n biopsies from the same group of patients. Differences between mean values were evaluated using one-way ANOVA, followed by post hoc comparison of means. Correlation analysis of these data with the three parameters of inflammatory activity (acute and chronic inflammation and H. pylori titer) after pathology examination was performed using Spearman’s rank correlation. P 
0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Expression of CRH-like peptides was studied in antral biopsies from human gastric mucosa using RT-PCR in total RNA preparations (Fig. 1
). Using primers designed to target the human Ucn gene, a unique RT-PCR product was amplified in RNA preparations from biopsies of both normal and inflamed gastric mucosa. The size of the DNA band was the same as that amplified from a human placenta RNA sample, used as a positive control (13). No PCR product was detected in the negative control samples performed in parallel, using no reverse transcriptase enzyme or no cDNA template, excluding the possibility of genomic or other DNA contamination of the samples. In contrast, when RT-PCR was performed using primers for the human CRH gene, no PCR products were amplified in the RNA samples from normal and inflamed gastric mucosa, as in the placenta sample that resulted in a DNA band of the predicted size (Fig. 1). The quality of the RNA preparations from these samples was assured by RT-PCR amplification for the actin gene. These results revealed the presence of Ucn, but not CRH, gene transcripts in the gastric mucosa of the human stomach.
|
Our immunohistochemistry data are depicted in Fig. 2. Ucn was present in the epithelial cells of faveolars and the mucus-secreting glands (antral glands) in patients with H. pylori infection (Fig. 2D). Positive staining was also localized to capillaries and inflammatory elements scattered in the gastric mucosal stroma, mostly plasma cell aggregates. Human term placenta was stained as a positive control (Fig. 2B). In the placental sections, specific positive staining was observed in the trophoblastic epithelial cells in contrast to the adjacent stroma-negative villii, confirming the specificity of the method. Replacement of the primary antibody by nonimmune IgG or inactivation of the antibody by excess Ucn peptide before the procedure resulted in uniformly negative immunostaining in both tissue types (Fig. 2, A and C).
|
The levels of ir-Ucn in the biopsies of human gastric mucosa were correlated to the degree of local inflammatory activity, as described in Materials and Methods. Patients were grouped as follows: group a, subjects with no active gastritis, i.e. no evidence of acute or chronic inflammation or H. pylori infection (n = 8); group b, patients with diagnosed gastritis with chronic and acute inflammation of the gastric mucosa and H. pylori infection (n = 15); group c, responders after a 2-month therapy for H. pylori eradication with pathologically confirmed regression of inflammation and no elements of H. pylori infection (n = 10); and 4) nonresponders with remaining elements of inflammation (chronic or acute) and H. pylori infection (n = 5). Ucn was significantly elevated (P < 0.001) in the group of H. pylori gastritis patients (group b, 10.4 ± 1.8 pg/µg total protein) compared with nongastritis subjects (group a, 2.0 ± 1.3 pg/µg total protein; Fig. 3A). Ucn levels were further increased in the group of responders to treatment of H. pylori eradication (group c, 43.1 ± 9.8 pg/µg total protein; P < 0.001) compared with all other groups (a, b, and d). No such elevation was observed in the group of nonresponders to treatment (group c, 18.7 ± 12.3 pg/µg total protein; Fig. 3B). Correlation of the RIA data from the gastritis biopsies (n = 30) with each of the pathology parameters examined (acute and chronic inflammation and degree of H. pylori infection) revealed a significant negative correlation between the levels of Ucn and the pathological staging of gastritis by means of both acute and chronic inflammation and H. pylori infection (Fig. 4), confirming an increase in ir-Ucn levels during regression of the inflammatory activity and H. pylori infection.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our data suggest that this differential response of upper vs. lower tract may also extend to the distinctive and GI site-dependent response to noxious agents. Our data indicate that the Ucn/CRH-R2 system in the upper GI tract plays a completely different role in gastric inflammatory response compared with that of the CRH/CRH-R1 system in the inflammatory response in colon. It should be noted here that Ucn exerts a unique and complex effect on immune and inflammatory responses (16). Thus, the expression of Ucn in human lymphocytes (17) and rat thymus and spleen (18) further supports a distinctive role of Ucn in immune phenomena quite distinct from that of CRH expressed in enterochromaffin cells of human colon (3) and in the colonic mucosa of patients with ulcerative colitis, where it has a strong proinflammatory role (7). It should be noted that Ucn has been detected in human colon lamina propria macrophages (8). Positive correlation between Ucn and inflammatory activity has been documented in several other inflammatory conditions, including rheumatoid arthritis and osteoarthritis (19). Of course, CRH apart from its direct paracrine effects on several inflammatory responses, also exerts an indirect immunosuppressive effect via its stimulation of pituitary ACTH and the production of cortisol. On the other hand, Ucn appears to exert direct immunosuppressive effects, for instance after thermal skin injury (20). In addition, Ucn directly suppress LPS-induced TNF production both in vivo and in vitro (21). It also inhibits autoimmune encephalomyelitis (22).
To elucidate the biological role of Ucn in the human gastric mucosa and to examine its possible involvement in local inflammatory processes, the content of ir-Ucn was measured by RIA in gastric biopsies obtained from patients with diagnosed gastritis due to H. pylori infection, a common cause of gastric inflammation. The ir-Ucn levels were significantly elevated in biopsies taken from patients with active gastritis (assessed by morphological criteria) compared with gastric biopsies of noninflamed tissues. This finding can be attributed to the accumulation of Ucn-positive inflammatory cells in the inflamed gastritis tissues. The presence of Ucn-expressing plasma cells demonstrated by immunohistochemistry in tissue sections from H. pylori gastritis patients further supports this hypothesis. However, when Ucn was measured in gastric biopsies from patients responding to H. pylori eradication treatment, i.e. pathology-confirmed regression of their inflammation and of the H. pylori titer, the levels of Ucn were further increased. Indeed, the elevation of Ucn was directly related to a successful eradication outcome and was completely absent in the group of patients who did not respond to H. pylori eradication treatment (nonresponders) and in the group of patients with significant remaining elements of inflammation and H. pylori infection. Correlation analysis between levels of ir-Ucn in biopsies from patients with gastritis and degree of inflammation was performed separately for the three pathology parameters, acute and chronic inflammation and H. pylori infection. Spearman’s rank correlation analysis clearly demonstrated a significant negative correlation between levels of ir-Ucn and grading of gastritis for each of the three parameters examined, suggesting that regression of gastritis resulted in a simultaneous increase in ir-Ucn content. As regression of gastritis resulted in a decrease in the number of inflammatory cells the simultaneous increase in Ucn suggests a simultaneous increase in Ucn by the parietal cells of gastric epithelium. Based on these data we conclude that Ucn production is directly related to defense mechanisms activated locally to protect gastric mucosa from noxious stimuli. This hypothesis is strongly supported by a number of recently published reports on Ucn actions. An active involvement of Ucn in the cytoprotective mechanisms of gastric mucosa is suggested by findings showing colocalization of its expression with tyrosine hydroxylase in the rat gastric mucosa (6). It is of note that dopamine inhibits gastric acid secretion (23, 24) and enhances cytoprotective bicarbonate and mucus production (25), hence protecting against ulcer formation. Furthermore, Ucn induces prostaglandin E2 production in a number of tissues, including human placenta (26) and rat brain (27). Of course, prostaglandin E2 plays a key role in the maintenance of gastrointestinal tissue integrity, repair of gastric mucosal injury, and ulcer healing through induction of both cytoprotective bicarbonates and mucus glycoproteins (28, 29). Ucn also exerts a cytoprotective effect in multiple tissues. Thus, exogenous Ucn is more potent than CRH in protecting cardiac myocytes from necrotic and apoptotic death induced by ischemia (30). It should be emphasized that in this tissue, CRH receptor antagonists drastically compromise the cytoprotective effect of ischemia-preconditioned medium (30). Furthermore, ischemia, causing necrotic and apoptotic death of neonatal rat cardiac myocytes, is associated with increased expression of Ucn mRNA and peptide production (31). Finally, Ucn exerts a potent protective effect on cultured rat hippocampal neurons with concentrations in the range of 0.5–5.0 pM, increasing the resistance of these cells to oxidative and excitotoxic stresses (32). Indeed, Ucn is 10-fold more potent than CRH in protecting hippocampal neurons from these stresses. Thus, Ucn appears to exert multiple cytoprotective effects against a whole array of noxious stimuli.
In conclusion, our data suggest that a paracrine circuit is present in human stomach, composed of Ucn and CRH-R2. The equivalent circuit in human colon, composed of CRH and CRH-R1, appears to be missing in human stomach. Our data further suggest that gastric Ucn is involved in local antiinflammatory processes in the stomach. This effect of Ucn is opposite that exerted by CRH in human colon, promoting its inflammatory response. These differences between upper and lower GI tracts, vis-à-vis their CRH paracrine circuits, may partially explain the lack of severe chronic inflammatory diseases in stomach compared with colon.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: CRH-R, CRH receptor; GI, gastrointestinal; ir-, immunoreactive; Ucn, urocortin.
Received June 3, 2002.
Accepted October 15, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. Chorny and M. Delgado Neuropeptides Rescue Mice from Lethal Sepsis by Down-regulating Secretion of the Late-Acting Inflammatory Mediator High Mobility Group Box 1 Am. J. Pathol., May 1, 2008; 172(5): 1297 - 1307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kimura, T. Amano, H. Uehara, H. Ariga, T. Ishida, A. Torii, H. Tajiri, K. Matsueda, and S. Yamato Urocortin I is present in the enteric nervous system and exerts an excitatory effect via cholinergic and serotonergic pathways in the rat colon Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G903 - G910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E Zoumakis, D K Grammatopoulos, and G P Chrousos Corticotropin-releasing hormone receptor antagonists Eur. J. Endocrinol., November 1, 2006; 155(suppl_1): S85 - S91. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Kokkotou, D. Torres, A. C. Moss, M. O'Brien, D. E. Grigoriadis, K. Karalis, and C. Pothoulakis Corticotropin-releasing hormone receptor 2-deficient mice have reduced intestinal inflammatory responses. J. Immunol., September 1, 2006; 177(5): 3355 - 3361. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E Gonzalez-Rey, A Fernandez-Martin, A Chorny, and M Delgado Therapeutic effect of urocortin and adrenomedullin in a murine model of Crohn's disease Gut, June 1, 2006; 55(6): 824 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gonzalez-Rey, A. Chorny, N. Varela, G. Robledo, and M. Delgado Urocortin and Adrenomedullin Prevent Lethal Endotoxemia by Down-Regulating the Inflammatory Response Am. J. Pathol., June 1, 2006; 168(6): 1921 - 1930. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tsatsanis, A. Androulidaki, T. Alissafi, I. Charalampopoulos, E. Dermitzaki, T. Roger, A. Gravanis, and A. N. Margioris 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., February 1, 2006; 176(3): 1869 - 1877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. la Fleur, E. C. Wick, P. S. Idumalla, E. F. Grady, and A. Bhargava From the Cover: Role of peripheral corticotropin-releasing factor and urocortin II in intestinal inflammation and motility in terminal ileum PNAS, May 24, 2005; 102(21): 7647 - 7652. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
