Osteoimmunology: Interactions of the Immune and Skeletal Systems
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Osteoimmunology: Interactions of the Immune and Skeletal Systems, Second Edition, explores the advancements that have been made in the field during the last 40 years, including valuable information on our understanding of the interactions between hematopoietic, immune, and bone cells, now known as the field of osteoimmunology.
This comprehensive work offers the most extensive summaries of research trends in the field and their translation into new therapeutics.
Early chapters deal with the development of osteoblasts, osteoclasts, hematopoietic stem cells, T and B-lymphocytes, and communications between these cellular elements, while later sections contain discussions of the signaling pathways by which RANKL influences osteoclast development and function. Subsequent chapters explore the effects that estrogen has on bone and the immune system, the development of pathologic conditions, and the growing research around osteoporosis, Paget’s disease, the genetics of bone disease, and bone cancer metastasis.
- Explains the intricate interaction between the immune system and bone
- Features detailed discussions of the key cellular and molecular mechanisms governing the homeostasis of the individual systems
- Facilitates greater understanding of osteoimmunologic networks, their environments, and how this understanding leads to better treatments for human diseases involving both systems
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Osteoimmunology - Joseph Lorenzo
Osteoimmunology
Interactions of the Immune and Skeletal Systems
Second edition
Edited by
Joseph Lorenzo MD
Department of Medicine and Orthopaedics, University of Connecticut Health Center, Farmington, CT, USA
Mark C. Horowitz PhD
Department of Orthopaedics and Rehabilitation, Yale School of Medicine, New Haven, CT, USA
Yongwon Choi PhD
Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Hiroshi Takayanagi PhD
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO) Program, Takayanagi Osteonetwork Project, Tokyo, Japan
Georg Schett MD
Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany
Table of Contents
Cover
Title page
Copyright
List of Contributors
Foreword
Preface
Chapter 1: Overview: The Developing Field of Osteoimmunology
Abstract
Chapter 2: The Origins of the Osteoclast
Abstract
First descriptions of the osteoclast
Early controversies: are osteoclasts capable of bone resorption?
Early controversies: hematopoietic or mesenchymal origin of the osteoclast?
Osteoclast: a hematopoietic cell
Osteoclasts: cells of the myeloid lineage
Advancing the field: culturing osteoclasts in vitro
Identification of RANKL and OPG
Defining osteoclast precursors within myeloid cell development
Heterogeneity among osteoclasts
Origins of the osteoclast through the lens of evolution
Conclusions
Acknowledgments
Chapter 3: Trafficking of Osteoclast Precursors
Abstract
Introduction
A century-long search for the identity of osteoclast precursors
Intravital two-photon imaging of bone tissues
Osteoclast precursors are motile and circulate throughout the body
Guidance cues sensed by osteoclast precursors in bone marrow
S1P-dependent migratory control of osteoclast precursors
Differences between osteoclast precursor and mature osteoclast migration mechanisms
Control of osteoclast migration and function by Rho GTPases
Role of integrins in osteoclast precursor migration
Control of osteoclast precursor differentiation by GPCR-mediated inhibition of cell migration
Unanswered questions in osteoclast precursor trafficking and differentiation
Chapter 4: Osteoclast Biology: Regulation of Formation and Function
Abstract
Introduction
RANKL and RANK: an osteoclastogenic cytokine and its receptor
TRAF6: the multifunctional signaling molecule activated by RANK
What happens downstream of TRAF6?
The role of NF-κB in osteoclast differentiation
The critical role of AP-1 transcription factors
MAPKs activated by RANKL
NFATc1 is a master transcription factor of osteoclast differentiation
Autoamplification of NFATc1 and its epigenetic regulation
Inhibition of NFATc1 induction
Transcriptional control governed by NFATc1
Costimulatory receptor signals for RANK: FcRγ and DAP12
The ligands for the costimulatory receptors
Importance of ITAM costimulatory signals in humans: Nasu-Hakola disease
Additional costimulatory signals involved in osteoclastogenesis
Receptors signaling through DAP12
The inhibitory signals for costimulatory signals
Src family kinases: activation of ITAM signaling
Syk kinase: downstream of DAP12/FcRγ?
PLCγ2: enzyme and adaptor molecule
Tec kinases: integrating RANK and ITAM signaling
Negative regulatory role of DAP12
M-CSF and c-Fms: a road to proliferation and survival
M-CSF signaling
Erk, PI3K, and c-Fos signaling
The osteoclast’s job: bone resorption
Osteoclast cytoskeleton: the podosomes and the sealing zone
Osteoclast cytoskeleton: the microtubules and the sealing zone
Osteoclast functional structure: the ruffled border
Osteoclast and bone matrix: role of αvβ3 integrin
Integrin-associated proteins
M-CSF and the osteoclast cytoskeleton
Coupling factors released in osteoclastic bone resorption
Stimulation of bone formation by clastokines
Inhibition of bone formation by clastokines
Coupling by cell–cell interaction between osteoclasts and osteoblasts
Conclusions
Chapter 5: Osteoimmunology and the Osteoblast
Abstract
Advantages of immune-osteoblast interaction
Immune-osteoblast interaction in fracture repair
Dual role for TNF-α
TNF is an inhibitor of Wnt signaling
Coupling of skeletal homeostasis with innate and acquired immunity
Osteoblast support of hematopoiesis
Osteoblast support of B cell differentiation
Osteoblasts support hematopoietic stem cells
Conclusions
Chapter 6: The Variety of Osteocyte Function
Abstract
Introduction
The osteocyte network
New tools to study osteocyte function
Osteocytes and bone remodeling
Osteocytes and mineral homeostasis
Osteocytes as mechanosensors
Osteocytes and hematopoiesis
Conclusions
Chapter 7: Bone Marrow Hematopoietic Niches
Abstract
Introduction
Hematopoiesis occurs within the bone marrow and is closely linked to skeletal development
A role for the osteoblast lineage in supporting hematopoietic stem cells
A perivascular niche for HSCs involves mesenchymal progenitors
Signaling pathways implicated in microenvironment-HSC communication
Perivascular osteoblast precursors support hematopoiesis
Other components of the hematopoietic niche
Clinical implications
The bone marrow HSC microenvironment is complex
Chapter 8: RANK and RANKL of Bones, T Cells, and the Mammary Glands
Abstract
RANK and RANKL in bone
Downstream signaling of RANK/RANKL
Rank/Rankl mutations in human patients
Osteoimmunology
RANK and RANKL in the organogenesis of the immune system
Immunotolerance
RANK/RANKL and metastases
RANK/RANKL and the mammary gland
RANK and RANKL and their function in mammary stem cell biology
Breast cancer
Is there even more?
Denosumab, a rational treatment for bone loss
Conclusions
Chapter 9: The Effects of Immune Cell Products (Cytokines and Hematopoietic Cell Growth Factors) on Bone Cells
Abstract
Receptor activator of nuclear factor-κB ligand (RANKL), receptor activator of nuclear factor-κB (RANK) and osteoprotegerin (OPG)
Colony-stimulating factor-1
Additional colony stimulating factors
Interleukin-1
Tumor necrosis factor
Additional TNF superfamily members
Interleukin-6
Additional interleukin-6 family members
Interleukin-7
Interleukin-8 and other chemokines
Interleukin-10
Interleukin 12
Interleukin 15
Interleukin 17, Interleukin 23, and Interleukin 27
Interleukin 18 and interleukin 33
Interferons
Additional cytokine
Conclusions
Chapter 10: Coupling: The Influences of Immune and Bone Cells
Abstract
Introduction: bone remodeling and the concept of coupling
Modeling and remodeling in anabolic therapy for the skeleton
Osteoclast-derived factors that promote osteoblast differentiation
What is the target cell of osteoclast-derived factors that may promote bone formation?
How do osteocytes contribute to coupling?
Promotion of bone formation in the BMU during the reversal phase
The influences of T and B lymphocytes on the coupling process
Signals between the bone surface and the vasculature
Isolation of the remodeling site by the bone remodeling canopy
Conclusions
Chapter 11: The Role of the Immune System in the Development of Osteoporosis and Fracture Risk
Abstract
Introduction
Connections between bone and the immune system
Bone remodeling
Periarticular bone structure and bone loss in inflammatory arthritis
Bone involvement in rheumatic diseases
From fracture risk evaluation to fracture prevention: a 5-step plan
Differential diagnosis
Fracture prevention in inflammatory joint diseases
Follow up
Conclusions
Key messages
Chapter 12: The Role of Sex Steroids in the Effects of Immune System on Bone
Abstract
Introduction
Estrogen and other sex steroids
Interactions of sex steroids and immune cells
Effects of sex steroid-modulated immune cells on bone cells
Conclusions
Chapter 13: The Role of the Immune System in the Local and Systemic Bone Loss of Inflammatory Arthritis
Abstract
Introduction
Bone disease associated with RA
Bone changes in spondyloarthritis and psoriatic arthritis
Conclusions
Acknowledgments
Chapter 14: Osteoarthritis and the Immune System
Abstract
Introduction
Physiological structural organization of periarticular bone
Periarticular bone changes in osteoarthritis
Regulatory mechanisms involved in OA bone pathology
Bone marrow lesions and targeted bone remodeling
Calcified cartilage, bone, and articular cartilage interactions in OA
Osteophytes
The role of synovium in OA cartilage and bone pathology
Conclusions
Chapter 15: Inflammatory Bowel Disease and Bone
Abstract
Introduction
Pathophysiology of IBD and osteo-immune connections
IBD and osteoimmunology
Conclusions
Chapter 16: The Role of the Immune System and Bone Cells in Acute and Chronic Osteomyelitis
Abstract
Introduction
Mechanism of microbial infection in the pathogenesis in osteomyelitis
Bacterial persistence in chronic osteomyelitis
The host response to osteomyelitis
Osteoblasts and their multiple roles in bone infections
Cellular responses to acute and chronic osteomyelitis
Osteoclast mobilization
Osteoclasts as immune cells
DCs and osteoclasts in infection
The role of B-cells in bone infection and the potential of passive immunization
Chapter 17: The Role of the Immune System in Fracture Healing
Abstract
Bone repair as a postnatal regenerative process
Fracture healing cascade
Role of mesenchymal stem cells in the modulation of immune function
Cytokines involved in fracture healing
RANK, RANKL, and OPG
Phase-specific roles of cytokines in fracture healing
Role of nonsteroidal anti-inflammatory drugs in fracture healing
Biological effects of COX-2 inhibition
Clinical effects of COX inhibitors on fracture healing
Chapter 18: The Role of the Immune System in the Effects of Cancer on Bone
Abstract
Introduction
The vicious cycle of bone metastasis
Bone as the preferred site for metastasis
Role of mesenchymal stromal cells in bone metastasis
T lymphocytes and bone metastasis
T regulatory cells
Role of macrophages and macrophage-derived cells in bone metastasis
Tumor associated macrophages
Myeloid derived suppressor cells (MDSC)
Dendritic cells (DC)
B Cells
Conclusions
Chapter 19: Osteoimmunology in the Oral Cavity (Periodontal Disease, Lesions of Endodontic Origin, and Orthodontic Tooth Movement)
Abstract
Introduction
Periodontal diseases
Lesions of endodontic origin
Orthodontic tooth movement
Conclusions
Acknowledgment
Chapter 20: Marrow Adipose Tissue and its Interactions with the Skeletal, Hematopoietic, and Immune Systems
Abstract
Introduction
Adipose development and expansion
Measurement of marrow adipose tissue
The cellular origin of bone marrow adipocytes
Molecular regulation of BM adipogenesis
Cellular interactions between adipocytes, bone, hematopoietic and immune cells
Conclusions
Acknowledgments
Subject Index
Copyright
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List of Contributors
Antonios O. Aliprantis MD, PhD, Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Sheila N. Bello-Irizarry PhD, Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA
Ryan Berry PhD, Department of Orthopaedics and Rehabilitation, Yale School of Medicine, New Haven, CT, USA
Karine Briot MD, PhD, Department of Rheumatology, INSERM U1153, Paris Descartes University, Cochin Hospital, Paris, France
Julia F. Charles MD, PhD, Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Yongwon Choi PhD, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
John L. Daiss PhD, Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA
Jean-Michel Dayer MD, Faculty of Medicine, Centre Medical Universitaire, Geneva, Switzerland
Karen L. de Mesy Bentley BS, MS
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester
Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
Thomas A. Einhorn MD, Department of Orthopaedic Surgery, Boston University School of Medicine, Boston, MA, USA
Roberta Faccio PhD, Department of Orthopedics, Washington University in St. Louis School of Medicine, Saint Louis, MO, USA
Jackie A. Fretz PhD, Comparative Medicine and Molecular, Cellular and Developmental Biology, Yale School of Medicine, New Haven, CT, USA
Gustavo P. Garlet DDS, MS, PhD, Department of Biological Sciences, School of Dentistry of Bauru, University of Sao Paulo, Bauru, Brazil
Louis C. Gerstenfeld PhD, Department of Orthopaedic Surgery, Boston University School of Medicine, Boston, MA, USA
Piet Geusens MD, PhD, Department of Internal Medicine, Subdivision of Rheumatology, Maastricht University Medical Center, Maastricht, The Netherlands
Mary B. Goldring PhD, The Hospital for Special Surgery and Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY, USA
Steven R. Goldring MD, The Hospital for Special Surgery and Department of Medicine, Weill Cornell Medical College, New York, NY, USA
Ellen M. Gravallese MD, Department of Medicine, Division of Rheumatology, University of Massachusetts Medical School, Worcester, MA, USA
Dana T. Graves DDS, DMSc, Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA
Danka Grčević MD, PhD, Department of Physiology and Immunology, University of Zagreb School of Medicine, Zagreb, Croatia
Mark C. Horowitz PhD, Department of Orthopaedics and Rehabilitation, Yale School of Medicine, New Haven, CT, USA
Masaru Ishii MD, PhD
Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Osaka
CREST, Japan Science and Technology Agency, Tokyo, Japan
Rayyan A. Kayal BDS, DSc, Department of Periodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia
Junichi Kikuta MD, PhD
Department of Immunology and Cell Biology, Graduate School of Medicine, Osaka University, Osaka
CREST, Japan Science and Technology Agency, Tokyo, Japan
Anne Klibansky MD, Department of Medicine, Neuroendocrinology, Massachusetts General Hospital, Boston, MA, USA
Natasa Kovačić MD, PhD, Department of Anatomy, University of Zagreb School of Medicine, Zagreb, Croatia
Henry M. Kronenberg MD, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
Sun-Kyeong Lee PhD, Department of Medicine, UCONN Center on Aging, University of Connecticut Health Center, Farmington, CT, USA
Joseph Lorenzo MD, Department of Medicine and Orthopaedics, University of Connecticut Health Center, Farmington, CT, USA
Ormond MacDougald PhD, Departments of Molecular and Integrative Physiology and Internal Medicine (Metabolism, Endocrinology & Diabetes Division), University of Michigan School of Medicine, Ann Arbor, MI, USA
T. John Martin MD, DSc
St. Vincent’s Institute of Medical Research, Fitzroy
Department of Medicine, St. Vincent’s Hospital, Melbourne, The University of Melbourne, Fitzroy, Victoria, Australia
Mary C. Nakamura MD
Department of Medicine, Division of Rheumatology, University of California, San Francisco
Arthritis/Immunology Section, Veterans Administration Medical Center, San Francisco, CA, USA
Mark S. Nanes MD, PhD
Veterans Affairs Medical Center and Division of Endocrinology, Metabolism, and Lipids
Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
Erin Nevius PhD, Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
Kohei Nishitani MD, PhD, Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA
Charles A. O’Brien PhD, Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences; the Central Arkansas Veterans Healthcare System, Little Rock, AR, USA
Thomas Oates DMD, PhD, Department of Periodontics, University of Texas Health Science Center, San Antonio, TX, USA
Josef Martin Penninger MD, Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
João P. Pereira PhD, Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
Julian M.W. Quinn PhD, The Garvan Institute, Darlinghurst, New South Wales, Australia
Matthew S. Rodeheffer PhD, Comparative Medicine and Molecular, Cellular and Developmental Biology, Yale School of Medicine, New Haven, CT, USA
Garson David Roodman MD, PhD, Division of Hematology Oncology, Indiana University School of Medicine, Indianapolis, IN, USA
Clifford J. Rosen MD, The Center for Clinical and Translational Research, Maine Medical Center Research Institute, Scarborough, ME, USA
Christian Roux MD, PhD, Department of Rheumatology, INSERM U1153, Paris Descartes University, Cochin Hospital, Paris, France
Georg Schett MD, Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany
Edward M. Schwarz PhD
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester
Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester
Department of Orthopaedics and Rehabilitation, University of Rochester Medical Center, Rochester, NY, USA
Verena Sigl MA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
Rebecca Silbermann MD, Division of Hematology Oncology, Indiana University School of Medicine, Indianapolis, IN, USA
Natalie A. Sims PhD
St. Vincent’s Institute of Medical Research, Fitzroy
Department of Medicine, St. Vincent’s Hospital, Melbourne, The University of Melbourne, Fitzroy, Victoria, Australia
Brandon M. Steen MD, Department of Orthopaedic Surgery, Boston University School of Medicine, Boston, MA, USA
Francisco A. Sylvester MD, Department of Pediatrics, University of North Carolina at Chapel Hill, NC, USA
Hiroshi Takayanagi PhD
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO) Program, Takayanagi Osteonetwork Project, Tokyo, Japan
Steven L. Teitelbaum MD, Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, Saint Louis, MO, USA
Anthony T. Vella PhD, Department of Immunology, University of Connecticut Health Center, Farmington, CT, USA
Joy Y. Wu MD, PhD, Division of Endocrinology, Stanford University School of Medicine, Stanford, CA, USA
Foreword
"The way that can be spoken of
Is not the constant way;
The name that can be named
Is not the constant name.
The nameless was the beginning of heaven and earth;
The named was the mother of the myriad creatures."
From Tao Te Ching
by Lao Tzu
Of course, many researchers had noticed the intimate relationship between the skeletal system and the immune system before the birth of the field of osteoimmunology
. For example, osteoclast-activating factor, which turned out to be interleukin (IL)-1β, is generated by immune cells, and stimulates pathological bone resorption by osteoclasts in tumor-induced bone destruction. Meanwhile, rheumatoid arthritis (RA) is an autoimmune inflammatory disorder that results in massive bone and joint destruction with disease progression, and various proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-6, produced by immune cells are critically implicated in its pathogenesis. However, it was not until the name osteoimmunology
was coined by Arron and Choi in 2000 that the currents of the research in these two fields merged to become a large stream. Osteoimmunology became a great platform for bone biologists and immunologists to get together and collaborate. In other words, the named was the mother of the myriad creatures
.
The finding of RANKL was undoubtedly an important driving force in the field of osteoimmunology. RANKL was originally identified as a membrane-bound cytokine within the TNF-α family that is produced by activated T cells and prolongs the survival of dendritic cells through the activation of NF-κB pathways. However, subsequent studies demonstrated that RANKL is an essential cytokine for osteoclast differentiation together with macrophage colony-stimulating factor, which is produced by osteoblasts in response to calciotropic hormones. Moreover, RANKL was found to be involved in the pathological bone resorption observed in osteoporosis, RA, cancer metastasis, and so on (see Chapters 13 and 18). Several signal transduction pathways turned out to regulate both RANKL-induced osteoclast differentiation pathways and immunological reactions. In addition to its critical roles in bone biology and immunology, RANKL has been found to play many other unexpected roles in various types of cells and tissues (see Chapter 9).
After the finding of RANKL as an initial bridge between bone biology and immunology, several further bridges have been built between these two fields that have markedly contributed to recent advances in clinical medicine. However, it is important to point out that the flow of osteoimmunology is not simply one-way from basic science to clinical medicine, and that important progress in osteoimmunology has been brought about by clinical medicine. For example, biological agents including anti-TNF-α and anti-IL-6 receptor antibodies have dramatically changed the therapeutic strategies for RA and other inflammatory disorders, and in turn clinical information using these agents has provided great insights into the pathogenesis of human diseases, and greatly enhanced our knowledge of human immunology. The catch ball
between basic research and clinical practice has been the driving force of osteoimmunology.
This 2nd edition of Osteoimmunology is composed of 20 chapters, which beautifully summarize the current state of the art in osteoimmunology. I believe that this edition will provide a comprehensive overview of the recent advances in the field of osteoimmunology, and foster further research efforts leading to better understanding of the mystery of biological systems, as well as better treatment of patients.
The ancient Chinese philosopher Lao Tzu departed to the west after composing the 81 verses of the Tao Te Ching. Where will osteoimmunology lead us from here?
Sakae Tanaka MD, PhD
Professor and Director
Department of Orthopaedic Surgery
Faculty of Medicine
The University of Tokyo, Tokyo, Japan
President of The Japanese Society for Bone and Mineral Research
Preface
The editors welcome readers to the second edition of this book on the topic of osteoimmunology. The importance of the interactions of bone and immune cells was first appreciated a little over 40 years ago. In addition, the term osteoimmunology was first used in an editorial in Nature by Arron and Choi just 15 years ago. Hence, as disciplines go, osteoimmunology is relatively young. However, over its short existence, it has seen great progress. Perhaps its most important discovery was the identification of RANKL as the master regulator of osteoclasts. This TNF superfamily member was first isolated and cloned because of its ability to regulate the interactions between T-lymphocytes and antigen presenting dendritic cells. However, after its original description, it was soon found to be the critical signal for bone resorption. In addition to RANKL, a large number of cytokines and immune cells are now known to influence bone cell function and bone mass. This has become important for understanding how bone loss develops in diseases like inflammatory arthritis, inflammatory bowel disease, periodontal disease and after organ or bone marrow transplant. Conversely, bone is now known to provide important signals to the hematopoietic and immune system. Hematopoietic stem cells colonize the marrow and initiate the production of all blood and immune cells. It is well appreciated that these cells exist in the bone marrow because they receive critical signals from bone. In addition, the bone marrow is the site where memory immune cells reside. These T and B-lymphocytes are central for the development of immunity from repeat infection. It is highly likely that bone cell-derived signals also regulate the various niches
that support these important functions. This book is designed to bring the reader a broad overview of the latest knowledge about these interactions in a wide variety of areas. It is hoped that both experienced investigators and those just learning about this field will find the information useful as a reference for their own studies in this area. The editors and the authors have tried to be as comprehensive as possible and to provide a detailed list of references in this area where the reader can find additional information about this topic.
Chapter 1
Overview: The Developing Field of Osteoimmunology
Joseph Lorenzo MD*
Yongwon Choi PhD**
Mark C. Horowitz PhD†
Hiroshi Takayanagi PhD‡
Georg Schett MD§
* Department of Medicine and Orthopaedics, University of Connecticut Health Center, Farmington, CT, USA
** Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
† Department of Orthopaedics and Rehabilitation, Yale School of Medicine, New Haven, CT, USA
‡ Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan; Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO) Program, Takayanagi Osteonetwork Project, Tokyo, Japan
§ Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany
Abstract
It has been over 40 years since the initial observations that cells of the immune system could influence the functions of bone cells. Since that time, significant strides have been made in our understanding of the interactions amonghematopoietic, immune, and bone cells, which is now known as the field of osteoimmunology.
In this introductory chapter, we will briefly establish some of the key features of osteoimmunology, which are described in greater detail in the subsequent chapters of this book and provide an overview of the changes in the first and second editions.
Keywords
bone
immune cells
osteoblasts
osteoclasts
It has been over 40 years since the initial observations that cells of the immune system influence the functions of bone cells. Since that time, significant strides have been made in our understanding of the interactions among hematopoietic, immune and bone cells, which is now known as the field of osteoimmunology.
This introductory chapter will establish briefly some of the key features of osteoimmunology, which are described in greater detail in the subsequent chapters of this book.
Bone cells originate from two lineages: osteoclasts and osteoblasts. Osteoclasts, which are responsible for bone resorption, are large, multinucleated cells that are uniquely capable of removing both the organic and mineral components of bone. Osteoclasts share a common origin with cells of the myeloid dendritic cell and macrophage lineages, and because of this respond to and produce many of the cytokines that regulate macrophage and dendritic cell function. The discovery of a tumor necrosis factor (TNF) family member, receptor activator of NFB ligand (RANKL), on activated T cells and its subsequent identification as one of the key differentiation and survival factors for osteoclasts, provided critical evidence for a potential link between normal immune responses and bone metabolism.
Bone is formed by osteoblasts that originate from mesenchymal stem cells (MSC). Osteoblast-lineage cells carry out at least three major functions: (1) they secrete bone matrix that mineralizes over time to form new bone; (2) they regulate osteoclast differentiation; (3) they support hematopoietic cell growth and differentiation. In addition some osteoblasts differentiate into osteocytes that are a specialized bone cell that senses mechanical force on bone and influences a variety of functions including osteoclast activity through the production of RANKL. It is now well accepted that MSC can differentiate into a variety of lineages including osteoblasts, adipocytes, muscle cells, and hematopoiesis-supporting stromal cells.¹ Osteoblast-lineage cells, which are sometimes referred to as stromal cells, produce a variety of cytokines that are critical for hematopoietic cell differentiation.
The first observation that immune cells could influence the activity of bone cells came from the finding that supernatants from phytohemagglutinin-stimulated peripheral blood monocytes of normal humans contained factors that stimulated bone resorption.² This activity was named osteoclast-activating factor (OAF). When it was eventually purified and sequenced, the principal stimulator of bone resorption in these crude OAF preparations was identified as the cytokine interleukin-1 (IL-1).³ In addition to its ability to stimulate osteoclast formation and resorbing activity, IL-1 is a mediator of a variety of inflammatory responses and a potent stimulus of prostaglandin synthesis, which independently increases bone resorption. It also is an inhibitor of osteoblast activity and bone formation.
Subsequent to the identification of IL-1 as a bone resorption stimulus, TNF⁴ and interleukin-6 (IL-6)⁵ were also found to potently regulate bone cell function. Like IL-1, these cytokines are critical mediators of inflammatory responses. It has now been demonstrated that a long list of cytokines can have both positive and negative effects on bone mass and bone cell activity.
Production of cytokines by immune cells has been linked to human diseases that involve bone. Perhaps the most extensive studies have been of the role of cytokines in the development of the bone loss and lytic lesions that occur in inflammatory arthritis, inflammatory bowel disease, and periodontal disease.⁶–⁸ Here, production of RANKL from a variety of cell types mediates osteolysis by stimulating osteoclastic activity. In addition, production of proinflammatory cytokines such as IL-1, TNF, and IL-6 enhances the response of osteoclasts to RANKL.
Estrogen withdrawal after menopause is also associated with a rapid and sustained increase in the rate of bone loss. This phenomenon seems to result from an increase in bone resorption that is not met by an equivalent increase in bone formation. It was initially demonstrated that conditioned medium from cultured peripheral blood monocytes from osteoporotic women with rapid bone turnover contained more IL-1 activity than did conditioned medium from the cells of women with lesser amounts of bone turnover or normal controls.⁹ In rodents, treatment with inhibitors of IL-1 and TNF abrogated the bone loss that occurred with ovariectomy. In addition, ovariectomy was not associated with bone loss in mice that were genetically prevented from responding to IL-1¹⁰ and TNF¹¹ or unable to produce IL-6.¹²,¹³ These findings strongly link the bone loss of estrogen withdrawal to the effects of estrogen on the production and/or activity of proinflammatory cytokines. In addition, it was shown that inhibitors of IL-1 and TNF reduced the rate of bone resorption in postmenopausal women.¹⁴
The role of cytokines in the bone disease that occurs with malignancy has also been studied extensively.¹⁵ In hematological malignancies such as lymphomas or multiple myeloma, which are associated with increased osteoclast formation and activity, a variety of cytokines have been implicated as mediating the bone loss that can occur in these conditions. Unlike the bone disease of solid tumors, which is typically mediated by parathyroid hormone–related protein (PTHrP), hematological malignancies are often characterized by an uncoupling of resorption from formation and development of purely lytic bone lesions.
The immune system is also involved in normal fracture healing and the response of bone to infections (osteomyelitis). Understanding the interactions of bone and immune cells during these events is best accomplished by an osteoimmunologic approach that integrates an appreciation of the crosstalk between these two organ systems.¹⁶,¹⁷
The question of whether the immune system influences normal skeletal development and function is not well answered. Ontogenically, skeletal development precedes early immune-system development. Therefore, it is unlikely that the immune system influences early skeletal formation. However, bone homeostasis and remodeling occur throughout life. Anatomically, bone-marrow spaces are loosely compartmentalized, which allows immune and bone cells to interact and influence each other. Hence, bone homeostasis is often regulated by immune responses, particularly when the immune system has been activated or becomes pathologic.
It is not difficult to imagine that crosstalk occurs throughout life between activated lymphocytes and bone cells because all mammals are constantly challenged by a variety of infectious agents that produce some level of sustained low-grade immune system activation. Furthermore, as we age, there is an accumulation in the bone marrow of memory T cells, which can express RANKL on their surface.¹⁸,¹⁹ The role that these cells play in skeletal homeostasis is unknown. However, it is conceivable that they might influence bone turnover and be responsible for some of the changes that occur in the skeleton with aging.
Immunologists and hematologists are well aware that, at least in adult mammals, the development of the immune system depends on the normal function of hematopoietic stem cells (HSCs) that are now known to reside in close association with bone cells. It is not surprising to learn that the development of the immune system in the bone marrow is dependent on the production of a facilitative microenvironment by bone cells. This fact was made clearer by data demonstrating that osteoblast-lineage cells provide key factors that regulate HSC development. There is also accumulating evidence that bone continues to play a role in adaptive immunity, beyond its influence on lymphocyte development. It is now known that long-lived memory T and B cells return to specialized niches in the bone marrow. These cells are capable of circulating throughout the organism. However, the questions of why they remain in specific areas of bone marrow and what factors draw them there remain unanswered. It is likely that the answers to these questions will come from experiments that are designed in the context of osteoimmunology by investigators who have knowledge of both the immune system and bone.
In this second edition of this book we have expanded the number of chapters to 20 and added chapters on new topics in osteoimmunology. These are: Chapter 3, Trafficking of Osteoclast Precursors, Chapter 6, Osteocyte Biology, Chapter 8, The Functions of RANKL Beyond the Osteoclast, Chapter 12, The Role of Sex Steroids in the Interactions of the Immune System with Bone, Chapter 14, Osteoarthritis and the Immune System, and Chapter 20, The role of the Immune System in the Interactions of Fat and Bone. The remaining chapters are on topics covered in the first edition of the book. However, many are the works of new authors who have brought different perspectives to their topics. Chapters on topics from the first edition that have new authors are: Chapter 2. The Origins of the Osteoclast, Chapter 5, Osteoblasts and the Immune System, Chapter 11, The Role of the Immune System in the Development of Osteoporosis and Fracture Risk, and Chapter 18, The Role of the Immune System in the Effects of Cancer on Bone.
The remaining chapters, which retain authors from the first edition, have been extensively revised to reflect the latest published data. The topics of the chapters in this book span the breadth and depth of our current knowledge of osteoimmunology. In this volume, the contributions are organized according to their scientific messages, though these connections are not absolute.
After reading this book, one will hopefully appreciate the intricate interaction between the immune system and bone. However, despite the progress that has already been made toward understanding the cross-regulation between bone and the immune system, the biological implications of such interactions are only beginning to be identified. The fields of immunology and bone biology have matured sufficiently so that key cellular and molecular mechanisms governing the homeostasis of the individual systems are extensively described. Hence, progress toward understanding osteoimmunologic networks will likely be greatly facilitated by creating an environment conducive to its study. It is hoped that this endeavor will lead to better treatments for human diseases involving both systems.
Many of the pathologic processes of the skeletal and immune systems are major targets for therapeutic intervention. However, the search for novel treatments for these conditions is often pursued in the absence of a solid scientific understanding of the molecular and cellular pathways that underlie these processes. According to the US Surgeon General Report on Bone Health and Osteoporosis, by 2020 one in two Americans over the age of 50 will be at risk for fractures from osteoporosis or low bone mass. These health concerns become more prominent as people live longer and expect to remain active as they age. Future interventions to prevent and treat bone diseases will require a high degree of specificity, especially because these therapies are often tailored for a segment of the population that is already suffering from or vulnerable to other age-related ailments. These issues place osteoimmunology in a position of unique clinical significance and make its study highly relevant.
References
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3. Dewhirst FE, Stashenko PP, Mole JE, et al. Purification and partial sequence of human osteoclast-activating factor: Identity with interleukin 1 beta. J Immunol. 1985;135:2562–2568.
4. Bertolini DR, Nedwin GE, Bringman TS, et al. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature. 1986;319:516–518.
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7. Sylvester FA. IBD and skeletal health: children are not small adults! Inflamm Bowel Dis. 2005;11(11):1020–1023.
8. Taubman MA, Valverde P, Han X, et al. Immune response: the key to bone resorption in periodontal disease. J Periodontol. 2005;76(11s):2033–2041.
9. Pacifici R, et al. Spontaneous release of interleukin 1 from human blood monocytes reflects bone formation in idiopathic osteoporosis. Proc Natl Acad Sci USA. 1987;84:4616–4620.
10. Lorenzo JA, et al. Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy. Endocrinology. 1998;139(6):3022–3025.
11. Ammann P, et al. Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency. J Clin Invest. 1997;99(7):1699–1703.
12. Jilka RL, et al. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science. 1992;257:88–91.
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14. Charatcharoenwitthaya N, Khosla S, Atkinson EJ, et al. Effect of blockade of TNF-alpha and interleukin-1 action on bone resorption in early postmenopausal women. J Bone Miner Res. 2007;22(5):724–729.
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17. Marriott I. Osteoblast responses to bacterial pathogens: a previously unappreciated role for bone-forming cells in host defense and disease progression. Immunol Res. 2004;30(3):291–308.
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Chapter 2
The Origins of the Osteoclast
Antonios O. Aliprantis MD, PhD*
Julia F. Charles MD, PhD*
Mary C. Nakamura MD**
* Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
** Department of Medicine, Division of Rheumatology, University of California, San Francisco; Arthritis/Immunology Section, Veterans Administration Medical Center, San Francisco, CA, USA
Abstract
Bone-resorbing osteoclasts are key mediators of skeletal health and disease. Physiologic osteoclast activity facilitates bone growth and repairs skeletal microdamage. Excessive osteoclastic bone resorption results in diseases, including osteoporosis, arthritis, and bone cancer. Reduced osteoclast activity is also pathologic, highlighted by the severe phenotype of patients with osteopetrosis due to mutations that compromise osteoclast formation or resorption. In this chapter, the origin of the osteoclast
is considered from multiple viewpoints. First, we discuss the publications that led to the identification of the osteoclast as a bone-resorbing cell and how the controversy regarding the cellular origin of the osteoclast was resolved. Further, we explore how key molecules important for osteoclast differentiation were discovered and our current understanding of OCP. Finally, the biology of the osteoclast is considered from an evolutionary perspective. Overall, this chapter provides a broad understanding of the historical, cellular, molecular, and evolutionary origins of osteoclast.
Keywords
osteoclast
OCP
RANKL
osteopetrosis
hematopoietic differentiation
First descriptions of the osteoclast
The cellular origin of the osteoclast from the myeloid lineage and the critical ability of this cell to resorb bony matrix are taken for granted. All modern chapters and review articles on osteoclasts are crafted under the auspice of these facts. However, the story of the discovery of the osteoclast is fraught with controversy and intrigue spanning three centuries. The original identification of a multinucleated cell in close anatomic proximity to bone has been ascribed to C.H. Robin in 1849.¹,² He named these cells myeloplaques
and remarked that their function was unknown. Subsequently, Rollet in 1870, identified cells with many nuclei during a microscopic dissection of endochondral ossification.³ Three years later, Rindfleisch found multinucleated cells in erosion pits in bone termed Howship’s lacunae, which were described originally in 1817.⁴,⁵ None of these investigators linked the presence of these cells to the process of bone resorption. In 1872, Kolliker predicted that these myeloplaque cells would be the primary driving force for bone resorption and he coined the term "Ostoklast.⁶ For the next century, progress in the understanding of bone resorption was, as stated by N.M. Hancox in a review article on osteoclasts in 1949, dictated by
opinions (and polemics) based upon the subjective interpretation of histologic appearances."⁷ In the following two sections, we will describe the controversy regarding whether osteoclasts were capable of bone resorption, and the debate that raged for decades on the cellular origin of the osteoclast.
It should be highlighted that much of the discussion of the early work on osteoclasts presented here relies upon a wonderfully clear and comprehensive review article written by N.M. Hancox in 1949.⁷ Dr Hancox summarized over a century of publications written in multiple languages and gave an unbiased interpretation of many controversial and conflicting reports. Therefore, the 1949 Hancock review is used to frame early controversies in the osteoclast field that were ultimately resolved over the subsequent 40 years.
Early controversies: are osteoclasts capable of bone resorption?
As surprising as it may seem today, there was much debate, even until the 1970s, as to whether osteoclasts were capable of bone resorption.⁷ The primary reason for this debate was the fact that investigators were limited to the interpretation of static histologic images to illuminate a dynamic process. Early evidence in favor of the viewpoint that osteoclasts resorb bone came from three sets of observations.⁷ First, it was the striking association of osteoclasts with Howship’s lacunae. Second, it was the localization of large numbers of osteoclasts to areas where copious bone erosion was known to take place, such as in developing bones and above erupting teeth. The third piece of evidence came from models where bone was inserted into transparent rabbit ears.⁸ Serial imaging showed fragmentation of bone and closely associated osteoclasts, though transfer of matrix from the former to the latter was not demonstrated. Conversely, some very prominent investigators, including Von Reklinghausen, known for his descriptions of neurofibromatosis and hemochromatosis, held an opposing viewpoint. In his interpretation, resorption began with the death of bone cells, followed by softening of the ground substance (the organic component of bone), called thrypsis,
and, finally, the leaching out of bone salts. Osteoclasts were then viewed as fusion products of these dying bone cells and termed thryptic giant cells.
⁹ In weighing these viewpoints, Hancox made the astute point that because of the ephemeral
nature of osteoclasts, whose lifespan is limited to a few days,
when these cells are absent from a resorption site it is possible that they might have been present a few days previously.
⁷
A series of in vivo and in vitro studies published in the late 1950s and early 1960s left little doubt as to the ability of osteoclasts to resorb bone. In 1957, Arnold and Jee gave rats an intravenous injection of radioactive plutonium.¹⁰ Within hours, the plutonium was incorporated rapidly onto bone surfaces. Subsequently, plutonium was detected within osteoclasts, with both the number of positive cells and the intensity of the radiolabel increasing between 1 day and 4 days after injection. Interestingly, they observed a rough correlation between the size and number of nuclei in an osteoclast, and its localization to areas of intense versus slow bone resorption. Those that doubted osteoclasts resorb bone cited the lack of demonstrable bone crystal in osteoclasts. Improvements in electron microscopy (EM) technology, including the ability to generate ultra-thin sections of undecalcified bone, facilitated the studies that addressed this argument. Using EM, Scott and Pease were the first to show the presence of bone salt crystals in cytoplasmic vacuoles of osteoclasts at the growth plate of young cats (Fig. 2.1A).¹¹ Their study also beautifully demonstrated the ultrastructure of the osteoclast ruffled border,
which serves to increase the effective contact surface area between the cell and the bone surface (Fig. 2.1B,C). Moreover, they found that the bone approximating the osteoclast ruffled border assumed a frayed appearance (Fig. 2.1C), which together with the identification of bone crystals within cytoplasmic vacuoles, was strong evidence that osteoclasts resorb calcified bone matrix. Two other studies confirmed these EM observations in human, rat, and avian bone samples.¹⁴–¹⁶
Figure 2.1 Early EM imaging of the osteoclast sheds light on function.
(A) High magnification EM image demonstrating crystals of mineral (arrows) within osteoclast vacuoles. (B) High power magnification EM image of the ruffled border of an osteoclast. (C) Low power EM image of an osteoclast in contact with the bone surface. Note the ruffled border (rb) and frayed appearance of the bone, call the osseous fringe (of). (D) Resorption pit with osteoclast isolated from a neonatal rabbit and cultured on dentin. (E) Resorption trail formed by and osteoclast after longer culture as in (D). (F) Schematic representation of the osteoclast-mediated release of inorganic (⁴⁵Ca) prior to organic (³H-proline) components from labeled bone fragments in the experiment of Blair et al.¹² This experiment led to the concept that mineralized matrix components are resorbed prior to degradation of the organic matrix. Reproduced with permission from Scott and Pease (A–C) and Jones et al. (D, E).¹¹–¹³
The final piece of evidence came from in vitro studies that measured directly the resorption of physiologically relevant calcified matrices by osteoclasts. Luben et al. isolated two populations of cells from mouse calvaria, one of which had a higher proportion of multinuclear cells.¹⁷ They termed these CT cells and seeded them on devitalized calvaria isolated from pups labeled in utero with ⁴⁵Ca and ³H-proline. The CT cells released ⁴⁵Ca and ³H-proline from the calvaria, indicating they both dissolve bone mineral and hydrolyze bone protein. Interestingly, this activity could be blocked by calcitonin, a hormone known to inhibit bone resorption in vivo. Burger et al. reported a novel culture system in 1982, in which murine embryonic metatarsal rudiments were cocultured for 7 days with either fetal liver or bone marrow cells as a source of osteoclast precursor (OCP). Osteoclasts formed within the rudiments and were shown by light and EM to resorb the matrix. Moreover, when rudiments were isolated from pregnant dams treated with ⁴⁵Ca, the osteoclasts liberated the radioactive ion.¹⁸ This study also shed light on the identity of the OCP, as neither mature macrophages nor irradiated bone marrow cells generated osteoclasts in this system. Ali and coworkers were among the first to culture osteoclasts directly on calcified tissue.¹³,¹⁹ Using scanning EM, they demonstrated clear resorption tracks when either avian or mammalian osteoclasts, isolated by mechanical disruption of long bones, were cultured on dentin (Fig. 2.1D,E).¹³ Subsequently, Blair et al. cultured avian osteoclasts on bones isolated from rats labeled with ³H-proline or ⁴⁵Ca. Interestingly, mobilization of ⁴⁵Ca reached a maximal rate after 2 h, whereas liberation of ³H-proline took 12–24 h to reach a peak rate, suggesting that optimal proteolysis required mineral dissolution (Fig. 2.1F).¹² They also showed that osteoclast collagen proteases were only active at acidic pH. Taken together, these data were the foundation of our modern understanding of osteoclastic bone resorption whereby mineral mobilization by lacunar acidification precedes, and is necessary for, cleavage of the organic components of bone by osteoclast proteases that are optimally active at low pH. Since these early studies, seeding osteoclasts isolated either directly from bones, or generated by coculture with osteoblasts or with recombinant cytokines, onto calcified tissue to test their bone-resorbing activity has become a standard laboratory assay to study this remarkable cell.
In conclusion, decades of research, fueled by advances in microscopy and cell culture techniques, culminated in the firm conclusion that osteoclasts are not mere bystanders in the bone resorption process, but rather the demolition team.
Early controversies: hematopoietic or mesenchymal origin of the osteoclast?
Similar to the debate regarding whether osteoclasts resorb bone, the cellular origin of the osteoclast was also a long-standing controversy. This issue was difficult to resolve until modern cell transfer and chimera techniques were developed. At the time, Hancox wrote his comprehensive review on osteoclasts in 1949⁷ that a consensus on the origin of the osteoclast had not been reached, and it would be over 20 years until the issue was resolved.
Using the Hancox review as a starting point for this discussion, it was generally agreed upon in 1949 that the multinuclearity
of osteoclasts derived from cell fusion, rather than incomplete mitosis where cell fission fails to occur after nuclear replication.⁷ However, the identity of the precursor that underwent cell fusion was not known. Three major viewpoints were held among bone researchers working in the late nineteenth and first half of the twentieth century.⁷ The first viewpoint suggested that osteoclasts arose from the fusion of sessile connective tissue cells such as osteoblasts, fibroblasts, or other mesenchymal cells. In this case, the word sessile
refers to the relatively fixed in place
nature of these cells to contrast them with wandering
or circulating phagocytes (macrophages). Kolliker held this opinion. The second hypothesis suggested that osteoclasts represent a syncytial mass
of chondrocytes and osteocytes liberated from calcified matrix as bone is dissolved. This hypothesis was an extension of Von Recklinghausen’s concept of thrypsis
discussed earlier. The fact that osteoclasts could be found around implanted devitalized bone made this concept difficult to accept and hence it had few supporters. The last viewpoint held that osteoclasts develop from mononuclear wandering cells
(i.e., monocytes/macrophages) of the hematopoietic lineage. A number of attributes shared by osteoclasts and phagocytes supported this idea, which ultimately proved correct. These shared attributes include robust motility, an undulating membrane and similar staining patterns with vital dyes. Moreover, it was well established that other multinucleated giant cells formed by phagocyte fusion.⁷
Despite strong evidence for the last viewpoint by 1949, the issue remained controversial. In the early 1960s, a series of papers debated the cellular origin of the OCP. Tonna used cytologic evidence and ³H-thymidine to label dividing cells and concluded that osteoclasts arose from the fusion of osteoblasts.²⁰–²² This conclusion was reached largely based on the appearance of aggregates of osteoblasts in close proximity to formed osteoclasts, and the fact that ³H-thymidine was not observed in the nuclei of osteoclasts until seen in osteoblasts. At the same time, Schmidt published an image showing an osteoclast phagocytosing an osteocyte as it resorbed bone.²³ This process, he suggested, may contribute to osteoclast multinuclearity. In retrospect, these studies were handcuffed by attempting to study a dynamic process with static images and a lack of cell lineage markers that could be used to correctly identify mesenchymal or hematopoietic cell types.
The close proximity of osteoblasts and osteoclasts observed by Tonna was likely a histologic representation of the coupling
of bone resorption to bone formation, a theory conceptualized at the time in an important study by Hattner et al., published in Nature in 1965.²⁴ Examining trabecular bone, they found that over 95% of cement lines, which distinguish the interface between old and more recently deposited bone, had a scalloped appearance, indicative of a previous osteoclast mediated resorption event. The implication from this finding was that bone resorption by osteoclasts occurred before bone formation by osteoblasts, which made the hypothesis that osteoblasts give rise to osteoclasts less likely. In this context, Rasmussen and Bordier proposed a new view on the origin of osteoclasts and osteoblasts.²⁵ Working under the assumption that both cells were mesenchymal derived, they suggested that at bone sites destined for remodeling, a pool of mesenchymal cells becomes activated and proliferates into preosteoclasts, that fuse to give rise to bone resorbing osteoclasts. After a resorptive phase of one to a few weeks, the multinuclear osteoclast undergoes fission to generate mononuclear osteoblasts, which synthesize new bone over the course of months. Although Rasmussen and Bordier were ultimately wrong about the origin of the osteoclast and its ability to separate into osteoblasts, their overall vision of the bone remodeling unit,
a termed coined in their 1973 publication, was insightful.²⁵ We continue to use the term bone remodeling unit
to describe the coordinated sequence of rapid activation of bone resorption by osteoclasts followed by a slower osteoblastic bone formation phase.
Other investigators working at the same time as Tonna, Rasmussen and Bordier, but using different experimental systems, reached the conclusion that osteoclasts derive from hematopoietic derived mononuclear phagocytes. In 1962, Fischman and Hay took advantage of the ability of newts to regenerate limbs after amputation. They reported that osteoclasts appear 10–20 days after limb amputation and serve to resorb the distal bone stump before proliferating mesenchymal cells regenerate the limb. When newts were injected with ³H-thymidine after amputation, the label was never found in osteoclasts, despite intense labeling in mesenchymal cells. In contrast, when ³H-thymidine was injected before limb amputation, at a time when mesenchymal cells are quiescent but hematopoietic cells are proliferating at their basal rate, the label was incorporated into many of the osteoclasts that appeared 10–20 days later. The following year, Jee and Nolan reported corroborating results using another experimental approach. They injected the femoral artery of rabbits with bone charcoal. The charcoal was lodged in downstream arterioles and capillaries serving two purposes. First, phagocytes rapidly took up the charcoal, which acted to label these cells. Second, arterial blockade resulted in partial limb ischemia and an increase in osteoclastic bone resorption. The investigators reported that charcoal was seen initially within days in phagocytic macrophages. A progressive increase in charcoal laden osteoclasts was observed 12–30 days later. Charcoal was never seen in osteoblasts. These publications provided evidence that mononuclear phagocytes of hematopoietic origin are the precursors of osteoclasts.
Osteoclast: a hematopoietic cell
The definitive proof that osteoclasts derive from hematopoietic precursors came in the early to mid 1970s, in large part due to the efforts of Dr Donald Walker working on mouse models of osteopetrosis. Osteopetrosis, also called marble bone disease, is a congenital condition characterized by extremely dense but brittle bones.²⁶,²⁷ The clinical presentation is variable, depending on the particular genetic defect, and is most severe in its recessive form.²⁷ In the late 1960s, there was a consensus that osteopetrosis arose from a defect in bone resorption.²⁸ However, the mechanism underlying this defect was controversial. Explanations ranged from perturbations in the parathyroid hormone pathway, to increased secretion of calcitonin from thyroid parafollicular cells,²⁹ to abnormalities in cartilage matrix rendering it resistant to resorption ³⁰ and, finally, to intrinsic defects in osteoclasts.³¹ To resolve these possibilities, Dr Walker performed a series of experiments with profound implications illustrated in Figure 2.2. He joined the circulation of osteopetrotic grey-lethal (gl/gl) or microphthalmic (mi/mi) mice to their wild-type littermates by parabiosis.³² When parabiosis was established 10 days after birth, excess skeletal matrix was resorbed in the mutant by 6 weeks of age (Fig. 2.2A). This result indicated that osteopetrosis is neither driven by intrinsic defects in bone matrix nor elevated levels of a serum factor that inhibits osteoclast activity. Dr Walker concluded from this study that a humoral substance from the healthy parabiont was the curative factor.
Figure 2.2 Parabiosis and bone marrow chimera experiments prove the hematopoietic origin of the osteoclast.
(A) Parabiosis of osteopetrotic (gl/gl) mice with wildtype (WT) littermates led to resolution of osteopetrosis, demonstrating that a transferable factor determined the bone phenotype. (B) A brief period of parabiosis also results in eventual resolution of osteopetrosis, suggesting that the transferable factor is likely a long-lived cell. (C) Radiation chimeras prove the hematopoietic origin of the osteoclast. Transfer of gl/gl splenocytes into irradiated WT hosts leads to osteopetrosis. In contrast, transfer of WT splenocytes into irradiated gl/gl hosts results in resolution of osteopetrosis. Similar results were obtained for each of these experiments when mi/mi mice were used instead of gl/gl mice. Based on Refs 32–36.
In a follow up study, Walker limited the duration of parabiosis to 2 weeks, a time point at which little, if any, excess matrix was resorbed from the osteopetrotic parabiont ³³ (Fig. 2.2B). Surprisingly, during the subsequent months of observation, and in the absence of continued parabiosis, histologic signs of osteopetrosis vanished. This meant that a humoral factor from the normal littermate was highly unlikely to be driving the reversal of osteopetrosis. Rather, Walker concluded that only cells could have survived long enough after the severing of parabiosis to resolve the osteopetrosis.³³ To prove this hypothesis, Dr Walker first showed that osteopetrosis in gl/gl and mi/mi mice could be cured by lethal irradiation followed by rescue with wild-type bone marrow or spleen cells (Fig. 2.2C).³⁴,³⁵ Conversely, he demonstrated that wild-type mice transplanted with spleen cells from either gl/gl or mi/mi mice developed osteopetrosis (Fig. 2.2C).³⁵,³⁶ Taken together, these experiments proved that osteopetrosis was due to a defect in a hematopoietic precursor that rendered bone resorption dysfunctional. Two side notes should be appreciated in the context of Dr Walker’s studies. First, understanding of the cellular nature of osteopetrosis resulted in the first successful bone marrow transplantation of an infant with malignant osteopetrosis,³⁷ a therapy that has become standard of care.³⁸ Second, Dr Walker performed all of the experiments himself, and his five seminal articles ³²–³⁶ were all single author reports.
Walker’s parabiosis experiments indicated that osteoclasts were derived from circulating cells in the blood stream. This was also the conclusion of studies using quail-chick embryonic chimeras performed concurrently with Dr Walker’s work.³⁹ Implantation of a quail limb into chick chorioallantoic membrane led to deposition of osteoclasts identified to be of host (chick) origin, suggesting that precursors were derived from the circulation.³⁹ Ash et al. took Walker’s experiments one-step further.⁴⁰ They transplanted lethally irradiated mi/mi mice with bone marrow cells from beige (bg/bg) mice, which are not osteopetrotic but contain giant lysosomes in their myeloid cells. As expected, bg/bg bone marrow rescued the osteopetrosis in mi/mi mice. Most importantly, the osteoclasts that developed were marked by giant lysosomes, strongly suggesting the myeloid origin of osteoclasts.
Osteoclasts: cells of the myeloid lineage
Although the myeloid origin of osteoclasts was indisputable by the early 1980s, the relationship of osteoclasts to other myeloid lineages was yet to be worked out. The phenotypic similarity of osteoclasts to myeloid derived phagocytic and multinucleated giant cells led to early proposals that monocytes were the circulating hematopoietic precursor cell for osteoclasts.⁴¹,⁴² Osteoclasts and macrophages shared phenotypic features such as high numbers of lysosomes and the ability to adhere to glass in the presence of trypsin.⁴³ However, they were also noted to have distinct differences in their cell surface marker expression,⁴⁴ response to calcitonin,⁴⁵,⁴⁶ and ability to resorb bone.⁴⁶ Some doubt was cast on the myeloid origin of osteoclasts when macrophage infusion failed to rescue bone resorption in osteopetrotic rats and mice.⁴⁷ However, by the early 1980s, significant advances in studies of macrophage biology had led to recognition of tissue macrophage heterogeneity and the role of monocyte influx in macrophage differentiation.⁴⁸ Tinkler tested the early hypothesis that multinucleated osteoclasts arise from monocytes with an elegant in vivo transfer experiment. ³H-thymidine labeled peripheral blood monocytes were infused into hosts treated with 1,25(OH)2D3 and demonstrated to form labeled osteoclasts.⁴⁹ The formation of labeled osteoclasts from labeled mononuclear precursors also suggested that multinucleated osteoclasts are formed by fusion rather than division without cell separation.⁴⁹ Zambonin-Zallone et al. used time lapse microscopy to confirm, by direct visualization, that peripheral blood monocytes fuse into osteoclasts in vitro.⁵⁰
The identification of the causative genetic mutations in mice with severe osteoclast-poor osteopetrosis provided further verification of the myeloid origin of osteoclasts. Osteopetrotic (op/op) mice have very few macrophages, monocytes, and osteoclasts, a phenotype that mapped to a nonsense mutation in the gene encoding macrophage colony stimulating factor-1 (M-CSF or CSF1).⁵¹–⁵³ Moreover, all aspects of the phenotype were reversed by exogenous administration of M-CSF.⁵⁴ Similarly, mice deficient for the M-CSF receptor show severe osteopetrosis.⁵⁵ Tondravi et al. subsequently demonstrated the absence of osteoclasts and osteopetrosis in mice lacking PU.1, the ETS domain transcription factor essential for initial monocyte/macrophage differentiation.⁵⁶ Thus, these studies showed that the genes required for early myeloid and monocyte development were required for normal osteoclast formation in vivo.
Initial attempts to differentiate osteoclasts from myeloid cells in vitro required organ cultures. Ko and Bernard demonstrated osteoclast formation in vitro from mononuclear bone marrow cells cocultured with osteoclast-free fetal-mouse calvaria.⁵⁷ Similarly, Burger et al. differentiated osteoclasts from cultures of proliferating mononuclear phagocytes incubated with embryonic mouse long-bone primordia. Neither blood monocytes nor peritoneal macrophages, developed into osteoclasts in this system. From these studies it was concluded that monoblasts and promonocytes¹⁸ were the most likely OCP cells. Other laboratories helped confirm the myeloid origin of osteoclast progenitors. Kukita and Roodman generated a monoclonal antibody to osteoclasts that also bound mononuclear precursors in the bone marrow.⁵⁸ Fujika et al. differentiated osteoclasts in vitro from circulating human monocytes confirming that the cellular origin of the osteoclast in humans is similar to previously studied species.⁵⁹ Quinn et al. generated functional osteoclasts in vitro from murine mononuclear phagocytes.⁶⁰
Taken together, most of the data supported the model that osteoclasts derive from monocyte/macrophage precursors. However, it was extremely difficult to study osteoclast development in vitro using organ cultures on bone. The discovery of