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

ARTICLE

Maternal Microchimerism in Underlying Pathogenesis

of Biliary Atresia: Quantification and Phenotypes of

Maternal Cells in the Liver

Toshihiro Muraji, MDa_{, Naoki Hosaka, MD}b_{, Naoki Irie, PhD}c_{, Makiko Yoshida, MD}d_{, Yukihiro Imai, MD}e_{, Kohichi Tanaka, MD}f_, Yasutsugu Takada, MDg_{, Seisuke Sakamoto, MD}g_{, Hironori Haga, MD}h_{, Susumu Ikehara, MD}b

Departments ofa_{Surgery and}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 ofg_{Surgery and}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}

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

ABSTRACT

OBJECTIVE.The goal was to examine whether microchimerism plays a crucial role in the pathogenesis of biliary atresia; we analyzed the localization of maternal microchi-meric 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 under-lying pathogenesis in biliary atresia. The findings support the recently postulated mechanisms of alloautoimmune and/or autoalloimmune responses.

B

ILIARY 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 al}8_{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}

immunohistochemi-cally, with maternal anti-HLA antibody, in the hepatocytes and bile duct epithelia in BA. However, maternal microchi-merism 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

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

www.pediatrics.org/cgi/doi/10.1542/ peds.2007-0568

doi:10.1542/peds.2007-0568

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

BA were fixed in 10% formaldehyde solution and

em-bedded in paraffin. Serial tissue sections of 2-␮m

thick-ness 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, hemangioendo-thelioma, 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 (di-luted 50-fold; Dako), CD56 (predi(di-luted; 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,

Down-ers Grove, IL) were used, and the 4⬘

,6-diamino-2-phe-nylindole/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, includ-ing 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-posi-tive) 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 chro-mosome-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 (predi-luted; 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-posi-tive 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

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 lympho-cytes were immunohistochemically positive for CD8 or FIGURE 1

CD79a, few for CD4, and none for CD34 or CD56. Counts of maternal cells with a set of double X chromo-somes 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).

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.

Strik-ingly, 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

num-ber 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

im-mune responses proceeding in the liver samples from patients with BA.

DISCUSSION

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 10}4 _{to 10}5 _{cells). More}

impor-tantly, 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

con-troversial, with some reports indicating CD4⫹ T cells5,7

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 mater-nal 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,

TABLE 1 Analysis of Maternally Derived XXⴙ_{, CD8}ⴙ_{, and CD4}ⴙ_{T Cells in Liver Specimens From Patients With or Without BA}

Case Age at

Operation, mo

No. of Cells per 103_{Cells Proportion of XX}⫹_CD8⫹

Cells in XX⫹_{Cells, %}

Proportion of XX⫹_CD8⫹

Cells in CD8⫹_{Cells, %}

XX⫹ _CD8⫹ _XX⫹_CD8⫹ _CD4⫹ _XX⫹_CD4⫹

BA

1 2 6 8 0 0 0 0.0 0.0

2 2 16 2 2 0 0 12.5 100.0

3 2 50 7 1 1 0 2.0 14.3

4 2 7 17 3 0 0 42.3 17.6

5 3 13 13 2 0 0 15.4 15.4

6 3 32 32 3 1 0 9.4 9.3

7 3 16 15 9 0 0 56.3 60.0

8 4 19 15 1 0 0 5.3 6.6

Mean⫾SE 19.9⫾5.2a _13.6⫾_3.2b _2.6⫾_1.0c _0.3⫾_0.2d _0.0⫾_{0.0 18.0}⫾_{7.2 27.9}⫾_12.4e

Control

9 4 3 21 0 102 0 0.0 0.0

10 1 2 32 1 3 0 50.0 3.1

11 4 2 28 0 1 0 0.0 0.0

12 6 2 41 0 4 0 0.0 0.0

13 5 3 36 1 63 0 33.3 2.7

14 4 1 13 0 26 0 0.0 0.0

Mean⫾SE 2.2⫾0.3 28.5⫾4.2 0.3⫾0.2 33.2⫾16.7 0.0⫾0.0 13.9⫾9.1 1.0⫾0.6

Patients with BA received Kasai’s operation and those without BA received orthotopic liver transplantation; liver specimens were obtained at that time. The basic diseases in control cases were as follows: case 9, Crigler-Najjar syndrome; case 10, tyrosinemia; case 11, hemangioendothelioma; case 12, tyrosinemia; case 13, congenital biliary dilation; case 14, congenital absence of portal vein. a_P⫽_{.0007, compared with patients without BA, by Mann-WhitneyUtest.}

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 sug-gests that maternal cells might give rise to the transdif-ferentiated 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 immuno-competent maternal microchimeric cells could cause GvHD-like symptoms in patients with severe combined

immunodeficiency.14,15_{In addition, Landing et al}16

char-acterized BA as one of the immunologic deficiency dis-eases 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 mater-nal microchimerism, we hypothesize that matermater-nal chi-meric 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}

im-munologically competent cells” is satisfied by maternal

CD8⫹ effector cells being located in the biliary

epithe-lium. (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 be-tween mothers and fetuses is likely to be a key

determi-nant contributing to maternal microchimerism,18_the

re-sults 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 histocom-patibility antigens are rarely identical to the HLA anti-gens found in the mother (graft) that could thus recog-nize 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 ma-ternofetal microchimerism. Similarly, mama-ternofetal allo-autoimmune or autoalloimmune mechanisms might un-derlie 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 cyto-kines, which would make BECs more susceptible to T cell attack.25

ACKNOWLEDGMENTS

This work was supported by research grant C from Kan-sai Medical University.

We thank Kumiko Hayashi, Kyoto Laboratory, Mit-subishi Kagaku, for her contribution to FISH prepara-tion.

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DOI: 10.1542/peds.2007-0568

2008;121;517

Pediatrics

Tanaka, Yasutsugu Takada, Seisuke Sakamoto, Hironori Haga and Susumu Ikehara

Toshihiro Muraji, Naoki Hosaka, Naoki Irie, Makiko Yoshida, Yukihiro Imai, Kohichi

Quantification and Phenotypes of Maternal Cells in the Liver

Maternal Microchimerism in Underlying Pathogenesis of Biliary Atresia:

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DOI: 10.1542/peds.2007-0568

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Tanaka, Yasutsugu Takada, Seisuke Sakamoto, Hironori Haga and Susumu Ikehara

Toshihiro Muraji, Naoki Hosaka, Naoki Irie, Makiko Yoshida, Yukihiro Imai, Kohichi

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