Deposition of eosinophil granule major basic protein in eosinophilic gastroenteritis and celiac disease

GASTROENTEROLOGY

1992;103:137-145

Deposition of Eosinophil Granule Major Basic Protein in Eosinophilic Gastroenteritis and Celiac Disease NICHOLAS HERSCHEL

J. TALLEY, GAIL M. KEPHART, THOMAS A. CARPENTER, and GERALD J. GLEICH

W. MCGOVERN,

Division of Gastroenterology and Internal Medicine, Division of Allergic Diseases and Internal Medicine, Section of Surgical Pathology, and Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota

Degrees of eosinophil infiltration and eosinophil degranulation, as evidenced by localization of the eosinophil granule major basic protein (MBP), were compared in patients with eosinophilic gastroenteritis, patients with celiac disease, and healthy controls using a specific indirect immunofluorescence technique for the localization of MBP. Formalin-fixed, paraffin-embedded biopsy specimens from the mucosa of the stomach and small intestine of 11 patients with eosinophilic gastroenteritis, from the small intestine of 4 patients with celiac disease, and from the stomach and/or upper small intestine of 18 healthy asymptomatic volunteers were tested. Degrees of eosinophil infiltration and extracellular deposition of MBP were graded by two blinded observers; each section was given a score from 0 (nil) to 4 (marked). In the small bowel biopsy specimens, both eosinophil infiltration and extracellular MBP deposition scores were significantly greater in patients with eosinophilic gastroenteritis and in patients with celiac disease than in controls. In the gastric biopsy specimens, extracellular MBP deposition scores were significantly increased in patients with eosinophilic gastroenteritis compared with controls even though eosinophil infiltration scores did not differ significantly at this site. The results support the hypothesis that the eosinophil, through toxic cationic proteins such as MBP, plays a role in the pathogenesis of these diseases. issue eosinophilia is the hallmark of eosinophilic gastroenteritis’“‘; however, the role of the eosinophil in eosinophilic gastroenteritis is not understood. Morphometric studies have shown that lymphocytes, eosinophils; and mast cells are greatly increased in the mucosa of patients with celiac disease,5 although there is no evidence that cytotoxic lymphocytes cause epithelial cell damage.6 Eosinophils contain a number of cationic proteins including the major basic protein (MBP), eosinophil-derived

T

neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EP0).7 MBP has been localized to the core of both guinea pig’ and human eosinophil granulesg; in the guinea pig, MBP accounts for more than 50% of the eosinophil granule pr0tein.l’ In guinea pigs, MBP is toxic to intestinal epithelial cells in vitro.l* Therefore, we hypothesized that eosinophil degranulation in the gut is an important cause of tissue damage in eosinophilic gastroenteritis and celiac disease. It has been shown that eosinophil degranulation can be identified accurately in formalin-fixed, paraffin-embedded tissues by localization of the eosinophil granule MBP.‘2,‘3 In the present study we compared levels of eosinophil infiltration and degranulation in patients with eosinophilic gastroenteritis and patients with celiac disease with the levels in healthy controls. Materials and Methods Tissue Specimens All studies were approved by the Institutional Review Board of the Mayo Clinic. Tissue specimens obtained from two groups of patients were studied (Table 1). In the first group of 12 patients, eosinophilic gastroenteritis was diagnosed between 1966 and 1986, and the clinical details of these cases have been described elsewhere.4 Tissue specimens were from the stomach in 6 patients and the small intestine in 6. Their mean age was 38 years (range, 13-59) and 7 were male. Extensive evaluation had revealed no other cause for their eosinophilia. All but 1 of the patients had histologically confirmed mucosal-layer disease. One patient (patient 12) had only muscle-layer tissue available for study.4 The 4 patients in the second group had newly diagnosed celiac disease by clinical and histological criteria in 1987 and 1988; all 4 specimens were from the small intestine. The patients had not received treatment before the biopsy specimens were obtained, and independent review of the 0 1992 by the American Gastroenterological 0016~5065/92/$3.00

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Table 1. Clinical Data and Description Celiac Sprue

of

Biopsy Specimens

From Patients

With Eosinophilic

Gastroenteritis

and With

Eosinophil infiltration and degranulation scores Patient no. 2

Age (Yd

Sex

Diagnosis

Biopsy site

Biopsy procedure

14 29

M M M M F M M F F F F M M M M F

EG EG EG EG EG EG EG EG EG EG EG EG cs cs cs cs

Small intestine Small intestine Small intestine Small intestine Small intestine Stomach Stomach Stomach Stomach Stomach Stomach Small intestineb Small intestine Small intestine Small intestine Small intestine

Endoscopic Surgical Endoscopic Surgical Endoscopic Endoscopic Surgical Surgical Endoscopic Endoscopic Surgical Surgical Endoscopic Endoscopic Endoscopic Endoscopic

26 4 5

59 27

6 7 8

48 52 56

9 10

58 21

11 12

13 50

13 14

10 54

15 16

55 59

Infiltration [mean (range]“]

Degranulation [mean (range)“]

0.9 (0.5-1.5) 1.4 (1.0-2.0) 1.5 (1.5)

2.1 (1.5-3.0) 2.4 (1.5-3.5)

2.0 2.3 0.9 1.4 1.0 1.6 0.4

(2.0) (1.5-3.0) (0.5-1.0) (1.0-1.5) (1.0) (0.5-4.0) (0.0-1.0)

1.3 (0.5-2.0) 2.1 (1.5-2.5) 1.3 (1.0-1.5)

1.5 2.5 2.2 1.9 2.9 1.1 2.4

(1.5) (2.5) (1.5-2.5) (1.5-2.5) (2.5-3.0) (O.O-2.01 (0.5-4.0)

4.0 (4.0) 3.5 (3.0-4.0) 2.0 (1.5-2.0)

1.2 (1.0-1.5)

2.7 (2.0-3.5) 2.3 (2.0-2.5)

1.3 (1.0-2.0) 0.9 (0.5-1.5)

2.1 (1.5-3.5) 1.7 (0.5-2.51

EG, eosinophilic gastroenteritis; CS, celiac sprue. “Range is the variation of scores in different biopsies performed at the same time. bMuscle layer tissue only: results are not included in the analyses shown in Table 3. Patients 2 and 4 had muscle tissue in addition to mucosal tissue. The data shown for patients 2 and 4 do not include scores of eosinophil infiltration or extracellular MBP deposition in the muscle tissue.

histological material confirmed findings consistent with celiac disease. Their mean age was 45 years (range, IO-59 years), and 3 were male. Healthy controls consisted of 18 volunteers (Table 2); these subjects had no history of gastrointestinal disease. Tissue specimens were obtained from the gastric antrum in 7 patients and/or the upper small intestine in 15 patients using standard endoscopy biopsy forceps. Their mean age was 47 years (range, 30-65 years), and 6 were male. All biopsy specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Three 6-pm serial sections from each block were cut and glued to glass

slides with LePage’s glue (Bramalea, Ontario, Canada). One section from each block was stained with H&E; the other two sections from each block were processed for immunofluorescence studies.

Immunojluorescence Localization of Eosinophil MBP and Scoring of Tissue Sections Tissue sections were stained for MBP by an indirect immunofluorescence procedure using affinity chromatography-purified antibody to MBP and protein A-purified normal rabbit immunoglobulin (Ig] G (NRIgG) as a control; the immunofluorescence procedure has been previously described in detail.‘z-‘4 All specimens stained for immunofluorescence were scored independently by two investigators who were unaware of the clinical status of the patients and controls, and the scores were averaged. The degree of

eosinophil infiltration was graded from 0 to 4 (0, none or an occasional eosinophil per 160X field; 4, confluent eosinophils per 160X field) at 0.5 increments. The extent of extracellular MBP deposition was similarly graded from 0 to 4 (0, none per 160X field; 4, marked deposition per 160X field) at 0.5 increments. These scores were based on comparison to Ektachrome slide standards representing each score from 1.0 to 4.0 (Figure lA, C, E, and G; Figure 2A, C, E, and G). The Ektachrome (Eastman Kodak Co., Rochester, NY) slide standards were selected on the basis of the immunofluorescent results, not on the morphology of the respective H&Estained serial sections. Initially, several specimens from both diseased and normal tissues were stained by immunofluorescence for MBP and photographed to establish the spectrum of eosinophil infiltration and extracellular MBP deposition. Second, immunofluorescence photographs (such as those shown in Figures lA, C, E, and G and 2A, C, E, and G) were selected to illustrate degrees of either primarily eosinophil infiltration or primarily eosinophil degranulation, as evidenced by extracellular MBP deposition. These photographs, arbitrarily assigned scores of 1.0-4.0 for either eosinophil infiltration or eosinophil degranulation, became the Ektachrome slide standards used in the scoring procedure. Finally, additional sections from each block were stained again by immunofluorescence and scored for eosinophil infiltration and degranulation using the slide standards described above. Most specimens from the endoscopic biopsies of healthy controls occupied two to four 160X fields. Surgical speci-

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Table 2. Clinical

Data and Description

of Biopsy Specimens

OF THE GUT

139

From Healthy Controls Eosinophil infiltration and degranulation scores

Patient no.

Age (yr)

Sex

Biopsy site”

1

40

M

2 3 4 5 6 7 8 9

47 40 36 65 56 45 62 59

M M M M M F F F

10 11

34 55

F F

12 13 14 15

41 30 35 56

F F F F

16 17 18

40 34 58

F F F

Small intestine Stomach Small intestine Small intestine Small intestine Stomach Stomach Small intestine Small intestine Small intestine Stomach Small intestine Small intestine Stomach Small intestine Small intestine Small intestine Small intestine Stomach Small intestine Small intestine Stomach

“All biopsy specimens

were obtained endoscopically

mens from diseased patients were considerably larger, and endoscopic biopsy specimens from these patients were often from more than one site. In these cases, the various areas were separately graded, and the variations in eosinophi1 infiltration and MBP deposition are shown as a range in Tables 1 and 2. Overall, the two investigators did not differ in their grading of individual areas by more than 0.5.

Statistical

Analysis

Nonparametric analyses were performed using the Wilcoxon rank sum test to compare eosinophil infiltration and degranulation scores in the mucosal layer among the groups at each biopsy site (stomach and small intestine). All P values calculated were two-tailed; the a level of significance was set at 0.05. Results The eosinophil infiltration and extracellular MBP deposition scores are summarized in Tables 1 and 2; examples of each score (from 1 to 4) are shown in Figures 1 and 2. The levels of eosinophil infiltration and MBP deposition varied considerably within a given biopsy specimen from patients with eosinophilic gastroenteritis and celiac disease (Table 1).Eosinophil infiltration and extracellular MBP deposition were not invariably associated within a given biopsy; in one case (patient 10, Table l), extensive

Infiltration [mean (range)] 0.8 0.5 0.8 1.0 0.5 1.0 1.0 0.8 0.5 1.0 0.5 0.8 1.0 1.0 0.8 1.0 0.8 1.3 0.5 0.8 0.5 1.3

(0.5-1.0) (0.5) (0.5-1.0) (1.0) (0.5) (1.0) (1.0) (0.5-1.0) (0.5) (0.5-1.5) (0.5) (0.5-1.0) (1.0) (1.0) (0.5-1.0) (0.5-1.5) (0.5-1.0) (1.0-1.5) (0.5) (0.5-1.0) (0.5) (1.0-1.5)

Degranulation [mean (range)] 0.8 0.5 1.5 0.8 2.3 1.8 1.5 1.0 0.5 1.0 0.5 1.5 1.8 1.5 1.5 1.5 1.0 1.5 1.5 1.5 0.5 1.3

(0.5-1.0) (0.5) (1.5) (0.5-1.0) (2.0-2.5) (1.5-2.0) (1.5) (1.0) (0.5) (1.0) (0.5) (1.5) (1.5-2.0) (1.5) (1.0-2.0) (1.5) (1.0) (1.5) (1.0-2.0) (1.5) (0.5) (1.0-1.5)

MBP deposition was seen in the virtual absence of intact eosinophils (Figure 2G and H). Charcot-Leyden crystals and extracellular MBP deposition were observed in one patient (patient 4, Table 1) with eosinophilic gastroenteritis (Figure 3). Impressive extracellular MBP deposition was also evident in the muscle layer of the small intestinal biopsy specimen from another patient (patient 2, Table 1) with eosinophilic gastroenteritis (Figure 4). Mean eosinophil infiltration and extracellular MBP deposition scores are summarized in Table 3. The degree of eosinophil infiltration was significantly greater in the small intestines of patients with eosinophilic gastroenteritis and patients with celiac sprue than in healthy controls (P < 0.02 and P < 0.05, respectively). Moreover, the degree of extracellular MBP deposition was significantly greater in the small intestines of patients with eosinophilic gastroenteritis and patients with celiac sprue than in controls (both P < 0.02). No significant differences in eosinophi1 infiltration or extracellular MBP deposition scores were detected in patients with eosinophilic gastroenteritis compared with patients with celiac sprue. In the gastric tissue specimens, the degree of extracellular MBP deposition was significantly greater in the stomachs of patients with eosinophilic gastroenteritis than in controls (P < 0.05). However,

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Table 3. Scores

ofEosinophil

Infiltration

and Extracellular

MBP Deposition

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141

in Patients With Eosinophilic Gastroenteritis,

Patients With Celiac Sprue, and Healthy Controls Eosinophil

Group

Site Small intestine

Stomach

Eosinophilic gastroenteritis (n = 5) Celiac sprue (n = 4) Healthy control (n = 15) Eosinophilic gastroenteritis (n = 6) Healthy control (n = 7)

infiltration

Extracellular

scores

Range of means

p”

Mean

MBP deposition

Median

Range of means

scores

p”

Mean

Median

1.6

1.5

0.9-2.3

to.02

2.1

2.2

1.5-2.5

<0.02

1.2

1.3

0.9-1.3

<0.05

2.2

2.2

1.7-2.7

<0.02

0.8

0.8

0.5-1.3

1.2

1.3

0.5-2.3

2.6

2.7

1.1-4.0

1.2

1.5

0.5-1.8

1.1

1.2

0.4-1.6

0.8

1.0

0.5-1.3

NS

<0.05

“Compared with healthy controls (Wilcoxon rank sum test).

eosinophil infiltration scores were not significantly different in these two groups. Discussion Once released from their site of production in the bone marrow, eosinophils circulate in the blood stream and localize in tissues.15 Studies of eosinophil distribution in rats have shown that the skin and the gastrointestinal tract, especially the ileum, are the organs most infiltrated by eosinophils.‘6 In humans, our studies had not shown eosinophil infiltration and degranulation in the normal tissues of any organ except the bone marrow.17 In contrast, the findings presented here show that eosinophil infiltration is normally present in the stomach and small bowel and that some eosinophil degranulation apparently also normally occurs in these sites. Studies are underway to determine whether the degranulation seen in the endoscopic biopsy specimens of the normal subjects is an artifact caused by the trauma of the endoscopic biopsy procedure. Because of the eosinophil infiltration and degranulation in the gut of normal individuals, comparison of eosinophil participation in specimens from patients with eosinophilic gastroenteritis and patients with celiac sprue to specimens from normal individuals was performed by immunofluorescence localization of MBP. MBP, a 14kilodalton protein, is rich in

arginine’8~‘g and composes the core of the eosinophil granule.*,’ MBP is a potent toxin to numerous targets,‘O including mammalian cells, parasites, and bacteria,‘l and also activates cells such as basophils and platelets. ” Extracellular deposition of MBP has been used as an indicator of eosinophil degranulation in numerous diseases.7**5~23’24 The presence of significant extracellular MBP deposition in tissues from patients with eosinophilic gastroenteritis compared with controls is in accord with several prior investigations. For example, Torpier et al. observed that the electron density of eosinophil granule cores had inverted or disappeared in damaged areas in the duodenum of a patient with eosinophilic gastroenteritisz5; this loss of core density suggests a reduction in the MBP content of the core and possibly release of MBP. Similar observations of loss of electron density in eosinophil granule cores and possible release of MBP were made in a study of Crohn’s disease.” Keshavarzian et al. found that the number of activated degranulating eosinophils correlated with the degree of histological damage in two patients with eosinophilic gastroenteritis (however, normal controls were not evaluatedJz7 Whereas possible selective release of MBP in eosinophilic gastroenteritis has been reported,25 the initiating factors are not defined, although documented food allergy and drugs have been implicated in some cases.28’2gStudies of immunoglobulin-mediated eosinophi1 degranulation indicate that IgE30s31 and especiallyse-

. Figure 1. Localization of eosinophil granule MBP in gastrointestinal biopsy tissue specimens: scoring of eosinophil infiltration. (A, C, E, and G) Sections stained with affinity-purified rabbit anti-human MBP; (B) section serial to A, stained with H&E; (D, F, and II) H&E counterstains of the same sections shown in C, E, and G, respectively. A is a small intestinal biopsy specimen from a healthy control (patient 15, Table 2). C and G are gastric biopsy specimens from patients 8 and 9 (Table l),respectively, and E is a small intestinal biopsy specimen from patient 5 (Table 1); all three patients had eosinophilic gastroenteritis. A, C, E, and G show representative examples of eosinophil infiltration scored as 1, 2, 3, and 4, respectively. Note that these photomicrographs were selected to show grades of eosinophil infiltration and not to demonstrate epithelial morphology. Sections serial to A, C, E, and G stained with NRIgG were negative (results not shown) (original magnification X160).

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Figure 3. Deposition of extracellular MBP and presence of Charcot-Leyden crystals in a small intestinal biopsy specimen from a patient with eosinophilic gastroenteritis (patient 4, Table 1). (A) Section stained with anti-MBP; (B) same section as in A, counterstained with H&E. Numerous eosinophils and moderate amounts of extracellular MBP are present in A. In B, note the impressive number of hexagonal, bipyramidal Charcot-Leyden crystals (arrows) in proximity to the intact eosinophils. A serial section corresponding to A stained with NRIgG was negative (results not shown) (original magnification X400).

cretory IgA and IgG are potent stimulants of eosinophi1 degranulation3’ Similarly, the presence of significant extracellular MBP deposition in tissues from patients with celiac disease compared with controls is also in agreement with results from previous studies; others have shown that a fourfold increase in ECP occurred in patients with celiac disease after gliadin challenge.33 Our results support the hypothesis that eosinophil degranulation, as evidenced by the extracellular deposition of MBP, commonly occurs in patients with eosinophilic gastroenteritis and patients with celiac sprue as compared with normal individuals; it is likely that other eosinophil proteins such as EDN, ECP, and EP07 are also released in these diseases. However, our studies investigated eosinophil degranulation at a single point in time; sequential studies are needed to clarify the evolution of changes in tissue eosinophilia. An important finding in the present study was the notable patchiness of the degranulation process in eosinophilic gastroenteritis; marked variability was noted within most biopsy specimens from the same patient. Furthermore, eosinophil infiltration and eosinophil degranulation were not invariably asso-

ciated in our patients. Indeed, in the gastric specimens many eosinophils had degranulated so that the eosinophil infiltration scores were not increased in patients compared with controls. The results imply that biopsy specimens need to be taken from multiple sites for accurate diagnosis4 particularly because radiological findings cannot be relied on for diagnosis.34 Moreover, an assessment of eosinophil involvement in this disease should not be based simply on the number of eosinophils present because they may not be evident; Figure 2G shows extensive deposition of MBP in the virtual absence of intact eosinophils. This evidence suggests that here, as in other conditions,13,35-38 simple inspection of tissues for the numbers of eosinophils is inadequate and tissues should be examined for evidence of eosinophil degranulation. In conclusion, we have shown that eosinophil degranulation, as evidenced by extracellular deposition of MBP, is a disease marker in eosinophilic gastroenteritis and celiac disease. We hypothesize that toxic cationic proteins released by eosinophils play a role in the tissue damage found in these diseases. Therefore, our results are in keeping with a role for

Figure 2. Localization of eosinophil granule MBP in gastrointestinal biopsy tissue specimens: scoring of extracellular MBP deposition. (A, C, E, and C) Sections stained with affinity-purified rabbit anti-human MBP; (B and D) serial sections to A and C, respectively, stained with H&E; (F and H) H&E counterstains of the same sections shown in E and G, respectively. A is a small intestinal biopsy specimen from the same healthy control (patient 15, Table 2) shown in Figure 1A. C is a small intestinal biopsy specimen from an additional healthy control (patient 4, Table 2). E and G are gastric biopsy specimens from two patients with eosinophilic gastroenteritis (patients 7 and 10, Table 1). A, C, E, and G show representative examples of extracellular MBP deposition scored as 1, 2, 3, and 4, respectively. Note that these photomicrographs were selected to show grades of eosinophil degranulation, as evidenced by extracell’ular deposition of MBP, and not to demonstrate epithelial morphology. A and Care histologically normal but show crush artifact, with only minimal degranulation. Sections serial to A, C, E, and G stained with NRIgG were negative (results not shown) (original magnification X160).

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Figure 4. Deposition of extracellular MBP in a small intestinal biopsy specimen from a patient with eosinophilic gastroenteritis involving the muscle layer (patient 2, Table 1). (A) Section stained with anti-MBP; (B) same section as in A, counterstained with H&E. Note the marked extracellular deposition in A (white arrows) surrounding the blood vessels (black arrows, B). A serial section corresponding to A stained with NRIgG was negative (results not shown) (original magnification ~400).

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Received October 15, 1991. Accepted January 7, 1992. Address requests for reprints to: Nicholas J. Talley, Ph.D., F.R.A.C.P., Gastroenterology Research Unit, Mayo Clinic, 200 First Street SW., Rochester, Minnesota 55905. Supported by grants from the National Institutes of Health, AI 15231, and the Mayo Foundation.