Hossain Md. GOLBAR

称号及び氏名博士（獣医学）

学位授与の日付

平成２４年３月３１日

論Pathological Studies on the Biliary Fibrosis with Special Reference

文

名to Macrophage Properties in Relation to Myofibroblast Development（胆管線維症に関する病理学的研究：特に筋線維芽細胞形成との関連におけるマクロファージの特性について）
論文審査委員

主査

山手丈至

副査

松尾 三郎

副査

中村 洋一

論文要旨
Introduction
There is growing impact of cirrhosis owing to public health burden
associated with significant morbidity and mortality worldwide. Parenchymal
injury induced by ischemia, viruses, chemicals and malnutrition is the key initialstarting point of hepatic fibrosis. Injured hepatocytes secrete cytokines, which canrecruit inflammatory cells into damaged sites. Infiltrating cells, in turn, orchestratea series of remodeling events that culminate in reparative fibrosis. However,hepatic fibrosis can progress towards cirrhosis, an end stage condition, depending
on degrees of injury or type of injurious agents. Biliary fibrosis, a subtype of
hepatic fibrosis, is developed due to cholangiocyte injury such as enteric bacterial
infection and autoimmunity. The pathogenic mechanism of biliary fibrosis ispoorly understood. Animal models for biliary fibrosis should represent a series ofevents of fibrogenesis in the Glisson’s sheath, and may serve as a useful tool toclarify the underlying mechanisms, as well as to assess efficacy of anti-fibroticagents.
Basically, fibrosis is evoked through various functions of reactive cells
including inflammatory cells, vascular cells, and mesenchymal cells. Out of them,macrophages, which are divided into exudate macrophages, resident macrophages
and dendritic cells, have been implicated to play pivotal roles in hepatic fibrosis.
Macrophages are heterogeneous in nature, and their functions easily changedepending on microenvironmental conditions.
Soluble factors secreted bymacrophages are capable of inducing myofibroblast development.
Themyofibroblasts are so-called contractile cells expressing α-smooth muscle actin(α-SMA), and contribute to excessive deposition of collagens. Fibrotic lesions may
be developed via interactions between macrophages and myofibroblasts. In
hepatic fibrosis, hepatic stellate cells (HSCs) are considered to be the principal
progenitor of myofibroblasts; in addition to the origin, however, cellular propertiesof myofibroblasts in biliary fibrosis remain to be determined. Understanding theproperties of macrophages and myofibroblasts in biliary fibrosis may provide a newavenue in exploring agents for cell type specific therapeutic interventions.
A series of studies were conducted in order to investigate the properties ofmacrophages in relation to myofibroblast development in biliary fibrosis. InChapter 1, immunophenotypical analyses were performed to clarify the distributionof hepatic macrophages in developing rat livers. To understand the appearance ofmacrophages in spontaneous lesions, in Chapter 2, macrophages andmyofibroblasts in Fasciola-infected cattle liver with biliary cirrhosis werecharacterized with immunohistochemistry. Based on results in Chapters 1 and 2,
to more clarify the pathogenesis of biliary fibrosis, the author established the acute(Chapter 3) and chronic (Chapter 4) animal models for biliary fibrosis induced inrats by α-naphthylisothiocyanate (ANIT; hepatotoxicant causing cholangiocyteinjury); detailed immunophenotypical characteristics of macrophages andmyofibroblasts were analyzed to shed some light behind the pathogenesis. Finally,in Chapter 5, the author compared in macrophage and myofibroblast properties
between acute and chronic ANIT-induced biliary fibrosis.
Chapter 1: Hepatic Macrophages in Developing Rat Livers
Little is known about hepatic macrophages in fetuses and neonates of rats.
To establish baseline data, liver samples of F344 rats obtained on gestational days
18-20, neonate days 1-21, and adult weeks 5-35 were analyzed
immunohistochemically (n=2-5/examination point).
Macrophages positive forCD68 (ED1; implying phagocytic activity) were mostly predominant at prenatal
period, suggestive of ingestion of apoptotic cells; macrophages positive for CD163(ED2; relating to production of proinflammatory factors), CD204 (SRA-E5; relatingto lipid metabolism) and major histocompatibility complex (MHC) class II (MHC II)(OX6) appeared mainly in postnatal life. CD163+ and CD204+ cells were present
along the sinusoids, indicating Kupffer cells; cells expressing MHC II were limitedin the Glisson’s sheath, indicative of interstitial dendritic cells. In developing
livers, macrophages can express heterogeneous immunophenotypes, suggestingthat macrophages with divergent properties participate in liver development andstructure formation.
Chapter 2: Properties of Macrophages and Myofibroblasts in Fasciola-infected
Cattle Biliary Cirrhosis

To understand what types of macrophages can take part in spontaneous
biliary fibrogenesis, Fasciola-infected cattle livers (n=8) with biliary cirrhosis wereanalyzed using antibodies to CD68, CD163 (AM-3K), CD204 (SRA-E5) and Iba-1 (aprotein involved in calcium signaling, phagocytosis and chemotaxis) for
macrophages, and to α-SMA, vimentin and desmin for myofibroblasts. CD68+ andCD163+ cells were significantly increased in cirrhotic lesions. Myofibroblasts
expressed α-SMA, vimentin and desmin in varying degrees; the desmin expressing
cells were relatively fewer in number. These findings indicated that CD68+ andCD163+ macrophages could take part directly in biliary fibrogenesis, and thatmyofibroblasts expressed various cytoskeletons, mainly vimentin and α-SMA.
Chapter 3: Immunohistochemical Analyses of ANIT-induced Acute Cholangiocyte
Injury Lesions in rats
Section I: Immunophenotypes of Macrophages and MyofibroblastsTo establish an animal model for biliary fibrosis, cholangiocyte injury in theGlisson’s sheath was induced in F344 rats by a single intraperitoneal injection ofANIT (75 mg/kg). Liver samples were obtained on post injection (PI) days 0(control), 1, 2, 3, 5, 7, 9 and 12 (n=3/examination point). Macrophages reacting toMHC II consistently increased during the entire observation period. CD68+ andCD204+ cells appeared later than MHC II+ cells from PI day 2 with significant
increase; these macrophages decreased gradually at mid and late stages. CD163+cells increased transiently on PI day 3. There were double positive cells to MHC
II/CD68 or MHC II/CD163. Vimentin+ and desmin+ myofibroblasts appeared atearly and mid stages, and α-SMA+ myofibroblasts were developed at mid and latestages. Myofibroblasts co-expressing both vimentin and α-SMA were greater innumber than those expressing both desmin and α-SMA.
Monocyte
chemoattractant protein-1 (MCP-1) and transforming growth factor-β1 (TGF-β1)
mRNA increments appeared to be related to macrophage infiltration at early stages
and myofibroblastic differentiation at mid and late stages, respectively. Depending
on progression stages (cholangiocyte injury, biliary fibrosis and healing),
macrophages and myofibroblasts display various immunophenotypes; particularly,
it was found that MHC II+ cells might have central roles, and that α-SMA+
myofibroblasts could be formed in mid and late stages.
Section II: Regenerative Potential of Biliary Fibrosis-involving Cells
By using immunohistochemistry with antibodies to nestin (an intermediatefilament protein used as a marker for progenitor of nerve cells) and Ki-67 (a markerof proliferating cells), regenerative potential was investigated in injured cells of theGlisson’s sheath with biliary fibrosis.
Nestin expression was seen incholangiocytes, myofibroblasts and endothelial cells; some of these cells reacted toKi-67. After injury, cells of these types might have potential to regenerate as theirpossible progenitors. Nestin would be used as an indicator for regeneration in
hepatotoxicity.

Chapter 4: Immunophenotypes of Macrophages and Myofibroblasts in
ANIT-induced Chronic Biliary Fibrosis in rats
By repeated injections of ANIT (30 mg/kg for 5 weeks followed by 75 mg/kg
until 19 weeks, intraperitoneally, once weekly) for 19 weeks, slowly progressive
biliary fibrosis model, resembling human counterpart lesions, was established inF344 rats. Liver samples were examined at post-first injection (PFI) weeks 3, 7, 10,13, 16 and 19 (n=4/examination point). MHC II+ macrophages significantly
increased consistently during the entire observation.
CD68+ and CD204+
macrophages showed a significantly increased number from PFI weeks 7 to 19,
being later than MHC II+ cells. CD163+ macrophages significantly increased onlyat late stages (PFI weeks 13-19). Interestingly, there were double positive cells toMHC II/CD68 or MHC II/CD163, and the distribution of MHC II+ cells and CD204+
cells was limited in the affected Glisson’s sheath and the sinusoids of hepaticlobules, respectively. Vimentin+ and α-SMA+ myofibroblasts appeared at mid andlate stages, corresponding with increased TGF-β1 mRNA; however, double positive
cells to vimentin and α-SMA were few, indicating that there was difference innature between them. Desmin expression was very faint. This study showed thatmacrophages exhibited heterogeneous properties depending on stages andlocations; in particular, MHC II+ cells might play crucial roles in biliary fibrosis.
In association with such macrophage appearance, myofibroblasts expressing
cytoskeletons (particularly, vimentin and α-SMA at mid/late stages) might
participate in biliary fibrosis.
Chapter 5: Comparisons of Macrophage and Myofibroblast Properties between
Acute and Chronic Rat Biliary Fibrosis Models
The properties of macrophages and myofibroblasts were compared between
ANIT-induced acute and chronic biliary fibrosis models. In both models, MHC II+macrophages consistently increased throughout the entire experiment period,
followed by increments of CD204+ and CD68+ macrophages. CD163+ macrophages
increased transiently in acute ANIT model and at the late stages in chronic ANIT
model. These findings indicate that MHC II+ macrophages, which may bestimulated by cholangiocytes injured by ANIT, can induce macrophages reacting toCD204 and CD68, and participation of CD163+ macrophages is slight in acute ANIT
model and delay in chronic ANIT model. Vimentin+ and desmin+ myofibroblasts
increased at early and mid stages in acute ANIT model, whereas vimentin
expression was almost consistent and desmin expression was faint in chronic ANIT
model; α-SMA was expressed in myofibroblasts at mid and late stages in bothmodels. Vimentin+ portal fibroblasts and desmin+ HSCs in acute ANIT model, andvimentin+ portal fibroblasts in chronic ANIT model may be regarded as precursorsof α-SMA+ myofibroblasts. As compared with macrophage properties (exclusivelyCD68+ and CD163+ macrophages) in perivenular fibrosis model previously reported,
it was found that antigen presenting cells (MHC II+ macrophages) in the Glisson’s
sheath can regulate the progressive biliary fibrosis.

Conclusions
1. Heterogeneous macrophage populations participate in rat liver formation;
they show different distributions and functions, depending on age.
2. In Fasciola-infected cattle livers with cirrhosis as spontaneous lesions,
CD68+ (phagocytosis) and CD163+ (proinflammatory factor production)
macrophages are more predominant in fibrotic lesions; myofibroblasts
variously express α-SMA and vimentin.
3. In ANIT-induced acute and chronic biliary fibrosis, macrophages exhibit
heterogeneous properties (phagocytosis, proinflammatory factor production,
lipid metabolism, and MHC II expression), demonstrable withimmunohistochemistry, depending on stages of lesion progress in thedamaged Glisson’s sheath. Of them, MHC II+ macrophages may havecentral roles in the lesion development as the first responder.
4. Additionally, myofibroblasts in ANIT-induced lesions in the Glisson’s sheath
display various cytoskeletons such as vimentin, desmin and α-SMA; inbiliary fibrosis, desmin (although the reactivity is faint) tended to be
expressed at early stages, and vimentin and α-SMA at mid and late stages.
5. Nestin expression would be used as an indicator for regeneration in injured
cells in the Glisson’s sheath.
6. Because ANIT-induced lesions in the Glisson’s sheath resemble humanbiliary fibrosis, these lesions would be beneficial to clarify the underlying
mechanisms of biliary fibrosis, as well as to test efficacy of anti-fibrotic
agents as animal models.

審査結果の要旨
ウイルス感染、中毒、虚血などによる肝細胞傷害後に生じる線維化は、肝硬変へと進展し、重度の健康障害を招く。胆管傷害後の線維化も進行すれば肝硬変に至る。肝硬変は慢性的に生じる線維化で、その形成には、反応するマクロファージとコラーゲンなどの細胞外基質を産生する筋線維芽細胞が中心的な役割を担う。しかし、線維化の病理発生機序の全貌は不明な点が多い。マクロファージは貪食活性、炎症性因子の産生、酸化ストレス因子の産生、脂質代謝、抗原提示能など多彩な特性を現す細胞群であり、筋線維芽細胞は α-平滑筋アクチン（α-SMA）などの細胞骨格を種々の程度で発現する拘縮性細胞で、常在する線維芽細胞に由来する。しかし、胆管線維化に出現するマクロファージの機能的性状や筋線維芽細胞の細胞特性は未だ明らかにされていない。これら細胞の基本的な性状を解明し、相互の関連性を追究することは、肝硬変を治療する方策に繋がる。本研究では、ラットの肝発生過程におけるマクロファージの特性と、胆管上皮を特異的に傷害するα-naphthylisothiocyanate （ANIT）をラットに投与することで作出した

急性と慢性の胆管線維化モデルを用いて傷害されたグリソン鞘に出現するマクロファージと筋線維芽細胞の特性を、主として免疫組織化学的に解析している。得られた成績の概要は以下の通りである。
第 1 章では、発生過程（胎子、新生子、成体）の肝における正常なマクロファージの特性を解析している。その結果、胎子では貪食活性の高い CD68 発現マクロファージが、新生子から成体では抗原提示（MHC クラス II 発現）マクロファージが主に出現すること、また肝常在マクロファージである CD163 発現クッパー細胞は生後に現れ始め、肝組織構築に係わるとともに脂質活性と関連する CD204 を発現することが分かった。さらに、MHC II 発現マクロファージはグリソン鞘に、CD163 と CD204 発現マクロファー
ジは類洞に沿って出現することを示した。肝発生過程で出現するマクロファージ機能は発生時期に依存して異なること、そして部位特異的なマクロファージが存在することを明らかにした。
第 2 章では、ウシの肝蛭感染による胆管線維症の病態を解析し、線維化部位では CD68と CD163 発現マクロファージが増加し、それに伴いビメンチンとα-SMA 陽性の筋線維芽細胞が形成されることを示した。寄生虫感染による胆管線維化に出現するマクロファージと筋線維芽細胞の特性を初めて明らかにした。
第 3 章では、ANIT の単回投与による胆管傷害後の急性の線維化モデルを作出し、12日間に亘り病態の推移を解析している。その結果、MHC II 発現マクロファージは初期から後期まで常に増加し、それに続いて CD68 と CD204 発現マクロファージがやや遅れて増し始めることを示した。なお、CD163 発現マクロファージは一過性に増加したのみであった。筋線維芽細胞は、前半はビメンチンとデスミンを、後半からはα-SMA を発現することが示された。さらに、MCP-1 はマクロファージの出現と、TGF-β1 はα-SMA陽性の筋線維芽細胞の形成と関連することを明らかにした。また、傷害後に再生する胆管上皮と筋線維芽細胞などの間質細胞がネスチンを発現することを示した。
第 4 章では、ANIT を 19 週間に亘って投与し、形成されるグリソン鞘の線維化病変を解析している。その結果、MHC II 発現マクロファージは観察期間を通じ常に高頻度で出現し、CD68 と CD204 発現マクロファージがそれに続いて増加し、CD163 発現マクロファージは後期のみに出現することが示された。また、デスミン陽性の筋線維芽細胞の出現は少なく、ビメンチンとα-SMA 陽性の筋線維芽細胞が線維化病変の形成に深く係わることを示した。
第 5 章では、ANIT 誘発の急性と慢性の胆管線維化病変とすでに報告されている肝細胞傷害後の実質線維化に出現するマクロファージの特性を比較し、ANIT 誘発の胆管線維化では MHC II 発現マクロファージが中心的な役割を演じるのに対し、実質線維化では CD68 と CD163 発現マクロファージが主体となることを示した。また、α-SMA 陽性の筋線維芽細胞が胆管線維化の進行に深く係わることを明らかにした。

以上の研究により、胆管線維化過程において、病変形成に伴い異なる機能のマクロファージが出現すること、さらにα-SMA 発現の筋線維芽細胞が線維化の進行において重要な役割を演じることが明らかとなった。本研究は、肝硬変、特に胆管線維化に起因する肝硬変の病理発生機序を解明する上で極めて重要な知見を提示しており、医学・獣医学の基礎・応用研究の更なる発展に資するものである。従って、最終試験の結果と併せて、博士（獣医学）の学位を授与することを適当と認める。