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Intrahepatic mast cell population correlates with clinical outcome in biliary atresia
Intrahepatic Mast Cell Population Correlates With Clinical Outcome in Biliary Atresia By Abul Faiz Kabir Uddin Ahmed, Haruo Ohtani, Masaki Nio, Nobuo Funaki, Hideyuki Sasaki, Hiroshi Nagura, and Ryoji Ohi Sendai, Japan
Purpose: To determine if mast cells influence the clinical outcome in biliary atresia (BA), the authors examined the intrahepatic mast cell population in BA. Methods: Mast cells were identified histochemically using Toludin Blue and immunohistochemically using antimast cell tryptase antibody in formalin-fixed paraffin-embedded sections from 21 cases of BA. Patients were divided into 3 groups; group I (n ⫽ 8) with good liver function, group II (n ⫽ 8) with moderate liver dysfunction, and group III (n ⫽ 5) with severe liver dysfunction. Liver biopsies from patients with choledochal cysts (CDC, n ⫽ 5), and normal liver (NL, n ⫽ 4) served as controls. The results were compared among the groups.
in the portal tracts. Mast cell numbers per medium power field (20⫻ magnification) were higher in BA than in the controls (15.03 ⫾ 2.25 v 3.85 ⫾ .65, [mean ⫾ SEM], P ⬍ .05, BA v CDC; 15.03 ⫾ 2.25 v 1.73 ⫾ .06, [mean ⫾ SEM], P ⬍ .05, BA v NL, immunohistochemical data). Clinical correlation showed an association between higher mast cell number and liver dysfunction (32.62 ⫾ .80 v 8.52 ⫾ .87 [mean ⫾ SEM], group III v group I; P ⬍ .05, immunohistochemical data).
Conclusion: Increased mast cell population in BA adversely affects liver function and raises the possibility that type I allergic reaction may play role in the pathology of BA. J Pediatr Surg 35:1762-1765. Copyright © 2000 by W.B. Saunders Company.
Results: Both histochemical and immunohistochemical methods showed similar data. Mast cells were seen mostly
INDEX WORDS: Biliary atresia, mast cells, clinical correlation.
M
BA. In this study we therefore attempted to analyze the mast cells in BA to determine what roles the intrahepatic mast cell population play in the pathobiology of BA.
AST CELLS ordinarily are distributed in normal connective tissue, mainly adjacent to blood and lymphatic vessels, nerves, and epithelial surfaces.1 Mast cells contain a variety of potent mediators of inflammation2 and cytokines,3 which are released after mast cells are activated. Mast cells act as critical effector cells in the inflammatory reactions underlying disorders of IgEdependent immediate hypersensitivity and in the expression of protective immunity involving IgE.4 In type I allergic reaction they act to precipitate an acute circulatory disturbance through capillary dilatation.5 In acute inflammation they cause endothelial cell contraction and increased capillary permeability,2 whereas in chronic inflammation they participate in fibrogenesis.6 Biliary atresia (BA) is a disease of unknown etiology in which obliteration of the extrahepatic bile duct results in intrahepatic cholestasis, edema, widening of the portal tracts, proliferation of bile ductules, and progression to fibrosis. We hypothesized that mast cells might play important roles in BA pathology. However, there is no study describing the intrahepatic mast cell population in
MATERIALS AND METHODS
Tissue Specimens With due approval from the Ethical Committee, specimens from 21 patients with BA (sampled during primary Kasai hepaticoportoenterostomy) were retrieved from the pathology file of Tohoku University School of Medicine. There were 9 boys and 12 girls with an age range from 12 to 128 days (54.14 ⫾ 25.15 days [mean ⫾ SD]). Specimens from choledochal cysts (CDC, n ⫽ 5) with an age range of 18 days to 10 months (4.1 ⫾ 3.5 months) and normal liver (NL, n ⫽ 4) with an age range from 11 months to 107 months (40.5 ⫾ 44.64 months) were used as controls. We obtained informed written consents from the patients’ parents, relatives, or patients themselves.
Tissue Preparation for Immunohistochemistry Tissue specimens were fixed in 10% formalin for up to 24 hours and were embedded in paraffin using the routine procedures. Embedded blocks were sectioned at 3 m thickness and mounted on silane-coated glass slides. Routine H&E staining was done in all the blocks.
Immunohistochemistry From the Departments of Pediatric Surgery and Pathology, Tohoku University School of Medicine, Aoba-ku, Sendai, Japan. Address reprint requests to Ryoji Ohi, MD, FAAP, Professor and Chairman, Department of Pediatric Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Copyright © 2000 by W.B. Saunders Company 0022-3468/00/3512-0016$03.00/0 doi:10.1053/jpsu.2000.19245 1762
Sections were pretreated with 1% trypsin before the 2-step indirect immunoperoxidase method was used using DAKO Envision system (DAKO, Glostrup, Denmark) as the detection agent. Nonspecific reactivity was blocked by treating the sections with 1% bovine serum albumin (BSA) for 10 minutes. Mouse monoclonal antimast cell tryptase antibody (clone AA1, dilution 1:200; DAKO) was applied for 18 hours at 4°C. To block the endogenous peroxidase activity, sections were treated with 0.3% H2O2 in methanol for 12 minutes. The DAKO
Journal of Pediatric Surgery, Vol 35, No 12 (December), 2000: pp 1762-1765
MAST CELLS IN BILIARY ATRESIA
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Table 1. Number of Mast Cells in Portal Tracts in Biliary Atresia and Controls No. of Mast Cells Case
Biliary atresia (n ⫽ 21) Choledochal cyst (n ⫽ 5) Normal liver (n ⫽ 4)
Toludin Blue Staining
Mast Cell Tryptase Immunostaining
9.15 ⫾ 1.87
15.03 ⫾ 2.25*
1.85 ⫾ .85 0
3.85 ⫾ .65 1.73 ⫾ .06
* P ⬍ .05 from choledochal cyst and normal liver group.
Envision reagent that recognizes both mouse and rabbit primary antibodies were applied for 1 hour at room temperature. For the negative control, primary antibodies were replaced by 0.5% BSA or other irrelevant antibodies. Sections were developed in 0.03% diaminobenzidine (DAB; Dojin, Kumamoto, Japan) containing 0.005% H2O2 and 0.065% NaN3 for 5 minutes and counterstained by hematoxylin or methyl green.
Histochemistry
Mast Cell Histochemistry Table 1 summarizes the results. A similar distribution of mast cells could be observed, although the absolute numbers were lower than those observed with immunohistochemistry. A range of cells from partially degranulated to fully granulated ones could be observed (data not shown). Clinical Correlation The number of mast cells was highest in group III (32.62 ⫾ .80, severe liver dysfunction, Fig 1A, Table 2), lower in group II (10.56 ⫾ .70, moderate liver dysfunction, Table 2), and lowest in group I (8.52 ⫾ .87, good liver function, Fig 1B, Table 2). DISCUSSION
Our study analyzes the mast cell population in BA and correlates the mast cell numbers to clinical parameters.
Mast cells were stained histochemically using 0.5% Toludin Blue, pH 0.5. Briefly, after deparaffinization the sections were kept in Toludin Blue for 30 minutes, then washed in water and finally air dried. No nuclear counterstaining was done.
Quantification for Immunohistochemistry Mast cells were counted according to the method described7 with some modifications. Briefly, 5 nonoverlapping fields were chosen randomly from the portal areas. Using ⫻20 objective lens mast cells were counted, and the data are expressed as mean ⫾ SEM per medium power field. An independent observer crosschecked the results.
Clinical Correlation The duration of follow-up ranged from 5 months to 137 months with a median of 23 months. Patients were divided into 3 groups as previously described with some modifications.8 Group I included 8 patients with good liver function (Serum bilirubin ⬍1.5 mg/dL, SGPT ⬍35 IU/L, SGOT ⬍40 IU/L); group II included 8 patients with moderate liver dysfunction (serum bilirubin ⬍1.5 mg/dL, SGPT ⬎35 IU/L, SGOT ⬎40 IU/L); and group III included 5 patients with severe liver dysfunction (serum bilirubin ⬎1.5 mg/dL, SGPT ⬎35 IU/L, SGOT ⬎40 IU/L). The number of mast cells per medium power field was compared among the groups.
Statistical Analysis The results were tested for statistical significance using the Bonferroni-Dunn test.
RESULTS
Mast Cell Immunohistochemistry In the controls few mast cells could be observed (1.73 ⫾ .06, NL; 3.85 ⫾ .65, CDC; Table 1). In contrast, a significantly higher number of mast cells could be detected in BA (15.03 ⫾ 2.25, Table 1). Most of the mast cells were observed in the portal tracts, whereas a few mast cells could be seen in the hepatic lobule. Some mast cells appeared round to ovoid in shape, whereas some others appeared elongated and spindle shaped.
Fig 1. Mast cell–specific immunohistochemistry in BA. (A) Section from a patient who later had sever liver dysfunction shows a large number of mast cells (arrows). (B) In contrast a few cells are seen in the section from a patient with good liver function. (H&E, original magnification ⴛ200.)
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AHMED ET AL
Table 2. Correlation of Mast Cell Number in Portal Tracts to Indicators of Liver Function No. of Mast Cells Per Medium Power Field (mean ⫾ SEM) Group
Toludin Blue Staining
Group I (n ⫽ 8) (T.Bil ⬍1.5 mg/dL, GOT ⬍40 IU/L, GPT ⬍35 IU/L) Group II (n ⫽ 8) (T.Bil ⬍1.5 mg/dL, GOT ⬎40 IU/L, GPT ⬎35 IU/L) Group III (n ⫽ 5) (T.Bil ⬍1.5 mg/dL, GOT ⬍40 IU/L, GPT ⬍35 IU/L)
Mast Cell Tryptase Immunostaining
4.72 ⫾ 1.26
8.52 ⫾ .87
5.9 ⫾ 2.21
10.56 ⫾ .70
21.44 ⫾ 2.42
32.62 ⫾ .80*
* P ⬍ .05 from group I and group II.
Both our immunohistochemical and histochemical data show that mast cell population is increased in BA as compared with the controls. It has been further shown that higher mast cell number is associated with poor liver function indicating their significant involvement in the pathobiology of BA. We do not have any explanation regarding the expanded mast cell population in BA. One investigation has shown transforming growth factor beta (TGF-) to be a potent mast cell chemotactic factor,9 and we have reported recently that there is overexpression of TGF- in BA.10 It is possible that TGF- increases the hepatic mast cell number. Moreover, bile acids also may contribute to the recruitment and proliferation of hepatic mast cells.11 However, further studies are required to determine if mast cells act as primary etiologic agents for BA or they represent the effects of the disease process itself initiated by other factors. We have observed large accumulations of mast cells in the portal tracts in BA. It is possible that mast cells in this location may lead to such intrahepatic changes in BA as edema and widening of the porta tracts and progressive fibrosis because mast cells have been identified to be capable of releasing histamine and prostaglandins5 as well as cytokines such as interleukin 1 (IL-1),12 tumor necrosis factor alpha (TNF-␣),13 TGF-,14 and basic fibroblast growth factor (bFGF).15 It has been shown that chronic cholestasis is associated with increased liver histamine content.16 Moreover, it is speculated that bile acids may directly act on the tissue mast cells to release histamine and other mast cell mediators,11 which, in turn, can cause disturbed tissue microcirculation leading to edema and widening of the portal tracts as seen in BA.
Association of increased mast cell numbers with liver fibrogenesis also have been reported by various investigators.17,18 Published reports indicate that histamine enhances the biosynthesis of collagen in fibroblasts,19,20 and increased liver histamine level correlates to fibrogenesis.21 Mast cells may promote fibrosis in 2 ways— by enhancing the production of extracellular matrix through releasing fibrogenic cytokines and growth factors and by decreasing matrix degradation through releasing inhibitors of proteases.17 The presence of significantly higher number of mast cells in BA patients who had severe liver dysfunction suggests that mast cells actively contribute to liver damage. Therefore, mast cells need to be assessed as part of any clinicopathologic evaluation of BA. Identification of mast cells long has been a difficult task because of mast cell’s close similarity to basophils. However, immunohistochemistry with mast cell–specific antitryptase antibody now renders its accurate identification possible.11 In this study, immunohistochemistry and histochemistry provided similar data suggesting that the simpler and quicker histochemical staining can be useful in the assessment of the mast cell population in BA. Increased mast cell numbers in BA also raises the possible involvement of type I allergic reaction. The clinical improvement after the use of antiinflammatory antiallergic steroid, seen at times when the patients present with deterioration of liver functions, also highlights this possibility. However, a further detailed study focusing on the distribution of IgE including the distribution of the IgE producing plasma cells in BA may help us determine if indeed type I allergic reaction does exist in BA.
REFERENCES 1. Galli SJ: New concepts about mast cells. N Engl J Med 328:257265, 1993 2. Cotran RS, Kumar V, Robins SL: Pathologic basis of disease. Philadelphia, PA, Saunders, 1994, pp 64-65 3. Gordon JR, Burd PR, Galli SJ: Mast cells as source of multifunctional cytokines. Immunol Today 11:458-464, 1990 4. Ishizaka T: Mechanisms of Ig E-mediated hypersensitivity, in Middleton JE Jr, Reed CE, Ellis EF, et al (eds): Allergy Principles and Practice. St. Louis, MO, Mosby, 1988, pp 71
5. Roitt IM: Essential Immunology. Oxford, England, Blackwell Scientific Publications, 1994, pp 35 6. Thompson HL, Burkelo PD, Babreil G, et al: Murine mast cells synthesize basement membrane components. A potential role in early fibrosis. J Clin Invest 87:619-623, 1991 7. Yamashiro M, Kuoda W, Kono N, et al: Distribution of intrahepatic mast cells in various hepatobiliary disorders an immunohistochemical study. Virchows Arch 433:471-479, 1998 8. Kobayashi H, Miyano T, Horikoshi K, et al: Prognostic value of
MAST CELLS IN BILIARY ATRESIA
serum procollagen III peptide and type IV collagen in patients with biliary atresia. J Pediatr Surg 33:112-114, 1998 9. Gruber BL, Marchese MJ, Kew RR: Transforming growth factor 1 mediates mast cell chemotaxis. J Immunol 152:5860-5867, 1994 10. Ahmed AFKU, Ohtani H, Nio M, et al: In situ expression of fibrogenic growth factors and their receptors in biliary atresia: Comparison between early and late stages. J Pathol 192:73-80, 2000 11. Farrel DJ, Hines JE, Wallis AF, et al: Intrahepatic mast cells in chronic liver diseases. Hepatology 22:1175-1181, 1995 12. Burd RR, Rogers HW, Gordon JR, et al: Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med 170:245-257, 1989 13. Gordon JR, Galli SJ: Mast cells as a source of both preformed and immunological inducible TNF-alpha/cachectin. Nature 346:274276, 1990 14. Gordon JR, Galli SJ: Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via FceRI: Role of mast cells derived transforming growth factor- and tumor necrosis factor-␣. J Exp Med 180:2027-2039, 1994 15. Qu Z, Liebler JM, Powers MR, et al: Mast cells are major source
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of basic fibroblast growth factor in chronic inflammation and cutaneous hemangioma. Am J Pathol 47:564-573, 1995 16. Gittlen SD, Schulman ES, Maddrey WC: Raised histamine concentrations in chronic cholestatic liver disease. Gut 31:96-99, 1990 17. Armburst T, Batusic D, Ringe B, et al: Mast cell distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis. J Hepatol 26:1042-1054, 1997 18. Rioux KP, Walace JL, Sharkey KA, et al: Proliferation of hepatic mast cells associated with biliary cirrhosis in the rat. Hepatology 20:216A, 1994 (suppl; abstr) 19. Hatamochi A, Fujiwara K, Ueki H: Effects of histamine on collagen gel synthesis by cultured fibroblasts derived from guinea pig skin. Arch Dermatol Res 277:60-64, 1994 20. Russel JD, Russel SB, Trupin KM: The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue. J Cell Physiol 93:389-393, 1977 21. Umezu K, Yuasa S, Sudoh A: Change of histamine content during hepatic fibrosis. Biochem Pharmacol 34:2007-2011, 1985
Purpose: To determine if mast cells influence the clinical outcome in biliary atresia (BA), the authors examined the intrahepatic mast cell population in BA. Methods: Mast cells were identified histochemically using Toludin Blue and immunohistochemically using antimast cell tryptase antibody in formalin-fixed paraffin-embedded sections from 21 cases of BA. Patients were divided into 3 groups; group I (n ⫽ 8) with good liver function, group II (n ⫽ 8) with moderate liver dysfunction, and group III (n ⫽ 5) with severe liver dysfunction. Liver biopsies from patients with choledochal cysts (CDC, n ⫽ 5), and normal liver (NL, n ⫽ 4) served as controls. The results were compared among the groups.
in the portal tracts. Mast cell numbers per medium power field (20⫻ magnification) were higher in BA than in the controls (15.03 ⫾ 2.25 v 3.85 ⫾ .65, [mean ⫾ SEM], P ⬍ .05, BA v CDC; 15.03 ⫾ 2.25 v 1.73 ⫾ .06, [mean ⫾ SEM], P ⬍ .05, BA v NL, immunohistochemical data). Clinical correlation showed an association between higher mast cell number and liver dysfunction (32.62 ⫾ .80 v 8.52 ⫾ .87 [mean ⫾ SEM], group III v group I; P ⬍ .05, immunohistochemical data).
Conclusion: Increased mast cell population in BA adversely affects liver function and raises the possibility that type I allergic reaction may play role in the pathology of BA. J Pediatr Surg 35:1762-1765. Copyright © 2000 by W.B. Saunders Company.
Results: Both histochemical and immunohistochemical methods showed similar data. Mast cells were seen mostly
INDEX WORDS: Biliary atresia, mast cells, clinical correlation.
M
BA. In this study we therefore attempted to analyze the mast cells in BA to determine what roles the intrahepatic mast cell population play in the pathobiology of BA.
AST CELLS ordinarily are distributed in normal connective tissue, mainly adjacent to blood and lymphatic vessels, nerves, and epithelial surfaces.1 Mast cells contain a variety of potent mediators of inflammation2 and cytokines,3 which are released after mast cells are activated. Mast cells act as critical effector cells in the inflammatory reactions underlying disorders of IgEdependent immediate hypersensitivity and in the expression of protective immunity involving IgE.4 In type I allergic reaction they act to precipitate an acute circulatory disturbance through capillary dilatation.5 In acute inflammation they cause endothelial cell contraction and increased capillary permeability,2 whereas in chronic inflammation they participate in fibrogenesis.6 Biliary atresia (BA) is a disease of unknown etiology in which obliteration of the extrahepatic bile duct results in intrahepatic cholestasis, edema, widening of the portal tracts, proliferation of bile ductules, and progression to fibrosis. We hypothesized that mast cells might play important roles in BA pathology. However, there is no study describing the intrahepatic mast cell population in
MATERIALS AND METHODS
Tissue Specimens With due approval from the Ethical Committee, specimens from 21 patients with BA (sampled during primary Kasai hepaticoportoenterostomy) were retrieved from the pathology file of Tohoku University School of Medicine. There were 9 boys and 12 girls with an age range from 12 to 128 days (54.14 ⫾ 25.15 days [mean ⫾ SD]). Specimens from choledochal cysts (CDC, n ⫽ 5) with an age range of 18 days to 10 months (4.1 ⫾ 3.5 months) and normal liver (NL, n ⫽ 4) with an age range from 11 months to 107 months (40.5 ⫾ 44.64 months) were used as controls. We obtained informed written consents from the patients’ parents, relatives, or patients themselves.
Tissue Preparation for Immunohistochemistry Tissue specimens were fixed in 10% formalin for up to 24 hours and were embedded in paraffin using the routine procedures. Embedded blocks were sectioned at 3 m thickness and mounted on silane-coated glass slides. Routine H&E staining was done in all the blocks.
Immunohistochemistry From the Departments of Pediatric Surgery and Pathology, Tohoku University School of Medicine, Aoba-ku, Sendai, Japan. Address reprint requests to Ryoji Ohi, MD, FAAP, Professor and Chairman, Department of Pediatric Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Copyright © 2000 by W.B. Saunders Company 0022-3468/00/3512-0016$03.00/0 doi:10.1053/jpsu.2000.19245 1762
Sections were pretreated with 1% trypsin before the 2-step indirect immunoperoxidase method was used using DAKO Envision system (DAKO, Glostrup, Denmark) as the detection agent. Nonspecific reactivity was blocked by treating the sections with 1% bovine serum albumin (BSA) for 10 minutes. Mouse monoclonal antimast cell tryptase antibody (clone AA1, dilution 1:200; DAKO) was applied for 18 hours at 4°C. To block the endogenous peroxidase activity, sections were treated with 0.3% H2O2 in methanol for 12 minutes. The DAKO
Journal of Pediatric Surgery, Vol 35, No 12 (December), 2000: pp 1762-1765
MAST CELLS IN BILIARY ATRESIA
1763
Table 1. Number of Mast Cells in Portal Tracts in Biliary Atresia and Controls No. of Mast Cells Case
Biliary atresia (n ⫽ 21) Choledochal cyst (n ⫽ 5) Normal liver (n ⫽ 4)
Toludin Blue Staining
Mast Cell Tryptase Immunostaining
9.15 ⫾ 1.87
15.03 ⫾ 2.25*
1.85 ⫾ .85 0
3.85 ⫾ .65 1.73 ⫾ .06
* P ⬍ .05 from choledochal cyst and normal liver group.
Envision reagent that recognizes both mouse and rabbit primary antibodies were applied for 1 hour at room temperature. For the negative control, primary antibodies were replaced by 0.5% BSA or other irrelevant antibodies. Sections were developed in 0.03% diaminobenzidine (DAB; Dojin, Kumamoto, Japan) containing 0.005% H2O2 and 0.065% NaN3 for 5 minutes and counterstained by hematoxylin or methyl green.
Histochemistry
Mast Cell Histochemistry Table 1 summarizes the results. A similar distribution of mast cells could be observed, although the absolute numbers were lower than those observed with immunohistochemistry. A range of cells from partially degranulated to fully granulated ones could be observed (data not shown). Clinical Correlation The number of mast cells was highest in group III (32.62 ⫾ .80, severe liver dysfunction, Fig 1A, Table 2), lower in group II (10.56 ⫾ .70, moderate liver dysfunction, Table 2), and lowest in group I (8.52 ⫾ .87, good liver function, Fig 1B, Table 2). DISCUSSION
Our study analyzes the mast cell population in BA and correlates the mast cell numbers to clinical parameters.
Mast cells were stained histochemically using 0.5% Toludin Blue, pH 0.5. Briefly, after deparaffinization the sections were kept in Toludin Blue for 30 minutes, then washed in water and finally air dried. No nuclear counterstaining was done.
Quantification for Immunohistochemistry Mast cells were counted according to the method described7 with some modifications. Briefly, 5 nonoverlapping fields were chosen randomly from the portal areas. Using ⫻20 objective lens mast cells were counted, and the data are expressed as mean ⫾ SEM per medium power field. An independent observer crosschecked the results.
Clinical Correlation The duration of follow-up ranged from 5 months to 137 months with a median of 23 months. Patients were divided into 3 groups as previously described with some modifications.8 Group I included 8 patients with good liver function (Serum bilirubin ⬍1.5 mg/dL, SGPT ⬍35 IU/L, SGOT ⬍40 IU/L); group II included 8 patients with moderate liver dysfunction (serum bilirubin ⬍1.5 mg/dL, SGPT ⬎35 IU/L, SGOT ⬎40 IU/L); and group III included 5 patients with severe liver dysfunction (serum bilirubin ⬎1.5 mg/dL, SGPT ⬎35 IU/L, SGOT ⬎40 IU/L). The number of mast cells per medium power field was compared among the groups.
Statistical Analysis The results were tested for statistical significance using the Bonferroni-Dunn test.
RESULTS
Mast Cell Immunohistochemistry In the controls few mast cells could be observed (1.73 ⫾ .06, NL; 3.85 ⫾ .65, CDC; Table 1). In contrast, a significantly higher number of mast cells could be detected in BA (15.03 ⫾ 2.25, Table 1). Most of the mast cells were observed in the portal tracts, whereas a few mast cells could be seen in the hepatic lobule. Some mast cells appeared round to ovoid in shape, whereas some others appeared elongated and spindle shaped.
Fig 1. Mast cell–specific immunohistochemistry in BA. (A) Section from a patient who later had sever liver dysfunction shows a large number of mast cells (arrows). (B) In contrast a few cells are seen in the section from a patient with good liver function. (H&E, original magnification ⴛ200.)
1764
AHMED ET AL
Table 2. Correlation of Mast Cell Number in Portal Tracts to Indicators of Liver Function No. of Mast Cells Per Medium Power Field (mean ⫾ SEM) Group
Toludin Blue Staining
Group I (n ⫽ 8) (T.Bil ⬍1.5 mg/dL, GOT ⬍40 IU/L, GPT ⬍35 IU/L) Group II (n ⫽ 8) (T.Bil ⬍1.5 mg/dL, GOT ⬎40 IU/L, GPT ⬎35 IU/L) Group III (n ⫽ 5) (T.Bil ⬍1.5 mg/dL, GOT ⬍40 IU/L, GPT ⬍35 IU/L)
Mast Cell Tryptase Immunostaining
4.72 ⫾ 1.26
8.52 ⫾ .87
5.9 ⫾ 2.21
10.56 ⫾ .70
21.44 ⫾ 2.42
32.62 ⫾ .80*
* P ⬍ .05 from group I and group II.
Both our immunohistochemical and histochemical data show that mast cell population is increased in BA as compared with the controls. It has been further shown that higher mast cell number is associated with poor liver function indicating their significant involvement in the pathobiology of BA. We do not have any explanation regarding the expanded mast cell population in BA. One investigation has shown transforming growth factor beta (TGF-) to be a potent mast cell chemotactic factor,9 and we have reported recently that there is overexpression of TGF- in BA.10 It is possible that TGF- increases the hepatic mast cell number. Moreover, bile acids also may contribute to the recruitment and proliferation of hepatic mast cells.11 However, further studies are required to determine if mast cells act as primary etiologic agents for BA or they represent the effects of the disease process itself initiated by other factors. We have observed large accumulations of mast cells in the portal tracts in BA. It is possible that mast cells in this location may lead to such intrahepatic changes in BA as edema and widening of the porta tracts and progressive fibrosis because mast cells have been identified to be capable of releasing histamine and prostaglandins5 as well as cytokines such as interleukin 1 (IL-1),12 tumor necrosis factor alpha (TNF-␣),13 TGF-,14 and basic fibroblast growth factor (bFGF).15 It has been shown that chronic cholestasis is associated with increased liver histamine content.16 Moreover, it is speculated that bile acids may directly act on the tissue mast cells to release histamine and other mast cell mediators,11 which, in turn, can cause disturbed tissue microcirculation leading to edema and widening of the portal tracts as seen in BA.
Association of increased mast cell numbers with liver fibrogenesis also have been reported by various investigators.17,18 Published reports indicate that histamine enhances the biosynthesis of collagen in fibroblasts,19,20 and increased liver histamine level correlates to fibrogenesis.21 Mast cells may promote fibrosis in 2 ways— by enhancing the production of extracellular matrix through releasing fibrogenic cytokines and growth factors and by decreasing matrix degradation through releasing inhibitors of proteases.17 The presence of significantly higher number of mast cells in BA patients who had severe liver dysfunction suggests that mast cells actively contribute to liver damage. Therefore, mast cells need to be assessed as part of any clinicopathologic evaluation of BA. Identification of mast cells long has been a difficult task because of mast cell’s close similarity to basophils. However, immunohistochemistry with mast cell–specific antitryptase antibody now renders its accurate identification possible.11 In this study, immunohistochemistry and histochemistry provided similar data suggesting that the simpler and quicker histochemical staining can be useful in the assessment of the mast cell population in BA. Increased mast cell numbers in BA also raises the possible involvement of type I allergic reaction. The clinical improvement after the use of antiinflammatory antiallergic steroid, seen at times when the patients present with deterioration of liver functions, also highlights this possibility. However, a further detailed study focusing on the distribution of IgE including the distribution of the IgE producing plasma cells in BA may help us determine if indeed type I allergic reaction does exist in BA.
REFERENCES 1. Galli SJ: New concepts about mast cells. N Engl J Med 328:257265, 1993 2. Cotran RS, Kumar V, Robins SL: Pathologic basis of disease. Philadelphia, PA, Saunders, 1994, pp 64-65 3. Gordon JR, Burd PR, Galli SJ: Mast cells as source of multifunctional cytokines. Immunol Today 11:458-464, 1990 4. Ishizaka T: Mechanisms of Ig E-mediated hypersensitivity, in Middleton JE Jr, Reed CE, Ellis EF, et al (eds): Allergy Principles and Practice. St. Louis, MO, Mosby, 1988, pp 71
5. Roitt IM: Essential Immunology. Oxford, England, Blackwell Scientific Publications, 1994, pp 35 6. Thompson HL, Burkelo PD, Babreil G, et al: Murine mast cells synthesize basement membrane components. A potential role in early fibrosis. J Clin Invest 87:619-623, 1991 7. Yamashiro M, Kuoda W, Kono N, et al: Distribution of intrahepatic mast cells in various hepatobiliary disorders an immunohistochemical study. Virchows Arch 433:471-479, 1998 8. Kobayashi H, Miyano T, Horikoshi K, et al: Prognostic value of
MAST CELLS IN BILIARY ATRESIA
serum procollagen III peptide and type IV collagen in patients with biliary atresia. J Pediatr Surg 33:112-114, 1998 9. Gruber BL, Marchese MJ, Kew RR: Transforming growth factor 1 mediates mast cell chemotaxis. J Immunol 152:5860-5867, 1994 10. Ahmed AFKU, Ohtani H, Nio M, et al: In situ expression of fibrogenic growth factors and their receptors in biliary atresia: Comparison between early and late stages. J Pathol 192:73-80, 2000 11. Farrel DJ, Hines JE, Wallis AF, et al: Intrahepatic mast cells in chronic liver diseases. Hepatology 22:1175-1181, 1995 12. Burd RR, Rogers HW, Gordon JR, et al: Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med 170:245-257, 1989 13. Gordon JR, Galli SJ: Mast cells as a source of both preformed and immunological inducible TNF-alpha/cachectin. Nature 346:274276, 1990 14. Gordon JR, Galli SJ: Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via FceRI: Role of mast cells derived transforming growth factor- and tumor necrosis factor-␣. J Exp Med 180:2027-2039, 1994 15. Qu Z, Liebler JM, Powers MR, et al: Mast cells are major source
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