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Intraepithelial Lymphocyte Eotaxin-2 Expression and Perineural Mast Cell Degranulation Differentiate Allergic/Eosinophilic Colitis From Classic IBD

Torrente, Franco*; Barabino, Arrigo; Bellini, Tommaso; Murch, Simon H.

Journal of Pediatric Gastroenterology and Nutrition: September 2014 - Volume 59 - Issue 3 - p 300–307
doi: 10.1097/MPG.0000000000000432
Original Articles: Gastroenterology

Objectives: Allergic colitis shows overlap with classic inflammatory bowel disease (IBD). Clinically, allergic colitis is associated with dysmotility and abdominal pain, and mucosal eosinophilia is characteristic. We thus aimed to characterise mucosal changes in children with allergic colitis compared with normal tissue and classic IBD, focusing on potential interaction between eosinophils and mast cells with enteric neurones.

Methods: A total of 15 children with allergic colitis, 10 with Crohn disease (CD), 10 with ulcerative colitis (UC), and 10 histologically normal controls were studied. Mucosal biopsies were stained for CD3 T cells, Ki-67, eotaxin-1, and eotaxin-2. Eotaxin-2, IgE, and tryptase were localised compared with mucosal nerves, using neuronal markers neurofilament protein, neuron-specific enolase, and nerve growth factor receptor.

Results: Overall inflammation was greater in patients with CD and UC than in patients with allergic colitis. CD3 T-cell density was increased in patients with allergic colitis, similar to that in patients with CD but lower than in patients with UC, whereas eosinophil density was higher than in all other groups. Eotaxin-1 and -2 were localised to basolateral crypt epithelium in all specimens, with eotaxin-1+ lamina propria cells found in all of the colitis groups. Eotaxin-2+ intraepithelial lymphocyte (IEL) density was significantly higher in allergic colitis specimens than in all other groups. Mast cell degranulation was strikingly increased in patients with allergic colitis (12/15) compared with that in patients with UC (1/10) and CD (0/1). Tryptase and IgE colocalised on enteric neurons in patients with allergic colitis but rarely in patients with IBD.

Conclusions: Eotaxin-2+ IELs may contribute to the periepithelial eosinophil accumulation characteristic of allergic colitis. The colocalisation of IgE and tryptase with mucosal enteric nerves is likely to promote the dysmotility and visceral hyperalgesia classically seen in allergic gastrointestinal inflammation.

*Department of Paediatric Gastroenterology, Addenbrooke's Hospital, Cambridge, UK

Department of Paediatric Gastroenterology, Gaslini Institute, Genoa, Italy

Division of Metabolic and Vascular, Warwick Medical School, University of Warwick, Warwick, UK.

Address correspondence and reprint requests to Professor Simon Murch, Division of Metabolic and Vascular Health, Warwick Medical School, Clifford Bridge Road, Coventry CV2 2DX, UK (e-mail:

Received 13 February, 2014

Accepted 2 May, 2014

The present study was supported in part by a grant to S.M. from the UK Food Standards Agency and by an unrestricted grant from the Eulie Peto Legacy.

S.M. has given lectures at meetings sponsored by the manufacturers of hypoallergenic formulae, including Danone and Mead Johnson, and has been a member of advisory panels for both companies. The other authors report no conflicts of interest.

Gastrointestinal hypersensitivity disorders include both immediate immunoglobulin E (IgE)–mediated and delayed non–IgE-mediated reactions, in which symptoms may not be so clearly linked to food ingestion. Non–IgE-mediated disorders in childhood include failure to thrive or chronic diarrhoea because of enteropathy or colitis, eczema, or rhinitis (1).

Allergic colitis is induced by non–IgE-mediated responses to dietary antigens. Its presentation with rectal bleeding and disruption of intestinal motility overlaps with classical inflammatory bowel diseases such as Crohn disease (CD) and ulcerative colitis (UC), but the clinical course is generally less severe. A well-recognised presentation is in young infants, who, although usually systemically well, pass loose stools containing significant amounts of blood (2,3). Endoscopic features are less severe than classical colitides, with prominence of swollen erythematous lymphoid follicles, whereas histological findings are dominated by eosinophilia within the lamina propria and epithelial compartment (2,4). Although some infants may have an associated systemic component, manifesting a partial anaphylactic response in the food protein–induced enterocolitis syndrome (FPIES), most remain systemically well and the condition may settle spontaneously (5). There is a characteristic associated minor developmental immunodeficiency with low IgA and IgG subclasses, which normalise with age (6,7). The underpinning immunological issue is the impaired development of regulatory T cells and cytokines, in particular TGF-β (8). Many infants sensitise in this manner despite exclusive breast-feeding, as a result of a primary deficiency in establishing oral tolerance to low doses, because of inefficient innate immune responses to the gut flora (9). Thus, clinical resolution has been established using the probiotic Lactobacillus rhamnosus GG (10).

A second peak of allergic colitis occurs later in childhood, and the disorder can also be identified in adults (11). A historical case of milk allergic colitis occurred in Charles Edward Stuart (Bonnie Prince Charlie), 18th-century Jacobite pretender to the English, Scottish, and Irish thrones, whose “bloody flux” resolved on a milk-free diet (12). In some patients there is an eosinophilic colitis limited to the rectum and sigmoid (11) and in others a more extensive colitis, which may overlap with UC (13). It is also notable that a significant proportion of people diagnosed as having UC show clinical response to cow's milk exclusion and may relapse on challenge (13), implying that a part of the mucosal pathogenesis in such people may include non–IgE-mediated responses to cow's milk. Tissue eosinophilia may variably be found in IBD, and it is notable that higher mucosal eosinophilia occurs in patients with UC with concomitant skin prick reactivity (14).

The concept of food allergic dysmotility has recently emerged, in which dietary antigens (commonly cow's milk, soya, or wheat) induce gastro-oesophageal reflux, constipation, or visceral hyperalgesia related to mucosal infiltration of mast cells and eosinophils (15). Induction of colonic sensitisation to ovalbumin in mice, followed by challenge, led to a state of visceral hyperalgesia, in which maximal activation of anterior cingulate cortex neurones occurred with minimal colonic distension (16). As a consequence of neural remodelling, such mast cell sensitisation may induce dysmotility persisting after the triggering food has been withdrawn (17).

The common histological characteristic of intestinal food allergic reactions is local eosinophilic infiltration, which can occur in the gastrointestinal tract and can involve all depths of the intestinal wall (18). Eosinophilic recruitment is regulated through a network involving TH2 T cells, cytokines such as IL-5 and IL-13, and the eotaxin group of chemokines (19). Recent evidence also implicates the involvement of invariant chain natural killer cells (iNKT) in TH2-type reactions, which respond in a CD1d-restricted manner to lipid airborne or dietary antigens (20,21).

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Diagnostic ileocolonoscopy was performed in investigation of patients referred to 2 units (Gaslini Institute, Genoa, Italy; and Royal Free Hospital, London, UK) who had gastrointestinal symptoms such as altered bowel habit, blood in stools, chronic unexplained abdominal pain, or weight loss to exclude IBD. In all patients, biopsies for histological assessment were taken along the colon and additional samples were immediately snap frozen for later analysis. Permission for taking additional research biopsies had been granted by the local research ethics committees, and in all cases informed consent was obtained from the parents.

The aim of the study was to characterise the mucosal changes in patients with allergic colitis compared with those in patients with classic IBD (CD or UC) and histologically normal controls. The children with allergic colitis (n = 15) were diagnosed on the basis of their endoscopic features, including erythema and prominent lymphoid follicles, a characteristic eosinophilic infiltrate on histology without features diagnostic of IBD, and a complete response to exclusion diet without medications (excluding milk alone or milk plus soya). Those presenting at younger than age 1 year (n = 4) had rectal bleeding and loose stools and did not manifest significant abdominal pain. They did not demonstrate the systemic features characteristic of food protein–induced enterocolitis syndrome. By contrast, those presenting after the first year (n = 11) usually had abdominal pain in addition to loose stools, without overt blood loss. Children with CD (n = 10) and UC (n = 10) were diagnosed by standard criteria, including a combination of endoscopic and histological findings. The histologically normal controls (n = 10) were children who underwent colonoscopy to exclude disorders such as IBD, in whom investigations proved to be normal and mucosal biopsies were within histologically normal limits.

Clinical details of the patients studied are provided in Table 1. Those with allergic colitis were similar in age to the histologically normal controls but were significantly younger than those with UC and CD. They showed similar mean haemoglobin to normal controls and were also not significantly different from the patients with UC and CD. The mean erythrocyte sedimentation rate of the allergic colitis group was significantly higher than controls but not significantly different from that in patients with UC and CD. Data on C-reactive protein could not be compared because of different assay systems and normal ranges in the 2 units. Values were normal in all children with allergic colitis and increased in all of those with CD. Compared with controls, the children with allergic colitis showed lower albumin concentrations (mean 38.2, range 23–46 g/L vs 42.1 g/L, 38–43, P < 0.05). Raised peripheral eosinophil counts (>0.4 × 109/L) were identified in 2 of 12 controls, 0 of 10 patients with UC, 0 of 10 patients with CD, and 4 of 15 patients with allergic colitis. The mean (retrospectively calculated) Pediatric Ulcerative Colitis Activity Index score for the children with UC was 33 (range 10–55), consistent with mild to moderate disease. The Pediatric Crohn's Disease Activity Index had not been calculated contemporaneously for the children with CD, and insufficient data are available for secure retrospective calculation. All were assessed clinically as showing mild or moderate disease activity. None of the patients were receiving corticosteroids before colonoscopy.



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Tissue Assessment

Serial sections of the snap-frozen tissues were cut and stained either by haematoxylin and eosin (H&E) or by immunohistochemistry. Inflammation in the H&E specimens was scored blinded using a modified O’Morain score, as reported earlier (22,23). “0” was histologically normal, “1” mononuclear cell infiltration, “2” mononuclear infiltration with crypt distortion or mucosal atrophy, “3” mild active inflammation ± crypt abscesses, mild goblet cell depletion, or architectural change, “4” moderate active inflammation with erosions and architectural changes, and “5” severe active inflammation with epithelial ulceration.

Biotin/avidin immunohistochemistry (Vectastain Elite, Vector Laboratories, Peterborough, UK) was used for peroxidase immunohistochemistry, with inactivation of endogenous peroxidase using hydrogen peroxide. Primary antibodies included monoclonal anti-human T cell CD3 (Dako, Cambridge, UK, dilution 1:40), the cell proliferation marker Ki-67 (Dako, 1:50), and polyclonal goat anti-human eotaxin-1 (R&D, Abingdon, UK, 1:20) and eotaxin-2 (R&D, 1/20). Immunofluorescence was used to examine distribution of IgE (rabbit anti-human IgE, Dako, 1:750), tryptase (rabbit anti-human tryptase, Biorbyt, San Francisco, 1:100), and eotaxin-2 within the colonic mucosa, using fluorescein isothiocyanate–conjugated secondary antibodies (Dako, rabbit anti-goat or goat anti-rabbit as appropriate), compared with that of the neuronal markers monoclonal anti-human neuron-specific enolase (NSE, Dako, 1:200), neurofilament protein (NFP, Dako, 1:75), and nerve growth factor receptor (NGFR, Dako, 1:100), followed by tetramethylrhodamine isothiocyanate–conjugated rabbit anti-mouse antibody (Dako). Colocalisation of fluorescent antibodies was assessed by double exposure.

Mucosal density of CD3 T cells and eosinophils was established on blinded slides by quantitating cells per high-power field (original magnification ×40 = 0.229 mm2), with figures multiplied by 4.37 to give tissue density per square millimeter. The numbers of Ki-67+ cells and eotaxin-2+ intraepithelial cells per 100 crypt colonocytes were determined by manual counting of blinded slides.

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Statistical Analysis

Data were assessed using Statgraphics 5 Plus software. Because not all of the data were normally distributed, comparison was made between the allergic colitis group and the other disease groups using the Mann-Whitney U test. P values of ≤0.05 were considered significant.

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Histological Assessment

H&E analysis was performed on matched frozen colonic sections to those used for immunohistochemical analysis. In the normal control group no significant histological abnormalities were identified, and biopsies were within normal limits, with low density of lamina propria mononuclear cells and with no evidence of epithelial abnormality. In the CD specimens, patchy focal inflammation was seen in 3 of 10, moderate acute and chronic inflammatory infiltration in 4 of 10, and dense inflammatory infiltration in 3 of 10 specimens. In the biopsies from children with UC, a moderate diffuse increase in cellularity was seen in 7 of 10 patients, with more dense infiltration and epithelial ulceration in 3 of 10 patients. The allergic colitis specimens showed changes milder than classic IBD. Histological assessment showed moderate active inflammation with mucosal ulceration in 6 children with a predominant eosinophilic infiltration. The remaining 9 patients showed milder inflammatory change, with patchy focal increase in lamina propria mononuclear cell and eosinophil density. Eosinophils were identified within crypt or surface epithelium in 13 of 15 children. Formal quantitation of inflammation for the different groups, using the modified O’Morain score, is shown in Figure 1. The median colitis score in the normal controls was 0 (range 0–1), patients with UC 3 (2–5), patients with CD 3 (2–5), and patients with allergic colitis 2 (1–3) (Fig. 2). The mucosal density of eosinophils was higher in patients with allergic colitis (mean ± standard error [SE], 82.4 ± 8.4/mm2) than in controls (32.7 ± 7.6, P < 0.003), patients with CD (43.7 ± 11.6, P = 0.015), and patients with UC (57.5 ± 11.3, P = 0.015) (Fig. 1).





The most striking finding in the allergic colitis group was the presence of well-circumscribed lymphoid follicles within the mucosa. This was seen on H&E-stained frozen section in 13 of 15 of children, with 7 children showing >1 aggregate on a single section. Single lymphoid follicles were seen in 4 of 10 normal controls, 3 of 10 patients with UC, and 3 of 10 patients with CD.

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Immunohistochemical Analysis

The density of CD3+ T cells within the lamina propria was increased in patients with allergic colitis (mean ± SE, 623.4 ± 28.4) compared with that in controls (392.5 ± 36.6, P < 0.001), and was similar to that in patients with CD (666.9 ± 63.5, P = 0.2), although less than that in patients with UC (788.5 ± 56.8, P < 0.02) (Figs. 1 and 2). Allergic colitis specimens were more similar to CD specimens in that aggregation of CD3+ cells tended to be patchy. There was no overall increase in CD3+ intraepithelial lymphocytes (IELs) in the IBD groups compared with controls.

The percentage of Ki-67+ proliferating cells within the colonic crypts was significantly higher in patients with allergic colitis (mean 45.6% ± 1.8%) compared with that in controls (mean ± SE, 28.5% ± 2.7%, P = 0.0005), with no significant differences between patients with allergic colitis and the other disease groups (CD 40% ± 3.3%, UC 50% ± 9.4%) (Fig. 1). Ki-67+ mononuclear cells within the lamina propria were rare in the normal controls, but were frequently noted in the IBD specimens, with moderate or dense infiltration noted in 7 of 10 UC, 6 of 10 CD, and 11 of 15 allergic colitis specimens.

Eotaxin-1 and -2 showed a similar distribution pattern in the normal controls, maximal on the basolateral crypt epithelial surface (Fig. 2). Punctate staining was also identified within the goblet cell and surface mucus. Occasional eotaxin-1+ and eotaxin-2 cells were noted within the lamina propria. Within the colitis groups, including allergic colitis, a similar distribution of eotaxin-1 and -2 was noted on basolateral crypt epithelium, although expression appeared weaker in areas of focal cryptitis. In addition, eotaxin-1 and -2 immunoreactive cells could variably be identified within the lamina propria in all colitides, with more dense clustering in patients with UC and allergic colitis than in patients with CD. In addition to their location within the lamina propria, eotaxin-2+ cells were identified within the epithelial compartment. Quantitation of eotaxin-2+ IEL identified significantly greater density in patients with allergic colitis (mean 14.2 ± 2 per 100 crypt colonocytes) than in controls (1.44 ± 0.5, P = 0.0001), patients with CD (0.67 ± 0.4, P = 0.0005), and patients with UC (4.8 ± 1.4, P < 0.004). Their density in patients with UC was increased compared with that in patients with CD (P < 0.05) (Figs. 1–3). These intraepithelial eotaxin-2+ cells were of variable morphology, some small and densely stained and others large and more diffusely stained (Fig. 3, arrows). In the allergic colitis group but no others, focal aggregates of eotaxin-2 immunoreactive cells surrounding the crypts were also observed, although these were more variable than the IELs.



Immunofluorescence (Fig. 4) identified increased numbers of IgE+ cells within the lamina propria in the allergic colitis group. IgE expression was seen in 2 cell types: plasma cells (uniform dense cytoplasmic staining, negative for tryptase on serial sections) and mast cells (intense focal granular staining, also tryptase+ on serial sections). It was notable that non–cell-associated IgE+ and tryptase+ granules could be seen throughout the lamina propria, particularly in the pericryptal region, in 12 of 15 patients with allergic colitis, but not in normal controls or the classic IBD groups. These extracellular granules were sometimes distributed randomly and sometimes in linear conformation. Double staining for the neural markers NFP, NSE, and NGFR showed that these structures colocalised with granular IgE+ and tryptase+ cells in normal controls, consistent with earlier findings of close approximation of mast cells and enteric nerves. In the 12 of 15 patients with allergic colitis in whom mast cell degranulation was identified, the degranulated tryptase and IgE could be identified to cluster predominantly along neurons. In addition, eotaxin-2 variably colocalised with pericryptal enteric nerves in patients with allergic colitis. IgE and tryptase neural colocalisation was not identified in normal controls or patients with CD and was minimally identifiable in 2 of 10 UC specimens.



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We have identified mucosal features of allergic colitis that appear distinct from classical inflammatory bowel diseases. The patients with allergic colitis manifested less systemic inflammatory response and had milder endoscopic and histological features, but stood out by the density of mucosal lymphoid follicles and eosinophilia, and of intraepithelial eotaxin-2 immunoreactive lymphocytes. In addition, they showed evidence of neural alterations, particularly in the pericryptal lamina propria.

The present study represents the first immunohistochemical characterisation of the mucosal lesion in allergic colitis. We were unable to examine differential messenger RNA expression because of lack of additional frozen specimens. Microarray analysis of a small number of specimens has also identified significant upregulation of eotaxin messenger RNA in infant allergic colitis (24). One potential weakness is inclusion of young infants with allergic colitis, with few symptoms apart from loose stools containing blood, together with older children with more complex symptoms of abdominal pain and dysmotility. We found no significant differences in their mucosal changes.

Our finding of multiple lymphoid follicles in patients with allergic colitis concords with evidence that colonic lymphoid nodular hyperplasia is strongly associated with food allergies (25). This propensity to lymphoid hyperplasia relates to the characteristic developmental immunodeficiency of infant allergy (6,7), itself in turn characterised by impaired generation of TGF-β–producing regulatory lymphocytes and a gap in oral tolerance for low-dose antigens (8,9,26). Reduced numbers of regulatory T cells have been identified in peripheral blood in patients with allergic colitis (27).

We found histological features of allergic colitis consistent with earlier reports, in which mucosal inflammation is characterised by eosinophil infiltration, particularly around crypt epithelium (4). Eotaxin-1 and -2 expressions have earlier been confirmed within colonic epithelial cell lines in response to TH2 cytokines (28), whereas in situ hybridisation studies have demonstrated localisation to the basolateral crypt epithelium, as we found (29). We noted variable immunoreactivity for eotaxin-1 and -2 within colonic mucus, not identified on staining with an isotype matched control antibody, but it remains possible that this reflects nonspecific antibody binding to mucus. We note that eotaxin-1 and -2 have been quantified within mucus in patients with allergic keratoconjunctivitis (30), so it is possible that this represents a coordinated mucus secretion pattern. Exposure of cultured epithelial cells to the probiotic Lactobacillus rhamnosus GG reduced epithelial production of eotaxin-1 (31). Thus, the beneficial effect of this probiotic in patients with allergic colitis (10) may arise through induction of regulatory T-cell populations or reduction in eotaxin-mediated eosinophil recruitment.

One striking finding was the detection of numerous eotaxin-2+ intraepithelial cells in the majority of patients with allergic colitis. These cells were of variable morphology, suggestive of different populations. Some were small darkly stained cells, similar to classic IELs, whereas others were larger and less densely stained. Emerging clinical evidence for a role of lipid antigens in triggering allergic colitis raises the question whether the latter cells may represent iNKT cells, which present lipid antigens in a CD1d-restricted manner (20,21). In murine milk allergy, full fat milk induces a more vigorous mucosal response than semi-skim milk (32). In human infants, amino acid formula containing no protein but soya lipid has induced allergic colitis (33,34).

Evidence for a role for iNKT cells in mucosal eosinophilia has been provided in eosinophilic oesophagitis (EoE) in both murine and human disease (35–37). In mice, targeted deletion of iNKT cells or eotaxins-1 and -2 protects against the induction of food or aeroallergen-induced EoE (35). In human children with EoE, peripheral blood iNKT cells produce significantly greater TH2 cytokines in response to milk sphingolipids than do those in controls, whereas oesophageal mucosal iNKT cell density is increased (36). Further study of the EoE lesion has identified significant overexpression within the mucosa of the iNKT chemokine CXCL16, the iNKT cell marker Vα24, and CD1d, reducing on successful but not unsuccessful treatment (37). iNKT cells are known to produce an array of chemokines including eotaxins (38) and contribute strongly to eosinophilic inflammation in sites such as the airway (39). Conversely, iNKT cell–deficient mice are protected from allergic disease (20). Circulating iNKT cells from children with milk allergy respond to milk sphingomyelin by proliferation and production of TH2 cytokines, implying a role for dietary lipids in allergic gastrointestinal disease (40). The expression of CXCL16 is regulated by the enteric flora, and germ-free mice accumulate excess mucosal iNKT cells, which exacerbate colitis, whereas normal colonisation in early life is protective (41).

The epithelial accumulation of eotaxin-2+ cells is likely to play a role in the pericryptal aggregation of both eosinophils and mast cells in patients with allergic colitis, mediated by interaction with the eotaxin receptor CCR3 (42). We found that eosinophils and mast cells have a high incidence of degranulation compared with classic IBD, and that tryptase, IgE, and eotaxin-2 colocalise with neural structures. Pericryptal eosinophil degradation is likely to alter epithelial permeability, favouring increased ingress of dietary antigen and other intraluminal contents. It is notable that myelin basic protein–deficient mice are protected from induced colitis (43).

Mast cell tryptase has been shown earlier to bind to neuronal proteinase-activated receptors and thus modulate enteric nerve function (44). In human milk–allergic infants, gastric mucosal challenge induced mast cell degranulation, with a similar localisation of tryptase on neural proteinase-activated receptors, leading to alteration in gastric myoelectrical activity and dyspepsia (45). It has also been demonstrated that eotaxin proteins colocalise with airway neurones following aeroallergen exposure (46). Our findings are thus consistent with those reported at other mucosal surfaces in allergic disease, suggesting that the relatively mild pathology of allergic colitis may nevertheless account for the development of dysmotility and visceral hyperalgesia in allergic gastrointestinal disease (47,48). Intriguingly, the iNKT cell chemokine CXCL16 is significantly upregulated within the mucosa of patients with irritable bowel syndrome (49), whereas human enteric submucosal neurones are activated in situ by exposure to supernatants of irritable bowel syndrome colonic biopsy specimens, mediated through serotonin, histamine, and tryptase (50). We thus suggest that it will be important in further studies to characterise expression of the CXCL16, Vα24 iNKT cell, and CD1d axis in patients with allergic colitis and also to determine whether neural localisation of mast cell and eosinophil products occurs in apparently functional disorders associated with abdominal pain and dysmotility.

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The authors thank Drs Mike Thomson and Robert Heuschkel for their help in specimen collection.

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allergic colitis; enteric neurones; eosinophils; eotaxin; mast cells

© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,