Published online February 29, 2008
PEDIATRICS Vol. 121 No. 3 March 2008, pp. 517-521 (doi:10.1542/peds.2007-0568)

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ARTICLE

Maternal Microchimerism in Underlying Pathogenesis of Biliary Atresia: Quantification and Phenotypes of Maternal Cells in the Liver

Toshihiro Muraji, MD^a, Naoki Hosaka, MD^b, Naoki Irie, PhD^c, Makiko Yoshida, MD^d, Yukihiro Imai, MD^e, Kohichi Tanaka, MD^f, Yasutsugu Takada, MD^g, Seisuke Sakamoto, MD^g, Hironori Haga, MD^h and Susumu Ikehara, MD^b

Departments of ^a Surgery
^d Pathology, Kobe Children's Hospital, Kobe, Japan
^b First Department of Pathology, Kansai Medical University, Osaka, Japan
^c Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
Departments of ^g Surgery
^h Clinical Pathology, Kyoto University Gradate School of Medicine, Kyoto, Japan
^e Department of Pathology, Kobe City General Hospital, Kobe, Japan
^f Institute for Biomedical Research and Innovation, Kobe, Japan

	ABSTRACT

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OBJECTIVE. The goal was to examine whether microchimerism plays a crucial role in the pathogenesis of biliary atresia; we analyzed the localization of maternal microchimeric cells and their phenotypes.

METHODS. Liver biopsy specimens from 8 male infants with biliary atresia and 6 control subjects with other liver diseases were investigated for maternal chimeric cells and their phenotypes through double-staining fluorescence in situ hybridization and immunohistochemical analyses.

RESULTS. Significantly larger numbers of maternal XX⁺ cells were found in the portal area and sinusoids of patients with biliary atresia, in comparison with control patients. In phenotypic analyses of XX⁺ cells, CD8⁺ T cells, CD45⁺ cells, and cytokeratin-positive cells were found, and the numbers and proportions among total CD8⁺ T cells were significantly higher than those in control patients.

CONCLUSIONS. Significantly more maternal chimeric CD8⁺ T cells in the livers of patients with biliary atresia suggest that maternal immunologic insults represent the underlying pathogenesis in biliary atresia. The findings support the recently postulated mechanisms of alloautoimmune and/or autoalloimmune responses.

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Key Words: biliary atresia • graft-versus-host disease • microchimerism • CD8

Abbreviations: BA—biliary atresia • GvHD—graft-versus-host disease • BEC—biliary epithelial cell • FISH—fluorescence in situ hybridization

Biliary atresia (BA) is an infantile liver disease of unknown cause in which the intrahepatic and extrahepatic bile ducts are damaged progressively by an ongoing fibrosing process, even in some patients with good bile drainage. Several factors have been proposed as contributors to the etiopathogenesis of the perinatal form of BA, including occult viral infections, defects in morphogenesis, vascular insults, toxic agents, and aberrant immune responses.^1,2 In 1988, on the basis of our HLA-DR immunohistochemical study of the biliary epithelium in patients with BA, we postulated that immune-mediated insults are involved in the development of BA.³ Other reports also suggested that immune-mediated bile duct injury plays an important role in BA.^4–7 Suskind et al⁸ demonstrated maternal microchimerism in patients with BA, which suggests that the etiopathogenesis involves graft-versus-host disease (GvHD) induced by maternal lymphocytes engrafted through the placenta. Recently, Kobayashi et al⁹ demonstrated maternal microchimerism immunohistochemically, with maternal anti-HLA antibody, in the hepatocytes and bile duct epithelia in BA. However, maternal microchimerism itself does not necessarily prove any etiopathogenesis involved in BA, because 2-way maternofetal cell trafficking is a common phenomenon. In an attempt to obtain additional information to clarify the immunologic process that is involved, we characterized phenotypes and located the maternal chimeric cells infiltrating the livers of patients with BA.

	METHODS

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Patients
All 8 patients in this part of the study were male infants. The patients’ age at the time of surgery, associated anomalies, and types of porta hepatis were reviewed retrospectively. Liver biopsy specimens from these infants with BA were fixed in 10% formaldehyde solution and embedded in paraffin. Serial tissue sections of 2-µm thickness were prepared. Control specimens were liver tissues biopsied or resected for liver transplantation, at Kyoto University Hospital, from 6 male patients <6 months of age with various diseases, including cholestatic disease (tyrosinemia, Crigler-Najjar syndrome, hemangioendothelioma, choledochal cyst, and congenital absence of the portal vein).

Immunohistochemical Analyses
Immunohistochemical examinations were performed by using an Envision kit (Dako, Kyoto, Japan). Primary antibodies used in this study were against cytokeratin (diluted 50-fold; Dako), CD4 (prediluted; Nichirei, Tokyo, Japan), CD8 (prediluted; Nichirei), CD34 (diluted 50-fold; Dako), CD56 (prediluted; Nichirei), and CD79a (prediluted; Dako).

Fluorescence in Situ Hybridization
The existence of the XX chromosome was detected by using the fluorescence in situ hybridization (FISH) method. Probes for X and Y chromosomes (Vysis, Downers Grove, IL) were used, and the 4',6-diamino-2-phenylindole/fluorescein isothiocyanate/Texas red triple-bandpass combination (Nikon, Tokyo, Japan) was applied. The slides were scanned at a magnification of 1000 with a fluorescence microscope (Nikon Eclipse 600) equipped with an epi-illumination system, including a 100-W mercury lamp with a set of filters. A total of 1000 nonoverlapping cells with nuclei with positive signals for both sex chromosomes (red: Y-positive; green: X-positive) were counted. Cells with truncation of the nuclei from sectioning or with single signals for sex chromosomes were all excluded from the counts. Locations of XX chromosome-positive cells were investigated separately on the slides stained with cytokeratin-specific antibody and on the consecutive slides stained with hematoxylin and eosin.

Double-staining for FISH and immunohistochemical analyses was performed for CD8, CD4, CD45 (prediluted; Nichirei), CD79a, and cytokeratin in all cases, and results were matched to the corresponding fields on the consecutive slides stained with hematoxylin and eosin to characterize phenotypically the XX chromosome-positive cells. Double-staining analysis of CD34 and CD56 with FISH for the XX chromosome was not performed, because such cells had not infiltrated the portal area. The investigation was approved by the institutional review boards at Kobe Children's Hospital and Kyoto University Graduate School of Medicine, and parental informed consent to participate in this study was obtained.

	RESULTS

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We first examined the histologic results for maternal chimerism and determined a phenotype of the chimeric cells in the liver samples from the patients with BA. In the specimens from all 8 patients with BA, lymphocytes had infiltrated the portal area; some had destroyed bile ducts with inflammatory edema (Fig 1A). The lymphocytes were immunohistochemically positive for CD8 or CD79a, few for CD4, and none for CD34 or CD56. Counts of maternal cells with a set of double X chromosomes in the specimens, which ranged from 6 to 50 cells per 1000 cells in patients with BA, were significantly higher than counts in samples from patients without BA (patients with BA: 19.9 ± 5.2 cells per 1000 cells; control patients: 2.2 ± 0.3 cells per 1000 cells; P = .0007) (Table 1).

View larger version (100K): [in this window] [in a new window]   FIGURE 1 Histologic and immunohistochemical analyses with FISH signals in the portal area in patient 7. A, A number of lymphocytes infiltrated the portal area and some lymphocytes destroyed the bile ducts (arrows) with inflammatory degeneration (hematoxylin and eosin staining; original magnification x200 and inset x400). B, Some cells with XX signal (black arrows) were present in nuclei situated intimately in the cytokeratin-positive (white arrow) bile duct (immunohistochemical staining with FISH signals; original magnification x400 and inset x800). C, Some CD8+ T cells (white arrow) showed XX signal in the nucleus (black arrow) (immunohistochemical staining with FISH signals; original magnification x1000). X signal is green, Y signal red, and immunohistochemical signal (B, cytokeratin; C, CD8) brown.

 

View this table: [in this window] [in a new window]   TABLE 1 Analysis of Maternally Derived XX+, CD8+, and CD4+ T Cells in Liver Specimens From Patients With or Without BA

 
In the double-staining analyses, some XX⁺ cells were present in the injured bile duct epithelium stained for cytokeratin (Fig 1B). Furthermore, the numbers of XX⁺ maternal CD8⁺ cytotoxic T cells in patients with BA were significantly higher than those in patients without BA (patients with BA: 2.6 ± 1.0 cells per 1000 cells; control patients: 0.3 ± 0.2 cells per 1000 cells; P = .020) (Fig 1C). In addition, 7 of 8 patients with BA (86%) showed infiltration of XX⁺ CD8⁺ T cells, although only 2 (33%) of 6 control patients showed such cells. Strikingly, the proportion of maternal CD8⁺ T cells in total CD8⁺ T cells was 10-fold higher than that in patients without BA (patients with BA: 27.9 ± 12.4%; control patients: 1.0 ± 0.6%; P = .008), whereas the total number of CD8⁺ T cells in patients with BA was lower than that in control patients (patients with BA: 13.6 ± 3.2 cells per 1000 cells; control patients: 28.5 ± 4.2 cells per 1000 cells; P = .029). Neither XX⁺ CD4⁺ T cells nor CD79a⁺ B cells were remarkable. These findings indicate that there are maternal CD8⁺ T cell-predominant immune responses proceeding in the liver samples from patients with BA.

	DISCUSSION

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Our quantitative analysis of maternal XX⁺ cells in the liver of male patients with BA indicated 1 cell per 10² to 10³ cells counted, with the stringent criterion that a pair of X signals be clearly included within a nontruncated nucleus. The frequency of these maternal cells in the patients with BA was much higher than levels of nonself cells found after solid organ transplantation¹⁰ or normal pregnancy¹¹ (1 cell per 10⁴ to 10⁵ cells). More importantly, the frequency of XX⁺ cells was nearly 10-fold greater than that in patients without BA. However, our investigation was based on a relatively small number of maternal cells in only 8 cases, which could lead to type II statistical errors. We avoided using neonatal hepatitis as a control condition because neonatal hepatitis is itself a disease of unknown cause and could be a mild type of GvHD.

In the phenotypic analysis, the significantly elevated number of maternal XX⁺ CD8⁺ cells and the proportion of XX⁺ CD8⁺ cells in total CD8⁺ T cells were noted. We found only a few total CD4⁺ T cells, including XX⁺ cells. The effector cells predominantly found in BA are controversial, with some reports indicating CD4⁺ T cells^5,7 but another indicating CD8⁺ T cells.¹² Above all, the high chimeric rate of maternal cells of immune origin (XX⁺ CD8⁺ T cells) indicates that patients with BA have a considerable gross amount of these immunologic maternal cells in their tissues, which suggests the possible involvement of these cells in the pathogenesis of BA.

In addition, these XX⁺ cells were located in the portal area, intimately situated with the cytokeratin-positive biliary epithelial cells (BECs), which indicates a close association between microchimeric maternal cells and the BA lesion site. However, our additional phenotypic characterization using CD45 and cytokeratin staining with FISH for XX signals in 4 patients (patients 2, 6, 7, and 8) revealed that cytokeratin and CD45 were both detectable in 28% of 14 XX signal-positive cells counted. This XX-positive signal in cytokeratin-positive cells suggests that maternal cells might give rise to the transdifferentiated BECs in the fetus if they are circulating stem cells¹³ and might work as targets in autoalloimmune (or host-versus-graft) responses, rather than GvHD.

Because microchimeric maternal cells are basically thought to be immunologically tolerant to the fetus, the immunologic environment in patients with BA should be examined carefully. It was reported that immunocompetent maternal microchimeric cells could cause GvHD-like symptoms in patients with severe combined immunodeficiency.^14,15 In addition, Landing et al¹⁶ characterized BA as one of the immunologic deficiency diseases of children attributable to the fact that the rapid progressive loss of Hassall's corpuscles of the thymus continues for >18 months after birth. On the basis of these previous observations and the existence of maternal microchimerism, we hypothesize that maternal chimeric cells could be involved in the pathogenesis of BA, acting in a role similar to a graft in a GvHD-like process.

We verified this hypothesis with the 3 requirements necessary for graft-versus-host reaction that Billingham formulated in 1966.¹⁷ (1) "The graft must contain immunologically competent cells" is satisfied by maternal CD8⁺ effector cells being located in the biliary epithelium. (2) "The host must be incapable of rejecting the transplanted cells," and our study clarified that patients with BA have significantly high levels of bidirectional compatibility at HLA class I with their mothers (T.M., N.I., K.T., S.S., and Y.T., manuscript in preparation). Notably, because a HLA-compatible relationship between mothers and fetuses is likely to be a key determinant contributing to maternal microchimerism,¹⁸ the results also indicate that patients with BA may have genetically favorable conditions for accepting maternal microchimeric cells. (3) "The host must express tissue antigens that are not present in the transplant donor" is satisfied by the fact that fetal major and minor histocompatibility antigens are rarely identical to the HLA antigens found in the mother (graft) that could thus recognize the fetal BECs as a foreign element. Even HLA-identical stem cell transplants can induce GvHD because of the presence of minor histocompatibility antigens.¹⁹ Because it seems to satisfy the 3 criteria described by Billingham,¹⁷ BA can be seen as a phenotype of the GvHD-like response caused by maternally transferred chimeric T cells.

Studies on human autoimmune diseases, including juvenile idiopathic inflammatory myopathies,²⁰ type 1 diabetes mellitus,²¹ and scleroderma,^22,23 have clarified an association with increased fetomaternal or maternofetal microchimerism. Similarly, maternofetal alloautoimmune or autoalloimmune mechanisms might underlie the pathogenesis of BA. This concept of a new disease spectrum called maternofetal immune disease does not contradict the previously proposed etiologic considerations, such as viral infections or the ductal plate malformation theory.²⁴ The viruses responsible may not necessarily be specific, because common hepatotrophic viruses would be sufficient to activate the immunologic competency of the BECs to secrete inflammatory cytokines, which would make BECs more susceptible to T cell attack.²⁵

	ACKNOWLEDGMENTS

 
This work was supported by research grant C from Kansai Medical University.

We thank Kumiko Hayashi, Kyoto Laboratory, Mitsubishi Kagaku, for her contribution to FISH preparation.

	FOOTNOTES

 
Accepted Aug 1, 2007.

Address correspondence to Toshihiro Muraji, MD, PhD, Ibaraki Children's Hospital, 3-3-1, Futabadai, Mito, Ibaraki, Japan 311-4145. E-mail: t-muraji{at}ibaraki-kodomo.com

The authors have indicated they have no financial relationships relevant to this article to disclose.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 

1. Sokol RJ, Mack C. Etiopathogenesis of biliary atresia. Semin Liver Dis.2001; 21 (4):517 –524[CrossRef][Web of Science][Medline]
2. Perlmutter DH, Shepherd RW. Extrahepatic biliary atresia: a disease or a phenotype? Hepatology.2002; 35 (6):1297 –1304[CrossRef][Medline]
3. Muraji T, Hashimoto K, Ifuku H, et al. Increased expression of HLA-DR antigen on biliary epithelium cells in biliary atresia. J Jpn Soc Pediatr Surg.1988; 24 (4):793 –796
4. Kobayashi H, Puri P, O'Brian S, et al. Hepatic overexpression of MHC class II antigens and macrophage-associated antigens (CD68) in patients with biliary atresia of poor prognosis. J Pediatr Surg.1997; 32 (4):590 –593[CrossRef][Medline]
5. Davenport M, Gonde C, Redkar R, et al. Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg.2001; 36 (7):1017 –1025[CrossRef][Medline]
6. Schweizer P, Petersen M, Jeszberger N, et al. Immunohistochemical and molecular biological investigations regarding the pathogenesis of extrahepatic biliary atresia. Eur J Pediatr Surg.2003; 13 (1):7 –15[Medline]
7. Mack CL, Tucker RM, Sokol RJ, et al. Biliary atresia is associated with CD4⁺ Th1 cell-mediated portal tract inflammation. Pediatr Res.2004; 56 (1):79 –87[CrossRef][Medline]
8. Suskind DL, Rosenthal P, Heyman MB, et al. Maternal microchimerism in the livers of patients with biliary atresia. BMC Gastroenterol.2004; 4 :14 –18[CrossRef][Medline]
9. Kobayashi H, Tamatani T, Tamura T, et al. Maternal microchimerism in biliary atresia. J Pediatr Surg.2007; 42 (6):987 –991[CrossRef][Medline]
10. Starzl T, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism and graft acceptance. Lancet.1992; 339 (8809):1579 –1582[CrossRef][Web of Science][Medline]
11. Lo DYM, Lo ESF, Watson N, et al. Two-way cell traffic between mother and fetus: biologic and clinical implications. Blood.1996; 88 (11):4390 –4395[Abstract/Free Full Text]
12. Shinkai M, Shinkai T, Puri P, Stringer M. Elevated expression of IL2 is associated with increased infiltration of CD8⁺ T cells in biliary atresia. J Pediatr Surg.2006; 41 (2):300 –305[CrossRef][Medline]
13. Körbling M, Katz RL, Khanna A, et al. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med.2002; 346 (10):738 –746[Abstract/Free Full Text]
14. Grogan TM, Broughton DD, Doyle WF. Graft-versus-host reaction: a case report suggesting GVHR occurred as a result of maternofetal cell transfer. Arch Pathol.1975; 99 (6):330 –334[Medline]
15. Müller SM, Ege M, Pottharst A, Schulz AS, Schwarz K, Friedrich W. Transplacentally acquired maternal T lymphocytes in severe combined immunodeficiency: a study of 121 patients. Blood.2001; 98 (6):1847 –1851[Abstract/Free Full Text]
16. Landing BH, Yutuc IL, Swanson VL. Clinicopathological correlations in immunologic deficiency diseases of children with emphasis on thymic histologic patterns. In: Immunodeficiency: Its Nature and Etiological Significance on Human Diseases: Proceedings of the International Symposium on Immunodeficiency. Tokyo, Japan: Japan Medical Research Foundation; 1978:15–17
17. Billingham RE. The biology of graft versus host reactions. Harvey Lect.1966; 62 :21 –78[Medline]
18. Berry SM, Hassan SS, Russell E, Kukuruga D, Land S, Kaplan J. Association of maternal histocompatibility at class II HLA loci with maternal microchimerism in the fetus. Pediatr Res.2004; 56 (1):73 –78[CrossRef][Medline]
19. Goulmy E, Schipper R, Blokland E, Falkenburg F. Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host-disease after bone marrow transplantation. N Engl J Med.1996; 334 (5):281 –285[Abstract/Free Full Text]
20. Artlett CM, Ramos R, Jiminez SA, Patterson K, Miller FW, Rider LG. Chimeric cells of maternal origin in juvenile idiopathic inflammatory myopathies: Childhood Myositis Heterogeneity Collaborative Group. Lancet.2000; 356 (9248):2155 –2156[CrossRef][Web of Science][Medline]
21. Nelson JL, Gillespie KM, Lambert NC, et al. Maternal microchimerism in peripheral blood in type 1 diabetes and pancreatic islet β cell microchimerism. Proc Natl Acad Sci USA.2007; 104 (5):1637 –1642[Abstract/Free Full Text]
22. Nelson JL, Furst DE, Maloney S, et al. Microchimerism and HLA-compatible relationship of pregnancy in scleroderma. Lancet.1998; 351 (9102):559 –562[CrossRef][Web of Science][Medline]
23. Johnson KL, Nelson JL, Furst DE, et al. Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis. Arthritis Rheum.2001; 44 (8):1848 –1854[CrossRef][Web of Science][Medline]
24. Tan CEL, Driver M, Howard ER, Moscoso GJ. Extrahepatic biliary atresia: a first-trimester event? Clues from light microscopy and immunohistochemistry. J Pediatr Surg.1994; 29 (6):808 –814[Medline]
25. Reynoso-Paz S, Coppel RL, Mackay RL, et al. The immunology of bile and biliary epithelium. Hepatology.1999; 30 (2):351 –357[CrossRef][Medline]

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