Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis

Plasma Cells and the Chronic NonsuppurativeDestructive Cholangitis of Primary Biliary Cirrhosis

Toru Takahashi,1 Tomofumi Miura,1 Junichiro Nakamura,1 Satoshi Yamada,1 Tsutomu Miura,1

Masahiko Yanagi,1 Yasunobu Matsuda,2 Hiroyuki Usuda,3 Iwao Emura,4 Koichi Tsuneyama,5

Xiao-Song He,6 and M. Eric Gershwin6

There has been increased interest in the role of B cells in the pathogenesis of primary biliarycirrhosis (PBC). Although the vast majority of patients with this disease have anti-mitochon-drial antibodies, there is no correlation of anti-mitochondrial antibody titer and/or presencewith disease severity. Furthermore, in murine models of PBC, it has been suggested that deple-tion of B cells may exacerbate biliary pathology. To address this issue, we focused on a detailedphenotypic characterization of mononuclear cell infiltrates surrounding the intrahepatic bileducts of patients with PBC, primary sclerosing cholangitis, autoimmune hepatitis, chronichepatitis C, and graft-versus-host disease, including CD3, CD4, CD8, CD20, CD38, andimmunoglobulin classes, as well as double immunohistochemical staining for CD38 and IgM.Interestingly, CD20 B lymphocytes, which are a precursor of plasma cells, were found inscattered locations or occasionally forming follicle-like aggregations but were not noted at theproximal location of chronic nonsuppurative destructive cholangitis. In contrast, there was aunique and distinct coronal arrangement of CD38 cells around the intrahepatic ducts in PBCbut not controls; the majority of such cells were considered plasma cells based on their expres-sion of intracellular immunoglobulins, including IgM and IgG, but not IgA. Patients withPBC who manifest this unique coronal arrangement were those with significantly higher titersof anti-mitochondrial antibodies. Conclusion: These data collectively suggest a role for plasmacells in the specific destruction of intrahepatic bile ducts in PBC and confirm the increasinginterest in plasma cells and autoimmunity. (HEPATOLOGY 2012;55:846-855)

Although considerable effort has been expendedon defining the pathophysiology of primarybiliary cirrhosis (PBC),1 an interesting void

remains, i.e., the relative role of distinct lymphoidpopulations in chronic nonsuppurative destructive chol-angitis (CNSDC) associated with chronic portal inflam-mation. Indeed, the most disease-specific serologic auto-antibodies in all of human immunopathology are theanti-mitochondrial antibodies (AMAs) found in more

than 95% of patients with PBC, primarily targeted atthe E2 component of the pyruvate dehydrogenase com-plex (PDC-E2).2 However, despite the significant valueof both AMAs and elevated serum IgM in the diagnosisof PBC, there is no correlation of serum AMA or IgMwith either disease severity or any other clinical feature.Furthermore, in murine models of PBC, the clinical useof anti-CD20 antibody, aimed at depleting B cells, hasnot been successful, and, in another murine model of

Abbreviations: ABC, avidin–biotin–peroxidase complex; AEC, 3-amino-9-ethylcarbazole; AIH, autoimmune hepatitis; ALP, alkaline phosphatase; ALT, alanineaminotransferase; APRIL, a proliferation-inducing ligand; AST, aspartate aminotransferase; AMA, anti-mitochondrial antibody; ANA, anti-nuclear antibody;BAFF, B-cell–activating factor, BD, bile duct; BSA, bovine serum albumin; CH-C, chronic hepatitis C; CNSDC, chronic nonsuppurative destructive cholangitis;CS-1, cell attachment-specific domain 1; EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; c-GTP, c-glutamyl transpeptidase;GVHD, graft-versus-host disease; HST, hematopoietic stem cell transplantation; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; IP-10, interferon c–inducible protein 10; LFA-1, lymphocyte function–associated antigen 1; LIL, liver-infiltrating lymphocyte; mAb, monoclonal antibody; MAdCAM-1, mucosaladressin cell adhesion molecule-1; MIG, monokine induced by interferon c; NK, natural killer; OLT, orthotopic liver transplantation; PBC, primary biliarycirrhosis; PBL, peripheral blood lymphocyte; PBS, phosphate-buffered saline; PDC-E2, E2 component of pyruvate dehydrogenase; PSC, primary sclerosingcholangitis; SDF-1, stromal cell–derived factor-1; TB, total bilirubin; TBS-T, Tris-buffered saline containing 1% Tween 20; TC, total cholesterol; TG,triglyceride; TP, total protein; UDCA, ursodeoxycholic acid.From the Divisions of 1Gastroenterology and Hepatology, 3Medical Technology, and 4Pathology, Nagaoka Red Cross Hospital, Nagaoka, Niigata, Japan; 2Division of

Human Physiological Science, Department of Medical Technology, School of Health Sciences, Faculty of Medicine, Niigata University, Niigata, Japan; 5Department ofDiagnostic Pathology, Graduate School of Medical and Pharmaceutical Research, Toyama University, Toyama, Japan; and 6Division of Rheumatology, Allergy, andClinical Immunology, University of California, Davis, CA.Received June 14, 2011; accepted October 3, 2011.Supported by National Institutes of Health grant DK39588.

846

PBC, depletion of B cells results in escalating liver dis-ease, suggesting that B cells suppress the inflammatoryresponse in mouse models.3,4

We have investigated the immunohistochemical dis-tribution of liver-infiltrating B lymphocytes in liver bi-opsy specimens from patients with PBC and controlliver diseases using monoclonal antibody (mAb)reagents specific for CD20 and CD38. We report herea unique coronal arrangement of CD38þ cells that isaccompanied by CNSDC. Furthermore, this coronalarrangement of CD38þ cells is positively correlatedwith AMA titer and is inversely correlated with serumc-glutamyl transpeptidase (c-GTP) levels. TheseCD38þ cells primarily express intracellular IgM orIgG, suggesting a pathogenic role of plasma cellsin PBC.

Patients and Methods

Patients. A total of 78 patients were enrolled in thisstudy. These included 26 patients with PBC, 20 ofwhom were positive for AMAs and six of whom werenegative, 27 patients with chronic hepatitis C (CH-C),eight patients with autoimmune hepatitis (AIH), eightpatients with primary sclerosing cholangitis (PSC), andnine patients with graft-versus-host disease (GVHD).The CH-C control group was age- and sex-matched

to the PBC group, which was randomly selected froma cohort of 136 candidates for interferon therapy forCH-C. The diagnosis of all cases was based on estab-lished criteria for PBC,5 AIH,6 PSC,7 and GVHD,8

respectively, or by detection of serum hepatitis C virusRNA by polymerase chain reaction for CH-C.

Table 1. Clinicopathological Profiles of Subjects With PBC, CH-C, AIH, PSC, and GVHD

PBC n ¼ 26 CH-C n ¼ 27 AIH n ¼ 8 PSC n ¼ 8 GVHD n ¼ 9

Age, years 57.2 6 9.3 57.9 6 11.3 59.5 6 9.5 46.0 6 24.6 45.9 6 14.2a

Male/female 7/19 7/20 1/7 7/1 7/2

Scheuer stage, 1/2/3/4 15/3/8/0 — — — —

Fibrosis score, 0/1/2/3/4 — 0/15/6/6/0 0/2/5/1/0 — 1/7/0/1/0

Activity score, 0/1/2/3 — 0/13/12/2 0/1/1/6 — 0/8/0/1

Ludwig stage, 1/2/3/4 — — — 5/1/1/1 —

Biopsy procedures, Echo/Laparo/Ope 1/24/1 27/0/0 1/7/0 6/2/0 9/0/0

Evaluated portal tracts, no. 12.2 6 4.7b 15.2 6 5.6c 13.0 6 4.2d 9.0 6 6.5 7.0 6 2.5

Evaluated bile ducts, no. 22.5 6 11.4e 25.6 6 11.6f 30.6 6 19.8g 17.5 6 15.1 12.0 6 4.1

AST, IU/L 99.3 6 141.1 55.7 6 40.9 332.1 6 522.2h 111.9 6 90.7 124.4 6 140.8

ALT, IU/L 105.2 6 159.4 58.7 6 42.4 333.4 6 367.2i 126.4 6 99.1j 217.4 6 289.0k

ALP, IU/L 775.7 6 389.4l 251.2 6 92.3 412.0 6 156.9m 560.8 6 627.2 434.2 6 348.4

c-GTP, IU/L 380.1 6 258.8n 48.7 6 43.4 155.1 6 36.4o 374.3 6 269.8p 572.3 6 668.7q

Data are expressed as mean 6 standard deviation. For abbreviations, see list in opening page footnote. Statistical significance with Mann-Whitney U test:aP ¼ 0.0326 compared with that in PBC; P ¼ 0.0269 compared with that in CH-C; and P ¼ 0.0377 compared with that in AIH.bP ¼ 0.0033 compared with that in GVHD.cP ¼ 0.0343 compared with that in PBC; P ¼ 0.0139 compared with that in PSC; and P ¼ 0.0003 compared with that in GVHDdP ¼ 0.0119 compared with that in GVHD.eP ¼ 0.0038 compared with that in GVHD.fP ¼ 0.0019 compared with that in GVHD.gP ¼ 0.0233 compared with that in GVHD.hP ¼ 0.0035 compared with that in PBC; and P ¼ 0.0004 compared with that in CH-C.iP ¼ 0.0015 compared with that in PBC; and P ¼ 0.0001 compared with that in CH-C and P ¼ 0.0460 compared with that in PSC.jP ¼ 0.0148 compared with that in CH-C.kP ¼ 0.0184 compared with that in CH-C.lP < 0.0001 compared with that in CH-C; P ¼ 0.0133 compared with that in AIH; and P ¼ 0.0082 compared with that in GVHD.mP ¼ 0.0085 compared with that in CH-C.nP < 0.0001 compared with that in CH-C; and P ¼ 0.0008 compared with that in AIH.oP ¼ 0.0002 compared with that in CH-C.pP ¼ 0.0004 compared with that in CH-C.qP < 0.0001 compared with that in CH-C; and P ¼ 0.0433 compared with that in AIH.

Address reprint requests to: Toru Takahashi, M.D., Director, Uonuma Hospital, Jonai 4-1-38, Ojiyashi, 947-0028, Niigata, Japan. E-mail: [email protected]; fax 81-258-83-4789.CopyrightVC 2011 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24757Potential conflict of interest: Nothing to report.

HEPATOLOGY, Vol. 55, No. 3, 2012 TAKAHASHI ET AL. 847

Informed consent in writing was obtained from eachpatient, and the study protocol was approved by theInstitutional Committee for Human Research ofNagaoka Red Cross Hospital. Patient clinical detailsare presented in Table 1. Liver tissue was availablefrom all patients either from laparoscopic liver biopsiesor ultrasound-guided needle liver biopsies.Liver Histology and Clinical Data Collection. -

Liver biopsy specimens were fixed in 10% formalin,dehydrated, and embedded in paraffin. Then 5-lmsections were cut in a microtome and subjected to sub-sequent routine histological staining using silverimpregnation, hematoxylin and eosin, and diastase-re-sistant periodic acid Schiff. Histological staging basedon established criteria for PBC,5 CH-C,9 AIH, andPSC10 was performed by a pathologist who wasblinded to all clinical data. Blood biochemical datawere obtained from each patient within 1 week of liverbiopsy. These included aspartate aminotransferase(AST), alanine aminotransferase (ALT), alkaline phos-phatase (ALP), and c-GTP. Total bilirubin, indirectbilirubin, total protein, serum albumin, total choles-terol (TC), triglycerides, serum levels of IgG, IgA, andIgM, AMA titers, and anti-nuclear antibody titers wereincluded in the PBC cohort. AMAs were examined byimmunofluorescence and by titration with enzyme-linked immunosorbent assay (ELISA) with known pos-itive and negative standards throughout. Anti-nuclearantibodies were determined by immunofluorescence onHep2 cells.Immunohistochemistry. Immunohistochemistry of

liver biopsy was performed with a Ventana HX SystemBenchMark/20 (Ventana Medical Systems Inc., Tuc-son, AZ), which uses avidin-biotin-peroxidase complex(ABC) coupled with a formulated unmasking pretreat-ment for each targeted antigen. For unmasking CDantigens and pankeratins, liver sections were soaked inTris-EDTA buffer, pH 8.5 (Ventana CC1 standard so-lution) at 100�C for 60 minutes. For unmaskingimmunoglobulins, liver sections were soaked in Prote-ase 1 solution (pronase 0.5 U/mL) for 8 minutes. En-dogenous peroxidase activity in liver tissue was blockedby soaking specimens in 3% H2O2 methanol solutionfor 10 minutes. Nonimmunized mouse IgG, used as anegative control in each experiment, did not result inany nonspecific staining signal.The following mouse monoclonal antibodies were

used: anti-CD3 (clone PS1), anti-CD4 (clone 1F6),anti-CD8 (clone 1A5), and anti-CD38 (clone SPC32)from Novacastla Laboratories (Newcastle upon Tyne,UK); anti-CD20 (clone L26) and anti-pankeratin(clone AE1/AE3) from Dako (Glostrup, Denmark);

and anti-human IgG (clone A57H), anti-human IgA(clone CB1-10.4/B8), and anti-human IgM (clone R1/69) from Nichirei Corp. (Tokyo, Japan). After incuba-tion with each of these primary antibodies at anappropriate dilution with 5% bovine serum albumin(BSA), slides were rinsed three times with phosphate-buffered saline (PBS), and then incubated with biotin-ylated anti-mouse IgG secondary antibodies. The slideswere rinsed three times and then incubated with ABCreagents for staining of CD antigens and pankeratin.For staining of immunoglobulins, we used standardperoxidase-labeled streptavidin-biotin. Diaminobenzi-dine hydrochloride was used as a substrate for colori-metric reaction.Portal tracts and bile ducts were counted per speci-

men with pankeratin staining using AE1/AE3 mono-clonal antibodies that specifically stain bile ducts andbile ductules. Only portal tracts with more than halfof its circumference and proper bile ducts werecounted, and ductular reactions were excluded fromthis count. The bile ducts with a coronal arrangementof CD38þ cells were enumerated per specimen, andthe frequency of this finding among the total countedbile ducts was thence calculated.For CD38 and IgM double immunohistochemical

staining, mouse monoclonal anti-CD38 (SPC32) andrabbit polyclonal anti-IgM (Pierce Biotechnology,Rockford, IL) were used as previously described.11 Af-ter deparaffinization, sections were soaked in target re-trieval–buffered saline (Tris, pH 6.1, Dako Cytoma-tion, Carpinteria, CA) in a plastic pressure cookercontaining no metals, irradiated in a microwave ovenfor 10 minutes, soaked in 3% H2O2 methanol solu-tion for 5 minutes, and then soaked in 5% BSA for 1minute. A cocktail of anti-CD38 and anti-IgM anti-bodies was diluted to a predetermined optimal concen-tration in PBS containing 5% BSA. The diluted anti-bodies were applied to tissue sections in a moistchamber and irradiated intermittently for 10 minutes(250 W, 4 seconds on, 3 seconds off ). After threewashes with Tris-buffered saline containing 1% Tween20 (TBS-T) for 1 minute, a cocktail containing perox-idase-conjugated (Envision System, Dako Cytomation)or alkaline phosphatase–conjugated secondary antibod-ies (Simple Stain System, Nichirei, Japan) was appliedto specimens in the moist chamber. Irradiation wasthen performed intermittently for 10 minutes, asdescribed above. After washing five times with TBS-T,the sections were immersed in Fast blue (alkaline phos-phatase substrate kit, SK-5300, Vector, Burlingame,CA) and 3-amino-9-ethylcarbazole (AEC; peroxidasesubstrate kit, Nichirei) and counterstained with

848 TAKAHASHI ET AL. HEPATOLOGY, March 2012

hematoxylin (Dako Cytomation). After the substratereaction, CD38þ cells were blue and IgMþ cells werered–brown.Statistics. The Mann-Whitney U test was used for

comparing the blood biochemical and serological dataamong PBC, CH-C, AIH, PSC, and GVHD groupsand between PBC patients with or without a coronalarrangement of CD38þ cells; P values under 0.05were considered statistically significant. The Chi-squaretest was used for comparing the occurrence of lymphfollicle–like infiltration and the infiltration of CD4þ

or CD8þ cells into cholangioepithelium among thePBC, CH-C, AIH, PSC, and GVHD groups.

Results

Clinicopathological profiles of PBC, CH-C, AIH,PSC, and GVHD subjects are summarized in Table 1.Therapy for the 26 PBC patients varied according

to the nature and severity of the disease. Ursodeoxy-cholic acid (UDCA) was administered to 25 patients(96.2%); the daily dose was 300 mg in one, 600 mgin 22, and 900 mg in two patients. Bezafibrate wasalso administered to four patients at a daily dose of400 mg, all of whom simultaneously took UDCA.Prednisolone was given to two patients in whom ahepatitic form of PBC was noted on liver pathology.None of the 26 PBC patients progressed to the ictericstage, which would need liver transplantation duringthe observation period through September 2011.Histologic evaluations of liver biopsy sections

revealed typical CNSDC and epithelioid cell granulo-mas in patients with PBC but not those with the con-trol liver diseases (Table 2). The incidence of CNSDCin PBC was 50% (13/26) and that of granulomas was23.1% (6/26). In contrast, lymph follicle–like infiltra-tion was most frequently found in CH-C livers

(12/27, 44.4%) but was also found at lower frequen-cies in patients with PBC (6/26, 23.1%) and AIH(2/8, 25%). Such infiltration was not found in PSC orGVHD.The distribution of lymphoid elements is summar-

ized in Table 2. In PBC, CD20þ B lymphocytes, theprecursors of plasma cells, were found either scatteredor aggregated within the lymphoplasmocytic infiltra-tion (Fig. 1A). Such CD20þ B cells occasionallyformed follicle-like aggregations but, importantly, theywere not observed in the proximity of CNSDC (Fig.2B). In contrast, an intense coronal arrangement of

Table 2. Histological and Immunohistological Characteristics of the PBC, CH-C, AIH, PSC, and GVHD Livers

PBC n ¼ 26 CH-C n ¼ 27 AIH n ¼ 8 PSC n ¼ 8 GVHD n ¼ 9

No. of cases % No. of cases % No. of cases % No. of cases % No. of cases %

CNSDC 13 (50.0) 0 0 0 0 0 0 0 0

Granuloma 6 (23.1) 0 0 0 0 0 0 0 0

Lymph follicle–like infiltration* 6 (23.1) 12 (44.4) 2 (25.0) 0 0 0 0

Infiltration of CD4þ cells into cholangioepithelium* 16 (61.5) 0 0 1 (12.5) 2 (25.0) 0 0

Infiltration of CD8þ cells into cholangioepithelium* 7 (26.9) 4 (14.8) 3 (37.5) 1 (12.5) 7 (77.8*)

Coronal arrangement of CD38þ cells 13 (50.0) 0 0 0 0 0 0 1 (11.1)

Coronal arrangement of IgGþ cells 5 (19.2) — — — — — — — —

Coronal arrangement of IgMþ cells 8 (30.8) — — — — — — — —

Coronal arrangement of IgGþ and IgMþ cells 3 (11.5) — — — — — — — —

For abbreviations, see list in opening page footnote.

*Statistical significance with Chi-square tests: P ¼ 0.0221 compared with PBC; P ¼ 0.0017 compared with that in CH-C; and P ¼ 0.0275 compared with that

in PSC.

Fig. 1. Immunohistochemical staining of CD20þ B lymphocytesand CD38þ cells in consecutive sections of a PBC liver. (A) CD20þ Blymphocytes are either aggregated in lymph follicle–like structures(white stars) or scattered around inflamed portal tracts. (B) Coronalarrangement (CA) of CD38þ cells surrounding the intralobular bileducts (arrows). Note that CD38þ cells are scarce in lymph follicle–likestructures. ABC method, original magnification, x10.


CD38þ cells was found around intrahepatic bile ductsin every specimen with CNSDC (Figs. 1B, 2C) butwas never found in the portal tracts with ductopenia.CD3þ pan-T cells were randomly scattered aroundCNSDC (Fig. 2A), an area where CD20þ B lympho-cytes had not been observed (Fig. 2B), whereas themost typical coronal arrangement of CD38þ cells wasfound (Fig. 2C). This coronal arrangement of CD38þ

cells was continuously present along with CNSDC, asshown by serial liver sections (Fig. 2D). CD4þ andCD8þ T lymphocyte infiltration was observed eitherin proximity to, or within, the degenerated cholangioe-pithelium, suggesting the participation of these cells inthe destructive processes of intrahepatic bile ducts (Fig.2E,F). This CD4þ and CD8þ T lymphocyte infiltra-tion into the cholangioepithelium was also observed inpatients with CH-C, AIH, PSC, and GVHD as a con-sequence of lymphocytic cholangitis (Table 2).To determine whether the coronal arrangement pat-

tern of CD38þ cells was specific for PBC, we exam-ined CD20þ and CD38þ cells in liver sections withother liver diseases. In CH-C, CD20þ B lymphocyteswere aggregated in a follicle-like fashion in theinflamed portal tracts (Fig. 3A) where intrahepatic bileducts were often centered (an arrow in Fig. 3A); incontrast, CD38þ cells were found at the periphery ofinflamed portal tracts but were not found around theintrahepatic bile ducts (arrow in Fig. 3B). Similarly, in

Fig. 2. Immunohistochemical staining of CD3þ pan-T lymphocytes,CD20þ B lymphocytes, CD38þ cells, CD4þ T lymphocytes, and CD8þ

T lymphocytes in two sets of consecutive PBC liver sections. (A-D) Inthe first consecutive section set, CD3þ pan-T lymphocytes are scat-tered around an intrahepatic bile duct (BD) with CNSDC (A); CD20þ Blymphocytes are not observed around this intrahepatic bile duct (BD;B); and CD38þ cells show a distinct coronal arrangement surroundingthis intrahepatic bile duct (BD) with CNSDC (C). The coronal arrange-ment of CD38þ cells is continuously observed along with CNSDC inserial sections (D). In subsequent serial consecutive sections, CD4þ Tlymphocytes (E) and CD8þ T lymphocytes (F) demonstrate the samepattern of infiltration (arrows) into the cholangioepithelium of an intra-hepatic bile duct (BD) with CNSDC. ABC method, original magnifica-tion �25.

Fig. 3. Immunohistochemical staining of CD20þ and CD38þ cells in liver sections. (A) In CH-C, CD20þ B lymphocytes are aggregated in afollicle-like structure in an inflamed portal tract (white stars). Note that an intrahepatic bile duct (arrow) is centered in such a follicle-like struc-ture. (B) In a consecutive section of CH-C liver (with A), CD38þ cells are primarily located in the periphery of inflamed portal tracts (black stars)apart from the intrahepatic bile ducts (an arrow). (C) In AIH, CD38þ cells are not observed in the proximity of intrahepatic bile ducts (BD,arrows) but are abundantly infiltrated in the area of interface hepatitis (black stars). (D) In PBC, CD38þ cells show the coronal arrangement sur-rounding intrahepatic bile ducts (BD) with CNSDC (arrows). (E) In a case of stage 4 PSC, CD38þ cells are surrounding the onion skin-like fibro-sis that is a characteristic of PSC (arrows). This pattern is termed a satellite-like arrangement (SA) of CD38þ cells surrounding concentricperiductal fibrosis. The SA pattern is different from the CA of CD38þ cells in PBC located just beneath the cholangioepithelium with CNSDC, asshown in Figs. 2C and 4D. (F) In a case of stage 1 PSC, CD38þ cells are concentrically scattered in the onion skin–like fibrosis (black stars)surrounding an intrahepatic bile duct (arrows). This pattern is also regarded as SA of CD38þ cells. ABC method, original magnification �25.


AIH, CD38þ cells were not observed in the proximityof intrahepatic bile ducts (BD, arrows in Fig. 3C) butwere abundantly infiltrated in the area of interfacehepatitis (Fig. 3C). In contrast to PBC livers, in whichCD38þ cells formed a coronal arrangement around anintrahepatic bile duct (BD) with CNSDC (Fig. 3D),such a pattern was not observed in the disease controlgroups including CH-C, AIH, and PSC; it wasobserved in only one bile duct of one patient withGVHD (Table 2), although the frequency of this find-ing was only 0.9% (1/107) of all bile ducts evaluatedin this group and was thought to be incidental. InPBC livers, the coronal arrangement of CD38þ cellswas observed in 21.5% (7.1%-41.0% per specimen) ofall evaluated bile ducts when it was present (69 bileducts among 321 bile ducts counted in the PBC groupwith a coronal arrangement of CD38þ cells). In PSClivers, CD38þ cells were found surrounding, or in anonion skin–like fibrosis, a pattern that is unique inPSC (Fig. 3E,F).To determine the identity of the CD38þ cells that

formed a coronal arrangement specifically in the PBC liv-ers with CNSDC (Fig. 2C), we first examined the expres-sion of immunoglobulin classes in consecutive sections ofPBC livers with CNSDC by staining for immunoglobu-lin classes. The majority (8/13, 61.5%) of the coronalarrangements had IgMþ cells (Fig. 4C), followed byIgGþ cells (5/13, 38.5%; Fig. 4A). Three patients(23.1%) had both IgGþ and IgMþ cells in the same coro-nal arrangement (Fig. 4) whereas two had only IgGþ cellsbut not IgMþ cells. IgAþ cells were not observed in a cor-onal arrangement (Fig. 4B). IgGþ cells were relativelyloosely scattered around CNSDC (Fig. 4A), whereasIgMþ cells showed a dense and prominent coronalarrangement around CNSDC (Fig. 4C). These results

indicate that the B cells expressing antibodies participatein the formation of the coronal arrangement in CNSDC.Next we used double immunostaining to examine

the colocalization of CD38 and IgM in the same cellsthat comprise the coronal arrangement. The majority(up to 70%) of CD38þ cells in coronal arrangementthat were specifically stained by Fast blue also showedpositive red–brown staining of intracellular IgM(arrows in Fig. 5). These results suggest that themajority of CD38þ cells were IgM plasma cells.Finally, we examined the correlation of coronalarrangement with blood biochemical and serologicalparameters in PBC (Table 3). Among 14 parameters,the presence of coronal arrangement was significantly

Fig. 4. Immunohistochemical staining of IgG, IgA, and IgM in consecutive liver sections of a PBC liver. (A) IgGþ cells demonstrate a relativelyloose coronal arrangement surrounding an intrahepatic bile duct (BD) with CNSDC. (B) IgAþ cells are not found in the vicinity of this intrahepaticbile duct (BD) with CNSDC. (C) IgMþ cells demonstrate a dense and distinct coronal arrangement surrounding this intrahepatic bile duct (BD)with CNSDC. Labeled streptavidin-biotin method, original magnification �25.

Fig. 5. Double immunostaining of CD38 and IgM in a PBC liver.The majority (approximately 70%) of CD38þ cells that are immunohis-tochemically identified by a blue color (Fast blue) demonstrate a red-dish brown color (AEC) in their cytoplasm indicating the presence ofintracellular IgM (arrows). BD, an intrahepatic bile duct with CNSDC,indirect double enzyme-antibody method, original magnification �100.


associated with higher titers of AMA (P ¼ 0.0153)and lower levels of c-GTP (P ¼ 0.0256) (Fig. 6).

Discussion

The study of plasma cells in autoimmune diseaseshas led to the hypothesis that plasma cells with patho-genic potential are long-lived and depend on finding aniche within a local microenvironment such as the bil-iary tract. Indeed, in murine lupus, B-cell–activatingfactor (BAFF), a proliferation-inducing ligand(APRIL), interleukin-6 (IL-6), and adhesion moleculesall modulate the survival of plasma cells and lead tofurther inflammation.12-16 In organ-specific autoim-mune diseases such as PBC, the role of plasma cellshas not attracted significant attention. Here we havedemonstrated a relatively nonspecific follicle-like aggre-gation in inflamed portal tracts of CD20þ B cells and,more importantly, a prominent coronal arrangement ofCD38þ plasma cells surrounding the intrahepatic bileducts with CNSDC. CD20 is a type III membranousprotein of 297 amino acids17; it is a representative B-lineage cell marker that disappears from the cell sur-face when B cells are differentiated into antibody-pro-ducing plasma cells. Therefore, the differential distri-bution of CD20þ and CD38þ cells in a target tissuereflects the disease-specific movement of these two celltypes during B-cell maturation in the course of achronically evolving inflammatory disease such asPBC.Our findings suggest a PBC-specific dynamic settle-

ment in B-cell lineage populations during the inflam-matory processes in portal tracts, which involves

migration of B lymphocytes from the portal tracts tothe intrahepatic bile ducts during the maturation pro-cess from CD20þ mature B cells to professional anti-body-producing CD38þ cells, or plasma cells. We didnot, however, examine CD38þ cells in cirrhotic liversof patients with PBC. Future studies should focus ona longitudinal analysis and/or detailed cross-sectionalanalysis of patients at different stages of disease. Also,this concept of plasma cells infiltrating environmentalniches needs further exploration in other liver diseases,such as expanding the PSC database and includingpatients with hepatic allograft rejection after orthotopicliver transplantation (OLT) and chronic GVHD afterhematopoietic stem cell transplantation (HST).The most important finding of this study is the cor-

onal arrangement pattern of CD38þ cells around theintrahepatic bile ducts with CNSDC. This pattern isspecific for PBC and is not observed in other autoim-mune liver diseases including AIH, PSC, and GVHD,or in CH-C. CD38 is a part of nicotinamide adeninedinucleotide cyclase.18 It is a type II membranous pro-tein of 300 amino acids. CD38 expression is limitedto the cell surface of T-cell progenitors, lymphoid stemcells, plasmablasts, and mature plasma cells. We con-clude that the majority of CD38þ cells observed

Table 3. Coronal Arrangement (CA) of CD381 Cells andClinical Data in PBC

CA (�) n ¼ 13 CA (þ) n ¼ 13 P

AST 88.6 6 91.4 110.0 6 181.3 0.8174

ALT 88.6 6 79.1 121.8 6 215.5 0.7778

ALP 789.8 6 422.4 761.7 6 370.3 1.0000

c-GTP 477.6 6 296.4 282.5 6 176.2 0.0256*

TB 0.63 6 0.22 0.71 6 0.18 0.2012

TP 7.67 6 0.5 8.02 6 0.77 0.1635

Albumin 4.22 6 0.31 4.35 6 0.31 0.2668

TC 223.6 6 34.7 195.1 6 32.4 0.1059

TG 116.2 6 56.8 111.5 6 50.1 0.9795

IgG 1,728.3 6 367.2 1,838.2 6 529.4 0.7779

IgA 266.4 6 79.4 298.5 6 112.3 0.3428

IgM 293.5 6 199.9 517.2 6 438.3 0.0858

AMA titer 67.9 6 61.8 119.5 6 58.2 0.0153*

ANA titer 433.8 6 791.3 163.1 6 355 0.7163

CA (�), without coronal arrangement of CD38þ cells; CA (þ), with coronal

arrangement of CD38þ cells. For other abbreviations, see list in opening page

footnote.

*P< 0.05 (signi¢cant) by the Mann-Whitney U test.

Fig. 6. AMA titers and c-GTP levels in coronal arrangement (CA)-positive and CA-negative PBC patients . (A) The titer of AMA is signifi-cantly higher in the CA-positive PBC patients compared with the CA-negative patients. (B) c-GTP level is significantly lower in the CA-posi-tive PBC patients compared with the CA-negative patients.


around the intrahepatic bile ducts are mature plasmacells rather than T cell subsets based on the followingobservations: 1) the distributions of CD38þ cells andof IgM- and/or IgG-bearing cells were nearly identical;2) the distributions of CD3þ, CD4þ, and CD8þ Tcells and that of CD38þ cells were very different; 3)mature plasma cells expressed higher levels of CD38than other CD38þ B-cell or T-cell subsets19,20; 4) thedistribution of CD138þ cells, a marker more specificfor mature plasma cells than CD38, was similar tothat of CD38þ cells (data not shown); and 5) mostimportantly, double immunostaining of CD38 andIgM in PBC livers clearly indicated that approximately70% of CD38þ cells expressed IgM. However, thereremains a possibility that the observed CD38þ popula-tion was a mixture of diverse cell types including acti-vated T cells, other B-lineage cell populations, naturalkiller (NK) cells, and basophils in addition to matureplasma cells.21 Even if this is the case, the fact thatCD38þ cells clearly form a coronal arrangement sur-rounding CNSDC strongly suggests that the coronalarrangement is related to the pathogenesis of PBC.The role of T-cell lineage populations in the patho-

genesis of PBC has been extensively studied.1,22-31 Incontrast, the role of B-cell lineage populations in PBCis not clear. Recently it has been shown that AMAs arerequired in the production of inflammation cytokinesby macrophages in the presence of apoptotic humanintrahepatic biliary epithelial cells.32 This could explainthe biliary specificity of autoimmune damage in PBC.It is possible that at different stages of PBC, B cellsplay different roles in the breakdown of tolerance anddevelopment of small bile duct pathogenesis. Althoughwe cannot exclude the possibility that the formation ofcoronal arrangement is a consequence of, rather than acontributing factor to, the destruction of small bileducts, our data are in agreement with the findings ofLleo et al.,32 which strongly suggest an active role ofAMAs in the inflammatory responses at the affectedlocal bile ducts. Future studies should focus on theantigen specificity of the IgM and IgG produced inthese coronal arrangement–comprising plasma cells.Although IgA transcytosis has been considered one ofthe contributing factors toward bile duct lesions inPBC,33,34 it is unlikely that the coronal arrangementwe observed in this study includes IgAþ plasma cells.However, we observed that IgA staining was often seenin the degenerated cholangioepithelium of CNSDC orat the apical margin of damaged bile duct cells (datanot shown).There are two steps in lymphocyte recruitment

and chemotaxis in PBC: ‘‘tethering’’ and transendo-

thelial migration of peripheral blood lymphocytes(PBLs) from vessels to the inflamed area of liver,and recruitment and settlement of liver-infiltratinglymphocytes (LILs) around bile ducts, where theyparticipate in the destruction process of targeted bileducts. Induced or up-regulated expression of mono-kine induced by interferon c (MIG) and interferonc–inducible protein 10 (IP-10) in portal tracts isthought to contribute to T-cell recruitment into thePBC liver.35 Once these cells have entered the portaltract, they often form lymphoplasmocytoid aggre-gates. Lymphoid neogenesis depends on the interac-tion between CCL2136 and mucosal adressin cell ad-hesion molecule-1 (MAdCAM-1)37 expressed onhigh endothelial venules and on lymphatic vesselsand CCR7 expressed on lymphocytes.38 The lym-phoplasmocytoids are then recruited to, and retainedaround, bile ducts by the combinational or sequen-tial action of several chemokines.35,39,40 In the caseof the B cells, only CXCL12 (stromal cell-derivedfactor 1 [SDF-1]) has been reported to participate inthe recruitment of CD19þ B cells41-43; however,CD19 is expressed in intermediate and mature Bcells but not in plasma cells.CXCL12 (SDF-1) is expressed constitutively in bile

duct cells and may have a role in retention of lympho-cytes via its ability to augment their adhesion to fibro-nectin by triggering a4b7-mediated binding ofLIL.38,44,45 Moreover, the biliary basement membranehas immunoreactive fibronectin in 80% of patientswith PBC but not in those with other diseases or innormal control livers.46 We previously found that fi-bronectin was abundantly expressed in necrotic andnewly fibrosing areas, or in the area of inflammation,47

suggesting that fibronectin may contribute to the che-motaxis of CD38þ cells in interface hepatitis in CH-Cand AIH liver as well as in coronal arrangement inPBC livers. In addition, we have shown that theexpression of alternatively spliced fibronectin contain-ing a cell attachment–specific domain (CS-1 fibronec-tin) was increased in fibrotic human liver48; hence itmay also contribute to the recruitment and retentionof LIL through not only the a1b4 but also the a4b7integrin. Taken together, these previous findings sug-gest that CXCL12 might be involved in the formationof the coronal arrangement of CD38þ cells aroundbile ducts. Other potential contributing factors for theformation of coronal arrangement include hepatocytegrowth factor,49 lymphocyte function–associated anti-gen-1 (LFA-1)/ intercellular adhesion molecule-1(ICAM-1),50,51 CXCL13, and CXCL5,52 whichshould also be explored in future studies.


Although AMAs are well established as a diagnosticcriterion for PBC, serum AMA titer has not been cor-related with any parameter of disease activity. Weobserve for the first time that AMA titer was correlatedsignificantly with the formation of a coronal arrange-ment by CD38þ plasma cells in the liver, which couldbe a bridge that links AMA and the inflammatoryreactions in PBC. Although the implications of thisnovel correlation need to be studied further, we pro-pose that plasma cells may be a source of both AMAsand elevated serum IgM. UDCA is reported todecrease the level of total IgM and IgM AMAs butnot of IgG AMAs.53 Thus, UDCA may preferablyaffect the CD38þ plasma cells that comprise the coro-nal arrangement. The relationship of reduced c-GTPwith the coronal arrangement is unclear becauseanother biliary enzyme, ALP, did not correlate withthe coronal arrangement. Finally, the concept of anenvironmental niche for long-lived plasma cells andtheir impact on inflammation has implications notonly for PBC, but, from a generic perspective, forother autoimmune diseases as well; this concept hasbeen recently reviewed.54

Acknowledgments: The authors thank Mr. KyujiIwamoto for his technical assistance in preparing histo-logical sections and immunohistochemistry.

References1. Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med

2005;353:1261-1273.2. Gershwin ME, Mackay IR, Sturgess A, Coppel RL. Identification and

specificity of a cDNA encoding the 70 kd mitochondrial antigen recog-nized in primary biliary cirrhosis. J Immunol 1987;138:3525-3531.

3. Dhirapong A, Lleo A, Yang GX, Tsuneyama K, Dunn R, Kehry M,et al. B cell depletion therapy exacerbates murine primary biliary cir-rhosis. HEPATOLOGY 2011;53:527-535.

4. Moritoki Y, Zhang W, Tsuneyama K, Yoshida K, Wakabayashi K, YangGX, et al. B cells suppress the inflammatory response in a mousemodel of primary biliary cirrhosis. Gastroenterology 2009;136:1037-1047.

5. Scheuer P. Primary biliary cirrhosis. Proc R Soc Med 1967;60:1257-1260.

6. Hennes EM, Zeniya M, Czaja AJ, Pares A, Dalekos GN, Krawitt EL,et al. Simplified criteria for the diagnosis of autoimmune hepatitis.HEPATOLOGY 2008;48:169-176.

7. Lindor KD, LaRusso NF. Primary sclerosing cholangitis. In: Schiff ER,Sorrell MF, Maddrey WC, eds. Schiff ’s Diseases of the Liver. 9th ed.Philadelphia: Lippincott Williams & Wilkins, 2003;673-684.

8. Alpini G, McGill JM, LaRusso NF. The pathobiology of biliary epithe-lia. HEPATOLOGY 2002;35:1256-1268.

9. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classifi-cation of chronic hepatitis: diagnosis, grading and staging. HEPATOLOGY

1994;19:1513-1520.10. Ludwig J. Surgical pathology of the syndrome of primary sclerosing

cholangitis. Am J Surg Pathol 1989;13(Suppl 1):43-49.11. Kumada T, Tsuneyama K, Hatta H, Ishizawa S, Takano Y. Improved

1-h rapid immunostaining method using intermittent microwave irradi-ation: practicability based on 5 years application in Toyama Medical

and Pharmaceutical University Hospital. Mod Pathol 2004;17:1141-1149.

12. Liu Z, Zou Y, Davidson A. Plasma cells in systemic lupus erythemato-sus: The long and short of it all. Eur J Immunol 2011;41:588-591.

13. Oracki SA, Walker JA, Hibbs ML, Corcoran LM, Tarlinton DM.Plasma cell development and survival. Immunol Rev 2010;237:140-159.

14. Cassese G, Lindenau S, de Boer B, Arce S, Hauser A, Riemekasten G,et al. Inflamed kidneys of NZB/W mice are a major site for the home-ostasis of plasma cells. Eur J Immunol 2001;31:2726-2732.

15. Mohr E, Serre K, Manz RA, Cunningham AF, Khan M, Hardie DL,et al. Dendritic cells and monocyte/macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablastsmature. J Immunol 2009;182:2113-2123.

16. Tokoyoda K, Zehentmeier S, Chang HD, Radbruch A. Organizationand maintenance of immunological memory by stroma niches. Eur JImmunol 2009;39:2095-2099.

17. Tedder TF, Engel P. CD20: a regulator of cell-cycle progression of Blymphocytes. Immunol Today 1994;15:450-454.

18. Howard M, Grimaldi JC, Bazan JF, Lund FE, Santos-Argumedo L,Parkhouse RM, et al. Formation and hydrolysis of cyclic ADP-ribosecatalyzed by lymphocyte antigen CD38. Science 1993;262:1056-1059.

19. Harada H, Kawano MM, Huang N, Harada Y, Iwato K, Tanabe O,et al. Phenotypic difference of normal plasma cells from mature my-eloma cells. Blood 1993;81:2658-2663.

20. Jackson N, Ling NR, Ball J, Bromidge E, Nathan PD, Franklin IM.An analysis of myeloma plasma cell phenotype using antibodies definedat the IIIrd International Workshop on Human Leucocyte Differentia-tion Antigens. Clin Exp Immunol 1988;72:351-356.

21. Terstappen LW, Hollander Z, Meiners H, Loken MR. Quantitativecomparison of myeloid antigens on five lineages of mature peripheralblood cells. J Leukoc Biol 1990;48:138-148.

22. Gershwin ME, Ansari AA, Mackay IR, Nakanuma Y, Nishio A, RowleyMJ, et al. Primary biliary cirrhosis: an orchestrated immune responseagainst epithelial cells. Immunol Rev 2000;174:210-225.

23. Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis: con-venient and inconvenient truths. HEPATOLOGY 2008;47:737-745.

24. Kamihira T, Shimoda S, Harada K, Kawano A, Handa M, Baba E,et al. Distinct costimulation dependent and independent autoreactiveT-cell clones in primary biliary cirrhosis. Gastroenterology 2003;125:1379-1387.

25. Kamihira T, Shimoda S, Nakamura M, Yokoyama T, Takii Y, KawanoA, et al. Biliary epithelial cells regulate autoreactive T cells: implicationsfor biliary-specific diseases. HEPATOLOGY 2005;41:151-159.

26. Kita H, Matsumura S, He XS, Ansari AA, Lian ZX, Van de Water J,et al. Quantitative and functional analysis of PDC-E2-specific autoreac-tive cytotoxic T lymphocytes in primary biliary cirrhosis. J Clin Invest2002;109:1231-1240.

27. Krams SM, Dorshkind K, Gershwin ME. Generation of biliary lesionsafter transfer of human lymphocytes into severe combined immunodefi-cient (SCID) mice. J Exp Med 1989;170:1919-1930.

28. Shigematsu H, Shimoda S, Nakamura M, Matsushita S, Nishimura Y,Sakamoto N, et al. Fine specificity of T cells reactive to human PDC-E2 163-176 peptide, the immunodominant autoantigen in primary bil-iary cirrhosis: implications for molecular mimicry and cross-recognitionamong mitochondrial autoantigens. HEPATOLOGY 2000;32:901-909.

29. Shimoda S, Ishikawa F, Kamihira T, Komori A, Niiro H, Baba E, et al.Autoreactive T-cell responses in primary biliary cirrhosis are proinflam-matory whereas those of controls are regulatory. Gastroenterology2006;131:606-618.

30. Shimoda S, Nakamura M, Ishibashi H, Kawano A, Kamihira T, Saka-moto N, et al. Molecular mimicry of mitochondrial and nuclear autoanti-gens in primary biliary cirrhosis. Gastroenterology 2003;124:1915-1925.

31. Zhang W, Sharma R, Ju ST, He XS, Tao Y, Tsuneyama K, et al. Defi-ciency in regulatory T cells results in development of antimitochondrialantibodies and autoimmune cholangitis. HEPATOLOGY 2009;49:545-552.


32. Lleo A, Selmi C, Invernizzi P, Podda M, Coppel RL, Mackay IR, et al.Apotopes and the biliary specificity of primary biliary cirrhosis. HEPATO-

LOGY 2009;49:871-879.

33. Fukushima N, Nalbandian G, Van De Water J, White K, Ansari AA,Leung P, et al. Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC. HEPATOLOGY

2002;36:1383-1392.

34. Malmborg AC, Shultz DB, Luton F, Mostov KE, Richly E, Leung PS,et al. Penetration and co-localization in MDCK cell mitochondria ofIgA derived from patients with primary biliary cirrhosis. J Autoimmun1998;11:573-580.

35. Borchers AT, Shimoda S, Bowlus C, Keen CL, Gershwin ME. Lympho-cyte recruitment and homing to the liver in primary biliary cirrhosis andprimary sclerosing cholangitis. Semin Immunopathol 2009;31:309-322.

36. Weninger W, Carlsen HS, Goodarzi M, Moazed F, Crowley MA, Baek-kevold ES, et al. Naive T cell recruitment to nonlymphoid tissues: arole for endothelium-expressed CC chemokine ligand 21 in autoim-mune disease and lymphoid neogenesis. J Immunol 2003;170:4638-4648.

37. Grant AJ, Lalor PF, Hubscher SG, Briskin M, Adams DH. MAdCAM-1 expressed in chronic inflammatory liver disease supports mucosallymphocyte adhesion to hepatic endothelium (MAdCAM-1 in chronicinflammatory liver disease). HEPATOLOGY 2001;33:1065-1072.

38. Grant AJ, Goddard S, Ahmed-Choudhury J, Reynolds G, Jackson DG,Briskin M, et al. Hepatic expression of secondary lymphoid chemokine(CCL21) promotes the development of portal-associated lymphoid tis-sue in chronic inflammatory liver disease. Am J Pathol 2002;160:1445-1455.

39. Isse K, Harada K, Zen Y, Kamihira T, Shimoda S, Harada M, et al.Fractalkine and CX3CR1 are involved in the recruitment of intraepi-thelial lymphocytes of intrahepatic bile ducts. HEPATOLOGY 2005;41:506-516.

40. Shimoda S, Harada K, Niiro H, Taketomi A, Maehara Y, TsuneyamaK, et al. CX3CL1 (fractalkine): a signpost for biliary inflammation inprimary biliary cirrhosis. HEPATOLOGY 2010;51:567-575.

41. Goddard S, Williams A, Morland C, Qin S, Gladue R, Hubscher SG,et al. Differential expression of chemokines and chemokine receptorsshapes the inflammatory response in rejecting human liver transplants.Transplantation 2001;72:1957-1967.

42. Shackel NA, McGuinness PH, Abbott CA, Gorrell MD, McCaughanGW. Identification of novel molecules and pathogenic pathways in pri-mary biliary cirrhosis: cDNA array analysis of intrahepatic differentialgene expression. Gut 2001;49:565-576.

43. Terada R, Yamamoto K, Hakoda T, Shimada N, Okano N, Baba N,et al. Stromal cell-derived factor-1 from biliary epithelial cells recruitsCXCR4-positive cells: implications for inflammatory liver diseases. LabInvest 2003;83:665-672.

44. Buckley CD, Amft N, Bradfield PF, Pilling D, Ross E, Arenzana-Seis-dedos F, et al. Persistent induction of the chemokine receptor CXCR4by TGF-beta 1 on synovial T cells contributes to their accumulationwithin the rheumatoid synovium. J Immunol 2000;165:3423-3429.

45. Wright N, Hidalgo A, Rodriguez-Frade JM, Soriano SF, Mellado M,Parmo-Cabanas M, et al. The chemokine stromal cell-derived factor-1alpha modulates alpha 4 beta 7 integrin-mediated lymphocyte adhesionto mucosal addressin cell adhesion molecule-1 and fibronectin.J Immunol 2002;168:5268-5277.

46. Yasoshima M, Tsuneyama K, Harada K, Sasaki M, Gershwin ME,Nakanuma Y. Immunohistochemical analysis of cell-matrix adhesionmolecules and their ligands in the portal tracts of primary biliary cir-rhosis. J Pathol 2000;190:93-99.

47. Takahashi T, Isemura M, Nakamura T, Matsui S, Oyanagi Y, AsakuraH. Immunolocalization of a fibronectin-binding proteoglycan (PG-P1)immunologically related to HSPG2/perlecan in normal and fibrotichuman liver. J Hepatol 1994;21:500-508.

48. Matsui S, Takahashi T, Oyanagi Y, Takahashi S, Boku S, Takahashi K,et al. Expression, localization and alternative splicing pattern of fibro-nectin messenger RNA in fibrotic human liver and hepatocellular carci-noma. J Hepatol 1997;27:843-853.

49. Holt RU, Fagerli UM, Baykov V, Ro TB, Hov H, Waage A, et al.Hepatocyte growth factor promotes migration of human myeloma cells.Haematologica 2008;93:619-622.

50. Yokomori H, Oda M, Ogi M, Wakabayashi G, Kawachi S, YoshimuraK, et al. Expression of adhesion molecules on mature cholangiocytes incanal of Hering and bile ductules in wedge biopsy samples of primarybiliary cirrhosis. World J Gastroenterol 2005;11:4382-4389.

51. Yokomori H, Oda M, Yoshimura K, Nomura M, Ogi M, WakabayashiG, et al. Expression of intercellular adhesion molecule-1 and lympho-cyte function-associated antigen-1 protein and messenger RNA in pri-mary biliary cirrhosis. Intern Med 2003;42:947-954.

52. Oo YH, Adams DH. The role of chemokines in the recruitment oflymphocytes to the liver. J Autoimmun 2010;34:45-54.

53. Kikuchi K, Hsu W, Hosoya N, Moritoki Y, Kajiyama Y, Kawai T,et al. Ursodeoxycholic acid reduces CpG-induced IgM production inpatients with primary biliary cirrhosis. Hepatol Res 2009;39:448-454.

54. Hiepe F, Dorner T, Hauser AE, Hoyer BF, Mei H, Radbruch A. Long-lived autoreactive plasma cells drive persistent autoimmune inflamma-tion. Nat Rev Rheumatol 2011;7:170-178.


Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis

Documents

Transcript of Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis