Presence of intraepithelial food antigen in patients with active eosinophilic oesophagitis

Abstract

Background

Although eosinophilic oesophagitis (EoE) is putatively mediated by an abnormal response to food antigen, the oesophagus is considered relatively impenetrable to large molecules due to limited exposure time and poorly absorbable squamous epithelium.

Aim

To assess whether food antigens penetrate the esophageal mucosa in patients with EoE.

Methods

Anti-gliadin staining was performed in 3 groups: active EoE, inactive EoE and EoE patients on a low or gluten free diet. To appraise the specificity of our results, we also performed gliadin staining on 6 patients without esophageal disease who were consuming gluten. The groups with EoE on gluten also underwent endoscopic infusion with gluten containing soy sauce and repeat biopsies during the endoscopy. We measured eosinophil density, dilated intercellular spaces (on a 0–4+ scale) and gliadin in esophageal mucosa by immunofluorescence.

Results

Patients with active EoE had significantly greater epithelial density of anti-gliadin staining when compared to inactive EoE (p< .0065) and gluten free patients (p<.0008) at baseline and after soy infusion. Gliadin was not detected in non-EoE control patients. The distribution of gliadin was both cytoplasmic and nuclear. There was good correlation of DIS grade and total gliadin staining intensity (r=0.577, p= 0.0077). Acute esophageal perfusion of a commercial gliadin rich soy sauce did not lead to an increase in gliadin staining in active or inactive EoE.

Conclusion

These findings suggest, although do not prove, that antigen penetration in active EoE might be facilitated by impairment of epithelial integrity.

Introduction

The esophageal mucosa has been considered to be relatively impermeable to antigen penetration and therefore it has been considered that the oesophagus is an organ in which no allergic diseases have been described until relatively recently^{1, 2}. Eosinophilic oesophagitis is a disease that is mediated by food antigen recognition and initiation of a Th-2 allergic reaction in the oesophagus^{3, 4}. Data that support this process include evidence of increased esophageal permeability in active disease⁵; transport of large molecular markers through esophageal tissue from patients with EoE⁶; resolution of the disease with withdrawal of all food antigens⁷, and restoration of normal esophageal barrier function with treatment⁸.

Nevertheless, the presence of food antigens in the esophageal mucosa has not been documented to date in any esophageal disease. Indeed, it is reasonable to question whether this is likely to occur, given the rapid transit of undigested food through the oesophagus limiting the likelihood of penetration into the squamous esophageal mucosa by complex food antigens. As a result, other studies have suggested that the small bowel, a far more permeable and immunocompetent organ, plays a role in antigen recognition in EoE⁶. Why the oesophagus is the site of major infiltration and disease is not clear unless the inciting or aggravating antigens are sufficiently present there to induce the effector arm of the immune response.

We aimed to detect the presence of wheat protein in the esophageal mucosa through anti-gliadin antibody staining in patients with active and inactive eosinophilic oesophagitis, and compare the findings in controls without EoE who were consuming a gluten diet. In addition to assessment of gliadin in esophageal mucosa by immunofluorescence, we measured eosinophil density, and dilated intercellular spaces.

Methods and Materials

Design

Patients undergoing clinically indicated standard upper endoscopy and biopsy participated in this study. Four groups of patients were studied: 1. Patients with active eosinophilic oesophagitis defined by consensus criteria⁹ with > 15 eos/HPF. 2. Patients with eosinophilic oesophagitis defined as inactive by consensus criteria with < 15 eos/HPF. 3. Patients with eosinophilic oesophagitis treated with a gluten free diet. 4. Patients without esophageal symptoms and with a normal appearing oesophagus on endoscopy on a regular diet containing gluten.

Ethical Approval

The study was approved by Mayo Clinic Institutional Review Board and patients signed informed consent to have 3 additional esophageal biopsies performed for research purposes.

Collections of biopsies

During esophagoscopy with clinical biopsies, an additional three biopsies were taken 10 cm above the gastroesophageal junction in all patients.

In groups 1 and 2 with active and inactive EoE respectively, 10 mL of commercial gluten rich soy sauce (KikkomanUSA, San Francisco, CA) was sprayed through an endoscopically placed catheter diffusely over the distal half of the oesophagus. The endoscope was then passed into the stomach with completion of the gastroduodenoscopy. Upon withdrawal of the endoscope into the oesophagus approximately 5 minutes after perfusion, three additional endoscopic biopsies were taken 10 cm above the gastroesophageal.

Histological Analysis

Routine biopsy specimens were stained with hematoxylin and eosin and first read by a single, GI pathologist (TCS) with expertise in EoE.

Eosinophil Counts

The area of greatest eosinophil density was first located by low powered review of all tissue submitted from a given location. The area of greatest eosinophil density on esophageal biopsies was used for analysis in each individual patient regardless of esophageal location. Eosinophils were then counted using a 40X objective, a field diameter of 0.625 mm and a field area of 0.307 mm2. The peak eosinophil count/high power field (eos/HPF) was reported. From the area of greatest eosinophil density under low powered review, five random fields were chosen. Peak eosinophil counts from these five fields were then used to calculate a mean eosinophil count.

Dilated intercellular spaces

Dilated intercellular spaces (DIS) were graded on the basis of the worst area, using a 0–4+ scale as previously described⁸. Briefly, DIS grading was assessed by the appearance of the tight junctions seen on biopsy and the degree of widening between epithelial cells (figure 3).

Figure 3:

Correlation of DIS score to intensity of gliadin staining (r=0.5771, p= 0.0077)

Grade 0: no intercellular space widening and intact tight junctions.

Grade 1: mild widening of intercellular spaces with attenuation and prominence of tight junctions.

Grade 2: Further widening of intercellular space with some disruption of tight junctions.

Grade 3: complete disruption of tight junctions with further widening of intercellular spaces, forming small “lakes” in the epithelium.

Grade 4: Coalescence of epithelial lakes.

Measurement of Gliadin in Esophageal Mucosa

Esophageal biopsies taken during endoscopy were immediately placed into Optimal Cutting Temperature compound (VSR cat#4692300) and snap frozen on dry ice. Cryosectioning of the frozen esophageal tissue was done for immunofluorescent staining to determine the distribution/localization of different cells and dietary proteins. We used anti-gliadin (Biorbyt catalog # orb157160) and AF594 anti rabbit (Jackson Immunoresearch Laboratories) for immunofluoresence. Intensity of staining was performed with a 0–2 grading system (figure 1) of multiple biopsy sections by two investigators (EVM, DAK) blind to the group studied. Dapi (4’,6-Diamidino-2-phenylindole; Sigma Aldrich) was used to stain nucleic acid. As a further control for gliadin staining specificity, anti-casein antibodies (Abcam catalog # ab91167) and AF594 anti rabbit (Jackson Immunoresearch Laboratories) were also applied to normal control patients with EoE. Nuclear and cytoplasmic scores were added to yield a total score calculated with blinding to the activity status of the patients.

Figure 1:

Anti-gliadin staining in esophageal epithelium from different patients (63x). Panels A, B, and C depict the different levels of staining observed in the cytoplasmic region. White arrows in panels A and B point out examples. Panels D, E, and F depict different levels of staining observed in the nucleus, and white arrows point out examples in panels D and E. Panels A and D from active EoE patient, panels B and E from EoE inactive patient, panels C and F from EoE control patient. Arbitrary units of 2 (A, D), 1 (B, E), and 0 (C, F) were assigned for the level of staining (2 highest, 0 none). Gliadin staining (yellow), Dapi (blue). Control patient example not shown due to similar negative staining.

Statistical Analysis

A non-parametric Mann-Whitney test was conducted for each comparison of staining intensity.

This study was approved by the Mayo Clinic Institutional Review Board.

Results

Participants and EoE Endoscopic Reference Score

There were ten patients each with active and inactive EoE (all treated with budesonide) without a significant difference in mean age (37, 18–56 and 42, 22–60) or gender (6M, 5M) (supplementary table 1). The mean EoE Endoscopic Reference Scores (EREFS) were 3.7(2–6) and 1.8(0–5) in the active and inactive groups, respectively. The five patients with EoE on a gluten free diet were mean age 44(29–69, 2M) and EREFS score 2.2(0–5). The six control patients on a regular diet without esophageal disease were median age 57 (25–81). All were Caucasian and three were men.

Eosinophil Counts and Dilated Intercellular Spaces

On histology, the active and inactive groups had 61.5+/− 37.0 and 1.6+/−2.2 eosinophils/HPF (p<.01) (Table 1). Control EoE patients had 20.6+/−25.1 eos/HPF (range 0–58). Control patients without esophageal disease had no esophageal eosinophils present. Active EoE patients had a DIS grade of 2.1(1–3) in contrast to 1.1 (0–2, p<.02) in the inactive group. Gluten free EoE patients had a DIS grade of 0.4 (0–1) and control non-EoE patients had a DIS score of 0.

Gliadin Staining

Positive gliadin staining occurred in the epithelium in some of the EoE patients (figure 1). The staining occurred both in the cytoplasmic and nuclear region. Overall staining scores demonstrated a significant increase in the epithelial gliadin staining in patients with EoE when compared to EoE patients on a gluten free diet (p<0.01) and control patients. Active EoE patients on a gluten containing diet had a significantly higher total intensity of gliadin staining than both EoE patients with inactive disease (p=0.0065) and gluten free EoE patients (p=0.0008)(figure 2). Control patients without esophageal disease demonstrated a complete absence of epithelial gliadin staining. The correlation of DIS grade and total gliadin staining intensity in active and inactive patients on a wheat containing diet was r=0.5771 (p= 0.0077) (figure 3).

Figure 2:

Comparison of total anti-gliadin staining (y-axis) in patients on a gluten free diet (GFD), control patients and patients with inactive and active EoE on gluten (x-axis, 63x). ***p<0.001 and **p<0.01. Inactive EoE patients on gluten had a borderline significant increase (p=0.0521) over patients on a gluten free diet.

Effect of perfusion of gliadin-rich soy sauce

After perfusion with soy sauce in the EoE patients consuming wheat, there was no consistent effect of topically applied wheat based solution on the gliadin staining levels measured though differences at baseline were maintained (supplementary figure 1). EREFS score did not change after perfusion (data not shown).

Discussion

Eosinophilic oesophagitis is considered to be a food antigen driven allergic disease, but to this point, the finding of food antigens in esophageal mucosa as the initiating event is lacking. In this study, we demonstrate, by anti-gliadin antibody staining, that gliadin is present in the oesophageal epithelial. Furthermore, we corroborated the accuracy of this technique by demonstrating the absence of anti-gliadin staining in control patients without oesophageal disease consuming gluten, in patients with eosinophilic oesophagitis on a gluten free diet and to a lesser degree in patients with inactive disease on a gluten diet. Recent data demonstrated that active EoE is characterized by dilated intercellular spaces in vivo¹⁰ that could facilitate the entry of dietary antigen. These changes are of sufficient magnitude to account for decreased esophageal impedance when measured with a point directed⁸ or catheter based impedance probe⁵. On routine and electron microscope imaging, the spaces are on the order of 0.7 microns in diameter, thereby allowing large molecule passage into the epithelium^{6, 11}.

Several questions need to be answered on the basis of this study. For example, is the positive staining seen in this study all cellular without gliadin distribution in the intercellular space. The globular distribution of the staining more likely represents cytoplasmic staining (Figure 1) than the lake like appearance seen with marked DIS. Nevertheless, the assumption is that at least some of cellular entry occurs through DIS rather than through direct entry through the epithelial cell luminal border. This is supported by correlation of DIS scores to gliadin staining although this does not rule out DIS as an epiphenomenon of the disease rather than a cause of antigen penetration. It is also supported by prior data using rabbit esophageal epithelium in which oesophageal mucosa exposed to acid and pepsin became permeable to epidermal growth factor and to a dextran as large as 20 kD¹². Gliadin proteins start at 20kD¹³.

A second question is for what duration does the esophageal epithelium retain wheat protein after dietary ingestion in EoE? From this study, this retention time must be at least 8 hours as patients fasted after midnight for this study. The variability in gliadin staining in those with active disease may also represent varying times of last wheat ingestion. It is also unclear for how long esophageal epithelium retains the food antigen which needs to be explored in further studies.

Third, how may a complex food product with putatively little breakdown and short esophageal exposure time lead to a complex allergic reaction? This may be explained by partial breakdown occurring from exposure to acid and pepsin in the stomach with subsequent antigen exposure to the oesophagus with reflux. Also some prior digestion of food proteins may occur as a result of oral cavity microbe digestion of gluten¹⁴. This need for preliminary breakdown may also explain why perfusion with additional wheat product did not lead to increased staining. This might also be potentially explained by epithelial saturation with gliadin or that gluten protein infiltration may require repeated exposures to occur. Even so, with oral allergy syndrome, an immediate-type IgE-mediated hypersensitivity results from cross-linking of IgE and its receptor by antigen leading to rapid release of pre-formed mediators from mast cells and basophils despite short-lived food antigen exposure. This parallels eosinophilic oesophagitis by data demonstrating the presence of increased IgE, mast cells and basophils in esophageal biopsies^{15, 16}. Nevertheless, the lack of a clear histologic reaction in response to acute antigen infusion supports the postulated delayed hypersensitivity of EoE in contrast to immediate increases in inflammatory cells and permeability demonstrated in patients with irritable bowel syndrome in response to acute food antigen exposure¹⁷.

Finally, how precisely the entry of food antigen initiates the pathway of ongoing eosinophilia and inflammation? Studies have demonstrated in esophageal mucosa of children, but not adults, with eosinophilic oesophagitis an increased number of Langerhans type CD1a dendritic cells that act as the presumed antigen presenting cells¹⁸,¹⁹. Demonstrating the ingress of food antigens into the mucosa may not represent the initial pathway of the sensitization which could occur via the NALT (nasopharynx-associated lymphoid tissue) or the small intestine but it could explain why the oesophagus is the subsequent dominant target. This study demonstrates that in a diseased condition, epithelial penetration of large molecule proteins through the esophageal epithelium may occur. Whether this is unique to EoE or occurs in other diseases characterized by dilation of intercellular spaces in the oesophagus such as gastroesophageal reflux disease needs to be explored.

This study has several limitations. The most striking was being assured of the specificity of the anti-gliadin staining for the presence of wheat and not non-specific inflammatory protein. This is particularly concerning given the unexpected nuclear staining for gliadin. On the other hand, patients with inactive disease defined by both low eosinophil counts still had positive staining. In contrast, three of five patients with eosinophilic oesophagitis on a gluten free diet did not demonstrate positive anti-gliadin staining. The two positive results are likely explained by noting that most patients on a gluten free diet likely consume low levels of gluten. If small amounts of consumed gluten were present in all patients, this suggests the need for either a threshold response or that the patient diet was devoid of gluten for a prolonged time period prior to endoscopy. Finally, anti-casein staining was also negative in the control group.

In conclusion, this study demonstrates that wheat-derived gliadin protein is capable of infiltrating the esophageal epithelium. Furthermore, patients with active eosinophilic oesophagitis have greater amounts of antigen epithelial penetration as reflected through more intense anti-gliadin staining and presumably through the mechanism of dilation of intercellular spaces. Further investigation is needed to better understand the timing of food antigen penetration and retention and how food antigen recognition occurs and initiates the allergic inflammatory response characteristic of eosinophilic oesophagitis.

Supplementary Material

Supp TableS1

Table 1: Demographic and Baseline Information on Study Patients

Supp figS1

Figure 1: Anti-gliadin staining intensity in esophageal mucosa before and after perfusion of soy sauce. Total scores were calculated and plotted. No significant increase in gliadin staining was observed after perfusion with soy sauce (p=0.6911).

Abbreviations

HPF

High power field

EoE

Eosinophilic oesophagitis

Eos

eosinophils

DIS

Dilated intercellular spaces