- Email: [email protected]
Enterocyte apoptosis and proliferation are increased in microvillous inclusion disease (Familial microvillous atrophy)
Enterocyte Apoptosis and Proliferation Are Increased in Microvillous Inclusion Disease (Familial Microvillous Atrophy) GABRIEL M, GROISMAN, MD, EDMOND SABO, MD, ALONA MEIR, MD, AND SYLVIE POLAK-CHARCON, PHD Microvillous inclusion disease (MID) is characterized by diffuse villous atrophy without inflammatory changes. While increased apoptosis has been related to mucosal flattening in celiac disease, the role of apoptosis in the pathogenesis of MID is unknown. The aim of this study was to assess the rates of apoptosis and cell proliferation in MID and to compare them with those of normal controls and celiac disease. Small intestinal biopsies from 5 infants with MID, 10 children with normal villous architecture, and 10 children with untreated celiac disease were stained with the terminal uridine deoxynucleotidyl nick end labeling (TUNEL) method to assess apoptotic activity, and with Ki-67 immunohistochemistry to assess cellular proliferation. TUNEL and Ki-67 positive enterocytes were counted in a minimum of 20 well oriented half crypts per section. The percentage of apoptotic cells per crypt (apoptotic index) in normal, MID, and celiac biopsies was 0.03 _+ 0.01%, 0.08 -+ 0.08%, and 0.16 -+ 0.3%, respectively.
Significant differences were found between normal and MID, and between normal and celiac cases. The percentage of Ki-67 positive cells per crypt (proliferation index) in normal, MID, and celiac cases was 14 -+ 2.5%, 28 -+ 9.2%, and 56 -+ 14%. Significant differences were found between the 3 groups. In conclusion, (1) enterocyte apoptosis and proliferation are increased in MID; (2) apoptosis appears to be an important factor of cell loss and may be, at least in part, responsible for villous atrophy in MID; and (3) crypts in MID are hyperplastic and not hypoplastic. HUM PATHOL 31: 1404-1410. Copyright © 2000 by W.B. Saunders Company Key words: microvillous inclusion disease, apoptosis, proliferation. Abbrvviations: TUNEL, terminal uridine deoxynucleotidyl nick end labeling; TPN, total parenteral nutrition; AI, apoptotic index; PI, proliferation index.
Microvillous inclusion disease (MID), also known as congenital or familial microvillous atrophy, is a specific disorder causing severe refractory diarrhea in neonatesA ,2 The vast majority of the cases present in the first few days of life with severe watery diarrhea; however, a late onset form of the disease, with better prognosis, has been also described, e T h e disease is transmitted as an autosomal recessive disorder. Several cases of parental consanguinity and disease affecting more than 1 sibling have been described. 1-3 Recently, a cluster of MID in the Navajo population of Arizona was reported. 4 Histologically, MID is characterized by diffuse villous atrophy without inflammatory changes. Special stains such as periodic acid-Schiff (PAS), carcinoembryonic antigen (CEA), and alkaline phosphatase show a strong apical cytoplasmic staining in surface enterocytes instead of the normal, linear brush-border reactivity. 3,5 The definitive diagnosis of the disease is reached by ultrastructural examination showing surface enterocytes with apical intracytoplasmic microvillous inclusions, poorly developed brush-border microvilli, and a marked increase in electron-dense secretory granules or vesicular bodies of various sizes. 1-3Although MID largely affects the small intestine, microvillous inclusions have been also described in the large intestine, stomach, gallbladder, and kidney. 1,2,6-s The prog-
nosis of the disease is extremely poor, as life can be sustained only by total parenteral nutrition (TPN), and infants usually die from complications secondary to long-term TPN. 1,2 The only potentially life-saving therapy is combined bowel-liver transplantation. 9,1° T h e etiology or basic defect in MID is unknown. Previous studies f o u n d decreased levels of myosin, actin, and villin, suggesting a failure of cytoskeletal and microfilament proteins to bind properly with other cellular elements, la,12 Cutz et al I postulated a defect in subcellular protein trafficking leading to aberrant assembly of microvilli within intracytoplasmic vesicles, rather than at the apical cell surface. Potential mechanisms leading to this aberration could be abnormal interaction between the cytoskeleton and vesicular transport, 1 inability of apically targeted vesicles to fuse with the apical surface, inefficient recycling of membrane components, 13 or a transport defect in an as yet unidentified exocytic pathwayA 4 Alternatively, it has been proposed that the inclusions may result from involution of p r e f o r m e d surface epithelium. This hypothesis was supported by the observations that most inclusions are located within the apical part of the cytoplasm and that some superficial inclusions appear to be in continuity with the surface membrane. 2,~5 It appears that to help unravel the pathogenic basis of MID it will be necessary to determine the nature of the secretory granules (vesicular bodies) and to find an aberrant protein blocking the intracellular transport of brush b o r d e r components. As mentioned, MID is characterized histologically by villous atrophy with normal or slightly increased crypt depth. 2 Villous atrophy is the characteristic response of the small intestinal mucosa to injury. This pathological change reflects a failure in the delicate
From the Hillel-Yaffe Medical Center, Hadera, Israel; Sheba Medical Center, Ramat Gan, Israel; and Carmel Medical Center, Haifa, Israel. Accepted for publication August 24, 2000. Address correspondence and reprint requests to Gabriel M. Groisman, MD, Department of Pathology, Hillel-Yaffe Medical Center, Hadera 38100, Israel. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3111-0012510.00/0 doi:10.1053/hupa.2000.19831
ENTEROCYTIC APOPTOSISAND PROLIFERATION IN MID (Groisman et al)
FIGURE 1. Small intestinal mucosa in MID showing villous atrophy without lamina propria inflammation (A) and characteristic abnormal accumulation of PAS-positive material in the apical cytoplasm of surface enterocytes. A goblet cell (arrow) shows normal intracytoplasmic staining (B). For comparison, a normal small intestinal villus is shown. Note the typical linear brush border staining of enterocytes and intracytoplasmic staining of goblet cells. (A: hematoxylin-eosin stain, original magnification x 100; B and C: periodic acid-Schiff stain, original magnifications: B x 600, C x 400.)
balance between cell proliferation and cell loss. Recent studies showed that in normal intestine and in celiac disease, enterocytes are lost from the intestinal surface not by passive exfoliation but instead by apoptosis or programmed cell. lc~ls To our knowledge, the presence and extent of apoptosis in MID and its relationship with cell proliferation have not yet been studied. Therefore, we analyzed the presence and numbers of apoptotic and proliferative cells in MID using the terminal uridine deoxynucleotide nick end labeling (TUNEL) method to assess apoptosis and Ki-67 immunohistochemistry to detect proliferating cells. For comparison, the same techniques were performed in small bowel biopsies within normal limits and with findings typical of celiac disease.
MATERIALS AND METHODS
disease, and 10 controls with normal small intestinal morphology that had undergone small bowel biopsies for the investigation of anemia. Three of the MID cases were reported previously. ~ The fourth case was a male of Jewish origin with no significant family history. The fifth case was a female of Arab origin, with consanguineous parents. Both patients developed severe watery diarrhea during the first day of life that persisted after oral feeding was discontinued. They were supported on continuous TPN. Small bowel biopsies were obtained at 1 month in patient 4 and at 3 months in patient 5. Both patients died from sepsis attributable to TPN at 3 and 6 months of age, respectively. All the specimens were fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin and periodic acid-Schiff (PAS) reagent. Further 4-um thick sections were stained by the TUNEL method and by Ki-67 (MIB-1) immunohistochemistry.
Patients, S p e c i m e n s , a n d Tissue P r e p a r a t i o n
TUNEL P r o c e d u r e a n d A s s e s s m e n t o f A p o p t o t i c Cells
We studied well oriented small bowel biopsy specimens from 5 patients with MID, 10 patients with untreated celiac
For in situ detection of apoptosis, we applied the TUNEL method using a commercially available kit (Apoptag; Oncor,
1405
.18.16
3
.14 O Q T--
.12.10-
FIGURE 2. A p o p t o t i c indices for normal, MID, a n d celiac groups. P = .0006 (normal vMID, normal vceliac).
.=_
.08O Q.
<
.06.04.020.00 Normal
MID
Celiac
Gaithersburg, MD). Briefly, paraffin sections were dewaxed with xylene, dehydrated with graded dilutions of ethanol, and then washed with distilled water. Subsequently, the tissue specimens were digested with 20 u g / m L Proteinase K (Sigma, Dorset, UK) at room temperature for 15 minutes. Following the application of an equilibrium buffer, sections were incubated in working strength TdT containing dUTP-digoxigenin at 37°C for 60 minutes. The reaction was stopped by a prewarmed working strength stop/wash buffer. After being washed in distilled water, sections were treated with antiantidigoxigenin-peroxidase for 30 minutes at room temperature. Color was developed in a 0.05% diaminobenzidine (DAB) substrate solution. Sections were counterstained with methyl green. Negative control sections were produced by omission of the TdT. This procedure resulted in uniformly negative staining. Positive control sections were produced by DNAse I (Sigma) digestion of the DNA, giving rise to a strong
nuclear staining of all cells. Apoptotic cells present within the intestinal lamina propria served as additional intrinsic positive controls. Cells were defined as apoptotic when the whole nuclear area of the cell labelled positively or when they contained small positively labelled globular bodies (apoptotic bodies).
Immunostaining with Ki-67 Sections were placed on positively charged glass slides and deparaffinized. They were then exposed to microwave pretreatment (in 10-mmol/L citrate buffer, pH 6 at 850 W for 20 minutes) to expose epitopes. Endogenous peroxidase activity was quenched with hydrogen peroxide. Detection of binding of Ki-67 (monoclonal; ready to use; Zymed, South San Francisco, CA) was achieved using a labeled streptavidinbiotin peroxidase complex method (Zymed Histostain Plus
60-
50O O m
FIGURE 3. Proliferation indices for normal, MID, and celiac groups. P < .0001 (normal v MID, normal v celiac, MID v celiac).
40-
30 r-
._o = 9
20-
j
Normal
1406
MID
Celiac
ENTEROCYTICAPOPTOSISAND PROLIFERATIONIN MID (Groisman et al) Kit; Zymed Laboratories). The formed complexes were visualized with aminoethyl carbazole (AEC) chromogen/substrate. Sections were then counterstained with hematoxylin, dehydrated, cleared, and permanently mounted. Positive controls consisted of formalin-fixed, paraffin-embedded tonsillectomy specimens. Negative controls were performed without the primary antibody.
Q u a n t i t a t i v e a n d Statistical Analysis Hematoxylin-eosin and PAS sections of all cases were examined without knowledge of the diagnosis and classified as being either normal or consistent with celiac disease or MID. In each biopsy, TUNEL and Ki-67 staining were quantitated by counting the number of positive epithelial cells in at least 20 well oriented half-crypt units and dividing this number by the total number of crypt cells (×100) to obtain the apoptotic index (AI) and the proliferation index (PI). The results are presented as means + SD. Differences between non-parametric or parametric groups were analyzed using the Kruskal-Wallis analysis of variance or the one way analysis of variance (ANOVA) respectively, followed by the Bonferroni multiple comparison post hoc test. A P value of <.05 was considered statistically significant.
RESULTS Hematoxylin-eosin sections of all MID cases displayed subtotal to total villous atrophy with slightly increased crypt depth. O n PAS staining there was an abnormal accumulation of PAS-positive material within the apical cytoplasm of surface epithelium instead of the normal linear brush-border staining (Fig 1). The results regarding apoptosis and proliferation in the 3 groups investigated are presented in Figures 2 and 3 and Tables 1 and 2. As shown, the apoptotic index and the proliferation index for MID, although lower than those of celiac disease, were significantly higher than those of the normal controls. In all 3 groups, the apoptotic signal was detectable in apoptotic bodies and in apparently normal-looking nuclei. In normal controls, apoptotic cells were located in the u p p e r third of the villi, whereas in MID and celiac disease, apoptotic cells were seen along the surface epithelium and within the crypts (Fig 4). As for proliferating cells, they were f o u n d in the base of the crypts in normal controls and in the base and also in the middle and u p p e r portions of the crypts in MID and celiac disease (Fig 5).
TABLE 1.
Apoptotic Indices in Normal, MID, and Celiac Groups
Normal (n = 10) MID (n = 5) Celiac (n = 10)
Apoptotic Index Mean +_ SD
95% CI for the M e a n
P Value (KruskalWallis ANOVA)*
0.03 + 0.01 0.08 --- 0 . 0 8 0.16 --_0.3
0.022-0.037 0.06-0.09 0.032-0.26
.0006
* Statistically significant differences are detected between normal versus MID, and between normal versus celiac.
TABLE 2.
Proliferation Indices in Normal, MID, and Celiac Groups
Normal (n = 10) MID (n = 5) Celiac (n = 10)
Proliferation Index Mean -+ SD
95% CI for the Mean
14 -+ 2.5 28 -+ 9.2 56 -+ 14
12.9-14.6 25.7-32.6 51.4-59.9
P Value (OneWay ANOVA)* <.0001
* Statistically significant differences are detected between normal versus MID, normal versus celiac, and between MID versus celiac.
DISCUSSION This study focused on the presence and extent of apoptosis and cell proliferation in MID. We f o u n d that both cellular events are increased in this disorder, alt h o u g h not to the degree seen in celiac disease. O u r study supports the findings of Phillips and Schmitz 2 who f o u n d increased cell proliferation in MID. They p e r f o r m e d m o r p h o m e t r y of mucosal dimensions and crypt cell counting and f o u n d that crypt depth and crypt cell c o u n t in MID were increased in comparison with normal controls and decreased in comparison with celiac disease. They proposed to call the changes in MID "crypt normoplastic villous atrophy." It seems to us that "mild hyperplastic villous atrophy" would be a more accurate term. In addition to MID and celiac disease, a hyperplastic crypt response has been f o u n d in other types of pediatric intestinal diseases. Savidge et al 1° investigated epithelial cell proliferation in allergic and infectious childhood enteropathies and f o u n d crypt hyperplasia in all the disease states examined. Therefore, it appears that adaptive crypt hyperplasia is a c o m m o n tissue response to mucosal damage in allergy, infections, and MID. Although apoptosis has been extensively studied in the small intestinal mucosa, we were unable to find studies on apoptosis in MID. In the small intestine, the normal architecture is t h o u g h t to be preserved by the balance between cell proliferation and cell loss (apoptosis, necrosis, a n d / o r shedding). 16,1s Luminal shedding has long been accepted as the p r e d o m i n a n t mechanism of epithelial cell loss, balancing the high levels of epithelial cell proliferation. However, Hall et a116 have shown apoptosis to be a numerically more significant and more energy-efficient route for cell disposal. It is t h o u g h t that apoptosis probably functions as a regulator of cell migration toward the villous surface, but it is not certain whether this form of p r o g r a m m e d cell death is responsible for epithelial cell sloughing from the villous tip. 2° In the normal small intestinal mucosa there are well defined zones of both proliferation and apoptosis; proliferative cells are f o u n d in the base of the crypts, and apoptotic cells are seen most frequently toward the tips of the villi. 16,2° O u r study corroborated these observations and showed that in MID and celiac disease, proliferating cells are also detected in the mid-
1407
HUMAN PATHOLOGY
Volume 31, No. 11 (November 2000)
FIGURE 5. (A) Ki-67 positive small intestinal epithelial cells in biopsies from a normal patient.
FIGURE 4. Superficial and cryptal apoptotic small intestinal epithelial cells (arrows) in biopsies from normal (A), MID (B), and celiac (C) patients. (TUNED original magnification × 600.)
dle and upper portions of the crypts, and apoptotic cells are seen along the surface epithelium and within the crypts. The clearance of apoptotic bodies appears to occur by engulfment by adjacent enterocytes and macrophages although some shedding probably takes place. 16,1s In MID, engulfment of apoptotic bodies by adjacent cells would permit reutilization of the cellular materials. In contrast, luminal shedding will prevent their reutilization by the malabsoptive mucosa. MID and celiac disease are characterized by a flat mucosa, despite increased cell proliferation. This paradox may be explained, at least in part, by the increased epithelial apoptosis present in both diseases. Moss et a117 reported increased epithelial apoptosis and proliferation in celiac disease and assumed that the rate of overall cell loss must exceed that of new cell formation, despite the numbers of proliferating cells being higher than normal. They hypothesized that increased apoptosis is a major factor in the increased cell loss of celiac disease although other forms of cell loss may also be increased. Based on our findings, we suggest the same hypothesis for MID. In both Moss's study and ours, the absolute numbers of proliferating cells surpassed those of apoptotic cells. However, apoptosis and proliferation
1408
ENTEROCYTICAPOPTOSISAND PROLIFERATION tN MID (Groisman et al)
FIGURE §. (Continued) Ki-67 positive small intestinal epithelial cells in biopsies from MID (B) and celiac (C) patients. (Ki-67 immunostaining, original magnification × 200,)
were evaluated by two different techniques being possible that the period of time during which an enterocyte reacts to the TUNEL m e t h o d is m u c h shorter than that during which Ki-67 is expressed. In conclusion, we have shown that enterocyte apoptosis and proliferation are increased in MID and that crypts in MID are hyperplastic and not hypoplastic. Apoptosis appears to be a major factor of cell loss and may be responsible, at least in part, for the villous atrophy in MID. Acknowledgment. T h e a u t h o r s t h a n k S. Younes a n d A. A t a m n a for t e c h n i c a l assistance.
REFERENCES 1. Cutz E, Rhoads JM, Drumm B, et al: Microvillous inclusion disease: An inherited defect of brush-border assembly and differentiation. N Engl J Med 320:646-651, 1989 2. Phillips AD, SchmitzJ: Familial microvillous atrophy: A clinicopathological survey of 23 cases. J Pediatr Gastroenterol Nutr 14: 380-396, 1992 3. Groisman GM, Ben-Izhak O, Schwersenz A, et al: The value of
polyclonal carcinoembryonic antigen in the diagnosis of microvillous inclusion disease. HUM PATHOL 24:1232-1237, 1993 4. Pohl JF, Shub MD, Trevelline EE, et al: A cluster of microvillous inclusion disease in the Navajo population. J Pediatr 134:103106, 1999 5. Lake BD: Microvillous inclusion disease: Specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Pathol 41:880-882, 1988 6. Cutz E, Sherman PM, Davidson GP: Enteropathies associated with protracted diarrhea of infancy: Clinicopathological features, cellular and molecular mechanisms. Pediatr Pathol Lab Med 17:335367, 1997 7. RhoadsJM, Vogler RC, Lacey SR, et al: Microvillous inclusion disease. In vitro jejunal electrolyte transport. Gastroenterology 100: 811-817, 1991 8. Schofield DE, Agostini RM, Yunis EJ: Gastrointestinal microvillous inclusion disease. A m J Clin Pathol 98:119-124, 1992 9. Herzog D, Atkison P, Grant D, et al: Combined bowel-liver transplantation in an infant with microvillous inclusion disease. J Pediatr Gastroenterol Nutr 22:405-408, 1998 10. Oliva MM, Perman JA, SaavedraJM, et al: Successful intestinal transplantation for microvillous inclusion disease. Gastroenterology 106:771-774, 1994 11. Carruthers L, Phillips AD, Dourmashkin R, et al: Biochemical abnormality in brush-border membrane protein of a patient with congenital microvillous atrophy. J Pediatr Gastroenterol Nutr 4:902907, 1985
1409
HUMAN PATHOLOGY
Volume 31, No, 11 (November 2000)
12. Cutz E, Thorner P, Sherman PM: Microvillous inclusion disease (MID)-- Generalized defect of brush border assembly. Lab Invest 58:3P, 1988 13. Fish EM, Molitoris BA: Alterations in epithelial polarity and the pathogenesis of disease states. N Engl J Med 330:15801588, 1994 14. Phillips A, Fransen J, Hauri H-P, et al: The constitutive exocytotic pathway in microvillous atrophy. J Pediatr Gastroenterol Nutr 17:239-246, 1993 15. Bell SW, Kerner JA, Sibley RK: Microvillous inclusion disease. The importance of electron microscopy for diagnosis. AmJ Surg Pathol 15:1157-1164, 1991
16. Hall PA, Coates PJ, Ansari B, et al: Regulation of cell number in the mammalian gastrointestinal tract: The importance of apoptosis. J Cell Sci 107:3569-3577, 1994 17. Moss SF, Attia L, ScholesJV, et al: Increased small intestinal apoptosis in celiac disease. Gut 39:811-817, 1996 18. Str/ker J, Koretz K, Gfmthert AR, et al: In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis. Gut 37:819-825, 1995 19. Savidge TC, Shmakov AN, Walker-SmithJA, et al: Epithelial cell proliferation in childhood enteropathies. Gut 39:185-193, 1996 20. Watson AJM: Necrosis and apoptosis in the gastrointestinal tract. Gut 37:165-167, 1995
1410
Significant differences were found between normal and MID, and between normal and celiac cases. The percentage of Ki-67 positive cells per crypt (proliferation index) in normal, MID, and celiac cases was 14 -+ 2.5%, 28 -+ 9.2%, and 56 -+ 14%. Significant differences were found between the 3 groups. In conclusion, (1) enterocyte apoptosis and proliferation are increased in MID; (2) apoptosis appears to be an important factor of cell loss and may be, at least in part, responsible for villous atrophy in MID; and (3) crypts in MID are hyperplastic and not hypoplastic. HUM PATHOL 31: 1404-1410. Copyright © 2000 by W.B. Saunders Company Key words: microvillous inclusion disease, apoptosis, proliferation. Abbrvviations: TUNEL, terminal uridine deoxynucleotidyl nick end labeling; TPN, total parenteral nutrition; AI, apoptotic index; PI, proliferation index.
Microvillous inclusion disease (MID), also known as congenital or familial microvillous atrophy, is a specific disorder causing severe refractory diarrhea in neonatesA ,2 The vast majority of the cases present in the first few days of life with severe watery diarrhea; however, a late onset form of the disease, with better prognosis, has been also described, e T h e disease is transmitted as an autosomal recessive disorder. Several cases of parental consanguinity and disease affecting more than 1 sibling have been described. 1-3 Recently, a cluster of MID in the Navajo population of Arizona was reported. 4 Histologically, MID is characterized by diffuse villous atrophy without inflammatory changes. Special stains such as periodic acid-Schiff (PAS), carcinoembryonic antigen (CEA), and alkaline phosphatase show a strong apical cytoplasmic staining in surface enterocytes instead of the normal, linear brush-border reactivity. 3,5 The definitive diagnosis of the disease is reached by ultrastructural examination showing surface enterocytes with apical intracytoplasmic microvillous inclusions, poorly developed brush-border microvilli, and a marked increase in electron-dense secretory granules or vesicular bodies of various sizes. 1-3Although MID largely affects the small intestine, microvillous inclusions have been also described in the large intestine, stomach, gallbladder, and kidney. 1,2,6-s The prog-
nosis of the disease is extremely poor, as life can be sustained only by total parenteral nutrition (TPN), and infants usually die from complications secondary to long-term TPN. 1,2 The only potentially life-saving therapy is combined bowel-liver transplantation. 9,1° T h e etiology or basic defect in MID is unknown. Previous studies f o u n d decreased levels of myosin, actin, and villin, suggesting a failure of cytoskeletal and microfilament proteins to bind properly with other cellular elements, la,12 Cutz et al I postulated a defect in subcellular protein trafficking leading to aberrant assembly of microvilli within intracytoplasmic vesicles, rather than at the apical cell surface. Potential mechanisms leading to this aberration could be abnormal interaction between the cytoskeleton and vesicular transport, 1 inability of apically targeted vesicles to fuse with the apical surface, inefficient recycling of membrane components, 13 or a transport defect in an as yet unidentified exocytic pathwayA 4 Alternatively, it has been proposed that the inclusions may result from involution of p r e f o r m e d surface epithelium. This hypothesis was supported by the observations that most inclusions are located within the apical part of the cytoplasm and that some superficial inclusions appear to be in continuity with the surface membrane. 2,~5 It appears that to help unravel the pathogenic basis of MID it will be necessary to determine the nature of the secretory granules (vesicular bodies) and to find an aberrant protein blocking the intracellular transport of brush b o r d e r components. As mentioned, MID is characterized histologically by villous atrophy with normal or slightly increased crypt depth. 2 Villous atrophy is the characteristic response of the small intestinal mucosa to injury. This pathological change reflects a failure in the delicate
From the Hillel-Yaffe Medical Center, Hadera, Israel; Sheba Medical Center, Ramat Gan, Israel; and Carmel Medical Center, Haifa, Israel. Accepted for publication August 24, 2000. Address correspondence and reprint requests to Gabriel M. Groisman, MD, Department of Pathology, Hillel-Yaffe Medical Center, Hadera 38100, Israel. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3111-0012510.00/0 doi:10.1053/hupa.2000.19831
ENTEROCYTIC APOPTOSISAND PROLIFERATION IN MID (Groisman et al)
FIGURE 1. Small intestinal mucosa in MID showing villous atrophy without lamina propria inflammation (A) and characteristic abnormal accumulation of PAS-positive material in the apical cytoplasm of surface enterocytes. A goblet cell (arrow) shows normal intracytoplasmic staining (B). For comparison, a normal small intestinal villus is shown. Note the typical linear brush border staining of enterocytes and intracytoplasmic staining of goblet cells. (A: hematoxylin-eosin stain, original magnification x 100; B and C: periodic acid-Schiff stain, original magnifications: B x 600, C x 400.)
balance between cell proliferation and cell loss. Recent studies showed that in normal intestine and in celiac disease, enterocytes are lost from the intestinal surface not by passive exfoliation but instead by apoptosis or programmed cell. lc~ls To our knowledge, the presence and extent of apoptosis in MID and its relationship with cell proliferation have not yet been studied. Therefore, we analyzed the presence and numbers of apoptotic and proliferative cells in MID using the terminal uridine deoxynucleotide nick end labeling (TUNEL) method to assess apoptosis and Ki-67 immunohistochemistry to detect proliferating cells. For comparison, the same techniques were performed in small bowel biopsies within normal limits and with findings typical of celiac disease.
MATERIALS AND METHODS
disease, and 10 controls with normal small intestinal morphology that had undergone small bowel biopsies for the investigation of anemia. Three of the MID cases were reported previously. ~ The fourth case was a male of Jewish origin with no significant family history. The fifth case was a female of Arab origin, with consanguineous parents. Both patients developed severe watery diarrhea during the first day of life that persisted after oral feeding was discontinued. They were supported on continuous TPN. Small bowel biopsies were obtained at 1 month in patient 4 and at 3 months in patient 5. Both patients died from sepsis attributable to TPN at 3 and 6 months of age, respectively. All the specimens were fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin and periodic acid-Schiff (PAS) reagent. Further 4-um thick sections were stained by the TUNEL method and by Ki-67 (MIB-1) immunohistochemistry.
Patients, S p e c i m e n s , a n d Tissue P r e p a r a t i o n
TUNEL P r o c e d u r e a n d A s s e s s m e n t o f A p o p t o t i c Cells
We studied well oriented small bowel biopsy specimens from 5 patients with MID, 10 patients with untreated celiac
For in situ detection of apoptosis, we applied the TUNEL method using a commercially available kit (Apoptag; Oncor,
1405
.18.16
3
.14 O Q T--
.12.10-
FIGURE 2. A p o p t o t i c indices for normal, MID, a n d celiac groups. P = .0006 (normal vMID, normal vceliac).
.=_
.08O Q.
<
.06.04.020.00 Normal
MID
Celiac
Gaithersburg, MD). Briefly, paraffin sections were dewaxed with xylene, dehydrated with graded dilutions of ethanol, and then washed with distilled water. Subsequently, the tissue specimens were digested with 20 u g / m L Proteinase K (Sigma, Dorset, UK) at room temperature for 15 minutes. Following the application of an equilibrium buffer, sections were incubated in working strength TdT containing dUTP-digoxigenin at 37°C for 60 minutes. The reaction was stopped by a prewarmed working strength stop/wash buffer. After being washed in distilled water, sections were treated with antiantidigoxigenin-peroxidase for 30 minutes at room temperature. Color was developed in a 0.05% diaminobenzidine (DAB) substrate solution. Sections were counterstained with methyl green. Negative control sections were produced by omission of the TdT. This procedure resulted in uniformly negative staining. Positive control sections were produced by DNAse I (Sigma) digestion of the DNA, giving rise to a strong
nuclear staining of all cells. Apoptotic cells present within the intestinal lamina propria served as additional intrinsic positive controls. Cells were defined as apoptotic when the whole nuclear area of the cell labelled positively or when they contained small positively labelled globular bodies (apoptotic bodies).
Immunostaining with Ki-67 Sections were placed on positively charged glass slides and deparaffinized. They were then exposed to microwave pretreatment (in 10-mmol/L citrate buffer, pH 6 at 850 W for 20 minutes) to expose epitopes. Endogenous peroxidase activity was quenched with hydrogen peroxide. Detection of binding of Ki-67 (monoclonal; ready to use; Zymed, South San Francisco, CA) was achieved using a labeled streptavidinbiotin peroxidase complex method (Zymed Histostain Plus
60-
50O O m
FIGURE 3. Proliferation indices for normal, MID, and celiac groups. P < .0001 (normal v MID, normal v celiac, MID v celiac).
40-
30 r-
._o = 9
20-
j
Normal
1406
MID
Celiac
ENTEROCYTICAPOPTOSISAND PROLIFERATIONIN MID (Groisman et al) Kit; Zymed Laboratories). The formed complexes were visualized with aminoethyl carbazole (AEC) chromogen/substrate. Sections were then counterstained with hematoxylin, dehydrated, cleared, and permanently mounted. Positive controls consisted of formalin-fixed, paraffin-embedded tonsillectomy specimens. Negative controls were performed without the primary antibody.
Q u a n t i t a t i v e a n d Statistical Analysis Hematoxylin-eosin and PAS sections of all cases were examined without knowledge of the diagnosis and classified as being either normal or consistent with celiac disease or MID. In each biopsy, TUNEL and Ki-67 staining were quantitated by counting the number of positive epithelial cells in at least 20 well oriented half-crypt units and dividing this number by the total number of crypt cells (×100) to obtain the apoptotic index (AI) and the proliferation index (PI). The results are presented as means + SD. Differences between non-parametric or parametric groups were analyzed using the Kruskal-Wallis analysis of variance or the one way analysis of variance (ANOVA) respectively, followed by the Bonferroni multiple comparison post hoc test. A P value of <.05 was considered statistically significant.
RESULTS Hematoxylin-eosin sections of all MID cases displayed subtotal to total villous atrophy with slightly increased crypt depth. O n PAS staining there was an abnormal accumulation of PAS-positive material within the apical cytoplasm of surface epithelium instead of the normal linear brush-border staining (Fig 1). The results regarding apoptosis and proliferation in the 3 groups investigated are presented in Figures 2 and 3 and Tables 1 and 2. As shown, the apoptotic index and the proliferation index for MID, although lower than those of celiac disease, were significantly higher than those of the normal controls. In all 3 groups, the apoptotic signal was detectable in apoptotic bodies and in apparently normal-looking nuclei. In normal controls, apoptotic cells were located in the u p p e r third of the villi, whereas in MID and celiac disease, apoptotic cells were seen along the surface epithelium and within the crypts (Fig 4). As for proliferating cells, they were f o u n d in the base of the crypts in normal controls and in the base and also in the middle and u p p e r portions of the crypts in MID and celiac disease (Fig 5).
TABLE 1.
Apoptotic Indices in Normal, MID, and Celiac Groups
Normal (n = 10) MID (n = 5) Celiac (n = 10)
Apoptotic Index Mean +_ SD
95% CI for the M e a n
P Value (KruskalWallis ANOVA)*
0.03 + 0.01 0.08 --- 0 . 0 8 0.16 --_0.3
0.022-0.037 0.06-0.09 0.032-0.26
.0006
* Statistically significant differences are detected between normal versus MID, and between normal versus celiac.
TABLE 2.
Proliferation Indices in Normal, MID, and Celiac Groups
Normal (n = 10) MID (n = 5) Celiac (n = 10)
Proliferation Index Mean -+ SD
95% CI for the Mean
14 -+ 2.5 28 -+ 9.2 56 -+ 14
12.9-14.6 25.7-32.6 51.4-59.9
P Value (OneWay ANOVA)* <.0001
* Statistically significant differences are detected between normal versus MID, normal versus celiac, and between MID versus celiac.
DISCUSSION This study focused on the presence and extent of apoptosis and cell proliferation in MID. We f o u n d that both cellular events are increased in this disorder, alt h o u g h not to the degree seen in celiac disease. O u r study supports the findings of Phillips and Schmitz 2 who f o u n d increased cell proliferation in MID. They p e r f o r m e d m o r p h o m e t r y of mucosal dimensions and crypt cell counting and f o u n d that crypt depth and crypt cell c o u n t in MID were increased in comparison with normal controls and decreased in comparison with celiac disease. They proposed to call the changes in MID "crypt normoplastic villous atrophy." It seems to us that "mild hyperplastic villous atrophy" would be a more accurate term. In addition to MID and celiac disease, a hyperplastic crypt response has been f o u n d in other types of pediatric intestinal diseases. Savidge et al 1° investigated epithelial cell proliferation in allergic and infectious childhood enteropathies and f o u n d crypt hyperplasia in all the disease states examined. Therefore, it appears that adaptive crypt hyperplasia is a c o m m o n tissue response to mucosal damage in allergy, infections, and MID. Although apoptosis has been extensively studied in the small intestinal mucosa, we were unable to find studies on apoptosis in MID. In the small intestine, the normal architecture is t h o u g h t to be preserved by the balance between cell proliferation and cell loss (apoptosis, necrosis, a n d / o r shedding). 16,1s Luminal shedding has long been accepted as the p r e d o m i n a n t mechanism of epithelial cell loss, balancing the high levels of epithelial cell proliferation. However, Hall et a116 have shown apoptosis to be a numerically more significant and more energy-efficient route for cell disposal. It is t h o u g h t that apoptosis probably functions as a regulator of cell migration toward the villous surface, but it is not certain whether this form of p r o g r a m m e d cell death is responsible for epithelial cell sloughing from the villous tip. 2° In the normal small intestinal mucosa there are well defined zones of both proliferation and apoptosis; proliferative cells are f o u n d in the base of the crypts, and apoptotic cells are seen most frequently toward the tips of the villi. 16,2° O u r study corroborated these observations and showed that in MID and celiac disease, proliferating cells are also detected in the mid-
1407
HUMAN PATHOLOGY
Volume 31, No. 11 (November 2000)
FIGURE 5. (A) Ki-67 positive small intestinal epithelial cells in biopsies from a normal patient.
FIGURE 4. Superficial and cryptal apoptotic small intestinal epithelial cells (arrows) in biopsies from normal (A), MID (B), and celiac (C) patients. (TUNED original magnification × 600.)
dle and upper portions of the crypts, and apoptotic cells are seen along the surface epithelium and within the crypts. The clearance of apoptotic bodies appears to occur by engulfment by adjacent enterocytes and macrophages although some shedding probably takes place. 16,1s In MID, engulfment of apoptotic bodies by adjacent cells would permit reutilization of the cellular materials. In contrast, luminal shedding will prevent their reutilization by the malabsoptive mucosa. MID and celiac disease are characterized by a flat mucosa, despite increased cell proliferation. This paradox may be explained, at least in part, by the increased epithelial apoptosis present in both diseases. Moss et a117 reported increased epithelial apoptosis and proliferation in celiac disease and assumed that the rate of overall cell loss must exceed that of new cell formation, despite the numbers of proliferating cells being higher than normal. They hypothesized that increased apoptosis is a major factor in the increased cell loss of celiac disease although other forms of cell loss may also be increased. Based on our findings, we suggest the same hypothesis for MID. In both Moss's study and ours, the absolute numbers of proliferating cells surpassed those of apoptotic cells. However, apoptosis and proliferation
1408
ENTEROCYTICAPOPTOSISAND PROLIFERATION tN MID (Groisman et al)
FIGURE §. (Continued) Ki-67 positive small intestinal epithelial cells in biopsies from MID (B) and celiac (C) patients. (Ki-67 immunostaining, original magnification × 200,)
were evaluated by two different techniques being possible that the period of time during which an enterocyte reacts to the TUNEL m e t h o d is m u c h shorter than that during which Ki-67 is expressed. In conclusion, we have shown that enterocyte apoptosis and proliferation are increased in MID and that crypts in MID are hyperplastic and not hypoplastic. Apoptosis appears to be a major factor of cell loss and may be responsible, at least in part, for the villous atrophy in MID. Acknowledgment. T h e a u t h o r s t h a n k S. Younes a n d A. A t a m n a for t e c h n i c a l assistance.
REFERENCES 1. Cutz E, Rhoads JM, Drumm B, et al: Microvillous inclusion disease: An inherited defect of brush-border assembly and differentiation. N Engl J Med 320:646-651, 1989 2. Phillips AD, SchmitzJ: Familial microvillous atrophy: A clinicopathological survey of 23 cases. J Pediatr Gastroenterol Nutr 14: 380-396, 1992 3. Groisman GM, Ben-Izhak O, Schwersenz A, et al: The value of
polyclonal carcinoembryonic antigen in the diagnosis of microvillous inclusion disease. HUM PATHOL 24:1232-1237, 1993 4. Pohl JF, Shub MD, Trevelline EE, et al: A cluster of microvillous inclusion disease in the Navajo population. J Pediatr 134:103106, 1999 5. Lake BD: Microvillous inclusion disease: Specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Pathol 41:880-882, 1988 6. Cutz E, Sherman PM, Davidson GP: Enteropathies associated with protracted diarrhea of infancy: Clinicopathological features, cellular and molecular mechanisms. Pediatr Pathol Lab Med 17:335367, 1997 7. RhoadsJM, Vogler RC, Lacey SR, et al: Microvillous inclusion disease. In vitro jejunal electrolyte transport. Gastroenterology 100: 811-817, 1991 8. Schofield DE, Agostini RM, Yunis EJ: Gastrointestinal microvillous inclusion disease. A m J Clin Pathol 98:119-124, 1992 9. Herzog D, Atkison P, Grant D, et al: Combined bowel-liver transplantation in an infant with microvillous inclusion disease. J Pediatr Gastroenterol Nutr 22:405-408, 1998 10. Oliva MM, Perman JA, SaavedraJM, et al: Successful intestinal transplantation for microvillous inclusion disease. Gastroenterology 106:771-774, 1994 11. Carruthers L, Phillips AD, Dourmashkin R, et al: Biochemical abnormality in brush-border membrane protein of a patient with congenital microvillous atrophy. J Pediatr Gastroenterol Nutr 4:902907, 1985
1409
HUMAN PATHOLOGY
Volume 31, No, 11 (November 2000)
12. Cutz E, Thorner P, Sherman PM: Microvillous inclusion disease (MID)-- Generalized defect of brush border assembly. Lab Invest 58:3P, 1988 13. Fish EM, Molitoris BA: Alterations in epithelial polarity and the pathogenesis of disease states. N Engl J Med 330:15801588, 1994 14. Phillips A, Fransen J, Hauri H-P, et al: The constitutive exocytotic pathway in microvillous atrophy. J Pediatr Gastroenterol Nutr 17:239-246, 1993 15. Bell SW, Kerner JA, Sibley RK: Microvillous inclusion disease. The importance of electron microscopy for diagnosis. AmJ Surg Pathol 15:1157-1164, 1991
16. Hall PA, Coates PJ, Ansari B, et al: Regulation of cell number in the mammalian gastrointestinal tract: The importance of apoptosis. J Cell Sci 107:3569-3577, 1994 17. Moss SF, Attia L, ScholesJV, et al: Increased small intestinal apoptosis in celiac disease. Gut 39:811-817, 1996 18. Str/ker J, Koretz K, Gfmthert AR, et al: In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis. Gut 37:819-825, 1995 19. Savidge TC, Shmakov AN, Walker-SmithJA, et al: Epithelial cell proliferation in childhood enteropathies. Gut 39:185-193, 1996 20. Watson AJM: Necrosis and apoptosis in the gastrointestinal tract. Gut 37:165-167, 1995
1410