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Are intestinal endocrine cells affected in Hirschsprung's disease? An immunohistochemical study with anti-leu 7 monoclonal antibody
Are Intestinal Endocrine Cells Affected in Hirschsprung's Disease? An Immunohistochelnical Study With Anti-Leu 7 Monoclonal Antibody By Yoshio Nakagawa and Elias Perentes
Charlottesville, Virginia 9 Specimens from 15 cases of Hirschsprung's disease and 22 control cases of normal guts were studied by the peroxidase-antiperoxidase (PAP) method with with antiLeu 7 (Leu 7) monoclonal antibody (MAb). In the normal human gut, some satellite cells in the enteric plexuses and a few nerve fibers and ganglion cells in the intestinal wall were stained with Leu 7 MAb, while endocrine cells in the intestinal mucosa showed consistent and intense cytoplasmic Lau 7 positivity. In Hirschsprung's disease, the number of Leu 7-positive endocrine cells was significantly low. The mean value of the number of Leu 7-positive endocrine cells per one microscopic field ( x 3 0 0 magnification) was 4.6 _+ 0.6 (_+SE) in controls and 0.7 _+ 0.3 (+SE} in Hirschsprung's disease (P < .001 by Student's t test). Our findings suggest that the development of endocrine cells in the intestinal mucosa may be closely related to the development of ganglion cells in the enteric plexuses, and that the anti-Leu 7 MAb may be useful for the diagnosis of Hirschsprung's disease when biopsy specimens are limited to the mucosa only. 9 1988 by Grune & Straiten, Inc. INDEX WORDS: Hirschsprung's disease: Leu 7 monoclonal antibody.
IRSCHSPRUNG'S DISEASE is characterized by the absence of ganglion cells in both the submucosal and the myenteric plexuses of the distal colon, accompanied by a thickening of the intramural nerve fiber bundles. 1'2 Since ganglion cells and nerve fibers can be easily demonstrated by immunohistochemical methods with specific markers? immunohistochemical studies using specific antibodies to nervous system antigens (such as antibodies against neurofilament polypeptides,4"6 neuron-specific enolase [NSE] ,7-10and S-100 protein 7's) have been proven to be important tools in helping the diagnosis of Hirschsprung's disease. Changes in the number of endocrine cells in several gastrointestinal (GI) tract diseases have been reported. H'12However, using the peroxidase-antiperoxidase (PAP) method, Tsuto et a113did not find significant changes of the number of somatostatin-containing endocrine cells in the aganglionic colon. Since anti-Leu 7 monoclonal antibody (MAb) had been reported to stain endocrine cells of the intestine, ~4'15 we studied rectal biopsies and surgical specimens of the colon from patients with Hirschsprung's disease, as well as control specimens with this MAb. In this paper, we describe the results obtained by using anti-Leu 7 MAb as a marker for the intestinal endocrine cells, and we
H
Journal of Pediatric Surgery, Vol 23, No 10 (October), 1988: pp 957-961
discuss its value in the diagnosis of Hirschsprung's disease. MATERIALS AND METHODS
Rectal biopsies and surgical specimens of the colon from 15 patients (aged 2 weeks to 24 years) with Hirschsprung's disease, as well as 22 control cases (patients aged from 2 weeks to 77 years) of unaffected intestinal tissue adjacent to neoplastic or inflammatory lesions, were obtained from the files of the Department of Pathology of the University of Virginia School of Medicine (Charlottesville). All specimens were fixed in 10% formalin, embedded in paraffin, and 6 #m-thick sections were used for all the immunohistochemical reactions. The diagnosis of Hirschsprung's disease was established by conventional histological criteria and by immunohistochemistry (data not shown) using antibodies against NSE, 9 S-100 protein, 7`s and the 200 kd neurofilament subunit.4'6 Sections of all specimens were immunostained using the PAP method of Sternberger et aP 6 and the mouse anti-Leu 7 MAb (Becton and Dickinson, Mountain View, CA). All sections were deparaffinized in xylene for ten minutes and delipidized in chloroform for 24 hoursJ 7 After rehydration through graded methanol to water and blockage of the endogenous peroxidase activity with hydrogen peroxide (0.5% in methanol for 30 minutes), the specimens were saturated with 10% normal swine serum for 15 minutes. The four-step PAP method that was used comprised, sequentially, mouse anti-Leu 7 MAb (dilution, 1:300; 0.67 #g of purified immunoglobulin/mL, 18 hours at 4~ rabbit antimouse immunoglobulins (Dako, Santa Barbara, CA) (dilution 1:50, 15 minutes at room temperature); swine antirabbit immunoglobulins (Dako) (dilution 1:50, 15 minutes at room temperature); and rabbit PAP-complex (Dako) (dilution 1:200, 20 minutes at room temperature). All reagents were dissolved in 0.05 M Tris buffered saline (pH, 7.6) containing 1% normal swine serum. The immunohistochemical reactions were developed in freshly prepared 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis), 5.0 mg in 6.5 mL of 0.05 M Tris buffered saline containing 0.015% of hydrogen peroxide. As positive controls, autopsy specimens of spinal nerve roots were stained in parallel. Negative controls were obtained by applying nonspecific mouse lgM K/k-1 myeloma immunoglobulins (same dilution as anti-Leu 7 MAb) (Miles Scientific, Naperville, IL) instead of the primary antibody. In some cases, successive serial sections were also immunostained
From the Division of Neuropathology, Department of Pathology, University of Virginia School of Medicine, Charlottesville. Supported by a grant from the Fondation Suisse de Bourses de Mbdecine et Biologie (EP) and by Research Grant No. CA 31271 from the National Cancer Institute, US Department of Health and Human Services. Address reprint requests to Yoshio Nakagawa, MD, Department of Neurosurgery, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo, Kyoto 602, Japan. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2310-0017503.00/0 957
958
NAKAGAWA AND PERENTES
Fig 1. Satellite cells in the myenteric plexus showing anti-Leu 7 positivity on the cell membranes and call processes surrounding ganglion cells (control case), Immunoperoxidase stain with antiLeu 7 MAb, counterstainad with hematoxylin (original magnification x 460).
with a polyvalent rabbit antimyelin basic protein (MBP) serum (Dako). Details of the procedures have been previously described.~8 RESULTS
In the normal human gut, some of the satellite cells in both the submucosal and the myenteric plexuses expressed Leu 7 positivity, usually on the cell membranes (Fig 1), and sometimes in their cytoplasm (Fig 2). A few isolated nerve fibers in the muscularis propria and some rare ganglion cells in the enteric
Fig 2. Cytoplasmic anti-Leu 7 positivity in a satellite cell (control case). Immunoperoxidasa stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x480).
Fig 3. Ganglion cells in the submucosal plexus showing strong intracytoplasmic anti-Lau 7 positivity (control case). Immunoparoxidase stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x460).
plexuses showed intracytoplasmic Leu 7 positivity as well (Fig 3). The endocrine cells in the crypts were intensely and consistently stained with anti-Leu 7 MAb and displayed granular cytoplasmic immunoreactivity (Fig 4). In the 15 cases of Hirschsprung's disease, eight of the 13 cases that were found to contain hypertrophic nerve fiber bundles in the submucosal and myenteric regions and the muscularis propria displayed Leu 7-positivity in the hypertrophic bundles (Table 1, Fig
Fig 4. Endocrine calls in normal intestinal mucosa displaying intense intracytoplasmic anti-Lau 7 positivity (control case). Immunoperoxidase stain with anti-Lau 7 MAb, counterstainad with hematoxylin (original magnification x300)o
ENDOCRINE CELLS IN HIRSCHSPRUNG'S DISEASE
959
Table 1. Leu 7 Immunoreactivity in the Human Gut Hirschsprung'd No. of cases Hypertrophic nerve fiber bundles
Normal Gut
Disease
22 0
15 13
Leu 7-negative Leu 7-positive No. of cases evaluated for endo-
5 8
crine cells* Average no. of Leu 7-positive endocrine cells per one micro-
18
13
4.6 _+ 0.6 t
0.7 _+ O.3 t
scopic field ( x 300; mean _+ SE)
*Cases that contained at least ten different fields under x 300 magnification were evaluated. t P < .001 by Student's t test.
5). These hypertrophic Leu 7-positive nerve fiber bundles did not stain for MBP. In most of the cases of Hirschsprung's disease, the number of Leu 7-positive endocrine cells in the mucosa was definitely low, and usually Leu 7-positive cells were absent (Fig 6). For the quantitative comparison of the distribution of endocrine cells in the normal gut and in Hirschsprung's disease, the average number of Leu 7-positive endocrine cells was calculated in cases that contained at least ten different fields of mucosa under microscopic examination (x300). Eighteen of 22 control patients and 13 of 15 Hirschsprung's disease patients were evaluated (Table 1). The average number of Leu 7-positive endocrine cells per field (x 300) varied from 0.7 to 9.5 in controls and from 0 to 3.2 in Hirschsprung's disease. The mean value was 4.6 _+ 0.6 (_+SE) in controls and 0.7 _ 0.3 (___SE) in Hirschsprung's
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Fig 5. Anti-Leu 7-positive hypertrophic nerve fiber bundle in the submucosa in a Hirschsprung's disease specimen. Immunoperoxidese stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification •
Fig 6. Absence of anti-Leu 7-immunoreactive endocrine cells in a case of Hirachsprung's disease. Immunoperoxidase stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x 200).
disease (Table 1). The difference of mean value was statistically significant (P < .001 by Student's t test). The number of Leu 7-positive endocrine cells in Hirschsprung's disease did not exceed five in any single field. DISCUSSION
On conventionally stained histological sections, the recognition of ganglion cells in the enteric plexuses may be difficult, especially in the neonatal patients. 7 Histochemical staining for aeetylcholinesterase in frozen tissue may also provide similar difficulties, and false-negative staining in the very young neonate, possibly due to the immaturity of the enzyme system, is known to occur. ~'3 Recently, immunohistochemical studies of Hirschsprung's disease using various markers (eg, antineurofilament MAb, anti-NSE serum, and anti-S-100 protein serum) have been carried out vigorously. 41~ The major advantage of these methods is to facilitate the recognition of hypertrophic nerve fiber bundles in the enteric plexuses and to permit the identification of immature ganglion cells? Endocrine cells in normal human intestinal mucosa have been reported to be Leu 7-positive, 14'~5as well as nerve fibers in the submucosa and the myenteric plexuses. Is In the present study, we observed intense and consistent intracytoplasmic Leu 7 positivity in the endocrine ceils, but in our control specimens only a few isolated nerve fibers stained for Leu 7. We also observed Leu 7 positivity in the satellite cells of the enteric plexuses, usually on their cell membranes, and in the cytoplasm of a few ganglion cells.
960
N A K A G A W A A N D PERENTES
In Hirschsprung's disease, Leu 7 positivity was found in some of the hypertrophic nerve fiber bundles. Since both human Schwann ceils and central and peripheral myelin sheaths are known to stain for Leu 7,17'19 in order to determine which elements of the hypertrophic nerve fiber bundles were immunostained, sections of all the cases containing Leu 7-positive nerve fibers were treated with an anti-MBP serum, an antibody against the major protein of nerve myelin sheaths. 2~The negative results obtained with the antiMBP serum indicate that the Leu 7 positivity on the hypertrophic nerve fiber bundles is probably due to the positive reaction of Schwann cells and not to the presence of myelin. Leu 7 positivity was observed in 62% of the cases of Hirschsprung's disease that were found to contain hypertrophic bundles. In a previous study, ~7 we noticed that proliferating Schwann cells in some traumatic neuromas (in two of five cases) showed weak Leu 7 positivity. Another important finding in our study is the significantly low number of Leu 7-positive endocrine cells in the Hirschsprung's disease cases. By quantification of these Leu 7-positive endocrine cells, we propose tentative criteria for the diagnosis of Hirschsprung's disease using anti-Leu 7 MAb (Table 2). Confidence limits of average number of Leu 7-positive endocrine cells per field (• 300) were statistically estimated. 21 Lower confidence limit in controls was 2.9, and upper limit in Hirschsprung's disease was 1.6. When hypertrophic Leu 7-positive nerve fiber bundles were found, or the average number of Leu 7-positive endocrine cells was 1.6 or less, the diagnosis of Hirschsprung's disease was given. When the average number was 2.9 or more, or the number of the positive endocrine cells in any single field exceeded five, the specimen was considered to be normal. Using these criteria, 11 of the 13 evaluated cases of Hirschsprung's disease were correctly diagnosed. Although the specimens were too small for the evaluation in the remaining two cases, the results that were obtained fit these criteria. Specimens from two Table 2. Tentative Criteria for the Diagnosis of Hirscheprung's Disease Using Leu 7 Immunoreaetivity
Hypertrophic Leu 7-positive nerve fiber bundles Average no. of Leu 7-positive endocrine cells per one microscopic field ( x 300)* M a x i m u m no. of Leu 7-positive endocrine
cells per any single field ( x 3 0 0 )
Normal Gut
Affected Gut
--
+
> 2.9 t
< 1.6 t < 5
*Average number should be calculated from at least ten different fields, and full thickness of mucosa must be present on the examined section. tLower and upper limits were statistically estimated. 21
incorrectly diagnosed cases were found to be from the transitional zones between aganglionic and normal areas. Sixteen of the 18 evaluated cases of control specimens were correctly diagnosed as normal. In the other two cases, the specimens contained large areas of superficial mucosa (no submucosa present) where a small number of endocrine cells were present. Thus, it appears that correct diagnoses of Hirschsprung's disease could be possible even with small biopsy specimens of rectal mucosa if the full thickness of mucosa is present. However, a few of our cases were incorrectly diagnosed when the biopsy specimens were from the transitional zones or when superficial mucosa was only available. Tsuto et al, 13using the PAP method, reported that in Hirschsprung's disease the number of somatostatinpositive endocrine cells in the affected gut was similar to that in the ganglionic gut in five patients. This discrepancy with our results could be explained by the fact that anti-Leu 7 MAb can probably stain not only the somatostatin-containing cells but also other types of endocrine cells, as It should also be noted that the population of the cells containing somatostatin is smaller than that of other endocrine cells in the large intestine. 22 All types of GI endocrine cells can be immunostained with a recently developed antibody against chromogranin. 23 Using this antibody, changes in endocrine cells in diseases of the GI tract have been reported, including coeliac disease H and Crohn's ileitis, ~2 but Hirschsprung's disease has not yet been investigated with this antibody. The origin of endocrine cells in the intestinal mucosa has been a matter of dispute. Pearse 24 initially postulated that endocrine cells in the intestinal mucosa might originate from the neural crest because they possess the same metabolic pattern and synthetic, storage, and secretion mechanisms as cells of the amine precursor uptake and decarboxylation (APUD) system, but subsequent embryologic studies have not favored this derivation. 25 However, because of their morphological and functional similarity to neurons and their close relation to nerve elements, the neuroectodermal origin of the endocrine cells of the intestinal mucosa still remains hypothetical) 6'27 Using immunocytochemical methods for the detection of gastrin, somatostatin, motilin, and gastric inhibitory polypeptide (GIP), Buchan et a128 demonstrated that the development of endocrine cells in the human fetal intestine has a cranial-caudal gradient similar to that of the ganglion cells in the enteric plexuses. In the present study, we found that the number of Leu 7-positive endocrine ceils in the enteric mucosa is significantly low in the cases of Hirschsprung's disease. Since the traditional hypothesis of the pathogene-
ENDOCRINE CELLS IN HIRSCHSPRUNG'S DISEASE
961
sis o f H i r s c h s p r u n g ' s d i s e a s e is t h e f a i l u r e of m i g r a t i o n a n d d e v e l o p m e n t of g a n g l i o n cells in t h e distal colon, 2 our observation concerning the decrease of endocrine cells in t h e c a s e s o f H i r s c h s p r u n g ' s d i s e a s e i n d i c a t e s t h e close r e l a t i o n s h i p b e t w e e n t h e d e v e l o p m e n t of g a n g l i o n cells in t h e e n t e r i c p l e x u s e s a n d t h e d e v e l o p m e n t of e n d o c r i n e cells in t h e i n t e s t i n a l m u c o s a . W h e t h e r this r e p r e s e n t s an a r g u m e n t t h a t f a v o r s t h e n e u r o e c t o d e r m a l d e r i v a t i o n of t h e e n d o c r i n e cells in
t h e i n t e s t i n a l m u c o s a , or i n d i c a t e s t h a t t h e p r e s e n c e o f i n t r a m u r a l g a n g l i o n cells is p r e r e q u i s i t e for t h e d e v e l o p m e n t o f t h e e n d o c r i n e cells of t h e gut, n e e d s f u r t h e r investigation. ACKNOWLEDGMENT
The authors thank Dr Henry Urich for suggestions and advice in preparing the manuscript and Elisabeth Perentes for technical assistance.
REFERENCES
1. Meier-Ruge W: Hirschsprung's disease: Its aetiology, pathogenesis and differential diagnosis. Curr Top Pathol 59:131-179, 1974 2. Weinberg AG: Hirschsprung's disease--A pathologist's view. Perspect Pediatr Pathol 2:207-239, 1976 3. Blisard KS, Kleinman R: Hirschsprung's disease: A clinical and pathologic overview. Hum Pathol 17:1189-1191, 1986 4. Kliick P, Van Muijen GNP, Van der Kamp AWM, et al: Hirschsprung's disease studied with monoclonal antineurofilament antibodies on tissue sections. Lancet 1:652-653, 1984 5. Kliick P, Ten Kate FJW, Van der Kamp AWM, et al: Pathologic explanation for postoperative obstipation in Hirschsprung's disease revealed with monoclonal antibody staining. Am J Clin Patho186:490-492, 1986 6. Kliick P, Tibboel D, Leendertse-Verloop K, et al: Diagnosis of congenital neurogenic abnormalities of the bowel with monoclonal antineurofilament antibodies. J Pediatr Surg 21:132-135, 1986 7. Hall CL, Lampert PW: lmmunohistochemistry as an aid in the diagnosis of Hirschsprung's disease. Am J Clin Pathol 83:177-181, 1985 8. Taguchi T, Tanaka K, Ikeda K: Immunohistochemical study of neuron specific enolase and S-100 protein in Hirschsprung's disease. Virchows Arch [A] 405:399-409, 1985 9. Vinores SA, May E: Neuron-specific enolase as an immunohistochemical tool for the diagnosis of Hirschsprung's disease. Am J Surg Pathol 9:281-285, 1985 10. Tam PKH: An immunochemical study with neuron-specificenolase and substance P of human enteric innervation--The normal development pattern and abnormal deviations in Hirschsprung's disease and pyloric stenosis. J Pediatr Surg 21:227-232, 1986 11. Pietroletti R, Bishop AE, Carlei F, et al: Gut endocrine cell population in coeliac disease estimated by immunocytochemistry using a monoclonal antibody to chromogranin. Gut 27:838-843, 1986 12. Bishop AE, Pietroletti R, Taat CW, et al: Increased populations of endocrine cells in Crohn's ileitis. Virchows Arch [A] 410:391-396, 1987 13. Tsuto T, Okamura H, Fukui K, et al: Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease. J Pediatr Surg 20:266-270, 1985 14. Caillaud J-M, Benjelloun S, Bosq J, et al: HNK-l-defined antigen detected in paraffin-embedded neuroectoderm tumors and those derived from cells of the amine precursor uptake and decarboxylation system. Cancer Res 44:4432-4439, 1984 15. Shioda Y, Nagura H, Tsutsumi Y, et al: Distribution ofLeu 7 (HNK-1) antigen in human digestive organs: An immunohisto-
chemical study with monoclonal antibody. Histochem J 16:843-854, 1984 16. Sternberger LA, Hardy PH Jr, Cuculis JJ, et al: The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333, 1970 17. Perentes E, Rubinstein LJ: Immunohistochemical recognition of human nerve sheath tumors by anti-Leu 7 (HNK-I) monoclonal antibody. Acta Neuropathol (Berl) 68:319-324, 1985 18. Nakagawa Y, Perentes E, Rubinstein LJ: Immunohistochemical characterization of oligodendrogliomas: An analysis of multiple markers. Acta Neuropathol (Bed) 72:15-22, 1986 19. Schuller-Petrovic S, Gebhart W, Lassmann H, et al: A shared antigenic determinant between natural killer cells and nervous tissue. Nature 306:179-181, 1983 20. Itoyama Y, Sternberger NH, Kies MW, et al: Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system. Ann Neurol 7:157-166, 1980 21. Miller SL: Populations and samples--Errors of sampling and measurement, in Miller SL (ed): Introductory Statistics for Dentistry and Medicine. Reston, VA, Reston, 1981, pp 93-112 22. Solcia E, Polak JM, Larsson L-I, et al: Update on Lausanne classification of endocrine cells, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 96100 23. Facer P, Bishop AE, Lloyd RV, et al: Chromogranin: A newly recognised maker for endocrine cells of the gastrointestinal tract. Gastroenterology 89:1366-1373, 1985 24. Pearse AGE: The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J Histochem Cytochem 17:303-313, 1969 25. LeDouarin NM, Fontaine-Perus J: The neural crest and the digestive tract: Developmental relationships, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 107-118 26. Fujita T, Kobayashi S: Structure and function of gut endocrine cells. Int Rev Cytol 6:187-233, 1977 (suppl) 27. Fujita T, Kobayashi S: The endocrine cell, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 90-95 28. Buchan AM J, Bryant MG, Polak JM, et al: Development of regulatory peptides in the human fetal intestine, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 119-124
Charlottesville, Virginia 9 Specimens from 15 cases of Hirschsprung's disease and 22 control cases of normal guts were studied by the peroxidase-antiperoxidase (PAP) method with with antiLeu 7 (Leu 7) monoclonal antibody (MAb). In the normal human gut, some satellite cells in the enteric plexuses and a few nerve fibers and ganglion cells in the intestinal wall were stained with Leu 7 MAb, while endocrine cells in the intestinal mucosa showed consistent and intense cytoplasmic Lau 7 positivity. In Hirschsprung's disease, the number of Leu 7-positive endocrine cells was significantly low. The mean value of the number of Leu 7-positive endocrine cells per one microscopic field ( x 3 0 0 magnification) was 4.6 _+ 0.6 (_+SE) in controls and 0.7 _+ 0.3 (+SE} in Hirschsprung's disease (P < .001 by Student's t test). Our findings suggest that the development of endocrine cells in the intestinal mucosa may be closely related to the development of ganglion cells in the enteric plexuses, and that the anti-Leu 7 MAb may be useful for the diagnosis of Hirschsprung's disease when biopsy specimens are limited to the mucosa only. 9 1988 by Grune & Straiten, Inc. INDEX WORDS: Hirschsprung's disease: Leu 7 monoclonal antibody.
IRSCHSPRUNG'S DISEASE is characterized by the absence of ganglion cells in both the submucosal and the myenteric plexuses of the distal colon, accompanied by a thickening of the intramural nerve fiber bundles. 1'2 Since ganglion cells and nerve fibers can be easily demonstrated by immunohistochemical methods with specific markers? immunohistochemical studies using specific antibodies to nervous system antigens (such as antibodies against neurofilament polypeptides,4"6 neuron-specific enolase [NSE] ,7-10and S-100 protein 7's) have been proven to be important tools in helping the diagnosis of Hirschsprung's disease. Changes in the number of endocrine cells in several gastrointestinal (GI) tract diseases have been reported. H'12However, using the peroxidase-antiperoxidase (PAP) method, Tsuto et a113did not find significant changes of the number of somatostatin-containing endocrine cells in the aganglionic colon. Since anti-Leu 7 monoclonal antibody (MAb) had been reported to stain endocrine cells of the intestine, ~4'15 we studied rectal biopsies and surgical specimens of the colon from patients with Hirschsprung's disease, as well as control specimens with this MAb. In this paper, we describe the results obtained by using anti-Leu 7 MAb as a marker for the intestinal endocrine cells, and we
H
Journal of Pediatric Surgery, Vol 23, No 10 (October), 1988: pp 957-961
discuss its value in the diagnosis of Hirschsprung's disease. MATERIALS AND METHODS
Rectal biopsies and surgical specimens of the colon from 15 patients (aged 2 weeks to 24 years) with Hirschsprung's disease, as well as 22 control cases (patients aged from 2 weeks to 77 years) of unaffected intestinal tissue adjacent to neoplastic or inflammatory lesions, were obtained from the files of the Department of Pathology of the University of Virginia School of Medicine (Charlottesville). All specimens were fixed in 10% formalin, embedded in paraffin, and 6 #m-thick sections were used for all the immunohistochemical reactions. The diagnosis of Hirschsprung's disease was established by conventional histological criteria and by immunohistochemistry (data not shown) using antibodies against NSE, 9 S-100 protein, 7`s and the 200 kd neurofilament subunit.4'6 Sections of all specimens were immunostained using the PAP method of Sternberger et aP 6 and the mouse anti-Leu 7 MAb (Becton and Dickinson, Mountain View, CA). All sections were deparaffinized in xylene for ten minutes and delipidized in chloroform for 24 hoursJ 7 After rehydration through graded methanol to water and blockage of the endogenous peroxidase activity with hydrogen peroxide (0.5% in methanol for 30 minutes), the specimens were saturated with 10% normal swine serum for 15 minutes. The four-step PAP method that was used comprised, sequentially, mouse anti-Leu 7 MAb (dilution, 1:300; 0.67 #g of purified immunoglobulin/mL, 18 hours at 4~ rabbit antimouse immunoglobulins (Dako, Santa Barbara, CA) (dilution 1:50, 15 minutes at room temperature); swine antirabbit immunoglobulins (Dako) (dilution 1:50, 15 minutes at room temperature); and rabbit PAP-complex (Dako) (dilution 1:200, 20 minutes at room temperature). All reagents were dissolved in 0.05 M Tris buffered saline (pH, 7.6) containing 1% normal swine serum. The immunohistochemical reactions were developed in freshly prepared 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma, St. Louis), 5.0 mg in 6.5 mL of 0.05 M Tris buffered saline containing 0.015% of hydrogen peroxide. As positive controls, autopsy specimens of spinal nerve roots were stained in parallel. Negative controls were obtained by applying nonspecific mouse lgM K/k-1 myeloma immunoglobulins (same dilution as anti-Leu 7 MAb) (Miles Scientific, Naperville, IL) instead of the primary antibody. In some cases, successive serial sections were also immunostained
From the Division of Neuropathology, Department of Pathology, University of Virginia School of Medicine, Charlottesville. Supported by a grant from the Fondation Suisse de Bourses de Mbdecine et Biologie (EP) and by Research Grant No. CA 31271 from the National Cancer Institute, US Department of Health and Human Services. Address reprint requests to Yoshio Nakagawa, MD, Department of Neurosurgery, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo, Kyoto 602, Japan. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2310-0017503.00/0 957
958
NAKAGAWA AND PERENTES
Fig 1. Satellite cells in the myenteric plexus showing anti-Leu 7 positivity on the cell membranes and call processes surrounding ganglion cells (control case), Immunoperoxidase stain with antiLeu 7 MAb, counterstainad with hematoxylin (original magnification x 460).
with a polyvalent rabbit antimyelin basic protein (MBP) serum (Dako). Details of the procedures have been previously described.~8 RESULTS
In the normal human gut, some of the satellite cells in both the submucosal and the myenteric plexuses expressed Leu 7 positivity, usually on the cell membranes (Fig 1), and sometimes in their cytoplasm (Fig 2). A few isolated nerve fibers in the muscularis propria and some rare ganglion cells in the enteric
Fig 2. Cytoplasmic anti-Leu 7 positivity in a satellite cell (control case). Immunoperoxidasa stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x480).
Fig 3. Ganglion cells in the submucosal plexus showing strong intracytoplasmic anti-Lau 7 positivity (control case). Immunoparoxidase stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x460).
plexuses showed intracytoplasmic Leu 7 positivity as well (Fig 3). The endocrine cells in the crypts were intensely and consistently stained with anti-Leu 7 MAb and displayed granular cytoplasmic immunoreactivity (Fig 4). In the 15 cases of Hirschsprung's disease, eight of the 13 cases that were found to contain hypertrophic nerve fiber bundles in the submucosal and myenteric regions and the muscularis propria displayed Leu 7-positivity in the hypertrophic bundles (Table 1, Fig
Fig 4. Endocrine calls in normal intestinal mucosa displaying intense intracytoplasmic anti-Lau 7 positivity (control case). Immunoperoxidase stain with anti-Lau 7 MAb, counterstainad with hematoxylin (original magnification x300)o
ENDOCRINE CELLS IN HIRSCHSPRUNG'S DISEASE
959
Table 1. Leu 7 Immunoreactivity in the Human Gut Hirschsprung'd No. of cases Hypertrophic nerve fiber bundles
Normal Gut
Disease
22 0
15 13
Leu 7-negative Leu 7-positive No. of cases evaluated for endo-
5 8
crine cells* Average no. of Leu 7-positive endocrine cells per one micro-
18
13
4.6 _+ 0.6 t
0.7 _+ O.3 t
scopic field ( x 300; mean _+ SE)
*Cases that contained at least ten different fields under x 300 magnification were evaluated. t P < .001 by Student's t test.
5). These hypertrophic Leu 7-positive nerve fiber bundles did not stain for MBP. In most of the cases of Hirschsprung's disease, the number of Leu 7-positive endocrine cells in the mucosa was definitely low, and usually Leu 7-positive cells were absent (Fig 6). For the quantitative comparison of the distribution of endocrine cells in the normal gut and in Hirschsprung's disease, the average number of Leu 7-positive endocrine cells was calculated in cases that contained at least ten different fields of mucosa under microscopic examination (x300). Eighteen of 22 control patients and 13 of 15 Hirschsprung's disease patients were evaluated (Table 1). The average number of Leu 7-positive endocrine cells per field (x 300) varied from 0.7 to 9.5 in controls and from 0 to 3.2 in Hirschsprung's disease. The mean value was 4.6 _+ 0.6 (_+SE) in controls and 0.7 _ 0.3 (___SE) in Hirschsprung's
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Fig 5. Anti-Leu 7-positive hypertrophic nerve fiber bundle in the submucosa in a Hirschsprung's disease specimen. Immunoperoxidese stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification •
Fig 6. Absence of anti-Leu 7-immunoreactive endocrine cells in a case of Hirachsprung's disease. Immunoperoxidase stain with anti-Leu 7 MAb, counterstained with hematoxylin (original magnification x 200).
disease (Table 1). The difference of mean value was statistically significant (P < .001 by Student's t test). The number of Leu 7-positive endocrine cells in Hirschsprung's disease did not exceed five in any single field. DISCUSSION
On conventionally stained histological sections, the recognition of ganglion cells in the enteric plexuses may be difficult, especially in the neonatal patients. 7 Histochemical staining for aeetylcholinesterase in frozen tissue may also provide similar difficulties, and false-negative staining in the very young neonate, possibly due to the immaturity of the enzyme system, is known to occur. ~'3 Recently, immunohistochemical studies of Hirschsprung's disease using various markers (eg, antineurofilament MAb, anti-NSE serum, and anti-S-100 protein serum) have been carried out vigorously. 41~ The major advantage of these methods is to facilitate the recognition of hypertrophic nerve fiber bundles in the enteric plexuses and to permit the identification of immature ganglion cells? Endocrine cells in normal human intestinal mucosa have been reported to be Leu 7-positive, 14'~5as well as nerve fibers in the submucosa and the myenteric plexuses. Is In the present study, we observed intense and consistent intracytoplasmic Leu 7 positivity in the endocrine ceils, but in our control specimens only a few isolated nerve fibers stained for Leu 7. We also observed Leu 7 positivity in the satellite cells of the enteric plexuses, usually on their cell membranes, and in the cytoplasm of a few ganglion cells.
960
N A K A G A W A A N D PERENTES
In Hirschsprung's disease, Leu 7 positivity was found in some of the hypertrophic nerve fiber bundles. Since both human Schwann ceils and central and peripheral myelin sheaths are known to stain for Leu 7,17'19 in order to determine which elements of the hypertrophic nerve fiber bundles were immunostained, sections of all the cases containing Leu 7-positive nerve fibers were treated with an anti-MBP serum, an antibody against the major protein of nerve myelin sheaths. 2~The negative results obtained with the antiMBP serum indicate that the Leu 7 positivity on the hypertrophic nerve fiber bundles is probably due to the positive reaction of Schwann cells and not to the presence of myelin. Leu 7 positivity was observed in 62% of the cases of Hirschsprung's disease that were found to contain hypertrophic bundles. In a previous study, ~7 we noticed that proliferating Schwann cells in some traumatic neuromas (in two of five cases) showed weak Leu 7 positivity. Another important finding in our study is the significantly low number of Leu 7-positive endocrine cells in the Hirschsprung's disease cases. By quantification of these Leu 7-positive endocrine cells, we propose tentative criteria for the diagnosis of Hirschsprung's disease using anti-Leu 7 MAb (Table 2). Confidence limits of average number of Leu 7-positive endocrine cells per field (• 300) were statistically estimated. 21 Lower confidence limit in controls was 2.9, and upper limit in Hirschsprung's disease was 1.6. When hypertrophic Leu 7-positive nerve fiber bundles were found, or the average number of Leu 7-positive endocrine cells was 1.6 or less, the diagnosis of Hirschsprung's disease was given. When the average number was 2.9 or more, or the number of the positive endocrine cells in any single field exceeded five, the specimen was considered to be normal. Using these criteria, 11 of the 13 evaluated cases of Hirschsprung's disease were correctly diagnosed. Although the specimens were too small for the evaluation in the remaining two cases, the results that were obtained fit these criteria. Specimens from two Table 2. Tentative Criteria for the Diagnosis of Hirscheprung's Disease Using Leu 7 Immunoreaetivity
Hypertrophic Leu 7-positive nerve fiber bundles Average no. of Leu 7-positive endocrine cells per one microscopic field ( x 300)* M a x i m u m no. of Leu 7-positive endocrine
cells per any single field ( x 3 0 0 )
Normal Gut
Affected Gut
--
+
> 2.9 t
< 1.6 t < 5
*Average number should be calculated from at least ten different fields, and full thickness of mucosa must be present on the examined section. tLower and upper limits were statistically estimated. 21
incorrectly diagnosed cases were found to be from the transitional zones between aganglionic and normal areas. Sixteen of the 18 evaluated cases of control specimens were correctly diagnosed as normal. In the other two cases, the specimens contained large areas of superficial mucosa (no submucosa present) where a small number of endocrine cells were present. Thus, it appears that correct diagnoses of Hirschsprung's disease could be possible even with small biopsy specimens of rectal mucosa if the full thickness of mucosa is present. However, a few of our cases were incorrectly diagnosed when the biopsy specimens were from the transitional zones or when superficial mucosa was only available. Tsuto et al, 13using the PAP method, reported that in Hirschsprung's disease the number of somatostatinpositive endocrine cells in the affected gut was similar to that in the ganglionic gut in five patients. This discrepancy with our results could be explained by the fact that anti-Leu 7 MAb can probably stain not only the somatostatin-containing cells but also other types of endocrine cells, as It should also be noted that the population of the cells containing somatostatin is smaller than that of other endocrine cells in the large intestine. 22 All types of GI endocrine cells can be immunostained with a recently developed antibody against chromogranin. 23 Using this antibody, changes in endocrine cells in diseases of the GI tract have been reported, including coeliac disease H and Crohn's ileitis, ~2 but Hirschsprung's disease has not yet been investigated with this antibody. The origin of endocrine cells in the intestinal mucosa has been a matter of dispute. Pearse 24 initially postulated that endocrine cells in the intestinal mucosa might originate from the neural crest because they possess the same metabolic pattern and synthetic, storage, and secretion mechanisms as cells of the amine precursor uptake and decarboxylation (APUD) system, but subsequent embryologic studies have not favored this derivation. 25 However, because of their morphological and functional similarity to neurons and their close relation to nerve elements, the neuroectodermal origin of the endocrine cells of the intestinal mucosa still remains hypothetical) 6'27 Using immunocytochemical methods for the detection of gastrin, somatostatin, motilin, and gastric inhibitory polypeptide (GIP), Buchan et a128 demonstrated that the development of endocrine cells in the human fetal intestine has a cranial-caudal gradient similar to that of the ganglion cells in the enteric plexuses. In the present study, we found that the number of Leu 7-positive endocrine ceils in the enteric mucosa is significantly low in the cases of Hirschsprung's disease. Since the traditional hypothesis of the pathogene-
ENDOCRINE CELLS IN HIRSCHSPRUNG'S DISEASE
961
sis o f H i r s c h s p r u n g ' s d i s e a s e is t h e f a i l u r e of m i g r a t i o n a n d d e v e l o p m e n t of g a n g l i o n cells in t h e distal colon, 2 our observation concerning the decrease of endocrine cells in t h e c a s e s o f H i r s c h s p r u n g ' s d i s e a s e i n d i c a t e s t h e close r e l a t i o n s h i p b e t w e e n t h e d e v e l o p m e n t of g a n g l i o n cells in t h e e n t e r i c p l e x u s e s a n d t h e d e v e l o p m e n t of e n d o c r i n e cells in t h e i n t e s t i n a l m u c o s a . W h e t h e r this r e p r e s e n t s an a r g u m e n t t h a t f a v o r s t h e n e u r o e c t o d e r m a l d e r i v a t i o n of t h e e n d o c r i n e cells in
t h e i n t e s t i n a l m u c o s a , or i n d i c a t e s t h a t t h e p r e s e n c e o f i n t r a m u r a l g a n g l i o n cells is p r e r e q u i s i t e for t h e d e v e l o p m e n t o f t h e e n d o c r i n e cells of t h e gut, n e e d s f u r t h e r investigation. ACKNOWLEDGMENT
The authors thank Dr Henry Urich for suggestions and advice in preparing the manuscript and Elisabeth Perentes for technical assistance.
REFERENCES
1. Meier-Ruge W: Hirschsprung's disease: Its aetiology, pathogenesis and differential diagnosis. Curr Top Pathol 59:131-179, 1974 2. Weinberg AG: Hirschsprung's disease--A pathologist's view. Perspect Pediatr Pathol 2:207-239, 1976 3. Blisard KS, Kleinman R: Hirschsprung's disease: A clinical and pathologic overview. Hum Pathol 17:1189-1191, 1986 4. Kliick P, Van Muijen GNP, Van der Kamp AWM, et al: Hirschsprung's disease studied with monoclonal antineurofilament antibodies on tissue sections. Lancet 1:652-653, 1984 5. Kliick P, Ten Kate FJW, Van der Kamp AWM, et al: Pathologic explanation for postoperative obstipation in Hirschsprung's disease revealed with monoclonal antibody staining. Am J Clin Patho186:490-492, 1986 6. Kliick P, Tibboel D, Leendertse-Verloop K, et al: Diagnosis of congenital neurogenic abnormalities of the bowel with monoclonal antineurofilament antibodies. J Pediatr Surg 21:132-135, 1986 7. Hall CL, Lampert PW: lmmunohistochemistry as an aid in the diagnosis of Hirschsprung's disease. Am J Clin Pathol 83:177-181, 1985 8. Taguchi T, Tanaka K, Ikeda K: Immunohistochemical study of neuron specific enolase and S-100 protein in Hirschsprung's disease. Virchows Arch [A] 405:399-409, 1985 9. Vinores SA, May E: Neuron-specific enolase as an immunohistochemical tool for the diagnosis of Hirschsprung's disease. Am J Surg Pathol 9:281-285, 1985 10. Tam PKH: An immunochemical study with neuron-specificenolase and substance P of human enteric innervation--The normal development pattern and abnormal deviations in Hirschsprung's disease and pyloric stenosis. J Pediatr Surg 21:227-232, 1986 11. Pietroletti R, Bishop AE, Carlei F, et al: Gut endocrine cell population in coeliac disease estimated by immunocytochemistry using a monoclonal antibody to chromogranin. Gut 27:838-843, 1986 12. Bishop AE, Pietroletti R, Taat CW, et al: Increased populations of endocrine cells in Crohn's ileitis. Virchows Arch [A] 410:391-396, 1987 13. Tsuto T, Okamura H, Fukui K, et al: Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease. J Pediatr Surg 20:266-270, 1985 14. Caillaud J-M, Benjelloun S, Bosq J, et al: HNK-l-defined antigen detected in paraffin-embedded neuroectoderm tumors and those derived from cells of the amine precursor uptake and decarboxylation system. Cancer Res 44:4432-4439, 1984 15. Shioda Y, Nagura H, Tsutsumi Y, et al: Distribution ofLeu 7 (HNK-1) antigen in human digestive organs: An immunohisto-
chemical study with monoclonal antibody. Histochem J 16:843-854, 1984 16. Sternberger LA, Hardy PH Jr, Cuculis JJ, et al: The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315-333, 1970 17. Perentes E, Rubinstein LJ: Immunohistochemical recognition of human nerve sheath tumors by anti-Leu 7 (HNK-I) monoclonal antibody. Acta Neuropathol (Berl) 68:319-324, 1985 18. Nakagawa Y, Perentes E, Rubinstein LJ: Immunohistochemical characterization of oligodendrogliomas: An analysis of multiple markers. Acta Neuropathol (Bed) 72:15-22, 1986 19. Schuller-Petrovic S, Gebhart W, Lassmann H, et al: A shared antigenic determinant between natural killer cells and nervous tissue. Nature 306:179-181, 1983 20. Itoyama Y, Sternberger NH, Kies MW, et al: Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system. Ann Neurol 7:157-166, 1980 21. Miller SL: Populations and samples--Errors of sampling and measurement, in Miller SL (ed): Introductory Statistics for Dentistry and Medicine. Reston, VA, Reston, 1981, pp 93-112 22. Solcia E, Polak JM, Larsson L-I, et al: Update on Lausanne classification of endocrine cells, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 96100 23. Facer P, Bishop AE, Lloyd RV, et al: Chromogranin: A newly recognised maker for endocrine cells of the gastrointestinal tract. Gastroenterology 89:1366-1373, 1985 24. Pearse AGE: The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. J Histochem Cytochem 17:303-313, 1969 25. LeDouarin NM, Fontaine-Perus J: The neural crest and the digestive tract: Developmental relationships, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 107-118 26. Fujita T, Kobayashi S: Structure and function of gut endocrine cells. Int Rev Cytol 6:187-233, 1977 (suppl) 27. Fujita T, Kobayashi S: The endocrine cell, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 90-95 28. Buchan AM J, Bryant MG, Polak JM, et al: Development of regulatory peptides in the human fetal intestine, in Bloom SR, Polak JM (eds): Gut Hormones, ed 2. Edinburgh, Churchill Livingstone, 1981, pp 119-124