Mast Cell-Dependent Excitation of Visceral-Nociceptive Sensory Neurons in Irritable Bowel Syndrome

Background & Aims: Intestinal mast cell infiltration may participate to abdominal pain in irritable bowel syndrome (IBS) patients. However, the underlying mechanisms remain unknown. We assessed the effect of mast cell mediators released from the colonic mucosa of IBS patients on the activation of rat sensory neurons in vitro. Methods: Colonic mast cell infiltration and mediator release were assessed with quantitative immunoflorescence and immunoenzymatic assays. The effect of mucosal mediators was tested on mesenteric sensory nerve firing and Ca2+ mobilization in dorsal root ganglia in rats. Results: Mediators from IBS patients, but not controls, markedly enhanced the firing of mesenteric nerves (14.7 ± 3.2 imp/sec vs 2.8 ± 1.5 imp/sec; P < .05) and stimulated mobilization of Ca2+ in dorsal root ganglia neurons (29% ± 4% vs 11% ± 4%; P < .05). On average, 64% of dorsal root ganglia responsive to mediators were capsaicin-sensitive, known to mediate nociception. Histamine and tryptase were mainly localized to mucosal mast cells. IBS-dependent nerve firing and Ca2+ mobilization were correlated with the area of the colonic lamina propria occupied by mast cells (r = 0.74; P < .01, and r = 0.78; P < .01, respectively). IBS-dependent excitation of dorsal root ganglia was inhibited by histamine H1 receptor blockade and serine protease inactivation (inhibition of 51.7%; P < .05 and 74.5%; P < .05; respectively). Conclusions: Mucosal mast cell mediators from IBS patients excite rat nociceptive visceral sensory nerves. These results provide new insights into the mechanism underlying visceral hypersensitivity in IBS. Background & Aims: Intestinal mast cell infiltration may participate to abdominal pain in irritable bowel syndrome (IBS) patients. However, the underlying mechanisms remain unknown. We assessed the effect of mast cell mediators released from the colonic mucosa of IBS patients on the activation of rat sensory neurons in vitro. Methods: Colonic mast cell infiltration and mediator release were assessed with quantitative immunoflorescence and immunoenzymatic assays. The effect of mucosal mediators was tested on mesenteric sensory nerve firing and Ca2+ mobilization in dorsal root ganglia in rats. Results: Mediators from IBS patients, but not controls, markedly enhanced the firing of mesenteric nerves (14.7 ± 3.2 imp/sec vs 2.8 ± 1.5 imp/sec; P < .05) and stimulated mobilization of Ca2+ in dorsal root ganglia neurons (29% ± 4% vs 11% ± 4%; P < .05). On average, 64% of dorsal root ganglia responsive to mediators were capsaicin-sensitive, known to mediate nociception. Histamine and tryptase were mainly localized to mucosal mast cells. IBS-dependent nerve firing and Ca2+ mobilization were correlated with the area of the colonic lamina propria occupied by mast cells (r = 0.74; P < .01, and r = 0.78; P < .01, respectively). IBS-dependent excitation of dorsal root ganglia was inhibited by histamine H1 receptor blockade and serine protease inactivation (inhibition of 51.7%; P < .05 and 74.5%; P < .05; respectively). Conclusions: Mucosal mast cell mediators from IBS patients excite rat nociceptive visceral sensory nerves. These results provide new insights into the mechanism underlying visceral hypersensitivity in IBS. Irritable bowel syndrome (IBS) is a chronic functional bowel disorder characterized by abdominal pain and disturbed bowel habits that are not accompanied by underlying structural or biochemical changes detectable with diagnostic techniques.1Thompson W.G. Longstreth G.F. Drossman D.A. Heaton K.W. Irvine E.J. Muller-Lissner S.A. Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2022) Google Scholar IBS is common in western societies including the United Kingdom and the United States, where prevalence approaches 20%,2Jones R. Lydeard S. Irritable bowel syndrome in the general population.BMJ. 1992; 304: 87-90Crossref PubMed Scopus (554) Google Scholar, 3Talley N.J. Zinsmeister A.R. Van Dyke C. Melton III, L.J. Epidemiology of colonic symptoms and the irritable bowel syndrome.Gastroenterology. 1991; 101: 927-934PubMed Google Scholar and IBS accounts for ∼10% of family physician visits and 30% of gastroenterology practice.4Harvey R.F. Salih S.Y. Read A.E. Organic and functional disorders in 2000 gastroenterology outpatients.Lancet. 1983; 1: 632-634Abstract PubMed Scopus (310) Google Scholar, 5Drossman D.A. Camilleri M. Mayer E.A. Whitehead W.E. AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar Thus, IBS is an economic burden to society for direct (eg, diagnosis, therapy) and indirect (eg, work absenteeism) costs.5Drossman D.A. Camilleri M. Mayer E.A. Whitehead W.E. AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar The pathophysiology of IBS is poorly defined, which explains the limited efficacy of current treatments. Abdominal pain, an essential symptom of IBS,1Thompson W.G. Longstreth G.F. Drossman D.A. Heaton K.W. Irvine E.J. Muller-Lissner S.A. Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2022) Google Scholar correlates with the severity of the disease6Sandler R.S. Drossman D.A. Nathan H.P. McKee D.C. Symptom complaints and health care seeking behavior in subjects with bowel dysfunction.Gastroenterology. 1984; 87: 314-318PubMed Scopus (282) Google Scholar and contributes to the patient’s poor quality of life.7Spiegel B.M. Gralnek I.M. Bolus R. Chang L. Dulai G.S. Mayer E.A. Naliboff B. Clinical determinants of health-related quality of life in patients with irritable bowel syndrome.Arch Intern Med. 2004; 164: 1773-1780Crossref PubMed Scopus (154) Google Scholar Both central (ie, central nervous system) and peripheral mechanisms (ie, gastrointestinal tract) are thought to contribute to the origin of abdominal pain in IBS.5Drossman D.A. Camilleri M. Mayer E.A. Whitehead W.E. AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar Peripheral mechanisms of abdominal pain may involve an increased sensitivity of the intestinal wall to local stimuli (ie, visceral hypersensitivity),8Delvaux M. Role of visceral sensitivity in the pathophysiology of irritable bowel syndrome.Gut. 2002; 51: i67-i71Crossref PubMed Scopus (125) Google Scholar which results in increased activation of splanchnic afferent nerves9Lembo T. Munakata J. Mertz H. Niazi N. Kodner A. Nikas V. Mayer E.A. Evidence for the hypersensitivity of lumbar splanchnic afferents in irritable bowel syndrome.Gastroenterology. 1994; 107: 1686-1696Abstract PubMed Google Scholar and erroneous activation of nociceptive brain regions.10Silverman D.H. Munakata J.A. Ennes H. Mandelkern M.A. Hoh C.K. Mayer E.A. Regional cerebral activity in normal and pathological perception of visceral pain.Gastroenterology. 1997; 112: 64-72Abstract Full Text PDF PubMed Scopus (497) Google Scholar However, the causes of visceral hypersensitivity are unknown. Mediators from intestinal mast cells may play a crucial role in visceral hypersensitivity of IBS patients.11Barbara G. De Giorgio R. Stanghellini V. Cremon C. Corinaldesi R. A role for inflammation in irritable bowel syndrome?.Gut. 2002; 51: i41-i44Crossref PubMed Scopus (225) Google Scholar, 12Barbara G. Stanghellini V. De Giorgio R. Corinaldesi R. Functional gastrointestinal disorders and mast cells: implications for therapy.Neurogastroenterol Motil. 2006; 18: 6-17Crossref PubMed Scopus (153) Google Scholar Mast cells are sentinels of the immune system that are strategically located at the host–environment interface13Metcalfe D.D. Baram D. Mekori Y.A. Mast cells.Physiol Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1789) Google Scholar in close proximity to sensory nerves.14Stead R.H. Dixon M.F. Bramwell N.H. Riddell R.H. Bienenstock J. Mast cells are closely apposed to nerves in the human gastrointestinal mucosa.Gastroenterology. 1989; 97: 575-585Abstract PubMed Scopus (375) Google Scholar Multiple factors (eg, bacteria, parasites, viruses, toxins, complements, cytokines, endogenous peptides) can activate mast cells to promote degranulation and immediate (minutes) release of preformed mediators from cytoplasmic granules (eg, histamine, tryptase, proteoglycans) or the de novo synthesis of mediators [eg, leukotriene C4, platelet activating factor, prostaglandin (PG)D2].15Galli S.J. Maurer M. Lantz C.S. Mast cells as sentinels of innate immunity.Curr Opin Immunol. 1999; 11: 53-59Crossref PubMed Scopus (332) Google Scholar, 16Stevens R.L. Austen K.F. Recent advances in the cellular and molecular biology of mast cells.Immunol Today. 1989; 10: 381-386Abstract Full Text PDF PubMed Scopus (27) Google Scholar These mediators can activate sensory nerves, including those innervating the gastrointestinal tract, leading to visceral hyperalgesia/allodynia.11Barbara G. De Giorgio R. Stanghellini V. Cremon C. Corinaldesi R. A role for inflammation in irritable bowel syndrome?.Gut. 2002; 51: i41-i44Crossref PubMed Scopus (225) Google Scholar, 12Barbara G. Stanghellini V. De Giorgio R. Corinaldesi R. Functional gastrointestinal disorders and mast cells: implications for therapy.Neurogastroenterol Motil. 2006; 18: 6-17Crossref PubMed Scopus (153) Google Scholar, 17Bueno L. Fioramonti J. Delvaux M. Frexinos J. Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations.Gastroenterology. 1997; 112: 1714-1743Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 18Vergnolle N. Ferazzini M. D’Andrea M.R. Buddenkotte J. Steinhoff M. Proteinase-activated receptors: novel signals for peripheral nerves.Trends Neurosci. 2003; 26: 496-500Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar We have recently demonstrated a marked increase in colonic mucosal area occupied by mast cells in IBS patients compared to healthy subjects. Mast cells in IBS patients were frequently degranulated, and there was increased spontaneous release of histamine and tryptase. The proximity of activated mast cells to mucosal nerve fibers correlated with the frequency and severity of abdominal pain.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar Nonetheless, mechanisms by which mast cells could induce visceral hypersensitivity in IBS patients are completely unknown. We investigated the mechanisms by which mast cells from IBS patients may activate visceral sensory nerves. To do so, we obtained mediators spontaneously released from the colonic mucosa of patients with IBS and healthy controls and tested their effects on mesenteric sensory nerve fibers supplying the gut and Ca2+ mobilization in isolated dorsal root ganglion cells (DRG) in rats in vitro. Twenty-nine consecutive patients with IBS (aged 19–70 years; mean 39.0 ± 10.4 years; 18 females) as well as 15 controls (aged 22–30 years; mean 25.7 ± 3.8 years; 9 females) participated in the study. IBS patients were all seen in the Department of Internal Medicine and Gastroenterology of the University of Bologna and diagnosed according to the Rome II criteria.1Thompson W.G. Longstreth G.F. Drossman D.A. Heaton K.W. Irvine E.J. Muller-Lissner S.A. Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2022) Google Scholar Controls were recruited by public advertisement and included in the study after thorough exclusion of gastrointestinal symptoms. Exclusion criteria included also the use of nonsteroidal anti-inflammatory drugs, corticosteroids, and mast cell stabilizers, major abdominal surgery, celiac disease (excluded by detection of antitransglutaminase and antiendomysial antibodies), allergic diseases, including asthma (family and personal history and specific anti-IgE antibodies) and other organic or severe psychiatric disorders as assessed by history taking and appropriate consultations and laboratory tests. Patients and controls gave written informed consent and the study protocol was approved by the local Ethic Committee and conducted in accordance with the Declaration of Helsinki. Each patient completed an Italian19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar modified version of the Bowel Disease Questionnaire to evaluate symptoms.20Talley N.J. Phillips S.F. Melton III, J. Wiltgen C. Zinsmeister A.R. A patient questionnaire to identify bowel disease.Ann Intern Med. 1989; 111: 671-674Crossref PubMed Scopus (428) Google Scholar Patients were asked to score frequency and severity of their symptoms over the last 2 weeks before interview, as previously described.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar All participants underwent left colonoscopy after cleansing of the distal colon with a 500-mL water enema performed the evening before and the morning of the procedure. In all cases we obtained 6 mucosal biopsies from the proximal descending colon. Two biopsies were used for routine H&E histology and immunohistochemistry. Four biopsies were used for histamine, tryptase, and PGE2 assays and for mucosal mediators release experiments (see below). Biopsies were fixed in buffered 10% formalin and processed for either H&E histology or immunofluorescence. For the latter, paraffin-embedded specimens were cut and processed for immunofluorescence as previously described.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar Mast cells were identified using mouse monoclonal antibodies directed against tryptase (1:2000 dilution; Dakopatts, Glostrup, Denmark). Histamine was detected using a polyclonal antibody (1:100 dilution; Acris Antibodies, Herford, Germany), and nerves were visualized using a rabbit polyclonal NSE (1:500 dilution; Dakopatts) antibody. All histologic sections were evaluated by an expert pathologist who was unaware of the diagnosis, for exclusion of overt mucosal inflammation or microscopic colitis. Quantification of inflammatory cells was performed using a previously validated method.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar The quantification of mast cells in close vicinity (<10 μm) of nerve fibers was carried out on sections immunolabeled for tryptase (mast cell marker) and neuronal specific enolase (NSE; general neuronal marker) adapting a previously published method.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar Briefly, microscopic fields were digitized and randomly sampled with the aid of a grid (0.5 mm2) located below the slide. A stereologic grid containing cross-shaped points was overlaid on the digitized sampled fields by the computer software and used to determine the area occupied by tryptase+ mast cells within 10 μm of NSE+ nerve fibers over that occupied by lamina propria (ie, number of points hitting the cells divided by the total number of cross-shaped points in the lamina propria). For the double-labeling technique, additional specificity studies were performed by incubating the primary antibodies (used at the concentration previously reported) with the supernatants at 4°C overnight before incubation on tissue slides. In addition, further appropriate controls for the double-labeling technique were performed to determine that the supernatants did not cross-react when mixed together with secondary antibodies and that the secondary antibodies recognized the appropriate antigen–antibody complexes. For electron microscopy studies, biopsies were processed as described in detail elsewhere.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar Spontaneous release of mucosal mediators from colonic biopsies was obtained using a previously described method.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar Briefly, upon removal, biopsies were rapidly immersed in hard plastic tubes containing 1 mL of Hank’s solution (Sigma, St. Louis, MO), continuously oxygenated (95% O2/5% CO2) at 37°C. After a 25-minute incubation, the bathing solution was removed. All samples were centrifuged at 200g for 10 minutes, and 150 μL of supernatant aliquoted and stored at –70°C until the assay. At the end of the release experiment, biopsies were blotted and weighed. Histamine and tryptase were measured using previously described methods.19Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. 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 (1163) Google Scholar PGE2 was measured from duplicate aliquots (50 μL) of the cleared supernatants using a competitive enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). This assay is based on the competition between PGE2 and a PGE2-acetylcholinesterase conjugate for a limited amount of PGE2 monoclonal antibody. Histamine and PGE2 were measured using a microtiter plate reader (absorbance 405 nm; Tecan’s Sunrise, Phoenix Research Products Candler, NC) and normalized to the weight of the biopsies. Male Sprague-Dawley rats (250–350 g) were used in all experiments. Rats were fed regular laboratory chow with free access to water and housed under controlled conditions with a 12:12-hour light/dark cycle. All procedures were approved by the institutional Animal Care Committees. The mesenteric afferent nerve recording of isolated rat terminal jejunum was performed in vitro as described previously.21Wang B. Glatzle J. Mueller M.H. Kreis M. Enck P. Grundy D. Lipopolysaccharide-induced changes in mesenteric afferent sensitivity of rat jejunum in vitro: role of prostaglandins.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G254-G260Crossref PubMed Scopus (19) Google ScholarActivity was continuously monitored in real time as spike discharge frequency (impulse/sec) and both the raw signal and spike discharge frequency were spooled to a computer running Spike-2 software (Cambridge Electronic Design Limited, Cambridge, UK). Experiments were conducted on preparations in which baseline afferent discharge was maintained for at least 10 minutes. In control experiments (n = 5), the arterial perfusion was interrupted for 2 minutes and replaced with Krebs solution, biopsy supernatants from controls, and then the sample from IBS patients, all infused at a rate of 150 μL/min (300 μL vol) in a sequential manner separated by an interval of 15 minutes. In 1 further experiment only Krebs solution and the IBS sample were administered. The reproducibility of nerve responses to the IBS samples was tested in ancillary experiments by the application of the same IBS sample twice on the same gut preparations (n = 3). The second application of same IBS sample was infused at least 10 minutes after the nerve response to the first application recovered to the baseline. Three groups of treatment were performed (n = 5 for each) by adding drug in the perfusion Krebs solution after the increased nerve discharge evoked by IBS sample had returned to the baseline and 10 minutes before the second application of the same IBS sample. The effects of the serotonin (5-HT) type 3 receptor antagonist granisetron (1 μmol/L), and the histamine H1 receptor antagonist pyrilamine (10 μmol/L; Sigma) were tested in individual experiments. Rat DRG (T1-L6) were removed and placed in cold Dulbecco’s modified eagle medium solution, containing (in mg/mL): 0.5 trypsin, 1 collagenase type IA, and 0.1 DNAse type IV (all from Sigma).22Rigoni M. Trevisani M. Gazzieri D. Nadaletto R. Tognetto M. Creminon C. Davis J.B. Campi B. Amadesi S. Geppetti P. Harrison S. Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin.Br J Pharmacol. 2003; 138: 977-985Crossref PubMed Scopus (96) Google Scholar Enrichment of the fraction of nociceptive neurons was obtained following the methods reported previously.23Gilabert R. McNaughton P. Enrichment of the fraction of nociceptive neurones in cultures of primary sensory neurones.J Neurosci Methods. 1997; 71: 191-198Crossref PubMed Scopus (26) Google Scholar Briefly, ganglia were rinsed, dissociated, and centrifuged (200g for 5 minutes). Cells were than plated on poly-[scap]l[r]-lysine (8.3 μmol/L, Sigma) and laminin (5 μmol/L, Sigma) coated 25-mm glass coverslips and kept for 5 to 8 days at 37°C. After 2 days, plated neurons were loaded with Fura-2-AM-ester (3 μmol/L, Sigma) for 40 minutes at 37°C and transferred to a chamber on the stage of Nikon eclipse TE300 microscope. The dye was excited at 340 nmol/L and 380 nmol/L to indicate relative [Ca2+]i changes by the F340/F380 ratio recorded with a dynamic image analysis system (Laboratory Automation 2.0, RCS, Florence, Italy). Biopsy supernatants (200 μL) obtained from controls and IBS patients were added to the experimental chamber to obtain a final volume of 500 μL (2.5 dilution coefficient). Some experiments were performed in the presence of the histamine H1 receptor antagonist, pyrilamine (0.1 μmol/L, Sigma), and the nonselective EP1 and EP2 receptor antagonist AH 6809 (5 μmol/L, Cayman Chemical, Ann Arbor, MI) injected 10 minutes prior to the stimuli.21Wang B. Glatzle J. Mueller M.H. Kreis M. Enck P. Grundy D. Lipopolysaccharide-induced changes in mesenteric afferent sensitivity of rat jejunum in vitro: role of prostaglandins.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G254-G260Crossref PubMed Scopus (19) Google Scholar The serine protease inhibitor FUT-175 (50 μg/mL; Calbiochem, Darmstadt, Germany) was added to the supernatants 10 minutes before their application to the DRG neurons. [Ca2+]i was expressed as percentage of the maximum increase detected in the presence of the calcium ionophore ionomycin (5 μmol/L, Sigma). The stock concentration of capsaicin (10 mmol/L) was prepared in 100% ethanol. Fura-2-AM-ester, and ionomycin were dissolved in 100% DMSO. All other drugs were dissolved in Krebs buffer solution and were administered in volumes not exceeding 1% vol/vol of the bath volume. Data are reported as mean values ± standard error of the mean (SEM). Biopsies from all the enrolled IBS patients (n = 29) and controls (n = 15) were processed for immunohistochemistry and quantitative experiments relative to mast cells and nerve–mast cell interactions. Due to limited availability of tissue, different n values apply to the other experiments carried out in the present study. For Ca2+ imaging studies: IBS patients, n = 19; controls, n = 15. For electrophysiologic measurement of sensory nerves: IBS patients n = 16; controls, n = 5. All data were analyzed by means of the Mann–Whitney U and the Yates’ corrected chi-squared tests, with the following exceptions. Data relative to [Ca2+]i that were analyzed by means of the Student t test or analysis of variance (ANOVA) and the Dunnett’s test when required. Mean nerve discharge rate was calculated off-line using Spike 2 from the instantaneous discharge rates that had been saved to disk. This was then normalized by subtracting the mean discharge for a 5-minute stationary baseline period from the mean discharge rates taken at 50 second intervals up to 450 seconds after the onset of intra-arterial injection. The nerve discharge data were analyses by 2-way ANOVA followed by Bonferroni post-tests. The effects of antagonists on the peak nerve discharge were analyzed by 1-way ANOVA and post hoc Tukey’s multiple comparison test. Correlations were analyzed using the Spearman rank correlation test. Analyses were done by running the SPSS/PC+ statistical package (SPSS, Chicago, IL) on a personal computer. Two-tailed P values < .05 were considered statistically significant. Twenty-nine patients referred to the Department of Internal Medicine and Gastroenterology (tertiary referral center) for gastrointestinal complaints were diagnosed with IBS according to the Rome II criteria1Thompson W.G. Longstreth G.F. Drossman D.A. Heaton K.W. Irvine E.J. Muller-Lissner S.A. Functional bowel disorders and functional abdominal pain.Gut. 1999; 45: II43-II47Crossref PubMed Scopus (2022) Google Scholar and were included in the study. Patient’s clinical characteristics are reported in Table 1. Fifteen healthy subjects served as controls.Table 1Characteristics of the Study PatientsTotal number of patients29Age (y)39.0 ± 10.4Sex (% females)62.1Abdominal pain scores Severity (1–4)2.4 ± 0.2 Frequency (1–4)2.3 ± 0.2Bowel habit Constipation (%)45.5 Diarrhea (%)54.5Bloating (%)90.9 Open table in a new tab To quantify mast cells and investigate their spatial relationships with nerve fibers in the colonic lamina propria, we immunostained tissues for tryptase and NSE. Tryptase+ mast cells were scattered throughout the mucosal lamina propria of controls and IBS patients (Figure 1A and D). The mean area of mucosa occupied by tryptase+ mast cells was 152% greater in IBS patients compared with controls (7.8% ± 0.6% vs 3.0% ± 0.3%, respectively; P < .001) (Figure 1D and K). An increased mucosal area occupied by tryptase+ mast cells (ie, greater than mean + 2 SEM of controls: 3.6%) was present in the vast majority (72.3%) of the IBS patients studied. In both controls and IBS patients, nerve fibers were identified in the lamina propria surrounding mucosal crypts (Figure 1B and E). The association of tryptase+ mast cells with nerve fibers was assessed by simultaneously localizing tryptase and NSE. The mean area of mucosa occupied by tryptase+ mast cells within 10 μm of nerve fibers in IBS patients was significantly increased by 188% compared with controls (4.9% ± 0.3% vs 1.7% ± 0.3%, respectively; P < .01) (Figure 1C, F, and L). Mast cell activation and mast cell–nerve interactions were assessed by electron microscopy (Figure 1G–J). In controls, granule-filled mast cells were scattered throughout the mucosa (Figure 1G). Features of active degranulation, including clearing of single granules and membraneous labyrinthic arrays were particularly frequent in the mucosa of IBS patients (Figure 1H–J). Membrane to membrane contacts between mast cells and nerve fibers were occasionally observed, and activated mast cells with degranulation polarized toward the nerves were often found in the close proximity (0–10 μm) of nerve trunks in IBS specimens (Figure 1H and I). Thus, in most IBS patients there is an increased colonic mucosa area occupied by mast cells, and a closer proximity of mast cells to nerve fibers. These results provide a structural basis for enhanced mast cell–nerve interactions in the colonic mucosa of IBS patients. We assessed the tissue source and the spontaneous release of mediators from the colonic mucosa of IBS patients and controls: the mast cell-specific mediator histamine and tryptase as well as the nonspecific mediator PGE2, which derives from mast cells and other cell types. Histamine, tryptase, and PGE2 can excite visceral sensory nerves. The tissue weight of biopsies obtained from IBS patients used for these experiments (as well as for the assessment of biopsy supernatants on the electrophysiology of mesenteric sensory nerves and DRG neurons, see below) was not different