Irritable bowel syndrome (IBS) is characterized by abdominal pain, bloating, and changes in bowel habits (diarrhea, constipation, or mixed) in the absence of any organic or structural abnormality. It is a prevalent functional disorder of the gastrointestinal (GI) tract (1), and has a significant effect on quality of life (2,3). IBS has been documented in about 35% of the children with recurrent abdominal pain and mainly affects children older than 5 years of age and adolescents (4). Various etiologies such as visceral hyperalgesia, disturbance of brain-gut interaction, autonomic and hormonal events, genetic and environmental factors, postinfectious sequels, and psychosocial disturbances have been implicated in IBS (1,5–8), but the precise pathophysiology remains unclear and difficult to determine in most cases.
It is worthy of note that a substantial proportion of cases are precipitated by GI infections, and postinfection IBS has been well documented (8–11). The authors speculated that enteric infection and mucosal inflammation leads to immune activation and may precipitate IBS in individuals with underlying genetic and psychosocial predisposition (9). Recent studies supported the hypothesis of immune activation in adults with IBS. These studies provided the following evidence: immune cell infiltration in the intestinal mucosa (12–14); enhanced expression of proinflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α (1,5,7,15,16); and significantly lower ratio of IL-10 to IL-12 levels in patients with IBS (1,16,17). Specific single nucleotide polymorphisms in TNF-α and IL-10 genes have been reported to appear more frequently in patients with IBS than in healthy controls (HCs), possibly resulting in an imbalance between the proinflammatory and anti-inflammatory cytokines (1,18,19).
Although immune activation in adults with IBS has been proposed, immune involvement in children with IBS remains to be determined. We aimed to explore the proinflammatory and anti-inflammatory cytokine profiles of children with IBS.
PATIENTS AND METHODS
Thirty-five children with IBS (15 girls and 20 boys; ages 7–18 years; mean age 13.5 years) and 25 healthy children as controls (11 girls and 14 boys; ages 6–16 years; mean age 12.5 years) were enrolled in the study. Informed consent was obtained from all of the participants.
Patients were recruited consecutively from the outpatient clinic of the Department of Pediatric Gastroenterology at the Chang Gung Memorial Hospital, Keelung and Taoyuan, whereas healthy volunteers were recruited through advertisements. All of the patients had chronic or relapsing symptoms of IBS consistent with the Rome II criteria (19). Patients were further categorized on the basis of their symptoms and predominant stool patterns (6). Patients with more than 3 bowel movements per day and loose/watery stool consistency were categorized as having diarrhea-predominant IBS (D-IBS, n = 17), patients with fewer than 3 bowel movements per week and hard or lumpy stools were categorized as constipation-predominant IBS (C-IBS, n = 7), and patients with an alternating bowel pattern were categorized as having mixed IBS (M-IBS, n = 11).
The symptoms were present for at least 3 months (range 3–58 months; mean 18 months). Patients with recent infections, major allergic diseases, food intolerance, or psychiatric disorders were not included in the present study. A comprehensive diagnostic workup including hemogram, biochemistry, abdominal sonography, and repeated stool testing did not reveal acute infections or any evidence of structural anomaly to be the cause of the symptoms. Patients who had used probiotics, antibiotics, analgesics, or immunosuppressive drugs within the last 1 month were also not included in the study.
The present study was approved by the Chang Gung Memorial Hospital human ethics committee (institutional review board).
Assessment of Symptoms
To assess abdominal symptoms, all of the study participants completed a self-report bowel disease questionnaire. The visual analog scale 0 to 10 was used to quantify the severity of pain in the last 3 months (20). We defined a scale of 0 to 3 as mild, a scale of 4 to 6 as moderate, and a scale of 7 to 10 as strong intensity pain. Questionnaires completed by the patients served as a record of their demographic data, bowel habits, IBS duration, symptom frequency per month (3–5 days, 6–10 days, 11–20 days, and >20 days), and symptoms related to IBS, including emotional changes, sleep disturbance, decreased physical/social functioning, appetite loss, and effect on school performance.
Isolation of Peripheral Blood Mononuclear Cells
Blood samples (8–10 mL) were obtained from each subject at enrollment. Human peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats on Lymphoprep (Nycomed, Oslo, Norway) gradients by centrifugation. After washing, the cells were resuspended at a concentration of 1 × 106cells per milliliter in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum.
The concentration of PBMCs was adjusted to 106 cells per milliliter in complete medium, and the cells were transferred to 24-well plates. The cells were either nonstimulated or stimulated with Escherichia coli lipopolysaccharide (LPS) (SIGMA, L4391). Duplicate cultures at a final LPS concentration of 1 and 5 ng/mL were prepared and incubated for 24 hours at 37°C in a humidified 5% CO2 atmosphere. The supernatants were collected, pooled, and stored at −20°C until analysis using enzyme-linked immunosorbent assay.
Enzyme-linked Immunosorbent Assay
Concentrations of IL-10, TNF-α, and IL-6 in the culture supernatants were determined using commercially available kits according to the manufacturer's instructions (R & D Systems: [Minneapolis, MN] IL-10, D1000B; TNF-α, DTA00C; IL-6, D6050). Optical density was measured at a wavelength of 450 nm and a reference wavelength of 590 nm, and values were correlated linearly with cytokine standards. The sensitivity limit of the assay was 5 pg/mL.
Statistical analyses were performed using 1-way analysis of variance to compare baseline and LPS-stimulated cytokine levels in the IBS subgroups and the HCs. Analysis of variance was also performed to compare baseline and various LPS-stimulated cytokine levels in patients with IBS categorized by sex, symptom frequency per month and other related symptoms, and pain severity. P < 0.05 was considered statistically significant. Box plots indicate median values at the 25th and 75th percentiles, respectively, and the error bars indicate the 10th and 90th percentiles. Statistical calculations were performed using SPSS 17.0 for Windows (SPSS Inc, Chicago, IL).
Demographic Data and Symptom Characteristics of Children With IBS
Sex, mean age, IBS duration, pain intensity, frequency, and IBS-related symptoms in each subgroup are shown in Table 1. Twelve of 35 (34%) patients reported strong pain intensity, and 11 of 35 (31%) reported an adverse effect on school performance. Every patient had at least 1 symptom related to IBS, including emotional changes, appetite loss, sleep disturbance, and effect on physical functioning (Table 1).
Baseline and E coli LPS–induced Cytokine Production
Patients with IBS had lower baseline IL-10 levels compared with HCs (82.6 ± 46.41 pg/mL vs 214.65 ± 76.23 pg/mL, P = 0.089). After separate stimulations with 1 and 5 ng/mL of E coli LPS, the patient group had significantly lower IL-10 levels compared with HCs (1485.94 ± 167.11 pg/mL vs 2699.75 ± 252.10 pg/mL, P = 0.001, and 1825.06 ± 184.77 pg/mL vs 2984.32 ± 246.88 pg/mL, P = 0.001, respectively). Children with IBS showed higher baseline and LPS-induced TNF-α and IL-6 levels compared with HCs, but the differences were not statistically significant (Table 2).
Analysis of the 3 IBS subgroups showed that IL-10 levels in each group were significantly lower under LPS-stimulated conditions compared with HCs (P < 0.05) (Fig. 1A); however, there was no difference between the patient subgroups. Moreover, there were no significant differences in TNF-α and IL-6 levels among the 3 IBS subgroups and HCs (Fig. 1B and C).
Association Between Cytokine Levels and IBS Symptoms
Compared with patients with mild/moderate pain intensity, patients reporting strong pain intensity displayed lower baseline IL-10 (6.84 ± 2.01 pg/mL vs 157.62 ± 67.17 pg/mL, P = 0.126), and 1 ng/mL LPS-induced IL-10 levels (1246.84 ± 100.46 pg/mL vs 1637.72 ± 245.16 pg/mL, P = 0.160) (Fig. 2A and B). The 5 ng/mL LPS-induced IL-10 levels in these patients were significantly lower (1328.57 ± 103.89 pg/mL vs 2025.27 ± 259.54 pg/mL, P = 0.025) (Fig. 2C). However, no differences were observed in TNF-α and IL-6 levels between patient groups with strong and mild/moderate pain intensity.
There were no significant differences in any of the 3 cytokine levels between patients and the HCs with respect to sex, symptom frequency per month, and other symptoms related to IBS, including emotional changes, sleep behaviors, physical functioning, appetite loss, social activity, and effect on school performance.
IBS is prevalent in adults as well as in children and adolescents (21,22). Girls are more frequently affected than boys in Western countries (sex ratio, female:male 1.2:3.1) (22); sex hormones are thought to play a role (15,23). However, our series in children did not demonstrate this female preponderance (female:male 15:20). Differences in ethnicity, food habits, external environment, and lower sex hormone levels in children possibly contributed to this discrepancy. Similar to previous reports, our result showed IBS to have a significant effect on a child's quality of life, with an interruption of normal school and social activities (2,24) (Table 1).
Recently, the role of infection, inflammation, and immune dysfunction in patients with IBS has been emphasized (5,10,15,16). PBMCs display responses similar to those of the lamina propria mononuclear cells, with regard to proliferation and cytokine secretion when exposed to bacterial antigens (25). In the present study, we used 1 and 5 ng/mL LPS for stimulation of PBMCs (106/mL) for 24 hours to investigate concentration-dependent effects of bacterial antigens on cytokine production. The results demonstrated mild increase in the levels of the proinflammatory cytokines (TNF-α, IL-6), and the anti-inflammatory cytokine (IL-10) secretion was lower in children with IBS versus the HCs. Because cytokines can influence the function of epithelial cells, smooth muscles, and the enteric nervous system, changes in the cytokine profile of patients with IBS can exacerbate changes in secretion, permeability, motility, and sensory output, and generate IBS symptoms (26,27).
IL-10 is considered to be an anti-inflammatory cytokine involved in the regulation of immune and inflammatory responses. A growing body of evidence has demonstrated that changes in the genetic makeup or expression of cytokines play a critical role in the inflammatory response in the gut (8,16,18). Thus, individuals predisposed to producing lower amounts of IL-10 may be at a higher risk for developing IBS symptoms. Several studies (8,18,28) have found that patients with IBS have a higher prevalence of the low-expressive IL-10 genotype compared with HCs. Although polymorphisms in the cytokine gene are less specific in patients with IBS, this information may be more valuable if taken in conjunction with the cytokine changes in PBMCs. Our study showed that LPS-stimulated PBMCs from children with IBS had markedly lower IL-10 levels, suggesting that children with IBS may have defects in the secretion of regulatory cytokines or in the recruitment of regulatory cells. Genetic predisposition may be important in this regard and warrants further investigation.
Recent studies (1,5,7,16,17) in adults have reported that the cytokine secretion in patients with IBS was skewed toward a proinflammatory profile (eg, TNF-α, IL-6, and IL-8). van der Veek et al (18) found that the heterozygous (G/A) genotype that encodes for high TNF-α secretion was significantly more prevalent (41% vs 26%) in the IBS group compared with controls. TNF-α and IL-6 levels in our IBS group were elevated both in baseline and LPS-stimulated PBMCs compared with HCs, although without statistical significance, favoring enhanced proinflammatory state.
It is recognized that stressful life events are associated with symptom exacerbation in a majority of patients with IBS (29–31), and that symptoms of heartburn, headache, backache, and fibromyalgia increase as the severity of IBS increases (6). Because production of cytokines by PBMCs has been shown to be altered by psychological stress (32,33), to determine the cause of enhanced proinflammatory cytokines, psychological stressors need to be considered along with GI tract inflammation. The shorter disease duration (most cases <2 years) and less stressful events in children may have contributed to the considerably weaker proinflammatory response than that in adults.
Several studies have attempted to evaluate the correlation between immune activation and clinical presentation of IBS. Shulman et al (34) demonstrated that GI inflammation correlated with the degree to which pain interfered with daily activities. Ohman et al found that patients with lower CD25+ T cells in the blood experienced more IBS symptoms (12); and PBMC-mediated cytokine production, in particular TNF-α and IL-6, has been reported to reflect the severity of inflammation in patients with IBS (35). However, the correlation of cytokine levels with clinical severity remains controversial (35,36). Immune activation and proinflammatory cytokine changes in patients with IBS have been reported to correlate with female sex, abdominal bloating frequency, more than 3 diarrhea occurrences per day, urgency, pain intensity, and anxiety (1,5,15). In our study, LPS-induced IL-10 levels were significantly lower in patients with severe pain intensity compared with patients with mild to moderate pain intensity, indicating that IL-10 may play a role in clinical severity. However, TNF-α and IL-6 levels did not correlate with pain intensities in our patient groups. Owing to the small sample size of our study, the correlation between IL-10 and pain intensity should be interpreted with caution. Further studies are required to confirm this result.
Our results show that children with IBS have lower baseline and significantly lower LPS-stimulated IL-10 levels. Additionally, 5 ng/mL LPS-stimulated IL-10 levels were significantly lower in patients with stronger pain intensities compared with milder cases. These results suggest that defects in immune modulation may be one of the mechanisms that contribute to the occurrence of IBS in children.
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