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Keywords:

  • functional bowel disorders;
  • irritable bowel syndrome;
  • mast cell;
  • tryptase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Mast cells (MC) release potent mediators which alter enteric nerve and smooth muscle function and may play a role in the pathogenesis of the irritable bowel syndrome (IBS). The aim of this study was to determine if MC were increased in the colon of IBS patients compared to controls. Biopsy specimens were obtained from the caecum, ascending colon, descending colon and rectum of 28 patients: 14 IBS (Rome criteria); seven normal; and seven inflammatory controls. Tissue was stained immunohistochemically using a monoclonal mouse antibody for human mast cell tryptase (AA1). Tissue area occupied by tryptase-positive MC (volume density of mast cells) was quantified by image analysis. The number of plasma cells, lymphocytes, eosinophils, neutrophils and macrophages were each graded semiquantitatively (0–4) in haematoxylin and eosin stained sections. Mast cell volume density was significantly (P < 0.05) higher in IBS (0.91 ± 0.18; CI 0.79; 1.0) than normal controls (0.55 ± 0.14; CI 0.40; 0.69) in the caecum but not at other sites. Apart from MC, there was no evidence of increased cellular infiltrate in the IBS group. MC were significantly increased in the caecum of IBS patients compared to controls. The multiple effects of the intestinal mast cell alone, or as a participant of a persistent inflammatory response, may be fundamental to the pathogenesis of IBS.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Poor understanding of the pathophysiology of the irritable bowel syndrome (IBS) has made the development of specific and effective treatment strategies notoriously difficult. Some of the most innovative and promising advances in our understanding of the underlying mechanisms, in at least a subset of IBS, come from work on gastrointestinal inflammation.1

After an acute inflammatory event such as gastroenteritis, 30% of individuals develop persistent IBS symptoms2, 3 that may be attributable to a persistent inflammatory response.4 Since overt intestinal inflammation precludes a diagnosis of IBS, the focus must be on markers of a low-grade5 or previous inflammatory event. Even mild inflammation6 or that at a remote site7, 8 can cause persistent changes in enteric nerve and muscle function.9 In IBS, numerous studies show physiological evidence of altered muscle and nerve function.10[11][12][13][14][15][16][17]–18

Mast cells release potent inflammatory and immune-modulating mediators that alter nerve19[20]–21 and muscle22, 23 function and are therefore promising candidates for involvement in IBS. Although traditionally seen in the context of the type I hypersensitivity response, mast cell degranulation and mediator release is also associated with factors such as stress,24 nerve damage,25, 26 infection27 and a variety of inflammatory events.28 The close proximity of the MC to the enteric nerves29[30][31]–32 offers important potential for inducing changes in nerve function and the development of heightened visceral sensitivity. Furthermore, substance P, for example, can increase the excitability of mast cells without causing degranulation. In IBS, the mast cell may well be an important mediator in a complex interaction between a physiological events (gut insult, inflammation, tissue injury, nerve injury) and psychological factors (stressful events, emotions).

Mast cells in IBS have not been fully explored. In 1962, Hiatt and Katz33 reported an increase in mast cell numbers in the colonic muscularis mucosa of four patients with ‘spastic colitis’. A key study by Weston et al.34 identified an increased number of MC at the terminal ileum of IBS patients. Increases in MC, however, have not been demonstrated in colonic mucosa in IBS.35, 36 Methodological differences including sample size, biopsy site and staining technique make the interpretation of these data difficult, nevertheless, when combined with a large body of theoretical and experimental data, they form a considerable basis for further investigation.

The aim of this study was to determine if (1) mast cells were increased in the colonic mucosa in IBS patients compared to controls; and (2) if lymphocytes, plasma cells, eosinophils, neutrophils or macrophages were increased in IBS.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Patient population

Twenty-eight consecutive adult patients (Table 1) undergoing colonoscopy for clinical reasons were recruited through the endoscopy unit at the Adelaide & Meath Hospital. All patients gave informed consent.

Table 1.   Demographic and clinical data Thumbnail image of

They were placed in three groups:

1 IBS; 14 patients with clinically confirmed IBS compatible with the Rome criteria, macroscopically and histologically normal colonic mucosa and no evidence of organic bowel disease. Eight patients were classed as diarrhoea-predominant, two constipation-predominant and four alternating diarrhoea/constipation.

2 Normal controls; seven patients were included with macroscopic and histologically normal colonic mucosa and no persistent bowel symptoms and no organic or functional bowel disease. Five of the seven were under investigation because of anaemia and two because of a family history of colonic cancer.

3 Inflammatory controls; a total of seven, consisting of four patients with a clinical diagnosis of ulcerative colitis (UC) and three patients with evidence of microscopic changes at one colonic site each. These patients had no fixed gastrointestinal diagnosis on independent clinical evaluation at time of study and were therefore classed as ‘nonspecific inflammation’ (NSI). Of the three patients, two were under investigation because of a family history of colon cancer and one due to haem-occult positive stools.

For all groups, patients with the following criteria were excluded: (i) history of atopy, food allergy or asthma based on detailed medical history; (ii) use of mast cell stabilisers or steroids in the month before the study (except in UC where previous steroid use was not an exclusion criterion); (iii) active diverticulitis (incidental haemorrhoids or diverticula were not considered exclusion criteria); and (iv) patients presenting with GI infection.

Demographic and clinical details

Age, gender, onset of symptoms, date of diagnosis, relevant medical history and current medications were documented. Physical examination, detailed medical history, routine haematological and biochemical profiles were performed on all patients. All diagnoses were made independently by a physician unaware of the study data, based on clinical assessment and histological reports. The bowel disease questionnaire (BDQ) score37 and a checklist of the Rome criteria were recorded for all IBS patients and normal control subjects.

Colonoscopy and biopsies

Colonoscopy was performed (Olympus, Germany) to the caecum and two pinch biopsies were taken at each of four anatomical sites; caecum, ascending colon, descending colon and rectum (yielding a total of 224 biopsy specimens). Tissue was fixed in neutral buffered formaldehyde and embedded in paraffin within a maximum of 72 h. The macroscopic appearance of the mucosa was documented at endoscopy.

Histological evaluation

Haematoxylin and eosin (H & E) stained sections were graded as normal or as having mild, moderate or marked inflammation by an independent pathologist, who had no knowledge of the study data. In further H & E stained sections, plasma cells, lymphocytes, eosinophils, neutrophils and macrophages were graded semiquantitatively on a scale of 0–4 by a single observer blinded to patient diagnosis. The inflammatory score was used as an estimate of cell numbers. Quantification was based on three nonoverlapping fields of lamina propria (excluding edge areas and glandular tissue) at ×40 magnification. This semiquantitative grading system was derived from cell counts performed on colonic tissue in a parallel IBS study (Tongue et al., unpublished). Each grade was based on the number of cells per field as follows: plasma cells: ≤1, 2–50, 51–100, 101–200, >201 cells; lymphocytes ≤1, 2–100, 101–200, 201–300, >301 cells; eosinophils and neutrophils ≤1, 2–10, 11–20, 21–30 and >31 cells. Actual cell counts were recorded for macrophages.

Staining for mast cells

Mast cells were stained using a monoclonal antibody against the human mast cell protease tryptase, the specificity of which has been demonstrated38, 39 in human tissue, including colonic. This technique has been shown to be effective in tissue fixed in either formalin or Carnoy’s, although the internal mast cell structure was reported to be better preserved with formaldehyde fixation.38

Inhibiting endogenous peroxidase activity

After thorough dewaxing, tissue sections were incubated with 0.5% hydrogen peroxide in methanol at 22 °C for 30 min and washed in running tap water for 15 min.

Primary antibody

Sections were treated with trypsin 0.1% in calcium chloride 0.1% (pH 7.6) for 10 min at 37 °C. Non-specific binding of protein was blocked by normal rabbit serum diluted 1:5 in TBS, pH 7.6 for 15 min. Slides were incubated overnight at 4 °C with the monoclonal mouse antihuman tryptase AA1 (DAKO M7052; Dako Ltd., Cambridge, UK), diluted 1:4000 with normal rabbit serum (IgG fraction).

Secondary antibody

Tissue sections were incubated for 30 min with biotinylated rabbit antimouse antibody (DAKO E0354; Dako), diluted 1:100 with normal rabbit serum. Slides were washed in TBS for 7 min before and after addition of secondary antibody.

Avidin–biotin complex

Slides were incubated with streptavidin-biotin complex horseradish peroxidase (DAKO K0377; Dako) for 30 min and washed in TBS for 7 min. Slides were developed in diaminobenzidine (DAB) solution for 5 min [DAB chromogen tablets (DAKO S3000; Dako), 3% hydrogen peroxide and Tris-HCl buffer pH 7.6], rinsed in TBS and washed in tap water for 5 min. Sections were counterstained in Mayer’s haematoxylin (10–20 s), dehydrated, cleared and mounted. Mast cells stained dark brown and nuclei stained light blue.

Quantifying mast cells

The area occupied by tryptase positive mast cells (mm2), in three consecutive nonoverlapping fields (including lamina propria and epithelium, but excluding muscle) at ×20 magnification, and the corresponding tissue area was quantified by image analysis. Data were expressed as tryptase area per mucosal area (volume density of mast cells). Prior to quantification, validity and reproducibility were confirmed (reproducibility – Pearson’s correlation, r=0.86; mean difference 0.16; standard deviation of the difference 0.24; limits of agreement to 0.33 to 0.55). The image analysis system comprised a photomicroscope (Axiophot microscope; Zeiss, Germany) attached to a colour video camera (Sony DXC-3000 A 3CCD) and computer (Pentium processor; Image Associates) running imaging software (KS400 version 2.0 iC/Windows Release 2.0; © 1995 Kontron Elektronik GmBH).

Statistics

The original response data, which were not normally distributed, were transformed using square-root transformation prior to analysis. Exploratory analysis (scatter graphs) and an analysis of variance showed that possible explanatory factors (age, sex, onset), other than site and patient group did not appear important to mast cell volume density. A one-way analysis of variance ( ANOVA), using the Scheffe multiple comparison procedure (significance level 0.05), was used to compare differences in mast cell volume density between patient groups on a per site basis.

Chi-squared (χ2) test with Yates’ correction was used to determine differences in grades of other inflammatory cells between the IBS and normal controls. Descriptive data were expressed as mean ± standard deviation or median (range). All analysis was performed using SPSS (Version 6.1.2 SPSS Inc.).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Demographic and clinical data

The majority of IBS patients (86%; 12/14) and normal controls (71%; 5/7) were female and of comparable age (42 ± 11 and 44 ± 8 years, respectively). Descriptive data is summarized in Table 1. As expected, the mean bowel disease questionnaire (BDQ) scores in IBS (729 ± 76) predicted a high probability (>75%) of having functional bowel disease. Scores of 515 ± 60 and 432 ± 66, for the normal controls and nonspecific inflammation group, respectively, suggested a low to moderate probability of having a functional bowel disorder. The predominance of females primarily contributed to the higher BDQ in the normal controls compared to the NSI group.

The majority of IBS patients (11/14) were not on medications for their bowel symptoms at time of study; three were taking an antispasmodic, two of four UC patients were taking sulphasalazine. In terms of nongastrointestinal medications, one IBS patient was taking a beta-blocker for angina and one control subject was on thyroxine for hypothyroidism.

Macroscopic and microscopic appearance of the mucosa

The caecum, colon and rectum of all IBS patients and controls were macroscopically normal and all biopsy specimens were deemed microscopically normal by an independent pathologist’s examination. Although macroscopically normal, the colonic mucosa of the three NSI patients had evidence of mild inflammatory changes at one site (ascending colon in each case) without tissue distortion. These patients did not have bowel symptoms and on independent clinical examination were not diagnosed to have a gastrointestinal disease at the time of study.

Mast cells in IBS

Mast cells (as represented by mean volume density of mast cells), were significantly higher in the caecum of IBS patients (0.91 ± 0.18; CI 0.79–1.0) compared to normal controls (0.55 ± 0.14; CI 0.40–0.69). No significant differences were found in the ascending colon, descending colon or rectum ( Fig. 1). Although values were higher in both ascending and descending colon in IBS than controls, this was not statistically significant.

image

Figure 1.  Mast cells in IBS, normal and inflammatory controls. *P < 0.05, IBS vs. normal controls; ++P < 0.05, inflammatory vs. normal controls. AC ascending colon, DC descending colon.

Download figure to PowerPoint

Inflammatory infiltrate

No differences could be detected in plasma cell, lymphocyte, eosinophil, neutrophil or macrophage infiltration in the lamina propria of H & E stained sections at any site in IBS compared to normal controls (χ2 test) (Table 2).

Table 2.   Summary of inflammatory cells in IBS compared to controls Thumbnail image of

Mast cells and inflammation

MC were significantly increased in the patients with nonspecific inflammation (NSI) compared to controls at the inflamed site (which was the ascending colon in each case). In addition, these patients showed significantly higher MC compared to controls in the descending colon and caecum, which had no evidence of microscopic inflammation. The NSIs had the highest mean tryptase positivities of all groups ( Fig. 1).

In ulcerative colitis, MC infiltration was not significantly different compared to either IBS or normal controls. The majority of UC tissue specimens (11/16) showed acute and/or chronic microscopic inflammation with architectural distortion in 9/16 specimens. No clear association between mast cells, distortion or active inflammation could be seen in this small sample. Although increases in MC would be expected in inflammatory tissue, the estimation of MC in UC is probably confounded by factors such as tissue distortion, severe inflammation, medications and the presence of an underlying chronic disease, which has previously been suggested.40

Mast cells and other variables

No association could be found between mast cells and other possible explanatory variables including age, sex, duration of symptoms, symptom score, IBS subtype or presence of diarrhoeal symptoms. The number of patients in each of our IBS subcategories was too small to allow meaningful subgroup comparisons, although the majority had diarrhoea as a symptom.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

We report for the first time an increase in mast cells in the caecal mucosa of IBS patients compared to controls. No significant differences in MC could be detected at the ascending colon, descending colon or rectum. Although mast cells were higher in IBS than controls at the ascending and descending colon, this was not significant. While there are few published studies on MC in IBS, our finding of increased MC support those of Weston et al.34 who documented elevated MC numbers in IBS at the terminal ileum. Other investigators,35, 36 however, found no differences in the degree of MC infiltration in IBS compared to controls, based on tissue from the rectum through to the ascending colon. Similarly, we could find no significant differences at these sites, suggesting that raised MC in IBS may be a feature specific to the ileo-caecal region. Interestingly, the ileo-caecum has been explored as a possible origin of pain, bloating and altered bowel habit, and increased sensitivity to balloon distention has been documented at this region.41

There is considerable potential for mast cell involvement in the pathophysiology of IBS. Potent chemicals derived from mast cells, including histamine, 5-HT, platelet activating factor, prostaglandins, cytokines and leukotrienes,42, 43 may be implicated in the generation of IBS symptoms via their effects on enteric muscle and nerve function. Although the classic stimulus for mast cell activation and proliferation is the type I hypersensitivity response, nonspecific release of mast cells occurs under other conditions including nerve damage,25, 26 mechanical irritation,44 stress,24 infection27 and a variety of inflammatory events.28 Once stimulated, mast cell products may encourage further proliferation, resulting in a self-perpetuating increase in mast cell numbers long after the initiating event has subsided. In IBS, we propose that the mast cell may be an important mediator in a complex interaction between a physiological event (gut insult, inflammation, tissue injury, nerve injury), the nervous system and psychological factors (stressful events, emotions).

Close proximity of the MC to the enteric nerves has been demonstrated in both animals29, 30 and humans.31, 32 Stead et al.29 showed that 67% of intestinal MC were touching subepithelial nerves while an additional 20% were within 2 μm, which provides a structural foundation for communication between the mast cell and the nervous system. Animal studies have demonstrated that mast cells mediate increases in the excitability of enteric neurones19[20]–21 and the communication between mast cells and nerves is thought to be bi-directional. Picomolar amounts of the neurotransmitter substance P can alter excitability of mast cells without causing degranulation.45 In IBS therefore, mast cells may play a role in the development of increased visceral hyperalgesia, a feature documented in the colon and small intestine of IBS patients,10[11][12][13]–14 including postinfectious IBS.3

Increased MC in our IBS group may well be evidence of a persistent inflammatory response originally triggered by an event such as gastrointestinal infection. Recently, a persistent inflammatory response has been reported in postinfectious IBS, as evidenced by increased gene expression for interleukin-1β in rectal biopsies.4 There is also preliminary evidence that stress reactivates inflammation.46 Apart from MC, we, however, could find no other evidence of an inflammatory response in the IBS group; numbers of lymphocytes, eosinophils, neutrophils, macrophages and plasma cells were not increased compared to controls. Since overt colonic inflammation precludes a diagnosis of IBS, however, documentation of inflammatory evidence in this condition probably requires the study of more sensitive markers of low-grade inflammation.

In theory, an inflammatory event or GI insult could cause an increase in mast cells, subsequent changes in enteric nerve and muscle function and IBS symptoms. This, however, is unlikely to be the full picture, as many individuals have inflammatory changes but do not develop IBS. For example, after an episode of gastroenteritis, 70% of individuals do not develop IBS.2, 3 Other predisposing cofactors such as psychological traits2 or stress must be involved, otherwise all patients with gut inflammation would be presumed to develop IBS, which is known not to be the case. In IBS, psychological stress has been implicated in precipitating or exacerbating symptoms.47[48][49]–50 Importantly, a link between the mast cell and stress has been observed. Exposure to stress in rats has been associated with mast cell degranulation in the colon.24, 51 MacQueen et al. found that after a Pavlovian conditioning regime in rats, the audio-visual conditioning stimulus alone could evoke the release of mast cell mediators52 suggesting a role for CNS involvement in mast cell activation. In a recent key study53 experimental stress induced the release of intestinal mast cell tryptase and histamine in normal subjects and patients with food allergy, providing a crucial link between stress and the intestinal mast cell.

The results relating to the nonspecific inflammatory (NSI) group may have application to IBS. As expected, MC were significantly increased at the inflamed sites in this inflammatory group compared to controls. In addition, there were significant increases in MC over normal controls at noninflamed sites remote from the inflamed regions. Release of mast cell products at the inflamed site may have induced mast cell proliferation/recruitment in noninflamed sites. The premise that an inflammatory event can generate effects in noninflamed regions has been previously demonstrated.7, 8 It is conceivable, therefore, that the increased mast cells at the uninflamed caecum of our IBS group could be associated with inflammation in the ileum (which was not studied).

Food allergy will continue to be proposed as a potential confounding factor in this and similar studies. Although food has been implicated in the pathogenesis of IBS54[55][56]–57 and improvements with exclusion diets55, 57[58][59]–60 or mast cell stabilisers61 have been suggested, most adverse reactions to food in this disorder are not thought to be immune-mediated.62[63]–64 True food allergy (IgE mediated), is a rare condition,65 its study is difficult and remains a contentious issue in IBS. In our study of consecutive Rome criteria IBS patients, none presented with classical symptoms of atopy or food allergy, but prospective investigations for food allergy were beyond the scope of this study. Interestingly, some propose that IBS could be a systemic condition;66 noncolonic manifestations are common in IBS67[68]–69 and increases in bladder mast cells70 and airway hyper-responsiveness71 have been reported. Indeed, patients with systemic mastocytosis exhibit a symptom complex not dissimilar from IBS, including symptoms of episodic diarrhoea, abdominal pain, headache, backache, fatigue and urinary frequency.72

The study of mast cells in IBS is confounded by many factors and comparison with the few published studies is difficult. Additionally, there are probably innate individual variations in mast cell numbers in the intestine. Although increases in mast cells have now been documented in the terminal ileum34 and caecum, further confirmatory studies are clearly required. Significant increases in MC have not been identified throughout colonic mucosal tissue.35, 36 Studies to date, including ours, are based on small samples ranging from 7– 25 IBS patients,35, 36 while a pivotal study by Weston et al.34 did not include colonic specimens. Methodological differences exist including patient selection, sample size, biopsy site and the mast cell method, for example, the staining employed by some36 does not allow detection of degranulated mast cells.40 In our study, the tryptase-based staining technique allowed identification of both intact and degranulated mast cells; the image analysis process used, however, did not allow these to be quantified separately. It is therefore not clear if there were increases in mast cell numbers (hyperplasia) or degranulation or both. There are many unanswered questions for future work on the role of mast cells in functional GI disorders.

In conclusion, MC were significantly increased in the caecum in IBS patients compared to controls in this study. Mast cells were also elevated in the ascending and descending colon but this was not statistically significant. Increases in numbers of lymphocytes, plasma cells, neutrophils or eosinophils were not seen. An increase in mast cell numbers may be important in pathogenesis of IBS; these cells may act as an important link between a GI event/insult, the enteric nervous system and stress. More work is clearly needed to further investigate the role of immune and inflammatory cells in IBS.

Footnotes
  1. This paper was an oral presentation at Digestive Disease Week, New Orleans, USA, 1998.

  2. These co-authors are employees of GlaxoWellcome; remaining authors have no financial links with this company.

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  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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