Macsween's Pathology of the Liver: Expert Consult: Online and Print

MacSween’s

Pathology of the Liver

Commissioning Editor: Michael Houston Development Editor: Martin Mellor Project Manager: Anita Somaroutu Design: Charles Gray Illustration Manager: Merlyn Harvey Illustrators: Oxford Illustrators, Ethan Danielson Marketing Manager: Gaynor Jones

MacSween’s

Pathology of theLiver SIXTH EDITION

Alastair D. Burt

BSc MD FRCPath FRCP FSB

Professor of Pathology and Dean of Clinical Medicine Newcastle University; Honorary Consultant Histopathologist Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, UK Bernard C. Portmann

Linda D. Ferrell

MD

Professor of Pathology Vice-Chair of Anatomic Pathology Director of Surgical Pathology Department of Anatomic Pathology University of California, San Francisco San Francisco, CA, USA

MD FRCPath

Professor Emeritus, King’s College London, School of Medicine; Former Lead Consultant Histopathologist, Institute of Liver Studies, King’s College Hospital NHS Foundation Trust; Consultant Histopathologist, The London Clinic London, UK

For additional online content visit expertconsult.com

Edinburgh  London  New York  Oxford  Philadelphia  St Louis  Sydney  Toronto  2012

An imprint of Elsevier Limited © 2012, Elsevier Limited. All rights reserved. First edition 1979 Second edition 1987 Third edition 1994 Fourth edition 2002 Fifth edition 2007 The right of A.D. Burt, B.C. Portmann and L.D. Ferrell to be identified as authors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). The chapter entitled ‘Hepatic injury due to drugs, herbal compounds, chemicals and toxins’ is in the public domain. Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-7020-3398-8 Printed in China Last digit is the print number:  9  8  7  6  5  4  3  2  1

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List of contributors Jorge Albores-Saavedra

MD

Consultant and Researcher Department of Pathology Instituto Nacional de Ciencias Medicas y Nutricion ‘Salvador Zubiran’ Mexico City, Mexico Arturo Angeles-Angeles

MD

Chief Department of Pathology Instituto Nacional de Ciencias Medicas y Nutricion ‘Salvador Zubiran’ Mexico City, Mexico Pierre Bedossa

MD PhD

Professor of Pathology; Head of Department of Pathology Beaujon Hospital and University of Paris-Denis Diderot Paris, France Henry C. Bodenheimer Jr

MD

Chairman Department of Medicine; Professor of Medicine Albert Einstein College of Medicine Beth Israel Medical Center New York, NY, USA Elizabeth M. Brunt

MD

Professor of Pathology and Immunology Washington University School of Medicine St Louis, MO, USA Alastair D. Burt

BSc MD FRCPath FRCP FSB

Professor of Pathology and Dean of Clinical Medicine Newcastle University Honorary Consultant Histopathologist Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle, UK

Andrew D. Clouston

MBBS PhD FRCPA

Associate Professor of Molecular and Cellular Pathology University of Queensland; Managing Partner Envoi Specialist Pathologists; Visiting Consultant Royal Brisbane and Women’s Hospital Brisbane, Queensland, Australia James M. Crawford

MD PhD

Professor and Chair Department of Pathology and Laboratory Medicine Hofstra North Shore-LIJ School of Medicine Senior Vice President of Laboratory Services North Shore-Long Island Jewish Health System Manhasset, NY, USA Linda D. Ferrell

MD

Professor of Pathology; Vice-Chair of Anatomic Pathology; Director of Surgical Pathology Department of Anatomic Pathology University of California, San Francisco San Francisco, CA, USA Zachary D. Goodman

MD PhD

Director Hepatic Pathology Research Center for Liver Diseases Inova Fairfax Hospital Falls Church, VA, USA Weei-Yuarn Huang

MD PhD FRCPC

Assistant Professor Department of Pathology, Dalhousie University Staff Pathologist, QEII Health Science Center Halifax, Canada

vii

List of contributors

Stefan G. Hübscher

MBChB FRCPath FSB

MD PhD

Professor of Medicine; Director, Division of Internal Medicine 2 and Center for Hemochromatosis University Hospital of Modena Modena, Italy

David E. Kleiner

Bernard C. Portmann

MD PhD

Director, Clinical Operations Laboratory of Pathology National Cancer Institute Bethesda, MD, USA James H. Lewis

MD FACP FACG AGAF

Professor of Medicine Division of Gastroenterology; Director of Hepatology Georgetown University Medical Center Washington, DC, USA Sebastian B. Lucas

Michael P. Manns

Alberto Quaglia

MD, PhD, FRCPath

Lead Consultant Histopathologist Institute of Liver Studies King’s College Hospital NHS Foundation Trust; London, UK Eve A. Roberts

MD MA FRCPC

Adjunct Professor of Paediatrics, Medicine and Pharmacology University of Toronto Division of Gastroenterology, Hepatology and Nutrition The Hospital for Sick Children Toronto, ON, Canada

MD

Professor and Chairman Department of Gastroenterology, Hepatology and Endocrinology Medical School of Hannover Hannover, Germany Yasuni Nakanuma

MD FRCPath

Professor Emeritus King’s College London, School of Medicine; Former Lead Consultant Histopathologist Institute of Liver Studies King’s College Hospital NHS Foundation Trust; London, UK

FRCP FRCPath

Professor of Clinical Histopathology King’s College London School of Medicine Department of Histopathology St Thomas’ Hospital London, UK

Luigi M. Terracciano

MD

Professor of Pathology Head of Molecular Pathology Division Institute of Pathology University Hospital Basel, Switzerland

MD PhD

Professor and Chairman Department of Human Pathology Kanazawa University Graduate School of Medicine Kanazawa, Japan Brent A. Neuschwander-Tetri

MD

Professor of Internal Medicine; Director, Division of Gastroenterology and Hepatology Saint Louis University St Louis, MO, USA

Neil D. Theise

MD

Professor of Pathology; Professor of Medicine (Division of Digestive Diseases) Beth Israel Medical Center, Albert Einstein College of Medicine New York, NY, USA Richard J. Thompson

BA BM BCh MRCP MRCPCH

Professor of Pathology Pathology Department Beaujon Hospital, Clichy, France

Senior Lecturer in Paediatric Hepatology; Honorary Consultant Institute of Liver Studies Division of Transplant Immunology and Mucosal Biology King’s College London School of Medicine at King’s College Hospital London, UK

Alan C. Paterson

Ian R. Wanless

Valerie Paradis

MD PhD

MB BCh PhD FCPath(SA)

Professor of Pathology Consultant in Liver and Gastrointestinal Pathology Ampath Trust Johannesburg, South Africa

viii

Antonello Pietrangelo

Leith Professor and Professor of Hepatic Pathology University of Birmingham; Consultant Histopathologist, University Hospital Birmingham NHS Foundation Trust, Birmingham, UK

MD CM FRCPC

Professor of Pathology Dalhousie University Halifax, Nova Scotia, Canada

List of contributors

M. Kay Washington

MD PhD

Professor of Pathology Vanderbilt University Medical Center Nashville, TN, USA Aileen Wee

Sherif R. Zaki

MD PhD

Chief Infectious Diseases Pathology Branch Centers for Disease Control and Prevention Atlanta, GA, USA

MB BS FRCPath FRCPA

Professor and Senior Consultant Department of Pathology Yong Loo Lin School of Medicine, National University of Singapore National University Hospital National University Health System Singapore

Yoh Zen

MD PhD FRCPath

Consultant Histopathologist Institute of Liver Studies King’s College Hospital NHS Foundation Trust; London, UK

ix

Preface Liver disease continues to be a major cause of morbidity and mortality worldwide. There are nevertheless, epidemiological changes. The effects of mass immunization against Hepatitis B are beginning to be seen in some parts of the world and the development of more effective antiviral agents are influencing the clinical expression of Hepatitis C infection. In parallel however, there has been an almost exponential growth in the prevalence of fatty liver disease, much of which can be attributed to the progressive rise in obesity levels. In some countries, there has been a decline in alcohol-induced fatty liver disease while in others, including the UK, there has been an increase. In terms of the diagnosis of liver disease there have been significant developments since the fifth edition of MacSween’s Pathology of the Liver. Non-invasive imaging of the liver has continued to develop at a phenomenal pace and we have seen the introduction of techniques such as transient elastography for the assessment of fibrosis. Furthermore, surrogate markers of liver fibrosis have been developed by a number of groups and there are algorithms utilizing serum levels of matrix proteins and metalloproteinases. While these are seen to be supplementary diagnostic modalities, none has replaced liver biopsy as the ‘gold standard’. We have continued to see substantial increases in our understanding of morphological changes across a whole range of liver diseases, and in particular since the fifth edition, there have been very significant advances in the interpretation of drug-induced liver injury; tumour precursor lesions; benign hepatic neoplasms; variant malignant tumours; and the interpretation of patterns of fibrosis. In addition, new anti­bodies have been developed and made commercially available to refine the diagnosis of tumours and metabolic disorders. We felt it was timely therefore for a further edition, drawing on the expertise of many of the top hepatopathologists from around the globe. In this sixth edition, we have made some further changes to the structure of the book. Chapter One now combines an outline of structure and function of the normal liver with an overview of the basic pathological processes that can be seen in injury. This chapter draws on the introductory chapters from previous editions and we are indebted to the authors of these earlier chapters and in particular to the input of Professors Valeer Desmet and Tania Roskams. Chapter Two was designed to take a fresh look at technical aspects of liver pathology, introducing some ‘new kids on the block’. Some of these approaches enhance existing morphological techniques, while some are likely to be complementary in the diagnostic arena. The editors took the decision in this edition to separate out disorders of iron overload from all other genetic and metabolic

diseases, given its importance. We are delighted to have the input of Antonello Pietrangelo, arguably the most experienced scientist in this field internationally. Here we have adopted a more fundamental approach to classification than that which is included in the fifth edition. We also took the decision to combine the coverage of alcoholic and non-alcoholic fatty liver disease. While there are some differences morphologically, there are more similarities and overlap, both in histological features and in underlying mechanisms. It is with great sadness that since the publication of the fifth edition, Pauline Hall passed away. Pauline had contributed to successive editions with outstanding chapters on alcoholic liver disease and Chapter Six is very heavily influenced by her thoughts on this disorder and the many contributions she made to the field. In the chapter including non-viral infectious diseases, we are delighted to have the additional input of Sherif Zaki, who has outstanding expertise and experience in unusual infectious diseases in the liver. In the current edition, we also welcome the combined strengths of Kay Washington and Michael Manns with their excellent overview of autoimmune hepatitis. Ian Wanless has done much to highlight the importance of vascular events in liver disease, and we are delighted to include an extended coverage of this and the importance of parenchymal extinction lesions in the development of cirrhosis in his chapter. The chapter on liver tumours has been written to be consistent with the recently published WHO classification. It considers our evolving understanding of precursor lesions and novel approaches to subtyping hepatocellular adenomas. We have expanded this chapter to ensure that it is as comprehensive as possible. The editors were also aware of the importance of cytopathology in the diagnosis of liver tumours particularly in Asian centres and we are delighted to have enlisted the help of Aileen Wee as a contributor to this chapter. All three of us continue to feel honoured to be responsible for this textbook and once again wish to pay tribute to the man behind the very first edition: Sir Roddy MacSween, whom we all continue to look upon as a great friend and mentor. We have all enjoyed working with our contributors in developing this sixth edition, which we hope will continue to influence the practice of patho­ logists and clinicians involved in the care of patients with liver disease. It has once again also been a pleasure to collaborate with the publishing teams in Elsevier. Alastair D. Burt Bernard C. Portmann Linda D. Ferrell

Newcastle London San Francisco

xi

Acknowledgements Acknowledgements of illustrations from previous publications or of modification to illustrations and diagrams and acknowledgement of original photographic material and microscopic material is appropriately made in each chapter and figure legends. We would like to take the opportunity to thank our administrative assistants who have helped with the preparation of manuscripts and figures and who have contributed to the chores of proofreading, and in particular we are grateful for the help of Emma Reynolds

(Newcastle), Elisabeth Portmann (London) and Caren Hale (San Francisco). As always, preparation of a book this size and type is a time-consuming business, and we would like to thank our respective families for their patience and support during the more fraught times in the development of this sixth edition. We would like to thank our publishers and typesetters for their help and encouragement at all times and in particular Martin Mellor, Michael Houston and Anita Somaroutu.

xiii

Anatomy, pathophysiology and basic mechanisms of disease James M. Crawford and Alastair D. Burt

CHAPTER CONTENTS Introduction Development of the liver General features Vascular arrangements Hepatocytes and sinusoidal cells The bile duct system Haemopoiesis Molecular control of liver development Macroanatomy of the liver Microanatomy of the liver Anatomy of the hepatic microcirculation Regulation of the hepatic microcirculation Functional heterogeneity in the liver Ultrastructural anatomy of the hepatocyte The hepatic sinusoid and the sinusoidal cells The biliary system Other constituent tissues General concepts of liver injury and repair Inflammation The innate immune system The adaptive immune system Recruitment and influx of inflammatory cells Inflammation of portal tracts Granulomas Hepatocellular injury Ballooning degeneration

©2012 Elsevier Ltd DOI: 10.1016/B978-0-7020-3398-8.00001-5

2 2 2 2 4 4 5 5 6 7 8 8 12 13 19 23 26 30 31 32 33 34 34 36 36 37

1 

Steatosis Cholestasis Mallory–Denk bodies Cell death Apoptosis Necrosis Regeneration Regeneration of mature liver cells The role of progenitor cells Fibrosis Hepatic stellate cells Portal tract fibrogenesis Epithelial–mesenchymal transition Bone marrow-derived myofibroblast precursors Regulation of fibrogenesis Vascular remodelling Endothelial porosity Vascular thrombosis Sinusoidal blood flow Angiogenesis, arterial and venous changes Zonation Cirrhosis Parenchymal extinction Reversibility of fibrosis/cirrhosis The liver in biopsy and autopsy specimens

37 38 41 42 42 44 45 45 48 49 50 51 51 51 52 54 54 54 54 56 56 57 58 58 59

1

MacSween’s Pathology of the Liver

Introduction The practice of hepatopathology requires a clear understanding of liver anatomy and physiology, as a prelude to understanding the expression of pathological processes in the liver. As an anatomical entity, the liver is deceptively simple. It is large, representing about 2% of the total body mass of an adult human and occupying most of the right upper quadrant of the abdomen. It has a roughly triangular profile, with incomplete clefts helping to define the different ‘lobes’ of the liver. It has only one point of vascular inflow, the porta hepatis. Blood exits through several venous orifices into the inferior vena cava, which traverses a deep groove in the dorsum of the liver. There are no ‘moving parts’ of the liver, with the exception of daily secretion of several litres of bile into the common hepatic duct which exits from the porta hepatis. Belying its macroscopic simplicity, the liver is home to biosynthetic and biodegradative metabolic pathways of unequalled complexity, generating enough metabolic heat to be a prime source of core homeostatic temperature maintenance. This chapter considers the embryology, macroanatomy and microanatomy of the liver and its basic response to injury. Finally, we consider the appearance of ‘normal’ liver in biopsy and autopsy specimens.

Development of the liver General features In human embryos, the liver first appears at the end of the 3rd week of development. The liver bud or hepatic diverticulum arises as a hollow midline outgrowth of endodermal tissue from the ventral wall of the future duodenum. The connective tissue framework of the liver into which the endodermal bud grows is of mesenchymal origin, and develops from two sources: (1) the septum transversum, a transverse sheet of mesenchymal cells which incompletely separates the pericardial and peritoneal cavities, and (2) cells derived from the mesenchymal lining of the associated coelomic cavity, which actively invade the septum transversum. The confluence of endodermal cells from the hepatic diverticulum growing into host mesenchyme creates the solid organ destined to become the liver (Fig. 1.1).

Hepatic trabeculae

Heart Liver

Forebrain

Figure 1.1  Haematoxylin and eosin-stained sagittal section of a human embryo at approx. 32 days of development (Carnegie Stage 13). (Courtesy of S Lindsay, MRC/Wellcome Trust Human Developmental Biology Resource)

2

During the ensuing 4th week, buds of epithelial cells extend radially from the hepatic diverticulum into the mesenchymal stroma. Between the epithelial cords, a plexus of vascular hepatic sinusoids develops. As the epithelial buds grow into the septum transversum, they break up into thick anastomosing epithelial sheets which meet and enmesh vessels of the hepatic sinusoidal plexus, forming the primitive hepatic sinusoids (Fig. 1.2). The intimate relation between hepatocytes and sinusoidal capillaries, so characteristic of the adult organ, is therefore, already anticipated in the 4-week-old embryo (Fig. 1.2). The hepatic diverticulum remains as a tether between the developing liver and the duodenum, ultimately becoming the extra­ hepatic biliary tree. The caudal part of this tethering diverticulum forms a secondary bud, constituting the epithelial primordium of the cystic duct and gallbladder. Once established, the liver grows rapidly, to become the largest single visceral organ (by mass) for the remainder of gestation. It bulges into the peritoneal cavity on each side of the midline, as right and left lobes, which are initially symmetrical. It also grows ventrally and caudally into the mesenchyme of the anterior abdo­ minal wall, extending down to the umbilical ring. Associated with these changes, the stomach and duodenum, which were initially in broad contact with the septum transversum, draw away from it, thus producing a midsagittal sheet of mesoderm, the ventral meso­ gastrium or future lesser omentum. As the duodenum withdraws from the septum transversum, the stalk of the original hepatic diverticulum is also drawn out to form, within the lesser omentum, the epithelial elements of the extrahepatic bile ducts. Simultaneously, the liver becomes partly freed from its originally broad contact with the septum transversum by extensions of the peritoneal cavity and its visceral and parietal mesothelial surfaces so that, in the adult, direct contact of the liver with the diaphragm persists only as the ‘bare area’ of the liver. This is bounded by the attachments of peritoneal reflexions, which form the coronary and falciform ligaments.

Vascular arrangements Once formed, the fetal liver parenchyma consists of anastomosing sheets of liver cells, each sheet being several cells in thickness and forming a ‘muralium multiplex’.1 Coursing between the liver cells is the vascular ‘sinusoidal plexus’. Initially, the afferent hepatic blood supply is via the symmetrically arranged vitelline veins returning from the abdominal region of the embryo. Blood is also received from the laterally placed right and left umbilical veins, which run in the body wall and carry oxygenated blood from the placenta directly to the paired horns of the sinus venosus of the cardiac primordium. Both the vitelline and umbilical sources of blood enter into the hepatic sinusoidal plexus. Blood draining from the sinusoidal plexus passes through symmetrical right and left hepato-cardiac channels, to enter the sinus venosus (Fig. 1.2).2 Once these vascular connections are made (in embryos of 5 mm, 5th week), the circulatory pattern within the liver changes rapidly. The left umbilical vein becomes the principal source of blood entering the liver, partly because it comes to carry all the blood returning from the placenta when the right umbilical vein withers and disappears (generating the ‘double artery : single vein’ umbilical cord of the term fetus), and partly because the initial volume of blood returning from the gut in the vitelline veins is small. The definitive vascular pattern of the fetal liver, already established in embryos of the 7th week (about 17 mm long), is shown in Figure 1.2.3 The originally paired vitelline veins have given way to a single portal vein which, on entering the liver, divides into right and left branches. Blood in the left umbilical vein traverses a venous extension in the falciform ligament and has a choice of three routes: (1) through the

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Mesenchymal lining of coelomic tract Left hepatocardiac channel Left umbilical vein

Common cardinal vein Sinus venosus Right hepato-cardiac channel Left umbilical vein

Hepatic sinusoidal plexus Liver

Vitelline veins

Septum transversum

A

B Gut

Sinus venosus Right umbilical vein

Inferior vena cava Right hepatic vein Hepatic sinusoidal plexus

Middle hepatic vein Left hepatic vein Ductus venosus (Dia. = 200µm)

Left umbilical vein

Origin of hepatic bud

Portal vein (Dia. = 100µm)

Left umbilical vein (Dia. = 600µm)

Right and left vitelline veins C

D

Figure 1.2  (A) Section through region of the hepatic bud in a human embryo of 25 somites (about 26 days). (Redrawn from Streeter2) (B) Vascular channels associated with the developing liver, in a human embryo of 30 somites. (Redrawn from Streeter2) (C) Vascular channels in the human liver at a slightly later stage, showing the further extensive development of the hepatic sinusoidal plexus. (Based on Streeter2) (D) Scheme of the portal hepatic circulation, in a human embryo of 17 mm (about 7 weeks). (Redrawn from Lassau and Bastian3)

liver in branches which enter the sinusoidal plexus of the left half of the liver; (2) through the sinusoidal plexus of the right half of the liver, by retrograde flow through its connection with the left branch of the portal vein; (3) through the ductus venosus traversing the short space between the porta hepatis to the inferior vena cava, to enter directly into the systemic venous circulation. By these routes, the converging bloodstreams from the definitive portal vein and indirectly from the umbilical vein enter the rapidly enlarging hepatic primordium through the porta hepatis. Intrahepatic mesenchyme condenses around the intrahepatic branching portal venous system, making up the ramifying portal tracts of the liver. The hepatic artery is derived from the celiac axis. Arterial sprouts grow into the hepatic primordium from the porta hepatis along the portal tracts, spreading to the periphery as the fetal liver enlarges. These arterial sprouts appear to be the organizing element for formation of the intrahepatic biliary tree, discussed below. The hepatic arterial system continues to proliferate and grow after birth,

reaching an adult form only at 15 years of age.4–7 In the adult, approximately four arteries supply the largest intrahepatic bile ducts.8 At the level of the terminal portal tracts there is a uniform 1 : 1 pairing of hepatic arteries and terminal bile ducts, and approximately two artery : bile duct pairs per single portal vein.9 The most terminal portions of the portal tree lose their portal veins, leaving only residual hepatic artery : bile duct dyads, which themselves disappear into the parenchyma.9,10 To complete this discussion, the rapid changes in hepatic circulation at the time of birth must be considered. A sphincteric mechanism closes the ductus venosus at its proximal end, blood flow ceases in the umbilical vein, and the left side of the liver receives blood which now flows from right to left through the left branch of the portal vein. The closed segment of the umbilical vein between the umbilicus and the liver regresses to form the ligamentum teres; the ductus venosus undergoes fibrosis and becomes the ligamentum venosum.

3

MacSween’s Pathology of the Liver

Hepatocytes and sinusoidal cells Primitive hepatocytes are derived exclusively from the endodermal outgrowths of the hepatic diverticulum. Hepatocellular synthesis of α-fetoprotein begins at the earliest stage of liver differentiation, some 25–30 days after conception, and continues until birth. Glycogen granules are present in fetal hepatocytes at 8 weeks; the maximal glycogen reserve is achieved at birth, but the rapid onset of glycogenolysis over 2–3 days postpartum depletes the storage to approximately 10% of term levels. Hepatocellular haemosiderin deposits appear early in development, become more marked as hepatic haemopoiesis decreases (see below), predominantly in periportal hepatocytes; these are also the storage sites for copper. Hepatocellular bile acid synthesis begins at about 5–9 weeks and bile secretion at about 12 weeks.11 Canalicular transport and hepatic excretory function, however, are still immature at birth and for 4–6 weeks postpartum and, therefore, exchange of biliary solutes across the placenta (especially bilirubin) is important in the fetus. Within the sinusoids, endothelial cells, Kupffer cells and hepatic stellate cells appear at 10–12 weeks.12

A

The bile duct system The extrahepatic and intrahepatic biliary system is best understood if the liver is regarded as an exocrine gland. The hepatic bud gives rise not only to the epithelial parenchyma – the future hepatocytes – but also to the epithelial lining of the branching bile duct system. The extrahepatic bile ducts, gallbladder and cystic duct are derived from the caudal portion of the hepatic diverticulum, the portion that does not invade the septum transversum but remains as a stalk connecting the foregut to the developing liver.13 A derivative diverticulum becomes the gallbladder. The epithelial lining of the extrahepatic bile ducts is continuous at its caudal end with the duodenal epithelium and at the cephalic end with the primitive hepatic sheets. The intrahepatic ducts develop from the limiting plate of hepatoblasts, which surround the branches of the portal vein. This has been known since the 1920s,14 but was confirmed using immunohistochemical methods and monoclonal antibodies to (cyto) keratins and cell surface markers.15–17 Normal adult hepatocytes express K8 and K18, whereas intrahepatic bile ducts, in addition, express K7 and K19. During the first 7–8 weeks of embryonic development, no intrahepatic bile ducts are evident and the epithelial cells express K8, K18 and K19. At about 9–10 weeks (27–30 mm embryos) pri­ mitive hepatocytes (hepatoblasts) surrounding large portal vein branches near the liver hilum express these keratins more intensely and form a layer of cells (Fig. 1.3), which ensheaths the mesenchyme of the primitive portal tracts to form the so-called ductal plate.14 This is followed by a second but discontinuous layer of epithelial cells which show a similar phenotypic change and so a segmentally double-layered plate is formed. The liver cells which do not form ductal plates lose K19 expression. From 12 weeks onwards, a lumen develops in segments of the ductal plates forming double-layered cylindrical or tubular structures (Fig. 1.3). Further remodelling of the plate occurs; invading connective tissue separates it from the liver parenchyma and the tubular structures become incorporated into the mesenchyme surrounding the portal vein branches. Such incorporation of tubules is always preceded by development of a branch of the hepatic artery,18 suggesting that the hepatic artery plays a key role in inducing formation of tubular structures in the ductal plate, the precursors of mature terminal bile ducts.13 This concept also gives explanation for the pairing of hepatic arteries and terminal bile ducts observed in the adult human

4

B Figure 1.3  Development of the ductal plate and of intrahepatic bile ducts. (A) Increased expression of keratins in primitive hepatocytes at the interface with the mesenchyme of the primitive portal tracts; human fetus of 12 weeks’ gestation. (B) Later stage showing a discontinuous double-layered plate of epithelial cells at the mesenchymal interface; note the formation of tubular structures (upper right) within this plate. Human fetus of 14 weeks’ gestation (immunoperoxidase staining); antibody (5D3) to low molecular weight keratins.

liver.9 An anastomosing network of bile ducts is formed, excess ductal epithelium undergoes resorption and bile ducts appear within the definitive portal tracts. Weak immunoreactivity for K7 is present in large bile ducts from about 20 weeks’ gestation. As the terminal bile ducts mature within the portal tract mesenchyme, epithelial tethers remain as a connection with the hepatic parenchyma.13 While traversing the portal tract mesenchyme, these structures are bile ductules, circumferentially lined by bile duct epithelial cells (cholangiocytes) derived from the ductal plate. As these channels impale the parenchymal interface, they become hemilunar, with half of the circumference as bile duct epithelial cells, and the other half as hepatocytes. These are the canals of Hering, which may penetrate the parenchyma for a distance of up to a third of the zonal distance to the terminal hepatic vein, or may skirt the portal tract interface as a residual short canal.19 The canals of Hering are thought to harbour resident stem cells throughout life, serving as a source for a robust proliferative response following liver injury (Fig. 1.4).20 Lastly, bile canaliculi between hepatocytes are first seen in human embryos of the 6th week, long before bile production begins at 12

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Portal vein Hepatic artery

Portal tract

Parenchyme Canal of Hering

Intralobular (variably present)

Bile ductule

Intraportal Terminal bile duct Figure 1.4  Schematic diagram of relationship between bile ducts, ductules and canals of Hering. (Based on Roskams et  al. 200421)

Lastly, a key anatomical term must be noted: the limiting plate. Referring back to embryological development, once the ductal plate has involuted, only canals of Hering remain at the portal tract : parenchymal interface as elements containing bile duct epithelial cells. The remainder of the interface is rimmed by mature hepatocytes, directly abutting the portal tract mesenchyme and representing the limiting plate. When liver injury occurs at the interface, involving destruction of hepatocytes and influx of inflammatory cells, the limiting plate is compromised or destroyed; this process is termed interface hepatitis. The canal of Hering : bile ductular compartment proliferates in response, giving rise to the aforementioned ductular reaction. These concepts, and terminology, are now well-entrenched in usage.21,27

Haemopoiesis weeks. They develop from membrane foldings between junctional complexes, and appear as intercellular spaces within sheets of presumptive hepatocytes, thereby constituting an ‘apical’ luminal channel between hepatocytes. The bile canaliculi drain centripetally from the perivenous zonal region towards the periportal zone, discharging their fluid into the hemilunar canals of Hering where the hepatocyte : bile duct epithelial cell connection occurs.13 At the level of hepatocytes, and in addition to the fluid pressure generated by active secretion of biliary solutes and fluid,22 contraction of the hepatocellular pericanalicular actin network provides a contractile mechanical force for propulsion of newly-formed bile downstream.23 The entire process of intrahepatic bile duct development progresses centrifugally from the porta hepatis and also from the larger to the smaller portal tracts. However, this process may not be complete at 40 weeks’ gestation and full expression of K7 is not found until about 1 month postpartum. Thus, the intrahepatic bile duct system is still immature at birth.13 Indeed, the liver doubles in size in the first year of life and continues to grow incrementally until adulthood, arriving at a mass 10 times that at birth. Hence, formation of the intrahepatic biliary tree is not fully complete until many years after birth.13 Failure of remodelling and resorption during fetal liver development produces the ‘ductal plate malformation’,24,25 which is significant in the production of various congenital malformations of the intrahepatic biliary tree. Failure of remodelling has been observed in HNF6 and HNF1β knockout mice, indicating that these transcription factors in tandem play a role in normal remodelling of the ductal plate (see below).16 Furthermore, injury to or destruction of the ductal plate in utero may be a factor in the development of intrahepatic biliary atresia (see Chapter 3).24 Despite their common ancestry, hepatocytes and ductal epithelium have been considered as distinct cell types and the epithelium of the terminal twigs of the biliary tree – the canals of Hering – includes typical hepatocytes and typical ductal cells, but no cells intermediate between the two.21 However, a change in differentiation from hepatocytes to bile duct cells – ductular reaction – occurs in liver injury of various aetiologies and, in particular, contributes to the morphological changes seen in cholestatic liver disease.21 Immunohistochemical investigations have shown that such ‘metaplasia’ is characterized by a phenotypic change, in which hepatocytes express K7 and K19 which, in the normal liver, are restricted to bile duct cells.21,26 This topic is complicated by the fact that such ‘transdifferentiating cells’ cannot be distinguished from progenitor cell progeny differentiating towards hepatocytes, resulting in ‘transitional cells’ expressing both cholangiocellular (K7,19) and hepatocellular (K8,18) keratins.

Hepatic haemopoiesis is a feature of the embryonic and fetal liver of mammals, including man. It begins at about 6 weeks (10  mm), when foci of haemopoietic cells appear extravascularly alongside the sheets of hepatocytes. By the 12th week, the liver is the main site of haemopoiesis, having superseded the yolk sac. Hepatic haemopoietic activity begins to subside in the 5th month of gestation, when the bone marrow becomes haemopoietic, and has normally ceased within a few weeks after birth. Parenchymal haematopoiesis is largely erythropoietic; haematopoiesis within portal tracts tends more towards to granulocytes, megakaryocytes and monocytes.

Molecular control of liver development As noted above, the first morphological indication of development of the liver is an endodermal proliferation in the ventral part of the foregut, just cranial to its opening into the yolk sac, at the 18th post-fertilization day in the human embryo. Tissue culture and transplantation experiments have shown that endodermal cells require two inductive interactions to form cells that express hepatocyte morphology (‘hepatic specification’). Prehepatic endoderm is induced first by cardiac mesenchyme via fibroblast growth factor (FGF) signalling, giving rise to proliferation of endodermal cells.28 These cells then interact with the mesenchyme of the septum transversum and subsequently differentiate into hepatocyte precursors.29 Many of the molecules and receptors involved in regulation of the hepatoblasts and subsequent hepatocyte and cholangiolar differentiation have now been identified.29,30 It has become increasingly apparent that cellular interactions with non-parenchymal cells play a key role in early hepatic development.30–32 The sheets of hepatoblasts that invade the septum transversum in the developing mouse liver express the transcription factor hepatocyte nuclear factor (HNF) 4α, while the surrounding mesenchyme expresses GATA4;30 the migratory properties of the hepatoblasts appear to require a homeobox gene Prox1. GATA6 also appears to be essential in the formation of the early liver bud, as do FoxA1 and FoxA2 (under the induction of HNF1β).33–35 Some of these factors such as GATA4 appear to be important in early stimulation of hepatocytespecific gene expression including α-fetoprotein, transthyretin and albumin; this occurs prior to morphological change toward a hepatocyte phenotype.30 The nature of the signalling pathways involved during hepatogenesis has been elucidated by examining mRNA expression in cultured mouse endoderm; this has demonstrated the importance of the local concentration of FGF1 and FGF2;36 bone

5

MacSween’s Pathology of the Liver

morphogenetic proteins may also be involved.30,37 Wnt signalling is now also known to play a role in liver induction; liver development requires its repression by secretion of Wnt inhibitors by the endoderm.38 These inductive cues control gene expression in the developing hepato­blasts through transcription factors of which in the mouse Hex appears to be essential.39 Studies utilizing embryonic stem cells and small interfering RNA technology have reinforced the role of FoxA2 in hepatocytic differentiation.40 The myriad of transcription factors and signalling molecules identified thus far that may be involved in early liver induction are summarized elsewhere.29,30 Vasculogenic cells (angioblasts) are also critical for these earliest stages of organogenesis, prior to blood vessel formation. In the mouse embryo, angioblasts were found as a loose necklace of cells interceding between the thickening hepatically specified endoderm and the mesenchyme of the septum transversum. This mesenchymal–epithelial interaction precedes the emergence of the liver bud and persists throughout further liver development. The essential role of the vascular compartment can be illustrated in the Flk knockout mouse which lacks mature endothelial cells and in which there is failure of development of the liver bud.32 These interactions are also thought to be important in the development of the human liver.41 During later fetal liver development there is continued expansion of the parenchymal cell mass. This involves both stimulatory signals and protection from tumour necrosis factor α-mediated apoptosis; these phenomena involve the AP-1 transcription factor c-Jun, hepatoma-derived growth factor (HDGF), the Wnt signalling pathway, the nuclear factor κB pathway, and the hepatocyte growth factor (HGF)-c-met pathway among others.30,42 Development and maintenance of hepatocytic differentiation and function is under the control of HNF4α.43 The mechanisms that control differentiation of hepatoblasts towards cholangiocytes are beginning to be better understood; it appears to involve the Notch-Jagged 1 pathway44 and is at least in part under the control of SOX9, HNF6, HNF1β and Hhex (see review by Lemaigre).30

• A small area on the left posterior, covered by peritoneum and apposed to the abdominal oesophagus. The traditional division of hepatic anatomy into right and left lobes (delineated by the midline falciform ligament), and caudate and quadrate lobes is of purely topographical significance. A more useful and important subdivision is made on the basis of the branching pattern of the hepatic artery, portal vein and bile ducts. As these are followed into the liver from the porta hepatis, each branches in corresponding fashion, accompanied by a branching tree of connective tissue, derived from the original mesenchyme of the developing liver. On this basis of vascular anatomy, the liver is divided into right and left ‘physiological’ lobes of about equal size. The plane of separation between these two ‘hemi-livers’ corresponds, on the visceral surface of the liver, to a line extending from the left side of the sulcus for the inferior vena cava superiorly to the middle of the fossa for the gallbladder inferiorly. This parasagittal plane lies approximately 2–3  cm right of the midline. Each lobe has been further subdivided into portobiliary-arterial segments. Within each hemi-liver, the primary branches of the portal vein divide to supply two main portal segments, each of which is further divided horizontally into superior and inferior segments. According to this scheme there are, therefore, eight segments, or nine if the dorsal bulge of the liver between the groove of the inferior vena cava and midline – the caudate lobe – is separately designated. Using the Couinaud system for designating segments (Fig. 1.5),45 the numerical assignments are: caudate lobe (1); left lobe – mediosuperior (2), medio-inferior (3), latero-superior (4a), latero-inferior (4b); right lobe – medio-inferior (5), latero-inferior (6), laterosuperior (7) and medio-superior (8). As segment 4b lies between the falciform ligament medially and the gallbladder fossa and groove for the inferior vena cava laterally, this region is also designated the quadrate lobe. Numerology aside, the caudate lobe stands at the watershed between right and left vascular and ductal territor­ ies; its right portion in particular may be served by right or left vessels and ducts, although its left part is almost invariably supplied by the transverse portion of the left branch of the portal vein.

Macroanatomy of the liver The liver lies almost completely under the protection of the ribcage, projecting below it and coming into contact with the anterior abdominal wall only below the right costal margin and the xiphisternum. It is moulded to the under surface of the diaphragm, the muscular part of which separates it on each side from the corresponding lung and pleural sac. It is separated by the central tendon of the diaphragm from the pericardium and the heart. The anterior dome of the liver and its medial, ventral and lateral aspects are covered by the Glisson capsule, the connective tissue sheath of the liver with its glistening peritoneal surface. The posterior surface of the liver is the least accessible and its relationships are of some clinical importance. It includes the following, from right to left:

Left lateral (posterior) sector

Right lateral (posterior) sector

7 2

8 1 6

3

• The ‘bare area’, which is surrounded by the reflections of peritoneum, which form the superior and inferior layers of the coronary ligaments. It lies in direct contact with the diaphragm, except where the inferior vena cava, the right adrenal and the upper part of the right kidney intervene. • The caudate lobe, which lies between the inferior vena cava on the right and, on the left, the fissure of the ligamentum venosum and the attachment of the lesser omentum. The caudate lobe projects into the right side of the superior recess of the lesser sac and is covered by peritoneum; behind it lies the right crus of the diaphragm, between the inferior vena cava and the aorta.

6

5

Right medial (anterior) sector

4

Left medial (anterior) sector

Figure 1.5  Couinaud system for designating hepatic segments (see text for description of anatomical boundaries).

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

These nine hepatic segments are separate in the sense that each has its own vascular pedicle (arterial, portal venous and lymphatic) and biliary drainage. Although there is substantial opportunity for vascular anastomoses between the different hepatic segments, there are no major intrahepatic vascular connections between the right and left hepatic arteries or portal vein systems. The virtually in­­ dependent vascular supply for each segment has been shown by studies in living humans, using computed tomography, magnetic resonance imaging and ultrasonography, together with intravenous contrast injections which allow ready recognition of the liver’s major vascular structures.46 This vascular anatomy underpins the surgeon’s ability to perform a partial hepatectomy and achieve haemostasis for the residual liver, despite the absence of defining connective tissue septa between segments.47 While the branches of the hepatic artery and portal vein and the tributaries of the hepatic ducts run together and serve segments of liver, the hepatic veins run independently and are intersegmental. Like the portal vein, they lack valves. The three major hepatic veins, the right, intermediate and left (the intermediate and left often forming a common trunk) enter the upper end of the retrohepatic segment of the inferior vena cava: the terminal portion of each is frequently at least partially exposed above the posterior surface of the liver, where they are vulnerable to being severed by blunt abdominal trauma. In addition to these major hepatic veins, several accessory hepatic veins (about five per liver) open into the lower part of the hepatic segment of the inferior vena cava.48 Since the caudate lobe regularly drains directly into the inferior vena cava, it may escape injury from venous outflow block.

Microanatomy of the liver Definition of the fundamental structural and functional ‘unit’ of the liver has been an elusive goal since the first description of liver lobules by Weppler in 1665 (see Bloch49). Over the years, several concepts of the basic structural organization of the liver have been operative. The hexagonal ‘lobule’ described by Kiernan in 1833,50 remains the standard by which hepatic microarchitecture is named (Fig. 1.6). ‘Portal tracts’ containing a portal vein, hepatic artery and bile duct constitute the periphery of the hexagonal lobule, occupying three of the six apices of the hexagon. The effluent hepatic vein is at the centre of the lobule, hence its name ‘central vein’.52 To this day, regions of the parenchyma are referred to as ‘periportal’ or ‘pericentral’. More useful are subdivisions of the classical hexagonal lobule into smaller functional physiological units. The most robust concept is the ‘liver acinus’ (Fig. 1.6), defined by Rappaport and his colleagues in 1954.51 In this formulation, the portal tract is at one point of the base of an isosceles triangle, and the effluent vein is at the sharp apex of the triangle. The vein is regarded the ‘terminal portal venule’. In the idealized hexagonal lobule, there are six acinar units. However, actual liver microanatomy is not ideal, and there are evervariable relationships between portal tracts and terminal hepatic venules across the two- and three-dimensional anatomy of the liver parenchyma. Regardless, a key element of the Rappaport ‘acinus’ is portal venules derived from portal veins of various sizes, which penetrate the parenchyma and traverse the base of the isosceles triangle. Blood emanating from these venules perfuses the parenchyma across a broad zonal front, converging at the apex of the terminal hepatic vein. The most proximal zone, defined by the blood flow, is ‘zone 1’, with a mid-zonal ‘zone 2’ and a terminal ‘zone 3’, recapitulating the ‘pericentral zone’ of the hexagonal lobule. Importantly, zone 1 is not ‘pericentral’ – it is an ellipsoid region of higher-oxygenated parenchyma with its long axis being

PT

PT Microcirculatory periphery PT

ThV

3

2 1

1

2 3

ThV

3'

‘Nodal’ point of Mall 1'

2'

‘Nodal’ point of Mall

PT

Figure 1.6  Diagrammatic representation of the simple acinus and the zonal arrangement of hepatocytes. Two neighbouring classic lobules are outlined by the discontinuous lines, and the acinus occupies adjacent sectors of these. Although only one channel is shown as forming the central core of the acinus, the acinus is arranged round the terminal branches of the portal vein, hepatic artery and bile ductule. Zones 1, 2 and 3 represent areas which receive blood progressively poorer in nutrients and oxygen; zone 3 thus represents the microcirculatory periphery, and the most peripheral portions of zone 3 from adjacent acini form the perivenular area. The nodal points of Mall represent vascular watershed areas where the terminal afferent vessels from neighbouring acini meet. PT, portal tract; ThV, terminal hepatic vein (central vein of ‘classic lobule’); 1, 2, 3, microcirculatory zones; 1′, 2′, 3′, microcirculatory zones of neighbouring acinus; dashed line, outline of ‘classic lobule’. (Adapted from Rappaport et  al. 195451)

the base of the isosceles triangle (remembering that acini emanate symmetrically on both sides from the penetrating venule). Likewise, the septal venule is orthogonal to a line drawn between parallel terminal hepatic veins coursing through the parenchyma. In three dimensions, the perfect geometry is an interlocking system of triangular acini that can be viewed as contributing to the larger hexagonal lobules. Recent years have seen other formulations for liver micro­ anatomy.53–56 The primary lobule of Matsumoto et al.,53 described in 1979, regarded the ‘vascular septum’ emanating from the portal tract (the ‘penetrating venule’ of the liver acinus) as being at the origin of the unit’s blood flow. A more recent concept is the cholehepaton,57 viewed as akin to the nephron and synthesizing concepts of a ‘choleon’52 and ‘hepatic microcirculatory unit’.58 A group of hepatocytes – again an isosceles triangle with the terminal hepatic vein at its apex – is drained by a single bile ductule : canal of Hering (to be discussed) at the base of the triangle.57 In this fashion a counter-current cholehepaton unit is envisaged, with blood moving through sinusoids from the base-to-apex of the triangle, and bile moving apex-to-base in return. Most of these concepts represent particular ways of looking at an organ of great structural complexity and with a multitude of functions. However, there is a basic difference between the liver acinus and the other ‘lobule-based’ concepts, which makes them mutually exclusive. In the early editions of this text it was stated that the concept of the liver acinus was proving to be of the greatest value to the pathologist in the interpretation of disordered structure and function. It represented the structural and functional liver

7

MacSween’s Pathology of the Liver

unit concept that allowed for an explanation of important histopathological features, such as portal-central bridging, hepatic necrosis and fibrosis. However, the concept of the liver acinus was based on injection of coloured gelatin-based infusion fluids. In 1979 and 1982, Matsumoto and his colleagues published their findings from meticulous vascular reconstruction of the liver, and presented the concept of the ‘primary hepatic lobule’.53,59 Among devoted hepatic anatomists, this last concept has gradually gained attention, and several other concepts including choleon, hepatic microcirculatory unit, choleohepaton and the single sinusoid hepatic functional unit can be considered as variants of or existing within the primary lobule. Accordingly, a separate section is devoted below to the Matsumoto primary lobule. However, the intrahepatic vascular microcirculation and biliary drainage must be examined in greater detail before returning to the primary lobule. Given all these considerations, the terminology of hepatic microanatomy is somewhat fluid, and most certainly mixed. The practice of liver histopathology lives mostly in the two worlds of the classical ‘lobule’ and Rappaport ‘acinus’. The reader hopefully will thus forgive the use of these mixed terminologies in subsequent chapters of this book.

Anatomy of the hepatic microcirculation Portal circulation Within the liver, the portal vein divides into successive generations of distributing or conducting veins, so called because they do not directly feed the sinusoidal circulation. According to their diminishing position in the hierarchy of branching, they may be classified as interlobar, segmental and interlobular. The smallest conducting veins are interlobular veins. Further branching of these produces the smallest portal vein branches which distribute their blood into the sinusoids. These succeeding branches are: (1) the preterminal portal venules which, microscopically, are found in portal tracts of triangular cross-section; and (2) the terminal portal venules which taper to about 20–30 µm in diameter, and are surrounded by scanty connective tissue in portal tracts of circular rather than triangular crosssection. From both the preterminal and terminal portal venules arise very short side branches, designated inlet venules, which have an endothelial lining with a basement membrane and scanty adventitial fibrous connective tissue, but no smooth muscle, in their walls. They pass through the periportal connective tissue sheath and enter into the parenchyma to open into the sinusoids. These inlets are reported to be guarded by sphincters composed of sinusoidal lining cells – the afferent or inlet sphincters.60 Some early branching of the smallest conducting veins may produce more than one portal vein within some portal tracts and these supply only those sinusoids which abut upon the portal tract. The portal tract derives its name from the ramifications of the portal venous system through the liver. The portal system also provides the tracts along which the hepatic artery system and biliary tree travel, giving rise to the alternate term ‘portal triad’. However, as the terminal portal tracts reach their own terminus, the portal vein system ends, ironically leaving a final portal tract ‘dyad’ lacking a terminal portal vein but containing hepatic artery : bile duct pairs before they, too, end after a short distance. In the periphery of the normal adult liver sampled by percutaneous liver biopsy, portal ‘triads’ containing portal vein, hepatic artery and bile duct profiles constitute only 70% of portal tracts. Careful histological examination will also reveal an additional 30% of diminutive ‘portal dyads’ containing only hepatic artery and bile duct profiles.9

8

Lastly, the portal system ramifies through approximately 17-20 orders of branches to supply the entire corpus of the liver.61 The subdivisions are not strictly dichotomous, however, in that one branch may ultimately have fewer sub-branches than another. Hence, the liver ultimately has an irregular lobular organization at the microscopic level.

Arterial circulation The hepatic artery supplies branches throughout the portal tract system. The terminal blood flow of the arteries is by three routes: into a plexus around the portal vein; into a plexus around a bile duct; and into terminal hepatic arterioles.61 The perivenous plexus is characteristically distributed around interlobar, segmental and interlobular portal vein branches within the portal area; it drains into hepatic sinusoids. Occasional arterioportal anastomoses between perivenous arterioles and terminal portal venules have been observed but the frequency of these in normal human livers is uncertain.60,61 By the level of the terminal portal veins, the arteriolar plexus is absent. A peribiliary arterial-derived plexus supplies all the intrahepatic bile ducts. Around the larger ducts, the peribiliary plexus is twolayered, with a rich inner, subepithelial, layer of fine capillaries and an outer, periductular, venous network which receives blood from the inner layer. The smallest terminal bile ducts have only a single layer of fine capillaries. Ultrastructural studies have shown that the capillaries are lined by fenestrated endothelium.62 The peribiliary plexus drains principally into hepatic sinusoids. The peribiliary plexus develops in parallel with the development of the intrahepatic bile ducts, spreading from the hepatic hilum to the peripheral area of the liver and becoming fully developed with the full maturation of the biliary system.63 In addition to providing the vascular supply to bile ducts, the peribiliary vascular plexus is thought to be involved in reabsorption of bile constituents (including bile acids) taken up apically by bile duct epithelial cells and secreted across their basal plasma membrane into the portal tract interstitium, constituting a ‘cholehepatic’ circulation.64 The cholehepatic reuptake of biliary substances may play a key adaptive role during times of downstream bile duct obstruction, as these solutes may be ‘dumped’ into the systemic circulation for disposal by the kidneys.65 Terminal hepatic arterioles have an internal elastic lamina and a layer of smooth muscle cells, and open into periportal sinusoids via arteriosinus twigs. Although some mammalian species exhibit hepatic arterioles which penetrate deeply into the parenchyma before entering sinusoids near to the hepatic veins, reports of such penetrating arterioles in humans have been disputed.65,66 Regardless, isolated parenchymal arterioles may sometimes be seen in liver biopsies. Ekataksin58 has suggested that these vessels supply isolated vascular beds in the parenchyma.

Venous drainage Having perfused the parenchyma via the sinusoids, blood enters the terminal hepatic venules (the ‘central veins’ of the classic lobules). Scanning electron microscopy has clearly demonstrated in the walls of terminal hepatic veins the fenestrations through which the sinusoids open,67 and the astute observer will see these sinusoidal openings into terminal hepatic veins in histological sections. The terminal vein branches unite to form intercalated veins which in turn form larger hepatic vein branches whose macroanatomy has already been described. The venous anatomy does not strictly parallel the distribution of the portal system, as hepatic veins traverse between portal system-defined lobules as the venous system exits

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

the liver. This is understandable, as ultimately the hepatic veins have to exit through the dorsum of the liver, whereas the portal system enters the liver ventrally. The hepatic venous system also does not ramify as extensively as the portal system, so that there is a slight preponderance of terminal portal tracts to terminal hepatic veins throughout the liver.

Regulation of the hepatic microcirculation Total hepatic blood flow in normal adults under resting conditions is between 1500 and 1900 mL/min or approximately 25% of cardiac output.68 Of this, roughly two-thirds is supplied by the portal vein and the remainder by the hepatic artery. Owing to variations in splanchnic blood flow, portal vein blood flow increases after feeding and decreases during exercise and sleep.69 Direct external regulation of hepatic blood flow is mediated only through the hepatic artery. In contrast, intrinsic regulation of blood flow within the liver is quite complex. This account is based immediately on the work of McCuskey which has been reviewed by him,70 but derives ultimately from the pioneering studies of Knisely et al.71 using quartz rod transillumination of living liver. Anatomical arter­ ioportal relationships are summarized in Figure 1.7, which shows a terminal (penetrating) portal venule from which a series of sinus­ oids originates, and a hypothetical accompanying terminal hepatic arteriole (internal diameter approximately 10 µm). As noted, whether this arteriole actually penetrates the parenchyma is disputed. Regardless, there are various connections between arteriole and sinusoid, all of them being found in the periportal areas, and all of internal diameter no greater than the diameter of an erythrocyte. In accordance with macroscopic blood flow, approximately twothirds of the intrahepatic blood supply ultimately comes from the portal venules, whose inlets are controlled by sphincters – afferent or inlet sphincters – composed of sinusoidal endothelial cells. For the remaining third of blood supply – arterial – blood flow to the sinusoids is intermittent, and determined by independently contractile smooth muscle sphincters in the walls of hepatic arterioles AS S AS PV

S

Matsumoto’s primary lobule

SP AS

S

HV

HA

Figure 1.7  Scheme to show relationships of hepatic arteriole (HA), portal venule (PV), sinusoids (S) and hepatic venule (HV). Arteriosinusoidal branches (AS) may open in various ways into the sinusoids and blood flowing to a group of sinusoids could therefore be arterial, venous or mixed. Blood flow through the sinusoids is determined by the activity of ‘sphincters’ (SP) in the arteriolar wall and of sphincteric mechanisms at the inlet and outlet of the sinusoids. (Based on McCuskey 198854)

and their arteriolosinusoidal branches. Blood flowing into a group of sinusoids could therefore be arterial, venous or mixed, depending upon sphincteric activity of entering venular and arteriolar channels, and the distance of the originating sinusoid from the portal tract along the penetrating venule. There also is heterogeneity in the blood flow through the sinusoids. In the upstream zone, the sinusoids form an interconnecting polygonal network. Downstream, however, the sinusoids become organized as convergent parallel channels which drain into the terminal hepatic venule, a convergent architecture that is evident histologically at medium power. In this downstream region, short intersinusoidal channels connect adjacent parallel sinusoids. Blood entering the hepatic venules passes through efferent or outlet sphincters which, like the inlet sphincters, are composed of sinus­ oidal endothelial cells. The precise mechanisms which regulate the hepatic microcirculation remain controversial.70,72 The potential morphological sites for regulating blood flow through the sinusoids include segments of the portal venules and hepatic arterioles, the sinusoids themselves and the hepatic venules. The portal and hepatic venules and the hepatic arterioles contain some smooth muscle cells in their wall and are therefore contractile. However, the principal site of regulation is thought to reside in the sinusoids themselves. The sinusoidal endothelial cells respond to a variety of vasoactive substances and by contracting or swelling they may vary the diameter of the sinus­ oid lumen. Thus, blood flow through individual sinusoids is variable. Where the lumen is narrowed, blood flow may be impeded by leukocytes that transiently plug the vessel, a feature which is more common in the narrower, more tortuous periportal sinusoids.73 It seems likely that flow through some sinusoids may be intermittent while others have relatively constant rates of blood flow. Arterial blood flowing into an individual sinusoid through a dilated arterio­ sinusoid may increase the rate of blood flow. Kupffer cells could affect the rate of blood flow through sinus­ oids, with particular attention also being given to a role for hepatic stellate cells. Hepatic stellate cells are responsive to vasoconstrictors such as endothelin-1.74 In addition, reduction of the portal blood flow has been shown by in vivo microscopy to reduce sinusoidal blood flow with a considerable reduction in sinusoidal diameter, changes which were reversed on restoring the portal blood flow.75 It has been suggested that the stellate cells, whose long slender processes surround the sinusoids, may be responsible for producing these changes.

We now return to the concept of the fundamental architectural unit of the liver. Matsumoto and colleagues53,59 performed a detailed study of the angioarchitecture of the human liver with graphic reconstructions from thousands of serial sections. They distinguished a conducting and a parenchymal portion of the portal venous tree. The conducting portion should ensure delivery of blood to the parenchyma, with a pressure drop small enough in initial sidebranches to allow for variable metabolic demand, while maintaining pressure in the conducting trunk. The conducting portion meets this functional demand by remaining within the macroscopic range throughout its course, thus rendering resistance to flow virtually insignificant. This physiological concept underlies the observed non-dichotomous branching of the portal system mentioned earlier, since terminal portal tracts may branch off conducting portal tracts without the latter losing their conducting capability. The terminal portal venous tree ramifies in three steps. The first side-branches from conducting portal veins arise in orderly rows.

9

MacSween’s Pathology of the Liver

Each of these first-step branches supplies a fairly definite mass of parenchyma (approximately 1.6 mm wide; 1.2 mm long; and 0.8 mm thick). For this first ramification, at least, the portal branches of a given order are generally equal in number to the hepatic veins of the corresponding order. For the second step, every first-step branch gives off at about right angles 11 second-step branches (with an average diameter of 70 µm). This sudden increase in branching frequency (limited to the portal vein, and not observed in corresponding levels of the hepatic vein) engenders a characteristic portohepatic constellation: about six portal vein branches embracing a certain amount of parenchyma with one hepatic vein as the central axis. This constellation underscores the time-honoured concept of the classic lobule of Kiernan.50 The third-step ramification occurs at about right angles from every second-step branch. At this step all branches virtually lack connective tissue sheets and their wall structure gradually changes over to that of sinusoid. However, they are easily recognizable by their precise interlobular course and their larger lumina. These branches are termed the septal branches, in that they traverse the parenchyma ‘between’ the regions of sinusoids they perfuse. This three-step arrangement leads to two distinct zones of sinus­ oidal perfusion: the septal zone and the portal zone. The septal zone is mainly defined by the above mentioned septal branches. They not only supply this zone with portal blood, but also support it as a vascular skeleton. The septal branches, located in the plane between two adjacent second-step branches (portal tracts), run a more or less parallel course and are quite regularly spaced about 200 µm apart over the lobular surface. Upwards and downwards, they give off a few branches which all break up into sinusoids, thus forming a septum-like sinusoidal network at the surface of the lobule (Figs 1.8, 1.9). The septum serves as a starting plane for the intralobular radial sinusoids that converge on the terminal hepatic vein. The characteristic of this vessel is that it soon breaks up into branches of sinusoidal order, which spread in almost transverse direction to the original axis of the vessel, thus taking a dish-like form (Fig. 1.10). From this extension, blood streams inward into the parenchyma. Although the sinusoids have numerous lateral anastomoses, the preferential paths of blood from the dish-like extension can be followed over some distance until it reaches the surface (surface Fp in Fig. 1.10), where these paths pass into radial sinusoids. Along this course the bundle of preferential sinusoids takes the form of a curved cylinder (Fig. 1.10). In contrast, the portal zone is part of a marginal zone that is in contact with the portal tract. The vessels that supply this zone are short venules (inlet venules) given off either directly from the first- or second-step branches of portal veins, or from the most proximal portion of each septal branch. The sinusoidal beds of the septal zone and of the portal zone together constitute a synthetic functional unit: a high potential pool from which portal blood pours uniformly into the radial sinusoids. On lobular cross-sections, this pool space appears as a sickle-shaped area (the sickle zone). The sickle edge represents a cut line of the vertically extending inflow-front, and represents a haemodynamic equipotential surface. The inflow-front appears to be composed of smaller unit areas, each tending to be concave inwards. About six to eight such units cover the entire surface of the lobule. These units of the inflow-front have their counterparts in the draining system: each central vein is found to comprise six to eight draining poles, disposed in such a way that each faces a corresponding unit of the inflow-front across a certain parenchymal distance. The cone-shaped mass of parenchyma thus sandwiched by this pair is designated the primary lobule in the sense that it represents the most elementary parenchymal unit (Fig. 1.11).

10

A

A

SP

SP

A

A

I Figure 1.8  Diagrammatically represented angioarchitecture of vascular septum: a figure generated by sectioning the vascular septum in Figure 1.10 with a plane perpendicular to both the septal branches and the Y–Z plane. SP, primary septal branch; A, accessory branch; I, line of intersection with the Y–Z plane in Figure 1.10. Note that each primary septal branch builds (the upper one downward and the lower one upward) two- to three-storied (in this figure, two-storied) sinusoidal loops, which form the bulk of the septum. Arrows = direction of blood flow. (Adapted from Matsumoto and Kawakami 198259)

All these findings point to a new aspect of the classical lobule: the classical lobule is made up of six to eight primary lobules, and is itself termed the secondary lobule. The most important structural feature of the secondary lobule is that the central vein of the classical lobule (terminal hepatic vein of the primary lobule) represents the longitudinal axis of the classical lobule while the terminal branches of the portal vein form its periphery.76 Conversely, the septal vein of the primary lobule forms the base of the isosceles triangle defining the Rappaport acinus. The concept of the primary lobule is at variance with the acinus concept in the following ways:53,59

• The lobule has a sickle-shaped inflow-front derived both from the penetrating septal venule and direct portal vein effluent, whereas the acinus is characterized by a linear inflow-source with radial symmetry only around the penetrating septal venule. • The septal venular branches (conceptual equivalents of the terminal portal venules of the acinus concept) do not run the entire length of the distance between two portal tracts, but taper off into a sinusoidal configuration near the middle of the interportal distance. In other words, the vascular

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

II

PV

I

A X

Z

A HV

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100 µm

O

III

III (III, I) = 80º

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Figure 1.9  Overall view showing how septal branches span vascular septum. PV, portal vein; I, II, III, primary septal branches; arrows = secondary septal branches; A, accessory branch; HV, hepatic vein. Inset: diagram indicating the orientation of septal branches relative to their parent portal vein. (Adapted from Matsumoto and Kawakami 198259)

1

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Figure 1.10  Vascular beds (*) depending mainly on the inlet venule 2. Fp, inflow-front: equipotential surface that gives rise to typical ‘converging sinusoids’. (Adapted from Matsumoto and Kawakami 198259)

watershed areas are not located at the nodal points of Mall (Fig. 1.6) but at the midpoint of the interportal distance (midseptum M of Zou et al.76). It also is important to note that the septal venules are not orthogonal to the portal tract, but in three dimensions follow a somewhat arcuate course into the parenchyma. • The lines 1, 2 and 3 (Fig. 1.6) represent the borders of the acinar zones in the concept of the liver acinus, in descending order of nutrient level. One can interpret these borderlines of acinar zones as cut lines of haemodynamic equipotential surfaces; hence, any line traversing successively and perpendicularly across these surfaces should represent the potential gradient in that direction. In chronic venous congestion, damage to the hepatocytes starts in the microcirculatory periphery, progressing in all directions up the potential gradient, so that in the case of severe congestion only the zone of highest potential can survive. According to the acinar concept, the surviving parenchyma of acinar zone 1 would appear clover-shaped with the portal tract at its centre, but still damaged. However, Matsumoto et al.53 claim that immediate periportal damage actually never occurs in vascular insufficiency or chronic venous congestion and that, typically, the liver biopsy always shows sickle zones standing in relief.

11

MacSween’s Pathology of the Liver

500 µm

Portal vein Hepatic vein Portal tract Septum-like inflow-front Borderline enclosing a classical lobule

Figure 1.11  Classical lobule with its constituents, primary lobules. The direction of blood flow is suggested by arrows. (Adapted from Matsumoto and Kawakami 198259)

• Zonal gradients of metabolic activities and of enzyme histochemical staining patterns also conform with the sickle-zone pattern, and not with the acinus diagram. Matsumoto et al.53 illustrate this with pictures of peripheral fatty change and the enzyme-histochemical pattern of glucose 6-phosphatase. The sickle-zone pattern has been confirmed by other authors.55,77,78

Functional heterogeneity in the liver Recognition of hepatocyte heterogeneity dates back to as early as the 1850s79 when the heterogeneous contribution of different hepatocytes to bile secretion was described. Many aspects of liver physi­ o­logy and metabolism exhibit a heterogeneous distribution along the portocentric axis. Hepatocytes in the periportal zone have a higher capacity for gluconeogenesis and fatty acid metabolism, whereas perivenous hepatocytes have a higher capacity for detoxification.80 This heterogeneity is reflected in hepatocyte ultrastructure, including increased smooth endoplasmic reticulum (SER) with its biotransforming enzymes in the pericentral region, and gene expression.81 The concept of metabolic zonation is therefore operative.82 As first proposed for carbohydrate metabolism, opposite metabolic pathways like gluconeogenesis and glycolysis can be carried out simultaneously by hepatocytes in the periportal and

12

centrilobular region respectively. While this possibility for ‘futile cycles’ may seem at first to be inefficient, the presence of dynamically opposed interlocking metabolic pathways within individual hepatocytes and between hepatocytes in different positions in the lobule allows for exquisitely rapid regulation of metabolic function, including rapid reversal of the metabolic output of the organ.83 The presence of metabolic zonation for essentially all liver functions reflects a potentially important level of overall metabolic control. Two types of zonal heterogeneity in the liver are the gradient versus compartment type of zonation, and the dynamic versus stable type of zonation.84 In the gradient type, all hepatocytes are able to express a particular gene, but the level of expression depends on the position of the hepatocyte along the portocentral radius. Examples include key regulatory enzymes of carbohydrate metabolism, cytosolic phosphoenol-pyruvate carboxykinase I (PCK) and glucokinase. In the compartment type of zonation, the expression of genes has been thought to be restricted to either the periportal or the pericentral compartment. A striking example of compartmental zonation is the key enzymes of ammonia metabolism, carbamoyl phosphate synthetase I (CPS) and glutamine synthetase (GS), which in normal liver exhibit strict localization to hepatocytes rimming the terminal hepatic vein. The dynamic type of zonation is characterized by adaptive changes in expression in response to

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

changes in the metabolic or hormonal state, regardless of where the enzyme is zonally located. Examples are PCK, tyrosine aminotransferase, CPS, and ornithine aminotransferase. The stable type of zonation, on the other hand, is characterized by the virtual absence of dynamic adaptive change, such as fructose-1,6-biphosphatase and GS. The molecular mechanisms regulating metabolic zonation are varied. In the case of bile formation, physiological availability of biliary solutes in periportal blood is a major determinant, independent of hepatocellular expression of relevant transporters.84 Likewise, gradients of oxygen and hormones across the lobule are thought to play key roles influencing expression of metabolic enzymes.80 Recently, it has been shown that the Wnt/β-catenin signalling pathway plays a key role in regulating liver zonation.85–87 Specifically, the precise zonal localization of several β-catenin regulated liver-specific genes, including GS, transporter-1 of glutamate (Glt1) and ornithine aminotransferase (Oat), are under Apc control. Targeted disruption of Apc inhibition, or constitutive activation of the Wnt/β-catenin signalling pathway, leads to panlobular expression of these genes involved in ammonia metabolism. The resultant severe metabolic perturbation is lethal in experimental conditions. Conversely, experimental deletion of β-catenin results in loss of GS expression irrespective of whether Apc is present.87 A role for dicer, an endoribonuclease III type enzyme required for microRNA biogenesis, in regulating liver zonation has also been proposed.88 Zonal heterogeneity is a feature that characterizes not only hepatocytes, but other components of liver tissue as well. Sinusoids have a more tortuous course, more frequent intersinusoidal anastomoses, and a narrower lumen in the periportal area, whereas they appear straighter with less intersinusoidal sinusoids and a broader lumen in centrilobular areas.89 The sinusoidal endothelial cells have higher porosity (by fenestrae) in the centrilobular region, display different wheat-germ agglutinin-binding patterns in periportal versus centrilobular endothelial cells, and show portocentral gradients in mannose receptor-mediated endocytosis, and in production of reactive oxygen metabolites.90 In a three-dimensional reconstruction study of parenchymal units in rat liver, Teutsch et al.91 emphasized the importance of considering three-dimensionality for an adequate functional interpretation of the metabolic heterogeneity of hepatocytes. If the three-dimensionality of the parenchymal units is not taken into consideration, calculations show that it is likely that changes at the origin of sinusoids are under-estimated, whereas those at the termination of sinusoids are over-estimated. This should also apply in the interpretation of sections from pathologically altered liver tissue.91 The hepatic stellate cells display marked heterogeneity in structure and function based on their zonal (portal-central) and regional (portal vs septal sinusoidal) distribution in the hepatic lobules.92 They have more cytoplasmic processes with thorn-like microprojections in the centrilobular zone, whereas their desmin immunoreactivity and vitamin A storage is higher in the periportal zone. Kupffer cells are located preferentially in periportal regions, and some functional and morphological heterogeneity has been ascribed to their location. Thus, Kupffer cells in periportal zones are larger, contain more heterogeneous lysosomes and are more active in phagocytosis than their centrilobular counterparts.93 In contrast, in areas around centrilobular veins Kupffer cells are smaller and more active in terms of cytokine production and cytotoxicity.94 The extracellular matrix components in the space of Disse may vary along the portal-central axis. Collagens IV and V and laminin predominate in the area of transition between the bile ductule : canal of Hering units and hepatocytes at the periportal lobular periphery. Fibronectin, and collagens III, IV and VI are the predominant components along the portovenous parenchymal axis.95

Ultrastructural anatomy of the hepatocyte Plasma membrane The hepatocyte is a polyhedral epithelial cell approximately 30– 40 µm in diameter and, in common with other epithelial cells, it is highly polarized with transport directed from its sinusoidal surface to the canalicular surface (Fig. 1.12). Within its plasma membrane, three specialized regions, or domains, are recognized: sinusoidal, which faces the sinusoid and the perisinusoidal space; lateral, facing the intercellular space between hepatocytes; and canalicular, bounding that specific part of the intercellular space constituting the bile canaliculus.96,97 Using a more generic terminology, the sinusoidal and lateral domains constitute the basolateral plasma membrane of the hepatocyte, and the canalicular domain is the apical domain. This polarity is largely maintained by the tight junctions formed between adjacent hepatocytes, that delineate the basolateral

A

B Figure 1.12  (A) Light microscopy. Liver cell plates cut longitudinally show: (i) a centrally placed nucleus and occasional binucleate cells; (ii) the sinusoidal surface against which Kupffer cell nuclei are abutting; (iii) the canalicular pole; (iv) the intercellular surface. (B) Scanning electron micrograph (SEM) showing several cell types. Hepatocytes (H) contain a nucleus (N), and at the junction between cells the bile canaliculus (bc) and the intercellular surface are clearly defined. The sinusoidal surface is seen and in the sinusoidal area a Kupffer cell (Kc), endothelial cell (Ec) and two perisinusoidal cells (psc) are present. (This SEM was kindly provided by Professor E. Wisse, Brussels. They are all preparations of rat liver).

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MacSween’s Pathology of the Liver

domain from the canalicular domain, and which create a barrier between fluid in the intercellular space and bile in the canaliculus.98,99 In addition to tight junctions there are also gap junctions and desmosomes in the lateral domain, and it is across lateral domain gap junctions that intercellular communication takes place. Stereological studies in the rat have shown that the basolateral, canalicular and lateral domains comprise approximately 70%, 15% and 15% of the total cell surface area.100 Various techniques (reviewed by Meier97) have been used to characterize these domains, mainly in the rat liver, but also more recently in human liver.101 The domains of the hepatocyte plasma membrane are not simply topographical entities. They are specialized to subserve different basolateral and apical functions of the hepatocyte. Molecular differences include composition of the lipids in the plasma membrane, the complement of membrane proteins, function of endocytic and exocytic compartments, and relationship to the cytoskeleton.84,92,93,102 Over and above direct regulation of membrane protein function, hepatocytes can dynamically regulate actual membrane lipid content and the concentration of specific proteins in each domain, providing a powerful additional mechanism for functional control of plasma membrane function.103,104

Sinusoidal domain The sinusoidal domain of the hepatocyte faces the perisinusoidal space of Disse: the tissue space between the hepatocytes and the endothelial sinusoidal lining cells. The hepatocyte sinusoidal surface is covered with abundant microvilli (Fig. 1.12) each measuring 0.5 µm long but not evident, even as a brush or striated border, by optical microscopy. Microvilli may protrude through the fenestrae of the endothelial cells and into the sinusoidal lumen. The surface specialization is related here, as elsewhere, to absorptive and/or secretory activity; it obviously increases the surface area, but by a factor smaller than one might expect – approximately six-fold when compared with the 40–50-fold increase in surface area imparted by the microvilli of the small intestinal absorptive enterocyte. In the hepatocyte, sinusoidal plasma membrane between the bases of the microvilli shows small surface indentations or pits.67 Some of these represent secretory vacuoles discharging fluid into the plasma by a process of exocytosis. Others are clathrin-coated pits involved in selective receptor-mediated endocytosis, or caveolae, invaginating membrane microdomains enriched in cholesterol and sphingolipids and the cholesterol-binding protein caveolin and responsible for selective membrane trafficking into the interior of the cell.105 Hepatocytes along the limiting plate surrounding portal tracts have a surface which abuts on the adjacent portal tract mesenchyme. These hepatocytes are irregularly covered with microvilli and may be moulded around connective tissue fibres, producing irregular indentations. The space of Mall is conceptualized as the fluid space between hepatocytes along the limiting plate in the periportal area, and portal tract fibrous tissue; it is in continuity with the peri­ sinusoidal space of Disse.

Lateral domain The lateral surface of the hepatocyte extends from the margin of the sinusoidal surface to the bile canaliculus, and is specialized for cell attachment and cell–cell communication. Although simple in contour, it is not entirely flat: microvilli may extend into it from the sinusoidal surface and protrude into narrow extensions of the space of Disse; there are occasional folds (plicae) and round-mouthed openings, which may represent pinocytic vesicles.67 There are also knob-like protrusions and corresponding indentations which, fitted into one another, would form the ‘press-stud’ or ‘snap fastener’ type of intercellular attachment long familiar in transmission electron microscopy (TEM).

14

There are also specialized areas called gap (or, better, communicating) junctions. These are seen by TEM as patches of close approximation of the two membranes and in freeze-fracture preparations as irregularly shaped aggregates of particles on the P-face. The gap between the two membranes is 2–4 nm wide and is bridged by the intramembrane particles (of protein or lipid), which project like ‘bobbins’ from the external surface of each of the two membranes. Since each ‘bobbin’ is perforated by a central pore and apposed ‘bobbins’ are in contact, communications are established which provide for the transfer of ions or metabolites, or both, between hepatocytes.

Canalicular domain The bile canaliculus is defined as an intercellular space formed by the apposition of the edges of gutter-like hemicanals delimited by tight junctions, on the facing surfaces of adjacent hepatocytes (Fig. 1.13). Canalicular diameter varies from 0.5 to 1.0 µm in the peri­ venular area and from 1 to 2.5 µm in the periportal zone, in accordance with flow of bile from the centrilobular region of the lobule towards the portal tract. The canalicular surface is unevenly covered by microvilli (see Fig. 1.23), which are more abundant along a ‘marginal ridge’ at each edge of the hemicanaliculus. In experimental biliary obstruction the canaliculi become dilated and the microvilli disappear, except along the marginal ridges. Intracellular subapical microfilaments are concentrated around the canaliculi, forming distinct, organelle-free pericanalicular sheaths and extending into the microvilli. This pericanalicular microfilament web is contractile, enabling hepatocytes to propel secreted bile along the canalicular channel. The presence of contractile elements in the pericanalicular zone can be demonstrated by indirect immunofluorescence (Fig. 1.13).106 The presence of ATPase can be demonstrated histochemically (Fig. 1.13). Accumulations of lipofuscin or haemosiderin (Fig. 1.13) may also outline the canalicular pole of the hepatocyte, reflecting the presence of pericanalicular lysosomes. The canalicular surface is isolated from the rest of the intercellular surface by junctional complexes (Fig. 1.13), desmosomes, intermediate junctions, tight junctions and gap junctions. The tight junctions constitute a permeability barrier to macromolecules between the bile canaliculus and the rest of the intercellular space. ‘Tightness’ is, however, a relative term; there seems to be a positive correlation between degrees of tightness and the number of strands forming the junction. On this basis the canalicular tight junctions are comparable with those elsewhere in the body (e.g. in the rete testis and vasa efferentia), which are regarded as only ‘moderately tight’.

Nucleus The hepatocyte nucleus shows characteristics one would expect in the nucleus of a cell actively engaged in protein synthesis: it is large, occupying 5–10% of the volume of the cell, spherical, with one or more prominent nucleoli and scattered chromatin. The nuclear membrane is double-layered and contains many pores (Fig. 1.14). At birth, all but a few hepatocytes are mononuclear, and of uniform size. In the adult liver there is considerable variation in both number and size of nuclei. About 25% of the adult hepatocytes are binucleate: the two nuclei are similar in size and staining properties. Hepatocyte nuclei fall into various sizes, with volumes in the ratio 1 : 2:4 : 8. This variation reflects polyploidy, the DNA content increasing correspondingly.107 At birth, in man, nearly all hepatocytes are diploid (and mononucleate). From the 8th year, when more than 90% of hepatocytes are diploid, the number of tetraploid nuclei (i.e. those with twice the normal DNA content) increases, to reach about 15% in children of 15 years.108 Tetraploid cells are thought to arise by mitosis of cells with two diploid nuclei. The DNA content

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

A

C

B

D

of each nucleus doubles, but the chromosomes are then arranged on a single mitotic spindle, so that division produces two daughter cells, each with a single tetraploid nucleus. The significance of polyploidy in hepatocytes is unknown. Since cell size is proportional to cell ploidy,109 polyploidy does not provide an increased amount of genetic material per unit volume of cytoplasm. Hepatocyte mitotic division provides for intrauterine and postnatal growth of the liver, which continues well into childhood. By adulthood the liver has a very low mitotic index with estimates ranging from one mitosis per 10–20 000 cells, to 2.2 mitoses per 1000 cells.

Endoplasmic reticulum (ER) The endoplasmic reticulum is a network of parallel, flattened sacs or cisternae on whose cytoplasmic surfaces may be attached polyribosomes to constitute the rough endoplasmic reticulum (RER) (Fig. 1.15). Clusters of RER are scattered randomly throughout the hepatocyte cytoplasm and constitute approximately 60% of the

Figure 1.13  (A) TEM. Bile canaliculus (bc). Note microvilli projecting into the lumen and the organelle-free pericanalicular cytoplasm (*). Tight junctions (broad arrow), intermediate junctions (thin arrows) and desmosomes (arrowheads) are present between the two adjacent hepatocytes (H). Human liver. (Print courtesy of Professor P. Bioulac-Sage) (B) SEM. On the surface of a number of neighbouring hepatocytes, an interconnected network of bile hemicanaliculi show bifurcations (arrows) together with blunt ends (asterisks). (C) Bile canaliculi in rabbit liver: immunofluorescence preparation in which the section was reacted with a human serum containing smoothmuscle antibody: the pericanalicular microfilaments have produced strong positive immunofluorescence. (D) Pattern of bile canaliculi shown by histochemical demonstration of ATPase in pericanalicular cytoplasm.

endoplasmic reticulum. The remaining 40% is the smooth endoplasmic reticulum (SER), lacking a ribosomal coating. The SER also forms anastomosing networks of tubules and vesicles of varying diameter which are continuous with the cisternae of the RER (Fig. 1.15). The outer nuclear membrane also has attached ribosomes and is continuous with the membrane of the RER. The SER is often found in the region of the Golgi apparatus and communicates with it. There is frequently a close topographical relationship of the SER with glycogen (Fig. 1.16). The ER occupies 15% of the total cell volume and its surface area – approximately 60 000 µm2 per hepatocyte – is more than 35 times the area of the plasma membrane. There is also zonality in the distribution of the ER; the surface area of SER in the centrilobular area is twice that in the periportal zone. The cell functions associated with the ER include: (1) protein synthesis, both of secretory proteins and some of the protein constituents of the cell and organelle membranes; (2) the metabolism of fatty acids, phospholipids and triglycerides; (3) the production and metabolism of cholesterol and, possibly, the production of bile acids; (4) xenobiotic metabolism; (5) ascorbic acid synthesis; and

15

MacSween’s Pathology of the Liver

(6) haem degradation. In the case of protein synthesis, polypeptides synthesized by RER ribosomes are retained in the plasma membrane or are ejected into the lumen of the RER for folding and post-translational modification. The protein products pass via the SER to the Golgi apparatus, for packaging and insertion into membranes throughout the cell (in the case of membrane proteins) or for exocytotic secretion across the basolateral or apical membranes of the hepatocyte. The cytochrome P-450 system is localized in the ER membrane; this is the system whereby the liver cell metabolizes and detoxifies xenobiotics. This enzyme system can be reversibly induced by certain xenobiotics such as phenobarbital and this is accompanied by the synthesis and hypertrophy of ER; the mechanisms involved in new membrane production are not clear. The preponderance of SER in the centrilobular region of the lobule and the presence of haem in cytochrome P-450 enzymes, explain the darker hue of the centrilobular region, which can be observed by the naked eye on inspection of cut liver sections. Glucose 6-phosphatase is localized on the ER, playing a key role in dephosphorylation of intracellular glucose 6-phosphate prior to release of glucose into the circulation by hepatocytes. As a correlate, the SER proliferates during synthesis of glycogen and hence is available for hepatocyte glycogenolysis when hepatocellular release of glucose is required for metabolic needs elsewhere. Figure 1.14  Freeze-fracture replica illustrating the inner leaflet (P face) and the outer leaflet (E face) of the nuclear membrane of a hepatocyte. Note the numerous nuclear pores. Human liver, ×44 800. (Courtesy of Professor R. De Vos)

A

Golgi complex Each hepatocyte contains as many as 50 Golgi zones (which may not be separate but rather form a tri-dimensional continuity),

B

Figure 1.15  (A) TEM illustrating cytoplasmic organelles, nucleus and bile canaliculus (C) between two adjacent hepatocytes; lysosomes (L); mitochondria (M). Human liver, ×23 000. (B) TEM illustrating rough endoplasmic reticulum (RER) and mitochondria (M) with matrix granules; also part of the nucleus (N) with inner and outer membrane. Human liver, ×36 800. (Courtesy of Professor R. De Vos)

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Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Figure 1.16  TEM illustrating networks of smooth endoplasmic reticulum (SER); Golgi apparatus (G) peroxisomes (P). Note also glycogen rosettes (GL). Human liver, ×28 800. (Courtesy of Professor R. De Vos)

situated most commonly beside the nucleus or in the vicinity of the bile canaliculus.110 Each complex appears as a stack of four to six curved, flattened parallel sacs often with dilated bulbous ends containing electron-dense material. The convex or cis surface is directed towards the ER and small vesicles in the cis-Golgi transfer synthesized proteins from the ER. The concave or trans surface is the origin of secretory vesicles. Vesicles break off from the ends of the sacs, and carry the contained secretory proteins, including lipoproteins, for discharge at the sinusoidal surface or, less commonly, at the canalicular surface. Membrane proteins destined for insertion into any of the plasma membrane domains also are routed through the Golgi complex. The complex and its associated cytoplasm constitute approximately 2–4% of the cell volume. In addition to its role in the secretion of proteins, the Golgi complex has a large complement of glycosylating enzymes, important in the glycosylation of secretory proteins and in the synthesis and recycling of membrane glycoprotein receptors.110

Lysosomes The existence of lysosomes was first predicted by De Duve on the basis of biochemical studies on liver homogenates,111 and it was he who subsequently identified them as the ‘peribiliary dense bodies’ found in electron micrographs of liver, and who established them as a new species of cell organelle. Their functions in health and disease have been reviewed,112,113 and are of particular importance to pathologists because of their involvement in a number of storage diseases (Chapter 4). Lysosomes present a variety of appearances in electron micrographs of liver, but basically they are vesicles, bounded by a single

membrane (Fig. 1.15), containing acid hydrolases: e.g. acid phosphatase, aryl sulphatase, esterase and β-glucuronidase. Their form is so variable that unequivocal identification depends on histochemical demonstration of one of the contained enzymes, most conveniently acid phosphatase. Lysosomes of this type are essentially storage granules for enzymes, sequestered by membranes from contact with the cytoplasm; the enzymes are elaborated by the usual mechanism of protein synthesis – RER-SER-Golgi complex. Lysosomes number about 30 per hepatocyte114 and are found particularly in the neighbourhood of the bile canaliculus. They are capable of discharging their contents into bile, forming one of the few mechanisms for entry of proteins into bile (the other being transcellular vesicular transport of soluble plasma proteins).115 Lysosomal pleomorphism reflects a variety of functions. First, although the liver cell is long-lived, there is evidence for turnover of its cytoplasm and organelles, and cytoplasmic constituents may become incorporated within, and digested by, the primary lysosome, forming an autophagic vacuole, forming a so-called secondary lysosome. Autophagic vacuoles therefore show fragments of organelles or of cell inclusions in various stages of digestion. Second, lysosomes also incorporate: lipofuscin pigment, which may accumulate, undigested, over long periods, forming so-called residual bodies: material of exogenous origin, including iron, stored as ferritin, which accumulates in large quantities in iron overload states; and copper which accumulates in copper overload conditions and cholestasis. Third, coated vesicles and multivesicular bodies result from receptor-mediated endocytosis.115 In a complicated sequence of intracellular events, ligand-receptor complexes in clathrin-coated pits on the basolateral cell plasma membrane are internalized to form endocytic vesicles, or endosomes. Soluble ligands which are internalized in this way include insulin, low-density lipoproteins, transferrin, IgA and asialoglycoproteins. Fusion of endosomes occurs to form multivesicular bodies. Some of these vesicles are responsible for transcytosis or intracellular transport from the basolateral domain to the canalicular domain; others fuse with primary lysosomes and their contents undergo partial degradation before being exocytosed at the canalicular or basolateral domain; still others undergo complete degradation and become increasingly electron dense with the formation of dense bodies.113,115 Micro­ tubules appear to have an important role in sorting the pathways along which endocytic vesicles move within the hepatocyte.116

Peroxisomes These are ovoid single membrane-bound granules 0.2–1.0 µm in diameter (Fig. 1.16). They were first described as microbodies by Rouiller and Bernhard in 1956.117 The properties of peroxisomes in liver have been reviewed elsewhere.118–120 Each hepatocyte may contain 300–600 peroxisomes and they comprise 1.5–2% of cell volume. There is morphological heterogeneity between species; in the rat, peroxisomes contain a paracrystalline striated core or nucleoid in which urate oxidase is concentrated; human peroxisomes lack a core.121 Peroxisomes contain oxidases which use molecular oxygen to oxidize a number of substrates with the production of hydrogen peroxide (hence the name of the organelle) which, in turn, is hydrolysed by peroxisomal catalase. Approximately 20% of the oxygen consumption of the liver is used in peroxisomal activity. The energy produced by this oxidation is dissipated as heat. An alcohol overload may be metabolized in the liver by peroxisomal catalase. Drugs, such as clofibrate which lowers blood lipids, cause a proliferation of peroxisomes, an increase that has been causally linked to the hypolipidaemic action.122 Alterations in hepatocyte

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MacSween’s Pathology of the Liver

peroxisomes have been reported in bacterial infections, viral hepatitis, Wilson disease and alcoholic liver diseases.123,124 A number of metabolic disorders have been described in which there is either an absence of peroxisomes or a deficiency of peroxisomal enzymes.120,123 The liver involvement in these is discussed in Chapter 4.

Mitochondria These are large organelles (1.5 µm in diameter and up to 4 µm long) numbering approximately 1000 per cell, and constitute about 20% of the cytoplasmic volume of hepatocytes.125 Mitochondria may fuse and are remarkably mobile organelles which move about in the cytoplasm, closely associated with microtubules. They show the features commonly regarded as characteristic of mitochondria in general: an outer membrane, separated by a gap from an inner membrane, from which highly convoluted cristal folds project into the interior of the organelles, the lumen of which contains a so-called matrix (Fig. 1.15). The cristae considerably increase the area of the inner membrane and, in liver cells, it constitutes about a third of the total membrane of the cell. Crystalloid structures may be present within the matrix, but are a nonspecific feature and not related to any disease. More importantly, the matrix contains electron-dense granules (Fig. 1.15), which may represent concentration of Ca1+, a small circular DNA and ribosomes. The latter elements are notable, since mitochondria carry their own genomic material126 which contributes to synthesis of their own protein complement. The larger proportion of the organelle’s protein, however, is encoded by nuclear DNA and then imported into the mitochondria. Mitochondria are self-replicating with a half-life of about 10 days. Mutations in the mitochondrial genome account for various mitochondrial myopathies.127 More recently, it has become apparent that specific biochemical abnormalities of mitochondria may play an important role in the pathogenesis of certain liver diseases and that genetic defects in mitochondrial proteins and enzyme systems may be the underlying cause of other liver and metabolic diseases. Because mitochondria possess a distinct and unique extranuclear genome a new class of maternally, or mitochondrially, inherited diseases has emerged (Chapter 4). The structural compartmentation of mitochondria provides for topographical localization of various enzyme systems, the details of which form almost a science in themselves. It need only be said here that the outer membrane is relatively unimportant as a locus for enzymes; it keeps the inner membrane together and contains porin, a transport protein which forms channels which are permeable to molecules less than 2 kD.125 The inner membrane and cristal lamellae support the respiratory chain enzymes concerned with oxidative phosphorylation which generates ATP. The matrix contains most of the components of the citric acid cycle, and the enzymes involved in β-oxidation of fatty acids and in the urea cycle. Mitochondria are randomly distributed within individual hepatocytes but are smaller and less numerous in centrilobular than in periportal cells,128 the zonal implications of which are beyond the scope of this chapter.

The cytoplasm The cytoplasm of any cell is a highly concentrated matrix of proteins and microfilaments, within which organic and inorganic solutes diffuse.129,130 Movement of larger components through the matrix, especially membrane-bound vesicles, involves directed transport along the cytoskeletal fibres,114,131 the composition of which are described immediately below. A major macromolecular complex in the cytoplasm is glycogen. A principal function of liver is the synthesis of glycogen (from glucose, or from lactic and pyruvic acids

18

or glycerol), its storage and its breakdown and release as glucose into the circulation. Hence, hepatocellular glycogen is depleted under fasting conditions, disappearing last from the centrilobular cells; on refeeding, it appears first in periportal cells and when fully restored is uniformly distributed throughout the lobule. In electron micrographs, glycogen appears as dense granules of two types, β particles, 15–30 nm in diameter and α particles, aggregates of the smaller particles arranged in rosettes (Fig. 1.16). Intranuclear glycogen usually appears as β particles.

The cytoskeleton The major components of the cytoskeleton of most eukaryotic cells comprise 6 nm microfilaments, 8–10 nm intermediate filaments and 20 nm microtubules; the definitive review of their structure and function in the hepatocyte remains that of Feldmann.132 These are structurally, chemically and functionally distinct linear macromolecules coursing through the cytoplasm. Intermediate filaments are relatively stable macromolecules, capable of modulation measured in minutes and longer time intervals. Microfilaments and microtubules are highly dynamic structures capable of rapid poly­merization and depolymerization on a second-to-second basis, and hence rapid adaptation in response to functional demands. For all of these macromolecules, polymerization and depolymerization of their constituent molecules is under the influence of various intracellular factors which include free Ca1+ ions, high energy compounds and associated proteins. In addition, there are a number of accessory proteins which modulate these components, and which link them to one another, to cell organelles and to the cell membrane; these are part of a microtrabecular lattice or cytomatrix. These structures interact to regulate internal organization, cell shape, movement, secretion and divisions.132–134

Microfilaments Microfilaments are double-stranded molecules of polymerized fibrous (F) actin; the monomeric form of the protein is globular (G) actin and these two forms exist in equilibrium in the cell. The microfilaments are present in bundles and form a three-dimensional intracellular meshwork. There is extensive intracellular binding and cross-linking with other intracellular proteins such as myosin, lamin and spectrin. The filaments are mainly located at the cell periphery; they attach to the plasma membrane and extend into microvilli. They are particularly concentrated in the pericanalicular zone forming a pericanalicular web135 and attach to the junctional complexes which limit the canaliculus. Four main functions are envisaged for the contractile microfilaments of the hepatocyte: (1) translocation of intracellular vesicles implicated in bile secretion, especially via insertion and removal of canalicular plasma membrane transport proteins; (2) coordinated contraction producing peristaltic movement in the canaliculus;136 (3) together with microtubules they exert transmembrane control over the topography of intrinsic proteins in the phospholipid bilayer of the cell membrane, thus influencing the protein mosaic and hence the functional differentiation of a particular membrane domain;137 (4) they may modulate the structure and tightness of the so-called tight junction and in this way regulate the permeability of the paracellular pathway.138,139 The functional roles for microfilaments involve cell membrane motility, endo- and exocytosis, secretion and vesicle transfer.

Microtubules Microtubules are a family of unbranched rigid tubules of variable length which are structurally similar in all cells. They are polymers composed of two subunits of tubulin, α and β. Polymerization and

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

growth takes place from organizing centres including centrioles. Microtubules are part of the mitotic apparatus and are therefore important in cell division. They are also present in cell cilia. Like the microfilaments, they attach to and cross-link a number of proteins. Microtubules are involved in the blood-bound secretion of several liver cell products including lipoprotein, albumin, retinolbinding proteins, secretory component, fibrinogen and other glycoproteins.140,141 As a cytoskeletal framework, they play a role in the intracellular translocation of vesicles containing IgA and horseradish peroxidase.141–143

Intermediate filaments Intermediate filaments are a family of self-assembling protein fibres.143 They are structurally similar in all cells, with component polypeptides arranged in an α-helical coiled-coil arrangement which confers tensile strength. These filaments comprise the central scaffold of cells, imparting structural stability to the threedimensional intracellular architecture. Unlike the microtubules and microfilaments which are specifically composed of their respective subunit classes, intermediate filaments are strikingly heterogeneous in subunit composition and antigenicity. Intermediate filaments are grouped into five general types whose distribution is cell-specific:144 keratin, desmin, vimentin, glial fibrillary acidic protein and neurofilaments, the expression of which is highly specific to cell type. Keratins are the intermediate filaments of epithelial cells and are present in hepatocytes and, in greater amounts, in bile duct epithelium. In hepatocytes, they are located just inside the plasma membrane and are particularly condensed as a pericanalicular sheath which extends into desmosomes. They are linked to desmosomes on the lateral plasma membrane of hepatocytes, and in so doing providing scaffolding for the bile canalicular region. They also attach to other components of the cytoskeleton, as for example serving as anchors for the contractile activities of microfilaments. Keratins also attach to organelles such as the RER and vesicles. Keratins function as ‘the mechanical integrators of cellular space’, since their firm attachment to desmosomes, and the latter to desmosomes on adjacent hepatocytes, provides an integrated continuity of stable three-dimensional architecture across a multicellular region.133 Studies using nickel to induce their disorganization indicate that, in the hepatocyte, they maintain structural polarity, provide a scaffolding for the bile canaliculus and provide a framework for the distribution of actin and endocytotic vesicles along the plasma membrane.143,145 In disease states the hepatocellular keratin network, in particular, may become highly disrupted, as discussed later.

The hepatic sinusoid and the sinusoidal cells The sinusoids (Fig. 1.17) have an average diameter of about 10 µm, but they may distend to about 30 µm. Periportal sinusoids are more tortuous than the perivenular ones.146 Four distinct types of sinusoidal cell can be identified (Fig. 1.18), each with its own characteristic morphology, topography and population dynamics:147 the lining of the sinusoids is formed by endothelial cells; the hepatic stellate cells are found in the space of Disse, which lies between the sinusoidal endothelial cells and the hepatocytes; the Kupffer cells and liver-associated lymphocytes lie on the luminal aspect of the endothelium.

Sinusoidal endothelial cells These form an attenuated cytoplasmic sheet (about 50–80 nm in maximum thickness) perforated by numerous holes (fenestrae). Unlike endothelial cells elsewhere, hepatic sinusoidal endothelial

A

B Figure 1.17  (A) SEM illustrating normal hepatic sinusoid in rat liver. Note the regular distribution of fenestrae in the sieve plates, which are separated by intervening cytoplasmic processes. H, hepatocyte. (B) SEM. Endothelial fenestrations (f) of about 0.1 µm are grouped together in sieve plates. Processes of endothelial cells show small holes, most probably representing the pinching off of micropinocytotic vesicles (arrows). SD, space of Disse.

cells apparently do not form junctions with adjacent endothelial cells (Figs 1.17B, 1.18A). The fenestrae are so abundant that, on SEM, the greater part of the cell has a net-like appearance, forming a tenuous barrier, reinforced here and there where adjacent endothelial cells overlap one another (Fig. 1.17B). The fenestrae vary greatly in size, but fall, in general, into two size categories: small fenestrae (0.1–0.2 µm in diameter) grouped in clusters, forming so-called sieve plates; and large fenestrae (up to 1 µm in diameter) which are more numerous at the distal end of the sinusoid. Thus, endothelial cell porosity is higher in the perivenular zone than in the periportal zone.148 The smaller fenestrae traverse individual endothelial cells. The larger are usually intercellular, and some workers consider that they may be artefacts due to fixation.147 Regardless, there is evidence that fenestrae are labile structures whose diameter may change in response to endogenous mediators (e.g. serotonin)149 and exogenous agents such as alcohol.150 The extracellular matrix in the space of Disse also modulates the fenestrae. For example, lack of cell– matrix interaction results in loss of fenestrae in cultured sinusoidal endothelial cells, whereas cells plated on human amnion basement

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MacSween’s Pathology of the Liver

A

B

C

D

Figure 1.18  (A) TEM illustrating a sinusoid (S) with its lining endothelial cells (E); SD, space of Disse; CO, collagen bundles. Human liver, ×11 500. (B) TEM showing a stellate cell (SC) in the space of Disse. Note cell processes, small fat droplet and rough endoplasmic reticulum. Human liver, ×11 500. (C) TEM of a Kupffer cell with numerous cytoplasmic lysosomes (L). Note irregular microvillous projections (MV); sinusoidal lumen (SL). Small rims of endothelial lining cells are observed at both sides of the Kupffer cell. Human liver, ×9200. (D) Liver associated lymphocyte within a sinusoid. Note dense granules in the cytoplasm (arrows). Human liver, ×18 400. (Courtesy of Professor R. De Vos)

20

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

membrane retain their fenestrae.151 The fenestrae lack diaphragms and since, in most species, a basement membrane is absent on the deep surface of the sinusoidal endothelium, there is direct continuity between the sinusoidal lumen and the perisinusoidal space of Disse. This unique porous structure allows the endothelial cells to coarsely filter the sinusoidal blood. Solutes pass freely through the fenestrae from the lumen into the space of Disse and come into contact with the basolateral plasma membrane of hepatocytes. Large particles such as newly generated chylomicrons, however, are excluded. Sinusoidal endothelial cells show a number of phenotypic differences compared with vascular endothelium.152 They do not bind the lectin Ulex europaeus and, in most species, do not express factor VIII related antigen (von Willebrand factor), although the cells assume these properties in chronic liver disease.151 Furthermore, they contain absent to low levels of other molecules characteristically found in vascular endothelium, such as E-selectin, CD31 and CD34,153 but do express Fcγ IgG receptors (CD16 and CDw32), CD4, CD14 and amino-peptidase N.154 They also exhibit membrane immunoreactivity for ICAM-1.154 The natural ligand for this adhesion molecule, LFA-1, is present on Kupffer cells; this receptor may therefore be involved in adhesion of Kupffer cells to the endothelial lining.154 Upregulation of intercellular adhesion molecule-1 (ICAM1) expression in sinusoidal endothelial cells may be important in ‘trapping’ lymphocyte associated antigen-1 (LFA-1) positive lymphocytes in inflammatory liver diseases.155 There is also sinusoidal endothelial cell heterogeneity within the acinus: the increased porosity in the perivenular zone has already been mentioned but, in addition, variation in cell size, heterogeneous lectin binding, and expression of various receptors, cytoplasmic density, endocytic capacity and surface glycosylation have also been demonstrated.150,154,156,157 Another unusual feature of sinusoidal endothelial cells is their high endocytotic activity.158,159 This process appears to be directed towards uptake and lysosomal degradation of compounds, rather than providing an alternative route for their transport from the sinusoidal lumen to the space of Disse. A large number of endogenous compounds may be endocytosed, some of which are effete molecules and are cleared from the circulation and others of which are modified and do appear to undergo transcytosis to hepatocytes, perhaps in a more selective fashion than macromolecular solutes passing only through the fenestrae.159 Thus, the sinusoidal endothelial cells have a role in removing soluble immune complexes (like the Kupffer cells) and have also been shown to store and metabolize serum immunoglobulin and to remove hyaluronic acid/chondroitin sulphate proteoglycans from the circulation.150,160,161 The sinusoidal endothelial cells also have synthetic activity and produce nitrous oxide (NO), endothelins and prostaglandins and possibly cytokines such as interleukin-1 and interleukin-6, all of which have potent effects on vascular tone and the functions of nearby cells.162–164

The space of Disse The space of Disse (Fig. 1.18A) lies subjacent to sinusoidal endothelial cells, and is the fluid space in direct contact with the basal plasma membrane surface of hepatocytes, from which abundant microvilli project into the space and may be of importance in keeping the space open. This space is not normally discernible in biopsy material but in autopsy liver the hepatocytes shrink from the sinusoids and the space is then characteristically evident. Studies with TEM show continuity of the space of Disse with the lateral intercellular space between adjacent hepatocytes (Fig. 1.18B), considerably expanding the perisinusoidal fluid compartment.165 The space of Disse forms, therefore, an extensive and almost unique

extravascular space. As noted earlier, the sinusoidal endothelium is not only discontinuous and extensively fenestrated, but in many species (including man) lacks a basement membrane. It is thus freely permeable to blood plasma, which enters the space of Disse and comes into direct contact with hepatocytes and hepatic stellate cells. This extravascular plasma constitutes the immediate medium of exchange between blood and hepatocytes, whose surface area of contact is increased by the abundant microvilli. The plasma is then presumed to flow towards the hepatic veins. However, there is evidence that fluid within the space of Disse may also be taken up by lymphatic spaces in the periportal zone in a retrograde fashion, either re-entering the sinusoidal blood or continuing within lymphatic channels to exit via the hilar lymphatics. The nature of the anatomical link between the periportal ends of the space of Disse and the lymphatics is discussed later. The extracellular matrix within the space of Disse is produced predominantly by the hepatic stellate cells, and constitutes the structural ‘reticulin’ framework of the liver. Hepatocytes may produce some collagen and some proteoglycans; the role of Kupffer cells and sinusoidal endothelial cells is mediated through the production of cytokines which modulate the synthetic activity of the stellate cells, but they may themselves produce small amounts of proteoglycans. The extracellular matrix plays a major role in the normal biology of the liver, influencing hepatocyte, sinusoidal endothelial cell and stellate cell function. Interaction between this matrix and these cells is of fundamental importance in maintaining their differentiation, growth and function.166–168 There is a gradient in the extracellular matrix in the space of Disse with variation in its amount and composition with increasing distance from the portal tracts.169,170 Thus, laminin, collagen type IV and heparan sulphate predominate in the periportal zone whereas in the perivenular zones fibronectin, collagen type III and dermatan sulphate are more abundant.169 In contrast to what happens in other organs, the extracellular matrix does not act as a diffusion barrier between the plasma and the hepatocytes. There is no identifiable basement membrane and this facilitates the two-way exchange which has to take place between the hepatocytes and the blood in the sinusoids. The matrix components interact with the hepatocyte and endothelial cell membranes through various surface integrins and other receptors. Hepatocytes have receptors for fibronectin, laminin, type I and type IV collagens,171–174 and they can also bind proteoglycans.175 The precise role of the integrins in the cell–matrix interactions is not fully understood. The proteoglycans can also act as adhesion and receptor molecules and act as an important reservoir for cytokines and growth factors.176–178

Hepatic stellate cells Within the space of Disse are stellate cells whose long cytoplasmic processes surround the sinusoids. Originally identified by Boll and von Kupffer in the 1870s, they were largely ignored until 1951 when Ito described their morphological features on light microscopy.179 They were subsequently referred to under a variety of terms – Ito cells, hepatic lipocytes, fat storing cells, stellate cells and parasinusoidal cells.180,181 The now accepted nomenclature for them is hepatic stellate cells (HSC).182 Their pathobiology and their role in regulating inflammation in the liver have been extensively reviewed.183–186 It is of interest that similar stellate cells, and which also share the property of vitamin A storage, exist in the human pancreas187 and there is also evidence that there is a more widespread distribution involving lung, kidney and gut.187,188 In the normal liver parenchyma, extracellular matrix may be produced by the perisinusoidal HSCs, hepatocytes, and sinusoidal

21

MacSween’s Pathology of the Liver

endothelial cells.189 Stellate cells, hepatocytes, and sinusoidal endothelial cells are capable of synthesizing types I and IV collagen. Type III collagen and fibronectin can also be elaborated by these cells.186 Stellate cells have several key functions: (1) retinoid storage and homeostasis; (2) remodelling of extracellular matrix by production of both matrix components and matrix metalloproteinases; (3) production of growth factors and cytokines; and (4) contraction and dilation of the sinusoidal lumen.186 Stellate cells are present in the subendothelial space of Disse and sometimes in the peri­ sinusoidal recess between hepatocytes. Stellate cells comprise less than 10% of total resident liver cells under normal conditions, and are regularly spaced along the sinusoids (approximately 40 µm from nucleus to nucleus).188 Despite their relative scarcity, they have long cytoplasmic processes which can cover the entire peri­ sinusoidal area.186 It is notable that autonomic nerve endings running in the space of Disse come into contact with stellate cells, and the stellate cells respond to α-adrenergic stimulation. Hepatic stellate cells are not readily visualized on light micro­ scopy (Fig. 1.19A) but they may be prominent in some pathological conditions.189–191 They are readily seen in very thin histological sections or by transmission electron microscopy (Fig. 1.17B). The cells resemble pericytes and they establish close contacts with adjacent hepatocytes. They contain many small lipid droplets (Fig. 1.17B)

which are rich in vitamin A; this can be demonstrated by fluorescence microscopy when excitation light of 328 nm length is used and by gold chloride impregnation. Rough endoplasmic reticulum and Golgi apparatus are well developed in these cells (Fig. 1.17B). Stellate cells express vimentin, desmin and GFAP in their quiescent state (Fig. 1.19B).192,193 A key feature of stellate cells is up regulation of alpha smooth muscle actin (α-SMA) when the cells are activated, imparting to them a contractile phenotype.186,194 Hepatic stellate cells have four main functions in the liver:186,195,196

• They produce the extracellular matrix proteins both in the normal liver and when activated in the process of liver fibrogenesis.186,188,196 • They act in a pericyte-like manner around the sinusoids and may have a role in the control of microvascular tone in the normal liver.181,197,198 Activated stellate cells have a definite contractile role in the injured liver owing to their upregulation of α-SMA, and they respond to vasoactive agents such as endothelin-1 and nitric oxide.197,198 • They are a major site of storage for vitamin A.186,198 Dietary retinyl esters reach the liver in chylomicron remnants. These pass from the sinusoidal lumen through the endothelial fenestrae and are taken up by hepatocytes. Most of the endocytosed retinol is rapidly transferred to the stellate cells for storage by an as yet poorly defined transport mechanism.199 The cells contain a high concentration of cellular retinoid-binding protein and cellular retinol-acid binding protein. • They play a role in hepatic regeneration both in the normal liver and in response to liver injury.184 They express hepatocyte growth factor184,200 and this can be enhanced in human hepatic stellate cells in response to insulin-like growth factor-2.201 The mechanisms of hepatic stellate cell activation, and consequences, are discussed in the section on Fibrosis, below.

Kupffer cells A

B Figure 1.19  Hepatic stellate cells. (A) These may occasionally be seen on optical microscopy; the fat globules are phloxinophilic in this section stained by Masson trichrome and the perisinusoidal location of the cell is readily appreciated. (B) GFAP immunoreactivity of hepatic stellate cells in normal rat liver. (Courtesy of Dr Liena Zhao)

22

Kupffer cells are hepatic macrophages and are present in the lumen of hepatic sinusoids (Fig. 1.18C). They belong to the mononuclear phagocytic system but manifest phenotypic differences which distinguish them from other macrophages. They are of considerable importance in host defence mechanisms and in addition have an important role in the pathogenesis of various liver diseases.202 On SEM, Kupffer cells have an irregular stellate shape203 and within the sinusoidal lumen the cell body rests on the endothelial lining (Fig. 1.20). They are more numerous in the periportal sinusoids204 and as noted above there is some evidence that, like hepatocytes, Kupffer cells also manifest functional heterogeneity in the lobule.204,205 They never form junctional complexes with endothelial cells, but they may be found in gaps between adjacent endothelial cells and their protoplasmic processes may extend through the larger endothelial fenestrae into the perisinusoidal space of Disse. The luminal surface shows many of the structural features associated with macrophages: small microvilli and microplicae and sinuous invaginations of the plasma membrane. Kupffer cells have been considered to be fixed tissue macrophages, but they appear capable of actively migrating along the sinusoids, both with and against the blood flow, and can migrate into areas of liver injury and into regional lymph nodes.206 They contain lysosomes and phagosomes, and the cisternae of their endoplasmic reticulum are rich in peroxidase. Their primary functions include the removal by ingestion and degradation of particulate and soluble material from the portal blood, and in this they

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

in the yolk sac.219 These data suggest that Kupffer cells may have a dual origin.

Liver-associated lymphocytes

Figure 1.20  SEM. Part of Kupffer cell lying in a sinusoid and showing characteristic microvilli projecting at the cell surface (arrows). Small rims of fenestrated endothelium can be observed at both sides of the Kupffer cell (f). A small bundle of collagen fibres (c) is situated in the space of Disse (SD) on the right.

discriminate between ‘self’ and ‘non-self’ particles. They act as scavengers of microorganisms, and degenerated normal cells such as effete erythrocytes, circulating tumour cells and various macro­ molecules. These functions are in part carried out nonspecifically, but they are also involved in the initiation of immunological responses and the induction of tolerance to antigens absorbed from the gastrointestinal tract. The efficiency of this clearance function is shown by the fact that removal of particulate material is limited only by the magnitude of hepatic blood flow; removal of particles may approach single-pass efficiency. Kupffer cells play a major role in clearance of gut-derived endotoxin from the portal blood and this is achieved without the induction of a local inflammatory response. It has been estimated that the portal blood concentration of endotoxin varies from 100 pg/mL to 1 ng/mL.207 The precise mechanisms involved are not fully understood, but there appears to be finely balanced autoregulation between the release of proinflammatory and inflammatory mediators such as interleukins 1 and 6, tumour necrosis factor α (TNFα) and interferon-γ, and mediators such as interleukin-10 which suppresses macrophage activation and inhibits their cytokine secretions.208–210 The role of Toll-like receptors (TLRs) in this response is considered elsewhere in the text. Several cytokines released by activated Kupffer cells are also thought to have local effects, modulating microvascular responses and the functions of hepatocytes and stellate cells.211 Although Kupffer cells can express class II histocompatibility antigens212 and can function in vitro as antigen-presenting cells, they appear to be considerably less efficient at this than macrophages at other sites.213 Their principal roles in the immune response therefore appear to be antigen sequestration by phagocytosis and clearance of immune complexes.214 There is firm evidence from bone marrow transplant and liver transplant studies that Kupffer cells are derived, at least in part, from circulating monocytes.215,216 However, Kupffer cells are capable of replication and their local proliferation accounts for a substantial part of the expansion of this cell population in response to liver injury.217,218 Furthermore, Kupffer cells appear in the fetal liver of the mouse before there are circulating monocytes and there is evidence that they are derived from primitive macrophages which first appear

Hepatocytes constitute 80% of the cells in the normal liver. Of the remaining 20%, bile duct epithelial cells (cholangiocytes) comprise 1–3%, sinusoidal endothelial cells 10%, Kupffer cells 4%, and lymphocytes 5%. The last include cells of the adaptive immune system (T and B lymphocytes) and innate immune system: natural killer (NK) and natural killer T (NKT) cells.220 NK cells comprise 31% of hepatic lymphocytes and NKT cells 26%. The liver is thus particularly enriched with cells of the innate immune system, compared to other parenchymal organs. While this has immediate value for dealing with foreign antigens released from the gut into the splanchnic circulation, it also means that the liver is well equipped for an immune response to neoantigens expressed within its substance.221,222 It has been estimated that an average normal human liver contains approximately 1 × 1010 lymphoid cells.223 These lymphocytes are predominantly located within portal tracts but are also scattered throughout the parenchyma, where they are found in loose contact with Kupffer cells or sinusoidal endothelial cells. In the peripheral blood 85% of the lymphocytes comprise T and B cells which possess clonotypic antigen-specific receptors. In contrast, up to 65% of lymphocytes within the liver are NK cells, T cells expressing γδ T-cell receptors (TCR) and T cells expressing NK molecules (NKT cells); clonotypic T and B cells are only a minority of intrahepatic lymphocytes. NKT cells appear morphologically as ‘pit cells’ – large granular lymphocytes that reside in the subendothelial interstices of the space of Disse.222,224 The liver contains the largest number of γδ T cells in the body.223,225 γδ T cells are also found in the skin, gut and respiratory mucosa and in the pregnant uterus, and accumulate at sites of infection.226 They secrete various cytokines and can lyse antigen-bearing target cells. Their precise function in the liver is not yet clear. Hepatic NKT cells co-express a T-cell receptor and NK activating and inhibitory receptors. They can be further subclassified on the basis of their expressing various types of TCRs and various NK receptors.227 Functional studies have demonstrated that hepatic NKT cells have numerous cytotoxic activities and produce multiple cytokines.227 Their major role therefore may be to effect local immunological reactions through the production of cytokines. The relative frequency of the various subpopulations of liverassociated lymphocytes varies between individuals, probably reflecting each individual’s immunological status; this in turn is affected by genetic background and both previous and current antigen exposure. It is also possible that the hepatic environment itself may influence the distribution of the various subsets. The presence of these cells in such large numbers indicates that they must subserve important roles in normal hepatic immune responses and immune homeostasis. The liver thus is probably comparable to the gastro­ intestinal tract which is now regarded as an important lymphoid organ.

The biliary system The intrahepatic biliary system was considered earlier with regard to hepatic embryology. We must now examine, in detail, the architecture of the biliary tree. First, the bile canaliculi which run between adjacent hepatocytes form a complicated intra-parenchymal polygonal network. Based on rat studies, canalicular diameter increases gradually from the pericentral region to the periportal region; their diameter enlarges physiologically during periods of high bile flow.228

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MacSween’s Pathology of the Liver

Table 1.1  Terminology of the biliary tree

Generation of branching

Diameter (µm)

Terminology

Remarks

Large bile ducts   First generation

>800

Left and right hepatic duct

  Second generation

400–800

Segmental ducts

  Third generation

300–400

Area ducts

Cylindrical epithelium surrounded by dense fibrous tissue and elastic fibres Peribiliary glands Cylindrical epithelium surrounded by dense fibrous tissue and elastic fibres Peribiliary glands Lower cylindrical epithelium Peribiliary glands

Small intrahepatic bile ducts (not grossly recognizable)

>100 15–100 40–100 15–40

Septal ducts Interlobular ducts Medium-sized Small

Located in periphery of portal tracts Cuboidal epithelium accompanied by artery Lined by 3–4 cuboidal cells

12th generation

100 µm in diameter, and which are lined by a simple tall columnar epithelium with basally situated nuclei. Portal tract fibrous tissue shows some condensation round these septal ducts, but there is no well-marked concentric orientation. The terminology and heterogeneity of the intrahepatic bile ducts as proposed by Desmet et al.229 is summarized in Table 1.1. A definitive quantitative computer-aided three-dimensional study of the human biliary system has been published.230 The larger ducts further anastomose to form the intrahepatic bile ducts, 1–1.5 mm in diameter, which give rise to the main hepatic ducts. Studies by Nakanuma and his colleagues231–233 have demonstrated the presence of glandular elements around the larger intrahepatic bile ducts (Fig. 1.23). These peribiliary glands are of two types: (1) intramural mucous glands, which communicate directly with the bile duct lumen, and (2) extramural mixed seromucinous glands which form branching tubulo-alveolar lobules and secretory ducts that drain into the bile duct lumen. Scanning electron microscopy has shown that hilar ducts may also have irregular side branches and pouches in which bile may be stored and probably modified.234 As described earlier, the intrahepatic bile ducts are supplied by an anastomozing peribiliary vascular plexus derived from the hepatic artery and which drains into periportal sinusoids.

Cholangiocytes Cholangiocytes account for 3–5% of the endogenous liver cell population,235 lining the intrahepatic and extrahepatic bile duct system. More than cells lining inert conduits, cholangiocytes modify the composition of bile during its transit through bile ducts,

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

A

C

Figure 1.23  Large intrahepatic bile duct near the hilum. Note the surrounding mucous and seromucinous peribiliary glands; one mucous gland opens into the duct lumen. (H&E)

B

Figure 1.22  (A) TEM of a cross-section of a bile duct. (L), Lumen; basement membrane (arrows). Human liver, ×5750. (B) TEM of epithelium of a bile duct. Note contorted intercellular space (IC); apical microvilli (MV); junctional complexes (J). Human liver, ×36 800. (Courtesy of Professor R. De Vos) (C) Cross-section of a normal medium sized bile duct of similar size to A. (H&E)

involving secretion and absorption of water, electrolytes and other organic solutes.24,62,236–238 Ultrastructurally, the cholangiocyte has a prominent Golgi complex, numerous cytoplasmic vesicles and short luminal microvilli.162 Studies in the rat suggest that 10–15% of basal bile flow is produced by ductal epithelium239 and it has been estimated that the corresponding contribution in humans is 40%. Secretion is under hormonal control (secretin and somatostatin); secretin is released from the duodenum following vagal stimulation and the presence of acid in the duodenum, and stimulates the secretion of bicarbonate-rich bile.237 That the duct epithelium also secretes IgA and IgM (but not IgG) has been shown immunohistochemically on human duct cells.240 Reabsorption involves water, glucose, glutamate and urate. Bile acids are reabsorbed via biliary epithelium and are recirculated by a cholehepatic shunt pathway via the peribiliary plexus; this recycling promotes bile-acid dependent bile flow in the ducts.57,241–243 Extensive studies of cholangiocytes over the past two decades have proven very fruitful in understanding both the normal formation of bile, and how bile duct biology alters during non-obstructive or obstructive cholestasis.236 Several concepts merit emphasis. First,

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MacSween’s Pathology of the Liver

cholangiocytes are capable of proliferating in response to liver injury or surgical reduction, a process enhanced by secretin receptor signalling and secretin-stimulated choleresis.244 Second, accumulation of bile acids during cholestasis – obstructive or non-obstructive – may have profound effects on cholangiocytes, including stimulation of proliferation and increasing secretin responsiveness.245 However, as cholangiocytes contain an apical sodium-dependent bile acid transporter (ASBT; official symbol NTCP2; gene symbol Slc10a2), they are capable of taking up bile salts from the bile duct lumen and returning them to the peribiliary circulatory plexus.246 While the resultant cholehepatic shunt can further stimulate bile secretion by hepatocytes,247 in the setting of obstructive cholestasis bile salts can dump into the systemic circulation for elimination in urine. Third, cholangiocytes are responsive to innervation, as discussed below. Bile duct epithelial cells have phenotypic characteristics which distinguish them from hepatocytes, and also display phenotypic heterogeneity along the different segments of the biliary tree.248–250 They express receptors for epidermal growth factor, secretin and somatostatin.251 Normally, they express class I major histocompatibility complex (MHC) antigens but not class II; cytokine-induced expression of class II antigen is seen in graft-versus-host disease, allograft rejection, primary biliary cirrhosis and primary sclerosing cholangitis and this may be important in the pathogenesis of the bile duct injury in these diseases. Indeed, BDEC may themselves secrete proinflammatory cytokines and contribute to the destructive events that occur in autoimmune or secondary inflammatory biliary diseases.252 Bile duct cells express more cell-matrix adhesion mole­ cules or integrins than hepatocytes, including α2 β1, α3 β1, α5 β1, α6 β1, and α6 β4 concurring with those expressed by most simple epithelial cells.253 Bile duct epithelium also expresses glutamyl transpeptidase, carcinoembryonic antigen and epithelial membrane antigen, representing other phenotypic differences when compared with hepatocytes, the precise significance of which is, as yet, uncertain.249,251 Thus, even with this short discussion it is clear that there is a very dynamic biology of the cells lining the biliary tree.254

Other constituent tissues The extracellular matrix The liver normally has only a small amount of connective tissue in relation to its size. Whereas in the human body collagen constitutes about 30% of the total protein, the corresponding figure for the liver is 5–10%. Underlying the visceral peritoneum of the liver, or serosa, there is a layer of dense connective tissue admixed with elastic fibres which varies in thickness from 40 to 70 µm. This constitutes Glisson’s capsule, irregular extensions of which extend as delicate fibrous septae up to 0.5 cm into the superficial parenchyma. These anatomically normal septae produce some architectural distortion and the appearance of ‘fibrosis’, which must not be misinterpreted when wedge biopsies are examined. Condensation of Glisson’s capsule occurs at the porta hepatis, and the fibrous tissue then extends into the liver supporting and investing the portal vein, hepatic artery and bile duct branches, thereby constituting the portal tracts. Some extension of the capsular tissue also accompanies the large hepatic vein branches, but there is no fibrous sheath surrounding terminal hepatic venules, which are in direct contact with perivenular hepatocytes. The components of the extracellular matrix are collagens, glycoproteins and proteoglycans (Table 1.2). In the normal liver parenchyma, interstitial collagens (types I and III) are concentrated in portal tracts and around terminal hepatic veins, with occasional bundles in the space of Disse. Delicate strands of type IV collagen (reticulin) course alongside hepatocytes

26

Table 1.2  Extracellular matrix components

Component Collagens Type I

Normal distribution Portal tract points of Portal tract Portal tract space of Portal tract Portal tract

matrix, hepatic veins, inflection in hepatic cords matrix, space of Disse basement membranes, Disse matrix, space of Disse matrix, space of Disse

Fibronectin Entactin (nidogen) Elastin Fibrillin

Portal tract space of Portal tract Portal tract Portal tract Portal tract

basement membranes, Disse matrix, space of Disse basement membranes matrix matrix

Proteoglycans Heparan sulphate

Portal tract basement membranes

Type III Type IV Type V Type VI Glycoproteins Laminin

Data from Martinez-Hernandez and Amenta 1993.255

in the space of Disse. This so-called reticulin network or framework is usually visualized by silver impregnation staining methods (Fig. 1.24).

Collagens Collagens are composed of three identical or similar polypeptide chains folded into a triple helix to give the molecule stability. Numerous types of collagen have been described and of these, types I, III, IV, V and VI are found in the liver. Collagen XVIII also has been reported.256,257 Types I and III comprise more than 95% of the collagen in normal liver, with IV, V and VI contributing approximately 1%, 2–5% and 0.1%, respectively.258 The types of collagens fall broadly into two categories – fibrillar and basement membrane collagens. Types I, III, and V of the fibrillar collagens and types IV and VI of the basement membrane collagens have been identified in the liver. Types I, III and V are confined mainly to the portal tract and terminal hepatic vein wall. Type I collagen corresponds to the doubly refractile mature collagen in portal tracts and around the walls of hepatic veins, and is evident in tissue sections simply by lowering the substage condenser to increase refraction. Type III collagen is also present in the reticulin framework of the sinusoids, associating with type IV collagen, to form a two-dimensional lattice.256 Type IV collagen also forms the basement membranes around bile ducts, arteries and veins. Type VI collagen, in contrast, is found in the interstitial matrix of the portal tract. It is absent from basement membranes but is frequently present near blood vessels and it may have a role in anchoring vascular tissue to the perivascular matrix.257

Glycoproteins and proteoglycans The collagens are intimately complexed and interwoven with glycoproteins and the proteoglycans to form the total supporting structure of the liver. The non-collagenous glycoproteins include laminin, fibronectin, entactin and elastin. Laminin is a large glycoprotein (1000 kD) produced by stellate cells and endothelial cells in the normal liver, and in increased quantities by stellate cells and hepatocytes in the diseased liver.259 Laminin promotes cell adhesion,

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

A

B

C

D

Figure 1.24  (A) Liver biopsy from a child of 17 months; note the twin-cell liver plates: these are better shown on a reticulin preparation at the same magnification in (B), in which the nuclei are seen in a perisinusoidal position; (C) adult liver showing normal single-cell liver plates with centrally placed nuclei; (D) adult liver at same magnification as (C), showing a regenerative response with twin-cell liver plates; as in (B), the nuclei tend to be in a perisinusoidal position; there is also rosette formation. (Gordon-Sweet reticulin)

migration, differentiation, and growth148,260–262 and is an important mediator of capillary formation by endothelial cells.263–265 The fibronectins represent a class of large molecular weight glycoproteins which exist in plasma and cellular forms. In extracellular matrix, fibronectin exists as thin filaments associated with collagen fibres.255 Entactin, also referred to as nidogen, is a highly sulphated, dumb-bell shaped glycoprotein restricted to basement membranes, and hence is generally absent from the space of Disse. Elastin fibres are normally scattered throughout portal tracts. Elastin is deposited in fibrous septa of the cirrhotic liver over time; the presence of elastin in fibrous septa thus provides some indication that the fibrous tissue has not been deposited recently. The proteoglycans are macromolecules consisting of a central protein core to which glycosaminoglycans and oligosaccharide side-chains are attached. The proteoglycans are classified according to the type of glycosaminoglycan present. They contain specific functional domains which interact with cell surface receptor mole­ cules. The proteoglycans include heparan sulphate, chondroitin sulphate, dermatan sulphate and hyaluronic acid. Proteoglycans have a core protein with a variable number of unbranched carbohydrate side chains which are composed of repeating sulphated disaccharide units. For heparan sulphate these are iduronic acid-N-acetylglucosamine, for dermatan sulphate iduronic acid-Nacetylgalactosamine, and for chondroitin sulphate glucuronic acidN-acetylgalactosamine. Hyaluronic acid is the exception, as it lacks a protein core and is formed as a non-sulphated polysaccharide from glucosamine and glucuronic acid. In the liver, heparan

sulphate is the most abundant and is present in portal tracts, in basement membrane and on the surface of hepatocytes. The strong anionic charge on the proteoglycans contributes to their binding to the other constituents of the extracellular matrix. Heparan sulphate particularly modulates the proliferative and secretory characteristics of mesenchymal cells266 and is an essential extracellular component of basement membranes.176 Laminin is the major glycoprotein in basement membrane and interacts there with type IV collagen; small amounts are normally present in the space of Disse. Fibronectin exists in two isoforms, one of which, plasma fibronectin, is produced by hepatocytes. Fibronectin is also produced by hepatic stellate and sinusoidal endothelial cells. It mediates cell adhesion to collagen. The proteoglycans can function as adhesion molecules267 and can act as receptor molecules on cell surfaces.177 As such, they have been identified as an important reservoir for cytokines and growth factors, by binding up these diffusible substances within the matrix. Remodelling of the extracellular matrix, as during regeneration, can release substantial quantities of cytokines and growth factors.268 Conversely, deposition of the extracellular matrix during fibrogenesis can increase the reservoir of stored cytokines and growth factors within the liver. Fibrillin-1 is a more recently described key matrix protein. Fibrillin-1 polymerizes into ordered microfibril aggregates, which form filamentous longer structures that provide much of the biomechanical tensile properties of tissues. Fibrillin microfibrils without associated elastin are found in the extracellular matrix of

27

MacSween’s Pathology of the Liver

non-elastic tissues. Fibrillin and elastin form elastic fibres.269 In the normal human liver, fibrillin-1 and elastin are found in the vessel walls and connective tissue of portal tracts and in the wall of terminal hepatic veins.270 At the portal : parenchymal interface, only fibrillin-1 is detectable;271 fibrillin-1 is not found in sinusoids. Under obstructive cholestatic conditions, fibrillin-1 expression increases markedly in the expanded portal tracts. Following toxic injury (as from CCl4), fibrillin-1 is found in perivenular areas.

Function of the extracellular matrix in the liver As well as providing the structural framework of the liver, there is evidence that the complex matrix in the space of Disse is essential for maintaining the integrity and function of hepatocytes and sinusoidal cells. In a general sense, the extracellular matrix provides the framework for regulation of cellular polarization, migration, proliferation, differentiation, cell survival, and cell death. This occurs via signalling triggered by interaction between the extracellular matrix and cellular receptors. For example, hepatocyte differentiation in a polarized state requires an extracellular matrix rich in laminin and containing type IV collagen, heparan sulphate, and entactin;272 contact only with collagen matrix leads to loss of hepatocyte polarity and dedifferentiation as measured by expression of hepatocytespecific proteins.261,273,274 Therefore, liver injury that disrupts the sinusoidal subendothelial matrix could result in loss of differentiated hepatocellular function. The sinusoidal subendothelial matrix also helps to preserve the functions and activities of endothelial cells and stellate cells. When stellate cells are maintained on a basement membrane-like gel, they remain spherical with extensive filamentous outbranchings and do not proliferate.275 When cultured on abnormal substrates, they transform into myofibroblasts and proliferate.275 Similarly, the fenestrated sinusoidal endothelial cells lose their fenestrations in association with alterations in the extracellular matrix.148 These events initiate a vicious cycle of reduced porosity of the sinusoidal barrier, impaired movement of solutes and macromolecules into and out of the space of Disse, and hence hepatocellular damage and impaired systemic homeostasis.

Cell–matrix interaction Receptors for components of the extracellular matrix have been identified on hepatocyte membranes.276 Among these is a family of receptors, the integrins, which possess a recognition site for molecules containing the tripeptide sequence arginine–glycine–aspartic acid (RGD). Laminin, fibronectin, entactin, tenascin and type I collagen are all known to have RGD sequences. Hepatocytes have integrin receptors for fibronectin and type I collagen,172,171 and nonintegrin receptors for laminin and type IV collagen.173,277 Hepatocytes also bind proteoglycans and this binding can be saturated, implying a receptor-mediated interaction.278 The extracellular matrix serves as a binding reservoir for key fibrogenic cytokines such as TGFβ, TNFα, PDGF and IL-6, and growth factors such as hepatocyte growth factor (HGF).279 Release of cytokines by the matrix reservoir facilitates rapid activation of stellate cells, even before cytokine synthesis is upregulated. The role of integrins in the interaction of stellate cells and sinusoidal endothelial cells with the matrix is poorly understood. Those receptors that have been characterized on these cells seem to be primarily involved in the clearance of ligands.280

Lymphatics The liver is the largest single source of lymph in the body, producing 15–20% of the overall total volume and 25–50% of the thoracic duct flow.281 Hepatic lymph has an unusually high protein content

28

Figure 1.25  Lymphatic vessels in a medium sized portal tract in human liver. The lymphatic endothelium is selectively labelled by immunohistochemistry using the monoclonal antibody D240.

(about 85–95% of that in plasma) and a high content of cells, of which about 80% are lymphocytes and the remainder macrophages. Indeed it has been calculated that, in the sheep, more lymphocytes migrate through the liver in the lymph than through any other nonlymphoid organ, and that about 2 × 108 macrophages leave the liver in lymph each day.282 The terminal twigs of the intrahepatic lymphatic tree are found in portal tracts as a fine, valved plexus of flattened endothelial tubes, associated with terminal branches of the hepatic artery (Fig. 1.25). Traced centripetally towards the porta hepatis, the lymphatic plexus enlarges and remains primarily periarterial, although in the larger portal tracts it becomes associated also with portal vein branches and bile duct tributaries, adding a fourth element to the traditional ‘portal triad’. Similar but much smaller and functionally less important lymphatic plexuses are associated with the hepatic vein branches. A third plexus, found in the capsule, forms significant anastomoses with intrahepatic lymphatics. Most of the collecting lymphatics leave the liver at the porta hepatis and drain into hepatic nodes located along the hepatic artery and thence to coeliac nodes. There are other important efferent routes: via the falciform ligament and the superior epigastric vessels to the parasternal nodes; from the bare area to posterior mediastinal nodes; and from the visceral surface to the left gastric nodes. As efferent collecting lymphatics leave the liver their walls suddenly thicken, through the acquisition of a muscle layer.283,284 The importance of the anastomoses between intrahepatic and capsular lymphatics is evident when hepatic venous pressure is increased. There follows a great increase in production of hepatic lymph, of protein content identical with that of plasma, indicating unrestricted leakage of protein into Disse space.285 The capsular efferent lymphatics enlarge in response to the increased lymph flow, and exudation of excess lymph from the capsular plexus forms protein-rich ascitic fluid.286 The function of lymphatics is to drain excess fluid and protein from the interstitial spaces of an organ. In the liver the interstitial space of Disse is the most prominent and it is assumed that hepatic lymph is mainly formed there with a small supplement from the peribiliary capillary plexus in the portal tracts. A protein-rich filtrate is produced in the space of Disse because of the free permeability of the sinusoidal endothelium and the consequent absence of a colloid osmotic block.287,288 A protein-poor filtrate is formed by the

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

less permeable peribiliary capillaries and this may dilute the protein of the sinusoidal filtrate.281 The route followed by interstitial fluid formed in the space of Disse to its entry into the first-order lymphatic plexus has long been controversial.281,288,289 It is agreed that lymphatic capillaries are absent within the parenchyma and that there are no direct channels, with a continuous lining, between this space of Disse and the primary lymphatics. Wisse and his colleagues290,291 have suggested that ‘endothelial massaging’ by blood cells may be of importance in causing retrograde fluid movement in the space of Disse. In mice, Mehal has documented retrograde migration of fluorescentlylabelled lymphocytes within the space of Disse towards the parenchymal : portal tract interface.292 Henriksen et al.287 suggested that, as in other tissues, the terminal lymphatics in the portal tracts had anchoring filaments between opposing endothelial cells which regulated the direction of flow from the interstitial space into the lymphatics. Electron microscopic studies289,291 have established the following pathway, by the use of natural markers (precipitated lymph protein and chylomicrons) and artificial tracers injected intravenously (horseradish peroxidase, ferritin, pontamine blue and monastral blue). Fluid formed in Disse space escapes at the periphery of the portal tract through gaps between hepatocytes of the limiting plate, either independently of blood vessels or alongside terminal blood vessels penetrating the limiting plate. These gaps contained hepatocyte microvilli, delicate ‘wicks’ of collagenous fibres and occasional slender processes of portal tract fibroblasts, extending into the parenchyma from the periportal space of Mall. Here, long flattened processes of fibroblasts formed discontinuous linings of ‘spaces’ which contained tracer material, occasional lymphocytes and macrophages, and collagen bundles. These ‘spaces’ were not true lymphatics, since they lacked an endothelial lining, but they appeared to function as prelymphatic channels, leading fluid towards the terminal twigs of the lymphatic tree. The fluid then enters into portal tract lymphatic vessels, to travel down the portal tree and exit through lymphatic vessels in the porta hepatis.

Hepatic nerves While a nerve bundle clearly enters the liver through the hilum, its role remained a source of puzzlement since the orthotopically transplanted liver, which has no innervation, functions well in the recipient.293,294 Recent studies have made clear that hepatic innervation indeed has considerable significance under specific conditions (Fig. 1.26). Autonomic nerve fibres reach the liver in two separate but intercommunicating plexuses around the hepatic artery and portal vein, and are distributed with branching vasculature.295–297 The nerve fibres include preganglionic parasympathetic fibres derived from the anterior and posterior vagi, and sympathetic fibres which are mostly postganglionic with cell bodies in the coeliac ganglia, and which receive their preganglionic sympathetic connections from spinal segments T7–T10. The hilar plexuses also include visceral afferent fibres and some phrenic nerve fibres, probably afferent in character.297 Immunohistochemical studies of human liver, using antibodies to common neural proteins such as protein gene product (PGP) 9.5 and N-CAM, have shown that nerve fibres not only are present around vascular structures in portal tracts but extend into the parenchyma, running along the sinusoids. Fluorescence histochemistry298 and immunohistochemistry using antibodies to dopamine-β-hydroxylase and tyrosine hydroxylase299 have shown that the majority of intrasinusoidal fibres are sympathetic; many contain neuropeptide tyrosine (NPY), a regulatory peptide commonly found in adrenergic nerves.299 Unmyelinated nerve fibres can

Efferent fibres Afferent fibres PV

Hc

HA

GJ

BD CoH

GJ HSC

HV

Nerve trunk

Figure 1.26  Schematic representation of hepatic afferent and efferent innervation. BD: bile duct; PV: portal vein; HA: hepatic artery; CoH: canal of Hering; Hc: hepatocyte; GJ: gap junction; HSC: hepatic stellate cell; HV: hepatic vein.

be seen in the space of Disse using TEM.300 They are frequently surrounded by Schwann cell processes but a few bare nerve endings or varicosities are found in close apposition to hepatocytes or hepatic stellate cells. Synaptic clefts have been identified at points of contact suggesting that there is direct innervation of these cells, although true synapses are not found.300 An extensive cholinergic (parasympathetic) network is found in rat liver.301 In human liver, Amenta et al.302 described parasympathetic cholinergic innervation of portal tract vessels with only limited innervation of the parenchyma. Immunohistochemical studies have also identified intrahepatic fibres containing two neuropeptides – substance P and calcitonin gene-related peptide (CGRP) – which are commonly found in afferent nerves.303,304 Such afferent fibres may be involved in chemo- and osmoreception as well as in vasomotor regulation.297 It would seem likely that the liver should have sensory receptors since it is exposed to the nutrient and solute load delivered via the portal circulation from the gut.305,306 Within the parenchyma, release of neurotransmitters from the intrasinusoidal sympathetic fibres may modulate hepatocyte, sinusoidal endothelial cell and hepatic stellate cell function.307 There is evidence that adrenergic nerves play a role in the control of hepatocyte carbohydrate and lipid metabolism, and in intrahepatic haemodynamics and response to metabolic stress.308 Stimulation of hepatic sympathetic nerves in vivo produces hyperglycaemia. This effect is caused by enhanced glycogenolysis in hepatocytes and appears to be under α-adrenergic control. The regulation of hepatic carbohydrate metabolism by sympathetic nerve fibres may be mediated further by gap junctional communication.309 BioulacSage and co-workers have speculated that adrenergic nerves may induce contraction in hepatic stellate cells, thereby regulating intrasinusoidal blood flow.300 This may have relevance to evidence from experimental animals and humans,310 that the liver’s normal response to hypovolaemic shock is impaired by denervation and this may result in hepatic ischaemic injury. Loss of intrasinusoidal fibres in the cirrhotic liver may contribute to impaired metabolic function.311 Whether this loss accounts for some of the abnormalities of portal blood flow is not clear.311–313 Within portal tracts, the neurobiology of cholangiocytes has garnered considerable interest.236 Cholangiocytes express the M3 acetylcholine (ACh) receptor. Acetylcholine, acting on M3

29

MacSween’s Pathology of the Liver

receptor subtypes, potentiates secretin-induced choleresis, and can stimulate cholangiocyte proliferation. Pharmacological or surgical denervation can induce cholangiocyte apoptosis, suggesting that adrenergic innervation has a key role in regulating cholangiocyte proliferation during regeneration.314 Cholangiocytes may also express receptors for the neurotransmitter histamine (the G-proteincoupled H3R receptor), for serotonin, and for the α-type calcitonin gene-related peptide 1 (α-CGRP), all of which may influence cholangiocyte proliferation.235 Recognizing that the cholangiocyte compartment of bile ductules and canals of Hering contains putative stem cells, it is therefore notable that the surgically denervated transplanted liver contains fewer progenitor cells, and has less capacity to generate a proliferative response after liver injury.315,316

General concepts of liver injury   and repair The morphology of liver disease reflects the convergent influences of liver damage and liver recovery. The liver is vulnerable to a wide variety of metabolic, toxic, microbial, circulatory and neoplastic insults. However, the liver has a remarkable capacity for self-repair, including complete restitution of liver mass after loss, either from necroinflammatory events or surgical resection. An understanding of liver pathology requires knowledge of the causes of liver damage and the mechanisms by which the liver responds. Table 1.3 presents the fundamental causes of liver injury. They fall into the general classes of infectious, immune-mediated, drug-induced (including alcohol), metabolic, mechanical and environmental. This table is not comprehensive, but provides a framework for understanding liver injury. An individual patient is frequently subject to more than one form of liver injury simultaneously. This is due in part to the number of injuries to which the liver is subject, and in part to the remarkable propensity of humans to place their liver at risk for simultaneous injury from multiple sources. Common companion conditions include: viral hepatitis (including simultaneous infection by mul­ tiple viruses); drug and environmental toxicity (including alcohol intake); and obesity and/or diabetes with risk for non-alcoholic fatty liver disease. The enormous functional reserve of the liver masks the clinical impact of early liver damage. With the rare exception of fulminant hepatic failure, liver disease from these various causes is an insidious process in which symptoms of hepatic decompensation may occur weeks, months or even years after the onset of injury. There is often a long time interval between disease initiation and clinical detection. Conversely, the liver may be injured and heal without clinical detection. Hence, patients with hepatic abnormalities who are referred to liver disease specialists most frequently have chronic liver disease. The clinician and pathologist alike are therefore obliged to consider a differential diagnosis of multiple potential causes of liver disease, few of which are mutually exclusive. The inciting cause(s) may have begun years previously. When liver tissue is obtained for histological analysis, the liver pathologist’s task is further made difficult by the fact that the liver has only a limited repertoire for morphological manifestation of injury, as given in Table 1.4. Specifically, the liver can become inflamed, it can cease to function properly, it can exhibit morphological manifestations of inadequate function (such as retention of bile), and portions of the liver can die. Likewise, the hepatic response to injury is quite limited. The liver can regenerate (which it does extremely well), or it can become fibrotic and scarred. The combination of extensive regeneration in the midst of scarring leads to cirrhosis, conceptually the end-stage

30

Table 1.3  Causes of liver injury Infectious Viral hepatitis – hepatotropic Viral hepatitis – opportunistic Bacterial Fungal Parasitic Helminthic Immune-mediated Autoimmune hepatitis Primary biliary cirrhosis Primary sclerosing cholangitis Transplant rejection Graft-versus-host disease Drug- and toxin-induced hepatotoxicity Alcoholic liver disease Therapeutic agents (including complementary medicines and drugs of abuse) Metabolic Inherited metabolic disease Acquired metabolic derangement Non-alcoholic fatty liver disease Mechanical Obstructive cholestasis Vascular disorders Environmental Environmental toxins Heat stroke

Table 1.4  Histological manifestations of liver injury Inflammation Portal tracts Interface between portal tracts and parenchyma Parenchyma (the ‘lobule’)a Hepatocellular injurya Ballooning degeneration Steatosis Cholestasis Inclusions Necrosis and apoptosisa Vascular remodelling Regeneration Fibrosisa Cirrhosis Neoplasia a

The distribution of inflammation, hepatocellular injury, necrosis/apoptosis and fibrosis within the lobule are critical observations: periportal, mid-zonal, or pericentral.

of chronic liver injury. Interestingly, with cessation of injury and the long passage of time, fibrous tissue can be resorbed and the cirrhosis partially ‘reversed’. For the most part, the events of inflammation, dysfunction, cell death, regeneration and fibrosis play out on a microscopic scale. The macroscopic anatomy of the liver is usually the lesser consideration in assessment of hepatitic, toxic, cholestatic or metabolic liver injury. The exceptions to this statement are when there is mechanical obstruction to larger bile ducts or to major hepatic blood vessels, requiring intervention to correct the obstruction; and when

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Table 1.5  Well-established risk factors for liver neoplasia

Neoplastic condition

Risk factors

Adenoma

Oral contraceptive exposure Glycogen storage disease type 1a (Von Gierke disease)

Hepatocellular carcinoma

Viral hepatitis   Hepatitis B infection   Hepatitis C infection Cirrhosis from other causes   Alcoholic liver disease   Hereditary haemochromatosis   Hereditary tyrosinaemia   a1-antitrypsin storage disorder   Wilson disease (rare)   Primary biliary cirrhosis (rare) Inherited disorders without obligate cirrhosis   Glycogen storage disease type 1a (Von Gierke disease)

Hepatoblastoma

Familial adenomatous polyposis (FAP)

Cholangiocarcinoma

Primary sclerosing cholangitis Fluke infection of the biliary tract

Angiosarcoma

Toxin exposurea   Vinyl chloride   Arsenic

Table 1.6  Histological examination of liver microarchitecture Native elements Hepatic parenchyma Hepatocytes Hepatocyte cytologic features Hepatocyte plate architecture Sinusoids Endothelial cells Kupffer cells Stellate cells Extracellular matrix Zonation Periportal zone Portal tract interface Mid-zonal region Perivenous zone (‘pericentral zone’) Portal tracts Extracellular matrix Portal veins Hepatic arteries Bile ducts Lymphatic channels (if visible) Added elements Inflammatory cells Infectious agents Infiltrating neoplastic cells Cellular inclusions Viral cytopathic change Storage disorders

a

Historically, Thorotrast exposure was a risk factor for hepatocellular carcinoma, cholangiocarcinoma and angiosarcoma.

macroscopic liver nodules are evident. In the latter instance, the presence of a larger liver nodule raises immediate concern about neoplastic conditions, as given in Table 1.5. Interpretation of liver pathology requires rigorous and systematic examination of the microarchitectural compartments of the liver, as given in Table 1.6. Intrinsic changes in native liver elements (hepatocytes, sinusoidal structures, portal tract structures) must be evaluated, and the tissue examined for additional elements that may be present: inflammation, infection, neoplasia. The order in which this is done is a matter of personal preference. Regardless, compre­ hensive examination of all tissue elements is imperative. Such information must be correlated with clinical information on liver status, as optimal interpretation of liver pathology requires all such information. In the discussion that follows, a constant reference point will be the morphological manifestations of these principles. Mechanisms of carcinogenesis are beyond the scope of this introductory chapter but are considered in Chapter 14.

Inflammation The predominant form of liver disease throughout the world is hepatitis. Major causes are hepatotropic viral infections, alcoholic and non-alcoholic steatohepatitis, autoimmune hepatitis, and druginduced liver injury. All feature inflammation of the liver, either as cause or effect. We now discuss the biology of hepatic inflammation; discussion of specific causes of inflammatory liver injury will be left to other chapters. A summary of the inflammatory cells that

accumulate during hepatic injury (and their abbreviations) is given in Table 1.7. Because chronic viral hepatitis is paradigmatic for hepatic inflammation, pertinent general concepts summarized in Table 1.8. The cellular immune response drives most hepatic inflammatory events.221 Brief comment must therefore be made regarding antigen presentation to inflammatory cells. As with the rest of the body, in the normal liver MHC class I ‘self’ antigens are constitutively expressed: on the cells lining the vascular sinusoids (endothelial cells and Kupffer cells); cholangiocytes; and to a lesser extent hepatocytes. MHC class II antigens are normally expressed only by Kupffer cells and by dendritic antigen-presenting cells (APCs; also termed ‘dendritic cells’, DCs) in the portal tracts. Cholangiocytes and large vessel endothelial cells express few or no detectable MHC class II antigen. A proinflammatory environment established by cytokines such as tumour necrosis factor-α (TNFα), interleukin-1β (IL-1β), or IL-6 promotes the expression of HLA class II molecules on cells that do not normally express them, including hepatocytes and cholangiocytes.317,318 Aberrant expression of antigens on native liver cells via MHC I or MHC class II antigens, especially on hepatocytes and cholangiocytes, makes these native cells targets of the host immune system. Hence, while offending agents such as viruses may be the underlying cause of liver injury, it is the action of cytocidal lymphocytes in response to antigens expressed on native liver cells that actually causes liver cell death (Fig. 1.27). These antigens may be viral in origin in an infected hepatocyte, or may also be neoantigens expressed as a result of other forms of liver injury as in alcoholdamaged hepatocytes. Two elements of the immune system are then relevant: the innate immune system – already resident in the liver – which is able to

31

MacSween’s Pathology of the Liver

Table 1.7  Inflammatory cells in hepatitis

Cell type Antigen-presenting cells   Kupffer cells (KC)   Dendritic cells (DC)

Innate immune system   Natural killer cells (NK)

Comment When activated, secrete TNF-α, IL-2, IL-12 and leukotriene B3 Express toll-like receptors (TLR); secrete IL-12, TNF-α, IFN-α, and IL-10 ‘Pit cells’; can be Th-1* or Th-2**

  Natural killer T cells (NKT) Adaptive immune system   B lymphocytes   T lymphocytes    CD4+ T cells     CD4+ T helper cells     CD4+ CD25+ T cells (T-regs)    CD8+ T cells     Cytotoxic T Cells (CTL)

Secrete immunoglobulin, generate plasma cells Multiple subsets Can be Th-1* or Th-2** Regulate activation of CD4+ and CD8+ T cells Activity is enhanced by Th-1 cytokines; can be cytolytic or non-cytolytic

Table 1.8  The immune response to viral hepatitis

Hepatitis B virus   The virus (HBV)   Dendritic cells (DC)   CD4+ and CD8+ T cells   Cytotoxic T cells (CTL)

Hepatitis C virus   The virus (HCV)

  Hepatocyte changes

  Dendritic cells (DC)   Innate immune system (NK, NKT)   Cytotoxic T cells (CTL)

32

HCV Hepatocyte TNFα

IFNγ

CTL

APC (DC)

CD4+ naive T cell

Comment Genetically stable, does not escape adaptive immune system Process HBV antigens for presentation to lymphocytes Respond to HBV antigens expressed on infected hepatocytes Clear infected hepatocytes through cytolysis or non-cytolytic antiviral action (via secretion of IFNγ) Genetically unstable, generates numerous quasispecies which escape the adaptive immune system Increased sensitivity to apoptosis, decreased responsiveness to antiviral action of IFNγ Potentially defective antigen processing and presentation Contribute to the inflammatory destruction of HCV antigenexpressing hepatocytes Chronic infection may arise because CTL response is inadequate to clear infected hepatocytes

IL-4

CD8+ T cell

NKT cell NK cell

CD4+ Th-2 T cell IL-4 IL-5 IL-10

IL-12

B cell CD4+ Th-1 T cell

IFNγ IL-2

*Th-1: proinflammatory, IFN-γ and IL-2 secreting **Th-2: anti-inflammatory, IL-4 and IL-10 secreting

Virus

TLR

HBV HCV

Antibodies IL-10 Plasma cell

Figure 1.27  Schematic of the host immune response to hepatitis viral infection. A hepatocyte infected with either hepatitis B virus (HBV) or hepatitis C virus (HCV) processes and presents antigens, via the major histocompatibility (MHC) proteins expressed on its surface, to antigen-presenting cells (APC), in this case a dendritic cell (DC). The dendritic cell is also capable of taking up HCV directly, and may also respond to chemical products of other infectious agents through its toll-like receptor (TLR). The dendritic cell activates the following lymphocytes: naive CD4+ T cells; CD8+ T cells; natural killer cells (NK); and natural killer T cells (NKT). The naive CD4+ T cell is stimulated by the cytokine interleukin-4 (IL-4) to differentiate into a Th-2 CD4+ T cell; this T cell secretes cytokines which stimulate B cells to mature into plasma cells and secrete clonotypic antibodies. Under stimulation by interleukin-12 (IL-12), the activated naive CD4+ T cell differentiates into a Th-1 CD4+ T cell; this T cell secretes interferon-γ (IFNγ) and interleukin-2 (IL-2), which stimulate the activated CD8+ T cells to become cytotoxic lymphocytes (CTL). The activated NK, NKT and CTL secrete IFNγ, which has antiviral effects in hepatocytes. These cells can also interact directly with infected hepatocytes to effect cytolysis (not shown). Tumour necrosis factor-α (TNFα) secreted by CTL also can induce hepatocellular apoptosis through death-signalling pathways. In this fashion, HBV infection can usually be cleared; an inadequate immune response underlies chronic HBV infection. In contrast, HCV infection is usually not cleared successfully, owing both to the genetic instability of HCV and development of quasispecies (which evade the adaptive immune response), and to the inadequacy of the innate immune response to clear virus from infected hepatocytes. Not shown in this schematic is the potential role of CD4+ CD25+ regulatory T cells (T-regs), whose role in the immune response to viral infection is in need of further clarification and key intracellular events related to viral replication (see Chapter 7). (Courtesy of Aleta R. Crawford and adapted from Crawford JM, Immunopathology of the liver. American Association for the Study of Liver Diseases Post-Graduate Course, 2005, San Francisco, with permission)

respond to antigenic stimuli ab initio, without the need for prior antigenic exposure; and the adaptive immune system, in which antigenic stimulation activates an immune response.

The innate immune system Kupffer cells and dendritic cells Antigen-presenting cells (APC), including Kupffer cells (KC) and circulating blood dendritic cells (DC), take up antigens, process them and present them to other immune cells.319,320 DCs express toll-like receptors (TLR) that recognize particular components of infectious organisms. The activation of TLR signalling pathways results in production of proinflammatory cytokines317 and endmaturation of DCs. The activated DCs and Kupffer cells stimulate

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Figure 1.28  Lobular inflammation in chronic hepatitis, in which hepatocyte debris has been phagocytosed by resident macrophages, leaving clumps of parenchymal macrophages; apoptotic bodies are also seen. (H&E)

NK cells, NKT cells, T cells and B cells through secretion of cytokines such as IL-12, TNFα, IFNα and IL-10 (not all of which can be illustrated in Fig. 1.27). The DC subsets that produce IL-12 and TNFα support generation of cytotoxic Th1 T lymphocytes. DCs producing IL-10 promote generation of Th2 T cells, which on further secretion of IL-4, IL-5, IL-10, and IL-13 stimulate B-cell antibody production. Kupffer cells play a role in all forms of hepatitis, as activated KCs release TNFα, IL-2, IL-12, and leukotriene B3. In addition to the local effects of such cytokines, these mediators also are involved in the hepatic recruitment of circulating neutrophils and cytotoxic T lymphocytes (CTL). Hepatocellular death of any sort is rapidly followed by Kupffer cell phagocytosis of the residual debris. For example, when an isolated hepatocyte undergoes apoptosis, it is routinely engulfed by a nearby Kupffer cell within 2–4 h.321 With smouldering hepatocellular apoptosis, clumps of macrophages can accumulate in the parenchyma (Fig. 1.28). Such macrophages can persist in the parenchyma for an extended period of time, probably weeks to months, serving as sentinels of prior hepatocellular injury and death. Hepatic damage more extensive than just apoptosis of isolated hepatocytes engenders recruitment of circulating macrophages. The most dramatic example is massive hepatic necrosis, in which the vast expanse of the hepatocellular parenchyma undergoes cell death. With survival of the patient over the ensuing hours and days, the hepatic parenchyma becomes a sea of macrophages amidst the cellular debris. Their phagocytic and migratory action facilitates removal of the non-viable material, clearing the way for regeneration and recovery of the liver tissue.

NK and NKT cells Liver-specific natural killer (NK) cells, residing in interstices of the space of Disse as ‘pit cells’, are cytotoxic lymphocytes that can also produce cytokines.220 Some NK cells also express dendritic cell markers, and share properties with these cells. NK cells play an important role in first-line, innate defence against viral infection. Intrahepatic natural killer T cells (NKT) are a heterogeneous group of T lymphocytes that recognize lipid antigens presented by the nonclassical MHC class I-like molecule CD1.220 NKT cells comprise up to 50% of intrahepatic lymphocytes in humans, and reside

within the sinusoids adherent to endothelial cells, capable of crawling rapidly along these vessel walls.322 Unlike T and B lymphocytes, which require clonotypic antigen receptors to recognize antigens, NK and NKT cells can participate in the immune response without prior antigenic stimulation.222,323 NK cells, and potentially NKT cells, can produce high levels of proinflammatory (Th1) and anti-inflammatory (Th2) cytokines.324 NK cells are major producers of IFNγ (a proinflammatory cytokine); NKT cells produce IFNγ or IL-4 (an anti-inflammatory cytokine). Stimulated populations of NKT cells can expand locally, and undergo phenotypic maturation. In so doing, NKT precursors convert from a stage which is Th2 and IL-4 secreting to a stage which is Th1 and hence IFNγ secreting.325 IFNγ enhances the dendritic cell expression of proteins involved in cellular antigen processing and presentation, including proteosome subunits and MHC molecules. In addition, IFNγ induces additional chemokines that enhance and direct the adaptive immunity of T and B cells.326 The expanded NKT cell populations are thus a very effective resident proinflammatory effector arm of the host immune system. Along with Kupffer cells and sinusoidal endothelial cells, the constitutive activation of NK and NKT cells in the liver – and their ability to rapidly produce copious amounts of cytokines, enables the liver to respond quickly to remove pathogens, toxins and noxious food antigens that enter the liver via splanchnic blood. This innate immune system also is key to eliminating hepatocytes expressing aberrant antigens. Specifically, after hepatitis B virus (HBV) or hepatitis C virus (HCV) infects the liver, viral replication within hepatocytes occurs and viral particles are continuously released into the circulation.327 Through the action of antigenpresenting cells (especially dendritic cells), activation of NK and NKT cells occurs. In addition to direct cytolytic action of NK and NKT cells on hepatocytes expressing viral antigens, the secretion of cytokines such as IFNγ by NK, NKT and cytotoxic T cells (CTLs) can inhibit replication of HBV and HCV in other hepatocytes through a non-cytolytic mechanism (Fig. 1.27).328 Expression of IFNα also occurs (by both NK and NKT cells, and by infected hepatocytes); this interferon initiates intracellular signalling involving the signal transducer and activator of transcription (STAT) pathway to increase expression of genes that block viral replication. During HBV infection, the innate immune cells contribute significantly to the suppression of viral replication.329 Unfortunately, the same is not true for HCV infection,330 as HCV is capable of blocking the antiviral IFNα effect, through inhibition of STAT signalling within hepatocytes. Whether HCV induces a cytopathic elimination of infected hepatocytes through activation of the TRAIL signalling system is an area of active investigation.331

The adaptive immune system Adaptive immunity plays a critical role in hepatitis. A general principle of adaptive inflammation is that leukocytes are recruited to sites of injury in order to perform their normal functions in host defence. This involves activation and proliferation, killing of bacteria and other microbes, and ingestion of offending agents, including the debris of host tissues. The lymphocytes that accumulate in most forms of hepatitis are mobilized to participate in antibody-mediated and cell-mediated immune reactions.

• Antigen-specific CD4+ and CD8+ T cells are involved in eradication of HBV and HCV infection, during both acute and chronic infection.332

• CD4+ T-helper cells recognize short antigenic peptides displayed in the antigen-binding groove of HLA class II

33

MacSween’s Pathology of the Liver

molecules; these peptides are derived from intracellular proteolytic cleavage of exogenous antigens such as viruses.333 • CD4+ T cells secrete lymphokines that modulate the activity of antigen-specific B cells (which then produce type-specific antibodies) and CD8+ T cells.334 • A CD4+ T-helper type 1 secretion profile (Th-1) consists of antigen-specific production of IL-2 and IFNγ. A T-helper type 2 secretion profile (Th-2) consists of IL-4 and IL-10 secretion. It is the Th-1 cytokine profile that enhances CD8+ T cell cytolytic activity.335 Cellular immunity against intracellular viral pathogens involves CD8+ cytotoxic T cells (cytotoxic lymphocytes; CTL) as the effector arm (see Fig. 1.27). CTL respond to viral peptides presented by infected cells in the antigen-binding groove of HLA class I molecules.336 CTL-mediated lysis of virus-infected host cells can lead to viral clearance. Cell death is not an obligatory outcome, as CTL can secrete antiviral cytokines to induce non-cytolytic inhibition of viral gene expression and replication.337,338 Regardless, if the CTL response is incomplete a smouldering infection ensues, with chronic tissue injury.

Regulatory T cells A population of lymphocytes garnering particular attention are CD4+ T cells constitutively expressing the IL-2-receptor α-chain (CD25) and the forkhead family transcriptional regulator box P3 (FoxP3): CD4+ CD25+ FoxP3+ T-cells or ‘T-regs’. T-regs represent about 5–10% of peripheral CD4+ T cells. They are highly differentiated T cells that have limited proliferative ability and are prone to apoptosis.339 T-regs regulate the activation of CD4+ and CD8+ T cells by suppressing their proliferation and effector function. This suppressive action is critical in preventing the activation of autoreactive T cells,340 and thus limiting collateral autoimmune damage during an immune response.341 Suppression occurs both through cell : cell contact and possibly through release of inhibitory cytokines.342 Once stimulated, T-regs act in an antigennonspecific fashion.343 Patients with autoimmune hepatitis have a reduced number of circulating T-regs at the time of diagnosis. The numbers are replenished during remission on immunosuppression but never reach normal values.344 While the presence of T-regs in the active phases of hepatitis makes intuitive sense,345 their persistence during chronic viral hepatitis and in autoimmune hepatitis raises as yet unanswered questions about whether they themselves are inhibited in their effectiveness in suppressing inflammation.341,346

Recruitment and influx of inflammatory cells Over and above the actions of the resident immune system in the liver, circulating leukocytes are recruited to the infected liver. Significant numbers of CD4+ T cells, CD8+ T cells, NKT cells, NK cells and T-regs can accumulate. Recruitment of the cytotoxic T cells (T-regs excluded) is driven by the proinflammatory chemokines, for example as secreted by hepatocytes infected by virus.347 Interestingly, toxic injury to the liver, as in alcoholic hepatitis, may also lead to recruitment of neutrophils, owing to secretion of potent chemoattractants such as IL-8.348 Neutrophils are also prominently featured in the immediate vicinity of portal tract bile ducts when obstruction to biliary outflow causes leakage of toxic biliary solutes into the portal tract mesenchyme. The vast circulation of the liver, with both splanchnic influx of venous blood and direct arterial perfusion, gives every opportunity for inflammatory cells to remain in the liver in response to inflammatory stimuli. Unlike most vascular beds elsewhere in the body,

34

however, the most important initial phase of inflammation – vasodilatation – does not appear to be a major regulatory factor in hepatic inflammation. The initial phases of liver inflammation appear to occur virtually independently of blood flow regulation. Likewise, the second principle of inflammation operative elsewhere in the body, vascular leakage, is almost irrelevant in the liver. The fenestrated sinusoidal endothelium ensures that there is free exchange of plasma fluid with the extravascular space within the hepatic parenchyma. Hence, the liver is not subject to interstitial oedema in the same sense as in other body tissues. Rather, if swelling of the liver during hepatitis, it is due to swelling of hepatocytes occurs themselves. Since hepatocytes comprise over 80% of the liver volume, relatively small changes in hepatocyte volume regulation can have a profound effect on the overall liver size. The key event in hepatic inflammatory cell recruitment and influx is margination and egress. As is well known, leukocyte extravasation involves the following sequence of events: expression of vascular adhesion molecules by activated endothelial cells; margination and rolling of leukocytes expressing the cognate ligands; adhesion of leukocytes to the endothelium; transmigration across the endo­ thelium; and migration within the extravascular space towards a chemotactic stimulus. The key intrahepatic endothelium appears to be the activated sinusoidal endothelium.349 Recruitment of lymphocytes, in particular, may be driven by expression of powerful chemoattractants not only by the sinusoidal endothelium but also by hepatocytes.350,351 In the case of macrophage recruitment, the chemokine macrophage inflammatory protein-1α (MIP-1α) mediates the recruitment of inflammatory NK cells.352 Intrahepatic production of MIP-1α is accomplished through IFNα and IFNβ stimulation of the innate immune system in the liver to generate monocyte chemoattractant protein-1 (MCP-1),353 which in turn recruits MIP-1α-producing inflammatory macrophages to the liver.354 During the acute phase of viral infection, lymphocytes first suffuse the hepatic parenchyma to varying extents; the portal tracts are relatively free of inflammatory cell aggregates (Fig. 1.29A). In the acute phase, the cytolytic action of lymphocytes is exerted on viral peptide-expressing hepatocytes scattered throughout the parenchyma. If viral clearance does not occur, parenchymal cytolysis smoulders on. As the infection settles into a chronic phase, portal tracts characteristically become populated with a mixed inflammatory cell population dominated by lymphocytes, with admixed macrophages and scattered granulocytes (Fig. 1.29B). Noting that activated stellate cells serve as myofibroblasts, and that there are resident myofibroblasts in portal tracts, liver myofibrolasts may play a key role in regulating the infiltration and positioning of lymphocytes during chronic hepatitis.355 In turn, the portal tract inflammatory infiltrate is capable of generating ‘interface hepatitis’, whereby destruction of hepatocytes occurs at the interface of the parenchyma with portal tracts (Fig. 1.29C). This is a characteristic feature of progressive chronic hepatitis.

Inflammation of portal tracts The accumulation of inflammatory cells within portal tracts during chronic hepatitis raises the question of whether leukocytes leave the circulation through the vasculature of the portal tract. In the case of the portal veins, the answer appears to be ‘no’. Rather, experimental data indicate that lymphocytes entering the liver via the portal vein undergo adhesion and extravasation at the level of the sinusoids.292 Their immediate point of arrival is therefore the space of Disse. Definitive evidence is not available on their subsequent journey, but it is reasonable to suggest that the lymphocytes then

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

A

B

C Figure 1.29  (A) Acute viral hepatitis. The sinusoids are suffused with lymphocytes and there is disruption of normal lobular hepatocyte architecture, with ballooning of damaged hepatocytes. There is confluent necrosis in this perivenular zone. (B) Chronic viral hepatitis (hepatitis C viral infection). A portal tract is expanded by a mononuclear inflammatory infiltrate, consisting almost entirely of lymphocytes. The interface between portal tract and parenchyma is intact. (C) Chronic viral hepatitis (hepatitis B viral infection). The interface between a portal tract and the parenchyma is extensively disrupted by inflammation, including encirclement of hepatocytes by inflammatory cells. (H&E)

travel in a retrograde fashion through the space of Disse to the interface of the portal tract; the time frame for such migration is not known. The principles governing the behaviour of lymphocytes once they arrive at the portal tract are not well understood. On the one hand, it is plausible that fluid and lymphocytes filtered into the space of Disse enter into the hepatic lymphatic system. The elusive space of Mall, a potential space between the portal tract mesenchyme and the limiting plate of hepatocytes at the portal edge, is thought by some to represent the most terminal reaches of the intrahepatic lymphatic system. Alternatively, collagen fibres continuous between the space of Disse and portal tracts may constitute a submicroscopic channel for percolation of lymph from the space of Disse into portal tract lymphatics. Retention of leukocytes within this reticular mesenchyme may then occur. An intriguing consideration is the potential role of fibrogenesis at the portal tract : parenchymal interface leading to retention of inflammatory cells as part of interface hepatitis.356 For those leukocytes that accumulate within portal tracts during chronic hepatitis, a mixture of lymphocytes, plasma cells, macrophages, neutrophils, eosinophils and even mast cells may occupy an expanded mesenchymal space within the portal tract. The stimuli for their retention are presumably chemotactic factors, although this is a presumption. Expression of attractant chemokines, such as CXCL2, on blood vessel endothelial cells within portal tracts, and within the lymphoid aggregates in portal tracts, may promote retention of lymphocytes within portal tracts, and potentially emigration of additional lymphocytes directly into the portal tract.357 The chronically inflamed liver also expresses mucosal addressin cell adhesion molecules (MAdCAM-1) on the portal vein and sinusoidal endothelium, serving as an additional stimulus for homing of T-lymphocytes directly to the portal tract.358,359 The role of MAdCAM-1 in inducing intrahepatic inflammation may be particularly relevant in liver disease associated with inflammatory diseases of the gut. Neo-expression of lymphotoxin-β and its cognate receptor is also likely to play a role in the formation of lymphoid aggregates within portal tracts, particularly in hepatitis C infection.360,361 The accumulation of inflammatory cells in the immediate vicinity of bile ducts and ductules may follow different principles.362 Throughout the portal tract system, hepatic arteries and bile ducts are paired, travelling in close vicinity and of similar calibre.13,363 The blood supply of the biliary tree is arterial, and bile ducts are invested by a hepatic artery-derived capillary bed.19,364 Expression of proinflammatory cytokines by damaged cholangiocytes365 can recruit mononuclear inflammatory cells or neutrophils, depending upon whether the inciting injury is autoimmune or toxic in nature, respectively (Fig. 1.30). In the first instance, a complex group of cytokines are expressed on portal veins and capillary beds in portal tracts, recruiting CD4+ and CD8+ T cells and macrophages,366 with adhesion to the biliary epithelium and accumulation of intraepithelial lymphocytes.367 In the second instance, leakage of bile from an obstructed biliary tree induces release of the potent neutrophil chemoattractant IL-8,368 which induces neutrophils to accumulate in a periductal location.369,370 It is plausible to assume that these neutrophils exit from the vascular space in the peribiliary capillary bed or its immediate postcapillary venules. As will be discussed, bile ductules which traverse the portal tract mesenchyme between terminal bile ducts and the canals of Hering can become markedly proliferative under conditions of bile duct obstruction or damage. When such ‘ductular reaction’ is due to biliary obstruction, neutrophils also accumulate in a periductular location, presumably also in response to the extrusion of bile with its toxic bile salts into the periductal mesenchyme.

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MacSween’s Pathology of the Liver

TNFα IFNγ CXC3 T cell

+ + +

+ +

Neutrophil Capillary

+

Damage

+

Bile duct

Bile leakage

+

Cytokine secretion

TNFα IL-6 CX3C

TNFα IL-6

Capillary Portal vein

Figure 1.31  Portal tract granuloma in sarcoidosis; the centre of the granuloma is composed of epithelioid macrophages and these are surrounded by a cuff of lymphocytes. (H&E)

Hepatic artery Table 1.9  Examples of hepatocellular injury

Form of injury Figure 1.30  Schematic of portal tract in longitudinal section, depicting bile duct inflammation. A bile duct, the periductal capillary plexus, the companion hepatic artery and a portal vein are depicted. First, proinflammatory cytokines tumour necrosis factor-α (TNFα), interferon-γ (IFNγ), and the chemokine CXC3 (fractaline) stimulate emigration of CD4+ and CD8+ T cells into the portal tract mesenchyme. Cytotoxic CD4+ T cells may adhere directly to bile duct epithelial cells via cytokine-induced aberrant expression of the major histocompatibility complex class II receptor (MHC-II, not shown). Second, the proinflammatory environment also stimulates emigration of neutrophils into portal tracts; release of noxious chemicals leads to bile duct epithelial cell damage and destruction. TNFα secreted by portal tract lymphocytes can cause bile duct epithelial cell apoptosis. Third, bile leakage, containing bile salts, is itself proinflammatory, inducing chemotaxis of neutrophils to a periductal location. Lastly, bile duct epithelial cells themselves can secrete TNFα, interleukin-6 (IL-6), and the chemokine CXC3. These proinflammatory cytokines can enter not only the portal tract space, but also may seep into the portal venous circulation, thus exposing the downstream parenchyma to the deleterious effects of inflammation.

Granulomas Accumulation of activated macrophages into a microscopic focus surrounded by a collar of mononuclear leukocytes or fibrous tissue is termed granulomatous inflammation (Fig. 1.31). The macrophages may exhibit a distinct enlarged cytoplasm with a pale eosinophilic appearance, giving them an ‘epithelioid’ appearance. Hepatic granulomas may arise in a vast number of clinical settings, including infection (e.g. Mycobacterium tuberculosis, with ensuant caseous necrosis of the granuloma centre), foreign body reaction (e.g. talc, with formation of giant cells), drug reaction and auto­ immune disease (especially primary biliary cirrhosis). The mechanisms for formation of granulomas are as diverse as the causes. A key underlying concept is proinflammatory cytokine perpetuation of the immune response, as with chronic local secretion of IL-2 and IFNγ, with a further role for overlapping temporal stimuli

36

Oxygen deprivation   Hypoxia   Ischaemia

Examples of a condition Shock Devascularization during liver transplantation

Ischaemia/reperfusion

Revascularization of organ

Chemical or drug injury

Acetaminophen toxicity

Infection

Cytomegalovirus infection

Immunologic injury

Hepatotropic viral hepatitis: HBV, HCV

Genetic misprogramming

a1-antitrypsin storage disorder

Metabolic imbalance

Non-alcoholic fatty liver disease

for recruitment of neutrophils and macrophages to the site of inflammation, coupled with a fibrogenic inflammatory environment.371 This environment keeps the macrophages in an activated state and is capable of transforming them into epithelioid cells and multinucleated giant cells.

Hepatocellular injury Cellular injury in the liver occurs in the following general settings: oxygen deprivation (either hypoxic or ischaemic); chemical or drug injury; infection; immunological injury; genetic misprogramming; and metabolic imbalance. Examples are given in Table 1.9. Later chapters in this book cover in detail these and related disease conditions. Underlying all of these is a characteristic set of morphological changes in hepatocytes. Short of hepatocellular death, these changes are essentially reversible phenomena, in that with removal of the offending agent or correction of the disease condition hepatocytes presumably can recover both morphologically and functionally. The pathogenesis of these changes is now briefly discussed.

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

A Figure 1.32  Alcoholic hepatitis, in which many hepatocytes are markedly ballooned, with rounded plasma membrane contours, cleared-out cytoplasm and clumped strands of intermediate filaments (some of which would qualify as Mallory–Denk bodies). Interspersed among the ballooned hepatocytes are inflammatory cells and fibrous tissue. (H&E)

Ballooning degeneration Hepatocyte volume change (swelling) is part of normal physiology, and plays a critical role in the regulation of hepatocellular metabolism and gene regulation in response to environmental changes such as ambient osmolarity changes, oxidative stress, intracellular substrate accumulation and hormones such as insulin.372,373 However, deranged cellular swelling is a fundamental feature of cellular injury. In the lexicon of liver pathology, ‘ballooning degeneration’ is the term used. Hepatocyte ballooning is the result of severe cell injury, involving depletion of ATP and a rise in intracellular Ca1+, leading to loss of plasma membrane volume control and disruption of the hepatocyte intermediate filament network.374 If severe enough, cell death occurs. Ballooning occurs in ischaemic liver cells, cholestasis, and in many other forms of hepatic toxicity.375 The morphological manifestation of hepatocellular ballooning degeneration is hepatocellular swelling, vacuolization of the cytoplasm, clumping of intermediate filaments (manifest in H&E-stained tissue sections as clumped strands of eosinophilic cytoplasmic material), swelling of mitochondria, and blebbing of the cell membrane (Fig. 1.32). These features, which affect the entirety of the hepatocyte cytoplasm, are to be distinguished from the well-formed spherical lipid vacuoles of the steatotic hepatocyte (see below). That said, hepatocyte ballooning degeneration is considered a hallmark of steatohepatitis (Chapter 6), marked by substantial decreases in or loss of immunostaining for the intermediate filament keratins 8/18.376 On a macroscopic scale, with hepatocellular injury and swelling, the liver may be transformed into a swollen and turgid organ. Pallor may be evident both because of the increased intracellular fluid and because of impending compromise of the hepatic circulation. As sensory innervation of the liver is confined mainly to the liver capsule, it is the swollen liver that, by exerting tension on the liver capsule, engenders localizing right upper quadrant pain.

Steatosis Accumulation of triglyceride fat droplets within hepatocytes is known as steatosis. Multiple tiny droplets that do not displace the

B Figure 1.33  (A) Microvesicular steatosis in the liver of a pregnant woman. Hepatocytes contain innumerable small fat droplets which do not displace the cell nucleus. (Masson trichrome) (B) Macrovesicular steatosis in an alcoholic patient. Hepatocytes are distended by single large fat droplets, which displace the cell nucleus to the side. As fat is dissolved during routine tissue processing, a clear space only remains. (H&E)

nucleus are known as microvesicular steatosis, and appear in such conditions as acute fatty liver of pregnancy and valproic acid toxicity (Fig. 1.33A). A single large droplet that displaces the nucleus is termed macrovesicular steatosis, and may be seen in hepatocytes throughout the livers of obese or diabetic individuals, and interestingly in scattered hepatocytes in patients with hepatitis C viral infection (Fig. 1.33B). Both microvesicular and macrovesicular steatosis may be present in alcoholic liver disease. In alcoholic hepatitis, hepatocellular steatosis is associated with hepatocyte ballooning degeneration and Mallory–Denk body formation (see below), an inflammatory infiltrate of the lobule (neutrophils mixed with lymphocytes, often wrapped around ballooned hepatocytes containing Mallory–Denk bodies), and usually peri­ sinusoidal fibrosis. Non-alcoholic fatty liver disease (NAFLD) also features hepatocellular steatosis; when hepatocyte ballooning degeneration, Mallory–Denk body formation, and inflammation are present, the term non-alcoholic steatohepatitis (NASH) is given. The accumulation of triglyceride within hepatocytes is the hallmark of fatty liver diseases. In the normal liver, lipid biosynthesis maintains cellular membranes, supports hepatic bile secretion

37

MacSween’s Pathology of the Liver

(which contains bile salts, phospholipids, and cholesterol), and is a key feature of the assembly of very low-density lipoprotein (VLDL) particles, which are exported from the liver into the circulation. The liver does not normally store lipids. Following alcohol exposure, hepatocellular metabolism of alcohol to acetaldehyde, and thence to acetate, generates reducing equivalents in the form of NADH + H+ (Chapter 6). With excess and/or sustained alcohol exposure, the reducing equivalents cannot be satisfactorily disposed of via mitochondrial oxidative metabolism. The NADH + H+ are therefore diverted into lipid biosynthesis: fatty acids and glycerol. Esterification of fatty acids with glycerol to form triacylglycerols completes the biosynthetic pathway, leading to accumulation of neutral lipid droplets in the cytoplasm of hepatocytes. These droplets can accumulate within hours of a major alcohol exposure. Impaired export of VLDL from the hepatocyte also leads to accumulation of lipoprotein lipid within a distended Golgi apparatus.377 Regardless of location, these droplets initially are only a micron or two in diameter (microvesicular), with a high surface : volume ratio. These droplets are thus amenable to rapid dissolution through the action of lipases, which occurs at the lipid : cytoplasmic interface. Over time (probably days), the droplets coalesce, forming droplets exceeding 20 µm in diameter (macrovesicular), displacing the hepatocyte nucleus. As the surface : volume ratio of these larger lipid droplets is exceedingly low, the action of surface lipases is of minimal impact. Macrovesicular lipid droplets can thus remain for months, even in the absence of further alcohol exposure. Eventually, should the alcoholic liver disease progress, the number of steatotic hepatocytes may eventually diminish. Lipid accumulation in non-alcoholic fatty liver disease (NAFLD) follows a different pathogenetic sequence, and is predominantly macrovesicular, with one or several lipid droplets occupying the entire hepatocellular cytoplasm (the mechanisms of triglyceride accumulation in this setting is considered in detail in Chapter 6). Ironically, in non-alcoholic steatohepatitis, the formation of trigly­ ceride lipid droplets may actually be a protective mechanism by reducing the free fatty acid pool in the cytoplasm.378

Cholestasis Bile constitutes the primary pathway for elimination of bilirubin, excess cholesterol (both as free cholesterol and as bile salts), and xenobiotics which are insufficiently water soluble to be excreted into urine. Bile facilitates the digestion and absorption of lipids from the gut. Because bile formation requires well-functioning hepatocytes and an intact biliary tree, this process is readily disrupted. Physiologically, cholestasis denotes an impairment of bile flow and failure to secrete the inorganic and organic constituents of bile. In particular, cholestasis arises from molecular and ultrastructural changes that impair the entry of small organic molecules, inorganic salts, proteins, and ultimately water into the biliary space. Clinically, the physical findings of jaundice and pruritus are accompanied by elevated serum concentrations of bilirubin, bile salts and alkaline phosphatase. A general formulation for causes of cholestasis is given in Table 1.10. Pathologists have marvelled at the profound hepatic alterations in jaundiced patients for almost two centuries, but only in the last 30 years have the molecular secrets of cholestasis been unlocked. The key events in normal bile duct transport are outlined in Figure 1.34. A number of key molecular events occur in the hepatocyte in both obstructive and non-obstructive cholestasis.379,380 These are summarized in Table 1.11. The main conclusion to draw from Table 1.11 is that a strategic subset of the hepatocyte transport system changes during acquired cholestasis.379,381,382 First, expression of the basolateral sodium bile salt transporter, NTCP, is markedly downregulated, thereby reducing uptake of bile salts into

38

Table 1.10  Causes of cholestasis (excludes inherited hyperbilirubinaemic syndromes) Cholestasis in the adult or adolescent Obstructive cholestasis Gallstones Malignancy: pancreatic cancer, bile duct adenocarcinoma Stricture of the common bile duct Bile duct diseases Primary biliary cirrhosis Primary sclerosing cholangitis Graft-versus-host disease: acute, chronic Transplant rejection: acute, chronic Transplant: infarction of the biliary tree secondary to hepatic artery obstruction Vanishing bile duct syndromes: ibuprofen, chlorpromazine Non-obstructive cholestasis Infection: viral hepatitis, endotoxaemia, sepsis Toxic: drug, total parenteral nutrition Paraneoplastic syndrome: Hodgkin disease Inherited disease: Wilson disease, familial cholestatic syndromes Pregnancy: intrahepatic cholestasis of pregnancy Infiltrative disorders: amyloidosis, metastatic cancer Cirrhosis (any cause) Cholestasis in the neonate or young child Obstructive cholestasis Biliary atresia: extrahepatic biliary atresia, ‘early severe’ biliary atresia Common bile duct obstruction: biliary sludge, gallstones Choledochal cyst with biliary sludge Inspissated bile/mucous plug Non-obstructive cholestasis Bacterial infection: Gram-negative enteric bacteraemia, syphilis, listeria, toxoplasma Viral infections: cytomegalovirus, herpesvirus (includes simplex, zoster, parvovirus B19, adenovirus), rubella, reovirus, enteroviruses, hepatitis B, hepatitis C Toxic: total parenteral nutrition, drugs Metabolic disease With biliary tract compromise: a1-antitrypsin storage disease, cystic fibrosis Without biliary tract compromise: galactosaemia, tyrosinaemia, fatty acid oxidation defects, lipid storage disorders, glycogen storage disorders, peroxisomal disorders Specific defects in biliary function: bile acid biosynthetic defects, PFIC1, PFIC2, PFIC3 Paucity of bile ducts Alagille syndrome, non-syndromatic paucity of bile duct syndromes Miscellaneous Shock/hypoperfusion Histiocytosis X Idiopathic neonatal hepatitis Cirrhosis (any cause, as derived from the above specific conditions) PFIC, progressive familial intrahepatic cholestasis. Data from Li and Crawford 2004.369

hepatocytes under a condition in which their secretion into bile is inhibited. Second, canalicular secretion of the many solutes that make up bile is variably reduced, including reduction in bile salt secretion via the bile salt export pump (BSEP). A key downregulatory mechanism is endocytotic internalization of canalicular transport systems with potential redistribution to the basolateral plasma

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Hepatocyte

NTCP Bile acids

OATPs Organic anions

Na+

MRP3

MRP2 Conj. bilirubin Glutathione

FIC1 Aminophospholipids

BSEP Bile acids

MDR1 Drugs

MDR3 Phosphatidylcholine

OSTαβ Bile acids Bile duct

ASBT Bile acids

MRP4 OSTαβ Bile acids

CYP7A1

Cholesterol Bile acids

Cl – Cl – Na+

CFTR Chloride AE2 Bicarbonate HCO3–

Figure 1.34  Hepatobiliary transporters. Bile acids are derived from cholesterol by de novo synthesis or via hepatocellular uptake from the sinusoidal blood containing bile acids undergoing enterohepatic circulation. Uptake into hepatocytes from the sinusoidal blood is mediated by a high-affinity Na+/taurocholate cotransporter (NTCP) and a family of multispecific organic anion transporters (OATPs). Canalicular excretion of bile via specific ABC transporters represents the rate-limiting step of bile formation. The canalicular membrane contains a bile-salt export pump (BSEP) for monovalent bile acids; a conjugate export pump MRP2 mediates excretion of various organic anions such as bilirubin and divalent bile acids. The phospholipid export pump MDR3 flops phosphatidylcholine from inner to outer leaflet, which then forms intraluminal mixed micelles together with bile acids and cholesterol. Cationic drugs are excreted by the multidrug export pump MDR1. The canalicular membrane also contains a P-type ATPase, FIC1, a putative aminophospholipid flippase. At the basolateral membrane additional bile acid export pumps, MRP3, MRP4 and the heterodimeric organic solute transporter OSTα/β are present as back-up pumps for alternative sinusoidal bile acid export. The biliary epithelium is also involved in the reabsorption of bile acids via an apical Na+-dependent bile-salt transporter ASBT and the basolateral counterpart OSTα/β. (Redrawn from Wagner M, Zollner G,

A

B

Trauner M. New molecular insights into the mechanisms of cholestasis. J Hepatol 2009; 51:565–580)

membrane. Specific changes in bile transport function may be causes of cholestasis, particularly in the inherited cholestatic conditions, or secondary manifestations of acquired hepatic conditions that are accompanied by cholestasis. Hepatocellular cholestasis, to the pathologist, is the visible manifestation of this broad array of pathophysiological derangements in hepatocellular uptake, transcellular transport, and canalicular secretion of biliary constituents. Notably, hepatocellular changes exhibit considerable overlap between diseases involving bile duct obstruction and those in which the cholestasis is purely hepatocellular in origin. Hence, the features described here for non-obstructive cholestasis are also found in obstructive cholestasis; it is only upon finding additional obstructive changes in portal tracts and bile ducts that mechanical obstruction can be inferred. The parenchymal changes occurring as a result of non-obstructive or obstructive impairment of hepatocellular bile formation are given in Table 1.12 and illustrated in Figure 1.35.383 The dominant

C Figure 1.35  (A) Hepatocellular cholestasis. Numerous hepatocytes are discoloured by brown pigmented material (arrowheads), indicative of retention of biliary constituents within hepatocytes. Note that bile salts are colourless; retained bilirubin and bilirubin glucuronides are responsible for the coloration. A Mallory–Denk body is also present (*). (B) Hepatocellular and canalicular cholestasis. In addition to the retention of bile by hepatocytes, there is prominent retention of bile in a distended bile canaliculus (arrowhead). (C) A Kupffer cell within a sinusoid has engulfed biliary material that has regurgitated into the circulation, through disruption of the intercellular junctions between hepatocytes. (H&E)

39

MacSween’s Pathology of the Liver

Table 1.11  Key hepatocyte molecular transport systems, with changes during cholestasis

Transport protein

Transport function

Inherited defect

Associations with acquired cholestasis

Basolateral membrane   NTCP (SLC10A1)   OATP2 (SLC22A7)

Bile salts Organic anions

None known

Markedly decreased in cholestasis Modestly decreased in cholestasis

Canalicular membrane   BSEP (ABCB11)

Bile salts

PFIC2 (Byler syndrome)

  MDR1 (ABCB1)   MDR3 (ABCB4)

Multispecific organic solutes Phosphatidylcholines

PFIC3

Decreased during cholestasis, intrahepatic cholestasis of pregnancy, drug-induced cholestasis Maintained during cholestasis Decreased in intrahepatic cholestasis of pregnancy, drug-induced cholestasis

  ABCA1   MRP1 (ABCC1)   MRP2 (ABCC2)

Cholesterol Organic anions Organic anions, bilirubin glucuronides Organic anions, bile salts Chloride Aminophospholipids

  MRP3 (ABCC3)   CFTR (ABCC7)   FIC1 (ATP8B1)

Tangier disease type 1 Dubin–Johnson syndrome Cystic fibrosis PFIC1 (Byler disease)

Increased during cholestasis, basolateral expression Decreased during cholestasis, intrahepatic cholestasis of pregnancy Increased during cholestasis, basolateral expression Decreased in primary sclerosing cholangitis Decreased in intrahepatic cholestasis of pregnancy

BSEP, bile salt export pump; MRP, multidrug resistance protein; NTCP, sodium-taurocholate cotransporting polypeptide; PFIC, progressive familial intrahepatic cholestasis. Data from Li and Crawford 2004,369 updated as per Zollner and Trauner 2008379 and Roma et al. 2008.381

Table 1.12  Histology of cholestasis

Histological feature

Description

Parenchyma   Bilirubin pigment accumulation

Morphological features during non-obstructive or obstructive cholestasis Hepatocyte cytoplasm pigmentation Bile canalicular dilatation with inspissated bile Regurgitated pigment: Kupffer cell pigmentation Acinar distribution: perivenular > periportal Feathery degeneration with flocculent cytoplasm; perivenular > periportal Ballooning degeneration with swollen hepatocytes; periportal > perivenular (‘cholate stasis’) Mallory–Denk body formation: periportal Bile infarcts: coalescent periportal necrosis with retained pigmented material Accumulation of copper and copper-associated protein: periportal Dilated bile canaliculi surrounded by more than two hepatocytes in a pseudotubular arrangement Coalescence of hepatocytes, with multiple nuclei and free-floating ‘canaliculi’

  Hepatocellular degeneration

  Metal accumulation   Liver cell rosettes   Giant cell transformation Portal tracts   Portal tract expansion   Bile ductular reaction   Bile duct dilatation

  Fibrosis

Morphological features during obstructive cholestasis Oedema, mixed pattern of inflammation with abundant neutrophils Racemose small ductular channels, extending to the periphery of the portal tract, sectioned obliquely in-and-out of the plane of section, with or without inspissated bile Prominently dilated marginal bile ductules with inspissated bile. Also occurs in nonobstructive sepsis (‘cholangitis lenta’) Ectatic interlobular and segmental bile ducts, either with a reactive inflamed epithelium, or an attenuated and potentially ulcerated epithelium with associated neutrophilic abscess Broad-based fibrosis expanding portal tracts and creating blunt fibrous septa that subdivide the liver parenchyma in a ‘jigsaw’-like pattern

themes are accumulation of substances normally secrete in bile and toxic degeneration of hepatocytes, without significant alterations in portal tracts. The most obvious hepatocellular feature is brown pigmentation from retained bilirubin. Pigmentation includes accumulation of bilirubin and its glucuronides in hepatocytes, inspissated bile in swollen canaliculi, and bile regurgitation into the sinusoidal space with phagocytosis by Kupffer cells. Such pigmentation occurs predominantly in the perivenular region of the hepatic lobule, as does dilatation of bile canaliculi. During severe

40

cholestasis of long duration, bilirubin deposition and canalicular dilatation may extend into the periportal region. Necrosis per se is not necessarily a feature of cholestatic liver disease, but hepatocellular apoptosis with appearance of acidophilic bodies is often present, attributed to the direct toxic effect of retained bile acids on hepatocytes.149 Hepatocytes also undergo toxic degeneration, manifest as swelling with a flocculent cytoplasm, so-called feathery degeneration. When this degeneration leads to confluent necrosis of hepatocytes, bile infarcts are produced

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

(also called bile lakes), in which large masses of acellular pigmented material are surrounded by a rim of necrotic hepatocytes or reactive mesenchyme. Bile infarcts are usually encountered only in obstructive cholestasis. A particular feature of cholestatic periportal hepatocytes is cholate stasis. First, periportal hepatocytes may undergo ballooning degeneration, evident as cellular swelling with cytoplasmic material clumped around the nucleus, and large lucent peripheral areas of the cell. Second, it is not unusual to find Mallory–Denk bodies (see below) in periportal areas. Lastly, as copper is a heavy metal normally secreted in bile, special stains for copper or copper-associated protein may demonstrate copper accumulation in periportal hepatocytes. All of these changes are attributed to the retention of biliary constituents normally secreted by periportal hepatocytes. In particular, retained bile salts are toxic, on the basis of both their detergent properties and their ability to activate programmed cell death pathways,384 hence the term ‘cholate’ stasis. This feature may occur in non-obstructive cholestasis; it is more prominent in obstructive cholestasis. Chronic cholestasis of neonates, children and rarely, adults, may give rise to so-called cholestatic liver cell rosettes, consisting of dilated bile canaliculi surrounded by more than two hepatocytes in a pseudotubular arrangement. These rosettes express some keratins characteristic of biliary epithelium, and by three-dimensional reconstruction they are found to communicate with small bile ducts and ductules.385 They are thus thought to be an adaptive mechanism to chronic cholestasis. A further feature of neonatal cholestatic syndromes is the frequent appearance of giant cells – foamy-appearing multinucleated hepatocytes with intracellular pigmented inclusions having the appearance of ‘floating’ bile canaliculi.386 On occasion, giant cell transformation may be seen in adult cholestatic livers. Although the origin of such cells is unclear, it is thought that the detergent action of retained bile salts leads to dissolution of the lateral plasma membranes and coalescence of adjacent hepatocytes. Such giant cell transformation also can occur in biliary atresia. In non-obstructive cholestasis, there are no distinctive changes in portal tracts other than those associated with the underlying cause (e.g. viral or drug-induced hepatitis). However, with obstructive cholestasis, a distinctive set of portal tract histological features emerge, as detailed in Table 1.12 and illustrated in Figure 1.36.370 These features occur upstream to either intrahepatic biliary obstruction (as from primary biliary cirrhosis or primary sclerosing cholangitis) or extrahepatic biliary obstruction (as from gallstone obstruction or pancreatic cancer). Particularly distinctive is proliferation of the small bile ductules (also termed ductular reaction), creating a racemose array of uniformly sized small ductular channels passing in and out of the plane of section, but remaining within the topological space of the portal tract. This response is to be distinguished from the ‘ductular reaction’ observed during liver regeneration from massive injury, in which irregular partial ductular structures are present at the interface and within the damaged parenchyma. The latter structures represent massive proliferation of the canal of Hering-based progenitor cell population of the liver.

Mallory–Denk bodies Differentiated hepatocytes exhibit a simple keratin expression pattern: the type I keratin K8, and the type II keratin K18.388 In keeping with the requirement that proper assembly of keratins requires the presence of at least one type I and one type II peptide, K8 and K18 assemble in equimolar ratios into intermediate filaments (IF), which form a filamentous network within the cytoplasm of hepatocytes. In a variety of disease conditions, alterations in hepatocellular keratin assembly occur. Ballooning of hepatocytes is

A

B

C Figure 1.36  (A) Portal tract in obstructive cholestasis secondary to post-transplant stricture. There is extensive bile ductular proliferation, evidenced by the racemose ductular structures within the portal tract space. The portal tract is expanded by fibrous tissue. (B) Chronic obstructive cholestasis. Bile ducts are surrounded by concentric fibrosis. Peribiliary fibrosis such as this can occur in diseases other than primary sclerosing cholangitis, owing to the brisk fibrogenic response of portal tract myofibroblasts during obstructive cholestasis. (C) Cholangitis lenta. A distinctive dilatation of marginal ductules by inspissated bile can occur in the setting of sepsis or endotoxaemia, without there being biliary obstruction. (H&E)

41

MacSween’s Pathology of the Liver

accompanied by a reduced density or even loss of the cytoplasmic IF network. Misfolded and aggregated keratins can accumulate, clumped together in a characteristic intracellular inclusion, Mallory– Denk bodies (MDBs, Fig. 1.32). MDBs exhibit aberrant crosslinking, increased phosphorylation, partial proteolytic degradation, and an increase of β-sheet conformation. MDBs accumulate in alcoholic and non-alcoholic steatohepatitis, Wilson disease, cholestatic conditions such as primary biliary cirrhosis, and following exposure to certain drugs (such as amiodarone). First described by Frank B. Mallory in 1911, they were re-named Mallory–Denk bodies very recently in recognition of the singular contribution of Helmut Denk to their pathogenesis in the years since his development of the first animal model of MDBs in 1975.387 A key degradation pathway for misfolded proteins is the ubiquitination–proteasome pathway. By mass spectrometry, MDBs consist of keratin, ubiquitinated keratin, the stress-induced and ubiquitin-binding protein p62, heat shock proteins (HSPs) 70 and 25, and other peptides.388 Three mechanisms appear to be involved in formation of MDB:389 epigenetic changes in gene expression following exposure of hepatocytes to a toxic environment; a shift from the 26s proteasome to an immunoproteasome; and chronic activation of the toll-like signalling pathways which stimulate proin­flammatory and cell growth pathways. In the case of the immunoproteasome, activity of the 26s proteasome is decreased, leading to lack of ubiquinated protein degradation and aggregation of the proteasome, polyubiquinated proteins and associated heat shock proteins.390 These three mechanisms combine to cause formation of the MDB intracellular aggregates. Interestingly, keratins are able to modulate TNF signalling pathways and the apoptosis pathway. Specifically, keratins can bind to the TNF receptor 2 (TNFR2), thereby influencing TNFα-induced activation of the death signalling pathway.391 TNFα is a potent inducer of neutrophilic inflammation, and ballooned hepatocytes containing MDBs are frequently surrounded by neutrophils. Thus, the accumulation of MDBs may not simply be a by-product of hepatocellular toxic damage, but may also contribute to the perpetuation or advancement of inflammatory injury.

Cell death While the cellular changes described in the previous section may be viewed as reversible, at some point the hepatocyte cannot compensate and recover. The onset of irreversible injury is accompanied by extensive damage to all cellular membranes, swelling of lysosomes, vacuolization of mitochondria, and extensive catabolism of cellular membranes, proteins, ATP, and nucleic acid. Cellular death takes two broad forms: apoptosis and necrosis. Apoptosis is the usual consequence of immunologically-mediated cell death. For the most part, necrosis is a feature of disease processes other than hepatitis, especially injury involving tissue ischaemia. Curiously, despite extensive scientific investigation of both mechanisms of cell death, morphology still provides the fundamental distinction between the two.392 In fact, the intracellular signalling pathways overlap to some extent. Other avenues for cell death include necroptosis, autophagy and cornification. In the liver, the most extensive research has been on apoptosis and necrosis of several key endogenous cell types: hepatocytes, cholangiocytes, sinusoidal endothelial cells, and stellate cells.149

Apoptosis Apoptosis is an active form of cell death in which cells exhibit cytoplasmic shrinkage, cell membrane blebbing, chromatin

42

Figure 1.37  Hepatocyte apoptosis. A large apoptotic body is present in the parenchyma of an acute hepatitis. (H&E)

condensation (pyknosis), nuclear fragmentation (karyorrhexis), and cellular fragmentation into small membrane-bound ‘apoptotic bodies’.393 These characteristic changes are the result of activation of caspases and endonucleases, which induce the cleavage of structural proteins and DNA, respectively.392 In the liver, hepatocellular apoptotic bodies have long been referred to as acidophilic bodies or Councilman bodies (Fig. 1.37).394 Identification of apoptotic bodies indicates current and ongoing hepatocellular apoptosis, since apoptotic hepatocytes are engulfed within a matter of hours by Kupffer cells or other macrophages, as noted earlier. Upon triggering of apoptosis (as by a cytotoxic T cell), a characteristic sequence of downstream events occurs: translocation of phosphatidylserine to the outer leaflet of the cell plasma membrane; caspase activation; activation of the mitochondrial permeability transition; cytochrome c release from the mitochondrion; and DNA fragmentation. Exposure of phosphatidylserine on the outer leaflet of the cell membrane is an early stimulus for phagocytosis of the apoptotic body. Simply speaking, apoptosis results in elimination of infected host cells from the host organism ostensibly without spillage of cellular contents into the extracellular milieu, and is therefore one mechanism for controlling infection. Two overlapping signalling pathways lead to apoptosis:394 the extrinsic ‘death receptor’ pathway; and the intrinsic ‘mitochondrial’ pathway (Fig. 1.38).395,396 The extrinsic pathway is mediated by cell surface receptors such as Fas and the TNFα receptor-1 (TNF-R1), interacting with cytotoxic T cells expressing Fas-ligand or releasing TNFα. The downstream signalling involves cross-linking of the receptor complexes with adapter proteins and procaspases, thereby cleaving the procaspases to their active forms. Caspases initiating apoptosis include 2, 8, 9, and 10. Initiator caspases 8 and 10 are involved in death receptor-mediated apoptosis, whereas caspase 9 initiates apoptosis following mitochondrial dysfunction.149 Caspase 2 has been implicated in cell death by endoplasmic reticulum stress and following DNA damage.397,398 The extrinsic pathway may also be initiated by perforin and granzyme B released from activated cytotoxic lymphocytes. Perforin allows granzyme B to enter the cytoplasm of target cells, and directly cleave intracellular proteins such as procaspases. In addition to Fas and the TNFα receptor-1 mediated pathways, TRAIL-mediated apoptosis can also occur in viral hepatitis and cholestatic liver disease.399 Executioner caspases such as caspases 3, 7 and 6 are activated by initiator caspases, either through the extrinsic death receptor-mediated or the intrinsic

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

DD

Perforin

TNFα

TRAIL

FasL

DD

+

Bile acids

DD

Ischaemia –reperfusion

Bid

O2

tBid

O2•–

Caspase 8/10 +

Granzyme

Cathepsin B Bak tBid Mitochondria

+

Mitochondrial permeability transition

Cytochrome C APAF-1

ATP

+

Bax

AIF

Caspase 9 Caspase 3

APOPTOSIS DNA fragmentation Figure 1.38  Schematic overview of receptor-mediated and mitochondria-mediated apoptotic signal transduction pathways. Receptors for Fas-ligand (FasL), tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and tumour necrosis factor-α (TNFα) are normally dispersed monomeric subunits in the plasma membrane. Binding of FasL, TRAIL, or TNFα to their respective receptors induces receptor trimerization and recruitment of accessory intracellular molecules (not shown) to the cytoplasmic ‘death domains’ (DD) of the receptors. This causes activation of caspases 8 and 10, which then activates a proapoptotic pathway. Both cathepsin and activated Bid (tBid) induce the mitochondrial outer membrane permeabilization (MOMP), resulting in release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria. Assembly of a cytochrome c/Apaf-1/ATP/caspase 9 complex activates caspase 3. Both caspase 3 and AIF converge to cause DNA fragmentation and apoptosis. Caspases 8 and 10 also may activate caspase 3 directly. Additional proapoptotic stimuli include: entry of granzyme into the cell through a perforin pore, following release of perforin/granzyme from T lymphocytes; bile acid induction of trimerization of the cell surface death receptors by ligand-dependent or ligand-independent mechanisms, leading to activation of the proapoptotic pathways; and ischaemia–reperfusion with generation of superoxide radicals (O2−), which can activate the MOMP directly.

mitochondrial pathways of apoptosis.149 These latter caspases activate caspase-activated DNAse (CAD) by cleaving ICAD, an inhibitor of CAD.400 CAD activation produces DNA cleavage at the internucleosomal linker regions of DNA, giving rise to the ‘ladder’ pattern of DNA cleavage: multiples of the 180-bp nucleosomal regions, characteristic of apoptosis. The intrinsic pathway for apoptosis arises from mitochondrial dysfunction or possibly endoplasmic reticulum stress arising from

response to unfolded proteins. An example of the latter is accumulation of misfolded polypeptides of the Z form of α1-antitrypsin, α1-ATZ, in the endoplasmic reticulum in patients with α1antitrypsin deficiency. A brisk autophagocytic response is triggered, generating mitochondrial injury, activation of caspase signalling cascades, and initiation of hepatocellular apoptosis.401 By any mechanism, oxidative damage to the mitochondrial inner membrane leads to the mitochondrial phase transition, termed mitochondrial outer membrane permeabilization (MOMP), and release of several activators of apoptosis into the cytosol, including cytochrome c, second activator of mitochondrial apoptosis (SMAC), endonuclease G, high temperature requirement A2 (HrtA2), and apoptosis inducing factor (AIF).149 Cytochrome c complexes with Apaf-1 to activate procaspase 9.402 This three-protein complex is termed an apoptosome, which then activates downstream caspases such as caspase 3, 6 and 7.394 Mitochondria also release endo G, which with caspases can cleave chromosomal DNA. Mitochondrial release of cytochrome c is a near universal event in apoptosis and a key marker of the intrinsic pathway of apoptosis.403 The Bcl-2 (B-cell lymphoma-2) family of proteins regulate mitochondrial dysfunction during apoptosis, and includes both pro- and anti­ apoptotic members which interact with each other and/or the mitochondria to control the integrity of the outer mitochondrial membrane.400 Apoptosis is a normal feature of hepatic development and remodelling, both for the parenchyma of the liver and the biliary tree.394 Notably, hepatocellular apoptosis is also a regular feature of the major inflammatory hepatic diseases: viral hepatitis, autoimmune liver disease, alcoholic liver disease, NAFLD, and drug-induced liver disease,394,403 attributable to the presence of cytotoxic lymphocytes acting upon hepatocytes. Apoptosis may also be seen in chronic cholestatic disorders owing to the toxic nature of retained bile salts on the mitochondria and their activation of death-receptor signalling;404 cholangiocyte apoptosis also occurs in these disorders. While hepatocellular apoptosis can be seen in the alloreactive disorders of graft-versus-host disease and liver allograft rejection, cholangiocyte apoptosis, is a key diagnostic feature for these two conditions.405 Although apoptosis occurs as part of normal development and tissue remodelling and avoids stimulation of inflammatory pathways, pathological apoptosis may not only result from inflammation but may actually amplify the inflammatory process.406 Following engulfment of apoptotic bodies, Kupffer cells also express the death ligands TNFα, TNF-related apoptosis-inducing ligand (TRAIL), and Fas-ligand (FasL).407 These ligands are capable of inducing death receptor-mediated apoptosis in hepatocytes directly, further aggravating liver inflammation. When activated hepatic stellate cells engulf hepatocyte apoptotic bodies, they produce profibrogenic cytokines such as TGFβ1, and collagenesis is stimulated.408 Apoptotic cells also release the nucleotides ATP and UTP, which bind purinergic receptors such as P2Y2 on macrophages and stellate cells,409,410 further activating inflammation and fibrogenesis. In the case of viral hepatitis, viral antigens released from damaged or dead hepatocytes are taken up and processed by dedicated antigenpresenting cells (APC) – dendritic cells or Kupffer cells. Co-expression of these processed antigens on the surface of APC in association with class I and class II major histocompatibility antigens (MHC) can activate the T-cell mediated death pathways.411 Hence, apoptosis is not just a mechanism for eliminating damaged or infected cells; it is proinflammatory and fibrogenic in its own right. Important regulators of the intracellular apoptotic signalling pathways are anti-apoptotic survival proteins and pro-apoptotic proteins.412 A key player is nuclear factor-κ B (NFκB), which is activated when the TNF-receptor-1 is ligated by TNFα.413 NFκB-induces

43

MacSween’s Pathology of the Liver

transcriptional activation of survival factors such as cFLIP, XIAP, c-IAP1, c-IAP2, Bfl-1/A1, Bcl-2, and Bcl-xL. These survival factors downregulate or inhibit the intracellular apoptotic signalling pathways at a number of points. Paradoxically, activated NFκB can transactivate death receptors such as Fas or tumour necrosis factorrelated apoptosis-inducing ligand D5 (TRAIL-D5).414 TRAIL, which cannot normally induce apoptosis, nevertheless becomes capable of triggering hepatocellular apoptosis during viral infection or under cholestatic conditions when bile acid levels are elevated.415,416 Under such conditions, there appears to be loss of NFκB-dependent Bcl-xL upregulation.417 NFκB thus has a dual role as mediator or inhibitor of cell death, whereby the modulation of apoptosis by NFκB appears to be largely determined by the nature of the death stimulus.96 Nitric oxide (NO) is capable of protecting the liver against apoptosis,418 as it is able to inhibit the apoptotic signal transduction cascade at multiple points.419 As nitric oxide synthase (NOS) is normally expressed by endothelial cells (eNOS, also called NOS3), and may be induced in endothelial cells (iNOS, also NOS2), hepatocytes, and Kupffer cells under a wide variety of conditions, there is ample opportunity for this potent mediator to influence the apoptotic outcomes of hepatic injury.

Necrosis Necrosis is different from apoptosis in that it involves cell swelling, vacuolation, karyolysis, and release of cellular contents (Fig. 1.39).392 In keeping with the earlier discussion on hepatocellular ballooning degeneration, the term ‘oncotic necrosis’ has been applied to this form of cell death, owing to the loss of osmotic regulation of ion content at the level of the plasma membrane.149 In addition to cellular swelling en route to plasma membrane rupture and cell death, plasma membrane blebs form which are devoid of organelles.420,421 Both with fragmentation of the membrane blebs, and outright cell rupture, there is complete release of cellular contents into the extracellular environment, quite distinct from apoptosis. Oncotic necrosis is the predominant mode of death in states of extreme ATP depletion such as occurs during ischaemia or hypoxic

Figure 1.39  Oncotic hepatocellular death in alcoholic hepatitis. Severely ballooned hepatocytes have lost their sharply defined cell borders, and have undergone cell death. In the centre of the field a residual Mallory–Denk body (*) and brown discoloration of cholestasis is also present. (H&E)

44

cell injury, and oxidative stress with formation of reactive oxygen species,419 although apoptosis also may contribute to cell death in the latter.422 Accordingly, organ ischaemia due to interruption of blood flow with subsequent reperfusion is a valuable experimental example of injury leading to hepatocyte necrosis. Ischaemia– reperfusion occurs during hepatic resection, liver transplantation, and hypotensive shock followed by recovery.423 The first key step in the process of injury is depletion of intracellular ATP during the ischaemic period. This then precipitates an increase in intracellular Ca1+, owing to decreased active extrusion of Ca1+ from the cell by Ca1+-ATPase, and an opening of voltage-dependent Ca1+ channels due to membrane depolarization caused by decreased activity of the Na+-K+ ATPase (the latter of which consumes approximately 25% of cellular ATP under normal conditions).424 The increase in intracellular Ca1+ destroys the cytoskeleton,425 and plays a critical role in the opening of the mitochondrial permeability transition pore, thereby stimulating the mitochondrial pathway of apoptotic cell death.426,427 The second key event in ischaemia–reperfusion injury is the generation of oxygen radicals during reperfusion.423 As initially hypo­ thesized by McCord in 1985 and Adkins et al. in 1986,428,429 during the ischaemic interval, cytosolic xanthine dehydrogenase is converted to xanthine oxidase. Meanwhile, degradation of adenine nucleotides leads to accumulation of hypoxanthine in the ischaemic organ. Upon reperfusion and introduction of oxygen substrate, xanthine oxidase generates massive amounts of superoxide anion. The resultant oxidative damage disrupts cytoplasmic and nuclear proteins, organelle membranes and proteins, and DNA. The cytoskeleton also undergoes proteolysis, and mitochondrial oxygen reduction is also impaired. The reactive oxygen species also act as intracellular second messengers, inducing cascade reactions through the transcriptional factors NFκB and AP-1. As the liver has the greatest amount of macrophages of any organ in the body, these macrophages can also secrete oxygen radicals as well as tissue-toxic cytokines such as TNFα and IL-1; the recruitment of inflammatory cells further exacerbates the tissue injury. Interestingly, in oncotic cell death, nitric oxide (NO) potentiates hepatocellular necrosis. This appears to be mediated by an NO-induced decrease in intracellular ATP.430 As NO is generated by endothelial cells activated during tissue injury and inflammation,431 the sinusoidal endothelium can be yet another contributor to hepatic injury. Necrosis frequently exhibits a zonal distribution. The most obvious is necrosis of hepatocytes immediately around the terminal hepatic vein, an injury that is characteristic of ischaemic injury and a number of drug and toxic reactions (Fig. 1.40). Pure midzonal and periportal necrosis are rare; the latter may be seen in eclampsia. Necrosis of entire lobules (submassive necrosis) or of most of the liver (massive necrosis) is usually accompanied by hepatic failure. It should be noted submassive and massive ‘necrosis’ may also have resulted from extensive apoptosis, as in fulminant viral hepatitis. A cardinal feature of oncotic necrosis is mitochondrial dysfunction, characterized by permeability of both the outer and inner mitochondrial membranes.421 This contrasts with apoptosis which is associated with selective permeabilization only of the outer mitochondrial membrane. Loss of the inner mitochondrial membrane barrier leads to collapse of ion gradients and loss of the mitochondrial membrane potential that drives oxidative phosphorylation.432 Cellular ATP levels then collapse further, membrane ion pumps fail, leading to cell blebbing, swelling, and rupture. The exact mechanisms underlying permeabilization of both the outer and inner mitochondrial membranes are not clearly understood.

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Mitotic activity Parenchyma Portal tract

Hepatocytes

Canal of Hering Bile ductule Bile duct A Mild parenchymal damage

Hepatocyte differentation

Figure 1.40  Ischaemic hepatocyte necrosis following hepatic artery occlusion post-transplant. In the upper part of this field, hepatocyte features are completely lost, leaving only indistinct eosinophilic remnants of the hepatocyte cords, occasional pyknotic hepatocyte nuclei, and partial preservation of some sinusoidal cells such as macrophages. In the lower part of the field there are more viable liver cells. (H&E)

Regeneration Mild injury to hepatocytes, cholangiocytes, or the endothelium and mesenchyme may be met with complete recovery of the organ. There may also be complete recovery of the liver following massive hepatocellular death provided that inflammatory and fibrogenic pathways are not initiated. Regeneration of liver cells is the key step towards recovery from mild and severe injury. Although the human liver recovers its mass slowly after massive injury or surgical resection, hepatic function can be maintained despite significantly reduced tissue mass. Following childhood, the normal liver becomes a stable organ with slow turnover of hepatocytes. However, upon injury or surgical reduction, the liver converts to a proliferative organ that can restore approximately three-quarters of its own mass within 6 months. In addition to mesenchymal cells and hepatic stellate cells, hepatocytes, cholangiocytes, and hepatic progenitor or stem cells maintain the potential to multiply throughout adult life.433 Depending on the type of injurious agent, the nature of the liver disease, and the extent of hepatic destruction, liver regeneration may occur by at least two mechanisms (Fig. 1.41).434–436 First, mature differentiated hepatocytes and cholangiocytes may undergo division and replication, remaining within their tissue compartment and responding quickly to liver damage associated with mild to moderate cellular loss. Second, more extensive or massive hepatic necrosis stimulates the proliferation of progenitor cells within the periportal region, involving ductular reaction and differentiation through hepatocyte intermediates to new hepatocytes; and through cholangiocyte intermediates to new bile duct elements.437

Regeneration of mature liver cells Over and above the oft-cited mythological capacity of the liver of Prometheus to regenerate daily,438–441 the definitive experimental demonstration of liver regeneration was shown in rodents following two-thirds partial hepatectomy.442 This is not ‘regeneration’ in the strictest sense, since the lobes of the liver are not regrown. Rather, there is a profound hyperplastic response involving replication of

Bile duct Mitotic activity Ductular proliferation vs. oval cell proliferation B Severe parenchymal damage Figure 1.41  Mechanisms of parenchymal regeneration in the liver. (A) With mild to moderate parenchymal damage, as in chronic hepatitis, mitotic activity of hepatocytes alone is sufficient to maintain liver mass. The architecture of bile ductules, which bridge between the portal tract bile duct and the canals of Hering, remains normal. (B) With severe parenchymal damage, either in the setting of massive hepatic necrosis or during severe bouts of chronic hepatitis, there is proliferation of progenitor cells within the liver. These are thought to be derived from bipotential cells of the bile ductule/canal of Hering compartment, evident histologically as ‘ductular reaction’ and/or from a stem cell or ‘oval cell’ compartment. Although the relationship between ductular reaction and oval cell proliferation is not fully established, hepatocyte differentiation of these progenitor cells leads to restitution of liver mass in the unscarred liver, or contributes to the nodular proliferation of the scarred liver characteristic of cirrhosis.

virtually all (95%) of the mature functioning hepatocytes in the residual liver. The regenerative process is compensatory, since it stops once the original mass of the liver has been restored. By far the quickest and most efficient way to restore liver mass is through replication of existing hepatocytes.443 These observations have subsequently driven the field of liver regeneration, since explanation of both the stimuli for regeneration and for its cessation is considered to have immense value for understanding tissue growth under normal and cancerous conditions (in which failure of ‘cessation’ is critical). The key questions to be answered are:439 (1) What are the signals that trigger the early events in the regenerative process? (2) How are the architecture and function of the liver retained during regeneration? (3) Which signals

45

MacSween’s Pathology of the Liver

are responsible for turning off the growth response once the mass of the liver is reconstituted? (4) What cells are involved in liver regeneration? An extensive literature is reviewed elsewhere,444–449 and is summarized briefly here. The discussion will focus on hepatocyte regeneration; cholangiocyte regeneration has been reviewed elsewhere.450,451 In rats subjected to two-thirds partial hepatectomy, about 12 h are required for the rate of hepatocyte DNA synthesis to increase, as they move from the G0 phase of the cell cycle (quiescent) to the S phase. Hepatocellular DNA synthesis peaks at around 24 h. The induction of DNA synthesis occurs later in the non-parenchymal cells: at about 48 h for Kupffer and biliary epithelial cells and at about 96 h for endothelial cells. Complete restoration of liver mass requires about 1.6 cycles of replication in all cells.439 In mice, the induction of DNA synthesis and cell division occurs slightly later, and varies between strains. It should be noted that the limited number of cell cycles required to restore liver mass does not mean that hepatocytes have limited replication capacity. Serial transplantation experiments demonstrate that hepatocytes can replicate ≥70 times,452 without depending upon stem cell populations.

Regenerative signals Polypeptide growth factors Circulating polypeptide growth factors are capable of inducing hepatocyte replication, and include hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor alpha (TGFα), amphiregulin, heparin-binding EGF-like growth factors and insulin-like growth factors. Human hepatocyte growth factor (HGF) is an 85-kD protein, which can act as a motogen (stimulating proliferation), morphogen (stimulating maturation), or motogen (stimulating cell movement) for many different cell types. HGF is synthesized by non-parenchymal cells, particularly stellate cells, and therefore affects hepatocytes in a paracrine manner.439 HGF is secreted as a precursor, pro-HGF, and is rapidly activated by proteases – urokinase-type plasminogen activator (uPA) and its downstream effector, plasminogen. HGF is a potent stimulator of DNA and protein synthesis in hepatocytes,453 and is 10 times more effective than other polypeptide growth factors, such as EGF, in promoting hepatocyte proliferation.454 HGF can be bound by extracellular matrix proteins, so that remodelling of extracellular matrix through local release of matrix metalloproteinases can elevate levels of HGF and other growth factors such as VEGF, FGF and TGFβ within the microenvironment.455–457 HGF elicits its biological functions upon binding with its membrane receptor, the tyrosine kinase encoded by the c-MET proto-oncogene. The c-Met receptor has a multifunctional docking site on the cytoplasmic domain, enabling the phosphorylation of different intracellular transducers. The balance between proliferation and differentiation elicited by HGF binding to the c-Met receptor depends upon which intracellular signalling pathways are recruited.458 Epidermal growth factor (EGF) and transforming growth factor alpha (TGFα) are closely related polypeptides. EGF is synthesized and released by a large number of tissue cells and is motogenic in many mesenchymal and epithelial structures, including the liver.459 It is very effective in stimulating liver regeneration and this activity is considerably enhanced by insulin. Its potency for the liver may be due to the high number of receptors on the hepatocyte plasma membrane. The liver thus has an extraordinary capacity to clear EGF from the blood.460 Although EGF may be a major, primary hepatic growth regulator, blood levels of EGF do not change much immediately after partial hepatectomy461 and there is no increase in messenger RNA for EGF receptors for the first 24 h.462 However, amphiregulin, a ligand for EGF receptor (EGFR) is expressed within

46

30 min of partial hepatectomy.463 Amphiregulin is a polypeptide growth factor of the EGF family, and is expressed normally in the human ovary and placenta but not in the healthy and quiescent liver parenchyma.464 Amphiregulin can be detected in cultured hepatocytes, and is detectable in the cirrhotic liver.463 In the regenerating liver, tyrosine phosphorylation (and hence activation) of the EGFR is dramatically upregulated at 60 min, despite the absence of an elevation in EGF levels. It appears that amphiregulin is uniquely involved in this activation, as other ligands for EGFR cannot serve as substitutes. Hence, the EGFR may play a key role in initiation of liver regeneration depending upon which agonist ligand is bound, with amphiregulin playing a specific and unique role.465 Messenger RNA for TGFα is undetectable in the normal liver but appears within 8 h of partial hepatectomy and increases rapidly over the next 24 h in parallel with DNA synthesis.466 Since release of TGFα may be stimulated by EGF, an early role for EGF in hepatic regeneration cannot be excluded.467 TGFα also could be derived from extrahepatic sources. Either way, TGFα may act as a potent autocrine regulator in promoting liver regeneration, in the midst of its fibrogenic effects on hepatitic stellate cells. Other polypeptides involved include heparin-binding growth factors (HBGF) and the insulin-like growth factors (IGF, somatomedins). HBGFs have also been termed fibroblast growth factors (FGF) and are motogens for a wide variety of cell types. There are two main subtypes, HBGF-1 and HBGF-2, and an increase in both is found in hepatocytes at early, though not the earliest, stages of liver regeneration.468,469 As with TGFα, they may have an autocrine role in helping to amplify the initial regenerative signals. Insulin-like growth factors (IGFs) are synthesized in the liver in response to growth hormone and represent the probable mechanisms through which growth hormone influences liver regeneration. A time lag of several hours elapses before enhancement of regeneration occurs in response to growth hormone, and these polypeptides probably have a modulatory rather than an initiating role.470 The intracellular signalling pathways induced by these various polypeptide growth factors are complex and highly inter-related; these complex processes are discussed elsewhere.455,457

Nutritional and hormonal regulation The regenerating liver faces a considerable metabolic challenge, since it must continue to regulate systemic energy levels while meeting its own demands for reparative biosynthesis.444 Hepatic regeneration depends upon the availability of the basic nutrient building blocks that are usually supplied via the portal venous blood from intestinal digestion and absorption. Fasting has been shown to delay and diminish regeneration in response to partial hepatectomy, but it does not abolish it.471 It has also been demonstrated that there is an increase in the circulating amino acid pool following partial hepatectomy472 and enhanced movement of amino acids occurs across the hepatocellular membrane.473 Infusions of a branched-chain-enriched mixture of amino acids have been shown to stimulate regeneration474 although, generally, infusions of amino acids, lipids, or carbohydrate have an inhibitory effect upon the response.475 However, dietary protein does enhance regeneration as increased mitotic activity can be demonstrated in hepatocytes when experimental animals are transferred from lowprotein to normal or high-protein diets.476 Unfortunately, the very amino acids which may be beneficial to hepatic regeneration in the setting of cirrhosis are likely to tip a human patient into hepatic decompensation due to the amino acid load. Virtually all hormones have been shown to influence regeneration in the liver. They include insulin and glucagon, thyroid and adrenal cortical hormones, parathyroid hormone, prolactin, vasopressin, prostaglandins, catecholamines, and sex hormones.477 Of

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

these, the most important seem to be insulin, glucagon, and the catecholamines. In the absence of insulin, regeneration is not diminished but it is delayed in onset and takes much longer.478,479 Conversely, the presence of insulin has the opposite effect.480 Glucagon acts synergistically with insulin but neither hormone alone or in concert is able to initiate, or by their absence prevent, regeneration.468 Various observations suggest that catecholamines are involved in the early stages of liver regeneration and could play a part in the regulation of liver growth. Blood catecholamine levels are known to increase within 2 h of partial hepatectomy, and DNA synthesis in the regenerating liver is significantly reduced by chemical or surgical sympathetic denervation.481 The rich sympathetic nerve supply to portal tracts and sinusoids is also greatly increased in the liver after regeneration.482 The response appears to be modulated through the α-adrenergic receptors, and β-blockers do not have any influence. The number of α1-adrenergic receptors on hepatocytes decreases by 30–40% in the 24 h after partial hepatectomy, while β-receptors increase dramatically.483,484 Noradrenaline enhances the effects of epidermal growth factor on DNA synthesis in cultured hepatocytes485 and counteracts the growth inhibitory effects of TGFβ.486 Thyroid hormones can also promote regeneration but a combination of triiodothyronine, amino acids, glucagon and heparin is even more effective.487

Normal Avascular parenchymal islands

Cytokines Cytokines themselves play a critical role in the regulation of liver regeneration. On the positive side, IL-6 is a key effector cytokine, as it acts upon its receptor, IL-6R, to stimulate motogenic intracellular signalling cascades, and can potentiate signalling induced by growth factors.488 In mice, normal liver regeneration requires IL-6,489,490 although whether IL-6 is the major pro-regeneration cytokine is open to debate.491,492 TNFα is itself required for a normal prolif­ erative response after partial hepatectomy,493 as it induces IL-6 expression. However, absence of TNFα does not impair liver regeneration.494 Rather, the ability of Kupffer cells to generate IL-6 as the primary source of IL-6 in the liver is stimulated by TNFα or other agents.495 As the innate immune system plays a key role in the local generation of TNFα and IL-6, there is clearly an interplay between the immune response to injury and the initiation of a hepatic regenerative response. Moreover, as liver regeneration in the clinical setting is more often in the context of inflammation (as opposed to following surgical reduction), it is notable that the NK cells of the innate immune system impede the regenerative response.496 With the previously known importance of the Wnt/β-catenin pathway in hepatocarcinogenesis and the more recent discovery that this pathway also is a central regulator of hepatic lobular zonation, attention also has been directed to liver regeneration.497 The Wnt/βcatenin pathway is activated relatively early during regeneration, and drives the expression of target genes that are critical for cell cycle progression and help initiate the regenerative process. Whether this pathway is redundant in liver regeneration remains to be seen, although an argument can be made that this pathway would be expected to help reestablish normal zonation of hepatocellular function, with possible modulation by the autonomic nervous system.498

Maintenance of liver architecture Maintenance of hepatic function during regeneration requires reassembly of the liver parenchymal microarchitecture. After partial hepatectomy, hepatocytes rapidly proliferate and form cell clusters of 10–14 cells.499 These clusters are devoid of extracellular matrix and vascular channels. Particularly important is the angiogenesis that must occur, whereby ingrowth of epithelial cell buds impales

Establishment of new sinusoidal channels Figure 1.42  Schematic of liver regeneration, whereby proliferation of hepatocytes generates avascular parenchymal islands and thickened hepatocyte cords. Angiogenesis with endothelial cell proliferation, infiltration of hepatocyte clusters, and re-establishment of sinusoidal architecture allows return of the parenchyma to a normal vascular architecture. (Based on concepts presented in Ross et  al. 2001500)

the cell clusters and enables re-establishment of normal sinusoids (Fig. 1.42).500,501 This is the result of highly coordinated spatiotem­ poral expression of angiogenesis growth factor receptors on sinusoidal endothelial cells within the regenerating cell clusters.500 These include receptors for VEGF, angiopoietin, PDGF, and EGF. As all receptors are tyrosine phosphorylated, they are in a peak state of activation during the angiogenic phase of regeneration. Replicating stellate cells produce extracellular matrix about 4 days after partial hepatectomy, re-establishing the contact between hepatocytes and matrix. Simultaneously, sinusoidal endothelial cells orchestrate a rich environment of paracrine trophogens that helps coordinate hepatocellular proliferation.502 The result is re-establishment of communicating sinusoids, with initially oversized intervening hepatocyte plates. Further remodelling is required before the final normal relationships between hepatocytes and the sinusoids are established. This requires elimination of superfluous hepatocytes through apoptosis.450 Activation of Hedgehog (Hh), a morphogenic signalling pathway that controls progenitor cell fate and tissue construction during embryogenesis, occurs during liver injury in the adult. Activation of this pathway also promotes liver regeneration, including hepatic accumulation of inflammatory cells, extracellular matrix deposition, and vascular remodelling, all of which can play a role in tissue remodelling.503

Cessation of the regenerative response The best known hepatocyte antiproliferative factors are TGFβ and TGFβ-related family members such as activin.450,503 TGFβ is

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produced primarily by hepatic stellate cells, and contributes to liver fibrosis (discussed below). Stimulation of the TGFβ signalling pathway can induce G1/S phase arrest and induction of apoptosis.504 However, hepatocellular responses to TGFβ are complex, in that hepatocytes can exhibit resistance to the antiproliferative effects of TGFβ,505 most probably through inhibition of the TGFβstimulated intracellular SMAD signalling pathways.505 Activin, in turn, is an apoptogen of the TGFβ family that blocks hepatocyte motogenesis.439 Activin can also induce a reduction in liver mass, as it promotes apoptosis in hepatocytes.450 What is most intriguing is how the termination system is engaged, and then disengaged. The termination sequence appears to require, and act in parallel with, restoration of normal sinusoidal architecture and repopulation of the subendothelial space with stellate cells.450 Stellate cells home to the newly formed space of Disse in response to PDGF, FGF, and EGF. TGFβ augments the chemotactic response of stellate cells to PDGF and FGF. Transient activation of stellate cells to myofibroblasts is required for synthesis of peri­ sinusoidal extracellular matrix and completion of regeneration within the parenchyma.506 These transiently activated stellate cells are capable of generating TRAIL, thereby generating a TRAIL receptor-dependent apoptosis signal affecting both hepatocytes and stellate cells themselves.507 There thus appears to be a negative feedback loop which is both autocrine (stellate cell–stellate cell) and paracrine (stellate cell–hepatocyte) to limit the regenerative surge. Inactivation of the termination sequence may involve two transcriptional repressors of the TGFβ/Smad pathway, SnoN and Ski, which antagonize TGFβ-mediated termination via binding to Smad proteins.505 Telomere length is important for the replicative potential of hepatocytes, as experimental knockout of telomerase (the enzyme that maintains telomere length) impairs DNA synthesis, shortens the lifespan of hepatocytes, and impairs their ability to regenerate.508 Wiemann et al.509 demonstrated that hepatocyte telomere shortening correlated with progression of fibrosis and development of cirrhosis in human chronic liver disease. It is not clear whether relative deficiencies of telomerase both limit the normal regenerative response, and contribute to chronic liver disease through a failure of nascent cells to maintain adequate rates of regeneration.

The role of progenitor cells Hepatocytes, cholangiocytes and endothelial cells maintain the potential to multiply during adult life.433 However, certain types of injury render these cells unable to replicate. Toxic injury may diminish or eliminate the capacity of hepatocytes to replicate. Experimental toxins include diplin, retrorsine, galactosamine, and monocrotaline.510 Above and beyond toxic injury, extensive hepatic necrosis, especially involving the periportal region of the parenchyma, stimulates the proliferation of progenitor cells.511,512 As noted in the earlier section on innervation, surgical or pharmacological denervation of the liver may severely impair the regenerative response of both hepatocytes and cholangiocytes. Identification of small cells with oval nuclei in rat models of hepatocarcinogenesis and in regenerative responses to liver injury led to the premise that there is a liver progenitor cell compartment.513 The term ‘oval cell’ was first coined in 1956 by Farber,514 to distinguish these cells in different models of rat hepatocarcinogenesis. Similar oval cells have been identified in the human liver,515 and are capable of differentiating into hepatocytes and cholangiocytes. Similarities to the ductal plate hepatocytes of the fetal liver suggested that adult progenitor cells reside in the periportal region,433,516 with extensive attention being given to the canal of Hering : ductular compartment at the portal tract : parenchymal

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Figure 1.43  Ductular hepatocytes. Significant parenchymal destruction, particularly at the interface with the portal tracts, engenders a brisk regenerative response featuring ‘ductular reaction’. These ductular structures can differentiate towards a bile ductular phenotype (arrowheads), or acquire the granular and larger cytoplasm of the hepatocellular phenotype (asterisks). In this particular image from autoimmune hepatitis, there is extensive fibrosis as well. (Masson trichrome)

interface.19,20 Proliferation of this compartment gives rise to ductular structures containing cuboidal cells and slightly larger cells with mitochondria-rich cytoplasm (Fig. 1.43). Immunohistochemical stains demonstrate keratin staining characteristic of cholangiocytes, simultaneously with albumin staining.433,516 With time, these cells are capable of maturing into definitive hepatocytes on the one hand, and repopulating damaged bile duct structures with mature cholangiocytes on the other.517 In massive hepatic necrosis, there appears to be an explosive proliferation of ductular structures which are all connected to individual canals of Hering and thence to terminal bile ducts within portal tracts.20,518,519 These structures comprise the parenchymal ‘ductular reaction’ in massive hepatic necrosis,20 to be distinguished from the portal tract-based ductular reaction observed in the bile duct-obstructed liver (see Fig. 1.36 and Table 1.12). The hypothesis can thus be advanced that the bipotential progenitor cells reside in, or at least close to, the canals of Hering.520,521 The ‘ductular’ compartment may also serve as a transit point between mature hepatocytes and cholangiocytes, as transdifferentiation of rat hepatocytes into biliary cells has also been demonstrated.522 Bile ducts themselves may also contain cells phenotypically capable of acting as liver progenitor cells.523 ‘Plasticity’ of the liver may therefore involve the regenerative capacity of progenitor cells within the mature liver parenchyma, an ‘oval cell’ compartment in, or immediately adjacent to, the canal of Hering, and the biliary tree itself.524 There is a growing literature addressing whether hepatic progenitor cells can be derived from non-hepatic precursors, particularly bone marrow stem cells.447,525–527 At the very least, ongoing parenchymal destruction in the diseased liver, particularly when it is occurring at the septal interface, appears capable of stimulating proliferation of the bipotential progenitor cell compartment at the portal tract interface. Whether entry of stem cells from the bone marrow into the human liver plays a substantive role in liver restitution remains unclear and highly controversial. Attention also is being given to whether therapeutic delivery of engineered stem cells may be of value in treatment of liver diseases.445,528,529

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Fibrosis While regeneration may be viewed as a beneficial step to recovery, a central and perhaps defining detrimental process in progressive chronic liver disease is fibrosis.530–533 Fibrosis may be regarded as the wound healing response of the liver to repeated injury.534 Specifically, following acute liver injury, surviving cellular elements may regenerate, with an accompanying inflammatory response to clean up debris, and limited deposition and remodelling of extracellular matrix. However, if the hepatic injury persists, liver regeneration may fail to restore lost tissue and matrix deposition becomes more extensive, akin to the formation of scar in the liver. As chronic liver disease advances, excess type I and III collagens are laid down not only in portal tracts but also in the lobule, creating both fibrous septal tracts and severe alterations to sinusoidal ultrastructure. In the parenchyma, the key events involve stellate cells, deposition of extracellular matrix, and alteration of the parenchymal microvasculature. In portal tracts, resident myofibroblasts, including those around bile ducts, play a key role, with a potential contribution as well from epithelial–mesenchymal transition of cholangiocytes to matrix-producing cells. The mechanisms of hepatic fibrosis will be discussed in this section; discussion of vascular changes follows. In the normal liver, the extracellular matrix comprises less than 3% of the relative area on a tissue section.535 In advanced liver disease, all types of collagens, glycoproteins, and proteoglycans can increase to three times or even more than the amounts normally found in the liver.536,537 Collagen levels per se can increase up to eight-fold, mainly because of increased collagen type I deposits.538 In addition to the collagen deposition that can occur in portal tracts, collagens and non-collagenous extracellular matrix proteins are also deposited in the space of Disse.170,257 These include collagen types III and IV, laminin, and fibronectin, which are normal components of basement membranes. Aberrant deposition of extracellular matrix within the hepatic parenchyma produces an environment in which: (1) scarring with type I collagen develops; and (2) extracellular matrix proteins normally present in basement membranes are deposited within the space of Disse, creating a major barrier for solute exchange.539 Studies in rats have shown that, on a percentage area basis, total extracellular matrix components can increase to 25–40% in cirrhosis.540 The space of Disse changes from containing delicate interspersed strands of fibrillar collagen (types III and IV) to a dense matrix of basement membrane-type matrix proteins, closing the space of Disse to protein exchange between hepatocytes and plasma. In combination with the loss of fenestrations in the sinusoidal endothelium, this process is called ‘capillarization’ of the sinusoids (Fig. 1.44).541 In general, abnormal matrix deposition within the space of Disse occurs in those parts of the parenchyma where cell injury and inflammation are greatest. The extracellular matrix changes have a profound effect on liver cell regeneration and vascular redistribution.

Hepatic stellate cells As discussed earlier, hepatic stellate cells, residing in the space of Disse, normally are quiescent, fat-storing cells. With repeated injury to the liver parenchyma, stellate cells become activated, lose their retinyl ester stores, and transform into myofibroblast-like cells which are positive immunohistochemically for α-smooth muscle actin (Fig. 1.45).186 The major parenchymal source of excess collagen under abnormal conditions is stellate cells.217 The key features of stellate cell activation are: (1) robust mitotic activity in areas developing new parenchymal fibrosis; (2) a shift from the restingstate ‘lipocyte’ phenotype to a transitional myofibroblast phenotype; and (3) increased capacity for synthesis and secretion of

extracellular matrix.186,540 It is predominantly the cytokines secreted by activated Kupffer cells and other inflammatory cells that stimulate the stellate cells to divide and to secrete large amounts of extracellular matrix.542,543 There is a marked increase in stellate cell expression of messenger RNAs for collagens I, III, IV and laminin.544,545 Although inflammation is a key activator of stellate cells, they are themselves contributors to the inflammatory response, by acting as antigen-presenting cells.546 The activated state of stellate cells is maintained by survival factors. In keeping with the apposition of autonomic nerve endings on stellate cells, the fibrogenic phenotype is also promoted by α-adrenergic stimulation.547 Involution of the activated stellate cell population occurs through apoptosis, when the state of inflammation and release of proinflammatory cytokines subsides.507,548–550 The contractile properties of stellate cells during fibrogenesis and in cirrhosis have a major impact on vascular resistance and ensuant liver blood flow. Endothelin-1 (ET-1) is the key contractile stimulus for activated stellate cells.186,550 This system acts in an autocrine loop of stimulation whereby ET-1 synthesis is upregulated in activated stellate cells, as is expression of its two receptors, ET-A and ET-B.550 In the normal liver, ET-1 induced vasoconstriction is in balance with nitric oxide (NO) mediated vasodilatation (via antagonism of ET-1). During liver fibrosis, there is an increase in ET-1 expression and a decrease in NO generation by the sinusoidal endothelium, shifting the balance towards stellate cell (myofibroblast) contractility.551 The greatest activation of stellate cells is in areas of severe hepatocellular necrosis and inflammation.552 The activation of stellate cells may also exhibit a zonal pattern. Perivenular sinusoidal fibrosis has been well documented in the early stages of liver injury from alcohol553,554 or toxins,555 in both animals and humans. This is accompanied by a decrease in sinusoidal density in the perivenular region.556 Because the size of acini does not change significantly during the development of fibrosis/cirrhosis,557 the reduction in sinusoidal density appears to represent a true loss of microvascular capacity, before complete nodule formation occurs. Moreover, there is an increase in stellate cells in the perivenular region prior to the development of fibrous septa.557,558 These findings indicate that simultaneous alterations in stellate cells, extracellular matrix, and the parenchymal microvasculature occur during the evolution of cirrhosis. Put differently, microvascular changes are not merely the consequence of fibrosis and nodule formation, but are part of the evolution of cirrhosis.556

Portal tract fibrogenesis The above discussion has focused on the role of hepatic stellate cells in parenchymal fibrosis. Increasing attention is being given to fibroblasts located in portal tracts, both encircling bile ducts and ductules, and elsewhere in portal tracts.559–563 Portal tracts contain the portal vein, the hepatic artery, and the bile duct. While the portal vein and hepatic artery have walls composed of smooth muscle cells, the cholangiocytes reside on a basement membrane, which is surrounded directly by periductular fibroblasts.269 Biliary fibrosis initiated by bile duct inflammation and bile duct obstruction occurs as a result of rapid activation of the peribiliary fibroblasts with acquisition of smooth muscle actin, generating a myofibroblast phenotype.562 The progressive enlargement of portal tracts in these circumstances involves extensive bile duct proliferation, massive proliferation of peribiliary (myo)fibroblasts, deposition of portal tract collagen, and oedema (Fig. 1.46).559,564 The fact that it is peribiliary fibroblasts that are primarily responsible for the evolving hepatic fibrosis is explanation for the observation that ‘biliary fibrosis’ does not characteristically subdivide the liver with parenchymal

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Bile canaliculus

Space of Disse with perisinusoidal stellate cell

Fibrillar collagen Myofibroblasts

Sinusoidal endothelial cell (fenestrated)

Sinusoid

Hepatocyte

Sinusoid

Hepatocyte

Twinning of hepatocytes

Sinusoidal endothelial cell (non-fenestrated) Basal lamina

Normal liver Fenestrated endothelium No basement membrane Fat-storing perisinusoidal stellate cell Sparse reticular collagen Microvilli on hepatocyte basal surface

Cirrhotic liver Loss of endothelial fenestrations Capillarization Acquisition of basal lamina Proliferation of myofibroblasts Deposition of extracellular matrix and fibrillar collagen Loss of microvilli on hepatocytes

Figure 1.44  ‘Capillarization’ of sinusoids. In this schematic, the normal sinusoidal microanatomy is depicted on the left, and cirrhotic liver on the right. The sinusoidal channel is bounded by sinusoidal endothelial cells, which normally are fenestrated but lose their fenestrations in the cirrhotic liver. The space of Disse normally contains scattered fat-storing perisinusoidal stellate cells; these proliferate and become myofibroblasts in the cirrhotic liver. There are normally only delicate reticular collagen fibrils in the space of Disse. Activated myofibroblastic stellate cells are the primary source of fibrillar collagen and other extracellular matrix proteins which are deposited in the space of Disse. Notably, a basal lamina is deposited under the non-fenestrated endothelial cells, completing the process of ‘capillarization’. Lastly, hepatocytes lose their abundant basal microvilli (facing the space of Disse); regeneration of hepatocytes leads to thickened hepatocellular plates (‘twinning’).

fibrous septa (which would derive from sinusoidal stellate cellderived myofibroblasts) until late in the disease. The myofibroblasts of bridging septa in cholestatic fibrotic livers strongly resemble the myofibroblasts of the portal field,565 suggesting that portal tract myofibroblasts migrate into the developing parenchymal septa.566 Indeed, fibrillin-1 expression in conjunction with elastin may help distinguish myofibroblasts derived from the portal tract (elastinpositive) vs sinusoidal stellate cells (elastin-negative). Specifically, activated stellate cells acquire smooth muscle actin expression and deposit fibrillin-1-positive but elastin-negative extracellular matrix, whereas portal tract-derived myofibroblasts deposit extracellular matrix containing both fibrillin-1 and elastin.270 An alternative view, however, is the fact that portal fibroblasts produce significant amounts of TGFβ2 and, unlike activated stellate cells, express all three TGFβ receptors and are growth inhibited by TGFβ1 and TGFβ2.567 Fibroblast growth factor (FGF)-2, but not PDGF, causes portal fibroblast proliferation. Regardless, these data suggest a mechanism whereby portal fibroblasts play the dominant role in biliary fibrosis.563 Portal tract fibrosis also occurs in the setting of hepatitis, and other populations of fibrogenic cells in portal tracts probably play

50

a role. These include myofibroblasts loosely placed around the portal vein and hepatic artery, and those in the loose connective tissue of the portal field, especially at the interface between the portal tract and parenchyma.568 These portal myofibroblasts may represent a convergence of phenotype from quiescent portal tract fibroblasts to activated myofibroblasts.270 However, portal tractderived myofibroblasts maintain some differences from hepatic stellate cell-derived myofibroblasts, on the basis of protein expression such as cellular retinol-binding protein-1 (CRBP-1).569 Smooth muscle cells in the wall of the portal tract blood vessels and second layer fibroblasts of the terminal hepatic vein are also capable of synthesizing extracellular matrix proteins, in keeping with the general observation that the main contributors to hepatic matrix production are mesenchymal cells.570

Epithelial–mesenchymal transition The process by which epithelial cells can change their phenotype and acquire mesenchymal properties is known as epithelial– mesenchymal transition (EMT),571 counterbalanced by a return to the epithelial phenotype, mesenchymal–epithelial transition

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Sinusoid

Endothelial cell Space of Disse

Activated stellate cells Quiescent stellate cell

Kupffer cell

Delicate collagen fibres

‘Myofibroblast’ proliferation contraction chemotaxis fibrogenesis

Activated Kupffer cell releases cytokines that promote: Proliferation: PDGF TNF

Contraction: ET-1

Chemotaxis: MCP-1 PDGF Fibrogenesis: TNFβ

Hepatocyte dysfunction and death

Hepatocytes

Bile canaliculus

Normal liver

Liver fibrosis

Apoptotic hepatocyte

Figure 1.45  Stellate cell activation and liver fibrogenesis (right), in comparison to the normal liver sinusoid (left). Inflammatory activation of Kupffer cells leads to secretion of multiple cytokines; cytokines may also be released by endothelial cells, hepatocytes, and other inflammatory cells of the innate immune system within the liver such as T lymphocytes (not shown). These cytokines ‘activate’ stellate cells, whereby the lipid droplets (present in the quiescent state) are lost, and the stellate cells acquire a myofibroblastic phenotype. Tumour necrosis factor (TNF) is a potent stimulant of the change to a myofibroblastic state. Stellate cell proliferation is stimulated in particular by platelet-derived growth factor (PDGF). Contraction of the activated stellate cells is stimulated by endothelin-1 (ET-1). Deposition of extracellular matrix (fibrogenesis) is stimulated especially by transforming growth factor-β (TGFβ). Chemotaxis of activated stellate cells to areas of injury, such as where hepatocytes have undergone apoptosis, is promoted by PDGF and monocyte chemotactic protein-1 (MCP-1) and CXCR3 ligands. Kupffer cells also are a major source of TNF released into the system circulation. (Schematic based on concepts presented in Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008; 134:1655–1669)

Figure 1.46  Portal tract and periportal parenchyma in a fibrotic reaction. The interface between portal tract with its hepatic artery (left) and the parenchyma (right) is indistinct. There are abundant, strongly staining, activated portal tract myofibroblasts and sinusoidal stellate cells. α-Smooth muscle actin immunohistochemistry.

(MET).572 In EMT, cells acquire an increased capacity to synthesize extracellular matrix, thereby contributing to fibrosis. EMT is normally seen during tissue morphogenesis in embryonic development, and also is involved in the progression of carcinoma by facilitating the generation of migratory neoplastic cells at the tumour invasive front. EMT also assists in tissue repair and fibrosis after injury, with abatement of the fibrogenic response as the

inflammatory response subsides. One of the master positive regulators of EMT is TGFβ1,573 with bone morphogenetic protein 7 (BMP-7) serving as a prototypical negative regulator of EMT.574 There is increasing evidence that EMT may contribute to the deposition of determinant fibrous tissue during chronic injury of organs,571 including the liver.575 Three types of adult liver cells can undergo EMT under experimental conditions: hepatocytes, cholangiocytes, and hepatic stellate cells.572 In the case of hepatocytes, the generation of EMT-derived fibroblasts may play a critical role in parenchymal fibrosis under certain conditions such as toxic injury.576,577 In the case of cholangiocytes, the fact that ductular reaction during chronic liver injury may include proliferation of liver epithelial cell progenitors, along with EMT to generate periductular mesenchymal cells, points towards the rich transitional environment at the portal tract interface during chronic liver injury.578,579 The peribiliary fibrogenesis observed in chronic bile duct destructive diseases may be generated both by pre-existing peribiliary myofibroblasts, and potential cells derived from cholangiocyte EMT.577 However, while EMT does appear to occur during liver injury and response, not all experimental data supports this concept,580 and the demonstration that EMT from hepatocytes and cholangiocytes is a key driver of chronic hepatic fibrogenesis in vivo remains controversial.572 An intriguing possibility is that EMT is more important for cell integrity and survival than a driver for fibrosis.581

Bone marrow-derived myofibroblast precursors Recalling the earlier discussion regarding bone marrow-derived stem cells in liver regeneration, experimental evidence also indicates

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that a potentially substantive portion of the hepatic stellate cell and myofibroblast population in fibrotic liver may be bone marrowderived.582,583 This is corroborated by identification of marrowderived myofibroblasts in the livers of human patients receiving gender-mismatched bone marrow or liver transplants.582 An alternative interpretation is that bone marrow-derived circulating mesenchymal cell precursors may enter the liver as fibrocytes and undergo myofibroblastic differentiation.584 The contribution of such circulating cells to liver fibrogenesis remains to be resolved.

Regulation of fibrogenesis At the outset, three conditions must be fulfilled for the general process of fibroblast to myofibroblastic differentiation: (1) increased tension in the extracellular matrix; and the production of (2) TGF-β and (3) fibronectin EDA. All three conditions may be fulfilled during liver injury.585,586

Extracellular matrix (ECM) The ECM provides architectural integrity, provides tensile strength and resilience, modulates diffusion of both small solutes and macromolecules through the extracellular space, and is a key regulator of cell movement.587 ECM proteins act as ligands to a vast array of receptors on many cell types, regulating cellular signalling, differentiation, and mobility. The ECM also binds bioactive factors, serving as reservoir of bound growth factors and cytokines which, upon disruption of the ECM, may be released into the local microenvironment. In the liver parenchyma, disruption of the ECM leads to changes in local mechanical tension, to which stellate cells are sensitive.587

TGFβ Chronic inflammation leads to production of inflammatory cytokines such as tumour necrosis factor alpha (TNFα) and beta (TNFβ), interleukin-1 (IL-1), TGFβ, platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF-1). TgFβ, in particular, is a potent stimulator of stellate cell activation.541

perpetuation. Proinflammatory cytokines, as released from Kupffer cells,595 render stellate cells more responsive to soluble factors which may perpetuate liver damage, as in acetaldehyde in alcoholic liver disease,596,597 and PDGF in many other forms of liver disease with induction of PDGF receptors on the stellate cells.186 The initiating activity of Kupffer cell medium has been attributed to the release of cytokines and in particular to TGFβ. The mechanisms controlling the release and activity of cytokines from Kupffer cells are part of a complex cascade of proinflammatory events.252,598 The matrix in the space of Disse also provides an extracellular reservoir of TGFβ which can be released and/or activated by matrix proteases. Stellate cells themselves may be capable of producing TGFβ, as the messenger RNA for this cytokine has been identified in these cells.186 TGFβ can also enhance the proliferation of stellate cells induced by epidermal growth factor (EGF) and PDGF.180 TGFβ thus would seem to be the most important initiating cytokine so far identified because it can stimulate both stellate cell transdifferentiation into myofibroblasts and collagen synthesis.599–601 Most of the other soluble cytokines simply modulate proliferation of the stellate cells and have little effect on matrix synthesis. Specifically, cytokines that have been shown to stimulate stellate cells into mitosis and migration without fibrogenesis include EGF, TGFβ, fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), IL-1, TNFα and PDGF.602 Retinoids may also play a key role in stellate cell activation. Chronic excess intake of vitamin A is associated with fibrosis and cirrhosis, but cirrhosis from other causes is associated with depletion of hepatic vitamin A.603 When stellate cells in tissue culture are activated they show a loss of retinoids, but the addition of exogenous retinoid to the tissue culture medium both inhibits cell proliferation and decreases type I collagen synthesis.604,605 Nuclear receptors for retinoids have now been shown to control expression of many genes within hepatocytes606 and it stands to reason that gene regulation in stellate cells is likewise regulated. Once stellate cells gain an activated phenotype, it is maintained and amplified through a perpetuation mechanism involving autocrine and paracrine mediators, including a potentially key role for osteopontin.607,608

Fibronectin EDA This is a splice variant of cellular fibronectin expressed both during development and in response to injury,588 and is crucial for myofibroblast phenotype induction by TGFβ.586,589,590 Fibronectin also increases survival of hepatic stellate cells.591 Sinusoidal endothelial cells are a key source of intrahepatic fibronectin EDA during injury, occurring early in the fibrogenic sequence, and preceding collagen deposition. TGFβ stimulates sinusoidal endothelial cells to gene­ rate fibronectin EDA; albumin modified with malondialdehydeacetaldehyde (an ethanol by-product) can stimulate this process as well.591 Hence, a mechanism is evident for linking both inflammatory and toxic liver injury to hepatic fibrogenesis. Stellate cells themselves, and potentially hepatocytes, may also produce fibronectin EDA, but usually later than the ‘first responder’ sinusoidal endothelial cells.592,593

Modulation by soluble mediators The changes in the extracellular matrix may be initiated by proteinases that are produced by stellate cells and Kupffer cells during liver injury, in response to a wide range of hepatic injuries.594 Proinflammatory cytokines also are produced by injured endogenous cells (Kupffer cells, endothelial cells, hepatocytes, and cholangiocytes). A variety of soluble mediators have been identified that are capable of influencing both stellate cells and Kupffer cells. These are separated into two broad functional categories of initiation and

52

Metalloproteinases Almost by definition, fibrogenesis involves deposition of excess extracellular matrix. Conversely, ‘reversal of cirrhosis’ requires a reduction of intrahepatic extracellular matrix. Cell surface-bound and soluble matrix metalloproteinases (MMP) and their endogenous tissue inhibitors (TIMPs) constitute an important system for regulating turnover of extracellular matrix.609 The proteinases are subdivided into collagenases which degrade fibrillar collagens, stromelysins which degrade many protein substrates including type IV collagen, gelatin, laminin, and fibronectin, and the type IV collagenase/gelatinases which have a specificity indicated by the name (Table 1.13). All of the MMPs are closely related,610 as members of the large family of zinc-containing endopeptidases.611 Interstitial collagenases (MMP-1, MMP-13) were first identified in 1962 in tadpole tails undergoing morphogenesis612 but they have since been identified in a wide variety of cells, including fibroblasts, smooth muscle cells, mononuclear phagocytes, and capillary endothelial cells. These enzymes are synthesized as a preproenzyme which is then activated by proteinases. The activated enzyme cleaves type I collagen at specific Gly–Ile and Gly–Leu bonds. The collagenases can also degrade type III collagen but have no demonstrable activity against type IV basement membrane collagen. Although there is no clear consensus on whether hepatic stellate cells, Kupffer cells, or

Chapter 1  Anatomy, pathophysiology and basic mechanisms of disease

Table 1.13  Matrix metalloproteinases

Enzyme

Metalloproteinase (MMP) number

Molecular mass (kD) Proenzyme

Active

Interstitial collagenase

MMP-1, MMP-13

57, 52

47, 42

Collagen type III > I > II

Neutrophil collagenase

MMP-8

72, 57

70, 50

Collagen type I > II > III

72 kD gelatinase/type IV collagenase

MMP-2

72

66

Collagen type IV, ? V, VII, X, gelatin

92 kD gelatinase/type IV collagenase

MMP-9

92

84

Collagen type IV, V, gelatin

Stromelysin-1

MMP-3

60, 57

45, 28

Collagen type III, IV, V, laminin, proteoglycan, fibronectin, gelatins

Stromelysin-2

MMP-10

53

47, 28

Collagen type II, IV, V, fibronectin, gelatins

Stromelysin-3

MMP-11

–

–

Not known

PUMP-1

MMP-7

28

21, 19

Collagen type IV, laminin, fibronectin, gelatin

Substrate specificity

Reproduced and updated from Friedman SL, Millward-Sadler GH, Arthur MJ. Liver fibrosis and cirrhosis. In: Millward-Sadler GH, Wright R, Arthur MJ, eds. Wright’s liver and biliary disease. 3rd ed. London: Saunders; 1992, p. 821–881.

even stem cells can produce these collagenases, a recent study suggests that exogenous macrophages are a key source of MMP-13, thereby playing a role in collagenolysis and recovery from liver injury.613 A neutrophil collagenase (MMP-8), which is very similar to interstitial collagenase, also exists. This has a substrate specificity for interstitial collagens but has a much greater avidity for degrading type I than type III collagen.614 The enzyme is released from the cytoplasmic granules of neutrophil polymorphs following activation, and may be relevant to hepatic conditions in which neutrophils are active (e.g. alcoholic hepatitis). Type IV collagenase/gelatinases (MMP-2, MMP-9). Two major proteinases in this category have been described, one is 92 kD and the other 72 kD in size. The larger 92-kD type IV collagenase/gelatinase is synthesized and released predominantly by macrophages and neutrophils.615,616 As its name implies, it actively degrades gelatin and collagen types IV and V but does not degrade the interstitial fibrillar collagens. The enzyme has been identified in Kupffer cells and is released on their activation.617 The 72-kD collagenase/ gelatinase is synthesized and released by tumour cells, fibroblasts, and osteoblasts, and to a lesser extent by mononuclear phagocytes. In the liver, activated stellate cells are a key source of this enzyme.618,619 Three stromelysins (MMP-3, MMP-10, MMP-11) and a closely related metalloproteinase, PUMP-1, have been identified from a variety of tissues. These enzymes have a broad range of matrix degradation activity. The potential role of this group of proteases in regulating the intrahepatic balance between fibrogenesis and fibro­ lysis is becoming increasingly recognized.620 While the MMPs are important players in degradation of extracellular matrix, more recently they are appreciated as playing critical roles as regulatory proteases for cellular signalling and regulation.621 They are capable of binding and processing hormones, cytokines, chemokines, adhesion molecules and other membrane receptors as well as intracellular proteins entering the extracellular milieu in settings of apoptosis and necrosis.622 Hence, there is great complexity in considering the intrahepatic biology of MMPs, as demonstrated by the fact that pharmacological inhibition of MMPs as an

anti-inflammatory intervention623 may aggravate development of liver fibrosis.609

Metalloproteinase inhibitors Specific tissue inhibitors of metalloproteinases (TIMPs) are stoichiometric inhibitors of MMPs, and hence must also be considered in the extracellular milieu of fibrolysis. While hepatic stellate cells clearly play a role in matrix protein synthesis, they also are able to regulate matrix degradation. In the early phases of activation, HSC release MMPs with the ability to degrade normal liver matrix. When HSC are fully activated, there is a net downregulation of matrix degradation, reflected in increased stellate cell synthesis and release of TIMPs-1 and 2.624,625 TIMP-1 is a small 28-kD glycoprotein secreted by a wide variety of cell types including fibroblasts. It binds irreversibly to active metalloproteinases of all types. Regulation of gene expression is influenced by the same growth factors and cytokines that are involved in the regulation of metalloproteinase gene expression. TIMP-2 is a smaller 21-kD protein with significant homology to TIMP-1 and with many similar properties. α2Macroglobulin is a high molecular weight plasma glycoprotein (725 kD) that is able to bind both interstitial collagenase and stromelysin.626 It is synthesized predominantly by hepatocytes but stellate cells also contain messenger RNA for this proteinase scavenger and can synthesize the molecule.627 There is thus a complex interplay of factors involved in the production and maintenance of the liver matrix that clearly involves metalloproteinases and their inhibitors. Initially, metalloproteinases can disrupt the normal liver matrix and thus create a microenvironment that stimulates stellate cell activation and matrix formation. The excess matrix is then accompanied by decreased activity of metalloproteinases, and elaboration of inhibitory TIMPs. The biochemical correlates are that intrahepatic collagenase activity seems to be greatest during the development of early and progressive fibrosis, but diminishes with advanced fibrosis.628 A full understanding of the interplay of all these factors could lead to treatments that slow or even halt the progression of chronic liver diseases towards cirrhosis.629

53

MacSween’s Pathology of the Liver

Vascular remodelling Vascular modifications are central to the development of chronic liver damage, leading to the cirrhotic state.630 Key vascular changes are in endothelial cell porosity, loss of vascular patency owing to thrombosis, and reorganization of the vascular blood flow of the liver at the sinusoidal level and in larger vessels.

Endothelial porosity In normal liver, sinusoidal endothelial cells lack a basement membrane, and exhibit fenestrations approximately 100 nm in diameter, occupying between 2 and 3% of the area of the endothelial cell (‘porosity’). Maintenance of the normal sinusoidal endothelial phenotype, with ample fenestrae, requires both paracrine production of VEGF by hepatocytes or stellate cells, and autocrine production of nitric oxide by the endothelial cells.631 Physiologic regulation of fenestral diameter involves both caveolin-1 and Rho guanidine triphosphatases (GTPases) acting on the actin cytoskeleton within the endothelial cells.632,633 Deposition of extracellular matrix in the space of Disse is accompanied by the loss of fenestrations in the sinusoidal endothelial cells.634 With progressive fibrosis, the diameter of residual endothelial fenestrations slightly decreases, but the overall area porosity drops to below 0.5%.541 In combination with the deposition of aberrant extracellular matrix in the space of Disse by stellate cells, the sinusoidal space comes to resemble a capillary rather than a channel for exchange of solutes between hepatocytes and plasma (Fig. 1.47). As a result, hepatocellular secretion of proteins (e.g. albumin, clotting factors, lipoproteins) is greatly impaired.

Vascular thrombosis The second vascular insult is thrombosis. In angiographic and ultrasonographic studies, portal vein thrombosis has been found in 0.6–16.6% of cirrhotic patients,635 and grossly visible portal vein fibrosis or thrombosis has been found in 39% of cirrhotic livers at autopsy.636 Veno-occlusive lesions of hepatic veins 46 serum albumin

96% NPV; 51% PPV

112

Fibrotest

FT 0.30

90% NPV, F2–4

80: 42 Hispanic, 38 Caucasian

Clinical

✓ ✓

✓

✓

Cirrh: Hispanic Cirrh: Caucasian

BAAT, Body mass index, age, ALT, triglyceride; TAs, transaminases; MetS, Metabolic Syndrome; HA, hyaluronic acid; CDT:Tf, carbohydrate deficient transferrin:transferring; NPV, Negative predictive value; Elev, elevated; PPV, positive predictive; Cirrh, cirrhosis; F3-4, METAVIR fibrosis stage 3-4; F2-4, Kleiner, et al, fibrosis stage 2-4 typo for transferring instead of transferrin.

Chapter 6  Fatty liver disease: alcoholic and non-alcoholic

Table 6.6  NASH fibrosis

Obesity/ BMI

Study

n

Test(s)

Shimada et al. (2007)748

85

Early stage NASH

Angulo et al. (2007)749

733

Clinical tests Model

Wong et al. (2008)750

162

NAFLD fib score

Qureshi et al. (2008)751

331

Miyaaki et al. (2008)752

Age

DM/IR

ALT or AAR

Other

Fibrosis

Adiponectin HOMA-IR TIV collagen

3 Markers: sensitivity 94%; specificity 74%

Platelets Albumin

93% NPV; 90% PPV

✓

✓

✓

✓

✓

✓

Platelets Albumin

NPV 91% (9% had adv. Fib)

NAFLD fib score

✓

✓

✓

✓

Platelets Albumin

NPV 99% (14% had adv. fib)

182

Nippon score

✓

✓

✓

✓

Female HTN

No. of factors predicts adv. fib

Harrison et al. (2008)553

827

BARD score

✓

✓

✓

Guha et al. (2008)753

196

ELF markers w/out age

Guha et al. (2008)753

196

ELF w/age

✓

✓

✓

96% NPV

✓

HA, TIMP1, N-type III procoll.

AUROC 0.9, 0.82, 0.76: severe, moderate, no fibrosis

Platelets Albumin

AUROC 0.98, 0.93, 0.84

Fib, Fibrosis; BARD, BMI, ALT:AST ratio, DM; ELF, European liver fibrosis; TIV, type IV; TIMP1, tissue; inhibitor of matrix protein-1; Procoll, procollagen; Adv, advanced.

Table 6.7  NASH (vs NAFLD) and fibrosis

Study

n

Test(s)

Dixon et al. (2001)734

105 bariatric patients

HAIR score

Ong et al. (2005)519

212 bariatric patients

Clinical tests

Palekar et al. (2006)754

80

Clinical tests

Diab et al. (2008)543

99 bariatric patients

Serum K18 fragments

RodriguezHernandez et al. (2008)755

108 women

Clinical tests

Obesity

Age

DM/IR

ALT or AAR

Other

Fibrosis

NASH

✓

✓

HTN

HTN

HAIR

✓

AST AST

Male Hepatocyte necrosis

✓

✓

Female HA > 55

HA > 45

2 of 3

252–275 U/L

Mod/sev.

✓

✓

✓

✓ ✓

✓

allograft liver biopsies from patients transplanted for cryptogenic cirrhosis,760–762 there has developed a broadly held conviction that cryptogenic cirrhosis represents burnt-out NASH. In reality, burntout autoimmune hepatitis and burnt-out alcoholic hepatitis may also result in similarly inactive cirrhosis. Furthermore, NAFLD has been documented to occur de novo in 31% of adult allografts in patients transplanted for non-NASH end-stage liver disease.763,764 By multivariate analysis, risk factors for steatosis similar to nontransplant risk factors included post-OLT obesity, diabetes, hyperlipidaemia, hypertension; factors unique to the transplant setting

✓

✓

✓

✓

included tacrolimus-based immunotherapy, pre-transplant graft steatosis, and alcoholic cirrhosis as underlying aetiology of liver failure.764 Currently, it is recommended that pathologists report accurate descriptions of the findings in cirrhosis, including features that may be suggestive of steatohepatitis.727,765,766 With clinical support for a particular aetiology, a diagnosis of steatohepatitis may be included in the report, but if not, the diagnosis of ‘cirrhosis, aetiology not known’ is appropriate, and possible differential diagnoses can be listed.686,766

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MacSween’s Pathology of the Liver

A A

B

B

Figure 6.48  (A) Non-alcoholic steatohepatitis with severe activity. (B) The same patient was re-biopsied 2 years later; there had been no interventional therapy in the interim but loss of steatosis. (H&E)

Histological resolution

C Figure 6.47  (A) Explant tissue from patient with ‘cryptogenic’ cirrhosis. (B) A review of a biopsy obtained 11 years prior to transplantation showed cirrhosis with evidence of active steatohepatitis. (C) This case illustrates that active lesions may persist in cirrhosis, that NASH may ‘burn-out’ and result in bland, nonsteatotic cirrhosis. (H&E)

332

Treatment trials with lifestyle intervention,767 medical therapy aimed at improvement of insulin resistance and/or oxidative stress768–770 or bariatric surgery,696,703,704 have predominantly shown early post-treatment efficacy, but of varying duration.667,679,771,772 A large randomized controlled trial utilizing ursodeoxycholic acid showed no changes between randomized placebo and treatment groups.773,774 Furthermore, the long-term efficacy and risk : benefit ratio of side-effects from medical therapy has been questioned,771,772 in contrast with successful reversal of NASH in some series of bariatric surgery. The successful trials have shown improvement of steatosis and ballooning; fibrosis improvement704 or alteration (dense to delicate)701 have been noted rarely. Paradoxically, there may be an increase in portal chronic inflammation in otherwise improved biopsies.775 The results from long-term studies with follow-up biopsies732,733 as well as large randomized control trials have documented histological improvement in a proportion of placebo group of patients;550,770 while it must be recognized the patients were being closely followed in the context of a trial, this finding nonetheless suggests that the histopathological features of NASH may improve ‘spontaneously’(Fig. 6.48). This concept was recently confirmed in an 8.5 year long-term population study in Italy in which one of every two NAFLD patients had regression of ultrasound-detectable steatosis.776

Chapter 6  Fatty liver disease: alcoholic and non-alcoholic

Reproducibility studies in NASH Two of three histological studies of inter- and intra-observer reproducibility have validated the significant lesions of NAFLD and NASH. In all, the pathologists met together prior to blinded reviews. The first777 included 53 biopsies from adults with the full range of lesions of NAFLD. The second693 included 30 adult biopsies and 18 paediatric biopsies submitted by the pathologists for the multicentre NIDDK-sponsored NASH Clinical Network Research and included a spectrum of biopsies submitted clinically for consideration of NAFLD or NASH. Both of these studies showed high kappa values for inter-observer correlations with extent of steatosis, hepatocellular ballooning, presence of perisinusoidal fibrosis and fibrosis grade in adult biopsies. Mallory–Denk bodies had higher inter-observer correlation in one.693 There was high agreement for intra-observer correlation and lobular inflammation was the variable with least agreement. The third study, comprising 21 adult Japanese liver biopsies separately reviewed and scored by eight liver pathologists who had pre-agreed upon criteria, showed moderate agreement for steatosis and fibrosis, whereas ballooning and lobular inflammation were poor.778

Hepatocellular carcinoma and NAFLD Hepatocellular carcinoma is a recognized complication of cirrhosis in NASH patients. This has recently been reviewed.779,780 In the series of 42 patients by Powell et al., HCC was reported in one.758 Shimada et al.781 reported that from 82 cases of biopsy-proven NASH, 13 were cirrhotic and six of these had HCC (7.3% of total cohort; 47% of cirrhotics). Kudo noted that greater length of follow-up may be necessary for determining prevalence of HCC; he further discussed the fact that Japanese patients are over-represented in reported cases of HCC secondary to NASH. Whether this may be a result of more vigilant screening for HCC in this patient population is not known. His review highlights the lower risk of NASH-related HCC (1.3–2.4fold) compared with that of HCV cirrhosis (13–19-fold).782 A recent study has drawn attention to the possible potentiating effects of moderate alcohol use in NAFLD in the development of HCC.783 In a survey of 641 cirrhosis-associated HCC, Bugianesi et al. found 44 cases arose in cryptogenic cirrhosis; compared with case– control for viral and alcoholic cirrhosis-associated HCC, the cases with cryptogenic cirrhosis showed higher prevalence of obesity, diabetes, indices of insulin resistance and elevated triglycerides. The authors concluded that the natural history of NASH should include cryptogenic cirrhosis and HCC.784 Two further studies of patients with cryptogenic cirrhosis found a range of 18%785 to 27%786 incidence of hepatocellular carcinoma; in the latter, only the obese cryptogenics showed an increased risk of HCC. Both groups noted the incidence to be similar to that of HCC in hepatitis C-related cirrhosis. In both studies, NAFLD was the inferred underlying pathogenesis of the cryptogenic cirrhosis, based on associated clinical features of metabolic syndrome. Possible mechanisms for hepatocarcinogenesis in NAFLD include the known presence of insulin-like growth factor receptors in HCC, hyperinsulinemia of NAFLD,787–789 increased susceptibility of the steatotic liver to lipid peroxidation, production of free radicals and subsequent DNA mutations,787 increased susceptibility to carcinogens due to impairment of ATP production,788 disordered energy and/or hormonal regulation in obesity,790 the known metabolic derangements of ‘metabolic syndrome’ in NAFLD,784 and aberrations in regenerative processes occurring in cirrhosis of any cause. Soga et al.791 reported a non-obese inbred mouse model with spontaneous fatty liver that develops steatohepatitis; with time,

many of the animals develop hepatocellular adenomas and hepatocellular carcinomas. These lesions show a gender predilection occurring in up to 40% of male mice but ,2,>3

Nobili et al. (2009)846

59 NAFLD

Bx, RBP4

TG

Dec RBP4, higher NAS*

Dec RBP4, high fibrosis

Nobili et al. (2009)847

203 NAFLD

Paediatric NAFLD Fibrosis index (0–10)

WC, age, TG levels

✓

✓; PNFI ≥ 9; PPV 95%

Alisi et al. (2010)829

40 NAFLD

TNFα, IL-6, endotoxin, PAI-1

Serum endotox, PAI-1

✓

Male

2 : 1

3.5 : 1

Z-BMI

ALT

IR

Other

NAFLD

✓

✓

✓

BP, OGTT, uric acid

✓

✓

✓

✓

Elev lipids* *Ethnicity, AN

✓

AlkPhos TG

TNFα ± leptin NAS > 5

GGT*

AST, GGT: grade

✓

✓

ALT AST*

✓

✓

✓

✓

2 : 1

✓

✓

✓

Fibrosis

AST, WBC, dec HCT: score of fibrosis

Bx: St 1,2,3

AUROC 0.977,0.992,1

Insulin

MetS WHR:NAS

WHR, MetS AUROC 0.92,0.98, 0.99

CRP, c-reactive protein; US, ultrasound; Bx, biopsy; Clin, clinical; WHR, waist:hip ratio; RBP4, retinol binding protein 4; PAI-1, plasminogen activator inhibitor-1; BP, Blood pressure; OGTT, oral glucose tolerance test; AN, acanthosis nigricans; GGT, gamma glutamyl transferase; WC, white count; Endotox, endotoxin; HCT, hematocrit; PNFI, Pediatric nafld fibrosis index; Dec, decreased.

et al. showed that an azonal distribution of steatosis in paediatric NAFLD was associated with ballooning although interestingly there was no relation between the amount or type of steatosis and degree of fibrosis.838,839 As in adult NAFLD, active investigations to derive clinical algorithms for prediction of advanced fibrosis in children are ongoing (Table 6.8).847

Grading and staging in NAFLD   and NASH Pathologists are familiar with the evolution of semi-quantitative scoring systems for evaluation of necroinflammatory activity (grade) and fibrosis architectural alterations (stage) in chronic viral hepatitis (see Chapter 7); methods currently in use vary, but are based on

335

MacSween’s Pathology of the Liver

assessments of portal chronic inflammation and interface activity, lobular necroinflammatory lesions, and extent of portal-based fibrosis. One of the primary goals of standardized assessments in any histopathological scoring system is the assurance that across the spectrum of observers, the same lesions are being evaluated and given similar diagnostic weight; a system of grading and staging, therefore ideally enhances reproducibility. The lesions of steatohepatitis are sufficiently distinct from the portal-based lesions of chronic hepatitis of viral and autoimmune origin to warrant specific methods of evaluation. A novel histopathological grading and staging system was developed from evaluation of 51 biopsies from well-characterized adult patients with NASH.692 The method was developed to score relevant histological lesions, to derive a semi-quantitative global ‘activity’ grade and to standardize reporting of the pattern of fibrosis progression in NASH. This represents an alternative to prior methods that combined assessment of activity with those of fibrosis and follows the accepted paradigm for chronic hepatitis established in all systems in the recognition that processes that result in necroinflammatory lesion, grade, fibrosis with architectural changes and stage, are separately evaluated and reported.724 Tables 6.9 and 6.10 describe the proposed method. Determination of grades 1–3 is based on the constellation of features, steatosis, hepatocellular ballooning, lobular inflammation, and portal inflammation; emphasis is placed on gradation of injury and inflammation rather than steatosis, as

Table 6.9  Brunt system: grade

Grade

Steatosis

Ballooning

Inflammation

Mild, grade 1

1–3 (up to 66%)

Minimal

L: 1–2 P: None–mild

Moderate, grade 2

2–3 (>33%; may be >66%)

Present

L: 2 P: Mild–moderate

Severe, grade 3

2–3

Marked

L: 3 P: Mild–moderate

L, lobular; P, portal. Steatosis grade 1: ≤33%; grade 2: >33% 0 favours ALD, whereas ANI zone 3    RES, punctuate, panacinar   Fibrosis: zone 3 perisinusoidal/ chickenwire/pericellular/    Dense, diffuse    Delicate   Perivenular fibrosis   Periportal fibrosis   Microvesicular steatosis   Sclerosing hyaline necrosis, zone 3   Veno-occlusive lesions   Canalicular cholestasis, cholestatic rosettes Cirrhosis   Gross characteristics

       

Active lesions of steatohepatitis ‘Cryptogenic cirrhosis’ Copper deposition Hepatocellular iron, RE iron, (non-HFE patient)   α1-AT globules

338

± ± + ± ±

± ± + ± ±; in ballooned hepatocytes when present Less likely More likely Less likely ±; may be prominent in ‘resolution’ − + ± ±

±; may be significant ± ±

±, usually mild ± ±

More likely Less likely + +, with ductular reaction, acute inflammation + + + +

Less likely More likely − ± +/− − − −

Liver firm throughout; may be shrunken, or may be quite large; may be cholestatic; grossly visible foci of parenchymal extinction + + ± Common

Not distinguishable from other hepatitic forms of cirrhosis

±

−

+ + Unlikely ±

Chapter 6  Fatty liver disease: alcoholic and non-alcoholic

Table 6.13  Common clinical features of non-alcoholic and alcoholic steatohepatitis

Non-alcoholic

Alcoholic

Symptoms

Typically none or just fatigue Right upper quadrant abdominal pain (from mild aching to severe sharp pains) in one-third to one-half of patient

Fatigue, anorexia

Signs

Occasional hepatomegaly that can be difficult to palpate due to obesity Acanthosis nigricans can be seen, especially in children

Fever Jaundice If cirrhotic: muscle wasting, spider angiomata, encephalopathy

Laboratory

ALT normal to 300 U/L ALT > AST, typically by 1.5 : 1 ratio AST > ALT if cirrhotic Alkaline phosphatase usually normal; can be up to 250 U/L Occasional patients have normal ALT and mild alkaline phosphate elevation Total bilirubin normal Prothrombin time normal unless decompensated cirrhotic WBC normal Normal erythrocyte MCV; ferritin may be elevated

AST typically 100–300 U/L AST > ALT, often by more than 2 : 1 ratio Alkaline phosphatase can be elevated Total bilirubin elevated Prothrombin time can be elevated Leukocytosis common, can even be in leukemoid range (>50 000/mm3) Elevated erythrocyte MCV

alcoholic hepatitis is diagnosable in the absence of steatosis, whereas in NAFLD, steatosis is present. In cirrhosis due to ALD, regions of parenchymal extinction (septa) are more pronounced than in NAFLD. Lesions noted to be significantly more common in NAFLD compared to ALD include marked steatosis, periportal glycogenated

nuclei and lipogranulomas. However, on an individual case basis, the usefulness of these particular features is questionable. There may also be qualitative differences in collagen deposition between NAFLD and ALD. For instance, type I collagen is more common in NAFLD-associated steatohepatitis, whereas type III is more common in ALD.854

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Chapter 6  Fatty liver disease: alcoholic and non-alcoholic

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on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). JAMA 2001;285:2486–97. 827. Schwimmer JB, Pardee PE, Lavine JE, et al. Cardiovascular risk factors and the metabolic syndrome in pediatric nonalcoholic fatty liver disease. Circulation 2008;118:277–83. 828. Patton HM, Yates K, Unalp-Arida A, et al. Association between metabolic syndrome and liver histology among children with nonalcoholic fatty liver disease. Am J Gastroenterol. 2010. 829. Alisi A, Manco M, Devito R, et al. Endotoxin and plasminogen activator inhibitor-1 serum levels associated with nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr 2010;50: 645–9. 830. Tazawa Y, Noguchi H, Nishinomiya F, Takada G. Serum alanine aminotransferase activity in obese children. Acta Paediatrica 1997;86: 238–41. 831. Fraser A, Longnecker MP, Lawlor DA. Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999–2004. Gastroenterology 2007;133:1814–20. 832. Kinugasa A, Tsunamoto K, Furukawa N, et al. Fatty liver and its fibrous changes found in simple obesity of children. J Pediatr Gastroenterol Nutr 1984;3: 408–14. 833. Bellentani S, Scaglioni F, Marino M, Bedogni G. Epidemiology of Nonalcoholic fatty liver disease. Dig Dis 2010;28:155–61. 834. Wiegand S, Keller KM, Robl M, et al. Obese boys at increased risk for nonalcoholic liver disease: evaluation of 16,390 overweight or obese children and adolescents. Int J Obes 2010;34: 1468–74. 835. Louthan MV, Theriot JA, Zimmerman E, et al. Decreased prevalence of nonalcoholic fatty liver disease in black obese children. J Pediatr Gastroenterol Nutr 2005;41:426–9. 836. Roberts EA, Yap J. Nonalcoholic fatty liver disease (NAFLD): approach in the adolescent patient. Curr Treat Options Gastroenterol 2006;9:423–31. 837. Feldstein AE, Charatcharoenwitthaya P, Treeprasertsuk S, et al. The natural history of non-alcoholic fatty liver disease in children: a follow-up study for up to 20 years. Gut 2009;58: 1538–44. 838. Carter-Kent C, Yerian LM, Brunt EM, et al. Nonalcoholic steatohepatitis in children: a multicenter clinicopathological study. Hepatology 2009;50:1113–20. 839. Carter-Kent C, Brunt EM, Yerian LM, et al. Relations of steatosis type, grade,

Chapter 6  Fatty liver disease: alcoholic and non-alcoholic

and zonality to histological features in pediatric nonalcoholic fatty liver disease. J Pediatr Gastroenterol Nutr 2011;52:190–7. 840. Sartorio A, Del Col A, Agosti F, et al. Predictors of nonalcoholic fatty liver disease in obese children. Eur J Clin Nutr 2007;61:877–83. 841. Mager DR, Ling S, Roberts EA. Anthropometric and metabolic characteristics in children with clinically diagnosed nonalcoholic fatty liver disease. Paediatr Child Health 2008;13:111–7. 842. Manco M, Marcellini M, Giannone G, Nobili V. Correlation of serum TNF-α levels and histologic liver injury scores in pediatric nonalcoholic fatty liver disease. Am J Clin Pathol 2007;127: 954–60. 843. Manco M, Marcellini M, De Vito R, et al. Metabolic syndrome and liver histology in pediatric nonalcoholic steatohepatitis. Int J Obes 2008;32:381–7. 844. Manco M, Bedogni G, Marcellini M, et al. Waist circumference correlates

with liver fibrosis in children with non-alcoholic steatohepatitis. Gut 2008;57:1283–7. 845. Nobili V, Parkes J, Bottazzo G, et al. Performance of ELF serum markers in predicting fibrosis stage in pediatric nonalcoholic fatty liver disease. Gastroenterology 2009;136:160–7. 846. Nobili V, Alkhouri N, Alisi A, et al. Retinol-binding protein 4: a promising circulating marker of liver damage in pediatric nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol 2009;7:575–9. 847. Nobili V, Alisi A, Vania A, et al. The pediatric NAFLD fibrosis index: a predictor of liver fibrosis in children with non-alcoholic fatty liver disease. BMC Med 2009;7:21. 848. Ishak KG, Baptista A, Bianchi L, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696–9. 849. Scheuer PJ. Scoring of liver biopsies: are we doing it right? Eur J Gastroenterol Hepatol 1996;8:1141–2.

850. Calvaruso V, Burroughs AK, Standish R, et al. Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient. Hepatology 2009;49: 1236–44. 851. Mendler MH, Kamel G, Govindarajan S. Proposal for a histological scoring and grading system for non-alcoholic fatty liver disease. Liver Int 2005;25: 294–304. 852. Dunn W, Angulo P, Sanderson S, et al. Utility of a new model to diagnose an alcoholic basis for steatohepatitis. Gastroenterology 2006;131:1057–63. 853. Sanderson SO. The use of protein tyrosine phosphatase 1B and insulin receptor immunostains to differentiate nonalcoholic from alcoholic steatohepatitis in liver biopsy specimens. Am J Clin Pathol 2005;123:503–9. 854. Nakano M. Histological study on comparison between NASH and ALD. Hepatol Res 2005;33:110–5.

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Acute and chronic viral hepatitis Neil D. Theise, Henry C. Bodenheimer Jr. and Linda D. Ferrell

Acute viral hepatitis

CHAPTER CONTENTS Acute viral hepatitis Clinical features Pathological features Evolution of the lesion Differential diagnosis of acute hepatitis Sequelae of acute hepatitis Chronic viral hepatitis Clinical features Pathological features Pathogenetic mechanisms Multiple chronic viral infections Differential diagnosis of chronic hepatitis Semiquantitative scoring in chronic hepatitis Dysplasia Individual types of viral hepatitis Type A hepatitis Type B hepatitis Type C hepatitis Type D (δ) hepatitis Type E hepatitis Other forms of viral hepatitis

361 361 362 368 368 370 370 370 371 375 376 377 377 380 381 381 382 388 391 392 393

The subject of this chapter is the pathological consequences of infection with hepatotropic viruses. These infections are responsible for viral hepatitis which is generally classified by viral type (Table 7.1). Distinction can also be made by the duration of infection and by the clinicopathological syndrome that develops (Table 7.2). Although some pathological features are unique to the type of virus responsible for infection, many aspects of the pathological injury and clinical progression are common to multiple types of hepatotropic viral infection. ©2012 Elsevier Ltd DOI: 10.1016/B978-0-7020-3398-8.00007-6

7 

Clinical features Acute viral hepatitis occurrence may be sporadic or epidemic. Transmission of disease is commonly via the faecal–oral route through contaminated food or water. Alternatively, transmission may be through sexual contact or parenteral exposure, such as by intravenous drug use, blood transfusion or occupational needle-stick exposure. The acute infection is often subclinical and usually anicteric. If symptoms are present, they may be nonspecific and not readily identifiable as due to viral hepatitis. Typical symptoms include fatigue, anorexia and nausea in the early stages, followed by dark urine and jaundice. Right upper quadrant abdominal pain or discomfort, arthritis, urticaria, pruritus and low-grade fever may be present. In patients without jaundice, the symptoms may be mistaken for gastrointestinal viral infection or another nonspecific viral syndrome.1 The severity of acute viral hepatitis may vary from a mild asymptomatic infection to fatal fulminant hepatic failure. The latter is characterized clinically by the development of hepatic encephalopathy within 8 weeks of the onset of clinical illness2 and pathologically by massive or severe bridging hepatocyte necrosis. The terms ‘subacute hepatitis’ and ‘subacute hepatic necrosis’ are used to describe serious injury developing after 8 weeks of illness. In patients with acute viral hepatitis, the surviving parenchyma may be characterized by varying degrees of regeneration. Nodular hyperplasia, two-cell-thick hepatocyte plates between sinusoids, and mitotic figures within hepatocytes may indicate liver cell regeneration and a potential for recovery. Serum markers of patient prognosis in fulminant hepatic failure include α-fetoprotein,3 Gc-protein4 and troponin levels.5 Additionally, measures of hepatic synthetic capacity such as factor VII,6 and computed tomography-derived liver volume7 may also be prognostic. Death may result from complications of infection or renal failure rather than from hepatic synthetic dysfunction directly. Portal hypertension and ascites are typically associated with chronic liver disease and rarely develop in patients with acute liver injury. These complications, in acute disease, may be the result of hepatocyte loss and consequent collapse of the sinusoidal network.8 Acute viral hepatitis is typically characterized by injury to hepatocytes and is identified by a marked rise in aminotransferase

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Table 7.1  Clinical characteristics of human hepatotropic viral agents

Viral agents

Characteristics

Hepatitis A (HAV)

RNA picornavirus Sporadic or epidemic occurrence with faecal–oral transmission, resulting in acute disease only

Hepatitis B (HBV)

DNA hepadnavirus Sporadic or endemic occurrence through sexual, perinatal and parenteral transmission Chronic disease persists in 5% of adults and in up to 90% of infants Chronic infection is associated with hepatocellular carcinoma

Hepatitis C (HCV)

RNA flavi-like virus Sporadic occurrence with parenteral transmission Perinatal and sexual spread is less common Chronic disease develops in 60–80% of persons infected and cirrhosis is associated with hepatocellular carcinoma

Hepatitis D (HDV)

RNA defective virus Sporadic or endemic disease occurs as coinfection with HBV Transmission is parenteral and sexual Chronic disease is seen in patients with chronic HBV HDV worsens the clinical severity of HBV infection

Hepatitis E (HEV)

RNA virus Sporadic or epidemic occurrence Transmission is faecal–oral, resulting in acute disease Mortality rate is 25% in pregnant women

Table 7.2  Clinical–pathological syndromes of viral hepatitis

Acute • Subclinical/anicteric • Symptomatic/icteric • Fulminant • Cholestatic • Atypical presentation in immunosuppressed patients (including fibrosing cholestatic hepatitis)

Chronic • Asymptomatic without hepatocyte necrosis • Chronic infection with hepatic injury • Chronic hepatitis compensated with cirrhosis • Decompensated chronic hepatitis with cirrhosis • Chronic post-transplant

activities. Rise in aminotransferase values precedes a rise in bilirubin and may peak prior to the development of jaundice. Jaundice, in the presence of significant elevation of aminotransferase values, indicates a severe degree of liver injury. A fall in hepatic synthesis of clotting factors may be reflected in a prolongation of prothrombin

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time and elevation of international normalized ratio (INR). This is another indication of severe hepatic injury. Aminotransferase values generally rise over a period of weeks, and in those individuals who subsequently clear the virus, may normalize over weeks to months. Bilirubin elevation follows the aminotransferase rise. The onset of jaundice is clinically recognized when bilirubin levels rise above approximately 2.5  mg/ dL. Jaundice or icterus may persist at lower levels of serum bilirubin during recovery due to bilirubin that is conjugated to albumin in tissue, particularly if hyperbilirubinaemia has been prolonged. This bilirubin and albumin complex has been termed δ bilirubin.9 Cholestatic hepatitis is a clinical variant of acute viral hepatitis in which biliary dysfunction predominates. This syndrome is characterized by elevation of alkaline phosphatase and bilirubin values with proportionately less elevation of aminotransferase activities. The cholestatic biochemical abnormalities may persist for weeks to months. The prognosis of this pattern of hepatic injury is generally favourable when not associated with prominent hepatocellular necrosis. Cholestatic hepatitis must be distinguished clinically from biliary obstruction by biliary imaging studies (Table 7.2).

Pathological features Acute viral hepatitis is characterized morphologically by a combination of inflammatory-cell infiltration, macrophage activity, hepatocellular damage and regeneration. The proportion and detailed nature of these components vary widely according to the particular virus responsible, the host response and the passage of time. The pathological features are considerably different from those of classic acute inflammation, because they primarily represent a response of the patient’s immune system to viral antigens displayed on cells, rather than a vascular and cellular response to injury. Some features of acute inflammation are nevertheless present to a limited extent; thus, for instance, the liver in acute viral hepatitis is swollen and tender, its capsule is tense and blood vessels are engorged. In addition, polymorphonuclear leukocytes may be seen as a secondary response to epithelial injury, and are often seen associated with a ductular reaction.

Macroscopic appearances Information on the macroscopic appearances of the liver in nonfatal acute hepatitis is mainly derived from laparoscopy10 and from liver transplantation. Initially the liver is swollen and red, its capsule oedematous and tense; exuded tissue fluid may be seen on the capsular surface. Focal depressions are the result of localized subcapsular necrosis and collapse. In patients with severe cholestasis, the colour of the liver is bright yellow or green. In fulminant hepatitis, the organ shrinks and softens as a result of extensive necrosis, and the capsule becomes wrinkled. The left lobe may be more severely affected than the right. If the patient survives for weeks or months, tan to yellow-green nodules of regenerating parenchyma may be seen protruding from the capsular surface or deep within the liver (Fig. 7.1), separated or surrounded by more haemorrhagic, or redder, necrotic areas. In other examples, necrosis is uniform throughout the organ. Of note, these nodules can be seen by radiographic imaging11 and at times mistaken for cirrhosis, or even metastatic carcinoma, which could adversely affect the patient’s status for liver transplantation. In patients dying of fulminant hepatitis, there is often damage to organs other than the liver and this may significantly contribute to the immediate cause of death.12 Findings at autopsy include pneumonia, septicaemia, cerebral oedema, gastrointestinal haemorrhage

Chapter 7  Acute and chronic viral hepatitis

Figure 7.2  Classic acute hepatitis. Hepatocyte swelling and lymphocytic infiltrates are seen in the lobule. (H&E)

Figure 7.1  Massive necrosis with regenerative foci in severe hepatitis that progressed to liver failure over a time period of several weeks. The tan nodular zones represent regenerative foci, and the red zones represent massive necrosis with no residual hepatocytes.

and pancreatitis. Such lesions help to explain death when liver-cell damage is limited in extent or when there has already been substantial regeneration.

Light-microscopic appearances: types of necrosis The histological classification of acute hepatitis given in this chapter is based on different patterns of hepatocellular necrosis. These patterns will therefore be briefly discussed before the microscopic changes of acute hepatitis are described in detail. More than one of the patterns of necrosis described below may be seen in different parts of the same liver, and even within a single biopsy specimen.

Spotty (focal) necrosis and apoptosis In this form of necrosis, which represents the fundamental lesion of acute viral hepatitis, individual hepatocytes within otherwise intact parenchyma die and are removed. The mode of death of hepatocytes probably includes both lytic necrosis and apoptosis (Chapter 1), but the relative contribution of each is uncertain. In the case of apoptosis (or formation of acidophilic or Councilman bodies in older terminology), it is likely that T lymphocytes play a role. The remnants of cells affected by either process are rapidly removed from the site by blood flow or phagocytosis.

Confluent and bridging necrosis This type of necrosis comprises groups of adjacent dead hepatocytes or the site where a group of hepatocytes has undergone previous necrosis and removal, so that areas of confluent necrosis are formed. These are often perivenular (centrilobular) in location. Confluent necrosis linking vascular structures and portal zones is known as bridging necrosis. Bridging at the periphery of complex acini links terminal hepatic venules (central veins) to each other, but does not involve portal tracts. This is called ‘central–central’ bridging in the lobular nomenclature. The other form of bridging necrosis links terminal hepatic venules to portal tracts (‘central–portal’ bridging in the lobular nomenclature). This form is best explained as necrosis of zone 3 of the simple acinus, as described by Rappaport,13 because this zone touches both terminal hepatic venule and portal

tract. Zone 3 bridges are sometimes curved, in keeping with the shape of the zone. When confluent necrosis is more extensive, involving zones 2 and 1 in addition to zone 3 such that entire acini are destroyed, the process is described as panacinar or panlobular necrosis.

Interface hepatitis Interface hepatitis, formerly known as piecemeal necrosis, can be defined as death of hepatocytes at the interface of parenchyma and the connective tissue of the portal zone, accompanied by a variable degree of inflammation and fibrosis. In the acinar concept, interface hepatitis typically involves zone 1 and leads to apparent widening of portal tracts. These are therefore more likely to be cut longitudinally by a microtome knife, and this is seen in two-dimensional sections as linking of adjacent portal tracts, that is to say portal– portal bridging. Interface hepatitis was a defining feature of the formerly used category of chronic active hepatitis,14 but very similar periportal necrosis is found in some cases of acute hepatitis.

Light microscopic appearance: patterns of acute hepatitis Classic acute hepatitis Features seen in the parenchyma in this form of acute hepatitis include liver-cell damage and cell death, liver-cell regeneration, cholestasis, infiltration with inflammatory cells and prominence of sinusoidal cells (Fig. 7.2). The necrosis may be spotty or confluent. The histological changes, especially confluent necrosis, are often most severe near the terminal hepatic venule. The reason for this zonal distribution has not been established, but possible explanations include metabolic and functional differences between hepatocytes in different zones, and the lower oxygen content of the blood in perivenular areas. There is a variable degree of condensation of the reticulin framework but, in the classic form of hepatitis, this does not amount to substantial alteration of structure and vascular relationships. Portal tracts are inflamed and bile ducts may be damaged. Macrovesicular steatosis is relatively uncommon in the fully developed stage of hepatitis. Little is known about the earliest changes of acute viral hepatitis in man. Available information suggests that Kupffer cells are prominent and may show mitotic activity, and that hepatocellular necrosis occurs early in the course of the disease.15 In the setting of acquired hepatitis after liver transplantation, hepatocyte necrosis is seen as an early indicator of hepatitis.16

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A Figure 7.3  Classic acute hepatitis. Swollen hepatocytes are present, together with a mononuclear infiltrate in the sinusoids. Disarray of the plate architecture is present due to loss of hepatocytes. (H&E)

Hepatocyte swelling is a common feature in acute hepatitis, and results mainly from dilatation of the endoplasmic reticulum. The swollen cells are pale-staining as a result of intracellular oedema (Fig. 7.3). Because of the combination of swelling and rounding of the affected cells, the term ‘ballooning degeneration’ is sometimes applied. It should be noted, however, that such ballooning degeneration is not a diagnostic feature of viral hepatitis, since it may also be seen in other circumstances, for example in alcoholic or druginduced hepatitis, and following liver transplantation as part of the spectrum of ischaemia and/or preservation injury. The nuclei of the affected cells are also swollen, because of the accumulation of proteins.17 Nucleoli of hepatocytes may be more prominent than usual, mitotic figures are occasionally seen, and multinucleation may be increased. In addition to swollen cells, there are hepatocytes with deeply acidophilic cytoplasm, in which the nucleus is sometimes seen to be undergoing pyknosis, or apoptosis. Such acidophilic hepatocytes are small in comparison with ballooned cells, but may occasionally be larger than normal hepatocytes. This suggests that the two types of cell change, ballooning and acidophilic change, could represent stages of cell degeneration rather than fundamentally different responses to injury. Acidophilic cells have round or irregular outlines, sometimes assuming rhomboid, angular shapes, apparently determined by the pressure from adjacent swollen hepatocytes. The formation of acidophil (Councilman) bodies (Fig. 7.4) is probably a later stage of this process. Acidophil bodies are generally rounded hepatocyte remnants, but some may be ovoid or have other irregular forms, may or may not contain nuclear remnants and may appear thick and refractile. Affected cells eventually undergo fragmentation, the whole process representing an example of apoptosis.13 Some acidophil bodies are in close contact with lymphocytes or Kupffer cells, while others appear to lie free within liver-cell plates or sinusoids, with no other cells in close proximity. Like ballooned hepatocytes, acidophil bodies are not specific for acute viral hepatitis, although they often constitute a striking histological feature of the disease. Loss of individual hepatocytes leads to localized defects in the liver-cell plates, with consequent distortion and condensation of the supporting reticulin framework, especially when the necrosis is confluent. While an overall increase in connective tissue fibres is probably slight in acute hepatitis and therefore difficult to detect by light microscopy (with conventional staining methods), immunocytochemical and ultrastructural evidence indicates synthesis of collagen fibres18 and associated proteins such as fibronectin, as

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B Figure 7.4  (A) Classic acute hepatitis. Focal apoptosis of hepatocyte with nuclear remnant present. Inflammatory cells are present next to the apoptotic cell within the sinusoid. Hepatocyte swelling is also present. (B) Classic acute hepatitis. Focal apoptosis of hepatocytes is present (rounded acidophil body) as well as inflammation of the lobule and disarray of the plate architecture. (H&E)

well as increased prominence of stellate cells. There may also be degradation of these components, so that there is presumably a dynamic balance which determines the degree of fibrosis in an individual case. The distortion of cell plates is accentuated by regeneration of hepatocytes. This is recognized by the appearance of mitotic figures, rare in normal liver, and by a change in the structure of the cell plates; these become more than one cell thick, or assume the structure of short cylinders known as liver-cell rosettes. The end result of focal necrosis, reticulin condensation and regeneration is a diagnostically helpful disarray of the liver-cell plates (Fig. 7.3). Cholestasis is common in acute hepatitis. It varies from the presence of scanty, small bile plugs in perivenular canaliculi to extensive bile plug formation with canalicular dilatation. Intracellular bile is more difficult to recognize because bile is easily confused with lipofuscin pigment. For this reason cholestasis should only rarely be diagnosed on the basis of routine stains in the absence of canalicular bile plugs. Morphological cholestasis may be accompanied by clinical and biochemical features of cholestasis, but this is variable. The inflammatory infiltrate of acute viral hepatitis is mainly composed of lymphocytes, plasma cells and macrophages in varying proportions (Figs 7.2, 7.4). Within the parenchyma, the infiltrate is most abundant where liver-cell damage is greatest, usually in the perivenular zones. Lymphocytes are often attached to endothelial

Chapter 7  Acute and chronic viral hepatitis

Figure 7.5  Classic acute hepatitis. Clumps of hypertrophied Kupffer cells are most prominently seen near the centrizonal region and stain strongly with PAS-diastase. (PAS-D)

Figure 7.6  Giant cell change of hepatocytes in an adult. Many of the hepatocytes in the centrizonal area contain large numbers of nuclei. (H&E)

cells of the central and portal venules.19 Mononuclear phagocytes, either activated Kupffer cells or circulating phagocytes, are seen as large irregular cells in areas of liver-cell drop-out (Fig. 7.5). They often show brown pigmentation, due to phagocytosis of bile or to the accumulation of lipochrome pigment. Iron is also abundant in some cases. The iron-containing phagocytes are sometimes found in the form of small clumps composed of several cells.20 Whatever the pigment, the periodic acid-Schiff (PAS) reaction after diastase digestion is usually strongly positive (Fig. 7.5). Most portal tracts in acute viral hepatitis are infiltrated with inflammatory cells to a greater or lesser extent and, in the classic form, lymphoid cells predominate. Cytotoxic T-cells (CD45RO+) typically are more prominent than B-cells (CD20+).21 Plasma cells are common, the majority containing IgG.22 Perls-positive, iron-rich macrophages may be present. Portal infiltration may be diffuse or focal. Mild bile duct damage is common, and is seen as minor irregularities of shape, size and arrangement of the epithelial nuclei of the small, interlobular ducts. Less commonly, the ductal epithelium becomes vacuolated and disrupted; such lesions are seen most often near or within lymphoid follicles, but granulomas are not associated with the duct lesion as in primary biliary cirrhosis. Irregular dilatation of ductules is not typical but has been described in type A hepatitis.23 In general, however, a finding of dilated, bilecontaining ductules or canals of Hering should arouse a suspicion of sepsis, a complication of severe acute hepatitis in some patients. Bile duct damage in acute hepatitis does not usually correlate with a prolonged cholestatic clinical course, probably because there is not the widespread and progressive destruction of the duct system found in disorders such as primary biliary cirrhosis. The outlines of the portal tracts may remain intact and sharply delineated in classic acute hepatitis with spotty necrosis. More often, infiltration by lymphoid cells and accompanying disruption of the limiting plate of hepatocytes leads to an irregular outline (or interface hepatitis). Severe periportal necrosis and inflammation in acute hepatitis is discussed below. All the above features of classic acute hepatitis with spotty (focal) necrosis vary in extent. There is therefore a wide range of appearances, from a mild hepatitis with little cell damage or inflammatorycell infiltration, to one with widespread changes throughout the parenchyma. In the latter case the diagnosis presents no great difficulty, but in the former the distinction from nonspecific reactive changes or from non-hepatitic cholestasis may be difficult or even impossible. These characteristics of classic acute hepatitis with spotty (focal) necrosis also form the basis of the other three forms

of acute hepatitis, which may be regarded as exaggerations of one or other component of the classic form.

Variants of classic acute viral hepatitis Multinucleation may be very prominent with the formation of giant hepatocytes (Fig. 7.6).24 When this change is widespread, the appearances resemble those of neonatal giant-cell hepatitis. In addition, cholestatic variants have been described, especially in association with hepatitis A and E. In these cases, the degree of necrosis may be minimal and the cholestasis quite prominent, mimicking biliary obstruction.

Acute hepatitis with confluent (bridging) necrosis In this form of acute hepatitis, the features described under classic acute hepatitis with spotty (focal) necrosis are seen but, in addition, bridging in the form of confluent necrosis linking central venules to portal tracts (‘central–portal’ bridging) or linking central venules to each other (‘central–central’ bridging) can also be present. Of these two forms of confluent necrosis, central–portal bridging has been considered by some to be possibly more significant for the progression of the lesion than the central–central necrosis. For example, in patients who develop chronic liver disease, central– portal confluent necrosis may hasten the onset of cirrhosis by creating early disruption of the normal architectural relationships upon conversion of the bridges into fibrous septa, which undergo contraction. In patients who do not develop chronic hepatitis, bridging necrosis of central to portal type may lead to a certain degree of scarring and distortion, sometimes seen in biopsy specimens taken many months after the acute attack. However, the prognostic significance of central–portal bridging necrosis remains somewhat controversial. Boyer and Klatskin25 considered that patients with this type of bridging necrosis were more likely to develop chronic hepatitis, a conclusion later supported by another study.26 However, both groups of authors included not only central–portal bridging as described above, but also bridges linking portal tracts, probably representing periportal necrosis. Others have not considered bridging as a good predictor of chronicity.27,28 It must nevertheless be regarded as an important histological feature seen in the more severe forms of acute hepatitis. The appearance of the necrosis varies according to the stage of the illness. In the early stages of bridge formation, there is death of substantial numbers of hepatocytes. This is followed by disappearance of the affected cells, leaving a loose connective tissue stroma

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Figure 7.7  Acute hepatitis with bridging necrosis. Curving bridges are formed as a result of confluent necrosis linking central and portal zones. (H&E)

Figure 7.8  Acute hepatitis with bridging necrosis. Deeply stained elastic fibres are present in the portal tract. Adjacent areas of collapse have no elastic tissue. (Orcein)

infiltrated with lymphocytes and macrophages (Fig. 7.7). With time, the stroma collapses to form more or less dense ‘passive’ septa, which intersect the liver tissue. Confluent/bridging necrosis can develop in the early weeks of acute hepatitis, but its absence on a biopsy sample does not exclude the possibility that it will develop later. The combination of necrosis, collapse and hepatocellular regeneration leads to architectural distortion which, in turn, can easily be mistaken for that of chronic hepatitis or cirrhosis. Helpful differentiating features include the presence of other lesions of acute viral hepatitis and staining properties of the septa; recently formed passive septa are virtually devoid of elastic fibres (Fig. 7.8) whereas the older septa of chronic hepatitis and cirrhosis contain increasing numbers of these fibres.29 In addition, the stroma in zones of recent necrosis will tend to show the residual structure of the cell plates on reticulin stain (Fig. 7.9); this is not readily seen in chronic hepatitis and cirrhosis. The stroma is often more haemorrhagic or contains more macrophages than in typical chronic hepatitis (Fig. 7.10). Trichrome stain may also show a two-tone appearance, with darker staining of established areas of scar due to thicker collagen bundles (comparable to the normal residual portal tract collagen),

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Figure 7.9  Acute hepatitis with bridging necrosis. The reticulin framework remains intact and has not significantly collapsed at this time point. (Reticulin)

Figure 7.10  Acute hepatitis with bridging necrosis. The centrizonal region shows large numbers of macrophages that stain light blue to grey. The wall of the vein, in contrast, is bright blue and lined by dense collagen bundles. (Trichrome)

as compared to lighter staining of areas of recent collapse, where the residual framework and lack of hepatocytes account for the staining. Extensive liver cell destruction in acute hepatitis with confluent necrosis may also lead to a significant ductular reaction within the periportal areas, which mimics bile duct obstruction, and a more florid portal inflammatory reaction than is normally found in classic acute hepatitis. Neutrophils may be more abundant, and often seen adjacent to the ductular structures.

Acute hepatitis with panlobular (panacinar) necrosis This form of acute hepatitis represents the most severe degree of necrosis, with complete or near-complete destruction of hepatocytes in entire lobules. When several adjacent lobules undergo necrosis, the term ‘multilobular’, or multiacinar is applicable. The term massive hepatic necrosis has been used by some when the liver shows extensive, diffuse panlobular (panacinar) and multilobular necrosis of >60–70% as noted on examination of the entire liver on explant, autopsy, or clinical visualization. This lesion is typically the morphological counterpart of the clinical condition of fulminant hepatitis (Table 7.2), or fulminant liver failure, which is often fatal. The term submassive necrosis has also been sometimes used for lesions that involve global necrosis of 30–70% of the entire liver.

Chapter 7  Acute and chronic viral hepatitis

A Figure 7.11  Acute hepatitis with panlobular necrosis. The hepatocytes have been destroyed, and the collapsed stroma is haemorrhagic. Ductular structures (ductular metaplasia) are prominent. (H&E)

For reasons as yet unknown, panlobular necrosis may spare large areas of the liver (Fig. 7.1), though the more viable zones usually show lesser degrees of damage, including bridging necrosis. Thus, determination of the degree of total loss of liver parenchyma based on needle biopsy alone is not recommended owing to the patchy nature of the necrosis that can occur in acute viral hepatitis.30 Panlobular necrosis on a smaller scale is not necessarily accompanied by severe disease clinically, and may be seen particularly in a subcapsular location (or rarely at other sites), adjacent to less severely damaged liver tissue. It is even likely that panlobular necrosis can occur in the entire absence of severe symptoms, because areas of old necrosis and collapse may be found incidentally in the livers of patients with no history of acute hepatitis. Conversely, some patients have severe clinical hepatitis in the absence of panlobular necrosis, and it must then be assumed that a large proportion of hepatocytes have sustained sublethal damage. The macroscopic appearances in this form of acute hepatitis have been described above. Microscopically, acute hepatitis with panlobular necrosis is characterized by extensive liver-cell loss, the presence of ductular-like structures around portal tracts, inflammatory-cell infiltration and collapse (Figs 7.11, 7.12). The degree of collapse may be judged by the extent of approximation of adjacent portal tracts, which increases with time, and by the density of the collapsed reticulin framework. As in the case of confluent/bridging necrosis, areas of recent collapse contain few if any elastic fibres whereas staining for these fibres becomes positive later.31 Haemorrhage in the collapsing zone may be prominent in some cases (Fig. 7.11). Inflammatory-cell infiltration is mixed and variable in degree. The main infiltrating cells are often macrophages (Fig. 7.12), which usually contain lipochrome pigment and should not be mistaken for residual hepatocytes. Mononuclear infiltrates in areas of surviving parenchyma are sometimes surprisingly mild in comparison with classic acute hepatitis.32 Many of the ductule-like structures contain both liver-cell and bile duct elements, and these have been designated as zones of ductular metaplasia, ductular reaction or neocholangioles.33 They may reflect proliferation of a hepatic stem cell/progenitor cell population34 or transformation of hepatocytes into ductular-like structures. These structures generally are most prominent in the periportal zone, and their location would correspond to the former canals of Hering now transformed into complex arborizing networks of cells.34 Venulitis of central veins can be seen (Fig. 7.13). Evidence of significant parenchymal cell regeneration is present even in fatal cases.35

B Figure 7.12  (A) Acute hepatitis with confluent necrosis. The necrotic zone is filled with macrophages. (H&E) (B) Acute hepatitis with submassive necrosis. The macrophages as seen in Figure 7.12A stain intensely for CD68. A few residual hepatocytes are present in upper left. (Immunoperoxidase for CD68)

Figure 7.13  Acute hepatitis with submassive necrosis and central venulitis. (H&E)

The common hepatotropic viruses can cause panlobular necrosis, but the mechanisms responsible for development of massive necrosis, instead of the more typical pattern of spotty necrosis, are unknown. Possibilities include overwhelming viral infection, superinfection with a second virus36 and microcirculatory failure.32 In

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Figure 7.14  Acute hepatitis with periportal necrosis. In this example of type A hepatitis, extensive inflammation and necrosis are seen at the edge of a portal tract. (H&E)

some instances, mutants of the hepatitis B virus have been implicated.37,38

Acute hepatitis with periportal necrosis In this pattern of hepatitis, the usual changes of classic acute hepatitis are seen to a greater or lesser extent, but there is additionally a substantial degree of necrosis in the periportal zones, accompanied by significant periportal inflammatory infiltration (Fig. 7.14). Lymphocytes and plasma cells usually predominate in the portal and periportal infiltrate. Ductular reaction may be present. One effect of the periportal necrosis is to cause apparent widening of the portal tracts, and linking of tracts as seen in two-dimensional sections. Changes in other parts of the parenchyma may be mild or severe. In some examples of hepatitis A, for instance, the portal and periportal changes may be accompanied by very little perivenular necrosis. In contrast, there are patients in whom biopsy shows a combination of periportal necrosis with central–portal bridging. In contrast to the periportal necrosis (interface hepatitis) of chronic hepatitis, trapped periportal hepatocytes are usually absent or scant in number in the necrotic zone. There is, nevertheless, a close resemblance between these two kinds of necrosis; biopsies from patients with acute hepatitis may thus be wrongly diagnosed as chronic hepatitis, an error usually avoidable if the patient’s history is both unequivocal and available to the pathologist, and if an examination is carried out for the typical parenchymal changes of acute hepatitis.

Evolution of the lesion As already noted, little is known about the early stages of acute hepatitis in man. The descriptions given in this chapter refer to the fully developed acute lesion, but the time over which this lesion develops varies widely from patient to patient. In some, the lesion begins to regress after a few weeks, while in others the course is one of many months. There is international agreement that the term ‘chronic hepatitis’ should be used for inflammation of the liver continuing without improvement for more than 6 months39 but there is overlap between acute and chronic disease: the former may last for more than 6 months in some instances, regressing slowly thereafter, while the latter possibly becomes established in the first few weeks or months of the disease.40

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Following the fully developed stage of acute hepatitis, there is a stage of regressing and finally residual hepatitis. During regression, necrosis diminishes or ceases and phagocytic activity predominates. Portal inflammation is still seen, and in severe hepatitis, there may be ductular reaction. In patients with marked cholestasis, bile stasis can persist after much of the inflammatory activity and necrosis has subsided, often in association with a cholestatic clinical course. Condensation of the reticulin framework marks zones of liver-cell loss, and necrotic bridges undergo collapse to form passive septa. The risk of confusion with chronic liver disease and/or cirrhosis thus increases. In this regressing stage, however, the lesion can still be recognized as acute hepatitis on the basis of hepatocellular changes, parenchymal inflammation and phagocytic activity. In severe forms of acute hepatitis with collapsing stroma, the absence of established scarring, as evidenced by lack of dense bundles of collagen and elastic fibres, can help distinguish acute from chronic disease. The stage of regression passes imperceptibly into a residual stage, which is much less characteristic and easily mistaken for a nonspecific reaction unconnected with viral hepatitis. Changes include slight alterations of architecture, minor degrees of septum formation, focal liver-cell regeneration as shown by variation in liver-cell size and appearance from one area to another, inflammatory infiltration and Kupffer-cell activation. Clumps of Kupffer cells containing iron or lipochrome pigment are often present. Cholestasis, if still present, is mild. Gradually, these residual changes fade and the liver returns to normal. Minor degrees of inflammation and phagocytic activity may be seen as long as a year or more after onset.

Differential diagnosis of acute hepatitis Acute viral hepatitis is only one cause of an acute hepatitis-like clinical syndrome, or acute hepatitis syndrome, and the typical clinical question upon presentation is the aetiology of new-onset hepatic injury. This challenge is generally a clinical question rather than a pathological differential because diagnostic liver biopsy is usually not pursued in the acute setting. If a biopsy is done, the lesions of acute hepatitis are sufficiently diffuse within the liver to be diagnosed with confidence on small specimens.41 This may not hold true for some examples of panlobular necrosis, but this exception rarely presents real diagnostic problems when clinical data are taken into account. It should be noted, however, that it is very difficult to distinguish a cause for acute hepatitis by morphological means alone, so clinical information and correlation are imperative. The most common causes of an acute hepatitis syndrome include hepatotropic viral infection, drug toxicity, toxin or alcohol exposure and hepatic hypoperfusion. Other aetiologies may present as an acute hepatitis syndrome but be the presenting feature of a chronic underlying disorder such as autoimmune hepatitis, hepatic lymphoma or Wilson disease. In general, acute viral hepatitis should be considered in any presentation of acute hepatitis and appropriate history obtained to assess possible exposure to each of the hepatotropic viruses. The aetiology of acute viral hepatitis is appropriately evaluated by assessing the presence of antibody to the respective viral types (see below). Acute hepatitis A infection is indicated by the presence of IgM anti-HAV antibody. Acute hepatitis B infection is identified by the presence of hepatitis B surface antigen (HbsAg) with coexisting IgM antihepatitis B core antibody (IgM anti-HBc). Acute hepatitis C infection is defined earliest by the presence of hepatitis C RNA but current assays for hepatitis C antibody (anti-HCV) are sensitive and able to detect anti-HCV after several weeks of infection.42 Acute infection with hepatitis D (HDV) is defined by δ antigen in the

Chapter 7  Acute and chronic viral hepatitis

serum. HDV always exists in the context of hepatitis B coinfection as the δ agent requires the metabolic capability of the hepatitis B virus to survive. The assay for IgM anti-HDV indicates acute δ infection, but is not generally available. Hepatitis E infection can be detected by the presence of an antibody to the virus (anti-HEV). Other viral infections, such as Epstein–Barr or cytomegalovirus may also be responsible for acute liver injury. Histological clues may be present to suggest these viral infections. In infectious mononucleosis (EBV-related), for example, liver-cell damage is usually absent or mild, and atypical lymphocytes are seen in sinusoids and portal tracts. In the other herpes-type viral infections, e.g. herpes simplex and cytomegalovirus, the necrosis is often confluent rather than single-cell, spotty type, and a minimal associated lymphoid infiltrate is present in the sinusoids. The typical viral inclusions can often be seen. The histological findings of these viral infections are discussed in Chapter 8. Other infectious agents including syphilis, dengue fever or yellow fever may be considered as the clinical history suggests. Acute cholestatic hepatitis may be induced by hepatotropic viral infection, but must be distinguished from acute large bile duct obstruction, which can present with prominent aminotransferase elevation during the first days of presentation. This pattern is generally followed by a rise in alkaline phosphatase and bilirubin values, especially when obstruction is high-grade and unremitting. The presentation is usually accompanied by right upper quadrant or abdominal pain but may be occult, particularly in patients with diabetes or in the elderly. Biliary imaging studies, such as ultrasound or magnetic resonance cholangiograms, are sensitive in defining this pathology. Other relatively common cholestatic hepatitis syndromes include drug or toxin injury and alcoholic hepatitis. Less commonly this syndrome may be simulated by cholestasis of pregnancy, benign recurrent cholestasis or the exacerbation of a chronic underlying cholestatic disorder such as primary sclerosing cholangitis or primary biliary cirrhosis. The bile duct lesion of acute hepatitis can usually be distinguished from that of biliary tract diseases, such as primary biliary cirrhosis, by the presence of the parenchymal features of acute hepatitis, lack of granulomatous duct lesions, ductopenia and other clinical and biochemical findings. Granulomas associated with bile duct lesions are almost never seen in acute hepatitis. The presence of lobular changes also helps to distinguish hepatitis from primary biliary cirrhosis; however, sinusoidal lymphocytic infiltrates are relatively common and even spotty necrosis has been noted in primary biliary cirrhosis,43 so clinical correlation is always advised if the histological changes are equivocal. In addition, bile ductular reaction tends to be less in hepatitis (except in more severe forms of hepatitis), and the periportal hepatocytes do not retain copper as in the chronic cholestatic diseases. Overall, the key features for separation of these two entities may lie in the examination for granulomatous duct lesions or loss of the interlobular bile ducts in primary biliary cirrhosis. In cholestasis from any cause, secondary changes in hepatocytes and accompanying inflammation may cause confusion with acute hepatitis. In cholestasis, the changes are generally confined to the cholestatic areas, and liver-cell plates show little or no disruption, although some regenerative changes can be present. The presence of spotty hepatocellular necrosis can be a key histological feature to differentiate a cholestatic hepatitis such as hepatitis A from bile duct obstruction. Furthermore, bile retention is almost never seen in early stages of primary biliary cirrhosis, so, in the absence of ductopenia, the presence of canalicular cholestasis would favour a hepatitis or obstruction. Portal changes vary according to the cause of the cholestasis, and may also help to establish a correct diagnosis,

but both hepatitis and biliary diseases may show varying degrees of portal mononuclear infiltrates and interface hepatitis. Drugs and toxic agents should always be suspected as a possible cause of acute hepatitis syndrome (see Chapter 13). Correlation of the temporal relationship of liver disease to the usage of the drug is essential in any type of possible drug reaction, either idiosyncratic or toxic. Hepatic injury is generally recognized at least 5 days after initiating drug exposure but may become evident during the first year or later of administration.44 The pattern of liver test abnor­ malities may be hepatocellular with prominent aminotransferase elevation, although some agents such as chlorpromazine generate a predo­minant cholestatic pattern of liver test results.45 Mixed patterns are also possible. Drugs, environmental toxins, ‘health’ supplements, vitamin preparations and over-the-counter therapies all need to be considered as possible causes when evaluating acute hepatic injury. Most recently, the growing use of alternative medicine has focused attention on the potential hepatotoxicity of ‘natural’ products, or herbal agents. Although systemic evidence of hypersensitivity, such as rash or eosinophilia, may support drug hepatotoxicity, these findings are not necessary for the diagnosis. Histopathological findings included injury of varying severity from bridging necrosis to massive necrosis.46 Features that should arouse a greater than average degree of suspicion for an idiosyncratic type of drug hepatitis include a poorly developed or absent portal inflammatory reaction, abundant neutrophils or eosinophils, granuloma formation, sharply defined perivenular necrosis with little inflammation, or a mixed pattern of hepatitic and cholestatic features with duct damage; the latter may be a prominent feature. Direct toxic reactions such as paracetamol (acetaminophen) toxicity typically have a uniform pattern of perivenular necrosis with a minimal inflammatory component. Acute alcoholic hepatitis is usually an exacerbation of a chronic condition. The degree of elevation of aminotransferase values is generally modest and does not reflect the severity of injury. Typically, AST levels are greater than ALT values and prominent elevation of GGT is typical.47 GGT activity is a reflection of enzyme induction rather than the severity of hepatotoxicity. Alkaline phosphatase and bilirubin values commonly rise as aminotransferase values improve following abstinence. In alcoholic hepatitis, ballooning of hepatocytes is usually seen in perivenular (central) zones, often accompanied by a predominantly neutrophilic infiltrate with or without the presence of new collagen fibres around affected hepatocytes; fatty change and Mallory–Denk bodies are often present (see Chapter 6). In contrast, non-alcoholic steatohepatitis (NASH, see Chapter 6) may have a more intense lymphocytic infiltrate and absent or less prominent Mallory–Denk bodies but this entity is not typically in the clinical differential diagnosis owing to the lack of jaundice and the milder, more chronic nature of the aminotransferase abnormalities. Autoimmune hepatitis can present as an acute hepatitis syndrome as well (see Chapter 9), with aminotransferase values more than five times the upper limit of normal. When this occurs, histological changes are similar to those of acute viral hepatitis with extensive hepatocyte necrosis and inflammatory infiltrates, especially in the perivenular (central) zone. Plasma cells are likely to be prominent in autoimmune hepatitis, but they may be variable in number in viral hepatitis, and have been noted to be a common component of the inflammatory infiltrate in acute hepatitis A, so the presence of these cells cannot be used as a definitive distinguishing feature. Serum markers for autoimmune antibodies as well as elevated γ-globulin levels above 2 g/dL are typically present to distinguish these lesions.48 Wilson disease (see Chapter 4) can also rarely present as fulminant liver failure, and, in these instances, the histology can be

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Table 7.3  Sequelae of acute hepatitis • Restitution to normal morphology • Death from severe liver damage or its complications • Posthepatitic scarring • Viral carrier state • Chronic hepatitis and cirrhosis • Hepatocellular carcinoma

similar to acute viral hepatitis. However, some of these patients may have underlying evidence of chronic injury such as established scarring, demonstrated by densely staining collagen bands in the form of septa and/or increased elastic fibre deposition in septa. Tissue from Wilson disease patients may also show copper deposits by histochemical stains (see Chapter 4), but quantitative copper studies on liver tissue, serum ceruloplasmin, or 24-h urinary copper studies are typically of more value in establishing the diagnosis. Additional causes of acute liver injury are multiple and are pursued according to evidence supplied within the medical history. Liver pathology evaluation may be of value in focusing consideration on unanticipated results and assessing the severity of injury. As many as 15–20% of patients with fulminant liver failure may have no known cause for the hepatitis. The histology of these cases is often similar to the types of changes described in this chapter for severe hepatitis with panlobular necrosis. Lastly, areas of collapse in acute hepatitis must be distinguished from the fibrous septa of chronic liver disease, as discussed above.

Sequelae of acute hepatitis The morphological consequences of acute viral hepatitis are shown in Table 7.3. Restitution to normal liver occurs in the classic forms of acute viral hepatitis, and the results of fulminant hepatitis are described under acute hepatitis with panlobular necrosis. The spectrum of lesions of chronic hepatitis is discussed later in this chapter. A viral carrier state without significant histological changes (other than ground-glass hepatocytes in hepatitis B virus carriers) may possibly develop in both hepatitis B and C as a temporary state,49 but some question remains whether a permanent, non-progressive infection by these two viruses exists. It is likely that cirrhosis may develop following acute hepatitis due to ongoing hepatitic changes or chronic hepatitis. The idea that cirrhosis can develop directly from massive hepatic necrosis, without the intervention of chronic hepatitis, is expressed in the old term ‘post-necrotic cirrhosis’. Karvountzis et al.49 followed 22 patients surviving acute hepatitis with coma, and concluded that such patients rarely if ever developed chronic hepatitis. On the other hand, Horney and Galambos32 reported that chronic hepatitis had developed in three of nine patients having follow-up biopsies 6–60 months after fulminant hepatitis. Certainly, nodular regeneration of surviving parenchyma is seen in patients dying some weeks or months after the acute attack, but this should not be considered as cirrhosis as the septa are formed by collapse of pre-existing fibres rather than by true new collagen deposition (fibrosis); in addition, the nodularity is not usually uniform or diffuse. It is possible that in a small number of patients who recover from the acute attack, there is sufficient nodularity, portal–systemic shunting, fibrosis and portal hypertension to warrant a diagnosis of inactive cirrhosis even in the absence of chronic hepatitis,28,50 but this is probably the exception rather than the rule. A role for vascular occlusion in the pathogenesis of chronic liver disease and cirrhosis has been proposed,51 and one could also

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speculate that similar lesions play a role in postnecrotic scarring following acute hepatitis. The mechanism for the injury would probably start with phlebitis of the hepatic venules (Fig. 7.13) or portal veins, with resultant vascular sclerosis. Occlusion of these veins, in turn, would lead to ischaemia or outflow obstruction in these areas, a process which would impair regeneration and enhance the formation of scar tissue in those zones with severe hepatocyte necrosis and dropout. Hepatocellular carcinoma (see Chapter 14) is a long-term complication of hepatitis B and C; it is usually (but not always) seen in end-stage disease associated with cirrhosis, and is generally not considered to be a direct sequelae of acute viral hepatitis.

Chronic viral hepatitis Clinical features Chronic viral hepatitis is a syndrome of persisting hepatotropic viral infection usually associated with chronic inflammation, hepatocyte injury and progressive fibrosis. By convention, infection for more than 6 months is considered evidence that spontaneous resolution of infection is unlikely and hepatitis is chronic. A clinical diagnosis of chronic viral hepatitis may be made on an initial clinical evaluation when clinical history or pathological findings suggest chronic infection even in the absence of earlier laboratory data. Chronic viral hepatitis is typically classified by the responsible infecting virus and modified by the extent of pathological injury and clinical compensation. Hepatotropic viruses responsible for this syndrome include hepatitis B, C and D. The injury from hepatitis A and E viruses may be severe and relapsing but represents acute injury rather than chronic hepatitis. The epidemiologies of the various forms of viral hepatitis share modes of transmission and thus a small, but significant, number of individuals may have chronic coinfection with multiple viruses. In addition to hepatotropic viruses, HIV also shares epidemiological features with the hepatitis viruses. Individuals with HIV coinfection may experience accelerated injury and progression of liver disease, particularly in the setting of impaired immune function. Progression of fibrosis may be similar to that in mono-infected patients in the HIV coinfected patient on effective antiretroviral therapy (Chapter 8).52 The hepatotropic viruses form a group of diverse agents which utilize liver tissue as a site of replication and major pathological injury. The liver, usually, is the predominant focus of injury but the course of disease may vary considerably from patient to patient. Many patients are asymptomatic and most are anicteric. Infection with hepatitis B or C may be discovered through screening, which is most fruitful in populations at high risk of exposure (Table 7.4).53,54 Symptoms of chronic hepatitis may be nonspecific with fatigue being the most common. Patients may have mild discomfort in the right upper quadrant, pruritus, joint pain or anorexia. As liver disease progresses, muscle atrophy, jaundice, fluid retention and loss of mental acuity may develop. The characteristic biochemical abnormality associated with chronic viral hepatitis is an elevation of aminotransferase values. Typically, aminotransferase activities range from normal to 20 times the upper limit of normal. In patients with hepatitis C, this hectic course of aminotransferase values is more commonly seen early in infection, and in patients with hepatitis B infection, a rise in aminotransferase value may be associated with seroconversion of HBe antigen positive to HBe antibody reactivity or δ superinfection.

Chapter 7  Acute and chronic viral hepatitis

Table 7.4  Populations at risk of exposure to hepatitis B or C51,52

Populations at risk for exposure to hepatitis B • Persons born in high endemic areas • Men who have sex with men • Injection drug users • Dialysis patients • Persons with HIV infections • Family/household and sexual contacts of HBV-infected patients

Populations at risk for exposure to hepatitis C • Injection-drug users • Persons with HIV infection • Haemophiliacs who received clotting factors before 1987 • Dialysis patients • Persons who received a transfusion of blood or blood products before 1992 • Persons who received an organ transplant before 1992 • Children born to HCV-infected mothers • Healthcare workers with needle-stick injury or mucosal exposure to HCV-positive persons

In many patients with chronic viral hepatitis, aminotransferase values are normal. Although the correlation with aminotransferase values and the degree of liver injury is not precise, generally asymptomatic individuals with consistently normal aminotransferase values have a more favourable prognosis than individuals with elevated test results.55 Patients, however, can have significant pathological abnormality, even cirrhosis, in spite of normal alanine aminotransferase (ALT) values. In one study, 16% of patients with persistently normal ALT values had significant necroinflammation or fibrosis.56 Disease progression has been demonstrated in patients with hepatitis C and persistently normal aminotransferase values.57 As chronic hepatitis progresses, evidence of hepatic synthetic dysfunction is demonstrated by a fall in serum albumin and rise of INR and serum bilirubin. The presence of cirrhosis may be suspected by the presence of signs of portal hypertension such as splenomegaly and laboratory results may show a fall in platelet count. The AST/ platelet ratio correlates with the presence of cirrhosis.58 Once a patient is defined as having chronic viral hepatitis due to a specific hepatotropic virus, it is important to assess the severity of illness and the presence or absence of complications such as portal hypertension or hepatocellular carcinoma. Liver biopsy is particularly useful in this assessment and currently forms the basis for the most secure assessment of the stage of disease and the urgency of initiating antiviral therapy. Serological methods of assessing severity of hepatic fibrosis are of interest due to the avoidance of liver biopsy, but these methods often lack precision in defining the amount of fibrosis.59 Novel technologies, such as transient elastography, measure hepatic fibrosis in patients with chronic hepatitis C with precision approaching liver biopsy with accuracy being highest in advanced stages of fibrosis.60 However, these technologies have limited availability.

Pathological features Chronic viral hepatitis is characterized by a combination of inflammatory cell infiltration, hepatocyte death, atrophy and regeneration, and fibrosis. These components, while all present in acute viral hepatitis to some degree, have a different relative proportion and

distribution in chronic viral infection. The proportion and distribution may also differ between one viral infection and another, as well as in the same patient with the same infection, over time. Patterns of inflammation and scarring will be discussed first, followed by descriptions of the types of scarring that may be seen, if and when the disease is progressive. The possible mechanisms of injury and repair responsible for these gross and light microscopic changes will then be summarized. Finally, practical issues regarding routine biopsy specimen assessment in chronic viral hepatitis, including grading/staging and recognition of common, concomitant diseases will be discussed.

Macroscopic appearances The macroscopic appearances of livers with chronic viral hepatitis in advance of cirrhosis have not been systematically described. However, anecdotal observations of such livers obtained at autopsy or in partial hepatectomy for tumours indicate that they may appear normal, have focal areas of fibrosis with a somewhat gritty texture, or have a diffuse or focally lobulated appearance indicative of fibrous septa and local regeneration (Fig. 7.15). The colour of the liver at these early stages is generally the normal beefy red as these patients are rarely cholestatic. Some yellow colour will indicate steatosis. As cirrhosis develops, there is increasingly diffuse nodularity and obvious fibrous scarring. Classically, cirrhosis associated with chronic viral hepatitis appears either macronodular or mixed microand macronodular, though purely micronodular cirrhosis may be seen. A predominantly macronodular cirrhosis seems to be most often present in patients younger than 40 years old, while strictly micronodular cirrhosis in chronic hepatitis should raise the question of concomitant alcoholic liver disease. Nodules may vary in colour, ranging from beefy red to dark green of cholestasis to yellow of fatty change. Some nodules may appear necrotic, due to compromised blood supply. Larger portal vein branches may be thrombosed.

Light microscopic appearances: patterns of necrosis, inflammation and fibrosis Hepatocyte injury and inflammation in chronic hepatitis is referred to as ‘activity’, which is ‘graded’ (see below). Distribution of inflammatory cells may vary from case to case or even in sequential biopsies from the same patient. However, all cases of chronic viral hepatitis are distinguished by a relatively dense monocytic infiltration of the portal tracts. If extra-portal inflammation and hepatocyte injury are scant to absent, the grade is sometimes informally referred to as ‘inactive’ (formerly ‘chronic persistent’61) hepatitis; when there is more obvious inflammation and apoptosis of hepatocytes involving the limiting plate and lobular hepatocytes, the hepatitis may be termed ‘active’ (although the phrase ‘chronic active hepatitis’61 should also be avoided).

Portal inflammation Mononuclear infiltration of portal tracts is the defining lesion of chronic hepatitis of any cause. Some or all portal tracts may be involved and the portal infiltrates are usually much denser than those seen in acute viral hepatitis. The portal tracts may be of normal size or appear widened by the influx of mononuclear cells. The infiltrate includes predominantly CD4+helper/inducer T lymphocytes with an admixture of plasma cells; γ-δ T cells appear to not be increased beyond their normally low levels.62–64 Some portal macrophages may also be seen to contain periodic acid-Schiff (PAS)-positive, diastase-resistant material and iron pigment, representing the removal of hepatocyte debris.

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A Figure 7.16  Phlebitis of a portal vein in a liver with chronic hepatitis C. The portal tract is expanded by a dense mononuclear cell infiltrate which focally encroaches on the vein. Damage to the endothelium is evidenced by focal lifting of the endothelium from its basement membrane and adherent inflammatory cells. Such damage has been hypothesized to lead to thrombosis of veins with subsequent ischaemia and extinction of dependent regions of parenchyma. (H&E) (Courtesy of Dr I Wanless)

B Figure 7.15  (A) Micronodular cirrhosis due to chronic hepatitis C. Nodules of regenerating hepatic parenchyma separated by fibrous bands are rarely >3 mm in greatest dimension. The two green nodules are foci of hepatocellular carcinoma. (B) Macronodular cirrhosis in chronic hepatitis B. Nodules vary greatly in size, though most are >3 mm in greatest dimension. (Courtesy of Dr I Wanless)

Portal inflammation will often fill and expand the portal fibrous stroma, pushing structures aside without obvious injury. Lymphoid aggregates or fully formed follicles may be seen; while most common in hepatitis C, they are also seen in other forms of hepatitis. However, inflammation with damage or even destruction of bile ducts may also be seen, particularly in hepatitis C.65–67 Inflammation may also encroach on the portal blood vessels, in particular the portal veins (Fig. 7.16), endotheliitis may be present,67 and there may be associated fresh or organizing venous thrombosis; such lesions may be particularly evident with trichrome-stain.68

Interface hepatitis The region of liver tissue where the hepatic parenchyma comes into contact with the mesenchymal stroma of the intact or scarred portal tract is referred to as the stromal-parenchymal interface. Thus, hepatocyte apoptosis and inflammation of this area are generally referred to as ‘interface hepatitis,’ the currently favoured term. Previously, this classical histological feature of active chronic hepatitis was

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called ‘piecemeal necrosis,’ referring to the way in which the limiting plate of hepatocytes was eroded in a piecemeal, that is focal, fashion (Fig. 7.17).69 In regions of interface hepatitis, there is a predominantly mononuclear infiltration, though in these regions, Fas-ligand positive CD8+ suppressor/cytotoxic T cells predominate.70 Close contact of hepatocytes with these lymphocytes, and with macrophages and plasma cells, is seen.71–76 Emperipolesis, the invagination of lymphocytes into hepatocytes, has been described.69 While the inflammation interweaving amongst hepatocytes at the interface is sufficient by itself for describing the lesion as interface hepatitis, apoptotic hepatocytes may also be seen in these areas.75,76

Lobular hepatitis and confluent necrosis Another form of activity in chronic viral hepatitis is found within the hepatic lobule away from the portal areas or septal scars. Such lesions may be referred to as ‘lobular hepatitis’ or ‘spotty necrosis’ (Fig. 7.18).29,77–81 The inflammatory infiltrates in these areas of activity are the same as those seen in interface hepatitis, but their relative importance to the development of scarring and progression in chronic viral hepatitis remains uncertain. Some foci of lobular hepatitis are relatively devoid of mononuclear cells. In these areas, there may be acidophil bodies, clustered macrophages containing PAS-positive, diastase-resistant material indicating prior cell death, or simply cellular debris and loosely aggregated collagen and reticulin fibres. Such lesions are similar to the lobular damage found in acute viral hepatitis. If large areas are involved, one may refer to the lesion as ‘confluent necrosis’ which typically is found around central veins, but, if still more severe, may span from there to other central veins or to portal tracts, when the term ‘bridging necrosis’ is used. Such bridging necrosis is considered the most ominous finding in terms of progression toward scarring and cirrhosis.79,80 In some cases, when one or more entire lobule has been destroyed, it may be referred to as panlobular or multilobular collapse. In these areas, portal tracts will be in abnormally close proximity, separated only by regions filled with necrotic debris, macrophages, loose or more mature collagen, and elastic fibres.29 A ductular reaction, which is now

Chapter 7  Acute and chronic viral hepatitis

A Figure 7.19  A ductular reaction, consisting of a disorganized array of cholangiocyte-like cells (arrows), is present at the margin of a fibrous septum in hepatitis B cirrhosis. Such cells can also be seen focally within the fibrous tissue. (H&E)

B Figure 7.17  (A) Interface hepatitis in chronic viral hepatitis. Previously referred to as ‘piecemeal necrosis’, this lesion can often be observed at low power, evidenced by irregular contours of a portal tract containing a dense mononuclear cell infiltrate eroding the limiting plate of hepatocytes in a piecemeal fashion. (B) Higher-power view of an inflamed portal tract in hepatitis C shows mononuclear cells extending beyond the limiting plate, surrounding individual or small clusters of hepatocytes. (H&E)

Figure 7.18  Lobular hepatitis in chronic hepatitis C. Foci of inflammation and parenchymal necrosis located in the hepatic parenchyma near the hepatic venule are a form of activity in chronic hepatitis. Another term for this lesion is spotty necrosis. (H&E)

recognized as activation of a hepatic stem cell compartment, is also present and is considered potentially both regenerative response to hepatocyte injury and senescence and, paradoxically, instigator of fibrogenesis (Fig. 7.19).81,82 Confluent necrosis in a patient with known chronic viral hepatitis may indicate specific clinical situations related to the known virus (e.g. in HBV, super-infection with HDV or serologic HBeAg to HBeAb conversion; in HCV, an acute, but self-limited flare), but other concomitant injuries should be considered, including immunosuppression and concomitant autoimmune hepatitis or drug/toxin-mediated injury. Clinical correlation is important to investigate all these possibilities when confluent necrosis is seen.

Fibrosis and hepatocyte regeneration While some patients with chronic viral hepatitis do not show fibrous scarring, most will have some. Increasing fibrosis is now assessed as advancing ‘stages’ of disease. The scarring will usually develop as an extension of the portal stroma, though perivenular and pericellular fibrosis may also be seen. The perivenular fibrosis usually develops following collapse and condensation of the reticulin meshwork in an area of confluent necrosis. Such perivenular scars are bland and generally acellular, often lacking extensive accompanying interface hepatitis and linking central veins to neighbouring portal tracts or to other hepatic veins in which case they probably represent healed bridging confluent necrosis. If these scars persist, mature forms of collagen will deposit. Elastin fibres will follow, usually beginning between 3 and 6 months and becoming denser afterward. Thus, staining of elastic fibres with orcein or Victoria blue stains is a useful tool for evaluating age of a scar.29 Fibrous expansion of the portal tract probably results from a more active process of injury and repair. There is as yet, little agreement on the use of the terms ‘periportal fibrosis’ and ‘portal fibrosis’. Whichever term is used, it refers to fibrous stroma extending from the portal tract beyond its usual boundaries. This fibrosis is usually conceptualized as following Rappaport’s acinus zone 1 and, thus, eventually leads to linking of one portal tract to another. These septa usually contain mononuclear inflammatory cells as described above and may be associated with interface hepatitis (Fig. 7.20). The fibrosis is usually mature, darkly staining with trichrome stains, and containing abundant type I collagen in addition to type III collagen (Fig. 7.21A) and reticulin fibres (Fig. 7.21B).83 Again, elastic fibres are easily demonstrated, indicating that the scars are of at least several months duration. Immunohistochemical staining for

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A Figure 7.20  A fibrous septum in hepatitis C cirrhosis with a dense mononuclear cell infiltrate and extensive interface hepatitis. (H&E)

α-smooth muscle actin highlights numerous activated stellate cells in these regions and these cells are thought to be largely responsible for most of the scarring (Fig. 7.21C),84 although portal myofibro­ blasts may also contribute. Regeneration of hepatocytes becomes increasingly evident in parallel with the formation of fibrous septa and advancing stages of disease. The thickening of liver-cell plates to two or three cells thick evidences this regeneration. Haematoxylin and eosin (H&E) or silver stains for reticulin can demonstrate these changes. An incomplete periportal nodular transformation may thus be noted around portal tracts that are becoming fibrotic. However, in the later stages of developing cirrhosis, hepatocyte regeneration markedly diminishes in parallel with an increasingly proliferative ductular reaction at the stromal–parenchymal interface. This reaction is believed to be activation of the intrahepatic facultative stem cell compartment as pre-existing hepatocytes reach replicative senescence in response to the chronic injury.81,82

B

Regression of fibrosis and cirrhosis Development of scarring in a chronically diseased liver is actually the result of a balance in favour of matrix deposition in a liver dynamically producing and degrading matrix at all time points. That fibrosis can regress with elimination of viral activity and infection has now been demonstrated in all forms of chronic viral hepatitis.85–91 Cirrhosis itself, however, has generally been thought to be an ‘end-stage’ of chronic liver disease and therefore not reversible. However, exceptions to this concept have been noted in many liver diseases over the years86,88 and, indeed, the definition of cirrhosis itself is considered by many to be ready for revision in this light.89 In 2000, Ian Wanless and colleagues examined sequential biopsies from a patient with chronic hepatitis B infection who, with successful antiviral treatment and elimination of viral infection, apparently reversed the scarring and re-established some degree of functional, non-cirrhotic parenchyma.88 These changes were documented on biopsy. Wanless’ suggestion that cirrhosis was reversible when the impetus to hepatic injury ceases was exceedingly controversial at the time. The formal paper was finally published in a particularly interesting fashion: all in the same volume of the journal there appeared his paper, responses/critiques written by each of the reviewers,92–95 and then his response to their comments.96 Despite the controversial nature of the proposal, however, the concept rapidly entered the realm of general knowledge as clinical papers exploring the impact of successful eradication of

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C Figure 7.21  (A) Trichrome stain of a fibrous septum in hepatitis C cirrhosis, lining two portal tracts, shows dark blue staining of a mature scar, abundant in type I and type III collagen. (Gomori trichrome) (B) Reticulin stain of the same scar demonstrates collapse of the reticulin meshwork in the scar and highlights the regenerative thickening of liver cell plates in adjacent nodules of parenchyma. (Gomori reticulin) (C) Immunohistochemistry for smooth-muscle actin demonstrates actin fibres depositing in the scar, but also highlights individual activated stellate cells within the scar and in adjacent sinusoids. These cells are the resident myofibroblasts in the liver and are primarily responsible for deposition of matrix proteins which make up the scar. (Immunoperoxidase, DAB)

Chapter 7  Acute and chronic viral hepatitis

HCV infection on stage of disease in patients with paired preand post-eradication biopsy specimens demonstrated the possibility of regression of fibrosis and, in some cases, regression of cirrhosis.86 Wanless and colleagues described eight histological features of regression as the ‘hepatic repair complex’, which can be grouped into features evincing three regenerative phenomena: fragmentation and regression of scar (e.g. delicate, perforated septa; isolated thick collagen fibres; delicate, periportal fibrous spikes; hepatocytes within or splitting septa), evidence of prior, now resolving, vascular derangements (e.g. portal tract remnants, hepatic vein remnants with prolapsed hepatocytes, aberrant parenchymal veins) and parenchymal regeneration in the form of hepatocyte ‘buds’.88 These hepatocyte buds are small clusters of hepatocytes, usually emerging from within portal/septal stroma; and represent regeneration from the stem cell niche rather than entrapment of parenchyma by active scarring.81

Pathogenetic mechanisms There appear to be general mechanisms of injury and repair common to all hepatotropic viral infections, some involving viral determinants, others defined by host response, the complex interplay of which determines the outcome of viral hepatitis.97–101 Most importantly, the continued testing and development of antiviral drugs and immunomodulators is allowing clinicians to increasingly influence the balance and, therefore, alter outcomes of these infections. Viral determinants include gene products that cause or inhibit apoptosis of infected or reacting cell populations. Direct cytotox­ icity of the hepatotropic viruses is probably a smaller contributor. The viruses may produce gene products that interfere with cellmediated immunity and cytokine actions. Mutations of the viruses may lead to escape from both humoral and cell-mediated immune responses. Infection of extrahepatic, immunologically tolerant or privileged sites may encourage viral persistence.101 On the other hand, the host has a wide range of possible responses to prevent infection or to clear virus from the liver or at least inhibit viral activity. Non-immunological responses include apoptosis of an infected cell.102 Immune responses may be virus specific or nonspecific and include those of humoral-, cell-mediated immunity, and cytokine release/activation by immunocytes, parenchymal and nonparenchymal liver cells.97–101 In acute hepatitis, the most appropriate response is one where the immune system can outpace the spread of the infection, eliminating viral replication from all infected cells, or eliminating the infected cells directly. With efficient regeneration of hepatocytes, either from mature hepatocyte division or from activation of stem/ progenitor-cell compartments, the liver can recover, re-establishing normal architecture and function. However, if the viral infection is not kept in check by an active immune response, progressive immune destruction of the liver results in fulminant hepatic failure. Alternatively, the immune system may be generally or selectively impaired in response to the specific virus. Patients with a generalized immunosuppression, such as those coinfected with HIV or patients receiving immunosuppressive therapy for unrelated diseases, are at increased risk for developing chronic infection following acute exposure to a hepatitis virus.103,104 Selective immune system defects may also occur, either pre-existent (perhaps genetically determined) or influenced or induced by viral factors.97–101 Regardless of the specific mechanisms involved, the virus is able to infect hepatocytes beyond the ability of the immune system to kill the infected cells or to induce suppression of the viral replication, thereby maintaining a chronic presence in the liver and

provoking, to variable degrees in different individuals, ongoing immune-mediated damage to the liver and its sequelae. In many patients, the outcome of this damage is development of scarring, sometimes leading to cirrhosis. There is also likely to be an increased risk for development of hepatocellular carcinoma related, at least in part, to increased hepatocellular turnover.105,106

The role of humoral immunity Virus-specific antibodies play a relatively limited role in the development of viral hepatitis. Specific antiviral antibodies can protect against infection in the first place; thus, vaccine-induced anti-HAV and anti-HBV antibodies can prevent these infections.107,108 Humoral immunity against hepatitis C, however, appears very limited.97,99,100 Once HCV infection has occurred, the humoral response serves primarily to help clear virus from the body, preventing its spread to uninfected cells. However, humoral responses to the viruses can lead to immune complex diseases such as glomerulonephritis, vasculitis and cryoglobulinaemia.109,110 Autoantibody production can also be induced, perhaps leading to an autoimmune component of the hepatitis or autoimmune diseases affecting other organs.111

The role of cellular immunity Cell-mediated immunity, particularly the cytotoxic T-lymphocyte (CTL) response, is thought to be central to elimination of viral infections of the liver (Chapter 1). Through specific cell-to-cell recognition of host (major histocompatibility complex, MHC class I) and viral antigens on the surface of infected cells, CTL-induced cell death eliminates infected hepatocytes. Activation of CD4+ T helper cells throughout the MHC class II pathway is also important for viral clearance, whether by stimulating CTL responses, inducing different patterns of cytokine production, or perhaps through some direct, Fas-mediated cytotoxic activity.97,98,112,113 Recruitment and activation of these immunocytes has been shown to be partly mediated and promoted by platelet aggregation in the hepatic microcirculatory system leading to release of platelet-derived substances such as serotonin.114 However, the number of potentially infected cells in the liver can far outweigh the available, though proliferating, virus-directed lymphocytes. Thus, though direct cell–cell interactions are probably important, diffusible cytokines produced or induced by these lymphocytes may be a more important factor in viral clearance. CD1dreactive T cells, variants of natural killer cells, are seen to play important roles in production of these diverse cytokines as well, mediating via both Th1 and Th2 pathways.112,113 In addition classical FOXP3+ CD4+ regulatory T cells, IL-10-producing Tr1 cells, TGFβ-producing Th3 and CD8+ regulatory T cells all may play important roles.100

The role of cytokines Cytokines may have direct antiviral activity and thus be important in modulation of the immune system.97–101,113,114 For example, the interferons have been shown to inhibit all viral infective/replicative processes, from entry into the cell, through uncoating, to viral gene translation and protein synthesis. Through these activities viral clearance from infected cells, even in the absence of cell death, can occur, extending the antiviral capabilities beyond the limits of cellmediated immunity. They also induce production of host-cell proteins, including HLA class I and class II antigens. Macrophages, natural killer cells, and cytotoxic T cells are also stimulated by these cytokines. Tumour necrosis factor (TNF), produced by macrophages and T cells, stimulates chemotaxis, activation of macrophages,

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induction of class I and class II antigens and T-cell activation, and modulates acute-phase protein transcription, amongst other functions. IL-1 is produced by multiple cell types, including epithelial cells, macrophages, dendritic cells, endothelial cells and B cells. Actions include enhancement of lymphocyte and fibroblast proliferation and activation, and induction of acute-phase reactants. IL-6, produced by monocytes, fibroblasts and endothelial cells, induces B-cell differentiation and acute phase proteins and is a growth factor for T cells. Profibrotic cytokines, including IL-4 and IL-13, may also play a role in fibrosis. Thus, these cytokines have overlapping as well as distinct effects. At the same time, all cytokines may not be favourable, with evidence now suggesting a role for IL-10 and TGFβ in supporting chronic viral persistence.113

The role of vascular injury While hepatocyte injury often precedes cirrhosis, it is insufficient per se for its development. Even after significant hepatocyte necrosis, the liver can regenerate to a normal architecture without permanent fibrosis. Moreover, despite distribution of significant hepatocyte injury evenly throughout the hepatic lobule in chronic hepatitis, the formation of fibrous septa only occurs in limited regions. Some fibrous septa probably develop as scar tissue to replace bridging necrosis due to direct hepatocyte injury. However, some septa form in response to regions of parenchymal extinction following injury and thrombosis of intrahepatic arteries and veins with resultant parenchymal atrophy.68 In chronic hepatitis this injury is probably a ‘bystander’ phenomenon, resulting from the inflammatory response directed against, but not limited to, the infected parenchymal cell. Once cirrhosis is established, stasis leads to further thrombosis of portal and hepatic veins and secondary parenchymal loss that is independent of the activity of the original disease.

Mechanisms of fibrogenesis and fibrotic regression As noted above, scarring during chronic liver disease represents a balance between new deposition of matrix and its resorption. As long as the disease persists, the balance favours deposition and scar formation. If the disease is inhibited or eliminated, then fibrosis and even cirrhosis can regress. The principal cell responsible for collagen deposition and scar formation in the liver is the hepatic stellate cell, although it is becoming clear that this class of cells is markedly heterogeneous and that other cells and processes, including portal myofibroblasts, epithelial-mesenchymal transition and marrow-derived cells also play possibly significant roles.115,116 Many factors probably initiate stellate cell activation though oxidative stress plays a prominent role. For example, in vitro lipid peroxidation products, such as malondialdehyde, can stimulate stellate cell activation, inducing collagen synthesis, and have been identified in the regions of hepatocyte injury and fibrous septum formation in chronic hepatitis.117 Once activated, stellate cells respond to a variety of cytokines that upregulate collagen synthesis and deposition, including TGFβ, IL-1 and IL-4, many of which are primarily or secondarily produced in inflammatory responses typical of chronic viral hepatitis.118 Fibrogenic cytokines may derive from autocrine, paracrine and matrixbound sources. Certainly, regression of fibrosis requires breaking up and digestion of collagen and elastin fibres which make up the principal components of the scar. Various metalloproteinases accomplish some, if not all, of these tasks and are produced by non-parenchymal cells, particularly those of macrophage lineages. However, the appearance of fractured, ‘perforated’ septa with hepatocytes within

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or completely interrupting the septa, usually in the absence of inflammation or activated macrophages, suggests that hepatocytes themselves may play a major role. Expression of various metalloproteinases by hepatocytes supports this concept.119

Multiple chronic viral infections Coinfection by multiple hepatotropic viruses Since hepatitis B, C and D viruses are all parenterally spread, it is not surprising that dual or triple infection of these viruses occurs.120 Viral interference appears to be most common in coinfections experimentally121 and in clinical settings, usually HCV interfering with HBV replication,122 though the reverse has also been reported when HBV infection followed HCV infection.123 The clinical course, however, does not seem to be consistently altered by such coinfections.120,124 Histopathologically, there are no specific findings to suggest multiple infections and the diagnosis usually depend on serological investigations. However, immunohistochemical investigations have revealed suppression of HBV core antigen by simultaneous HCV infection. It may be reasonable to suggest that, in a patient serologically positive for hepatitis B surface antigen (HBsAg), the absence of demonstrable tissue staining for HBV antigens indicates that clinical suspicion of coinfection not only with HDV, but also with HCV, should be raised by the pathologist.125

Coinfection by hepatotropic viruses and HIV Since HBV, HCV, HDV and the human immunodeficiency virus (HIV) are all parenterally acquired, and HBV and HIV are also frequently transmitted sexually, coinfection of these hepatitis viruses and HIV is common.126,127 Additionally, coinfection between HIV and HAV has been identified, though this does not seem to lead to a difference in HAV’s clinical course.126 HIV coinfection with HEV has been reported and transmission to the fetus and maternal mortality may be thereby increased.128 Although acute hepatitis B is less commonly icteric in the setting of untreated HIV infection, viral persistence and development of chronic infection is more common.127 Most studies indicate a diminished histological activity of chronic hepatitis B, although greater activity has also been reported. Reactivation of hepatitis B has also been reported with untreated HIV coinfection. Rarely, the fibrosing cholestatic variant of hepatitis B is seen, as it is in hepatitis C.129–131 As far as HDV is concerned, coinfection with HIV seems to worsen liver damage as evidenced by increased serum aminotransferase levels and worsened histological severity.132 Replication of both HBV by itself and together with HDV is increased and may be demonstrated by more diffuse staining for hepatitis B and δ antigens in tissue specimens.125 Coinfection of HCV and HIV is also common.127,133 As HIVinfected individuals are surviving longer with new antiviral therapies, the importance of HCV infection in these patients increases. Acute hepatitis C seems to be more often symptomatic and much more likely to result in liver failure when HIV is simultaneously acquired. Many studies indicate that untreated HIV with significant immunosuppression increases the severity and accelerates the course of chronic hepatitis C, though successful anti-HIV treatment accompanied by immune reconstitution seems to lead, generally, to a more typical HCV course. However, it has been reported that even with minimal or absent immunosuppression in such treated patients, fibrosis is still more prevalent and more rapid, though the mechanism is uncertain.134 Conversely, it has also been suggested that HCV is a poor prognostic indicator for HIV disease and patients

Chapter 7  Acute and chronic viral hepatitis

with coinfection, even with successful immune reconstitution, have increased mortality and morbidity due to viral interactions directly or to interactions between the large battery of medications required to treat both infections.127,133

Differential diagnosis of chronic hepatitis Many disease states can mimic chronic viral hepatitis and other, non-viral conditions can cause the same chronic hepatitic patterns of injury. To evaluate this fully in a biopsy or resection specimen, clinical history must be obtained. In all cases, if a diagnosis of chronic viral hepatitis is contemplated, serological tests for hepatitis B, C and D must be performed. In the absence of positive viral sero­logy, the following diseases must be considered.

Other causes of chronic hepatitis Infecting viruses are not the only aetiology of the typical histological features of chronic hepatitis. Autoimmune hepatitis is charac­ terized by autoantibodies such as antinuclear antibodies or anti-liver–kidney microsome (LKM) antibodies (see Chapter 9). Histologically, abundant plasma cells in the inflammatory infiltrate and severe activity, i.e. confluent necrosis (perivenular or bridging necrosis or parenchymal collapse), are hints that an autoimmune process rather than a viral infection is present.135 Serological studies demonstrating autoantibodies, in the absence of those confirming viral infection, should establish the diagnosis. However, one caveat is that in chronic hepatitis C and less commonly in chronic hepatitis B, serological studies may indicate the presence of circulating autoantibodies because chronic infection with HBV, HCV or HDV may lead to breakdown of self-tolerance and induction of autoantibodies.136 For example, chronic hepatitis B infection may stimulate the formation of circulating immune complexes of viral antigens and reactive antibody. These complexes may fix complement and induce tissue damage such as vasculitis, arthritis and glomerulonephritis.137 Chronic hepatitis C is also associated with autoimmune phenomena. HCV and less frequently HBV are associated with mixed cryoglobulinaemia.138,139 Metabolic diseases may also have chronic hepatitic patterns of injury. α1-Antitrypsin deficiency can have the same necroinflammatory lesions and pattern of scarring, but histological demonstration of α1-antitrypsin globules by H&E, PAS staining after diastase digestion, or immunohistochemistry will confirm the diagnosis. Typically, liver disease is seen in individuals with ZZ phenotype. Phenotypes such as SS or a heterozygous pattern are not predictably related to chronic liver disease. Care must be taken, however, to exclude concomitant viral infection serologically, as it appears that many chronic hepatitides in the setting of metabolic diseases are, in fact, due to a superimposed viral infection.140 Similarly, Wilson disease can cause the same pattern of injury, but histological confirmation, while suggested by abundant Mallory–Denk bodies, fatty change, or even some copper accumulation in periportal hepatocytes, can be achieved only in combination with clinical and biochemical findings.141 The diagnostic tests are complex but documentation of low levels of serum caeruloplasmin, increased urinary copper, or evidence of increased tissue stores of copper (by histochemical staining or, better, by quantitative assessment) are helpful. However, none of the test results is patho­ gnomonic for Wilson disease and may be abnormal in patients with other causes of abnormal copper retention such as primary biliary cirrhosis or primary sclerosing cholangitis. Wilson disease should be remembered as a confounding impostor and considered in the differential diagnosis of non-viral chronic hepatitis irrespective of age.

Some drugs can also cause chronic hepatitis, including: α-methyldopa, isoniazid, oxyphenisatin, nitrofurantoin and diclofenac (see Chapter 13). Some of these drugs may actually induce autoantibodies, suggesting induction of an autoimmune hepatitis. Again, the absence of serological markers of viral infection will help diagnostic accuracy and careful history-taking will often reveal the offending toxin.

Diseases that mimic chronic viral hepatitis Any disease leading to dense lymphoplasmacytic infiltrate in the portal tracts may mimic chronic viral hepatitis. For example, primary biliary cirrhosis (PBC) not only often has a dense portal infiltrate, but may also have foci of interface and lobular hepatitis. A diagnostic biopsy for this disease demonstrating granulomatous destruction of bile ducts is unlikely to cause this confusion. A later-stage lesion, with prominent ductular reaction and features of chronic cholestasis adjacent to fibrotic septa, is also unlikely to cause confusion. However, non-diagnostic early lesions of PBC may be difficult to distinguish from chronic viral hepatitis, particularly hepatitis C, as the latter may have marked bile duct damage and even loss.65 In these instances, clinical correlation is important, as PBC predominantly is characterized by marked elevation of alkaline phosphatase and γ-glutamyl transpeptidase, elevated serum IgM, and antimitochondrial (or other) autoantibodies. Aminotransferase values are generally less than five times the upper limit of normal, though they may fluctuate to higher levels. Similar confusion may be encountered, although less frequently, with a non-diagnostic biopsy specimen for primary sclerosing cholangitis. Again, clinical correlation is essential, evaluating possible chronic inflammatory bowel disease (usually ulcerative colitis) and characteristic bile duct abnormalities on imaging studies. Lymphoma or leukaemic infiltrates may also mimic the inflammatory infiltrate of chronic viral hepatitis, particularly when the infiltrate is most prominently portal with overflow into neighbouring sinusoids. In such cases, careful inspection of the lesion will reveal an absence of true interface hepatitis at the margins of portal tracts where the malignant cells extend past hepatocytes that are atrophic, but not truly apoptotic. Similarly, parenchymal hepatocytes may appear atrophic, but acidophil bodies are generally absent. The typical scarring of progressive chronic hepatitis is also not usually present in such specimens. Most important, of course, is that the infiltrating cells will have features suggesting a lymphoproliferative disorder, including monomorphism or marked atypia.

Semiquantitative scoring in chronic hepatitis Application of scoring systems The first scoring system specifically designed for the study of chronic hepatitis was that of Knodell and colleagues.142 Its modification by Ishak and colleagues in 1995 (Tables 7.5, 7.6)143 followed as did those of Batts and Ludwig144 (Table 7.7) and the METAVIR group (a cooperative group of French investigators) (Fig. 7.22).141 These remain the most commonly used and all have advantages and disadvantages.135,145 Scores, particularly those for staging, commonly form part of the evaluation of patients for possible antiviral therapy.146 There is then merit in using the same system for pre- and post-treatment evaluation. In general, the more complex schemes (e.g. Ishak et al.143) are likely to enable smaller therapeutic effects to be recognized in the context of studies of large populations, but such variations in following an individual with serial biopsies may just as likely be a result of sampling error; thus, for routine clinical use simpler

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Table 7.5  A complex grading system PMN=0

A. Periportal or periseptal interface hepatitis (piecemeal necrosis)          

Absent Mild (focal, few portal areas) Mild/moderate (focal, most portal areas) Moderate (continuous around ∼50% of tracts or septa) Severe (continuous around ∼50% of tracts or septa)

0 1 2 3 4

B. Confluent necrosis              

Absent Focal confluent necrosis Zone 3 necrosis in some areas Zone 3 necrosis in most areas Zone 3 necrosis + occasional portal–central (P–C) bridging Zone 3 necrosis + multiple P–C bridging Panacinar or multiacinar necrosis

0 1 2 3 4 5 6

C. Focal (spotty) lytic necrosis, apoptosis and focal inflammationa          

Absent 1 focus or less per 10× objective 2 to 4 foci per 10× objective 5 to 10 foci per 10× objective More than 10 foci per 10× objective

0 1 2 3 4

D. Portal inflammation          

None Mild, some or all portal areas Moderate, some or all portal areas Moderate/marked, all portal areas Marked, all portal areas

0 1 2 3 4

Note: other features such as bile duct damage should be noted but not scored. aDoes not include diffuse sinusoidal infiltration by inflammatory cells. After Ishak et al.143

Table 7.6  Example of a staging system After Ishak et al.143

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Architectural changes, fibrosis   and cirrhosis

Score

No fibrosis

0

Fibrous expansion of some portal areas, with or without short fibrous septa

1

Fibrous expansion of most portal areas, with or without short fibrous septa

2

Fibrous expansion of most portal areas with occasional portal to portal (P–P) bridging

3

Fibrous expansion of portal areas with marked bridging (portal to portal (P–P) as well as portal to central (P–C))

4

Marked bridging (P–P and/or P–C) with occasional nodules (incomplete cirrhosis)

5

Cirrhosis, probable or definite

6

PMN=1

LN=0

A=0

LN=1

A=1

LN=2

A=2

LN=0,1

A=1

LN=2

A=2

LN=0,1

A=2

PMN=2

LN=2

PMN=3

LN=0,1,2

A=3

Figure 7.22  Algorithm for the evaluation of histological activity: PMN, piecemeal necrosis; 0, none; 1, mild; 2, moderate; 3, severe; LN, lobular necrosis; 0, none or mild; 1, moderate; 2, severe; A, histological activity; 0, none; 1, mild; 2, moderate; 3, severe. (After Bedossa et al.147)

schemes are advised (e.g. modified Ishak et al.,143 Batts and Ludwig,144 METAVIR147). Whichever system is chosen for a particular purpose, the pathologist should ensure the diagnostic report must always include naming of the selected scheme since the same number may imply very different stages of disease depending on the schemes employed (Fig. 7.23).135 Alternatives to these semiquantitative approaches have been investigated. Morphometric analyse yield more precise quantifi­ cation, but have not as yet proven clinically useful.148,149 There is currently an active hunt for the ‘holy grail’ of non-invasive approaches to grading and, particularly, staging. However, none of these non-invasive approaches, using serological assessments or radiological/ultrasonographic measurements, have yet reached appropriate levels of sensitivity and specificity to make them clinically reliable for assessing all stages of liver fibrosis.150 Liver biopsy remains, therefore, a very important diagnostic tool, if not in fact the gold standard for the management of chronic viral hepatitis.151

Adequacy of biopsy sampling As summarized by Guido and Rugge,152 there are two basic issues which bear on the adequacy of tissue acquisition for grading and staging: (1) Can a small random sample adequately reflect the overall state of a diffuse liver disease, such as chronic hepatitis? (2) Can sample size affect the accuracy of histological assessments? The answer to both questions is a clear ‘yes’. If sufficient tissue is obtained, then the little needle biopsy core of tissue, estimated to be 1/50 000 of the total liver, can reflect the status of disease in the organ as a whole. However, what is ‘sufficient’ in routine hepatology and hepatopathology practice? Several studies suggest a clear answer: a total of 2.0 cm of liver tissue, containing 11–15 portal tracts, is necessary to avoid underscoring of grade and stage of disease. Less than that and there are likely to be significant inaccuracies in assessment.153–158 An additional sampling problem concerns small biopsy specimens obtained from the subcapsular region. For perhaps as much as 0.5 cm below the capsule, increased stroma, including septum formation and perhaps focal nodularity, may be within the spectrum of normal. Thus, such biopsy specimens may overestimate the stage of disease and such regions in a specimen should be excluded from evaluation of stage.152

Chapter 7  Acute and chronic viral hepatitis

Table 7.7  A simple scoring system

Portal/periportal activity

Score

Lobular activity

Score

None or minimal

0

None

0

Portal inflammation only

1

Inflammatory cells but no hepatocellular death

1

Mild interface hepatitis

2

Focal cell death

2

Moderate interface hepatitis

3

Severe focal cell death, +/++ confluent necrosis without bridging

3

Severe interface hepatitis

4

Damage includes bridging necrosis

4

Fibrosis

Score

None

0

Enlarged, fibrotic portal tracts

1

Periportal or portal–portal septa but intact architecture

2

Fibrosis with architectural distortion but no obvious cirrhosis

3

Probable or definite cirrhosis

4

Note: Separate scores are generated for portal/periportal and lobular activity. After Batts and Ludwig.144

Stages of fibrosis Modified Ishak ⇔ Batts-Ludwig 1

2

3

4

5

6

Ishak 1 focal

2 frequent

3 focal

4 frequent

2 focal

3 frequent

Metavir 1

Portal fibrosis

Fibrous septa

4

Transition to cirrhosis

Cirrhosis

Figure 7.23  Comparison of different schemes for staging disease progression in chronic viral hepatitis. Note that different numbers in the different stages may correspond to different histologic features; therefore, care should always be taken to report the staging scheme being used along with the numerical score itself.

It cannot be overemphasized, therefore, that the pathologist must assess samples of tissue provided either by hepatologists or radiologists for adequacy before supplying an assessment of grade (necroinflammatory activity) and stage (scarring) to the clinician and the patient. It behoves the pathologists to educate their

clinical colleagues to supply adequate samples, either by obtaining one long needle core or through repeat passes. It should particularly be made clear to radiologists that they should eschew the easier approach, into the left lobe, in order to avoid excessive subcapsular tissue in this often narrow segment; right lobe

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Table 7.8  Accuracy and reproducibility of scoring: helpful precautions • Two simultaneous observers if possible • Agreement on criteria before starting study • Only adequate biopsy specimens evaluated • Biopsies from same patient scored within a short period of time • Test of intra- and interobserver variation at end of study

sampling is necessary in conformity with historic clinico-pathologic standards.

Intra- and interobserver variation Histological evaluation of liver biopsies includes a subjective element and is therefore liable to both inter- and intraobserver variation; this must be taken into account when interpreting numbers generated by different pathologists or by the same pathologist at different times.145,159 Staging appears to be less subject to variation than grading.160,161 Moreover a simple scoring system proved more reliable than a complex one when intra- and interobserver variation were investigated.161 Variation was reduced in a study by the METAVIR group when biopsies were evaluated simultaneously by two observers. The precautions listed in Table 7.8 are recommended for the achievement of maximal reliability in scoring.

Handling data from semiquantitative analysis When semiquantitative scoring data are evaluated, it is important to bear in mind the nature of the generated numbers. The numbers represent categories rather than measurements, and cannot therefore be used as real numbers in statistical analyses.145,162–164 Even adding the numbers representing different grading components (e.g. interface hepatitis, confluent necrosis, lobular activity and portal inflammation) together to create a total grading score can lead to inaccuracies, because the scales for these components are non-linear, and differ from each other. At best, a total grading score serves only to give an approximate idea of the severity of a chronic hepatitis; it usually gives no information on the types of liver damage and inflammation involved. It follows that statistical evaluation must be appropriate for categories rather than measurements, and that correlations should be made with individual grading components rather than with grading totals.

Assessment of concomitant disease processes The relatively high incidence of viral hepatitis worldwide means that concomitant, though less common conditions may often be lurking behind the known clinical viral syndrome for which a patient undergoes liver biopsy. Moreover, the viruses themselves may be a cause some of these pathologies. The pathologist must therefore always be intent on not only diagnosing and scoring chronic viral hepatitis pathology, but keeping in mind the range of other pathologic findings that require assessment for the clinical benefit of the patient.135 As noted above, for example, the presence of any degree of confluent necrosis – the most severe form of necroinflammatory activity – may have clinical implications for understanding the evolution of chronic hepatitis in that patient, but may also indicate concomitant hepatitic conditions of non-viral origin. Other important concomitant conditions are summarized here.

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Fatty liver disease The increasing importance of metabolic syndrome and, hence, fatty liver disease worldwide, makes awareness of fatty change in chronic viral hepatitis biopsy specimens essential. The generally regarded cut-off for physiologic steatosis is approximately 5%.165 Above this in chronic hepatitis B (with or without HDV) should be considered an indication of fatty liver disease, alcoholic or non-alcoholic, and would necessitate a second diagnosis to be conveyed to the patient’s clinician. On the other hand, hepatitis C, particularly genotype 3, is well known to lead to steatotic change in hepatocytes.166 Distinguishing this steatosis from true fatty liver disease is often straightforward, since in hepatitis C-related fat, the fat is usually mild (