A Controlled Study of Colonic Immune Activity and β7 Blood T Lymphocytes in Patients With Irritable Bowel Syndrome

Background & Aims: The mechanisms behind irritable bowel syndrome (IBS) are incompletely understood. Recently several studies have suggested a low-grade colonic inflammation as initiator of the gut dysfunctions recorded in this patient group. The aim of this study was to characterize the phenotype and homing properties of colonic and peripheral blood lymphocytes in patients with IBS. Methods: Patients with IBS (n = 33), defined by the Rome II criteria, were compared with UC patients (n = 23) and control subjects (n = 15) without gastrointestinal symptoms. Colonic and peripheral blood lymphocytes were analyzed by flow cytometry. Secretion of IFN-γ from intestinal biopsies was determined by enzyme-linked immunosorbent assay, and immunohistochemical staining of colonic biopsies was performed. Results: IBS patients displayed an increased frequency of peripheral blood CD4+ and CD8+ T cells expressing the gut homing integrin β7. Accordingly, IBS and UC patients had an augmented frequency of lamina propria CD8+ T cells in the ascending colon as compared with control subjects. The frequency of intestinal T cells expressing integrin β7+ was unaltered in IBS and UC patients, although the expression of mucosal addressin cell adhesion molecule–1+ endothelium, the ligand for integrin β7, was increased in the ascending colon of IBS and UC patients as compared with control subjects. Conclusions: Patients with IBS exhibit an enhanced immune activity in the gut and an increased frequency of integrin β7+ T lymphocytes in the peripheral blood. Our data further support the hypothesis of IBS being at least partially an inflammatory disorder. Background & Aims: The mechanisms behind irritable bowel syndrome (IBS) are incompletely understood. Recently several studies have suggested a low-grade colonic inflammation as initiator of the gut dysfunctions recorded in this patient group. The aim of this study was to characterize the phenotype and homing properties of colonic and peripheral blood lymphocytes in patients with IBS. Methods: Patients with IBS (n = 33), defined by the Rome II criteria, were compared with UC patients (n = 23) and control subjects (n = 15) without gastrointestinal symptoms. Colonic and peripheral blood lymphocytes were analyzed by flow cytometry. Secretion of IFN-γ from intestinal biopsies was determined by enzyme-linked immunosorbent assay, and immunohistochemical staining of colonic biopsies was performed. Results: IBS patients displayed an increased frequency of peripheral blood CD4+ and CD8+ T cells expressing the gut homing integrin β7. Accordingly, IBS and UC patients had an augmented frequency of lamina propria CD8+ T cells in the ascending colon as compared with control subjects. The frequency of intestinal T cells expressing integrin β7+ was unaltered in IBS and UC patients, although the expression of mucosal addressin cell adhesion molecule–1+ endothelium, the ligand for integrin β7, was increased in the ascending colon of IBS and UC patients as compared with control subjects. Conclusions: Patients with IBS exhibit an enhanced immune activity in the gut and an increased frequency of integrin β7+ T lymphocytes in the peripheral blood. Our data further support the hypothesis of IBS being at least partially an inflammatory disorder. Irritable bowel syndrome (IBS) is a common bowel disorder in Western society1Agreus L. Svardsudd K. Nyren O. et al.Irritable bowel syndrome and dyspepsia in the general population overlap and lack of stability over time.Gastroenterology. 1995; 109: 671-680Abstract Full Text PDF PubMed Scopus (643) Google Scholar and is defined according to the Rome II criteria.2Thompson W.G. Longstreth G.F. Drossman D.A. et al.Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2041) Google Scholar Patients suffer from chronic abdominal pain, bloating, and altered bowel habits, despite the absence of identifiable organic disease explaining the sometimes severe symptoms. No ubiquitous etiologic mechanisms have so far been identified, although abnormal gastrointestinal motility,3Kellow J.E. Phillips S.F. Miller L.J. et al.Dysmotility of the small intestine in irritable bowel syndrome.Gut. 1988; 29: 1236-1243Crossref PubMed Scopus (222) Google Scholar, 4Simren M. Castedal M. Svedlund J. et al.Abnormal propagation pattern of duodenal pressure waves in the irritable bowel syndrome (IBS).Dig Dis Sci. 2000; 45 ([correction of (IBD)]): 2151-2161Crossref PubMed Scopus (79) Google Scholar visceral hypersensitivity,5Mertz H. Naliboff B. Munakata J. et al.Altered rectal perception is a biological marker of patients with irritable bowel syndrome.Gastroenterology. 1995; 109: 40-52Abstract Full Text PDF PubMed Scopus (921) Google Scholar, 6Slater B.J. Plusa S.M. Smith A.N. et al.Rectal hypersensitivity in the irritable bowel syndrome.Int J Colorectal Dis. 1997; 12: 29-32Crossref PubMed Scopus (36) Google Scholar altered colonic fermentation,7King T.S. Elia M. Hunter J.O. Abnormal colonic fermentation in irritable bowel syndrome.Lancet. 1998; 352: 1187-1189Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar abnormal gastrointestinal responses to stress,8Posserud I. Agerforz P. Ekman R. et al.Altered visceral perceptual and neuroendocrine response in patients with irritable bowel syndrome during mental stress.Gut. 2004; 53: 1102-1108Crossref PubMed Scopus (250) Google Scholar and nutrients9Simren M. Abrahamsson H. Bjornsson E.S. An exaggerated sensory component of the gastrocolonic response in patients with irritable bowel syndrome.Gut. 2001; 48: 20-27Crossref PubMed Scopus (182) Google Scholar are factors that might contribute to symptom generation. Recently, it has been proposed that a low-grade inflammatory process in various compartments of the small and the large bowel is associated with gut dysfunction in at least a subpopulation of IBS patients.10Tornblom H. Lindberg G. Nyberg B. et al.Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome.Gastroenterology. 2002; 123: 1972-1979Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 11Chadwick V.S. Chen W. Shu D. et al.Activation of the mucosal immune system in irritable bowel syndrome.Gastroenterology. 2002; 122: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar Different factors such as genetic susceptibility, altered gut flora, unrecognized food reactions, and infectious enteritis have all been suggested as possible mediators of an inflammatory response. IBS patients have been found to be genetically predisposed to produce low amounts of interleukin-1012Gonsalkorale W.M. Perrey C. Pravica V. et al.Interleukin 10 genotypes in irritable bowel syndrome evidence for an inflammatory component?.Gut. 2003; 52: 91-93Crossref PubMed Scopus (233) Google Scholar and have increased levels of the proinflammatory cytokine interleukin-1β mRNA after infectious gastroenteritis when compared with non-IBS gastroenteritis patients.13Gwee K.A. Collins S.M. Read N.W. et al.Increased rectal mucosal expression of interleukin 1beta in recently acquired post-infectious irritable bowel syndrome.Gut. 2003; 52: 523-526Crossref PubMed Scopus (374) Google Scholar Studies have also shown that patients with postinfectious IBS have an activated intestinal immune response, as defined by increased numbers of T cells and macrophages in the colonic mucosa.14Spiller R.C. Jenkins D. Thornley J.P. et al.Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome.Gut. 2000; 47: 804-811Crossref PubMed Scopus (1021) Google Scholar, 15Dunlop S.P. Jenkins D. Neal K.R. et al.Relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS.Gastroenterology. 2003; 125: 1651-1659Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar Moreover, one of the most frequently recognized immunologic features of IBS is increased numbers of mast cells in colonic lamina propria (LP) and muscularis propria.16Barbara G. Stanghellini V. De Giorgio R. et al.Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome.Gastroenterology. 2004; 126: 693-702Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar, 17Hiatt R.B. Katz L. Mast cells in inflammatory conditions of the gastrointestinal tract.Am J Gastroenterol. 1962; 37: 541-545PubMed Google Scholar, 18O'Sullivan M. Clayton N. Breslin N.P. et al.Increased mast cells in the irritable bowel syndrome.Neurogastroenterol Motil. 2000; 12: 449-457Crossref PubMed Scopus (437) Google Scholar, 19Salzmann J.L. Peltier-Koch F. Bloch F. et al.Morphometric study of colonic biopsies a new method of estimating inflammatory diseases.Lab Invest. 1989; 60: 847-851PubMed Google Scholar Indeed, high numbers of activated mast cells have been found in close proximity to nerves in the colonic mucosa, with a numeric correlation to abdominal pain perception.16Barbara G. Stanghellini V. De Giorgio R. et al.Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome.Gastroenterology. 2004; 126: 693-702Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar Food reactions might also contribute to the immune activation, because some patients with IBS symptoms benefit from specific elimination diets.20Atkinson W. Sheldon T.A. Shaath N. et al.Food elimination based on IgG antibodies in irritable bowel syndrome a randomised controlled trial.Gut. 2004; 53: 1459-1464Crossref PubMed Scopus (448) Google Scholar During intestinal inflammatory conditions, increased numbers of lymphocytes are recruited to the intestinal mucosa, partly by binding of selectins and integrins on the cell surface to addressins expressed on endothelial tissues. Homing of circulating blood lymphocytes to the gut mucosa is largely mediated by the expression of the α4β7 integrin that binds to its ligand, mucosal addressin cell adhesion molecule–1 (MAdCAM-1), which is constitutively expressed by intestinal LP venules and Peyer's patch high endothelial venules.21Briskin M. Winsor-Hines D. Shyjan A. et al.Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue.Am J Pathol. 1997; 151: 97-110PubMed Google Scholar, 22Berlin C. Berg E.L. Briskin M.J. et al.Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.Cell. 1993; 74: 185-195Abstract Full Text PDF PubMed Scopus (1297) Google Scholar UC patients have been reported to display an altered frequency of integrin β7+ T cells in blood and rectal mucosa,23Meenan J. Spaans J. Grool T.A. et al.Altered expression of alpha 4 beta 7, a gut homing integrin, by circulating and mucosal T cells in colonic mucosal inflammation.Gut. 1997; 40: 241-246PubMed Google Scholar but the gut homing pattern of lymphocytes in IBS has not previously been studied. Overall, a low inflammatory response in the intestinal mucosa is acknowledged in IBS, although the underlying mechanisms and various cell populations driving this immunologic response need to be further clarified. Therefore, the aim of this study was to determine and compare phenotype and homing properties of lymphocytes isolated from ascending and sigmoid colonic mucosa and peripheral blood in patients with IBS, UC patients, and control subjects. This study was approved by the Human Research Ethical Committee of the Medical Faculty, Göteborg University, and was performed after receiving written informed consent from all subjects. Study subjects were recruited among patients referred for colonoscopy at the Sahlgren's University Hospital, Göteborg, Sweden. Individuals with known celiac disease or food allergy were excluded from the study. Venous blood samples and 8 biopsies from the ascending (5 cm distal to the ileocecal valve) and the sigmoid colon, respectively, were assessed in each patient. IBS patients were defined according to the Rome II criteria2Thompson W.G. Longstreth G.F. Drossman D.A. et al.Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2041) Google Scholar and underwent routine diagnostic colonoscopy to exclude organic bowel disease. Biopsies were defined as noninflammatory by routine histology. Specially, no evidence for collagenous or lymphocytic colitis was detected on the basis of standard criteria.24Bogomoletz W.V. Collagenous, microscopic and lymphocytic colitis an evolving concept.Virchows Arch. 1994; 424: 573-579Crossref PubMed Google Scholar Thirty-three IBS patients were included (19 women; mean age, 42 ± 12 years; body mass index, 25.7 ± 5.2 kg/m2). Twenty patients had diarrhea-predominant IBS, 4 had constipation-predominant IBS, and 9 had alternating bowel habits.2Thompson W.G. Longstreth G.F. Drossman D.A. et al.Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2041) Google Scholar One of the IBS patients had less than 1-year duration of symptoms, 16 of the patients had a symptom duration of 1–5 years, and 16 of the patients included reported IBS symptoms for more than 5 years. Acute onset after supposed infectious diarrhea was described by 4 diarrhea-predominant IBS patients and 1 constipation-dominant IBS patient. Twenty-three UC patients, all with a well-defined disease (10 women; mean age, 42 ± 11 years; body mass index, 24.7 ± 3.2 kg/m2), underwent colonoscopy for evaluation of disease activity and dysplasia surveillance. Nine of the UC patients had an active inflammation, of which 5 had total colitis, and 4 had left-sided colitis. Four of the UC patients had less than 1-year disease duration, 4 of the patients had a duration time of 1–5 years, whereas 15 of the patients reported disease symptoms for more than 5 years. Patients with active UC were included as positive inflammatory controls, and UC patients in remission were also studied because these patients often report IBS-like symptoms.25Simren M. Axelsson J. Gillberg R. et al.Quality of life in inflammatory bowel disease in remission the impact of IBS-like symptoms and associated psychological factors.Am J Gastroenterol. 2002; 97: 389-396PubMed Google Scholar As control subjects, we included 15 individuals (7 women; mean age, 53 ± 8 years; body mass index, 23.4 ± 2.6 kg/m2) who underwent colonoscopy for investigation of anemia, rectal bleeding, or polyp surveillance. These subjects were free from gastrointestinal symptoms, and the colonoscopy was normal, both macroscopically and microscopically. The control subjects were significantly older than patients with IBS (P = .008) and UC (P = .008), but age did not differ significantly between the patient groups. LP lymphocytes were isolated as previously described in detail,26Lundgren A. Stromberg E. Sjoling A. et al.Mucosal FOXP3-expressing CD4+ CD25 high regulatory T cells in Helicobacter pylori-infected patients.Infect Immun. 2005; 73: 523-531Crossref PubMed Scopus (230) Google Scholar and peripheral blood lymphocytes (PBLs) were isolated from venous blood by density-gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Freshly isolated cells, 1 × 105 cells/sample, were stained for flow cytometry analysis of various surface markers by using combinations of the following antibodies: anti-CD4-PerCP, anti-CD8-APC, anti-CD45RA-FITC, anti-Integrinβ7-PE (BD Pharmingen, San Diego, CA). All cells were fixed in cellfix (BD Pharmingen) before fluorescence-activated cell sorter analysis, which was performed by using an LSR II (BD Pharmingen). At least 10,000 live lymphocytes per sample were analyzed, as defined by forward and side scatter. The data were analyzed by using Flow Jo software (Treestar Inc, Ashland, OR). Samples from 19 IBS patients, 13 UC patients, and 8 control subjects were used for fluorescence-activated cell sorter analysis. Endothelial cells were detected by staining against factor VIII (DakoCytomation; Glostrup, Denmark), and the endothelial expression of the addressin MAdCAM-121Briskin M. Winsor-Hines D. Shyjan A. et al.Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue.Am J Pathol. 1997; 151: 97-110PubMed Google Scholar was detected by using a specific monoclonal antibody (Bender Medsystems, Vienna, Austria). Serial tissue sections were stained with either factor VIII or MAdCAM-1. After blocking of endogenous peroxidase activity with .3% H2O2 in PBS, cryo-cut sections from biopsies were incubated with monoclonal antibodies against factor VIII or MAdCAM-1 in PBS with 5% human serum for 3 hours at room temperature. The reaction was developed by stepwise addition of biotinylated rat-anti-mouse immunoglobulin G antibody (BD Pharmingen) and an avidin–biotin–horseradish peroxidase complex (DakoCytomation) according to the manufacturer's instructions. Tissue sections were developed with chromogen 3-amino-9-ethylcarbazole containing H2O2(AEC substrate chromogen) (DakoCytomation) and counterstained with Mayer's hematoxylin (Histolab, Göteborg, Sweden). Slides were dehydrated with ethanol and xylen and mounted with Faramount Mounting Medium (DakoCytomation). An isotype antibody was included in each experiment as a negative control and did not give positive staining. Tissue sections were examined by light microscopy, and the specifically stained area in each section was determined by using Leica Qwin software (Leica, Wetzlar, Germany). The data were expressed as the frequency of MAdCAM-1 positive endothelial tissue (VIII+). Biopsies from 11 IBS patients, 8 UC patients, and 8 control subjects were used for MAdCAM-1/factor VIII analysis. The biopsies were washed in PBS and incubated in 24-well plates for 24 hours with or without the addition of 5 μg/mL phytohemagglutinin (Sigma, St Louis, MO) at 37°C, 5% CO2, in 400 μL of Iscove's medium (Seromed, Berlin, Germany) containing 10% heat inactivated fetal calf serum, 5 × 10−5 mol/L 2-mercaptoethanol, 1 mmol/L L-glutamine, and 50 μg/mL gentamicin. The production of interferon-γ (IFN-γ) per mg tissue was determined by PharMingen OptEIA Set cytokine kits (BD Biosciences, San Jose, CA), according to the manufacturer's recommendations. In the few cases in which the spontaneous production of IFN-γ exceeded the sensitivity limit of the assay, it was subtracted from the IFN-γ levels obtained by stimulation with phytohemagglutinin. Biopsies from 11 IBS patients, 6 UC patients, and 4 control subjects were used for cytokine analysis. Differences between the groups were assessed with the Kruskal–Wallis test, and in case of statistically significant differences, comparisons were performed groupwise with the Mann–Whitney U test. A P value <.05 was regarded as significant. The frequency of peripheral blood T lymphocytes expressing the gut homing marker integrin β7 was measured by flow cytometry. IBS patients had a significantly increased frequency of CD4+ and CD8+ Τ cells expressing integrin β7 in peripheral blood as compared with control subjects (Figure 1A). No significant difference in the frequency of integrin β7 expressing T cells was found between patients with IBS and UC. Neither UC patients in remission nor those with active disease showed any significantly altered frequency of integrin β7+ blood T lymphocytes as compared with control subjects (Figure 1A). Thus, IBS patients, but not UC patients, have a significantly increased frequency of circulating T cells expressing the gut homing marker integrin β7 as compared with control subjects. The endothelial expression of MAdCAM-1, the ligand for integrin β7, was examined in biopsies from the ascending and the sigmoid colon. The frequency of MAdCAM-1+ endothelium was significantly increased in the ascending colon of IBS patients and UC patients in exacerbation (UCa) and remission (UCr) as compared with the control subjects (Figure 1B). An augmented MAdCAM-1 expression could also be recorded in the sigmoid colon of IBS and UCr patients, although the increase was not statistically significant as compared with control subjects. Because the expression of MAdCAM-1 is up-regulated by the proinflammatory cytokine IFN-γ, the level of secreted IFN-γ in phytohemagglutinin-stimulated biopsy cultures was examined. IBS and UC patient biopsy cultures from the ascending colon tended to secrete more IFN-γ, although IFN-γ levels were not statistically increased in any patient group compared with control subjects (Figure 1C). Thus, an increased expression of MAdCAM-1 was recorded in IBS patients. The frequencies of the different lymphoid cell populations isolated from the ascending and the sigmoid colonic mucosa were investigated by flow cytometry. There was no statistically significant variation in the number of cells isolated from the diverse locations between the different subject groups. IBS patients displayed a significantly increased frequency of CD8+ T cells in the ascending colon LP (Table 1), as compared with control subjects. Furthermore, IBS patients tended to have an increased frequency of CD4+ LP T cells in the ascending and the sigmoid colon, similar to the frequency recorded in UC, although the frequencies were not significantly increased in either IBS or UC as compared with control subjects (Table 1). In addition, a subpopulation of more than 40% of the IBS patients displayed a frequency of ≥38% CD4+ LP T cells, which was the highest ratio of CD4+ LP T cells detected in any individual control subject (not shown). UCa and UCr patients showed an increased frequency of CD8+ LP T cells in the ascending colon. None of the mentioned cell populations in IBS or UC patients displayed any alterations in peripheral blood as compared with control subjects (Table 1). Thus, the lymphocyte distribution in the ascending colonic LP of IBS resembles the pattern recorded in UC, with an increased frequency of CD8+ T cells.Table 1The Frequencies of CD4+ and CD8+ T Cells Among Lymphocytes in the Ascending Colon LP (LPL A) and the Sigmoid Colon LP (LPL S) and PBLs as Determined by Flow CytometryCD4+ cells (%)CD8+ cells (%)MeanSDP vs CTRLMeanSDP vs CTRLLPL A IBS35.610.3NS16.95.9.01 UCr34.98.7NS20.45.1.01 UCa33.44.9NS16.46.9.05 CTRL27.68.710.64.4LPL S IBS38.615.8NS12.85.9NS UCr41.89.0NS20.83.6NS UCa38.411.9NS13.66.2NS CTRL29.79.810.96.1PBL IBS43.98.1NS27.16.3NS UCr41.55.2NS34.17.6NS UCa42.210.0NS29.111.1NS CTRL40.010.026.65.1NOTE. The results are shown as mean ± SD of 19 IBS patients, 6 UCr (in remission), 6 UCa (active disease) patients, and 8 controls. (NS > .05).SD, standard deviation; CTRL, control. Open table in a new tab NOTE. The results are shown as mean ± SD of 19 IBS patients, 6 UCr (in remission), 6 UCa (active disease) patients, and 8 controls. (NS > .05). SD, standard deviation; CTRL, control. When investigating the integrin β7 expression on LP T cells, we recorded that IBS patients had similar frequencies of integrin β7+ CD4+ T cells and integrin β7+ CD8+ T cells to control subjects (Figure 2). Also UCr and UCa patients had comparable frequencies of integrin β7+ T cells in the ascending and sigmoid colon LP to control subjects. Thus, the increase of blood T cells expressing the gut homing marker in IBS patients is not reflected by an altered integrin β7+ T cells frequency in the colonic mucosa. The majority (70%–99%) of all T cells isolated from the intestinal mucosa of all subject groups had a memory phenotype (CD45RA−) (not shown). The distribution of colonic naïve (CD45RA+) cells in IBS patients was comparable to control subjects in the LP T cell populations of the ascending and the sigmoid colon (Figure 3). However, a subgroup of IBS patients had an augmented frequency of naïve CD4+ and CD8+ T cells in the ascending and the sigmoid mucosa (Figure 3). The frequency of naïve LP CD4+ T cells in the ascending colon was significantly increased in UCa patients compared with control subjects (Figure 3). Also the sigmoid colonic LP compartment of UCa patients contained increased frequency of naïve CD4+ and CD8+ T cells, compared with control subjects (Figure 3). Thus, the frequency of naïve T cells in IBS patients as a group does not differ from control subjects, but a subgroup of IBS patients has an increased frequency of naïve T cells as compared with control subjects. We have shown that IBS patients have significantly increased levels of integrin β7+ T cells in peripheral blood. The integrin β7 is expressed on T cells activated in gut-associated lymph nodes27Farstad I.N. Norstein J. Brandtzaeg P. Phenotypes of B and T cells in human intestinal and mesenteric lymph.Gastroenterology. 1997; 112: 163-173Abstract Full Text PDF PubMed Scopus (56) Google Scholar and mediates homing to the gut mucosa by binding to its ligand, MAdCAM-1, expressed by endothelial cells in the gastrointestinal tract.28Hamann A. Andrew D.P. Jablonski-Westrich D. et al.Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo.J Immunol. 1994; 152: 3282-3293PubMed Google Scholar Thus, IBS patients have an increased frequency of circulating T cells destined to home to the gut. These cells have presumably encountered their specific antigen in a gut-associated lymph node and are theoretically homing to the intestinal mucosa to perform effector functions. This finding indicates that IBS patients have an augmented cell-mediated intestinal immune response to unidentified antigen/s. Our study shows that IBS patients as well as UC patients, in either remission or exacerbation, have augmented levels of MAdCAM-1+ endothelium in the ascending colon as compared with control subjects, implicating local immune activation and increased recruitment of β7+ T cells from the blood to the tissue. Accordingly, we found an increased frequency of CD8+T cells in all patient groups as compared with control subjects, supporting the notion of an increased recruitment of lymphocytes to the intestinal mucosa. However, neither IBS nor UC patients displayed increased frequencies of integrin β7+ cells at mucosal sites as compared with control subjects. Our UC data are in agreement with those of Meenan et al,23Meenan J. Spaans J. Grool T.A. et al.Altered expression of alpha 4 beta 7, a gut homing integrin, by circulating and mucosal T cells in colonic mucosal inflammation.Gut. 1997; 40: 241-246PubMed Google Scholar who reported decreased frequencies of β7+ cells in the colonic mucosa in UC patients with an active inflammation. Thus, in spite of an augmented migration of T cells into the colonic LP of IBS and UC patients as shown in our study, no increased frequency of mucosal lymphocytes expressing β7+ was recorded. This might result from a down-regulation of β7 after the cells have entered the mucosa, when the integrin-addressin interaction has fulfilled its purpose, or it might be due to a non–integrin-specific recruitment of cells to the mucosa. The latter hypothesis might possibly explain the finding of increased frequencies of naïve mucosal T cells in UCa patients and subgroup of IBS patients. However, it cannot be excluded that the blood β7+ cells also home to non-colonic regions or other compartments of the gut-associated lymphoid tissue expressing MAdCAM-1. The myenteric plexus of the small intestine might be suggested as an alternative destination for gut homing cells, which is supported by the recent report of low grade cell infiltrate in this compartment in IBS patients with severe symptoms.10Tornblom H. Lindberg G. Nyberg B. et al.Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome.Gastroenterology. 2002; 123: 1972-1979Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar The patient groups were significantly younger than the control group, and the age difference concurred with the alterations of integrin β7 and MAdCAM-1 expression recorded between the control group and patient groups. However, recent studies report unaltered or up-regulated frequencies of integrin and adhesion molecules29De Martinis M. Modesti M. Loreto M.F. et al.Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly.Life Sci. 2000; 68: 139-151Crossref PubMed Scopus (24) Google Scholar and unaltered expression of MAdCAM-130Ogino T. Miura S. Komoto S. et al.Senescence-associated decline of lymphocyte migration in gut-associated lymphoid tissues of rat small intestine.Mech Ageing Dev. 2004; 125: 191-199Crossref PubMed Scopus (11) Google Scholar in elderly individuals, as compared with adolescents. Thus, the increase of β7 and MAdCAM-1 in the patient groups as compared with the control subjects in our study is probably not due to the age difference of the groups. The possibility of IBS being an inflammatory-mediated disease has been suggested in recently published studies in which increased numbers of CD3+ T cells in the epithelial, LP, and muscularis plexus lymphoid compartments of the small and/or large intestine, as analyzed by immunohistochemistry, have been reported.10Tornblom H. Lindberg G. Nyberg B. et al.Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome.Gastroenterology. 2002; 123: 1972-1979Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 11Chadwick V.S. Chen W. Shu D. et al.Activation of the mucosal immune system in irritable bowel syndrome.Gastroenterology. 2002; 122: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar, 14Spiller R.C. Jenkins D. Thornley J.P. et al.Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome.Gut. 2000; 47: 804-811Crossref PubMed Scopus (1021) Google Scholar, 31Barbara G. De Giorgio R. Stanghellini V. et al.A role for inflammation in irritable bowel syndrome?.Gut. 2002; 51: i41-i44Crossref PubMed Scopus (226) Google Scholar Because the majority of previously published studies focused on alterations in the large bowel, we wanted to further identify different colonic cell populations and their immunologic status. In contrast to others,11Chadwick V.S. Chen W. Shu D. et al.Activation of the mucosal immune system in irritable bowel syndrome.Gastroenterology. 2002; 122: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar we recorded an increased frequency of CD8+ T cells in IBS as compared with control subjects in the LP of the ascending colon. This discrepancy might be due to the use of different methods and the fact that the increase in total numbers of CD8+ cells is moderate and might not be detectable when assessed by immunohistochemistry. Moreover, Spiller et al14Spiller R.C. Jenkins D. Thornley J.P. et al.Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irritable bowel syndrome.Gut. 2000; 47: 804-811Crossref PubMed Scopus (1021) Google Scholar reported increased numbers of rectal CD8+ T cells in postinfectious IBS patients, supporting our data. Additional investigations are needed to further identify various immune cell populations and their properties in other intestinal compartments as the small bowel and the myenteric plexus in patients with IBS. In conclusion, we report immunologic similarities in the ascending colon of patients with IBS and UC. IBS patients, in resemblance to UC patients, have an increased frequency of CD8+ T cells and increased MAdCAM-1 expression in the ascending colon. A subgroup of the IBS patients as well as UCa patients display an augmented frequency of naïve T cells in the ascending colonic mucosa. Moreover, an increased frequency of blood T cells expressing the gut homing integrin β7+ was recorded in IBS patients. Further studies in larger samples of IBS patients are needed to detect subgroup differences, as well as the relationship with the symptom profile of the patients.