• enteroendocrine cell;
  • irritable bowel syndrome;
  • visceral hypersensitivity


  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Abstract  The objective of this study was to determine whether or not the number of enteroendocrine cells (ECs) in the gut is related to visceral hypersensitivity in patients with diarrhoea-predominant irritable bowel syndrome (D-IBS). Twenty-five subjects with D-IBS (mean, 43.1 years; 16 women, nine men) were recruited into our study, along with 13 healthy controls (mean, 40.7 years; nine women, four men). Maximally tolerable pressures were evaluated via barostat testing, and the levels of ECs were immunohistochemically identified and quantified via image analysis. The numbers of ECs between the D-IBS subjects and the controls were not significantly different in the terminal ileum, ascending colon and rectum. However, the maximally tolerable pressures determined in the D-IBS subjects were significantly lower than those of the control subjects (P < 0.01), and we detected a significant relationship between the maximally tolerable pressures and the numbers of ECs in the rectum (r = −0.37, P < 0.01). Rectal sensitivity was enhanced to a greater degree in D-IBS patients exhibiting an elevated level of rectal ECs. This study provides some evidence to suggest that ECs play an important role in visceral hypersensitivity.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by chronic abdominal pain or discomfort, along with alterations in bowel habits. This disorder is very commonly encountered, and has been associated with alterations in gastrointestinal (GI) motility,1,2 enhanced stress responsiveness,3 and increased visceral perception.4–6

Recently, a more holistic mechanism of action, involving interactions between the brain and the gut, has been suggested as an explanation of the symptoms of IBS. Interactions between the brain and the enteric nervous system (ENS) occur via bidirectional neural pathways,7 and the ENS acts directly on effector systems to modulate both secretion and motility. However, these effector pathways can also be modulated by means of a vast array of neurotransmitters and neuromodulators, including serotonin, noradrenaline, dopamine and corticotropin-releasing hormone (CRH).7

Among these modulators, serotonin has received the greatest amount of attention. The majority of serotonin is synthesized and stored in the enteroendocrine cells (ECs). Released serotonin stimulates the 5-hydroxytryptamine (5-HT)3 and 5-HT4 receptors located on the primary afferent neurones of both the splanchnic and vagal fibres.8 Furthermore, colonic mucosal ECs also produce CRH.9 CRH is considered to be a primary mediator of stress responses on the brain-gut axis, and the intravenous administration of CRH has been shown to reduce rectal perceptual thresholds.10 These results suggest that ECs might function as an important regulator of physiological functioning within the GI tracts. Earlier studies have reported that the numbers of ECs were higher in patients with IBS11 and postdysenteric IBS12 than in normal subjects, and also that elevated levels of ECs and depression are both important independent predictors for the development of postinfectious irritable bowel syndrome (PI-IBS).13 However, the potential clinical significance of an increase in the numbers of ECs has yet to be completely elucidated.

Therefore, in the present study, we compared the numbers of ECs between IBS patients and normal subjects, and attempted to delineate the relationship between visceral hypersensitivity and the number of ECs in the colonic mucosa. Considering the fact that differences in rectal motor characteristics and visceroperception have been determined to exist between D-IBS and constipation-predominant IBS patients,14,15 we included only D-IBS patients as subjects in this study.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References


Twenty-five D-IBS patients in the Gastroenterology Department of our institution, all of whom exhibited symptoms that fulfilled the Rome II criteria,16 were recruited in the study. None of the study participants had a clear history of inflammatory bowel disease or PI-IBS, which is usually diagnosed when patients exhibit at least two of the following: fever, vomiting, diarrhoea, or a positive stool culture at the onset of IBS symptoms, when the patient's bowel habits have previously been within normal limits.17 Informed written consent was provided by all patients, and approval for the study was obtained from the local ethics committee at the hospital. We also included a group of normal control subjects in this study. These control subjects exhibited (i) macroscopically and histologically normal colonic mucosa; (ii) no persistent bowel symptoms; (iii) no organic or functional bowel diseases; and (iv) no history of chronic medical disease. Patients with the following criteria were excluded from both groups: (i) a history of atopy, food allergies or asthma on the basis of a detailed medical history; (ii) active diverticulitis; or (iii) GI infection.18 All subjects underwent colonoscopies after complete colonic cleansing with polyethylene glycol-electrolyte lavage solution, conducted on the morning of the procedure. In all cases, two pinch biopsies were obtained at each of three anatomical sites (terminal ileum, ascending colon and rectum) for routine haematoxylin and eosin (H&E) histological and immunohistochemical testing.


The participants were instructed to complete a questionnaire regarding their bowel symptoms and psychological distress over the 2 weeks prior to the interview. The questionnaire was designed to delineate the severity of the patient's abdominal pain or discomfort, which was scored as: 0 = none, 1 = mild (symptoms could be ignored if the patient did not think about it), 2 = moderate (symptoms could not be ignored, but did not influence daily activities), or 3 = severe (symptoms influenced daily activities). The questionnaire also included questions regarding the duration of IBS symptoms, as well as an assessment of the following supportive symptoms of IBS,16 as present or absent: fewer than three bowel movements a week, more than three bowel movements a day, hard or lumpy stools, loose (mushy) or watery stools, straining during a bowel movement, urgency (having to rush to have a bowel movement), feeling of incomplete bowel movement, passing mucus (white material) during a bowel movement, and abdominal fullness, bloating or swelling.

To evaluate the psychological well-being or lack thereof in the study subjects, all participants were instructed to complete a Beck Depression Inventory19 and a State–Trait Anxiety Inventory.20

Histology and immunohistochemistry

EC staining  Enteroendocrine cells were stained with a monoclonal antibody against chromogranin A (CgA).21 Biopsy specimens were fixed in 10% neutral buffered formalin, and processed for either H&E histology or immunohistochemistry.

For the latter, paraffin-embedded specimens were sliced to a thickness of 4 μm with a microtome, and attached to precoated slides (Dako, Glostrup, Denmark). After thorough dewaxing, the tissue sections were immunostained with anti-CgA antibodies, as described previously, using a Dako Techmate 500 Plus (DAKO, Carpinteria, CA, USA) and Techmate reagents (Dako). Primary antibody binding was detected using antimouse IgG rabbit antibody (Dako), via the avidin–biotin complex immunoperoxidase technique.

Quantification of ECs  All histological sections were evaluated via light microscopy by an expert pathologist. Immunohistochemically positive-stained ECs were counted in six consecutive non-overlapping microscopic fields (three sections × two biopsy samples) at a magnification of ×200, and the average number of ECs per field was calculated. Counting was conducted with the pathologist blinded to the origin of the histological sections.

Inflammatory infiltrate (microscopic inflammation and cell infiltration)  The grade of inflammation was evaluated by the same pathologist under the same light microscope, at a magnification of ×40, and the pathologist was kept unaware of the clinical diagnosis of each of the tissue sections. The number of plasma cells, lymphocytes, eosinophils and neutrophils were each semiquantitatively graded (0–4) on H&E-stained sections.18 Each of the grades was based on the number of cells per field as follows: plasma cells: ≤1, 2–50, 51–100, 101–200, >200 cells; lymphocytes: ≤1, 2–100, 101–200, 201–300, >300 cells; eosinophils and neutrophils: ≤1, 2–10, 11–20, 21–30, >30 cells.

Rectal barostat

Barostatic measurements were obtained separately from the other examinations, and no bowel preparation was conducted before the measurements were taken. A dual-drive barostat (Distender series IITM; G&J Electronic Inc., Toronto, Canada) and a polyethylene bag (600 mL, 12 × 15 cm) fixed to the end of a double-lumen barostat catheter (MUI Inc., Mississauga, Canada) were used in this procedure. The bag was tightly folded, inserted into the anus and positioned within the rectum. One lumen was then connected to the pressure transducer, and 200 mL of air was pumped through the second lumen. Afterwards, the bag was deflated, and the second lumen of the tube was connected to the barostat. The patients were positioned in the left lateral decubitus position, in a relaxed fashion. The bag was then inflated gradually with air via the stepwise cumulative ascending method (rate of pressure increase = 2 mmHg 2 min−1).22 The minimal distending pressure (MDP) was defined as the pressure measured in the bag when its volume reached 25 mL. The resting tone was determined from the volume of the bag at MDP plus 6 mmHg. The compliance value was obtained from the slope value of the pressure–volume curve via linear regression, assuming a simple physical property of elasticity.23 During the initial phase of distension, the subjects were instructed to report and classify their perceived sensations, with regard to the first sensation of distention, sensation of defecation, sensation of the urge to defecate, and the maximal tolerable distention caused by pain. The pressure was recorded at each of the self-reported steps. Discomfort and pain were both scored from 0 to 10 on a visual analogue scale. The end-point, at which the distensions were discontinued, was the reporting of a sensation of pain that reached at least 3 on a 10-point visual analogue scale.

Statistical analysis

All data are expressed as the mean ± standard deviations (SD). Two-tailed Student's t-tests and chi-square tests were used to compare statistical differences between the IBS subjects and the controls. The correlations were analysed with Pearson's correlation. The alpha level of significance was set at P < 0.05. All analyses were conducted using SPSS (version 11.5.0; SPSS Inc., Chicago, IL, USA).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Subject characteristics

Twenty-five referred subjects with D-IBS (mean, 43.1 years; 16 women, nine men) and 13 healthy controls (mean, 40.7 years; nine women, four men) were included in this study. The median symptom duration was 70 months (range 10–360 months), and there were no statistical differences between the groups with regard to either age or sex.

EC counts and relative quantification of inflammatory cells

The numbers of ECs were not significantly different between the groups in the terminal ileum, ascending colon or rectum (Fig. 1, Table 1).


Figure 1.  Immunoperoxide staining of enteroendocrine cells (ECs) in the rectal mucosa of (A) control subjects and (B) irritable bowel syndrome (IBS) patients (magnification ×200). The numbers of EC were not significantly increased in the terminal ileum, ascending colon or recta of IBS patients when compared with controls (P > 0.05).

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Table 1.   Comparison of enteroendocrine cell counts between IBS patients and control subjects
 IBS (n = 25)Controls (n = 13) P
  1. IBS, irritable bowel syndrome; ns, not significant. *Cell counts/high power field (HPF) (×200), expressed as mean ± SD.

Terminal ileum*10.9 ± 3.111.0 ± 2.4ns
Ascending colon*11.2 ± 5.49.3 ± 3.6ns
Rectum*16.6 ± 8.612.4 ± 3.8ns

The degree of infiltration of plasma cells, eosinophils and neutrophils in each of the colonic segments was also not statistically different between the groups. However, the numbers of lymphocytes in the rectum were significantly higher in the IBS subjects than in the controls (Table 2) (P < 0.05).

Table 2.   Relative quantification of inflammatory cells
No. cellsNo. biopsies in terminal ileumPNo. biopsies in ascending colonPNo. biopsies in rectumP
IBS (n = 25)Normal (n = 13)IBS (n = 25)Normal (n = 13)IBS (n = 25)Normal (n = 13)
  1. IBS, irritable bowel syndrome; ns, not significant.

Plasma cells

Rectal barostat test

The rectal barostat test was performed on all 38 subjects (13 women, 20 men). All female subjects except two (both were postmenopausal women) had normal menstrual cycles, and none described any history of chronic gynaecological symptoms. Each of the women was assessed during non-menses phases. As shown in Table 3, the mean pressure values for the ‘first sensation’ and ‘maximal tolerable distension’ steps in the IBS subjects were significantly lower than those of the controls (P < 0.05). However, on a multivariate analysis including psychological traits, only the pressure of maximally tolerable distension (in other words, the maximally tolerable pressure) proved to be a predictive factor for IBS, and the most efficient threshold of maximally tolerable pressure for the identification of IBS was determined to be 34 mmHg (sensitivity 62%, specificity 93%, positive predictive value 95%, negative predictive value 61%, accuracy 77%).6

Table 3.   Sensory thresholds to rectal distension
 IBS (n = 25)Controls (n = 13) P
  1. ns, not significant. *All values expressed as mean ± SD (mmHg).

Resting tone7.4 ± 1.38.7 ± 2.0ns
Compliance (mL mmHg−1)9.2 ± 3.110.6 ± 3.6ns
First sensation*8.3 ± 2.110.5 ± 3.5<0.05
Sensation of defecation*13.3 ± 3.815.2 ± 4.8ns
Sensation of urge to defecate*17.7 ± 4.720.5 ± 5.0ns
Maximally tolerable distension*31.9 ± 8.037.5 ± 3.8<0.01

Correlation between EC numbers and symptoms

All IBS subjects complained of abdominal pain, and the symptom most frequently associated with the onset of the condition was a change in stool frequency (87.5%). However, no significant correlations were existed between EC counts and D-IBS symptoms.

EC and rectal barostat parameters

For all participants, we noted a significantly negative correlation between maximally tolerable pressure and EC counts in the rectum (r = −0.37, P < 0.05) (Fig. 2). However, the EC counts in the terminal ileum and ascending colon were not significantly correlated with the maximally tolerable pressures. Therefore, to determine whether or not a significant difference could be detected between subjects with and without visceral hypersensitivity with regard to the number of ECs in the rectum, the D-IBS subject group was divided into rectal hypersensitivity (+) and (−) groups on the basis of a distension level of 34 mmHg, which was considered to be the most efficient threshold of maximally tolerable pressure for the identification of IBS. Rectal EC counts in the rectal hypersensitivity (+) group were significantly higher than those in the rectal hypersensitivity (−) group (95% confidence interval: 1.397–10.667, P < 0.05) (Table 4, Fig. 3).


Figure 2.  Correlation between the rectal enteroendocrine cell counts and maximally tolerable rectal pressure in all participants. The relationship between the two variables was negative and significant (P < 0.05).

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Table 4.   Comparison of enteroendocrine cell counts between rectal hypersensitivity (+) and (−) groups
 Rectal hypersensitivity (+) (n = 17)Rectal hypersensitivity (−) (n = 21) P
  1. *Cell counts/HPF (×200), expressed as mean ± SD.

Enteroendocrine cells in rectum*18.5 ± 8.812.5 ± 5.2ns

Figure 3.  Correlation between rectal enteroendocrine cell (EC) counts and rectal hypersensitivity. The irritable bowel syndrome (IBS) patient group was divided into rectal hypersensitivity (+) and (−) groups on the basis of a distension level of 34 mmHg, which was the most efficient maximally tolerable pressure threshold for the identification of IBS. EC counts were significantly higher in the rectal hypersensitivity (+) group than in the rectal hypersensitivity (−) group (P < 0.05). Hyper (−) = rectal hypersensitivity (−) group; hyper (+) = rectal hypersensitivity (+) group.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

In this study, we demonstrated that the numbers of ECs in the terminal ileum, ascending colon and rectum were not significantly different between the D-IBS patient group and the control group. However, rectal EC counts were significantly correlated with maximally tolerable pressures, and D-IBS patients exhibiting rectal hypersensitivity manifested significantly higher EC densities in their rectal mucosa.

Enteroendocrine cells are a plausible mediator of diarrhoea-predominant symptoms, as they clearly play a pivotal role in the control of gut motility and secretions.24 The only possible source of serotonin released in response to mucosal stimulation is the ECs of the intestinal mucosa.24 Recent studies have implicated alterations in serotonin signalling in GI functional disorders, including IBS, chronic constipation, diarrhoea and functional dyspepsia. Furthermore, alterations in the numbers of ECs have been reported in cases of ‘postdysenteric IBS’ involving recurrent abdominal pain and diarrhoea,12 and reduced numbers of ECs have been reported in patients with functional constipation.25 However, other studies have demonstrated similar alterations of EC and 5-HT cells in patients suffering from constipation, diarrhoea26,27 and IBS.28 We also determined that the numbers of ECs were not significantly higher in patients who suffered from D-IBS. A variety of factors may have contributed to the substantial discrepancies between different studies. The most probable reason for these discrepancies, however, is that the number of IBS subjects exhibiting visceral hypersensitivity may exert a substantial influence on the mean rectal EC counts. IBS patients may be heterogeneous with regard to visceral hypersensitivity, with hypersensitive and normosensitive groups.29 Therefore, when the relationship between EC count and visceral sensitivity in this study is considered, the interpretation of EC counts is expected to be different according to the portion of IBS patients with sensitive rectum. Other possible explanations for the discrepancies between different studies may include differences in the immunohistochemical EC-staining protocols in various studies, and the exclusion (in our study) of PI-IBS patients from the IBS patient group.

A putative mechanism underlying the relationship between EC counts and visceral sensitivity could be explained by the Gαq/PLC/Ca2+ signalling pathway.30

Enteroendocrine cells have mechanosensitive and chemosensitive elements that detect changes in force or contents of the intestinal lumen. Mechanical stimulation acts through the Gαq/PLC/Ca2+ signalling pathway to increase 5-HT release. And 5-HT acting on 5-HT2 and 5-HT4 receptors may contribute to hypersensitivity of primary afferent neurones in IBS patients.31 In addition, 5-HT release from EC is also modulated by endogenous adenosine.32 Mechanical stimulation releases ATP that acts as an extracellular mediator by binding to P2 nucleotide receptors coupled to the Gαq/PLC/Ca2+ pathway leading to 5-HT release.

Therefore, enhanced 5-HT release from increased ECs in IBS could lead to dysmotility and altered fluid secretion.

In our laboratory, we evaluated immunoreactivity using antihuman CgA antibody. The majority of neuroendocrine cells in the GI tract express CgA.33 Thus, the category of CgA-positive ECs included all serotonin-positive cells and most other cells that secrete peptides.26 In a previous study of PI-IBS, the levels of synaptophysin-positive cells, a universal enteroendocrine cell marker, were increased substantially along with an increase of serotonin-containing cells, in IBS patients,12 and D-IBS patients have been determined to have significantly higher postprandial 5-HT concentrations, and longer 5-HT peak durations, than do healthy volunteers.34 Therefore, on the basis of these findings, an increase in the levels of CgA-positive ECs might result from an increase in the number of serotonin-positive cells. However, given the multiplicity of the neuroactive factors present in ECs, this hypothesis requires further verification.

In this study, we have obtained some evidence supporting a relationship between visceral hypersensitivity and the levels of ECs in the rectum. However, no significant correlations between the two variables could be detected in any of the other segments. Possible explanations for this lack of a detectable relationship include the following. First, the numbers of ECs in the rectum were significantly higher than those measured in other sectors of the colon (P < 0.01). Therefore, assuming that the pain threshold is determined by the numbers of ECs in each of the colonic segments, the rectum would be the most susceptible segment to visceral hypersensitivity. Second, the EC counts in the ascending colon and terminal ileum might be related to hypersensitivity within their own segments, rather than that of the rectum. Although heightened sensitivity in the jejunum and stomach along with the rectum of IBS patients has been reported,35,36 it remains unclear whether all colonic segments are sensitive simultaneously. Finally, rather than being due to a simple increase in the number of ECs, increased levels of serotonin-positive cells and molecular changes in serotonergic signalling mechanisms might also contribute to the visceral hypersensitivity noted in IBS patients.

This study has some limitations in terms of establishing a relationship between EC and visceral hypersensitivity. One of these limitations is that the sample size of our IBS patients is small. This could have resulted in type 2 errors. And the other is our limited counting method of EC counts. EC would be better expressed by numbers per crypt or per 100 epithelial cells rather than per high power field.

The finding that IBS subjects exhibited higher numbers of lymphocytes in the rectum than normal subjects is interesting. This result is consistent with the results of previous studies, which have reported intraepithelial lymphocytosis in the jejunum of IBS patients,37,38 as well as increased numbers of ECs and T lymphocytes and increased gut permeability in patients with PI-IBS.12 This suggests that ECs and lymphocytes may both constitute a component of a continuing coordinated immune and motor response.12 However, despite our findings of increased rectal lymphocyte infiltration, we did not detect a relationship between visceral hypersensitivity and lymphocytes in this study. We also did not find that the numbers of lymphocytes in other sections of the colons of IBS patients were higher than those measured in the controls. Thus, the roles of EC and lymphocytes in the pathogenesis of IBS remain to be clearly and completely elucidated.

In summary, our results show that a significant relationship exists between rectal EC counts and visceral hypersensitivity. This study provides some evidence that ECs play an important role in IBS.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References
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