Allergic diseases have reached high proportions in the western world, affecting about 30% of the population. Notably, this increased prevalence is paralleled by an increase in the severity and spectrum of gastrointestinal hypersensitivity disorders, including cow’s milk allergy, eosinophilic esophagitis (EE), gastroenteritis, inflammatory bowel disease, and food protein-induced allergic diseases (Hogan and Rothenberg, 2004).
Eosinophils, a constitutive component of the gastrointestinal tract (GIT), play an essential role in allergic responses and parasitic infestations. The tissue density of these cells also increases in a variety of conditions of unclear etiology. With the exception of the esophageal squamous epithelium, in which no eosinophils are normally present, the population of normal eosinophils in the rest of the luminal gut is not well defined (Jawairia et al., 2012).
Primary eosinophilic gastrointestinal disorders are defined as disorders that selectively affect the GIT with tissue eosinophil-rich inflammation in the absence of known causes for tissue eosinophilia (e.g. drug reactions, parasitic infestations, and malignancy). These disorders include EE, eosinophilic gastroenteritis (EG), and eosinophilic colitis and are emerging with increasing frequency (Rothenberg, 2004). A major part in diagnosing these diseases is the finding of more than expected eosinophils in the gastrointestinal tissues, which usually constitutes an area of controversy, as eosinophilic inflammation of the GIT is considered to be a nonspecific finding (Masterson et al., 2011).
However, the presence of gastrointestinal eosinophilia does not always imply a pathogenic role for the eosinophils. There is increasing evidence that mast cells and eosinophils play a central role in mediating gastrointestinal allergic reactions. Gastrointestinal mast cells clearly play a role in many pathological effects associated with food hypersensitivity disorders (Brandt et al., 2003). It is well known that the sensitivity to food glycoproteins is caused by a series of interactions among T cells, B cells, antigen-presenting cells, and mast cells. Moreover, studies have demonstrated the presence of food-specific IgE antibodies and an increased number of gastrointestinal mast cells in patients with food hypersensitivity compared with normal controls (Ramsay et al., 2010). Furthermore, several studies have noted an increased number of mast cells in the mucosa of patients with gastrointestinal disorders such as irritable bowel syndrome, mastocytic enterocolitis, and systemic mastocytosis. The role of mast cells in the pathogenesis of these diseases has been fully appreciated and could provide avenues for new therapeutic options (De Winter et al., 2012).
We conducted this retrospective study to evaluate the mucosal mast cell density and activation and investigate its correlation with abnormal eosinophilic infiltration in pediatric EG.
Materials and methods
The material of the present study consisted of 45 cases diagnosed with EG derived from a group of Egyptian children. All cases were selected retrospectively from diverse sources during the period from March 2012 to March 2013. The studied material consisted of tissue specimens received by means of upper GIT endoscopic biopsies. The clinical data including age, sex, main complaint, and site of biopsy were recorded from the patients’ files. The paraffin blocks were recut at 5 µm thickness and stained with hematoxylin and eosin (H&E) for routine histopathological evaluation and eosinophil counting. Another section was prepared from each case and stained with mast cell tryptase (MCT) monoclonal antibody for mast cell identification and counting. In H&E sections, eosinophils were recognized by their brick red color, granular cytoplasm, and bilobed nucleus, which may appear small and unilobed or may even appear to be two minor nuclei depending on the level at which the bilobed nucleus has been sectioned. Some eosinophils can only be recognized by their cytoplasmic eosinophilic granules (Muller, 2008). In each section, eosinophils within the mucosa were counted in five random high-power fields (HPFs) at ×400 magnification with a light microscope, and the average number of eosinophils per HPF was calculated and recorded. The intensity of eosinophilic infiltration was graded as follows: mildly abnormal (10–20 eosinophils/HPF), moderately abnormal (21–50 eosinophils/HPF), and markedly abnormal (more than 50 eosinophils/HPF) (Whitington and Whitington, 1988).
For the immunohistochemical detection of mast cells, the standard avidin–biotin peroxidase complex technique was used. The unstained paraffin sections were cut on poly-L-lysine-coated slides and dried overnight. They were then deparaffinized in xylene and rehydrated in a series of graded alcohols (95, 85, and then 70%). The endogeneous peroxidase activity was blocked by adding 3% hydrogen peroxide for 5 min, followed by a wash with PBS for 5 min. To retrieve the antigens, the sections were boiled in a microwave oven in PBS at 100°C three times, 5 min each, and were allowed to cool for 30 min and then washed for 5 min with PBS. After treating the sections with a blocking serum for 20 min to block the nonspecific binding of immunoglobulins, they were incubated at room temperature with the primary antibody (antiMCT monoclonal antibody, clone AA1; Dako, Glostrup, Denmark) at 1 : 50 dilution. Next, the sections were washed twice for 5 min with PBS and incubated for 30 min with the biotinylated secondary antibody (Dako). Thereafter, the slides were washed twice for 5 min with PBS and incubated for 30 min in avidin–biotinylated peroxidase complex (Dako). Afterwards, the sections were washed twice with PBS for 5 min and treated with 3,3′-diaminobenzedine tetrahydrochloride (Dako) as a chromogen and incubated for 5 min at room temperature.
Finally, the sections were washed in distilled water, counterstained with hematoxylin, dehydrated, cleaned, and mounted. The positive control consisted of a tissue section of an allergic nasal polyp known to be positive for MCT, whereas the negative control was prepared by incubating a tissue section with PBS instead of the primary antibody.
The positively stained mast cells were identified by their brown cytoplasmic staining. Intact and degranulated mast cells were counted in five random HPFs at ×400 magnification using a light microscope and the average number of mast cells per HPF was calculated. The mast cells were classified as intact if they were dense and compact with unbroken cytoplasmic boundaries. Degranulated mast cells appeared with broken cell boundaries and discharged granules, which diffusely spread around the cells (Chang et al., 2009). The density of MCT-positive cells was scored as + (<20 cells/HPF), ++ (20–40 cells/HPF), and +++ (>40 cells/HPF). Moreover, the mast cell degranulation identified using MCT was graded as absent, mild (few extracellular granules), and extensive (moderate-to-marked degranulation in the maximally involved areas) (Chehade et al., 2007).
The numerical data of the present study were expressed as mean±SD. Statistical analysis was carried out using SPSS statistical package, version 11.5 (SPSS Inc., Chicago, Illinois, USA) and a P value less than 0.05 was set to represent statistical significance. The relationship between mast cell density and the studied parameters was evaluated using the χ2-test.
Among the 45 studied cases, there were 20 (44.4%) male and 25 (55.6%) female patients (male : female ratio 1 : 1.25). Their ages ranged from 3 months to 15 years, with a mean age of 5.9 years. The jejenum was the site of predominant localization of EG in our studied cases, representing 48.9% of cases, followed by the stomach (20%), duodenum (17.8%), and the terminal ileum (13.3%). The different clinical presentations among the studied cases were as follows: chronic diarrhea (53.3%), hematmesis (13.3%), failure to thrive (13.3%), persistent vomiting (11.1%), and abdominal pain (9%). Abnormal mucosal eosinophilic infiltration was mild in 33.3% of cases, moderate in 51.1% of cases, and marked in 15.6% of cases (Table 1 and Fig. 1).
Immunohistochemically, the mucosal mast cell density scores were + in eight (17.8%) cases with a mean mast cell count of 15.8±3.41, ++ in 28 (62.2%) cases with a mean count of 27.9±5.24, and +++ in nine (20%) cases with a mean count of 47.6±7.24 (Table 2 and Fig. 2). Mast cell degranulation was evident in 88.9% of the studied cases. Twenty-one (46.7%) cases showed mild degranulation, whereas 19 (42.2%) cases showed extensive degranulation (Table 3 and Fig. 3). There was a significant relationship between mucosal eosinophilic infiltration and mast cell density as identified by MCT (P=0.045) (Table 4). A statistically significant relationship was also found between mucosal eosinophilic infiltration and the degree of mast cell degranulation (P=0.026) (Table 5). Most cases with mild eosinophilic infiltration had absent and mild mast cell degranulation, representing 26.7 and 46.6%, respectively, whereas 85.7% of those with marked eosinophilic infiltration had extensive degranulation. No association was found in the present study between mucosal mast cell density and other clinical parameters such as age, sex, and anatomical site of EG.
Eosinophilic gastrointestinal diseases (EGID) are primarily polygenic disorders involving mechanisms that fall between IgE-mediated food allergy and cell-mediated hypersensitivity diseases (Rothenberg, 2004). In contrast to EE, the entity EG is not well characterized in children. It is considered a heterogeneous group of overlapping disorders with variation in the degree of disease involvement of the stomach and small intestine and further heterogeneity in the depth of involvement of the gut wall (Powell et al., 2010). In addition, many aspects of EG remain unclear. Thus, no definitive epidemiological features have been established, pathophysiological data are extremely limited, and therapeutic options are mainly based on empirical experience. There is a complete lack of controlled and randomized studies that clearly provide definitive information on EG (Lucendo and Arias, 2012), whereas in the case of EE, the most common EGID, evidence is emerging that the immunopathogenesis of EE involves a complex interplay of different cell types, including epithelial cells, lymphocytes, and mast cells (Vicario et al., 2010). From this point of view, this research was concerned with studying the mast cells in EG as regards their intensity, activation, and relation to eosinophilic infiltration.
From the 45 studied cases of EG, 37 cases representing 82.2% had more than 20 mast cells/HPF using MCT immunostaining, and according to a study conducted by Jakate et al. (2006), the presence of more than 20 mast cells/HPF was considered an increase in mast cells above normal levels. Brandt et al. (2003) have identified that eosinophils were not critical for the development of allergic diarrhea in their studied experimental cases and highlighted the critical role for mast cells instead, drawing attention to the analysis of mast cells in human gastrointestinal allergic disorders such as EGID, especially in patients with chronic diarrhea. Moreover, 88.9% of our studied cases had MCT-positive mast cell degranulation denoting their activation. The presence of activated mast cells suggests that the local mast cell-mediated inflammatory events contribute to the functional disturbances and clinical manifestations of EG and consequently their possible role in the studied disease (Shakoory et al., 2004). This finding is comparable to that of the study by Abonia et al. (2010), in which mast cell degranulation was also assessed by means of tryptase staining, and most of their studied EE patients (92%) demonstrated evidence of degranulation with ~20-fold increase in mast cell degranulation in EE patients versus normal individuals.
The current study revealed a statistically significant relation between mucosal eosinophilic infiltration and mast cell density (P=0.045). This is in agreement with the findings of Lombardi et al. (2007), who demonstrated a concomitant presence of mast cell infiltration in eosinophilic enteritis and interpreted this as the result of the local release of chemotactic factors by eosinophils. Moreover, Chehade et al. (2007) reported a similar finding in children with EE. In addition, several data suggest that mast cells can contribute to eosinophil-mediated inflammatory responses. Activation of mast cells leads to the release of leukotrienes, proteases, lipid mediators, and histamine that contribute to tissue inflammation and allow recruitment of eosinophils to the tissues. Moreover, eosinophils can regulate mast cell function in a paracrine manner. This two-way interaction between eosinophils and mast cells can pave the way for chronic inflammatory reactions in a variety of human diseases, including EG (Shakoory et al., 2004). Furthermore, this study revealed that the increase in mucosal infiltration with eosinophils was associated with a significant increase in MCT-positive mucosal mast cell degranulation (P=0.026), confirming the presence of intimate relation between mast cells and eosinophils and suggesting that not only eosinophils but also mast cells may play a role in the pathogenesis of EG. Similarly, Mir et al. (2014) reported the presence of augmented mast cell degranulation in patients with EGID in which increased eosinophils were present.
In agreement with our findings, Chehade et al. (2006) suggested that mast cells, especially the intestinal mast cells, have a potential role in EG and demonstrated that a potential role for eosinophils in increased intestinal permeability cannot be excluded. Moreover, mast cells have been shown to play a crucial role in increased intestinal permeability in various animal models. Mast cells have been shown to disrupt the intestinal barrier function during an enteric nematode infection, a disease with similarities to EG in its T-cell-mediated mechanism of inflammatory reaction and intestinal eosinophilia (McDermott et al., 2003). Moreover, Jakate et al. (2006) concluded that, in chronic intractable diarrhea, duodenal or colonic biopsy specimens may appear unremarkable on routine H&E staining, but increased mast cells may be demonstrated by MCT immunostaining, with the novel term mastocytic enterocolitis describing this condition, and added that similar increases in mast cells are not apparent in control populations or in patients with other specific diseases that cause chronic diarrhea.
The role of mast cells and their mediators have been studied in a number of gastrointestinal diseases that cause chronic diarrhea, such as inflammatory bowel disease (He, 2004), diarrhea predominant irritable bowel syndrome (Lee et al., 2013), and celiac disease (Frossi et al., 2016). Considering the role of mast cells in the pathogenesis of these diseases according to the diffuse tissue distribution, its close relationship with the enteric nervous system is inevitable. Mast cells in the mucosa of the terminal ileum, colon, and rectum have been proposed to be significantly elevated in most patients with irritable bowel syndrome and could be responsible for the altered visceral perception found in these patients (Barbara et al., 2004). The close proximity of the mucosal mast cells to the enteric nerves suggests that they have a potential involvement in the induced changes in nerve function and the development of visceral hypersensitivity (Park et al., 2006). In another disease such as Crohn’s disease, increased mast cells were associated with impaired motility, often due to the deleterious effects of mast cell proteases on the interstitial cells of Cajal (Wang et al., 2008) or the enteric nervous system (Barbara et al., 2006). As the end effector of the brain–gut axis, mast cells can translate the stress signals into liberation of proinflammatory mediators that can stimulate gastrointestinal nerve endings and affect its perception, alter intestinal motility, cause intestinal hyperpermeability, and in susceptible individuals with hyperreactive intestinal immune systems modify the inflammation (Farhadi et al., 2007).
In addition, the mast cell density has been evaluated in other forms of gastritis. Mysorekar et al. (2003) have reported that mast cell counts were significantly higher in the antral mucosa in chronic Helicobacter pylori gastritis. However, Moorchung et al. (2006) reported that mast cells did not have a significant role to play in chronic gastritis, whereas the severity of chronic gastritis was significantly correlated with the eosinophilic score. However, Mahjoub et al. (2009) have proposed a new entity termed mast cell gastritis for a group of children complaining of chronic abdominal pain with histologically normal gastric mucosal biopsies except for the increase in mast cells and recommended that the mast cell density should be addressed in any gastric biopsy.
The present study pointed that mast cells may play a role in EG. The presence of increased numbers of gastrointestinal mast cells together with eosinophils could serve as a useful tissue criterion in EGID. This provides a rationale for further evaluation of these potent effector cells and their possible role in EGID and raises the potential for mast cell-targeted therapy for EGID.
Conflicts of interest
There are no conflicts of interest.
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