|Year : 2017 | Volume
| Issue : 3 | Page : 385-389
|Levels of interleukins 2, 6, 8, and 10 in patients with irritable bowel syndrome
Shaileshkumar R Patel1, Anshul Singh1, Vatsala Misra1, Sri Prakash Misra2, Manisha Dwivedi2, Pawan Trivedi1
1 Department of Pathology, M L N Medical College, Allahabad, Uttar Pradesh, India
2 Department of Gastroenterology, M L N Medical College, Allahabad, Uttar Pradesh, India
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|Date of Web Publication||22-Sep-2017|
| Abstract|| |
Irritable bowel syndrome (IBS) was previously considered as a psychosomatic disorder. But recent studies indicate that inflammation plays a significant role. The present study was undertaken to evaluate role of pro-inflammatory (IL 2, IL 6 and IL 8) and anti-inflammatory (IL 10) cytokines in clinically diagnosed patients of IBS. 51 patients and 29 controls were included in this study. On the basis of history of gastrointestinal infection, patients were divided into Post Infectious (PIIBS) and Non Post Infectious (NPIIBS) groups. All subsequently underwent colonoscopy and a rectosigmoid biopsy as well as measurement of levels of IL 2, 6, 8 and 10. The levels of IL 2 and IL 8 were significantly raised in IBS patients compared to controls with the mean level of IL 2,6 and 8 higher in PIIBS group than NPIIBS group but statistically significant for IL 8 only. The mean level IL-10 was reduced in patients compared to controls but statistically insignificant. Present study shows that Interleukin levels are altered in patients suffering from IBS and may have a key role in its pathogenesis.
Keywords: Cytokines, inflammation, interleukins, irritable bowel syndrome, pathogenesis
|How to cite this article:|
Patel SR, Singh A, Misra V, Misra SP, Dwivedi M, Trivedi P. Levels of interleukins 2, 6, 8, and 10 in patients with irritable bowel syndrome. Indian J Pathol Microbiol 2017;60:385-9
|How to cite this URL:|
Patel SR, Singh A, Misra V, Misra SP, Dwivedi M, Trivedi P. Levels of interleukins 2, 6, 8, and 10 in patients with irritable bowel syndrome. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Dec 7];60:385-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/3/385/215391
| Introduction|| |
Irritable bowel syndrome (IBS) is a common gastrointestinal condition encountered in clinical practice characterized by abdominal pain and cramps, changes in bowel movements (diarrhea, constipation, or both), gassiness, bloating, nausea, and other symptoms. Diagnostic criteria have evolved since 1979 when Manning et al. first published their criteria. The subsequent changes have included the Rome I, II, III and now the most recent Rome IV criteria. According to the updated Rome III criteria, IBS is a clinical diagnosis and presents as one of the three predominant subtypes: (1) IBS with constipation (IBS-C); (2) IBS with diarrhea (IBS-D); and (3) mixed IBS (IBS-M).
In the past, IBS was considered a psychosomatic disorder; hence, it was a diagnosis of exclusion. However, now, many factors have been suggested to be implicated in its pathogenesis viz-altered gastrointestinal motility, visceral hypersensitivity, postinfectious reactivity, brain-gut interactions, alteration in fecal microflora, bacterial overgrowth, food sensitivity, carbohydrate malabsorption, and intestinal inflammation. Evidence regarding the role of immune activation in the etiology is consistently being proven, mainly by the studies investigating mechanisms of postinfectious IBS (PIIBS).
Cytokines play an important role in inflammation. The fact that a disbalance between pro- and anti-inflammatory cytokines may have a role in the pathogenesis of IBS has been substantiated by the results of many studies done worldwide.,, However, studies on cytokines from IBS patients are very few from India. Hence, this study was carried out to assess the levels of interleukins (ILs) in patients presenting clinically as IBS and to compare it with the levels observed in apparently normal controls and see if there is any significant difference between the two.
| Materials and Methods|| |
The study was conducted in the Department of Pathology in collaboration with Department of Gastroenterology over 1 year. Ethical clearance was taken from college committee for the study and informed consent was obtained from all patients and controls.
The study included 29 controls who were apparently healthy staff members and family members of patients, and were included in study after proper information and willful agreement, without any compulsion to undergo screening colonoscopy, biopsy and routine blood investigations (biochemical and hematological) free of cost to rule out any incidental pathology, of which they may be completely unaware.
Fifty-one patients were selected on the basis of clinical presentations fulfilling Rome III criteria after proper counseling and explanation of the study procedure. All controls and patients underwent flexible colonoscopy. Any individual with an abnormal finding on colonoscopy was excluded from the study. Colonic biopsy from the rectosigmoid area was taken from those who were selected for study to rule out any other microscopic pathology.
Patients were divided into two groups of Post infectious IBS (PIIBS) and Non post-infectious IBS (NPIIBS) based on the previous history of gastrointestinal infection. PIIBS is defined as acute onset, Rome criteria positive IBS, developing after an infectious etiology, which is characterized by two or more of the following: fever, vomiting, acute diarrhea, positive stool culture, and no symptoms suggestive of IBS before this episode. Although, no specific time gap between episodes of infection and onset of IBS has been mentioned in the literature, in the present study, only those patients, who gave a history of previous gastrointestinal infection at least 2 weeks back were enrolled with the hope that their colonic pathology and cytokine profile would have reverted back to normal by then.
Blood samples were collected in the plain vial from both patients and controls at the time of colonoscopy. Serum was separated and levels of IL-2, 6, 8, and 10 were measured by ELISA method using human ELISA kit from “GEN-PROBE” California, USA.
Expected cut-off values provided by the manufacturer were 07, 02, 29, and 05 pg/ml for IL-2, IL-6, IL-8, and IL-10, respectively.
Student's unpaired t-test and Chi-square test with Yates' correction were applied as required.
| Results|| |
Twenty-nine controls were included with mean (standard deviation [SD]) age of 38.3 (13.6) years and male to female ratio of 2.75:1. All had a normal colonoscopy. On histopathology, biopsies showed normal colonic mucosa. The lining epithelium was made of columnar cells and goblet cells with normal-appearing crypts. Lamina propria showed loose fibro-connective tissue with few scattered mixed inflammatory cells comprising lymphocytes, eosinophils and mast cells [Figure 1].
|Figure 1: Normal biopsy - Section show surface epithelium, crypts, and lamina propria with scattered Intra Epithelial Lymphocytes and mast cells (H and E, ×400)|
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Fifty-one patients of IBS were included, 13 of these belonged to postinfectious category, whereas the rest 38 patients were of non post infectious subgroup. The mean (SD) age of presentation was 37.4 (13.7) years. Male to female ratio was 3.8:1. All had a normal colonoscopy.
The histopathological evaluation revealed findings of nonspecific colitis (NSC) in 75% of patients, whereas it was normal in 25% of patients. In cases diagnosed as NSC, sections showed mainly normal epithelial lining and crypts with edematous lamina propria showing mild to moderate infiltration by inflammatory infiltrate comprising mainly of lymphocytes (mean value being 4.25/100 epithelial cells), mast cells (mean value being 2.85/hpf) and few eosinophils. A thin strip of muscularis mucosae was also seen [Figure 2],[Figure 3],[Figure 4]. The cut-off value for exclusion was <20/100 epithelial cells for lymphocytes (to differentiate it from microscopic colitis) and <20/hpf for mast cells (to differentiate it from mastocytic enterocolitis).
|Figure 2: Non post-infectious irritable bowel syndrome. Patient - Section show mildly raised IEL's (black arrow) and occasional mast cell (blue arrow) (H and E, ×400)|
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|Figure 3: Postinfectious irritable bowel syndrome. Patient - Section shows increased IEL's in the surface, crypts as well as lamina propria (H and E, ×400)|
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|Figure 4: Postinfectious irritable bowel syndrome. Patient - Section show increased mast cells in lamina propria (H and E, ×400)|
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Serum IL levels in patients [Figure 5] and controls are shown in [Table 1],[Table 2],[Table 3],[Table 4].
|Figure 5: Line diagram showing the levels of interleukins 2, 6 and 8 in irritable bowel syndrome patients|
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|Table 1: Number of controls and irritable bowel syndrome patients showing increased interleukin levels|
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|Table 2: Number of controls and subgroups of irritable bowel syndrome patients showing raised interleukin levels|
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|Table 3: Comparison of interleukin levels in controls and irritable bowel syndrome patients|
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|Table 4: Comparison of interleukin levels in controls and subgroups of irritable bowel syndrome patients|
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The number of IBS patients (irrespective of PIIBS and NPIIBS) showing increased levels of IL s was conspicuously higher than the number of controls, but it was statistically significant for IL-2 and IL-8 only [Table 1]. The number of PIIBS group patients showing increased ILs were significantly high when compared to controls and NPIIBS group [Table 2].
It was also observed that the mean levels of IL-2, 6, and 8 were increased in patients as compared to controls, whereas, the mean level of IL-10 was reduced in patients as compared to controls. The difference was statistically significant for IL-2 and 8 only [Table 3].
On comparing mean levels of each of the 4 ILs between controls, PIIBS and NPIIBS groups, it was observed that for IL-2, levels were significantly high in both the IBS subgroups in comparison to controls whereas for IL-8, the difference was significant between controls and PIIBS group only. For IL-6 and IL-10, the difference in mean levels was insignificant in all the subgroups [Table 4].
| Discussion|| |
The magnitude of patients with functional gastrointestinal disorders visiting outpatient clinics is high. The rapid socioeconomic development in the past 20 years has also created a transition in the health and environment situation in Asia. The prevalence of IBS varies from 4% to 20% in different Asian nations. Studies from India have reported a prevalence from 4% to 8%.,
IBS was considered a psychosomatic disorder in the past, but now strong evidence are being found that favor it to be an organic disorder. The role of chronic persistent inflammation in its pathogenesis has been studied extensively in the past few years and is now considered as one of the leading mechanisms in the development of the cardinal manifestations of IBS, along with various other etiologies such as altered gut microflora, food allergies, psychological factors, and genetic predisposition.
Epidemiological studies suggest that enteric infections, especially bacterial, is one of the most important risk factors for developing IBS, labeled as PIIBS. The IBS symptoms which develop are often regarded by the patient as a continuation of the initial illness. Most of them meet the Rome criteria for IBS-D or IBS with mixed bowel habit (IBS-M). The rest are classified as NPIIBS.
The significance is that PIIBS is said to have a better prognosis and different treatment protocol as compared to NPIIBS. Furthermore, it is mainly this subgroup in which significant changes in the mucosal immune system and altered ratio of anti and pro inflammatory cytokines have been reported.,,
Routine histological examination reveals none to subtle mucosal abnormality in the majority of cases, but techniques such as special stains, immunohistochemistry, and ultrastructure analysis reveal morphologic changes involving lymphocytes, mast cells, enterochromaffin cells, and antigen presenting cells.
Low-grade inflammation in the intestine in IBS patients is associated with the activation of T lymphocytes and mast cells as well as increased expression of lipopolysaccharide stimulated proinflammatory cytokines such as IL-6 and IL-8, in peripheral blood mononuclear cells, especially in IBS-D patient.,
There is considerable difference of opinion regarding the blood cytokine profile in IBS, with some suggesting a predominating TH1 phenotype (↑tumor necrosis factor-α [TNF-α], IL-1β, IL-12, IL-6, and ↓IL-10) whereas, others showing a predominating TH2 type phenotype (↑IL-5, IL-10, IL-13, and ↓TNF-α). Blood levels of IL-1β, TNF-α, and IL-6 have been observed to correlate positively with IBS symptoms, whereas negative correlations have been observed for IL-10 and IL-12.
Scully et al. documented increased plasma levels of IL-6 and IL-8 in their study on IBS patients while Dinan et al. found similar findings along with no alteration in the anti-inflammatory cytokine IL-10 levels. However, Schmulson et al. observed that at least some patients with IBS produce lower amounts of the anti-inflammatory cytokine IL-10. In another study done by Rana et al., the IL 6 and TNF α levels were found to be significantly increased in diarrhea predominant subjects, whereas the levels of IL-10 were not significantly different. Similar findings were observed in another study conducted on Asian population by Bashashati et al. In the present study also, pro-inflammatory cytokines IL-2 and IL-8 were found to be significantly increased in IBS patients, whereas anti-inflammatory cytokine IL-10 was found to be decreased although non significantly. However, we found no significant difference in the levels of IL-6 between controls and patients which is in quite contrast to many studies done previously.
Gwee et al. also found that patients developing PIIBS exhibit greater IL-1β mRNA expression, both during and after the infection compared to NPIIBS. Similarly, in another study, enhanced IL-1α and β production was observed in IBS patients with small intestinal bacterial overgrowth.
Genotyping studies done on IBS patients indicate them to more likely have alleles associated with excessive production of IL-6, IL-2, TNF-α, and decreased production of IL-10, supporting the theory of imbalance between pro- and anti-inflammatory cytokines in the etiopathogenesis of IBS. Macsharry et al. reported decreased expression of chemokine-producing genes namely IL-8, CXCL-9, and MCP-1 in IBS patients. A few studies observed certain IL-10 polymorphisms to be significantly prevalent in IBS patients as compared to controls which could be responsible for lower levels of this cytokine in IBS patients.,
In another study, frequency of single nucleotide polymorphisms in five cytokine genes regulating inflammation was assessed in IBS patients and was found to be significantly higher for IL-6 promoter when compared to age- and sex-matched apparently healthy controls.
No correlation of changes in IL levels and type of causative organism of pre-IBS infection was possible as most of the patients had initial treatment at smaller center or they had self medication without detailed microbiological examination. Similarly, no correlation with the effect of treatment could be done as many patients were lost to follow-up after relief from symptoms. To the best of our knowledge, there is no well-documented study where these factors have been studied in detail.
| Conclusion|| |
As regards to the Indian subcontinent, studies on cytokines as well as cytokine producing gene polymorphisms in IBS patients are very limited.,, Our study, though with a small sample size, can be considered a small step forward in that direction. In our study, the number of IBS patients showing increased levels of ILs as well as the levels of these ILs were significantly high in PIIBS group (2 weeks after the onset of past infection episode) as compared to NPIIBS subgroup, a finding echoed by other studies.,
However, many more studies, with large sample sizes including the molecular ones, need to be done to evaluate the role of various cytokines, apart from the established ones in IBS patients with special reference to the Indian population which can aid in development of better understanding of the etiopathogenesis, targeted therapy and better management of these patients.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, M L N Medical College, Allahabad - 211 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]
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