More than ten years ago, O’Sullivan et al[26] evaluated the number of plasma cells, lymphocytes, eosinophils, neutrophils, and macrophages in a case-control study. Specifically, each cell type was semiquantitatively graded in hematoxylin-and-eosin-stained sections of the entire colon, and possible increases in the number of mast cells (MCs) in the colon of IBS patients compared with controls were examined using a monoclonal mouse antibody for human MC tryptase (AA1). Other than MCs, increases in cellular infiltrate were not observed in the IBS group, and the number of MCs was significantly increased in the cecum of IBS patients compared with controls.

Similarly, in 2008, Piche and coworkers[27] aimed to examine associations between fatigue, depression, and the MCs of the colonic mucosa in IBS by comparing the numbers of CD3-positive intraepithelial T lymphocytes, MCs, plasma cells, eosinophils, and neutrophils in cecal biopsies taken during colonoscopy. There was not a significant difference in the numbers of intraepithelial lymphocytes, plasma cells, eosinophils, or neutrophils between IBS patients and healthy controls, but the MC numbers per high-power field were significantly higher in IBS patients than in healthy controls (9.3 vs 4.0, P = 0.001). Furthermore, the number of MCs correlated with the severity of fatigue and depression scores in IBS patients but not in healthy controls[28].

With regard to the small bowel, Walker et al[29] examined the MC, eosinophil, and intraepithelial lymphocyte populations in duodenal biopsies of subjects with IBS and functional dyspepsia. Their study showed a significant increase in the number of intraepithelial lymphocytes in biopsies from the duodenum in patients with IBS-C. However, this increase was not observed in the second part of the duodenum. Nevertheless, MC counts were also higher in IBS cases in both the first and second parts of the duodenum, but this difference was only significant for constipation-predominant IBS[28]. Interestingly, the eosinophil counts in this study did not differ between IBS patients and controls in either the first or second part of the duodenum[28].

To date, a significant difference in the numbers of plasma cells, neutrophils, or eosinophils has not been demonstrated among IBS cases[28]. Importantly, increases in eosinophils were not identified in IBS, but the eosinophil counts were elevated in individuals with functional dyspepsia[29]. Functional dyspepsia and IBS demonstrate significant overlap in cross-sectional surveys[30], despite attempts to classify them separately, and a biomarker to predict the presence of IBS remains elusive[31]. Therefore, this histopathologic marker may serve to distinguish the two conditions[28].

MCs are innate immune cells involved in food allergies, wound healing, and protection against pathogens[32]. Their functional activation consists of a degranulation process, leading to the release of various compounds, such as histamine, tryptase, and chymase[32].

More than 40 years ago, Hiatt and Katz[33] were the first to demonstrate MC infiltration in the muscular layer of full-thickness colonic biopsy samples from four patients with “spastic colitis”[34]. Increases in the number of mucosal MCs have been observed in IBS patients in the rectum[35,36], rectosigmoid colon[37,38], descending colon[39-44], ascending colon[36], cecum[45], terminal ileum[25,36,46], jejunum[47-49], and duodenum[29,50] (Figure 2).

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Figure 2 Immunohistochemistry for tryptase showing increases in the number of mast cells in the colonic mucosa in inflammatory bowel disease. A: Irritable bowel syndrome patient; B: Control (× 40 magnification). Courtesy of Giancarlo Pompei, personal data.

However, discrepancies in data obtained from these studies could be due to sex-specific differences, bowel preparation artifacts, fixation protocols, tissue orientation, sample size, or IBS-related recruitment criteria[32,43,44,51]. Furthermore, clinical studies have also yielded conflicting evidence correlating MC numbers with the onset of abdominal pain[32].

The number of functionally active MCs (exhibiting changes in the release of tryptase and histamine), rather than the absolute number of MCs, plays a pivotal role in IBS[43,50]. Consequently, the role of MCs in IBS may be affected by cells that are functionally active and form close connections with enteric and extrinsic nerve terminals, thus determining visceral hypersensitivity and altered gut function[47].

The aforementioned inflammatory changes described in the mucosa of IBS patients show that immune activation may play a role in IBS pathophysiology[34]. Mucosal T- and B-type lymphocytes are also part of the gut adaptive immune response to pathogens[32].

An increased density of T lymphocytes in the mucosa of IBS patients has been widely demonstrated. Specifically, the T-cell density is higher in the rectum[35,36,52], rectosigmoid colon[53], colon[38,48,54], cecum[26], jejunum[55], and duodenum[56]. Like for MCs, several studies showed that IBS patients exhibit a normal lymphocyte density in different intestinal tissue segments[19,36,38,53,57-61]. These discrepancies may be due to differences in the immunostaining techniques, quantification methods, and IBS-related recruitment criteria (Figure 3)[32].

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Figure 3 Immunohistochemistry for CD3 showing increase in the number of intraepithelial T-lymphocytes in the large bowel in inflammatory bowel disease. A: Irritable bowel syndrome patient; B: Normal distribution of T-lymphocytes, which are mainly distributed within the lamina propria of the large bowel of a control patient (× 20 magnification). Courtesy of Giancarlo Pompei, personal data.

Conversely, the density of B-cells in the rectum[19,36], colon[19,43,53,62], cecum[19,20], ileum[36], or jejunum[60] did not differ in IBS patients. However, Forshammar and coworkers[62] and others[32] found a decrease in secretory B cells in the colon (Figure 4).

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Figure 4 Immunohistochemical staining for CD20. An equivalent distribution of B lymphocytes in the lamina propria was seen in the large intestine. A: Patient with irritable bowel syndrome; B: Control patient (× 20 magnification). Courtesy of Giancarlo Pompei, personal data.

With regard to intraepithelial lymphocytes, several discrepancies have been reported in studies assessing the density of these cells in IBS patients. An increase in density has been demonstrated in the rectum[35,36,57], colon[57], jejunum[55,49], and duodenum[56], but these increases were not confirmed by other groups[20,32,45,49,59,61].

Dendritic cells are antigen-presenting cells that are usually located at the surveillance interfaces of the human body, such as the skin or mucosa, and play a pivotal role in the generation and regulation of immune responses[63]. In fact, they represent the link between allergen uptake and the clinical manifestations of intestinal inflammation[64,65]. Furthermore, the gut also harbors abundant macrophages. These cells do not function as typical antigen-presenting cells and lack the cellular machinery for the production of pro-inflammatory cytokines and induction of potent adaptive immune responses. However, they show very potent phagocytic activity[65].

In a Trichinella spiralis mouse model of PI-IBS, Long and coworkers[66] reported numerical and phenotypic alterations in the lamina propria dendritic cells following acute T. spiralis infection. In their study, the lamina propria dendritic cells expressed increased levels of costimulatory molecules and exhibited a greater ability to migrate and induce CD4⁺ T-cell proliferation[66]. Consequently, these changes favored increased levels of pro-inflammatory interferon-γ, interleukin-23, and tumor necrosis factor-α production in the so-called “PI-IBS stage”[66,67].

With regard to macrophages, the numbers of resident CD68⁺ macrophages are reduced in PI-IBS cases following Campylobacter jejuni infection, probably due to the cytotoxic nature of the pathogen inside host cells[50]. Similarly, Shigella spp.[68,69] and Salmonella infections have also been shown to be involved in PI-IBS, and both of these organisms are intracellular pathogens that induce phagocytosis by macrophages[70,71]. Furthermore, Salmonella seems to be less cytotoxic to macrophages[72] and also causes a marked interleukin-18 response[72] with important implications in exerting paracrine effects on surrounding immune cells (inducing interferon-γ expression). These changes result in increased levels of activated T cells in the infected intestine[47,52,54,55,57].

Enteroendocrine cells (residing among the epithelial cells of the mucosa in all gut segments, with the exception of the esophagus) secrete multiple regulatory molecules that control several functions, such as postprandial secretion and motility[12,73,74]. Animal experimental studies have demonstrated abnormalities in the function of enteroendocrine cells in the setting of gastrointestinal infection[73]. Enteroendocrine cells seem to be involved in visceral hypersensitivity, disturbed gastrointestinal motility, and abnormal gut secretion[12] that patients with IBS usually present[75-77].

In fact, visceral hypersensitivity has been shown in the colon of IBS patients[78-85], but the correlation of this disturbance with the severity of abdominal pain is currently poorly understood[12]. Some authors hypothesize the involvement of a peripheral mechanism in visceral hypersensitivity in IBS[86-88]. Because the gut mucosa can produce high levels of serotonin[88], a reduction in serotonin impairs intracellular uptake and degradation in the gut epithelial cells and consequently increases serotonin availability of in the gut mucosa[89-92]. Therefore, the amount of serotonin available at its receptors is markedly increased[12,87,88]. Due to this mechanism, the development of visceral hypersensitivity in PI-IBS patients may be due to the increase in serotonin at the 5-hydroxytryptamine 3 receptors of the sensory neurons of the enteric nervous system.

Dysmotility has also been shown in the small and large bowel of IBS patients, as evidenced by the involvement of cholecystokinin, ghrelin, secretin, serotonin, and peptide YY[12]. Both esophageal motility abnormalities and abnormal gastric emptying have been observed in IBS patients with conflicting results[93-108]. Specifically, IBS-C patients exhibit delayed gastric emptying, whereas accelerated gastric emptying was observed in IBS-D patients[77,98]. In this setting, ghrelin was shown to stimulate gastric and small- and large-bowel motility[106-117]. Conversely, serotonin relaxes the stomach through a nitrergic pathway and consequently delays gastric emptying[118-120]. Moreover, cholecystokinin[121-123] and secretin[124,125] were shown to relax the proximal stomach, which inhibited gastric emptying in a manner similar to secretin; furthermore, small-bowel transit was also found to be delayed overall in IBS-C patients and accelerated in IBS-D patients[126-131], but conflicting results have been reported[132-139].

Ghrelin, which is involved in the stimulation of small-bowel motility, and peptide YY, a regulator of the ileal brakes[140-145], play pivotal roles in gastric emptying by stimulating the absorption of water and electrolytes and inhibiting prostaglandin E2 and vasoactive intestinal polypeptide[146-148]. Therefore, ghrelin cell density is reportedly low in the stomach, and that of peptide YY is reported to be high in the ileal mucosa of IBS-C patients, whereas the ghrelin cell density is reported to be high in the stomach, and that of secretin is reported to be low in the duodenum of IBS-D patients[12]. Furthermore, colorectal transit was found to be delayed in IBS-C patients and accelerated in IBS-D patients[80,125,126,149-154], but contradictory results have been reported[12,105,152,154-176].

Finally, abnormal gastrointestinal secretion is common in IBS patients. Among the abnormalities in the enteroendocrine cells in IBS patients, low levels of duodenal cholecystokinin (which stimulates the secretion of digestive enzymes from pancreatic exocrine glands) and secretin (which stimulates pancreatic bicarbonate and fluid secretions)[122,123], as well as high levels of ileal peptide YY (which stimulates the absorption of water and electrolytes), were reported in IBS-C patients[12].

The term “mucosal barrier” was adopted by Cummings et al[177] in 2004 to describe “the complex structure that separates the internal milieu from the luminal environment, consisting of the vascular endothelium, the epithelial cell lining, and the mucus layer, next to which digestive secretions, immune molecules, cell products such as cytokines, inflammatory mediators, and antimicrobial peptides, are found, mainly produced by Paneth cells in the crypts of the small intestine”[177,178].

Conversely, impaired intestinal permeability is defined as “an altered permeability being nontransiently changed compared to the normal permeability leading to a loss of intestinal homeostasis, functional impairments and disease”[178]. Increases in the numbers of MCs in the gut of IBS patients were found to be related to changes in gut permeability[32,179-181]. In IBS patients, the assessment of permeability via the urinary recovery of orally administered markers has demonstrated increases in the permeability of the small and in the large bowels[45,182-184], but results have been contradictory[185-187]. Furthermore, rectal permeability was also reportedly increased in IBS-D patients following exposure to MC tryptase[188]. Finally, recent studies of the permeability of the epithelial barrier have reported a decrease in the colonic expression of the tight junction proteins occludin, claudin-1, and zonula occludens-1 in IBS patients[42,189] (Figure 5).

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Figure 5 Immunohistochemistry for chromogranin A showing increased expression in nerve terminals at the level of the basal membrane in the large intestine in inflammatory bowel disease. A: Irritable bowel syndrome patient; B: Control patient (× 40 magnification). Courtesy of Giancarlo Pompei, personal data.

Several aliments, as well as microbiota and bile acids, have been proposed to cause low-grade inflammation and altered permeability in IBS. In fact, in some patients, IBS was related to food allergy[190]. Furthermore, endogenous triggers, such as MC-derived histamine, proteases, and eicosanoids, could increase intestinal permeability, either directly or via the stimulation of neurons of the enteric nervous system[183]. Moreover, serotonin was also identified as an endogenous trigger of pain, inflammation, and increased permeability in IBS[40]; therefore, LX1031, an oral inhibitor of tryptophan hydroxylase, the principal enzyme needed for mucosal serotonin synthesis, has been successful for treatment of patients with non-constipating IBS[191].

Few studies have investigated the role of calcitonin gene related peptide and substance P. Wang et al[37] investigated the incidence of IBS in patients who had recovered from bacillary dysentery by focusing on neuroimmunologic changes, including changes in interleukins, MCs, neuropeptides, and the relationship between MCs and intestinal nerves[50]. The density of substance P-immunoreactive fibers was increased in both the ileal and the rectosigmoid samples of IBS patients[37], but the density of calcitonin gene related peptide-containing fibers remained unchanged. Palsson and coworkers[192] reported similar findings, and Kerkhoffs and coworkers[193] reported an increase of rectal substance P. However, these findings have not been universal[192,194], possibly reflecting region-specific discrepancies[51].

Neuronal plasticity in the enteric nervous system has also been investigated[195]. Akbar et al[38] investigated the capsaicin receptor transient receptor potential vanilloid 1-immunoreactive nerve fibers in colonic biopsies from patients with IBS. Specifically, they demonstrated that the number of nerve fibers exhibiting immunoreactivity for substance P and transient receptor potential vanilloid 1 was increased in IBS patients. Moreover, the number of these fibers did not differ by IBS subtype, but significantly correlated with patient pain scores[50,196].

Nerve growth factor has also been suggested to play a central role in promoting the growth and differentiation of primary afferent fibers[50]. Specifically, its expression was found to be markedly increased in rectal biopsies from pediatric[197] and adult IBS patients[198], suggesting both the sprouting of sensory afferent fibers expressing transient receptor potential vanilloid 1 and increases in receptor sensitivity in IBS, which consequently induced visceral hyperalgesia[50]. More recently, Dothel et al[199] also showed that nerve fiber density and sprouting, as well as the expression of nerve growth and neurotrophic tyrosine kinase receptor type 1, are significantly increased in the mucosal tissues of patients with IBS. Mucosal mediators participate in these neuroplastic changes.

Finally, the morphology of enteric glia, which are known to regulate intestinal barrier integrity and neuronal activity[200] has only been examined in one study of human intestinal biopsy samples from IBS patients and was found to be unchanged[50,201].