The association of mast cells and serotonin in children with chronic abdominal pain of unknown etiology
- Tara J Taylor†1,
- Nader N Youssef†2,
- Ravi Shankar†1,
- David E Kleiner†3 and
- Wendy A Henderson†1Email author
© Henderson et al; licensee BioMed Central Ltd. 2010
Received: 6 October 2010
Accepted: 21 October 2010
Published: 21 October 2010
Abdominal pain of unknown origin affects up to 20% of school-aged children. Evaluation of children is symptom-based without clear guidelines to investigate molecular mechanisms of abdominal pain. Aberrant molecular mechanisms may increase intestinal permeability leading to interactions between the immune and nervous systems, subclinical inflammation, and visceral pain. This study evaluated the association between interleukin-6 (IL-6), mast cell infiltrates, and serotonin (5-HT) levels in gastrointestinal (GI) biopsies, with perceived abdominal pain in a pediatric cohort.
Clinical data and biopsy samples from pediatric patients (n = 48) with chronic abdominal pain, with and without inflammation were included. Formalin-fixed paraffin-embedded GI biopsies were sectioned and immunohistochemistry performed for IL-6 and 5-HT; mast cells were identified with toluidine blue stain. Histological findings were compared to self-reported abdominal pain between groups.
There was significantly greater IL-6 immunoreactivity in biopsies with confirmed histologic inflammation (p = 0.004). There was a greater number of mast cells per HPF in non-inflammatory biopsies (3.5 ± 2.9) compared to the inflammatory biopsies (2.6 ± 1.8) p = 0.049. The non-inflammatory biopsy group was significantly less likely to respond to standard treatment as evidenced by higher pain reports (p = .018). Mast cells (p = .022) and 5-HT (p = .02) were significantly related to abdominal pain scores.
A potential association between self-reported abdominal pain, number of mast cells, and 5-HT levels, which may contribute to perceived GI pain in pediatric patients may exist.
Unspecified chronic abdominal pain in children that has no identified biologic marker or known organic cause and has occurred for greater than two months is defined by Rome III criteria as chronic abdominal pain . These criteria assume that no metabolic or structural causes can be related to the continued symptoms of chronic abdominal pain for an eight week period over the previous year. Current Rome III criteria divide pediatric abdominal pain diagnosis into several subsets which include functional abdominal pain syndrome and irritable bowel syndrome (IBS) . Approximately 15-20% of children  and adults living in the United States suffer from chronic abdominal pain [4, 5]. Children with chronic abdominal pain experience lessened quality of life in comparison to their healthy peers which leads to numerous absences from school for medical care [6, 7]. Chronic abdominal pain in pediatric patients is a significant burden on the health system which warrants further investigation on the pathogenesis of disease to identify novel targets for intervention.
Recognition of the role of inflammation and its interaction with the neuro-immune system of the gastrointestinal tract is an emerging area of interest in patients with chronic abdominal pain . The mechanisms of chronic abdominal pain of unknown origin may be related to interactions between the immune and nervous system in the gut thereby leading to visceral hypersensitivity of the intestinal mucosa . Mast cells have been shown to interact with colonic nerve endings by the secretion of tryptase, histamine, and possibly serotonin (5-hydroxytryptamine, 5-HT) in humans . There is evidence of closer proximity of mast cells to nerve fibers in the colon of IBS patients leading to augmented visceral sensitivity . A possible relationship between the number of mucosal mast cells and rectal sensitivity has also been demonstrated in humans . There is evidence of a significant increase in mast cell numbers in patients with IBS . Along with increased mast cell counts there is support that mast cell numbers directly correlate with abdominal pain in IBS patients . Recently, a study conducted by Mahjoub, et al. found increased mast cell density in pediatric patients with recurrent abdominal pain . They propose that mast cell density measurements be incorporated with routine GI biopsies due to a significant correlation between increase GI complaints and mast cell count. Inflammation and increased permeability of GI tract mucosa may also induce pain in pediatric patients with functional abdominal pain .
Chronic abdominal pain of unknown origin may be due to low grade inflammatory changes in the GI tract [16, 17]. Low level inflammation may result in increased permeability across the mucosal barrier; which in turn permits the entrance of antigens into the intestinal wall . Other potential mechanisms include altered function at the level of neurotransmitter receptors of 5-HT, increased visceral hypersensitivity, and impaired colonic mucosal permeability . Alleviation of symptoms with medication that target 5-HT receptors suggest that 5-HT is involved in gut motility regulation .
Increased permeability and its role in pediatric abdominal pain has recently been the focus of work by Shulman (2008). They explored the relationship of subclinical inflammation and its relationship to chronic pain of the GI tract. In a well-designed prospective controlled study investigating the difference in GI permeability and fecal calprotectin (marker for intestinal inflammation) concentration in children with abdominal pain versus control . Proximal GI permeability, colonic permeability, and fecal calprotectin were found to be significantly greater in the abdominal pain group compared to the control group. Fecal calprotectin concentrations correlated with pain interference with activities. However, there was no correlation between GI permeability and pain-related symptoms. This study highlighted that more research is needed to examine the interactions at the molecular level between mast cells and 5-HT.
These converging lines of evidence suggest that mast cells and 5-HT contribute to increased colonic permeability and thereby lead to chronic abdominal pain. We assessed this hypothesis at the molecular level by exploring the relationship between mast cell and 5-HT levels and perceived abdominal pain in a pediatric cohort with and without the diagnosis of an inflammatory GI disorder.
Demographic and clinical indicators of sample.
Overall (n= 48)
All subjects were pain positive
( n = 26)
( n = 22)
Age (M ± SD)
11.9 ± 2.9
11.9 ± 2.4
12 ± 2.9
BMI (M ± SD)
18.9 ± 4.3
19.5 ± 5.2
18.2 ± 4.3
Clinical data collection
All abdominal pain reports (categorized as no pain, mild, moderate, or severe) were recorded before and after 6 months of endoscopic examination and medical treatment as a part of routine clinical care. All patients reported abdominal pain for more than 6 months prior to endoscopy. Pre-existing data were extracted and re-coded into a secure database without personal identifiers. Clinical data included sex, race, age, body mass index (BMI), and lactase deficiency. Data on race were collected by electronic medical record review and categorized as Caucasian, Asian, African American, Hispanic, and mixed race. BMI was calculated from the initial outpatient visit information with the formula age (years) by weight (kilograms)/height (meters) squared. Medical record data of confirmed lactase deficiency was collected and categorized as positive or negative based on a duodenal biopsy disaccharidase assay (Women's and Children's Hospital of Buffalo, Buffalo, NY).
Biopsies were stained with hematoxylin and eosin, toluidine blue and immunohistochemical markers at the National Cancer Institute, Science Applications International Corporation (Frederick, MD). Microscopic histologic review of stains was performed by either a pathologist or trained technicians who were blinded to clinical information. Biopsies were categorized as either inflamed (with evidence of chronic mucosal changes, villous/mucosal atrophy, ulceration, cryptitis, crypt abscesses, or crypt destruction) or not inflamed.
Mast cells identification was performed after the biopsies were sectioned (5-6 microns), de-paraffinized and rinsed with 60% ethanol. Slides were then stained in toluidine blue for 2 minutes, rinsed, and dehydrated in acetone (2 times for 2 minutes). Slides were cleared in xylene and mounted. Mast cell number was recorded as the number of cells per 40× high power field (HPF) then averaged 10 fields containing the maximum number of mast cells.
For 5-HT immunohistochemistry, colonic GI tract biopsies were sectioned (7 microns), de-paraffinized, and incubated with 2% normal horse serum (Vector Laboratories Inc. Burlingame, CA) for 20 minutes followed by incubation with anti-5-HT antibody diluted 1:40 (Vision Biosystems, Newcastle, UK) for 30 minutes at room temperature. Slides were rinsed and biotinylated horse anti-mouse IgG antibody (Vector Laboratories Inc., Burlingame, CA) was applied for 30 minute incubation. Immunoreactivity of 5-HT was identified by positive stained entrochromaffin (EC) cells. The slides were scored as: minimal (1-2 EC), mild (3-5 EC), moderate (6-10 EC), or marked (more than 16 EC) by a pathologist.
For IL-6 immunohistochemistry, biopsies were sectioned (7 microns), de-paraffinized and blocked with 2% normal goat serum (Vector Laboratories Inc. Burlingame, CA) for 20 minutes followed by incubation with anti-IL-6 antibody diluted 1:400 (Vision Biosystems, Newcastle, UK) at room temperature for 30 minutes. Slides were then incubated for 30 minutes with biotinylated goat anit-rabbit IgG (Vector Laboratories Inc., Burlingame, CA). The level of IL-6 immunoreactivity was scored as: minimal, mild, moderate, or marked by a pathologist.
Data were collected, coded and doubly entered into SPSS version 15.0. Statistical analysis included: means, frequencies, standard deviation, independent t test and Chi square analysis.
This study was approved by the NIH Office of Human Subject Research (protocol #3906) and the Institutional Review Board of Goryeb Children's Hospital- Atlantic Health (IRB #R07-09-009).
The overall sample included pediatric patients (n = 48), 54% female, with a mean age of 11.9 ± 2.9 yrs (range 5-17 yrs). After histological review, 22 pediatric GI biopsies were categorized as inflammatory and 26 pediatric GI biopsies as non-inflammatory which coincided with the original clinical diagnosis of disease (inflammatory bowel disease, gastritis, IBS, or functional abdominal pain). There was no significant difference between the phenotype (non-inflammatory and inflammatory GI mucosa) with regard to sex, race, age, BMI or lactase deficiency (Table 1).
At baseline self-reported pain scores of patients with non-inflamed biopsies were 35% mild, 45% moderate, and 20% severe, compared to patients with inflamed biopsies 23.5% mild, 53% moderate, and 23.5% severe (Figure 1A). Post-treatment self-reported pain scores of patients with non-inflamed biopsies were 47% no pain, 32% mild pain, 21% moderate pain; patients with inflamed biopsies 95% reported no pain, 5% reported mild pain (Figure 1B). The difference between pre and post treatment self-reported pain scores for patients with non-inflamed biopsies were 12.5% reported no change in pain, 62.5% reported a mild change in pain, and 25% reported moderate change in pain. The difference in pre and post treatment self-reported pain scores of patients with inflamed biopsies were 23.5% mild change in pain, 58.8% reported moderate change in pain, 17.6% reported in a complete change in pain (Figure 1C).
In this study, we illustrate for the first time in the pediatric population with chronic abdominal pain of unknown origin that increased self-reported abdominal pain is correlated with higher levels of mast cells and 5-HT. All patients, non-inflammatory and inflammatory, presented with abdominal pain prior to endoscopic evaluation. Patients with inflammation responded better to standard care as evidenced by lower self-reported abdominal pain scores post endoscopic evaluation. Patients with decreased pain after standard treatment had lower mast cell and 5-HT levels. The non-inflammatory cohort who were unresponsive to standard medical care presented with higher pain, had increased mast cells, and 5-HT levels compared to the inflammatory cohort. This early work may begin to explain some of the elusive etiology related to chronic abdominal pain of unknown origin in children.
Current proposed mechanisms of IBS give additional information which may be related to the mechanism behind chronic abdominal pain of unknown origin. For example, in post infectious IBS there may be a translocation of microbiota due to increased mucosa permeability allowing for the interaction between inflammatory mediators and the enteric nervous system thereby stimulating smooth muscle motility . Patients with chronic abdominal pain may be affected by a similar mechanism at the molecular level of the GI mucosa. Increased permeability across the mucosal barrier may occur, in turn leading to hypersensitivity and augmented pain.
There are some limitations to this study including the retrospective nature of data collection using medical record review. Additionally, the limited sample size makes the findings not necessarily generalizable to the greater population of patients with chronic abdominal pain of unknown origin. Furthermore, because of the retrospective nature only associations are presented here and care should be taken in the interpretation of these findings as they do not imply direct causality. A future study could include both direct objective and subjective measures of abdominal pain as well as real-time measures of 5-HT, IL-6, and mast cell activation.
Recent advances on the role of 5-HT in the enteric nervous systems and its relationship to intestinal visceral hyperalgesia have contributed significantly to the understanding of abdominal pain and IBS in adults. This increased knowledge has helped shift the paradigm that these disorders are exclusively behavioral in nature and that pathophysiologic disturbances at the cellular level exist. To date, the consideration of inflammation and intestinal pain has been traditionally reserved for conditions such as Crohn's disease and ulcerative colitis. The preliminary findings presented in this manuscript indicate that similar relationships may exist in the pediatric population with chronic abdominal pain of unknown origin. Future research is needed in order to uncover the associated causes of abdominal pain without inflammatory pathology in pediatric patients. Novel medical treatments can arise by unveiling the role of mast cells and 5-HT in the pathophysiology of chronic abdominal pain of unknown origin in children.
irritable bowel syndrome.
The authors would like to acknowledge support from the National Institute of Nursing Research (NINR) Intramural Research Program (PI: W. Henderson, 1 ZIA NR000018-01 SML, 2009). Additionally, support from SAIC-Fredrick contractor to National Cancer Institute (NCI Contract No. HHSN261200800001E). The options expressed herein are those of the authors and do not represent the position of the NIH or the US Government. The authors would like to thank Dr. Miriam Anver, Ms. Donna Butcher, and Dr. Mones Abu-Asab for immunohistochemical support. The authors would also like to thank Dr. Jessica Gill for manuscript review and Ms. Annette Langseder for clinical support.
- Drossman D, Corazziari E, Delvaux M, Spiller RC, Talley NJ, Thompson WG, Whitehead WE: Rome III; The Functional Gastrointestinal Disorders. 2006, Degnon Associates, Inc., ThirdGoogle Scholar
- Hahn B, Saunders W, Maier W: Differences between individuals with self-reported irritable bowel syndrome (IBS) and IBS-like symptoms. Dig Dis Sci. 1997, 42 (12): 2585-2590. 10.1023/A:1018889318063.PubMedView ArticleGoogle Scholar
- Petersen S, Brulin C, Bergström E: Recurrent pain symptoms in young schoolchildren are often multiple. Pain. 2006, 121 (1-2): 145-150. 10.1016/j.pain.2005.12.017.PubMedView ArticleGoogle Scholar
- Creed F, Ratcliffe J, Fernandez L, Tomenson B, Palmer S, Rigby C, Guthrie E, Read N, Thompson D: Health-Related Quality of Life and Health Care Costs in Severe, Refractory Irritable Bowel Syndrome. Ann Intern Med. 2001, 134 (9_Part_2): 860-868.PubMedView ArticleGoogle Scholar
- Russo MW, Gaynes BN, Drossman DA: A National Survey of Practice Patterns of Gastroenterologists With Comparison to the Past Two Decades. Journal of Clinical Gastroenterology. 1999, 29 (4): 339-343. 10.1097/00004836-199912000-00009.PubMedView ArticleGoogle Scholar
- Youssef NN, Murphy TG, Langseder AL, Rosh JR: Quality of life for children with functional abdominal pain: a comparison study of patients' and parents' perceptions. Pediatrics. 2006, 117 (1): 54-59. 10.1542/peds.2005-0114.PubMedView ArticleGoogle Scholar
- Whitehead W, Burnett C, Cook E: Impact of IBS on quality of life. Dig Dis Sci. 1996, 41: 2248-2253. 10.1007/BF02071408.PubMedView ArticleGoogle Scholar
- De Giorgio R, Barbara G: Is irritable bowel syndrome an inflammatory disorder?. Curr Gastroenterol Rep. 2008, 10 (4): 385-390. 10.1007/s11894-008-0073-0.PubMedView ArticleGoogle Scholar
- Barbara G, Stanghellini V, De Giorgio R, Cremon C, Cottrell GS, Santini D: Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology. 2004, 126 (3): 693-702. 10.1053/j.gastro.2003.11.055.PubMedView ArticleGoogle Scholar
- Barbara G, Wang B, Stanghellini V, de Giorgio R, Cremon C, Di Nardo G: Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome. Gastroenterology. 2007, 132 (1): 26-37. 10.1053/j.gastro.2006.11.039.PubMedView ArticleGoogle Scholar
- Park JH, Rhee PL, Kim HS, Lee JH, Kim YH, Kim JJ: Mucosal mast cell counts correlate with visceral hypersensitivity in patients with diarrhea predominant irritable bowel syndrome. J Gastroenterol Hepatol. 2006, 21 (1 Pt 1): 71-78. 10.1111/j.1440-1746.2005.04143.x.PubMedView ArticleGoogle Scholar
- Piche T, Saint-Paul MC, Dainese R, Marine-Barjoan E, Iannelli A, Montoya ML, Peyron JF, Czerucka D, Cherikh F, Filippi J: Mast cells and cellularity of the colonic mucosa correlated with fatigue and depression in irritable bowel syndrome. Gut. 2008, 57 (4): 468-473. 10.1136/gut.2007.127068.PubMedView ArticleGoogle Scholar
- Akbar A, Yiangou Y, Facer P, Walters JR, Anand P, Ghosh S: Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain. Gut. 2008, 57 (7): 923-929. 10.1136/gut.2007.138982.PubMed CentralPubMedView ArticleGoogle Scholar
- Mahjoub FE, Farahmand F, Pourpak Z, Asefi H, Amini Z: Mast cell gastritis: children complaining of chronic abdominal pain with histologically normal gastric mucosal biopsies except for increase in mast cells, proposing a new entity. Diagn Pathol. 2009, 4: 34-10.1186/1746-1596-4-34.PubMed CentralPubMedView ArticleGoogle Scholar
- Shulman RJ, Eakin MN, Czyzewski DI, Jarrett M, Ou CN: Increased gastrointestinal permeability and gut inflammation in children with functional abdominal pain and irritable bowel syndrome. J Pediatr. 2008, 153 (5): 646-650. 10.1016/j.jpeds.2008.04.062.PubMed CentralPubMedView ArticleGoogle Scholar
- Barbara G: Mucosal barrier defects in irritable bowel syndrome. Who left the door open?. Am J Gastroenterol. 2006, 101 (6): 1295-1298. 10.1111/j.1572-0241.2006.00667.x.PubMedView ArticleGoogle Scholar
- Spiller R, Garsed K: Postinfectious irritable bowel syndrome. Gastroenterology. 2009, 136 (6): 1979-1988. 10.1053/j.gastro.2009.02.074.PubMedView ArticleGoogle Scholar
- Crowell MD: Role of serotonin in the pathophysiology of the irritable bowel syndrome. Br J Pharmacol. 2004, 141 (8): 1285-1293. 10.1038/sj.bjp.0705762.PubMed CentralPubMedView ArticleGoogle Scholar
- Costedio MM, Hyman N, Mawe GM: Serotonin and its role in colonic function and in gastrointestinal disorders. Dis Colon Rectum. 2007, 50 (3): 376-388. 10.1007/s10350-006-0763-3.PubMedView ArticleGoogle Scholar
- Barbara G, Cremon C, Pallotti F, De Giorgio R, Stanghellini V, Corinaldesi R: Postinfectious irritable bowel syndrome. J Pediatr Gastroenterol Nutr. 2009, 48 (Suppl 2): S95-97. 10.1097/MPG.0b013e3181a15e2e.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.