oulu 2019 d 1532 university of oulu p.o. box 8000 fi-90014
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UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND
A C T A U N I V E R S I T A T I S O U L U E N S I S
University Lecturer Tuomo Glumoff
University Lecturer Santeri Palviainen
Senior research fellow Jari Juuti
Professor Olli Vuolteenaho
University Lecturer Veli-Matti Ulvinen
Planning Director Pertti Tikkanen
Professor Jari Juga
University Lecturer Anu Soikkeli
Professor Olli Vuolteenaho
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-2363-6 (Paperback)ISBN 978-952-62-2364-3 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)
U N I V E R S I TAT I S O U L U E N S I S
MEDICA
ACTAD
D 1532
AC
TAJani Luukkonen
OULU 2019
D 1532
Jani Luukkonen
OSTEOPONTIN AND OSTEOCLASTSIN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU
ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 5 3 2
JANI LUUKKONEN
OSTEOPONTIN AND OSTEOCLASTS IN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defence inthe Leena Palotie auditorium (101A) of the Faculty ofMedicine (Aapistie 5 A), on 9 November 2019, at 12noon
UNIVERSITY OF OULU, OULU 2019
Copyright © 2019Acta Univ. Oul. D 1532, 2019
Supervised byProfessor Petri LehenkariProfessor Juha TuukkanenDoctor Sanna Turunen
Reviewed byProfessor Mikko LammiDocent Anna-Marja Säämänen
ISBN 978-952-62-2363-6 (Paperback)ISBN 978-952-62-2364-3 (PDF)
ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2019
OpponentProfessor Timothy Arnett
Luukkonen, Jani, Osteopontin and osteoclasts in rheumatoid arthritis andosteoarthritis. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center OuluActa Univ. Oul. D 1532, 2019University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Rheumatoid arthritis and osteoarthritis are two chronic joint diseases, which cause two of thelargest socioeconomical burdens among all joint diseases according to the World HealthOrganization. Both diseases are associated with changes in bone structure and bone cell, especiallyosteoclast, function. The etiology or pathogenesis of these diseases are not completely understood.Traditionally, osteoarthritis is seen as a disease resulting from mechanical wear of cartilage andbone, and rheumatoid arthritis as an autoinflammatory disease of synovial tissue. However, alsoin osteoarthritis chronic inflammation is present in synovial tissue, and in rheumatoid arthritislarge changes in bone structure are seen. The field of study focusing on this connection betweeninflammation and bone is called osteoimmunology and it can explain many features of thesechronic diseases linking joint health to disturbances in bone homeostasis.
Here, the study focused on the function of osteoclasts in normal and pathologicalenvironments, and on the factors that have an effect on bone resorption, with a special emphasison the protein osteopontin. Samples of synovial fluid and serum from rheumatoid arthritis andosteoarthritis patients were analyzed for factors affecting osteoclasts, and in vitro cell cultures ofhuman derived osteoclasts were used to analyze osteoclast function in normal and pathologicalenvironment.
The phosphorylation of osteopontin was found to be increased in rheumatoid arthritis, alongwith multiple other inflammatory factors that also affect osteoclasts, such as IL-6, IL-8 and VEGF.Osteoclast cell cultures showed how the use of different patient samples significantly affectedosteoclastogenesis, due to so-called inflammatory osteoclastogenesis. Additionally, we show thatosteoclasts deposit osteopontin into the resorption lacunae during bone resorption.
Based on the results, the inflammatory component present in both osteoarthritis andrheumatoid arthritis significantly affects osteoclast function, and its further study in the future mayreveal new therapeutic possibilities. Especially the new discoveries of osteopontin’s role in normalosteoclast function and its changes seen between osteoarthritis and rheumatoid arthritis may proveto have therapeutic potential.
Keywords: bone resorption, osteoarthritis, osteoclasts, osteoimmunology, osteopontin,rheumatoid arthritis, tartrate-resistant acid phosphatase
Luukkonen, Jani, Osteopontiini ja osteoklastit nivelreumassa ja nivelrikossa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter OuluActa Univ. Oul. D 1532, 2019Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Nivelreuma ja nivelrikko ovat kroonisia nivelsairauksia, jotka Maailman terveysjärjestön(WHO) mukaan aiheuttavat eniten sosioekonomista haittaa. Molemmissa sairauksissa luidenrakenteessa ja luusolujen, erityisesti osteoklastien, toiminnassa tapahtuu muutoksia. Kumman-kaan taudin etiologiaa tai patogeneesiä ei täysin tunneta. Perinteisesti ajatellaan, että nivelrikkojohtuu rusto- ja luukudoksen mekaanisesta kulumisesta ja nivelreuma nivelkalvon autoinflam-matoorisesta tulehduksesta. Kuitenkin nivelrikossa nähdään myös selkeä nivelkalvon krooninentulehdus ja nivelreumassa suuria luun rakenteen muutoksia. Tutkimusala, joka tutkii tulehduk-sen ja luun yhteyttä, on nimeltään osteoimmunologia.
Tässä väitöskirjassa tutkitaan osteoklastien toimintaa ja niihin vaikuttavia tekijöitä, erityises-ti proteiini osteopontiinia, normaalissa ja tautiympäristössä. Analysoin osteoklasteihin vaikutta-via tekijöitä nivelrikko- ja nivelreumapotilaiden näytteistä sekä osteoklastien toimintaa soluvil-jelmissä. Soluviljelmissä käytettiin nivelreuma- ja nivelrikkopotilaiden näytteitä mahdollisim-man totuudenmukaisen ympäristön luomiseksi osteoklasteille.
Tutkimuksessa osoitettiin, kuinka osteopontiinin fosforylaatio on lisääntynyt nivelreumapoti-laiden nivelnesteessä. Myös useiden muiden osteoklasteihin vaikuttavien tekijöiden, kuten IL-6:n, IL-8:n ja VEGF:n, havaittiin lisääntyneen nivelreumassa. Osteoklastien soluviljelmissähavaittiin selkeät erot siinä, miten eri potilasnäytteet vaikuttavat osteoklasteihin ja erityisestitulehduksen aiheuttamaan osteoklastien syntyyn. Osoitan myös, miten osteoklastit erittävätosteopontiinia luunhajotuskuoppaan luun hajotuksen aikana.
Tutkimustulosten mukaan krooninen tulehdustila nivelrikossa ja nivelreumassa vaikuttaahuomattavasti osteoklastien toimintaan. Uskon, että lisätutkimukset tällä saralla voivat paljastaauusia hoidollisia mahdollisuuksia. Erityisesti uudet löydökset osteopontiinin roolista osteoklas-tien toiminnassa sekä muutoksista nivelrikossa ja nivelreumassa vaativat jatkotutkimuksia, jottaproteiinin kliininen merkittävyys saadaan selvitettyä.
Asiasanat: luun hajotus, nivelreuma, nivelrikko, osteoimmunologia, osteoklasti,osteopontiini, tartraatti-resistantti hapan fosfataasi
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Acknowledgements
This study was carried out at the Department of Anatomy and Cell Biology, Cancer
and Translational Research Unit, Medical Research Center Oulu, University of
Oulu. This study was financially supported by the Emil Aaltonen Foundation and
Oulu Duodecim Society. I owe my deepest gratitude to all the people who have
taught, helped and supported me during these years, and to all the rheumatoid
arthritis and osteoarthritis patients who have given their samples to be studied.
I am deeply grateful to my supervisors Professor Petri Lehenkari, Professor
Juha Tuukkanen and Doctor Sanna Turunen for guidance. I thank Petri and Juha
for allowing me the opportunity to join their research groups as a young medical
student. Petri’s enthusiastic attitude towards medicine and all science in general is
truly remarkable, and he has shown how to combine the careers of an orthopedic
surgeon and a researcher. Juha’s calm supportive positive attitude has been uplifting
at many times of need. Sanna’s enthusiasm towards cells and small details is
astonishing, and without her guidance this thesis would not have reached its best
form.
I also owe thanks to my research colleagues Doctor Elina Kylmäoja and Doctor
Antti Koskela, whose footsteps I have followed on this thesis path. Thanks also to
the rest of Anatomy department’s staff, with special thanks to our laboratory
technicians Miia Vierimaa and Tarja Huhta, whose hands-on guidance and help has
been crucial.
I thank all collaborators and co-authors who took part in this study, with special
thanks to Professor Göran Andersson, and follow-up group Professor Juhana
Leppilahti and Doctor Anna Karjalainen. Professor Mikko Lammi and Docent
Anna-Marja Säämänen are thanked for the careful review of this thesis and the help
to finalize it, and Professor Deborah Kaska for reviewing the English language.
Helka-Maaria Lieslehto is thanked for reviewing the Finnish language.
I am truly honored that Professor Timothy Arnett has accepted the role of
opponent.
My warmest gratitude goes to all my friends from all parts of life. Without you
life would be boring. Thank you for the merry times and adventures in school, work
and leisure activities. May the adventures on and off the mountains never end.
Thanks to my classmates and PhD colleagues Johannes, Joni and Tuomas for
discussions and instructions, your success with your own theses has been a great
motivation.
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Finally, I want to thank my family. Johanna and Kalle, I thank you for the love
and support, and raising me to believe that hard work eventually pays off. You laid
the base for everything. Dear Tuija, thank you for believing in me.
12.8.2019 Jani Luukkonen
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Abbreviations
ACPAs anti-citrullinated protein antibodies
BMP Bone morphogenetic protein
BMU Basic Multicellular Unit
BSP Bone sialoprotein
CD Cluster of differentiation
CRP C-reactive protein
CTX C-terminal telopeptide
DAMPs damage-associated molecular patterns
DMP1 Dentin matrix protein 1
DSPP Dentin sialophosphoprotein
ELISA Enzyme-linked immunosorbent assay
FE-SEM Field emission scanning electron microscopy
FGF Fibroblast growth factor
G-CSF Granulocyte colony stimulating factor
Gm-CSF Granulocyte-macrophage colony stimulating factor
HSC Hematopoietic stem cell
IFN Interferon
IL Interleukin
IL-1ra Interleukin-1 receptor antagonist
IP-10 Interferon gamma-induced protein 10
MCP-1 Monocyte chemoattractant protein 1
M-CSF Macrophage colony-stimulating factor
M-CSFR Macrophage colony-stimulating factor receptor
MEPE matrix extracellular phosphoglycoprotein
MIP-1-α Macrophage inflammatory protein 1 alpha
MIP-1-β Macrophage inflammatory protein 1 beta
MMP Matrix metalloproteinase
NCP Non-collagenous protein
OA Osteoarthritis
OPG Osteoprotegerin
OPN Osteopontin
PDGF-BB platelet-derived growth factor
PTH Parathyroid hormone
QuOP Cell cycle arrested quiescent osteoclast precursor
RA Rheumatoid arthritis
10
RANK Nuclear factor kappa-Β
RANKL Nuclear factor kappa-Β ligand
RF rheumatoid factor
Runx2 Runt-related transcription factor
SIBLING Small Integrin-Binding Ligand, N-linked Glycoprotein
Th T helper cell
TNF-α Tumor necrosis factor-alpha
TRAcP Tartrate-resistant acid phosphatase
VEGF Vascular endothelial growth factor
WHO World Health Organization
Wnt Wingless and integration
αvβ3 Alpha-v beta-3 integrin
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List of original publications
This thesis is based on the following publications, which are referred to throughout
the text by their Roman numerals:
I Luukkonen, J., Pascual, L. M., Patlaka, C., Lång, P., Turunen, S., Halleen, J., Nousiainen, T., Valkealahti, M., Tuukkanen, J., Andersson, G., Lehenkari, P. (2017). Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio. Plos One, 12(8). doi:10.1371/journal.pone.0182904.
II Luukkonen, J., Hilli, M., Nakamura, M., Ritamo, I., Valmu, L., Kauppinen, K., Tuukkanen J., Lehenkari, P. (2019). Osteoclasts secrete osteopontin into resorption lacunae during bone resorption. Histochemistry and Cell Biology. doi:10.1007/s00418-019-01770-y.
III Luukkonen J., Huhtakangas J., Turunen S., Tuukkanen J., Vuolteenaho O., Lehenkari P. (2019). Bone resorption and osteoclastogenesis are stimulated by synovial fluid from osteoarthritis and rheumatoid arthritis patients in vitro. Manuscript.
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Contents
Abstract
Tiivistelmä
Acknowledgements 7
Abbreviations 9
List of original publications 11
Contents 13
1 Introduction 15
2 Review of the literature 17
2.1 Bone ........................................................................................................ 17
2.1.1 Bone matrix .................................................................................. 19
2.1.2 Bone cells ..................................................................................... 21
2.1.3 Osteoclasts and bone resorption ................................................... 24
2.1.4 Bone remodeling .......................................................................... 30
2.2 Joint ......................................................................................................... 32
2.3 Osteoimmunology ................................................................................... 33
2.4 Osteopontin (OPN) ................................................................................. 35
2.5 Tartrate-resistant acid phosphatase (TRAcP) .......................................... 38
2.6 Rheumatoid arthritis (RA) ...................................................................... 41
2.6.1 Osteoimmunology in RA .............................................................. 45
2.7 Osteoarthritis (OA) ................................................................................. 47
2.7.1 Osteoimmunology in OA ............................................................. 50
3 Aims of the study 53
4 Materials and methods 55
5 Results 57
5.1 Analyses of OPN and TRAcP in RA synovial fluid and tissue (I) .......... 57
5.1.1 Phosphorylation of OPN in synovial fluid (I) .............................. 57
5.1.2 TRAcP 5A and 5B concentrations in synovial fluid (I) ................ 58
5.1.3 Localization of OPN and TRAcP in RA and OA patient
synovial tissue (I) ......................................................................... 58
5.2 Analyses of OPN in resorption lacunae (II) ............................................ 60
5.2.1 Detection of OPN in resorption lacunae using FE-SEM
(II) ................................................................................................ 60
5.2.2 Analysis of proteins enriched in the resorbed bone slices
(II) ................................................................................................ 61
5.2.3 Analysis of glycan epitope in the resorption lacunae (II) ............. 61
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5.3 Immunohistology of OPN and TRAcP in the areas of increased
bone metabolism in OA (II) .................................................................... 62
5.4 Analyses of the effect of RA serum and synovial fluid on
osteoclastogenesis and bone resorption (III) ........................................... 64
5.4.1 Effect of novel untreated RA patient serum on
osteoclastogenesis and bone resorption ........................................ 65
5.4.2 Effect of RA and OA patients’ synovial fluid on
osteoclastogenesis and bone resorption ........................................ 66
5.4.3 Cytokine analyses of the patient samples ..................................... 66
6 Discussion 69
6.1 Decreased TRAcP 5B/5A ratio leads to increased
phosphorylation of OPN in RA synovial fluid. ....................................... 69
6.2 OPN is one of the key proteins secreted into the resorption
lacunae during bone resorption ............................................................... 71
6.3 OPN is found in the calcified cartilage ...................................................... 74
6.4 RA synovial fluid and serum increase osteoclastogenesis and
bone resorption in vitro ........................................................................... 74
6.5 Future aspects .......................................................................................... 78
7 Conclusions 81
List of references 83
Original publications 101
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1 Introduction
Bones give humans and other vertebrates their distinct appearance and form. Other
tissues and organs have their own specific place within or outside the skeleton. The
bones are connected to each other by joints, which allows us to move.
For a long time, bone tissue was considered only as an inert tissue functioning
as a scaffold supporting and protecting other tissues and vital organs. However later,
based on the advances in research of human physiology, it was discovered that bone
is a living and constantly changing tissue, which functions as the body’s primary
reservoir of calcium and phosphate ions and serves many other vital functions, such
as hematopoiesis.
During the last decades, osteoimmunology, bone’s function in regulating the
body’s immunological processes, has been a topic of high interest. Recent advances
in the field have shown how the bone tissue immune-system connection plays a
part in the pathological process of many chronic diseases, such as rheumatoid
arthritis (RA), osteoarthritis (OA) or osteoporosis (Okamoto & Takayanagi, 2019).
The discoveries have led to the rapid development of drugs that effect the bone
tissue immune-system interaction, such as denosumab or adalimumab. However,
much work is still needed to gain a deeper understanding in this relatively new field
of study.
According to the World Health Organization (WHO), two chronic rheumatic
conditions that cause the greatest impact on society are RA and OA (Briggs et al.,
2018). The prevalence of both diseases is on the rise, and they cause a huge amount
of morbidity and disability to the affected individuals and a significant economic
burden on societies throughout the world. Unfortunately, the pathogeneses of these
diseases are not completely understood and treatments even with modern advances
are limited.
RA and OA are both diseases characterized by typical pathological changes in
bone metabolism, local bone loss and synovitis. This study was conducted to
examine the osteoimmunological mechanisms, especially the ones leading to bone
loss, in the two diseases, and osteoclast function in disease and normal
environments. I focus on how the diseases affect osteoclastogenesis and possible
mechanisms behind it, with a special emphasis on osteopontin (OPN) protein.
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2 Review of the literature
2.1 Bone
The human skeleton consists of around 270 bones at the time of birth. During
growth multiple bones fuse together and therefore the number of bones in an adult
skeleton is around 206 (Gray, 1918). The main function of the skeleton is to act as
a scaffold and to protect other tissues of the human body. Other vital functions of
bone tissue are hematopoiesis in bone marrow and serving as the body’s calcium
and phosphate reservoir.
Based on a bone’s gross appearance two forms of bone can be distinguished,
compact bone and trabecular bone. To the naked eye a cross section of compact
bone appears as a solid mass and trabecular bone as a network of thin bone
trabeculae. Generally compact bone is found at the shaft, diaphysis, of long bones,
such as the femur, where it forms a hollow cylinder with the central cavity called
the bone marrow cavity. The ends of long bones, metaphysis and epiphysis, are
made of trabecular bone, which is covered by a thin layer of compact bone.
(Kierszenbaum & Tres, 2015)
Under a microscope, bone can be divided into two types based on the
organization of the extracellular matrix: lamellar bone, typical in mature bone, and
woven bone, typical in developing bone. In typical compact lamellar bone, the
organized bone matrix is seen as layers, lamellae, surrounding a Haversian or
Volkmann’s canal. Haversian canals are longitudinal blood vessel channels within
the compact bone tissue. Volkmann’s channels are similar but transverse or oblique
and can connect the Haversian channels. The combination of the lamellae and the
canal is called an osteon, which is separated from other osteons by a cement line
and interstitial lamella. In trabecular bone the lamellae are parallel to the trabecular
surface, as there are no blood vessel channels and the bone cells receive nutrients
directly from the bone marrow. The anatomical structures of bone are presented in
figure 1. (Kierszenbaum & Tres, 2015; Repp et al., 2017; Weatherholt, Fuchs, &
Warden, 2011)
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Fig. 1. The architecture of a long bone. Anatomically a bone can be divided into three
sections, diaphysis, metaphysis and epiphysis, and by macroscopic appearance into
compact and trabecular bone. Diaphysis is the shaft of a long bone. Metaphysis and
diaphysis are divided by the growth or epiphyseal plate during growth, and in adults by
the epiphyseal line after the cartilage in the growth plate turns to bone. Trabecular bone
is sponge-like bone present at the ends of a long bone, which is covered by a thin layer
of compact bone. The diaphysis is composed only of compact bone at the perimeter
and a hollow bone marrow cavity in the middle. Microscopically, trabecular bone is
made of small bone trabeculae, which are surrounded by bone marrow. In compact
bone, osteons and Haversian channels can be seen with a microscope. The bone is
surrounded by periosteum on the outer surface and by endosteum on the inner surface.
Modified from Kierszenbaum et al., 2015 and Weatherholt, Fuchs, & Warden, 2011.
Bone tissue consists of extracellular matrix (90%) and bone cells (10%) (Mansour,
Mezour, Badran, & Tamimi, 2017). The extracellular matrix is composed of
inorganic (65%) and organic parts (25%), and water (10%) (Boskey, 2013). The
bone tissue is a live tissue, which is constantly degraded and rebuilt by bone cells.
The process is called bone remodeling or bone metabolism. In adults it should be
in equilibrium. A disturbance in the process can lead to the onset of diseases, such
as osteoporosis when bone degradation is increased or osteopetrosis when bone
deposition is increased pathologically (Hadhidakis & Androulakis, 2006).
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2.1.1 Bone matrix
Ninety percent of bone is extracellular matrix which is 65% inorganic mineral,
mostly carbonated hydroxyapatite (Ca10(PO4)6(OH)2); the organic part
constitutes roughly around 25% of the extracellular matrix and the rest is water
(Boskey, 2013). The organic part is made of mostly type I collagen (90%) and small
amounts of collagen type III and V, non-collagenous proteins (NCPs) (8%) and
lipids (2%) (Young, 2003).
Bone mineral density is the main component of bone’s strength and stiffness
(Seeman & Delmas, 2006). Carbonated hydroxyapatite is the main mineral
substrate in bone and the basis of bone’s stiffness. It forms hexagonal crystals in
the scaffold created by collagen fibers and NCPs to form the rigid bone matrix
(Florencio-Silva, Sasso, Sasso-Cerri, Simões, & Cerri, 2015). Along with
hydroxyapatite the bone mineral matrix contains also significant amounts of
bicarbonate, sodium, potassium, citrate, magnesium, carbonate, fluoride, zinc,
barium, and strontium, which also affect the mechanical properties of the bone
matrix (Florencio-Silva et al., 2015). The correct bone mineral density is important
for the bone function, if the mineral density is too low the bones are weak, but if
the mineral density is too high the bone loses its elasticity and becomes brittle
(Seeman & Delmas, 2006). Also, the bone mineral density varies between species
and bones depending on their function. For example, the stapes of the human ear is
98% mineral so it is very stiff and can transfer sound better, whereas in the average
human bone the mineral density is around 60% to make it more elastic and damage
resistant (Boskey, 2013).
To create the scaffold for the bone minerals, collagen type I, which is produced
by the bone forming cells, osteoblasts, self assembles into a triple helix form (three
separate molecules, two alpha-1 chains and a one alpha-2 chains) and groups triple
helices form a microfibril via covalent cross linking (Stock, 2015). Multiple fibrils
come together to form a fiber. Water stabilizes the collagen structure by forming
hydrogen bonds between the fibers (Stock, 2015). If the fibril formation is
interrupted or faulty, the scaffold will not be able to stay together and hold the
hydroxyapatite, as can be seen in a genetic disease osteogenesis imperfecta (Young,
2003). In certain types of osteoporosis, a faulty collagen scaffold is also thought be
a factor (Young, 2003).
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Non-collagenous proteins
Bone tissue contains various non-collagenous proteins (NCPs) produced by
different bone cells among its collagen fibers. Most of the NCPS are glycoproteins
or proteoglycans, either located at the bone cell surfaces or in the extracellular
matrix (Young, 2003). The NCPs exert various important functions in the bone, e.g.,
they bind into the collagen fibers and hydroxyapatite and take part in the
mineralization process, act as paracrine signal molecules in bone, or as cytokines
for inflammatory cells in bone or further away in other tissues as endocrine
molecules (Florencio-Silva et al., 2015; Gramoun et al., 2010). Most NCPs are not
bone specific proteins and their expression is found throughout the human body
which, along with the fact that many of them have multiple overlapping functions
also in bone, has made them hard to study and their functions are not thoroughly
understood (Morgan, Poundarik, & Vashishth, 2015; Young, 2003).
An interesting group of bone NCPs are the SIBLING proteins (Small Integrin-
Binding Ligand, N-linked Glycoprotein), which all contain an RGD-sequence
(Arg-Gly-Asp). Integrin receptors found at the cell surfaces of, e.g., osteoclasts and
osteoblasts, can bind the RGD sequence (Qin, Baba, & Butler, 2004). Proteins that
belong in this group are osteopontin (OPN), bone sialoprotein (BSP), dentin matrix
protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular
phosphoglycoprotein (MEPE). Common for these proteins is that they have
multiple post-translational modifications, such as phosphorylation, glycosylation,
and proteolytic processing, which have significant effects on their structure and
biological functions (Qin et al., 2004). The various modifications present in the
proteins has made it hard to solve their various distinct functions. The proteins were
originally identified in bone, and accordingly have their particular roles in
biomineralization and bone metabolism, but many of them have also been found to
play various interesting roles in other human tissues. OPN, for example, is an
important mediator of inflammation, but also has been detected in various cancers
(Morimoto, Kon, Matsui, & Uede, 2010; Pietras et al., 2014).
Other NCPs of interest in bone are fibronectin, osteonectin, osteocalcin and
sclerostin. Fibronectin is one of the most abundant NCPs in bone and also contains
the same RGD sequence as the SIBLING proteins (Young, 2003). Fibronectin
accumulates around sites of new bone formation, where it likely acts as an
attachment point for bone cells and regulates especially osteoblast function and
bone mineralization (Young, 2003). Osteonectin is another protein that is found in
large quantities in bone; it is believed to act as a connector molecule between the
21
mineral crystals and collagen, and also to take part in regulating osteoblast and
osteoclast activity (Rosset & Bradshaw, 2016). Osteocalcin is an interesting NCP
produced by osteoblasts. Its primary biological functions are actually outside of
bone, where it has been shown to play only a small role in regulating bone mineral
density and its main functions are thought to be as an endocrine molecule that
regulates the body’s glucose homeostasis, brain development, cognition, and male
fertility (Moser & van, 2019). Sclerostin is a natural inhibitor of osteoblast function
and bone formation produced by osteocytes. This discovery has led to the rapid
research and development of new promising osteoporosis drugs (Bhattacharyya,
Pal, & Chattopadhyay, 2018).
Along with the few NCPs mentioned above, various other NCPs can also be
found in bone, for example, bone morphogenetic proteins (BMPs), various growth
factors and small leucine-rich proteoglycans, such as decorin, biglycan, lumican
and osteoaderin etc., many of their biological functions or clinical relevance are
still unknown (Florencio-Silva et al., 2015).
2.1.2 Bone cells
Bone cells make up 10% of bone tissue (Mansour et al., 2017). The bone cells come
from two different lineages; the osteoprogenitor cells, osteoblasts and osteocytes,
are of mesenchymal origin and the osteoclasts derive from the monocyte-
macrophage line of hematopoiesis (Kierszenbaum & Tres, 2015). In general,
osteoblasts are bone forming cells, osteoclasts resorb bone and osteocytes control
the bone metabolism.
Osteoblasts
The osteoblasts account for 4-6% of all bone cells (Capulli, Paone, & Rucci, 2014).
They are derived from the mesenchymal stem cells residing near bone surfaces,
which first evolve into osteoprogenitor cells, and then into mature osteoblasts
capable of bone formation (Roeder, Matthews, & Kalajzic, 2016). The process is
controlled by various cytokines and BMPs. During osteoblast maturation specific
signaling pathways are activated: the three most important and specific for
osteoblastogenesis are Wnt (Wingless and integration), Runx2 (Runt-related
transcription factor) and osterix (Sp7) (Roeder et al., 2016). Knockout-out mice
without these genes exhibit severely decreased bone formation due to impaired
osteoblastogenesis (Roeder et al., 2016).
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During bone formation the mature osteoblasts create the organic matrix which
is then mineralized. The osteoblasts secrete the collagens, mostly type I collagen,
and multiple NCPs, such as osteocalcin, osteonectin, BSP and OPN, to create the
organic scaffold, which is then mineralized (Capulli et al., 2014; Roeder et al.,
2016). During the mineralization, osteoblasts produce alkaline phosphatase, which
degrades the mineralization inhibitor pyrophosphate, and thus allows
mineralization and secretion of matrix vesicles containing hydroxyapatite crystals
that are incorporated into the extracellular organic matrix (Capulli et al., 2014;
Roeder et al., 2016). After actively forming the bone matrix the osteoblasts can
undergo apoptosis, become embedded in the matrix and differentiate into
osteocytes, or become quiescent bone lining cells depending on their location and
other bone cells present.
Osteoblasts have various other functions besides bone formation. Particularly
they control osteoclastogenesis and bone resorption by secreting three of the most
important cytokines that affect osteoclast differentiation and activation; receptor
activator of nuclear factor kappa-Β ligand (RANKL), macrophage colony-
stimulating factor (M-CSF) and osteoprotegerin (OPG) (Sato et al., 2013).
Osteoblasts also affect processes outside of bone by secreting osteocalcin, which
acts as an endocrine molecule as explained earlier (Moser & van, 2019), and
participate in the regulation of the hematopoietic system by regulating
hematopoietic stem cell homing, mobilization and number, and especially B-cell
lymphopoiesis (Kode et al., 2014).
Osteocytes
Osteocytes are spider shaped cells embedded inside the bone matrix, which account
for 90-95% of all bone cells (Capulli et al., 2014). The area around the cell body is
called a lacuna, and the osteocytes are connected to each other via canaliculi, where
adjacent cells are connected to each other by gap junctions. Via this lacuna-
canalicular system oxygen and nutrients are transported to the cells living in the
encapsulated environment (H. Chen, Senda, & Kubo, 2015). During the
transformation process from an osteoblast to an osteocyte the cell volume and
number of cell organelles are considerably decreased. The expression of key
osteoblast proteins is decreased or stops and the expression of key osteocyte
proteins begins. Such proteins have been identified to include DMP1, MEPE,
fibroblast growth factor (FGF), dickkopf-related protein 1 and sclerostin, which
have been shown to affect bone formation and mineralization and phosphate
23
metabolism (H. Chen et al., 2015). Of these proteins, especially sclerostin has been
of great interest recently. Sclerostin inhibits the Wnt signaling pathway in the
osteoblasts, which decreases bone formation. Inhibition of sclerostin’s action with
anti-sclerostin drugs have shown promise as new osteoporosis treatments in animal
models and clinical trials are currently being conducted (Bhattacharyya et al., 2018).
The main function of an osteocyte is to act as a regulator of bone metabolism
by regulating both osteoclasts and osteoblasts (Florencio-Silva et al., 2015).
Osteocytes act as mechanoreceptors in bone. They sense load and possible fractures
or other changes, and transmit signals to other bone cells via the lacuna-canalicular
system (Capulli et al., 2014; Florencio-Silva et al., 2015). Mechanical stress
increases bone mass and strength, while rest decreases them (Robling & Turner,
2009). The regulation of bone metabolism by osteocytes is not completely
understood, but it seems that osteocyte apoptosis under load is one crucial factor
(Capulli et al., 2014; H. Chen et al., 2015). Too little or too great mechanical
stimulus causes osteocyte apoptosis (Robling & Turner, 2009). The apoptotic
osteocytes release various cytokines, most importantly RANKL, tumor necrosis
factor-alpha (TNF-α), interleukin-6 (IL-6) and IL-11 (Capulli et al., 2014; H. Chen
et al., 2015). The cytokines cause an increase in osteoclast differentiation and
activity resulting in increased bone turnover (Amarasekara et al., 2018). There is
controversial evidence as to whether osteocyte apoptosis decreases or increases
osteoblast activity (Capulli et al., 2014; H. Chen et al., 2015). However, the
evidence that bone mass and strength increase under load is consistent (Robling &
Turner, 2009). It has been suggested that under load sclerostin production in
osteocytes is decreased, which results in increased osteoblast activity (Bullock,
Pavalko & Robling, 2019). Further research is needed on this subject before
conclusions can be drawn on how osteocytes regulate osteoblast function, but the
consensus seems to be that osteocytes are the link between osteoclast and osteoblast
action (Capulli et al., 2014; H. Chen et al., 2015).
Osteocytes also have effects outside the bone. They function as endocrine cells
by secreting FGF-23, which controls phosphate and D-vitamin metabolism by
affecting kidney cells (Bonewald & Wacker, 2013). The osteocytes have also been
shown to have immune- and fat metabolism regulatory functions, as osteocyte
deficient mice had severe lymphopenia due to impaired T- and B-cell
lymphopoiesis and a total lack of white adipose tissue (Sato et al., 2013).
24
2.1.3 Osteoclasts and bone resorption
Osteoclasts are defined as multinucleated tartrate-resistant acid phosphatase
(TRAcP) expressing giant cells capable of resorbing mineralized bone (Pierce,
1991). They differentiate from the monocyte-macrophage line of hematopoiesis by
fusing from mononuclear precursor cells into multinucleated TRAcP positive cells
(Boyle, Simonet, & Lacey, 2003). Most common human bone diseases are due to
excess osteoclast activity, which is a result of an imbalance of factors regulating
bone metabolism (Rodan & Martin, 2000). Such diseases include osteoporosis,
osteoarthritis, rheumatoid arthritis, multiple myeloma and metastatic cancers.
Like the other bone cells, the osteoblasts and the osteocytes, the osteoclasts
also have various functions other than just their primary job as bone degraders. The
osteoclasts take part in the regulation of bone metabolism by affecting the
osteoblasts, as well as the regulation of hematopoiesis and angiogenesis in bone
(Charles & Aliprantis, 2014). The osteoclasts also have important immunological
functions. The term osteoimmunology was introduced around the turn of the
millennium, almost twenty years ago now, when osteoclasts and immune cells,
especially T-cells, were found to have a tight relationship regulating each other’s
function (Arron & Choi, 2000; Takayanagi et al., 2000).
Osteoclastogenesis
During osteoclastogenesis, monocytes from the myeloid line of hematopoiesis fuse
together to create the osteoclasts. The pre-osteoclast cells capable of fusing together
have been suggested to be cells of the monocyte linage expressing cluster of
differentiation (CD) molecule CD14 (Costa-Rodrigues, Fernandes, & Fernandes,
2011; Hemingway et al., 2011; Sørensen et al., 2007). In the presence of M-CSF
the cells start expressing RANK, a signaling receptor for RANKL, and this
activation is the main promoter of differentiation and fusion of pre-osteoclasts into
osteoclasts (Park-Min, 2018).
Osteoclast precursor cells can be found in bone marrow and circulating in
peripheral blood. It has been suggested that after initial differentiation from
monocytes to pre-osteoclasts in the bone marrow or in another hematopoietic organ
the cells undergo cell cycle arrest and circulate in the blood stream as cell cycle
arrested quiescent osteoclast precursors (QuOPs) (Kotani et al., 2013; Mizoguchi
et al., 2009). The QuOPs circulate in the blood until they eventually reach the bone
surfaces, where they undergo the final steps of osteoclastogenesis by fusing (Kotani
25
et al., 2013; Mizoguchi et al., 2009). Figure 2 presents a schematic presentation of
osteoclast precursor differentiation and homing.
Fig. 2. Osteoclast precursor differentiation and homing. The pre-osteoclasts proliferate
and differentiate from progenitor cells of the monocyte line in hematopoietic organs,
such as bone marrow and spleen. After differentiation, the pre-osteoclasts undergo cell
cycle arrest then circulate in the blood stream and home towards bone, where they
undergo fusion and differentiation into the osteoclasts in the presence of
osteoclastogenesis promoting factors, such as M-CSF and RANKL produced by the
osteoblasts and the osteocytes.
The generation of the osteoclasts from the pre-osteoclasts is most importantly
controlled by two cytokines M-CSF and RANKL, which both are produced by cells
of the osteoblast lineage, the osteoblasts and the osteocytes, of which the
osteoblasts are thought to be more important producers of osteoclastogenesis
regulating factors (Sato et al., 2013). First M-CSF signaling activates Akt and ERK
signal pathways inside the pre-osteoclasts, which causes an increase of cell surface
receptor RANK production and induction of the main osteoclast marker TRAcP
production (Amarasekara et al., 2018; Park-Min, 2018). RANKL activates the
RANK receptor and causes multiple other signaling pathways to be induced in the
pre-osteoclast and production of the key osteoclast proteins, which promote cell
fusions and the final differentiation into the mature osteoclasts (Amarasekara et al.,
2018; Park-Min, 2018). RANKL is the most important factor of osteoclastogenesis,
26
as it has been shown in knockout mice models that in the absence of either RANK
or RANKL osteoclastogenesis does not happen (Dougall et al., 1999; Kong et al.,
1999; J. Li et al., 2000). During the last stage of osteoclast differentiation,
production of proteins involved in the adhesion of the cell to the bone matrix and
resorption activity, such as alpha-v beta-3 integrin (αvβ3) and cathepsin K, begins
(Boyle et al., 2003; Soysa & Alles, 2016). The cells attach themselves to the bone
matrix and become polarized as their cytoskeleton is reshaped creating the unique
morphology of an osteoclast (Boyle et al., 2003; Soysa & Alles, 2016).
As explained earlier in chapter 2.1.2, osteocytes act as mechanosensors in bone
and produce signals in areas where increased bone turnover is needed. Therefore,
it has been suggested that they control the location of osteoclast activation.
However, the specific mechanisms are as yet unknown, but osteocyte apoptosis has
been suggested to be key factor as it causes production of osteoclastogenesis
promoting factors, such as RANKL, in the surrounding viable osteocytses (Capulli
et al., 2014; H. Chen et al., 2015).
The main controlling cell of osteoclastogenesis is the osteoblast, which along
with the production of M-CSF and RANKL also produces OPG, a competitive
inhibitor of RANKL, which is the most important inhibitor of osteoclastogenesis
(Park-Min, 2018). OPG knockout mice suffer from severe osteoporosis, and
transgenic mice overexpressing OPG suffer from osteopetrosis (Bucay et al., 1998;
Simonet et al., 1997). Reciprocally osteoclasts also affect osteoblast function by
secreting so-called clastokines, which either increase or decrease osteoblast activity
(Cappariello, Maurizi, Veeriah, & Teti, 2014). Clastokines that are believed to
increase osteoblast activity include TRAcP, sphingosine-1-phosphate, BMP6,
wingless-type 10b, hepatocyte growth factor and collagen triple helix repeat-
containing protein 1, and clastokines thought to decrease osteoblast activity include
platelet derived growth factor BB (PDGF BB) and sclerostin (Teti, 2013). However,
the effect of osteoclasts on osteoblasts is not as well understood as the osteoblasts’
effect on the osteoclasts. Further studies are needed to gain a full understanding of
the osteoclast-osteoblast connection and effect on the bone metabolism.
Various other factors which influence osteoclastogenesis less significantly than
M-CSF, RANKL and OPG are also known. Various inflammatory cytokines have
been shown to increase osteoclastogenesis either by the RANKL pathway or
independently. These include TNF-α, IL-1, IL-6, IL-7, IL-8, IL-11, IL-15, IL-17,
IL-23, IL-34, C-reactive protein (CRP) and vascular endothelial growth factor
(VEGF) (Amarasekara et al., 2018; H. Kim, Kim, Kim, Cho, & Lee, 2015; K. Kim,
Kim, Moon, Lee, & Kim, 2015). Reciprocally various cytokines are also shown to
27
decrease osteoclastogenesis, which include interferon-α (IFN-α), IFN-β, IFN-γ, IL-
3, IL-4, IL-10, IL-12, Il-27 and IL-33 (Amarasekara et al., 2018). Increased
inflammatory osteoclastogenesis can be seen in inflammatory diseases, such as RA,
but interestingly the phenotype of inflammatory osteoclasts has been found to differ
from the normal osteoclast. The inflammatory osteoclasts are described to be
smaller and contain fewer nuclei, and thus present a more macrophage like
phenotype as a result of a strong inflammatory stimulus (Suzuki et al., 2001; Tsuboi
et al., 2003).
Important physiological factors that regulate osteoclastogenesis are pH and
hypoxia. Low pH and oxygen levels have been shown to increase
osteoclastogenesis (Arnett, 2010; Yuan et al., 2016). The optimal pH for the
osteoclasts has been suggested to be around 6.8 (Arnett, 2010; Yuan et al., 2016),
while the normal arterial blood pH in the human body is around 7.4. In normal
tissue the interstitial oxygen pressure is at a minimum of around 3%, but optimal
osteoclastogenesis has been shown to occur in 1-2% oxygen (Arnett, 2010).
Bone resorption
To resorb bone the mature osteoclasts must first attach themselves to the matrix and
become polarized. First the osteoclast forms a sealing zone, a structure which
attaches the osteoclasts to the extracellular matrix (Takito, Inoue, & Nakamura,
2018). As the sealing zone is formed the osteoclast becomes polarized. The cell
membrane area inside the sealing zone is called a ruffled border membrane and the
cell membrane on the opposite side of the cell is called the functional secretory
domain.
The sealing zone and the ruffled border are formed as the cell attaches to the
matrix with multiple podosomes, foot-like short cellular protrusions present at the
cell membrane (Rucci & Teti, 2016). Each podosome is made of an actin
microfilament core and integrins that protrude outside of the cell (Rucci & Teti,
2016; Takito et al., 2018). The αVβ3 integrin has been suggested to be the main
integrin facilitating osteoclast attachment, as it recognizes the RGD amino acid
sequence present in various bone NCPs, such as fibronectin, vitronectin and the
SIBLING proteins (OPN, BS, DMP1, DSPP, MEPE) (Qin et al., 2004; Ross et al.,
1993). The round sealing zone completely segregates the extracellular area between
the osteoclast and the resorbed matrix from the rest of the environment. This
microenvironment is called the resorption lacuna or pit. The origin of the proteins
to which the αVβ3 integrin attaches is not fully understood. There are proteins
28
already present in bone, but the osteoclasts have also been suggested to produce
them (Dodds et al., 1995), and interestingly, the osteoclasts have been shown to
resorb also carbonated hydroxyapatite free of pre-deposited proteins (Nakamura,
Hentunen, Salonen, Nagai, & Yamashita, 2013).
After attaching to the matrix and creating the ruffled border, the cells start
secreting hydrochloric acid (HCl) into the resorption area, as separate protons (H+)
and chloride ions (Cl-), which acidifies the lacuna (Cappariello et al., 2014). The
acidic environment dissolves the hydroxyapatite crystals leaving the bone’s organic
component exposed to be proteolytically processed (Vaananen, Zhao, Mulari, &
Halleen, 2000).
The organic bone matrix is degraded by lysosomal enzymes, which function in
the acidic conditions. The organic matrix degradation process is not completely
understood. The enzymes that have been suggested to be secreted into the
resorption lacunae are cathepsin K, TRAcP and multiple matrix metalloproteinases
(MMPs) (Henriksen et al., 2006; Rucci & Teti, 2016; Vaananen et al., 2000).
Cathepsin K is the primary cathepsin secreted by the osteoclasts and is thought to
be the main degrader of the bone’s main organic component the collagen type I
(Clarke, Drake, Oursler, & Khosla, 2017). Elevated cathepsin K levels are
associated with increased osteoclast activity and bone destruction (Skoumal et al.,
2004). TRAcP is the main intracellular osteoclast marker and it has also been shown
to function as a marker of osteoclastic activity in serum (Andersson et al., 2003;
Halleen, Tiitinen, Ylipahkala, Fagerlund, & Vaananen, 2006; Janckila, Takahashi,
Sun, & Yam, 2001). It has been suggested that osteoclasts secrete TRAcP into the
resorption lacuna, where it is believed to affect at least OPN and BSP function by
dephosphorylating them, which may affect the osteoclast attachment to the bone
matrix and migration (Andersson et al., 2003). The specific functions of the
multiple MMPs secreted into the resorption lacunae are not completely understood.
In general, MMPs have been suggested to take part in processing of both the
collagenous and NCPs of the bone matrix, as well as directly regulating osteoclasts
and other bone cells (Paiva & Granjeiro, 2017; Rucci & Teti, 2016).
During the matrix degradation, the degradation products and some of the
degrading enzymes are endocytosed into the osteoclast. Inside the cell the
endocytotic vesicles further fuse with more TRAcP containing intracellular vesicles,
and the degradation process continues (Halleen et al., 2006). The vesicles are then
transported to the functional secretory domain at the opposite side of the cell, where
they are exocytosed into the surrounding extracellular environment (Soysa & Alles,
29
2016; Vaananen et al., 2000). A diagram of a resorbing osteoclast is shown in figure
3.
Fig. 3. A schematic presentation of a bone resorbing osteoclast. Osteoclasts attach to
the bone matrix and form the sealing zone. A resorption pit is formed under the ruffled
border membrane. Osteoclasts acidify the resorption pit by secreting H+-ions, and
matrix degrading enzymes to degrade the bone matrix. The matrix degradation products
are endocytosed and trafficked transcellularly to the functional secretory domain at the
opposite side of the cell and excreted. Modified from Cappariello et al., 2014 and Soysa
& Alles, 2016.
The transcytosis of degradation products allows osteoclasts to migrate and resorb
huge amounts of matrix during a resorption cycle when compared to their actual
size. It has been suggested that osteoclast migration during resorption is controlled
by the calcium concentration inside the resorption lacuna; after a certain
concentration is reached the cell moves slightly forward and starts resorbing again
(Cappariello et al., 2014; Teitelbaum, 2007). During an osteoclast’s life span the
cycle is repeated multiple times, and the osteoclast creates a resorption trail behind
it, as shown in figure 4. After multiple resorption cycles, at the end of their lifespan,
the osteoclasts undergo natural apoptosis (Rucci & Teti, 2016; Soysa & Alles, 2016).
The factors affecting or causing the osteoclast apoptosis are not known. Anti-
osteoporotic drugs such as zoledronic acid have been shown to cause osteoclast
apoptosis (Tai et al., 2017).
30
Fig. 4. Electron microscope image of a resorbing osteoclast on a bone slice. On bone
osteoclasts migrate during resorption and leave a resorption trail behind.
2.1.4 Bone remodeling
Bone remodeling is fundamental to the quality of bone and maintenance of the
body’s mineral homeostasis throughout human life, as old bone is constantly being
degraded by osteoclasts and new bone produced by osteoblasts. A disturbance in
the balance may lead to a metabolic skeletal disease. As in osteoporosis, a common
disease, in which the balance is shifted towards increased osteoclastic activity
(Hadhidakis & Androulakis, 2006).
Bone remodeling is described to happen in cycles, which consist of five parts:
activation, resorption, reversal, formation and termination (Katsimbri, 2017;
Kenkre & Bassett, 2018). The cycles happen in an anatomical unit called the Basic
Multicellular Unit (BMU), which is composed of osteoclasts, osteoblasts, lining
cells and a capillary blood supply (Katsimbri, 2017; Kenkre & Bassett, 2018).
The bone remodeling activation and resorption parts consist of osteoclast
precursor migration to the BMU, osteoclastogenesis, osteoclast activation, and
bone resorption, with the local factors affecting them, as described earlier in chapter
2.1.3. The activation and reversal phases are also controlled by endocrine
hormones which control the body’s calcium and phosphate metabolism, such as
parathyroid hormone (PTH) and vitamin D. PTH and the active vitamin D
31
(1α,25(OH)2D3) induce RANKL production in cells of the osteoblast lineage, thus
resulting in increased osteoclastogenesis (Silva & Bilezikian, 2015; Takahashi,
Udagawa, & Suda, 2014). Another important hormone is estrogen, which induces
apoptosis in the osteoclasts and is anti-apoptotic in the osteoblasts (Krum et al.,
2008). Estrogen promotes overall bone formation, and it has been shown that
estrogen deficiency in post-menopause is the major cause of osteoporosis in women
(Krum et al., 2008). Other important endocrine factors associated with bone
metabolism are glucocorticoids, calcitonin, growth hormone, thyroid hormone and
androgens (Kenkre & Bassett, 2018).
During the reversal phase, the pre-osteoblasts arrive at the location of resorbed
bone and differentiate into the osteoblasts that start producing new bone. This phase
of bone remodeling is not as well understood as the others (Katsimbri, 2017;
Kenkre & Bassett, 2018). It is likely that the before their apoptosis the osteoclasts
produce cytokines and other factors, the so-called clastokines, which attract cells
of the osteoblast lineage into the area to prepare the bone surface for new bone
formation by the mature osteoblasts (Cappariello et al., 2014). The first arriving
cells, called reversal cells, are of the osteoblast lineage and can further mature into
osteoblasts. However, they also have important functions of their own. The reversal
cells have been suggested to remove unmineralized collagen matrix, and then
deposit non-collagenous mineralized matrix, the “cement line”, in its place to
enhance the attachment and activity of the mature osteoblasts (Delaisse, 2014).
Unfortunately, the nature and function of the clastokines that affect cells of the
osteoblast lineage are not very well understood, as it has been apparent that even
though many cytokines produced by the osteoclasts increase osteoblastogenesis
and activity, many others decrease it, as explained earlier in 2.1.3 (Matsuo & Irie,
2008; Teti, 2013). Further research on this subject is needed.
After osteoblasts have arrived and matured at the BMU they begin the bone
formation phase. During this phase the removed old bone is completely replaced
with new bone. A disturbance in the osteoblast function at this point may lead to
decreased bone volume or density, e.g., post-menopausal estrogen deficient
osteoporosis. After enough new bone is formed, the final bone remodeling phase
termination is reached. During termination, the osteoblasts either undergo apoptosis,
become embedded in the matrix and differentiate into the osteocytes, or become
quiescent bone lining cells depending on their location and other bone cells present,
as described earlier in 2.1.2 (Katsimbri, 2017; Kenkre & Bassett, 2018).
32
2.2 Joint
A joint is a site where two bones come together. In the human body there are two
kinds of joints, solid joints and synovial joints. In a solid joint two bones are
attached to each other by fibrous connective tissue or cartilage (Gray, 1918). The
movement in a solid joint is restricted. Such joints can be found, for example, in
skull sutures or at the pubic symphysis.
A synovial joint is a connection between two moving bones, which are
separated by an articular cavity, for example, a knee or a hip (Gray, 1918). The
articular cavity is formed by a joint capsule (Iwanaga, Shikichi, Kitamura, Yanase,
& Nozawa-Inoue, 2000). Usually the ends of bone are covered by articular cartilage,
and the joint capsule attaches to the margin of bone and cartilage (Iwanaga et al.,
2000). Other structures that can be found within the synovial capsule include
articular discs, fat pads, ligaments etc. (Gray, 1918). The shape of the synovial joint
varies according to its function from ball joints, which allow movement in all
directions, to plane joints, which allow only small sliding of the bones (Gray, 1918).
The articular cartilage which covers the end of the bones within the joint is
hyaline cartilage (Bhosale & Richardson, 2008). The cartilage, which is lubricated
by synovial fluid, creates a sliding surface between the opposing bones, (Bhosale
& Richardson, 2008). The hyaline cartilage is composed of chondrocytes, mostly
collagen type II fibers (90-95%) and other extracellular matrix proteins (5-10%)
(Kierszenbaum & Tres, 2015). Sixty-five to eighty percent of cartilage weight is
water (Bhosale & Richardson, 2008).
Chondrocytes are the main cartilage cells. They are responsible for the
production of the extracellular matrix (Kierszenbaum & Tres, 2015). The
chondrocytes are trapped within the cartilage, and because the hyaline cartilage is
avascular, they receive nutrients only by diffusion through the extracellular matrix
from the bone and the synovial fluid (Kierszenbaum & Tres, 2015).
The other extracellular matrix proteins, than collagens, are mainly
proteoglycans. They maintain the fluid and electrolyte balance within the cartilage
(Bhosale & Richardson, 2008). The proteoglycans can be divided into two: large
aggrecans and small proteoglycans, such as decorin, biglycan and fibromodulin
(Bhosale & Richardson, 2008).
The articular cartilage can be divided structurally and by chondrocyte
morphology into four layers from bone to articular cavity: calcified, deep, middle
and superficial zones (He et al., 2014). The extracellular matrix of the calcified
33
cartilage is rich in calcium, and it acts as a transition zone between the bone and
the cartilage (He et al., 2014).
The joint capsule is formed by an inner synovial membrane and outer fibrous
membrane. The inner synovial membrane consists only of one to two layers of
synovial cavity lining cells on top of loose connective tissue, which is highly
vascularized and rich in lymphatic vessels (He et al., 2014). Further away from the
joint cavity the loose connective tissue merges into the denser connective tissue of
the outer synovial membrane (He et al., 2014). There are two types of synovial
lining cells: type A macrophage-like synovial cells and type B fibroblast-like
synovial cells (Iwanaga et al., 2000). The synovial cells do not have a continuous
base membrane or attach to each other tightly (Iwanaga et al., 2000). Normally, the
macrophage-like cells are in the minority, but in inflammation they can account for
up to 80% of the lining cells (Smith, 2011). The origin of the synovial macrophages
is suggested to be from the myeloid-monocyte line of cells, and the origin of the
fibroblast-like cells is thought to be mesenchymal (Kurowska-Stolarska &
Alivernini, 2017; Smith, 2011).
The synovial cells control the volume of the synovial fluid through the
production of synovial fluid glycoproteins and hyaluronic acid (Smith, 2011).
Synovial fluid is a viscous ultrafiltrate of plasma and proteins produced by the
synovial lining cells and the chondrocytes (Bennike et al., 2014). The main function
of synovial fluid is to work as a lubricant for the sliding articular surfaces, but it
also facilitates the transportation of nutrients and waste products between the
synovial lining and cartilage (Bennike et al., 2014).
2.3 Osteoimmunology
The interaction between the bone and the immune system was first discovered in
1972, when it was discovered that activated peripheral blood leukocytes release
agents that stimulate osteoclastic bone resorption (Horton, Raisz, Simmons,
Oppenheim, & Mergenhagen, 1972). Later multiple factors that regulate both the
bone and the immune cells have been found. Probably the most notable revelation
to date has been the discovery of RANK and RANKL, which were first discovered
as mediators of T-cell activation and dendritic cell function, but later were found to
be the most important regulators of osteoclastogenesis and osteoclast function
(Kong et al., 1999). The term osteoimmunology was first presented in 2000 to name
the field of study that investigates the connections between the bone and the
immune system (Arron & Choi, 2000). During this millennium osteoimmunology
34
has been a rapidly growing and progressing field of study, which has resulted in
many great advances that have even led to production or on-going research of new
drugs, such as denosumab, an OPG analog, for treating osteoporosis, and multiple
biological RA drugs, for example, the TNF-α antibody adalimumab.
As the immune and the bone cells originate both from the same precursor cells
off bone marrow it is no wonder that complex signaling occurs between them. So
far, in inflammation the major interacting cells have been identified to be the
osteoclasts and the T-lymphocytes (Terashima & Takayanagi, 2018). During
inflammation the T-cells, and other inflammatory cells, activate and produce
multiple cytokines that either directly affect osteoclastogenesis or cause other cells,
such as fibrocytes or macrophages, to produce RANKL or other pro-osteoclastic
substrates, thus increasing the osteoclastogenesis (Okamoto & Takayanagi, 2019).
Cytokines that affect the osteoclastogenesis were discussed earlier at chapter 2.1.3.
The cytokines present in inflammation reflect its etiology and differ in different
situations, such as in OA or RA, and hence the effect on osteoclasts is likely to
differ between the diseases.
The osteoclastogenesis resulting from the increased inflammatory stimulus on
the pre-osteoclasts is called inflammatory osteoclastogenesis, which can especially
be seen in inflammatory joint diseases, such as RA, but is has also been suggested
to be behind secondary osteoporosis in chronic inflammatory diseases (Clayton &
Hochberg, 2013; Greisen et al., 2015; Suzuki et al., 2001). Interestingly the
inflammatory osteoclasts have been described to present a more macrophage-like
phenotype than the regular osteoclasts, as they are smaller and contain fewer nuclei
(Suzuki et al., 2001; Tsuboi et al., 2003).
The osteoclasts can also reciprocally affect the T-cells, but the total effect is
not fully understood and needs further research. Some studies have shown anti-
inflammatory regulation of T-cells by osteoclasts in both inflammation and cancer
(An et al., 2016; H. Li et al., 2014), but also reports of the osteoclasts further
activating the T-cells have been published (Pappalardo & Thompson, 2015). The
phenomenon is likely as complex as the combined effect of various cytokines either
increasing or decreasing the osteoclastogenesis.
The bone cells are crucial for the differentiation of the blood cells from
hematopoietic stem cells (HSCs) of the bone marrow. Osteoclasts provide the space
for the bone marrow and hematopoiesis within the bone by keeping the bone
remodeling in balance with the osteoblasts. It has been shown that osteopetrotic
mice suffer from high levels of extramedullary hematopoiesis due to lack of bone
marrow within the bones, and osteopetrosis is also associated with increased risk
35
of inflammation and anemia (Lowell, Niwa, Soriano, & Varmus, 1996; Terashima
& Takayanagi, 2018). The osteoblasts also participate in HSC maturation and
differentiation by affecting the HSC homing, mobilization and number by
providing a bone platform to maintain them and by secreting different growth
factors that regulate the process (Kode et al., 2014; Okamoto & Takayanagi, 2019).
At this moment, advances in immune, osteoimmune and disease biomarker
research have led to discoveries of numerous proteins that may affect both the bone
cells and the immune cells, but their clinical significance in disease is uncertain,
whether they are actually an active player or an innocent by-stander
(Krishnamurthy et al., 2016; Lotz et al., 2013; J. W. Seol, Lee, Kim, & Park, 2009;
Yamamoto et al., 2003). In this study, we focus on the effects of the proteins OPN
and TRAcP, and their post-translational modifications, in inflammation and bone.
2.4 Osteopontin (OPN)
OPN is a phosphorylated N- and O-glycosylated non-collagenous extracellular
matrix protein (Christensen, Nielsen, Haselmann, Petersen, & Sorensen, 2005). It
belongs to the SIBLING protein family (Qin et al., 2004). Originally OPN was first
identified as a bone matrix protein in 1985 (Franzen & Heinegård, 1985), but later
it has been detected in almost all human tissues, and it is expressed by a large
variety of cells including all the bone cells (osteoblasts, osteocytes and osteoclasts),
macrophages, dendritic cells, chondrocytes, fibroblasts, endothelial cells, and
smooth muscle cells (Icer & Gezmen-Karadag, 2018; Sodek, Ganss, & McKee,
2000).
OPN regulates various cellular functions, e.g., activation, differentiation,
migration and adhesion throughout the human body, and plays a part in
inflammation, biomineralization, cardiovascular diseases, cancer, diabetes and
renal stone disease through different mechanisms (Brown, 2012; Ge et al., 2017;
Icer & Gezmen-Karadag, 2018; L. Wang et al., 2008). The known major biological
roles of OPN are explained in figure 5. OPN’s biological activity is controlled by
its post-translational modifications, especially phosphorylation, which is crucial for
most of its functions, such as immune cell or osteoclast activation (Addison, Masica,
Gray, & McKee, 2010; Kazanecki, Uzwiak, & Denhardt, 2007; Nyström, Dunér, &
Hultgårdh-Nilsson, 2007; Weber et al., 2002). OPN’s phosphorylation is controlled
extracellularly by TRAcP (Andersson et al., 2003).
36
Fig. 5. The known major biological roles of OPN. Modified from Icer & Gezmen-Karadag,
2018.
The OPN protein consists of 300 amino acids, with some N- and O-linked
oligosaccharides. The protein contains an RGD sequence, two heparin binding
domains, one thrombin binding and one calcium binding domain (Kariya et al.,
2014; Christensen et al., 2005; H. Li et al., 2015). It has a total of 36
phosphoresidues; 34 phospho-serine and two phospho-threonine groups
(Christensen et al., 2005). OPN is secreted extracellularly in a phosphorylated form
after being phosphorylated intracellularly for example by Fam 20C kinase
(Christensen et al., 2005). It is known to have signaling functions through various
cell surface integrins, such as αvβ3 which can bind OPN’s RGD sequence, and a
CD 44 receptor (Kariya et al., 2014; Christensen et al., 2005; H. Li et al., 2015).
OPN can be cleaved into two active parts by thrombin, the N- and C-terminal ends,
which abilities are also dependent on the extent of their phosphorylation (Weber et
al., 2002). The C-terminal fragment contains the CD 44 receptor binding part and
it induces macrophage chemotaxis, while the N-terminal fragment binds integrins
and affects other cell spreading and activation (Weber et al., 2002).
In bone, OPN is known to affect osteoclast differentiation, activation and
migration (Ek-Rylander & Andersson, 2010; Ross et al., 1993; Sodek et al., 2000).
OPN is suggested to have an essential role in the adhesion of the osteoclasts to the
bone matrix and the generation of the ruffled border zone during bone resorption.
It has been shown that OPN can bind to bone hydroxyapatite (Goldberg, Warner,
37
Li, & Hunter, 2001). The osteoclasts are believed to attach themselves to the
unresorbed bone through the αvβ3-integrin receptor and it is thought that the RGD-
sequence containing OPN is one of the key ligands for the αvβ3 in the bone matrix
(Ek-Rylander & Andersson, 2010; Ross et al., 1993; Sodek et al., 2000). It has been
shown that tge osteoclasts themselves can produce OPN, and it has been suggested
that the osteoclasts could facilitate their own attachment to hydroxyapatite by
producing OPN to function as a connecting molecule (Dodds et al., 1995; Maeda,
Kukita, Akamine, Kukita, & Iijima, 1994).
Osteoblasts are also capable of OPN production, but OPN’s extracellular
effects on osteoblasts are not well understood (Icer & Gezmen-Karadag, 2018).
OPN is likely to play an important role in osteoclast-osteoblast communication, as
the mineral content and crystallinity of bone in OPN knockout mice is increased
(Boskey, Spevak, Paschalis, Doty, & McKee, 2002). The mineral deposition by
OPN knockout osteoblasts has been shown to increased, but it can be decreased by
addition of extracellular phosphorylated OPN (Holm et al., 2014). Another study
showed that phosphorylated OPN might have suppressive effects on osteoblasts
that are under mechanical stress (Kusuyama et al., 2017). However, a study using
an osteoblast-like cell line (SaOS-2 cells) suggests that the suppressive effects of
OPN are also likely regulated by OPN’s phosphorylation. Dephosphorylated OPN
did not have the same effect as phosphorylated OPN on the SaOS-2 cells (Halling
Linder et al., 2017).
Along with OPN’s cellular functions, it also regulates biomineralization in its
free extracellular form (Gericke et al., 2005; Hoac et al., 2017; L. Wang et al., 2008).
The regulation happens in bone, but also in other sites involving calcification, such
as aortic valve calcification or urinary stone formation (Gericke et al., 2005; Hoac,
Nelea, Jiang, Kaartinen, & McKee, 2017; Kohri et al., 2012; Sainger et al., 2012).
Also, OPN’s effect on biomineralization is regulated by its phosphorylation, which
is required for its biomineralization inhibitory effect (Gericke et al., 2005; Hoac et
al., 2017; L. Wang et al., 2008). During mineralization OPN can effectively bind
ionic and crystalline calcium, and thus inhibit it. The key properties behind this
function are the protein’s large number of phosphorylation sites, highly acidic
nature, and intrinsically disordered structure (Hoac et al., 2017).
OPN’s expression is elevated during inflammation. It works as an important
chemoattractant especially for macrophages, neutrophils, dendritic cells, and T
cells (K. X. Wang & Denhardt, 2008). During inflammation OPN production is
increased in macrophages, activated T cells, epithelial and endothelial cells, and
smooth muscle cells (Icer & Gezmen-Karadag, 2018). Studies with OPN knockout
38
mice have shown that OPN’s main immunoregulatory function is to regulate T-cell
activation and drive it towards a Th1 (T helper cell 1) cell-mediated response
instead of a Th2 (T helper cell 2) humoral response (Ashkar et al., 2000; K. X.
Wang & Denhardt, 2008). Th1 type T-cells produce the proinflammatory responses
responsible for killing intracellular microbes and for perpetuating autoimmune
responses. They regulate especially macrophage-monocyte function, as the Th2
responses balance these by promoting a humoral response which is more targeted
against extracellular parasites, by regulating eosinophils, basophils and mast cells
(Berger, 2000). Excessive Th1 T-cell activation is associated with increased tissue
damage during inflammation (Berger, 2000). OPN has also been shown to increase
Th17 cell differentiation and activity (G. Chen et al., 2010). Th17 cells differ from
Th1 and Th2 by predominantly producing IL-17 family cytokines, regulating
neutrophils, and they mainly target extracellular microbes and fungi (Ouyang,
Kolls, & Zheng, 2008).
It has been shown that in OPN null mice the severity of Herpes simplex,
Mycobacterium bovis BCG and Listeria monocytogenes-induced infections are
increased (Ashkar et al., 2000; Nau et al., 1999). Interestingly, OPN knockout mice
have a better survival from sepsis than normal mice (Fortis, Khadaroo, Haitsma, &
Zhang, 2015; Piao & Yuan, 2017). The increased survival is suggested to be a result
of an excessive inflammatory response in normal mice, and it has been suggested
that OPN’s protective effects in infection are overbalanced by its pro-inflammatory
capability. Similar results have been seen in inflammatory joint diseases. In RA
OPN levels decrease after one year of successful treatment (Izumi, Kaneko,
Hashizume, Yoshimoto, & Takeuchi, 2015). In addition, OPN deficiency has been
shown to reduce disease severity in arthritis mouse models (Yumoto et al., 2002).
In conclusion, research has shown that OPN plays an important role in many
biological functions and is an extremely versatile molecule present throughout the
human body. Especially, OPN has been of special interest to researchers working
on osteoimmunology, as it affects both bone and immune cells. However, due to
the extensive post-translational modifications and their effect on the protein’s
functions it has so far proven hard to completely understand the multiple functions
of OPN in the human body.
2.5 Tartrate-resistant acid phosphatase (TRAcP)
TRAcP is considered the main regulator of extracellular OPN’s phosphorylation
(Andersson et al., 2003), and thus the main regulator of OPN’s biological activity.
39
A schematic presentation of how impaired TRAcP function in bone may lead to
pathological changes by affecting OPN’s phosphorylation is shown in figure 6. Two
forms of TRAcP can be found in the human body, TRAcP 5A and 5B. TRAcP is
synthesized as a relatively inactive monomer loop peptide pro-enzyme, TRAcP 5A.
The 5A form can proteolytically cleaved into a two-subunit active dimer form,
TRAcP 5B, by proteinases, such as cathepsin K, increasing its activity at least ten
times (Fagerlund et al., 2006; Ljusberg et al., 2005; Lång & Andersson, 2005). In
general, the TRAcP 5A form is thought to be produced by macrophages and
dendritic cells, and the 5B form by osteoclasts, thus 5A is the dominant form in
serum and 5B in bone (Halleen et al., 2006; Janckila & Yam, 2009).
TRAcP 5B is the main osteoclast marker and its levels in serum correlate with
osteoclast activity and number (Halleen et al., 2006; Janckila, Takahashi, Sun, &
Yam, 2001a). Elevated TRAcP 5B levels have been found in diseases associated
with increased bone metabolism and destruction, such as OA, RA, osteoporosis and
cancer with bone metastases (Chao, Wu, & Janckila, 2010; Halleen et al., 2002; J.
W. Seol et al., 2009; Tsuboi et al., 2003).
In bone TRAcP is suggested to control the osteoclast migration by regulating
their ability to attach to the bone matrix bound OPN by regulating OPN’s
phosphorylation status (Andersson et al., 2003; Ek-Rylander & Andersson, 2010).
By regulating OPN’s phosphorylation TRAcP also regulates the mineralization as
explained previously in chapter 2.3. TRAcP also partakes in the resorption and
degradation of bone matrix both in the resorption lacunae and intracellularly by
generating reactive oxygen species (Fagerlund et al., 2006; Andersson et al., 2003).
After secretion fro the osteoclasts TRAcP has been suggested to function also as a
clastokine which marks the newly resorbed resorption pit and acts as a docking
point for the osteoblasts and stimulates their activity (Cappariello et al., 2014;
Janckila & Yam, 2009).
TRAcP knockout mice exhibit a mildly osteopetrotic phenotype (Hayman et
al., 1996), as a likely result of weakened osteoclast activity. Transgenic mice
overexpressing TRAcP show increased bone turnover, but no change in bone
density or quality has been found (Hayman et al., 1996). The TRAcP inhibitors
developed have been suggested to possibly work as new osteoporosis drugs, but
are still to be clinically tested (Hussein, Feder, Schenk, Guddat, & P McGeary,
2018).
40
Fig. 6. TRAcP regulates extracellular OPN’s biological activity by dephosphorylating it.
Impaired TRAcP activity may lead to pathological changes due to hyperphosphorylation
of OPN. In bone and related tissues phosphorylated OPN increases the activity of
immune cells and osteoclasts, decreases the activity of osteoblasts, inhibits
biomineralization, and increases the production of cartilage degrading enzymes in
chondrocytes. These pathological changes are seen in many bone related diseases,
such as RA, OA or osteoporosis.
TRAcP 5A’s clinical significance is not as well understood as 5B, but as it accounts
for the major part of TRAcP in serum it can be assumed to be effective, even though
it is not as potent an enzyme as the 5B form. 5A levels have been shown to correlate
with inflammation markers, such as CRP, ferritin, and triglycerides, and as the 5A
form is produced by macrophages it is suggested to be a special marker of their
activity (Janckila, Lederer, Price, & Yam, 2009). In recent research TRAcP 5A
levels have been correlated with chronic inflammation seen in cancer or in
metabolic syndrome (Y. Chen et al., 2015; Huang et al., 2017).
TRAcP is not only a marker of inflammation, but an active pro-inflammatory
regulator of cell function. However, TRAcP’s effects on inflammatory cells are not
as well understood as OPN’s. TRAcP null mice have been shown to have delayed
macrophage response to Staphylococcus aureus (Bune, Hayman, J Evans, & Cox,
2001) and impaired maturation of dendritic cells, which leads to deficient antigen
promotion, and thus impaired Th1 responses in T-cells (Esfandiari et al., 2006).
41
In conclusion, TRAcP is the main regulator of OPN phosphorylation and its
most notable effects are mediated through its effects on OPN’s phosphorylation.
However, TRAcP also has independent functions in bone and inflammation that are
not well understood yet, and more studies are needed on both the 5A and 5B forms
of the enzyme. Further studies of TRAcP may reveal even a new therapeutic
prospective.
2.6 Rheumatoid arthritis (RA)
Rheumatoid arthritis is a chronic autoimmune inflammatory disease that affects
synovial joints. The characteristic pathological changes seen in RA are synovial
tissue inflammation, cartilage degradation, subchondral bone erosions, and
increased production of inflammatory cytokines (Weissmann, 2004). Typically, RA
is described as a symmetrical inflammation of small or intermediate synovial joints,
e.g., metacarpophalangeal joints in hands or wrist joints. It causes a substantial
burden on both the society and the patient. The patient’s burden results from
musculoskeletal deficits, and disease related comorbidities, such as cardiovascular
diseases or osteoporosis, which result in decreased quality of life and can be
debilitating (Smolen, Aletaha, & McInnes, 2016). The socioeconomic burden is a
result of direct medical costs, but also a consequence of functional disability
leading to decreased work and societal participation (Smolen et al., 2016).
The incidence and prevalence of RA varies between geographic areas and time.
In North America and Northern Europe the annual incidence rate is 20-50 per 100
000 inhabitants, while in Southern Europe the incidence is lower, 9.5-36 per
100 000 inhabitants (Scublinsky & Gonzalez, 2016). In Finland the incidence rate
is 41.6 per 100,000 inhabitants (Kononoff et al., 2017). The worldwide prevalence
of RA is considered to be just under 1%, with lower values in Mediterranean and
Scandinavian countries, 0.19-0.94 % (Scublinsky & Gonzalez, 2016). In Finland
the prevalence in the adult population is around 0.8% (Aho, Kaipiainen-Seppänen,
Heliövaara, & Klaukka, 1998). RA is two to three times more common in females
than men, and after pregnancy the incidence is increased by 2.3 times for the next
two years (Wallenius et al., 2010).
The etiology of RA is unclear, but it is thought be affected by both genetic and
environmental factors. The heritability of RA is estimated to be around 60%, of
which the HLA-antigens contribute 11-37% (Kurko et al., 2013). The
environmental factors suggested to play a part in RA are smoking, low economic
status, periodontitis, infectious agents such as Proteus mirabilis, Escherichia coli
42
and Epstein-Barr virus, and changes in the bronchial or gut microbiome (Smolen
et al., 2016).
RA patients can be divided into two subgroups, by the level of rheumatoid
factor (RF) or anti-citrullinated protein antibodies (ACPAs), seropositive and
seronegative (Aletaha et al., 2010). The seropositive group is positive for either of
the biomarkers (Aletaha et al., 2010). The seropositive group is considered a more
homogenous group, due to a more similar disease etiology between the patients and
more aggressive symptoms (Aletaha et al., 2010). RA is associated with increased
mortality, especially from cardiovascular diseases (Dadoun et al., 2013). However,
with modern treatments the mortality has decreased close to the normal level today
(Listing et al., 2015), but RA still seriously affects an individual’s quality of life.
The pathogenesis of RA is not completely understood. The combinatory effect
of the environmental and the hereditary factors leads to the onset of the disease
(McInnes & Schett, 2011). The biomarkers of RA, such as ACPAs, RFs or increased
inflammatory markers have been shown to be circulating in the blood stream years
before the onset of the disease, especially in seropositive disease (Deane, 2014).
This condition is called preclinical RA, which is thought to be a result of altered
post-transcriptional modifications of different proteins, such as citrullination,
which leads to the generation of RA biomarkers (Deane, 2014). Various cells
implicated in RA pathogenesis, such as monocytes and osteoclasts, express the
citrullinated peptides on their cell surfaces, which are targeted by ACPAs (Shim,
Stavre, & Gravallese, 2018). This is suggested to lead to a loss of innate (e.g.,
macrophages, dendritic cells and neutrophils) and adaptive immunity (e.g., B- and
T-lymphocytes) tolerance (Calabresi, Petrelli, Bonifacio, Puxeddu, & Alunno, 2018;
McInnes & Schett, 2011). Later, an unknown trigger, an infection or other
environmental factor, leads to an onset of inflammation in the synovial tissue
(Calabresi, Petrelli, Bonifacio, Puxeddu, & Alunno, 2018; McInnes & Schett, 2011).
The joint swelling in RA is a result of the synovial tissue inflammation after
immune activation, which is characterized by leukocyte infiltration into the
synovial tissue. The inflammatory cells, such as T-cells and macrophages, produce
inflammatory cytokines, such as TNF-α, ILs and granulocyte-macrophage colony
stimulating factor (Gm-CSF), which lead to activation of synovial fibroblasts and
enhanced inflammatory cytokine production, which eventually leads to an
activation of osteoclasts (McInnes & Schett, 2011; Smolen et al., 2016). The
activation of osteoclasts leads to cartilage and bone damage, which causes a release
of pro-inflammatory substances and damage-associated molecular patterns
(DAMPs) (Orlowsky & Kraus, 2015). These in turn stimulate the innate immune
43
system and immune cells even more, further increasing the synovial tissue
inflammation, and thus the osteoclasts activity, creating a vicious circle of
inflammation (Orlowsky & Kraus, 2015). Also, the areas of inflammation are
hypoxic and acidic, which promotes osteoclastogenesis and bone resorption (Arnett,
2010; Yuan et al., 2016).
There are no absolute diagnostic criteria for RA. The diagnosis is done
according to the symptoms and classification criteria by the American College of
Rheumatology/European League Against Rheumatism (Aletaha et al., 2010). The
typical clinical symptoms of RA, which are used to diagnose RA are symmetrical
synovial joint inflammation, typically in small or intermediate size joints, morning
stiffness and soreness, increased inflammatory parameters, and elevated ACPA or
RF levels in seropositive RA (Aletaha et al., 2010). In progressed disease typical
bone erosions can be seen especially in smaller joints at the bone areas which are
in contact with the synovial tissue (Schett & Gravallese, 2012). The erosions can
be visualized with native x-ray imaging (Aletaha et al., 2010). In larger joints, such
as knees, the erosions are not as large in relation to smaller joints, but a rapid
progression of so-called secondary OA can be seen, as shown in figure 7. It is
related to inflammation and the mechanism is different than in normal OA.
44
Fig. 7. Native X-ray images of an RA patient’s knees. The patient was diagnosed with
RA in 2005. From 2009 to 2014 there has been a rapid progression of secondary OA in
the patient’s right knee. Due to the severity of the symptoms the patient underwent a
total knee replacement operation of the right knee.
After diagnosis anti-rheumatic immunosuppressive treatment is started
immediately. The goal of the treatment is remission of all symptoms within three
to six months (Rheumatoid Arthritis. Current Care Guidelines, 2015). The first line
of treatment suggested according to the Finnish Current Care Guideline is a
combination of methotrexate, sulfasalazine, hydroxychloroquine, small dose oral
glucocorticoid and local glucocorticoid injections in the affected joints. After a
long-sustained remission is reached the medication may be decreased slowly step-
by-step. Other conventional synthetic RA drugs include leflunomide, cyclosporin,
azathioprine, cyclophosphamide, mycophenolate and gold injections (Rheumatoid
Arthritis. Current Care Guidelines, 2015). If a combination of different
conventional synthetic drugs is not enough, a biological drug may be used
(Rheumatoid Arthritis. Current Care Guidelines, 2015; Smolen et al., 2017). A
45
biological drug can be combined with methotrexate for better effect (Smolen et al.,
2017). The biological drugs for RA have five mechanisms of action: TNF-α
inhibition, interleukin 6 or 1 receptor inhibition, T-cell co-stimulation blockade,
and B-cell depletion (Smolen et al., 2016). Use of all biological drugs significantly
increases the risk of serious infections (Smolen et al., 2017).
Operative treatments are considered if conservative treatments fail, and their
goal is to relieve pain and improve function (Nikiphorou, Konan, MacGregor,
Haddad, & Young, 2014). They mainly include synovectomies, arthrodesis and
joint replacement operations. Operative synovectomy is done when a joint becomes
so inflamed that its movement and function are impaired, and it does not respond
to conservative treatments and the cartilage is still relatively healthy (Rheumatoid
Arthritis. Current Care Guidelines, 2015; Trieb & Hofstaetter, 2009). If the
cartilage is destroyed and operative treatment is needed the choice is usually
between arthrodesis and joint replacement depending on the affected joint and the
patient’s personal needs (Rheumatoid Arthritis. Current Care Guidelines, 2015;
Trieb & Hofstaetter, 2009).
Extra-articular co-morbidities associated with RA include osteoporosis,
cardiovascular disease, interstitial lung disease, vasculitis, secondary amyloidosis
and lymphoma (Rheumatoid Arthritis. Current Care Guidelines, 2015; Smolen et
al., 2016). The co-morbidities are often believed to be a result of both the chronic
inflammation and adverse effects of the treatments (Clayton & Hochberg, 2013;
Smolen et al., 2016). The increased mortality seen in RA is due to the increased
risk of cardiovascular diseases (Chung et al., 2005). However, with successful
treatment the risk can be reduced as the risk caused by the chronic inflammation is
higher than that of the treatments (Chung et al., 2005). Also, the risk of secondary
osteoporosis can be reduced with effective treatment (Clayton & Hochberg, 2013).
2.6.1 Osteoimmunology in RA
In RA the balance of bone metabolism between catabolism and anabolism is
heavily shifted towards catabolism, which is seen in the form of inflammatory bone
erosions caused by increased osteoclast number and activity (Favero et al., 2015).
The inflammatory synovial tissue is heavily infiltrated by myeloid cells of the
monocyte-macrophage lineage, which can differentiate into osteoclasts under the
stimulation of M-CSF and RANKL (Favero et al., 2015). The inflamed synovial
tissue cells at the bone-synovium interface express RANKL (Pettit, Manning,
Walsh, Goldring, & Gravallese, 2006), and even spontaneous generation of
46
osteoclasts in synovial tissue and synovial monocyte cultures in vitro has been
reported (Greisen et al., 2015; Suzuki et al., 2001).
RANKL knockout mice are protected from bone erosions in inflammatory
arthritis models (Pettit et al., 2001), and it is a general consensus that RANKL is
the main factor behind the inflammatory osteoclastogenesis and increased
osteoclastic activity in RA (Favero et al., 2015; Okamoto & Takayanagi, 2019;
Shim et al., 2018). Other inflammatory factors, such as inflammatory cytokines and
proteins, affect osteoclast differentiation and activity either by driving the
inflammatory or synovial cells to produce RANKL, by affecting RANK/RANKL
signaling in the pre-osteoclasts or by direct cellular mechanisms (Favero et al.,
2015; Okamoto & Takayanagi, 2019; Shim et al., 2018). An example of factors that
can directly affect osteoclasts are ACPAs, which can directly bind into osteoclasts
or their precursors and increase their differentiation and activity, and high blood
ACPA levels correlate with aggressive disease phenotype and bone erosions (Harre
et al., 2015).
Most of the important cytokines increased in RA are also associated with
increased osteoclast activity, such as TNF-α, IL-6, IL-8, IL-17, VEGF etc.
(Amarasekara et al., 2018; Barra, Summers, Bell, & Cairns, 2014; Hampel,
Sesselmann, Iserovich, Sel, Paulsen, & Sack, 2013). Also increased pro-
inflammatory cytokine levels have been shown to correlate with the disease
severity (Barra et al., 2014; Hampel, Sesselmann, Iserovich, Sel, Paulsen, & Sack,
2013). However, as in all inflammation, there is also an active anti-inflammatory
component in RA, and many cytokines shown to be increased in RA are also
associated with decreased osteoclast function, such as IFN-α, IFN-γ, IL-3, Il-10,
IL-27 and IL-33 (Amarasekara et al., 2018; Khan, 2018; Lai et al., 2016; Pavlovic,
Dimic, Milenkovic, & Krtinic, 2016; Sellam et al., 2016; Zhao, Huang, & Zhang,
2016). Overall, the pro-osteoclastic stimulus is stronger, as can be seen in the
clinical bone erosions. However, because the RANKL effect on osteoclasts is so
much stronger than the other factors, it has been hard to evaluate the combined
effect of the other factors, even though the RA drugs target factors other than
RANKL.
In our study OPN is of special interest. OPN’s levels have been shown to be
elevated in serum, synovial fluid and urine of RA patients (Hasegawa et al., 2009;
Iwadate et al., 2013; Shio et al., 2010). Serum OPN levels have also been shown to
correlate with bone resorption markers, such as TRAcP 5B and C-terminal
telopeptide (CTX), and as such to correlate with disease severity (Iwadate et al.,
2013). OPN expression has been indicated in RA synovial tissue (Hasegawa et al.,
47
2009). OPN knockout mice suffering from inflammatory arthritis, were shown to
be protected from articular cartilage and inflammatory bone loss (Yumoto et al.,
2002). Overall, OPN plays a crucial role in the inflammatory joint diseases, such
as RA, but its clinical significance is not well understood yet.
In synovial tissue, overexpression of OPN during inflammation, especially in
synovial fibroblasts and T-cells, leads to further inflammatory cell activation, which
leads to elevated production of pro-inflammatory cytokines, including OPN itself
(G. Xu et al., 2005). In RA OPN is shown to upregulate Th1 and Th 17 T-cell
activation and function (G. Chen et al., 2010). Both Th1 and Th17 activation and
the related cytokine production activation are associated with the autoinflammatory
pathogenesis and increased bone degradation in RA (Bazzazi et al., 2018). Other
inflammatory cells that OPN activity directly increases are macrophages,
neutrophils and dendritic cells (K. X. Wang & Denhardt, 2008). Also, OPN directly
increases osteoclast differentiation, migration and activation, as previously
discussed in chapter 2.4 (Ek-Rylander & Andersson, 2010; Ross et al., 1993; Sodek
et al., 2000).
TRAcP levels have been shown to be elevated in RA (Iwadate et al., 2013;
Janckila, Neustadt, & Yam, 2008). As previously discussed in chapter 2.4, OPN’s
biological activity is dependent on its phosphorylation, which is controlled by
TRAcP. Interestingly, even though both TRAcP and OPN levels increase in RA, the
effect of TRAcP on OPN’s phosphorylation, and, thus on its biological activity has
not been assessed. In this study, one of our goals was to evaluate the
phosphorylation of OPN in RA.
The therapeutic properties of anti-OPN treatments have been investigated and
are under research in RA animal models, and some results have been encouraging
(Zhang, Luo, Li, Gao, & Lei, 2015), but more studies are necessary for further
clarification of OPN’s role in RA and the development of OPN-targeted RA therapy.
2.7 Osteoarthritis (OA)
Osteoarthritis is the most common joint disease in the world. It affects 10% of men
and 18% of women over 60 years of age and causes a huge socioeconomical burden
(Woolf & Pfleger, 2003). In Finland the age adjusted prevalence of knee OA is 6.1%
in men and 8.0% in women over 30 (Arokoski et al., 2007). In the age group 75-
84, 15.6% of men and 32.1% of women have knee OA (Arokoski et al., 2007).
Clinical knee OA is rare in people under 45 years, as in the age group 30-44 the
prevalence is only 0.3% in men and 0.4% in women (Arokoski et al., 2007).
48
Traditionally OA has been considered to be a result of prolonged mechanical
wear on joint articular surfaces, which results in a loss of articular cartilage,
pathological changes in subchondral bone areas and reactive synovial tissue
inflammation. However, advances in modern research have revealed that OA is
actually a very complex disease of the whole joint associated with genetic,
biological, and biomechanical components, and the etiological factors are joint
specific (Glyn-Jones et al., 2015). Most often OA affects weight bearing joints,
such as knees and hips, or smaller joints, such as finger joints that are extensively
used, but any synovial joint in the body can be affected. To date the fundamental
etiology of OA is unknown.
The main risk factor of OA is age, as the other primary factors that cause OA
must generally affect over a longer period of time. Factors that have been associated
with the risk of OA are female gender after menopause, obesity, genetics, diet,
trauma, abnormal or extensive loading of joints and structural abnormalities
(Georgiev & Angelov, 2019; Palazzo, Nguyen, Lefevre-Colau, Rannou, &
Poiraudeau, 2016). Interestingly, obesity does not only increase the risk of OA in
weight bearing joints but also in hands, indicating that obesity has metabolic and
inflammatory systemic effects also on the joints (Palazzo et al., 2016). The genetic
factors have been suggested to account for 60% of hand and hip OA and 40% of
knee OA (Palazzo et al., 2016). Diseases that increase the risk of OA are generally
associated with metabolic syndrome, such as diabetes, cardiovascular diseases and
depression (Georgiev & Angelov, 2019). OA is not associated with increased risk
of osteoporosis (Clayton & Hochberg, 2013; Im & Kim, 2014).
In early OA, the synovial cells are triggered to produce inflammatory cytokines
and mediators, which activate the chondrocytes and make them to produce cartilage
degrading enzymes, such as MMPs, and more inflammatory cytokines, such as IL-
1β, IL-6 and TNF-α (Glyn-Jones et al., 2015; Sulzbacher, 2013). The inflammatory
mediators in cartilage can ultimately cause chondrocyte hypertrophy and apoptosis
(Sulzbacher, 2013). The cartilage degradation causes a release of pro-inflammatory
substances and DAMPs into the synovial fluid. This in turn stimulates the innate
immune system and immune cells in the synovial tissue, which further increases
the synovial tissue inflammation. The circle of inflammation in OA joint very much
like that seen in RA, but in OA the inflammation is generally significantly weaker
(Orlowsky & Kraus, 2015). Along with the cartilage degradation and the synovial
tissue inflammation, OA is characterized by changes in the subchondral bone
remodeling and resorption in the form of local subchondral bone sclerosis, and by
the formation of osteophytes and cysts. Osteophytes are exostoses that form along
49
the joint margins. In OA, the production of RANKL and various other signals that
affect osteoclasts are also upregulated in synovial tissue and their levels increased
in synovial fluid (Hampel, Sesselmann, Iserovich, Sel, Paulsen, & Sack, 2013;
Pilichou et al., 2008). The changes in the subchondral bone start already in early
OA and can advance throughout the disease (Glyn-Jones et al., 2015; Sulzbacher,
2013). In advanced OA, the articular cartilage can be completely degraded and the
joint may become ankylosed (Kellgren & Lawrence, 1957).
The most characteristic symptom of OA is joint pain, which typically is worse
during exercise and eases at rest, but in advanced disease the pain can become
continuous even at night (Knee and Hip Osteoarthritis. Current Care Guidelines,
2018). Morning stiffness of the affected joints, especially in the hands is also typical
(Knee and Hip Osteoarthritis. Current Care Guidelines, 2018). In advanced disease
a joint’s range of motion can become decreased. The stronger the symptoms are the
more they limit the patient’s daily activities and severe OA symptoms can be
debilitating.
The typical bone changes in OA are increased cortical bone thickness,
deformation and flattening of the articular shape, decreased mass of subchondral
trabecular bone, bone marrow lesions, subchondral cysts, tidemark duplication and
osteophyte formation (Favero et al., 2015). Bone marrow lesions are subchondral
bone oedema, so-called bone bruises, which can be seen with magnetic resonance
imaging in early OA. (Alliston, Hernandez, Findlay, Felson, & Kennedy, 2018).
Tidemark duplication means the advancement of the calcified cartilage further
away from bone in OA (Alliston et al., 2018). The pathological bone changes in
OA are either osteolytic or sclerotic, which gives OA two types of phenotypes,
osteoporotic or osteosclerotic, that can both co-exist in the same patient even at
different parts of the same joint (Favero et al., 2015). Subchondral bone turnover is
increased 20-fold in OA (Bailey, Mansell, Sims, & Banse, 2004). The increased
subchondral turnover is a result of increased osteoclastic and osteoblastic activity,
which are thought to be results of increased mechanical stress and inflammation in
the joint (Hügle & Geurts, 2016). However, in OA the subchondral bone is
hypomineralized and of lesser quality compared to normal subchondral bone
(Bailey et al., 2004).
The diagnosis of OA is done from native x-ray images (Knee and Hip
Osteoarthritis. Current Care Guidelines, 2018). The radiologic findings in OA are
joint space narrowing, osteophytes, subchondral bone sclerosis and bone
deformities in advanced disease (Kellgren & Lawrence, 1957). The most common
radiological grading used for staging OA is the Kellgren-Lawrence grade, which
50
grades the radiological severity of OA as grades I-IV according to the above
radiologic changes (Kellgren & Lawrence, 1957).
As the etiology of OA is unknown and the pathogenesis is not completely
understood, there are no specific curative or disease progression preventing
treatments available. The treatments are focused on managing the symptoms and
improving the patients’ quality of life. Conservative drug free treatments are the
basis of OA treatments. Lifestyle changes that promote healthy lifestyle, exercise
and weight loss are recommended for each patient (Glyn-Jones et al., 2015; Knee
and Hip Osteoarthritis. Current Care Guidelines, 2018). Physical or manipulative
therapy may be used to help symptoms (Knee and Hip Osteoarthritis. Current Care
Guidelines, 2018). Joint pain can be treated with paracetamol, non-steroidal anti-
inflammatory drugs, intra-articular corticosteroid or hyaluronate injections, and
opioids according to the severity of symptoms (Knee and Hip Osteoarthritis.
Current Care Guidelines, 2018). Surgical treatments, such as prosthesis operation,
osteotomies to correct a joint’s mechanical axis or osteosynthesis can be performed
in patients with progressed disease and difficult symptoms according to the
required standard of function and affected joint (Glyn-Jones et al., 2015; Knee and
Hip Osteoarthritis. Current Care Guidelines, 2018). Biological drugs, such as anti-
TNF-α, used for RA have been suggested and tested as new drugs for OA, but they
have shown only mild benefit and are not recommended to be used at present, while
further studies on biological drugs are conducted (Dimitroulas, Lambe, Klocke,
Kitas, & Duarte, 2017).
2.7.1 Osteoimmunology in OA
The synovial inflammation seen in OA is very much like that seen in RA or other
inflammatory arthritis (Dar, Azam, Anupam, K Mondal, & Srivastava, 2018; Hügle
& Geurts, 2016). In OA, as in RA, there is also an abundance of inflammatory
monocytes/macrophages in the synovial tissue (Mathiessen & Conaghan, 2017).
As earlier discussed, it is generally thought that some triggering event causes an
activation of the innate immunity in the synovial tissue, which leads to the
activation of the inflammatory cells (Orlowsky & Kraus, 2015). Various studies
have revealed that the inflammatory T-cell subtypes activated in OA resemble the
ones activated in RA, with elevated percentages of Th1, Th9 and Th17 and
decreased T-regulatory cells in OA patients as compared to healthy controls (Dar et
al., 2018). The activation of inflammatory cells and synovial cells due to
inflammation leads to the production of inflammatory cytokines and related
51
proteins. The proinflammatory cytokines most importantly associated with OA are
IL-1β, TNF-α, IL-6, IL-15, IL-17, and IL-18 (Wojdasiewicz, Poniatowski, &
Szukiewicz, 2014). The production of RANKL in the synovial cells and its
concentration in synovial fluid is also increased in OA, when compared to healthy
controls (Monasterio et al., 2018; Pilichou et al., 2008). As in all inflammation, also
increases of anti-inflammatory cytokine levels, most notably IL-4, IL-10, and IL-
13, are seen in OA (Wojdasiewicz et al., 2014). Overall, the combined effect of
increases of inflammatory cytokines along with the increased RANKL levels
increase osteoclast differentiation and activity in a similar fashion as in RA.
However, the strength of the inflammation and the levels of inflammatory cytokines
in RA are generally significantly higher than in OA (Hampel, Sesselmann,
Iserovich, Sel, Paulsen, & Sack, 2013). Thus, their effect on bone cells is stronger
and the changes seen in the bone often happen faster in RA and are more clearly
seen at locations in contact with the synovial tissue, especially in smaller joints. In
OA, the bone turnover is increased in wider areas throughout the joint and the
clinical changes seen in bone generally develop over a longer time.
OPN levels have been shown to be elevated in both the synovial fluid and the
serum of OA patients when compared to healthy controls (Cheng, Gao, & Lei,
2014), and increased OPN levels have been shown to correlate with the severity of
joint lesions and inflammation in OA (Gao et al., 2010; Honsawek, Tanavalee,
Sakdinakiattikoon, Chayanupatkul, & Yuktanandana, 2009). The synovial
fibroblasts have been shown to produce OPN in OA (Zhang et al., 2013). The
increased OPN levels in the OA synovial tissue are likely to function in a similar
fashion as in RA, where they increase the inflammatory cell function, especially
Th1 and Th 17 cells (G. Chen et al., 2010). This would also result in the increases
of the cytokines associated with OA. OPN has also been suggested to directly
interact with the chondrocytes and cause a release of cartilage degrading MMPs
(Cheng et al., 2014). Interestingly, OPN deficient mice have been shown to be more
prone towards joint instability and aging related OA (Matsui et al., 2009), even
though OPN knockout mice suffering from inflammatory arthritis were shown to
be protected from the articular cartilage and the inflammatory bone loss (Yumoto
et al., 2002), which may indicate an inadequate response of bone and cartilage cells
towards mechanical stress.
The expression of OPN’s phosphatase, TRAcP, in the damaged chondrocytes
and concentration in the synovial fluid of OA patients has been shown to be
increased (Seol et al., 2009). The phosphorylation of OPN in synovial fluids of OA
patients has not been assessed. However, increased phosphorylation of OPN in the
52
articular cartilage has been shown, when compared to healthy cartilage, and the
increased phosphorylation of OPN to lead to increased MMP production in the
chondrocytes (Xu et al., 2013).
Anti-OPN treatments for OA have been suggested, and they have been shown
to possibly decrease inflammation, cartilage degradation and bone changes (Cheng
et al., 2014), but elucidating the therapeutic aspect of OPN needs further studies
and better knowledge of OPN’s role in the pathogenesis of OA.
53
3 Aims of the study
Osteoimmunology is a field of study which focuses on the connection of the bone
and the human immune system. Rheumatoid arthritis and osteoarthritis are
common joint diseases which cause enormous amounts of human suffering and
huge economical burdens throughout the world. Their pathogenesis is not fully
understood, and we are limited to treating the symptoms of the diseases as no
curative treatment is known. The etiology and pathogenesis of the diseases are
different, but both are associated with changes in bone metabolism and bone
destruction. To study how the diseases affect bone metabolism, one must know how
the bone cells function. Therefore, the basic functions of osteoclasts and different
proteins that affect them were studied, along with the effect of the studied diseases
on osteoclasts.
The specific aims of the study were to examine:
1. The phosphorylation of OPN in RA synovial fluid.
2. Secretion of OPN by human osteoclasts into the resorption pits on human bone
during resorption.
3. The effect of RA and OA patients’ synovial fluid and serum on
osteoclastogenesis and bone resorption in vitro.
54
55
4 Materials and methods
The methods used in this thesis are summarized in the table 1 below and described
in detail with references in the original articles I-III.
The patient samples for the studies were collected in Oulu University Hospital.
All the patients were informed of the study and gave a written approval for the use
of their samples. The healthy control samples were collected from healthy informed
volunteer donors. The protocol followed the Helsinki Declaration principles in full,
and the Northern Ostrobothnia Hospital District Ethical Committee provided an
approval for the study and tissue collection. The process of sample and material
acquisition for each individual study is described in detail in the original articles I-
III. The number of patient samples for each study are listed in the results. Other
materials used are described in the original articles.
For study I, synovial tissue biopsies and fluid were obtained from RA and OA
patients. The phosphorylation of OPN in synovial fluid was analyzed using the
western blot method and the concentrations of TRAcP 5A and 5B forms were
analyzed using an enzyme-linked immunosorbent assay (ELISA).
Immunohistochemistry and light microscopy were used to assess OPN and TRAcP
5A/5B forms in the synovial tissue.
In study II, human peripheral blood and bone marrow derived osteoclasts were
cultured on human bone and carbonated hydroxyapatite slices. Bone resorption and
OPN were quantified from the surface of the resorbed slices using field emission
scanning electron microscopy (FE-SEM) and gold labeling of OPN. A variety of
fluorescently labeled lectins were used to stain the glycan epitopes present in the
resorption pits. Mass spectrometry was used to analyze the proteomics of the
resorbed bone slices. The effect of enzymatic removal of α2,3 and α2,6 sialic acids
from the surface of pre-resorbed bone slices on osteoclasts was also analyzed.
Finally, immunohistochemistry of osteoarthritic femoral heads was done to analyze
the localization of OPN and TRAcP in sites of increased bone metabolism in vivo.
In study III, human peripheral blood monocyte derived osteoclasts, obtained
from a healthy donor, were cultured on bone slices with the synovial fluid samples
acquired from RA and OA patients undergoing knee surgery, and with serum from
untreated RA patients collected at the time of diagnosis and healthy volunteer
controls. Supraphysiological RANKL concentrations were used to evaluate the
effect of other inflammatory factors. The osteoclast differentiation and the bone
resorption were evaluated using light and laser microscopy. The concentrations of
27 different cytokines and related proteins were analyzed from the RA and OA
56
patient synovial fluids, and the untreated RA patient and the healthy control serums
with a multiplex assay. The synovial fluid OPG and RANKL concentrations were
analyzed using a simplex assay.
Table 1. Methods. The materials used are explained in each study.
Level Method Used in
Protein Western blot I
ELISA (Enzyme-linked immunosorbent assay) I, III
Multicytokine assay III
Cells and tissues Cell culture II, III
Preparation and staining of tissue samples and cells I, II, III
Mass spectrometry II
Electron microscopy II
Laser microscopy III
Confocal microscopy II, III
Light microscopy I, II, III
Other Statistical analyses I, II, III
The statistical analyses for the articles were done using IBM SPSS Statistics
versions 20 and 24 (IBM, NY, USA). A specialist statistician was consulted about
the analyses. Depending on the distribution of the values, parametric or non-
parametric tests were used. In study I non-parametric Mann-Whitney U-test and
Spearman’s correlation were used due to the low number of study subjects and non-
normal distribution of results. In study II the results were normally distributed, and
student’s t-test was used. In study III nonparametric tests, Independent Samples
Median Test and Mann-Whitney U test, and Fisher’s exact test for nominals were
used due to the low number of samples. In all studies a p-value less than 0.05 was
considered statistically significant.
57
5 Results
5.1 Analyses of OPN and TRAcP in RA synovial fluid and tissue (I)
Synovial fluid and tissue biopsies were acquired from RA and OA patients during
knee prosthesis operations. OPN’s phosphorylation level and the concentrations of
TRAcP 5A and 5B forms were analyzed from the synovial fluid, and
immunohistochemistry was conducted to show the presence of OPN and TRAcP in
synovial tissue. The hypothesis was that OPN is more phosphorylated in RA
synovial fluid, which leads to the stronger immune cell and osteoclast activation as
seen in RA when compared to OA. The samples analysed are shown in table 2.
Table 2. Patient samples analyzed in the study I.
Study Sample n
OPN in synovial fluid (Fig 1A, B and C) seropositive RA patient synovial fluid 9
seronegative RA patient synovial fluid 7
OA patient synovial fluid 5
TRAcP 5A/5B in synovial fluid (Fig 2D, E and F) seropositive RA patient synovial fluid 14
seronegative RA patient synovial fluid 10
OA patient synovial fluid 24
Synovial tissue immunohistochemistry (Fig 3) Representative RA biopsies 5
Representative OA biopsies 16
In study I tissue biopsies and synovial fluids were taken from the same patients, but
unfortunately many of the biopsies, especially the samples from RA patients, were
of poor quality and the number of representative biopsies that could be analyzed
was reduced. The difference in the number of samples analyzed for OPN and
TRAcP is due to the low volume and availability of samples at the time of analysis.
5.1.1 Phosphorylation of OPN in synovial fluid (I)
The western blotting method was used to analyze the level of OPN’s
phosphorylation in the synovial fluids of OA and RA patients. Full-length OPN was
significantly more phosphorylated in both seropositive and seronegative RA
patients’ synovial fluid, when compared to OA patients’ synovial fluid (Fig 1A in
I). The total levels of OPN did not differ between the patient groups (Fig 1B in I).
58
We also showed that the thrombin cleaved C-terminal end of the OPN fragment
was similarly more phosphorylated in both RA groups when compared to OA (Fig
1C in I).
5.1.2 TRAcP 5A and 5B concentrations in synovial fluid (I)
To explain the differences seen in OPN’s phosphorylation ELISA was used to
measure the concentrations of TRAcP 5A (Fig 1D in I) and 5B (Fig 1E in I) forms
from the synovial fluid samples of RA and OA patients. The overall combined
amount of both TRAcP forms in the synovial fluids was the same between all
sample groups; seropositive and seronegative RA, and OA (Fig 1F in I). However,
in the seropositive RA samples the ratio of TRAcP 5B/5A forms was decreased
when compared to OA samples. Seropositive RA patients had more less active 5A
than active 5B form (mean TRAcP 5B/5A ratio 0.714±0.259). The OA synovial
fluids had more active 5B than less active 5A form (mean TRAcP 5B/5A ratio
1.384±0.275) (Fig 2A in I).
When comparing the samples in which the TRAcP 5B/5A ratio and the
phosphorylation of OPN were both measured, a significant negative correlation
between the phosphorylation of OPN and the TRAcP 5B/5A ratio was found (Fig
2B in I). A decreased TRAcP 5B/5A ratio led to increased phosphorylation of OPN
in RA synovial fluid.
5.1.3 Localization of OPN and TRAcP in RA and OA patient synovial
tissue (I)
OPN was localized mostly in the extracellular matrix in the synovial tissue samples.
The staining for OPN was the most intense in the sublining layer of synovial tissue,
with lighter staining present in the lining layer of the joint cavity and deeper stroma
(Table 2, Fig 3J and K in I). Areas containing large quantities of inflammatory cells,
such as lymphocytes and macrophages, also showed strong intracellular staining.
No differences in staining were found between RA and OA samples (Table 2 in I).
Visualization of TRAcP in synovial tissue was done at the same time with two
different antibodies, one binding both 5A and 5B forms (Fig 3A, B, E and F in I)
and the other capable of binding specifically the TRAcP 5A form (Fig 3C, D, G and
H in I). Staining for both antibodies was strongest at the synovial lining layer and
weakest at the sublining layer. Average staining was present also in the deeper
stroma. The staining of the sublining layer was slightly stronger with the TRAcP
59
antibody binding both forms in OA than in RA samples: no other differences were
found between RA and OA samples (Table 2 in I). This small difference may
indicate that there is more TRAcP 5B in the sublining layer of OA synovial tissue
when compared to RA, which would be in accord with the measured TRAcP 5B/5A
ratio differences between OA and RA synovial fluid samples. However, this is only
speculative based on the histological samples, and no definite conclusions can be
drawn. No unspecific staining was seen in control samples, for which the staining
protocol was done without the primary antibodies, as shown in figure 8.
Fig. 8. Synovial tissue section staining without primary antibody. No unspecific
staining was seen.
An EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse, HRP.
Rabbit/Mouse (DAB+) kit was used according to the manufacturer’s instructions
for the stainings, and the samples were studied visually under a light microscope.
60
5.2 Analyses of OPN in resorption lacunae (II)
The effects of extracellular OPN on osteoclasts are far better known than those of
endogenous osteoclast OPN. Earlier studies have suggested that osteoclasts deposit
OPN into the resorption pit during bone resorption to facilitate their attachment into
the resorbed matrix, as explained in chapter 2.3. However, no previous studies with
human derived cells on human bone have been conducted to investigate OPN’s
deposition by osteoclasts.
In study II, human peripheral blood and bone marrow derived osteoclasts were
cultured on human bone and carbonated hydroxyapatite slices to study the
deposition of OPN by the osteoclasts during resorption. Carbonated hydroxyapatite
is a material that osteoclasts can resorb, but which is free of pre-deposited proteins
to avoid confounding by revelation of pre-deposited bone osteopontin during bone
resorption.
The glycan epitope at the bottom of the resorption pit was also studied to reveal
other signals present after resorption. Proteomics analysis of the resorbed bone
slices was conducted to distinguish and measure the proteins increased in the
resorbed bone.
Table 3. Samples analysed in study II.
Study Sample n
FE-SEM detection of OPN in resorption pit Resorbed bone slices 4
(Fig 1 in II) Resorbed carbonated hydroxyapatite slices
Total number of resorbed slices
2
6
Proteomics analysis of resorbed bone slices Resorbed bone slices 2
(Fig 3 in II) Number of mass spectrometry runs per slice 6
Lectin stainings of resorbed bone slices Resorbed bone slices per lectin 3
(Fig 2 a-l in II)
Osteoclast differentiation on enzymatically treated
human bone slices (3 cultures from different bone
marrow donors) (Fig 2 m-n in II)
Total number of resorbed bone slices
Resorbed bone slices
Un-resorbed bone slices
21
9+6+4
9+6+4
5.2.1 Detection of OPN in resorption lacunae using FE-SEM (II)
FE-SEM and immunodetection of OPN with gold particles were used to study the
deposition of OPN into the resorption pits on human bone and carbonated
hydroxyapatite by human osteoclasts. Actively resorbing osteoclasts (Fig 1a in II),
both bone marrow and peripheral blood monocyte derived, created multiple
61
resorption lacunae on both the bone (Fig 1c and e in II) and the hydroxyapatite (Fig
1b and d). OPN was significantly enriched in the resorption pits on both resorbed
substrates indicating that osteoclasts deposit OPN into the resorption lacunae
during resorption (Fig 1e, d and f in II). On bone, the number of OPN particles in
the electron microscope view with 20k magnification inside the pit was increased
2.0-3.7 times and on carbonated hydroxyapatite 2.8-12 times. The real difference
is likely larger as the bottom of the resorption pit is not even and the area on which
the electron microscope can focus is reduced as seen in Fig 1d and e in II. Due to
the low number of samples that it was possible to produce for analysis at a single
time, the study was repeated with similar results.
5.2.2 Analysis of proteins enriched in the resorbed bone slices (II)
Proteomics analysis of the resorbed bone was conducted using mass spectrometry
to distinguish the proteins that are enriched in the resorbed bone, and to measure
how much the proteins are enriched. The cells were mechanically removed from
the bone slice before the analysis. Un-resorbed bone slices were used as controls.
Multiple proteins where found enriched, of which OPN was the most enriched
(Fig 3b in II). Along with OPN, the proteins that were notably enriched in the
analyzed bone slice samples were prolargin, asporin and mimecan. OPN was
increased ten fold, prolargin and asporin seven fold, and mimecan four fold. OPN’s
phosphorylation was not studied, and it may be possible that heavily
phosphorylated forms of OPN may not have been detected.
5.2.3 Analysis of glycan epitope in the resorption lacunae (II)
The glycan epitope at the bottom of resorption pits on human bone resorbed by
human bone marrow and peripheral blood derived osteoclasts in vitro was studied
using different test plant lectins and light microscopy (Fig 2a-l in II). The cells were
mechanically removed from the bone slices before the analyses. An abundance of
α2,3 and α2,6 -linked sialic acids were found at the bottom of resorption lacunae.
The sialic acid residues were the highest in O-glycans. OPN is an O-glycosylated
α2,3 and α2,6 -sialic acids containing protein, and being the most enriched protein
in resorbed bone slices, it is most likely the protein presenting the sialic acids the
lectins bind.
When the α2,3 and α2,6 sialic acids were enzymatically removed from the
resorbed bone slice surface and osteoclasts re-cultured on them, the number of
62
osteoclasts was doubled, when compared to cultures where the sialic acids were
still present on the surface of the pre-resorbed bone slices (Fig 2m and n in II). The
α2,3 and α2,6 sialic acids containing protein, such as OPN, present at the resorption
pits decreased osteoclastogenesis on already resorbed bone slices.
5.3 Immunohistology of OPN and TRAcP in the areas of increased
bone metabolism in OA (II)
To relate the in vitro data of how the osteoclasts produce OPN during bone
resorption to in vivo conditions, immunohistochemistry was conducted to show the
presence of OPN and TRAcP in the areas of increased bone metabolism and
damage in osteoarthritic human cadaver femoral head samples.
Altogether biopsies were acquired from 32 femoral head samples. The biopsies
were taken from the damaged cartilage and sclerotic bone underneath it, trabecular
bone and cortical bone of the femoral neck. The samples were stained using an
EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse, HRP. Rabbit/Mouse
(DAB+) kit according to the manufacturer’s instructions and the samples were
visually quantified under a light microscope.
OPN was present in the extracellular matrix of all bone areas. However, the
staining was most intense in the osteoarthritic sclerotic bone areas, where bone
metabolism is increased, as expected, due to increased osteoclast activity (Table 2
in II).
Two different TRAcP antibodies were used. One specific for TRAcP 5A and
the other binding both 5B and 5A forms. TRAcP was also found in the extracellular
bone matrix. Both TRAcP antibodies stained the cortical bone slightly more than
sclerotic bone or trabecular bone, as shown in figure 9 (Table 2 in II). No
differences between the antibodies were seen in bone.
63
Fig. 9. TRAcP 5A + 5B localization in the cortical bone (A) in the femoral neck and in the
trabecular bone (B) in the OA femoral head.
64
Interestingly, as the samples also contained sections of damaged cartilage, staining
of the tight junction line between the non-calcified and the calcified cartilage was
TRAcP positive (Fig 4c and d in II). The staining was equally strong with both
TRAcP antibodies. When using the OPN antibody the line itself did not stain, but
intense staining was found in the calcified cartilage (Fig 4b in II). In non-calcified
cartilage the OPN stain would fade out further away from the tight junction. As a
control the staining protocol was done without the primary antibodies. No
unspecific staining was seen, as shown in figure 10.
Fig. 10. OA femoral head section section staining without primary antibody. No
unspecific staining was seen in cartilage, bone or bone marrow.
5.4 Analyses of the effect of RA serum and synovial fluid on
osteoclastogenesis and bone resorption (III)
To evaluate the combined effect of all inflammatory factors present in RA patients’
serum and synovial fluid samples, human peripheral blood derived osteoclasts were
cultured with them to study their effect on osteoclastogenesis and bone resorption.
65
Healthy volunteers’ serum and OA patients’ synovial fluid were used as controls.
The number of osteoclasts on bone slices was counted after the culture period and
their morphology analyzed under a light microscope, and the resorbed volume of
bone slices was analyzed with a laser microscope.
In study I, OPN and its phosphorylation were analyzed with TRAcP 5A/5B
form concentrations. In study III, a multiplex analysis was conducted to quantify
other inflammatory factors in RA and OA that affect osteoclasts.
The number of samples used for each study are shown in table 3. The synovial
fluids were pooled due to the low volume of samples available and known high
variation of quality and biological diversity, to obtain standardized RA and OA
synovial fluids that represents the diseases in general and not the situation only
within a single joint.
Table 4. Patient samples analyzed in the study III.
Study Sample n
Osteoclast culture (Fig 1, 2 and Table 1 in III) seropositive RA patient synovial fluid 3 pooled
OA synovial fluid 3 pooled
RA serum 3
healthy serum 3
Multicytokine assay (Fig 3 and 4 and Supplement 1 in III) seropositive RA patient synovial fluid
OA patient synovial fluid
10
10
RA serum 6
Healthy serum 9
5.4.1 Effect of novel untreated RA patient serum on
osteoclastogenesis and bone resorption
Untreated RA patient serum increased the number of osteoclasts and volume of
resorbed bone significantly when compared to healthy control serum (Fig 1A in III).
The number of osteoclasts and the volume of resorption increased in the same ratio,
(~1.5 times) indicating that the increase in resorption was a direct result of
increased osteoclastogenesis (Fig 1C in III). No differences were seen in osteoclast
morphology in cultures with serums.
66
5.4.2 Effect of RA and OA patients’ synovial fluid on
osteoclastogenesis and bone resorption
RA synovial fluid increased the osteoclastogenesis (~6 times) and the volume of
resorbed bone (~8 times) significantly when compared to healthy serum (Fig 1B
and D in III). Interestingly, OA synovial fluid increased the number of osteoclasts
and the resorption significantly more than RA synovial fluid (Fig 1B and D in III).
OA roughly doubled the number of osteoclasts and resorption, when compared to
RA. As in the serum assay, the increases in the bone resorption were a direct result
of the increased number of osteoclasts.
Synovial fluid changed the osteoclasts’ morphology (Fig 2 and Table 1 in III).
Both synovial fluids increased the osteoclasts’ size and the number of nuclei
significantly when compared to the healthy serum. However, the cells cultured with
OA synovial fluid were even larger, had more nuclei and were irregularly shaped
when compared to cells cultured with RA synovial fluid. On average the cells
cultured with healthy serum had two or three nuclei, the ones cultured with RA
synovial fluid had four to seven nuclei and the ones cultured in the presence of OA
synovial fluid had more than eight nuclei. Interestingly, the change in phenotype
did not affect the osteoclasts’ resorption capacity.
5.4.3 Cytokine analyses of the patient samples
To distinguish the factors behind the changes seen in the osteoclastogenesis and the
bone resorption 27 different cytokine and related protein concentrations (IL-1-β,
IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-12, IL-13, IL-15, IL-17, eotaxin, FGF, granulocyte colony stimulating factor (G-
CSF), granulocyte-macrophage colony stimulating factor (Gm-CSF), IFN-γ, IP-10,
MCP-1, MIP-1-α, MIP-1-β, platelet-derived growth factor (PDGF-BB), RANTES,
TNF-α, and VEGF) were analyzed using a multicytokine assay kit from the
synovial fluids and serums. RANKL and OPG concentrations were also analyzed
from the synovial fluid samples.
In RA serum multiple cytokines associated with RA were found increased,
many of which are associated with increased osteoclastogenesis, thus explaining
the cell culture result. Significantly increased concentrations of IL-6 (3.5 fold
increase), IFN-gamma (1.5 fold increase), IP-10 (4.5 fold increase) and IL-9 (4.5
fold increase) were found in RA serum, and other cytokines that were considerably
increased in RA serum but did not reach statistical significance due to the wide
67
distribution of results were IL-1ra (5 fold increase), TNF-α (2 fold increase),
RANTES (3 fold increase), IL-1b (5.5 fold increase), IL 13 (3 fold increase), MCP1
(4.5 fold increase) and IL-7 (5.5 fold increase) (Fig 3 and 4 in III).
In both OA and RA synovial fluids all tested cytokine concentrations were
generally increased when compared to either tested serum. When comparing the
synovial fluids, an increase of almost all cytokines in RA was found. As expected,
the most increased cytokines were associated with RA and increased
osteoclastogenesis, as in serum samples. Statistically significantly increased
cytokines between RA and OA synovial fluids were IL-1ra (85 fold increase), IL-
1b (10 fold increase), MCP-1 (30 fold increase), IL-8 (70 fold increase), IL-10 (15
fold increase), IL-15 (10 fold increase) and IL-17 (10 fold increase), and other
cytokines that were considerably increased but did not reach statistical significance
due to the wide variation in the measurements were MIP1b (30 fold increase), IL-
6 (10 fold increase), IFN-gamma (10 fold increase), GMCSf (10 fold increase),
TNF-α ( 30 fold increase), RANTES (5 fold increase) and IP-10 (40 fold increase)
(Fig 3 and 4 in III).
It is noteworthy, that in the serum samples the mean changes in the cytokine
concentrations were approximately one to six-fold, while in the synovial fluids the
changes in the cytokine concentrations were considerably larger, from five to
eighty-fold.
No differences were found in OPG concentrations in the synovial fluids of RA
and OA patients. Unfortunately, in all samples, all but one RA RANKL measure
fell under a detection level of 7.04pg/ml. However, this indicates that the 20ng/ml
concentration used in cell cultures is supraphysiological. Thus, the natural RANKL
in the samples does not cause confounding effect and the differences seen in the
osteoclast differentiation and the bone resorption are a result of other cytokines
present in the samples.
68
69
6 Discussion
During this millennium the interaction between the bone and the immune system
has proven to be crucial in the pathogenesis of RA and OA. New discoveries in the
field of osteoimmunology have led to the rapid development of new biological RA
drugs, which have proven very effective. However, these diseases are still often
debilitating for the patients and cause an enormous socioeconomical burden. New
treatment targets and treatments are sought rigorously to improve the patients’
quality of life.
In this thesis osteoclast function and factors that affect it in RA and OA were
studied. Especially OPN and its phosphorylation, which is controlled by TRAcP,
was studied in detail. Also, to understand the function of the osteoclasts in disease
one must better understand their function in the normal environment, hence basic
research on bone resorption by osteoclasts in a normal controlled environment was
conducted.
The phosphorylation of OPN was found to be increased in RA in study I, which
may lead to increased inflammatory cell and osteoclast activation along with the
other inflammatory factors, as shown in study III. In study II, the deposition of
OPN into the resorption pits on human bone by human osteoclasts was shown. In
the resorption pit OPN may affect mineralization and act as a signal for other cells
in the normal situation, but also in disease.
The results are discussed in detail in the next chapters. The clinical significance
of these new discoveries is to be discovered in future studies.
6.1 Decreased TRAcP 5B/5A ratio leads to increased
phosphorylation of OPN in RA synovial fluid.
In study I, the TRAcP 5B/5A isoenzyme concentration ratio’s relation to the OPN’s
phosphorylation was assessed in OA and RA synovial fluid. It was evident that the
decreased TRAcP 5B/5A ratio correlated with the increased phosphorylation of
OPN in RA. Earlier increased levels of OPN have been associated with a more
severe inflammatory disease phenotype (Gao et al., 2010; Iwadate et al., 2013; Shio
et al., 2010). However, the phosphorylation of OPN in inflammatory diseases has
not been studied before, even though it is the key regulator of OPN’s functions,
such as its role as a proinflammatory cytokine or inhibitor of mineralization
(Addison, Masica, Gray, & McKee, 2010; Ek-Rylander & Andersson, 2010;
Nyström et al., 2007; Sainger et al., 2012; L. Wang et al., 2008; Weber et al., 2002).
70
OA patient synovial fluid was used as a control because acquiring synovial
fluid from healthy individuals is thought unethical and is very difficult due to the
low volume of fluid in a healthy joint. However, in a single study OPN’s
phosphorylation has been compared between synovial fluids of OA patients and
healthy controls, and shown that OPN is more phosphorylated in OA synovial fluid
(Xu et al., 2013). In respect to this study, according to our results it can be said that
OPN’s phosphorylation is significantly higher in RA synovial fluid than in healthy
synovial fluid.
TRAcP is the main regulator of extracellular OPN’s phosphorylation level and
thus the main regulator of its function, as it is believed, that OPN is secreted in its
phosphorylated form after being phosphorylated by intracellular kinases, such as
Fam20C (Andersson et al., 2003; Christensen et al., 2005). However, Fam20C
kinases have also been shown to be secreted extracellularly (Tagliabracci et al.,
2012). The combined effect of TRAcP and extracellular kinases has not been
studied. Total absence of Fam20C kinase leads to a rare disease called Raine
syndrome, which is characterized by congenital sclerosing osteomalacia and
cerebral calcification, which are partly thought to result from the loss of
biomineralizationinhibitory factors, such as phosphorylation of OPN (Whyte et al.,
2017).
TRAcP production has been shown to be elevated in both RA and OA (Janckila
et al., 2008; Seol et al., 2009). We did not find a difference in the overall TRAcP
concentrations between the diseases, but the TRAcP 5B/5A ratio was lower in RA
synovial fluid when compared to OA, and the decreased ratio correlated
significantly with the increased phosphorylation of OPN. We hypothesize that in
RA the production of phosphorylated OPN is upregulated during inflammation
(Lund, Giachelli, & Scatena, 2009), and insufficient TRAcP 5B levels in the
synovial fluid are not enough to dephosphorylate it to the normal level. The
increased phosphorylation of OPN in the synovial fluid leads to increased
inflammatory cell and osteoclasts activation, which leads to increased synovitis and
bone destruction, thus playing a part in the vicious circle of chronic inflammation
(Fig 4 in I).
It is worth mentioning that the synovial fluid and tissue samples were collected
during a knee prosthesis operation from patients suffering joint destruction, so the
duration of the chronic inflammation is rather long in both disease groups and this
situation may be different from the early stages of the diseases. The late stage
cytokine profile of the OA synovial fluid may resemble more RA than in the early
71
stages of OA. However, the heterogeneity of the samples was reduced as the
patients must have met strict criteria for the knee prosthesis operation.
Earlier, TRAcP positive mono- and multinucleated cells have been found in the
OA and RA synovial tissue (Yamaguchi et al., 2014; Tsuboi et al., 2003). We did
not detect clearly multinucleated cells in our histological samples of OA or RA
synovial tissue, but we found that the synovial lining layer cells were strongly
TRAcP positive. An antibody detecting both TRAcP 5A and 5B forms, and another
antibody detecting only the 5A form was used. The staining in the lining layer was
identical with both antibodies and we suggest that most of the TRAcP in the lining
layer cells is in the less active 5A form. A slightly stronger staining with the TRAcP
antibody binding both forms was observed in OA than in RA samples, which may
indicate that there is more TRAcP 5B in the area, in accordance with the measured
TRAcP 5B/5A ratios in the synovial fluid, but further studies of TRAcP expression
in the synovial tissue are needed.
TRAcP 5A is proteolytically processed into the 5B form by cathepsin K and
other proteinases (Fagerlund et al., 2006; Lång & Andersson, 2005; Ljusberg et al.,
2005). Interestingly, in earlier studies the levels of these proteinases have been
shown to be higher in RA than in OA synovial tissue or fluid (Hou et al., 2002;
Pozgan et al., 2010). In relation to the data, it seems possible that some mechanism
may prevent TRAcP 5A from being processed in RA into the 5B form or the
conversion mechanism does not function properly. In future studies it would be
interesting to further look into what is the cell in the synovial tissue that produces
TRAcP. It was speculated that most of the TRAcP positive cells in the synovial
lining are synovial macrophages, but the general staining of the cells suggests that
synovial fibroblasts could also be TRAcP positive. This raises a question on the
role of TRAcP in fibroblast-like synoviocytes and its origin. It has been suggested
that the synovial cells can differentiate into bone resorbing cells under the right
circumstances (Suzuki et al., 2001), but as the standard synovial cells are mostly
TRAcP 5A positive, not 5B positive as are the osteoclasts, synovial cells are
unlikely to resorb bone or cartilage themselves.
6.2 OPN is one of the key proteins secreted into the resorption
lacunae during bone resorption
In study II, the secretion of OPN into the resorption lacunae by human osteoclasts
during bone resorption was verified and different glycan epitopes at the bottom of
the resorption pit were identified. Additionally, the resorption and deposition of OPN
72
on pre-deposited protein free carbonated hydroxyapatite was also analyzed. The
presence of OPN and its phosphatase TRAcP was also evaluated histologically
from cartilage and subchondral bone biopsies of patients suffering from OA to
show their presence in the areas of increased mineral metabolism.
Various diseases and situations affect extracellular OPN levels and post
translational modifications, such as phosphorylation, as shown in study I. The
effects of exogenous extracellular OPN on osteoclasts are better understood than
the function of endogenous osteoclast OPN. Previous studies have investigated the
secretion of OPN into the resorption lacunae during bone resorption but have used
either animal cells or bone (Dodds et al., 1995; Maeda et al., 1994; Shimazu, Nanci,
& Aoba, 2002). In study II, the deposition of OPN by osteoclasts was investigated for
the first time using human cells on human bone and analyzed with modern FE-SEM
methods. The main discovery of this study was that OPN is specficly secreted by both
human bone marrow and peripheral blood derived osteoclasts into the resorption
lacunae on human bone and on protein-free carbonated hydroxyapatite. The proteomics
analyses of the resorbed bone slices showed that OPN was the most increased protein
in the resorbed bone. The glycan epitope analyses from the bottom of resorption lacunae
showed an increase of α2,3 and α2,6 -linked sialic acids, which were mostly in O-
glycans. This supports the discovery that OPN is the most abundant protein secreted
into the resorption pit, as the other enriched proteins do not contain both of the
sialic acid residues. Also, as the mineral content and crystallinity of bone in OPN
knockout mice is increased, this supports the hypothesis that OPN has a key role in
the regulation of bone metabolism, by likely acting as an important mediator of
activity for both the osteoclasts and the osteoblasts (Boskey et al., 2002).
The proteins secreted into the resorption pit function as signals for other bone
cells; osteoclasts, osteoblasts and osteocytes (H. Li et al., 2016; Lotinun et al.,
2013). Osteoclasts are thought to attach themselves to bone during resorption
through the αvβ3-integrin receptor, which is shown to be able to bind OPN’s RGD-
sequence (Ek-Rylander & Andersson, 2010; Ross et al., 1993; Sodek et al., 2000).
We support this hypothesis, as we found that OPN is secreted into the resorption
pit also on carbonated hydroxyapatite, and it has been previously shown that OPN
can bind to hydroxyapatite (Goldberg, Warner, Li, & Hunter, 2001). Thus, our
suggestion is that OPN acts as a connecting molecule between the resorbed
hydroxyapatite and the cell surface receptor.
TRAcP is the primary osteoclast marker, and it has also been shown to be
secreted into the resorption lacunae during bone resorption (Andersson et al.,
2003). Since OPN’s ability to inhibit biomineralization is dependent on its
73
phosphorylation (Gericke et al., 2005; Hoac et al., 2017; Wang et al., 2008), it would
seem paradoxical that OPN is dephosphorylated by TRAcP in the resorption pit during
the resorption. And, it has been shown that osteoclasts’ migration towards OPN is
dependent on its phosphorylation (Ek-Rylander & Andersson, 2010). We suggest that
OPN may be dephosphorylated during or at the late stage of resorption in order to mark
the resorbed area not to be resorbed again by other osteoclasts.
Extracellular OPN’s effects on osteoblasts are not fully understood (Icer &
Gezmen-Karadag, 2018). However, various other signals secreted by osteoclasts
that affect osteoblasts, the so-called clastokines, are known: they either increase or
decrease osteoblast activity (Cappariello et al., 2014). A phosphorylated form of
OPN is suggested to decrease osteoblast activity in bone, and, thus, have also cell
mediated inhibitory effects on the mineralization (Holm et al., 2014; Kusuyama et
al., 2017). However, only a single study so far has studied the effect of
phosphorylated and de-phosphorylated OPN on osteoblasts. On SaOS-2 cells, an
osteoblast-like cell line, phosphorylated OPN decreased their mineralization
activity but de-phosphorylated OPN did not (Halling Linder et al., 2017). Our
research group’s hypothesis was, that OPN is dephosphorylated during bone
resorption to enable new mineralization by osteoblasts.
It is known that the resorbed area on bovine bone can be recognized using
WGA-lectin (Selander, Lehenkari, & Vaananen, 1994), which binds to β1,4-linked
N-acetylglucosamine (Allen, Neuberger, & Sharon, 1973). In this study, WGA and
multiple other lectins were used to study the glycan epitope in the the resorption pits
on human bone. Strong binding of lectins indicating an abundance of α2,3- and α2,6-
linked sialic acids was found in the resorption lacunae, with most of the sialic residues
in O-glycans. This is consistent with the hypothesis that the major protein found at the
bottom of the resorption pit is OPN. When the sialic residues were enzymatically
removed from the resorbed bone slices and osteoclasts re-cultured on them, the number
of osteoclasts doubled when compared to untreated pre-resorbed bone slices. This may
mark that osteoclastogenesis is increased because more α2,3/6-linked sialic acid or a
protein containing it, such as OPN, is required on the bone. Or the protein’s inhibitory
effect on osteoclast differentiation is lost after de-sialylation.
It has been suggested that exogenous OPN may actually inhibit osteoclastogenesis
in certain conditions, but during osteoclastogenesis from monocytes, OPN expression
is increased, and it still acts as an important mediator of migration and activity (Ge et
al., 2017). As discussed earlier, the hypothesis is that osteoclasts may deposit OPN, or
another protein containing the above sialic residues, on bone to act as marker in
resorbed areas for other osteoclasts not to resorb the same areas again. This effect may
74
be dependent on the level OPN’s phosphorylation, which would be interesting to test
in future studies.
6.3 OPN is found in the calcified cartilage
In study II, immunohistochemistry of OPN, and TRAcP was conducted to
investigate their presence in areas of increased bone metabolism in OA. Areas from
subchondral, trabecular and cortical bone were analyzed. OPN staining was slightly
stronger in the subchondral sclerotic bone, than in the other areas. In general, the
bone metabolism is thought to be increased in the sclerotic bone. However, the
staining for OPN was the most intense in the calcified cartilage, a transition area
between the non-calcified cartilage and the subchondral bone. The line between
normal cartilage and calcified cartilage is called the tidemark.
Both TRAcP antibodies stained the cortical bone slightly more than the
sclerotic or trabecular bone. No increase of TRAcP in the areas of increased bone
metabolism in the samples was seen. No differences in bone staining between the
antibody specific for 5A or the other binding both 5A and 5B forms were seen.
Interestingly, both TRAcP antibodies used showed a strong staining of the
cartilage tidemark, while no TRAcP was detected in either calcified cartilage or
normal cartilage. The presence of TRAcP in the tidemark may indicate a change in
the phosphorylation of OPN between normal and calcified cartilage. Our
hypothesis is, that TRAcP dephosphorylates OPN at the tidemark, which causes its
inhibitory effect on biomineralization to be lost (Gericke et al., 2005; Hoac et al.,
2017; L. Wang et al., 2008), which leads to increased calcification of the cartilage
at the bone side of the tidemark, the calcified cartilage. Unfortunately, the
phosphorylation of OPN cannot be easily quantified histologically, as there is no
specific antibody available to detect phosphorylated or de-phosphorylated OPN.
6.4 RA synovial fluid and serum increase osteoclastogenesis and
bone resorption in vitro
In study III, the effect of inflammatory factors present in RA synovial fluid and
serum on osteoclast differentiation and bone resorption was investigated. OA
synovial fluid and healthy serum were used as controls. As in study I, OA patients’
synovial fluid was used as a control due to ethical issues with acquiring healthy
controls’ synovial fluid. Supraphysiological RANKL concentrations were used in
all cell cultures to elucidate the effect of other factors affecting the
75
osteoclastogenesis. It was evident that RA synovial fluid and serum both increased
the osteoclast differentiation, when compared to healthy controls. Surprisingly OA
synovial fluid increased the osteoclastogenesis even more than RA synovial fluid.
Inflammatory cytokines from the serums and synovial fluids were analyzed
with a 27-cytokine multiplex assay. A general increase of the inflammatory
cytokines was found between RA and healthy control serums, and RA and OA
synovial fluids. Surprisingly, the higher concentrations of inflammatory cytokines
in RA synovial fluid did not lead to increased osteoclastogenesis and activity when
compared to OA. However, morphological differences between the osteoclasts
cultured with different synovial fluids were found, as the cells cultured with OA
synovial fluid were significantly larger and contained more nuclei. No differences
in the osteoclast morphology were seen in cultures with serum samples.
Previous studies of osteoclastogenesis in the areas of inflammatory bone
erosions in RA have suggested that the pro-inflammatory cytokines present in RA
synovial tissue, along with increased RANKL stimulus, cause so-called
inflammatory osteoclastogenesis in the inflamed RA synovial tissue adjacent to
bone (Schett & Gravallese, 2012; Suzuki et al., 2001; Tsuboi et al., 2003). However,
the osteoclasts generated by the inflammatory osteoclastogenesis have been
described to be smaller and contain fewer nuclei, and thus present a more
macrophage like phenotype, than the osteoclasts in normal bone (Suzuki et al., 2001;
Tsuboi et al., 2003). We believe that in our experiment with the synovial fluids the
strong pro-inflammatory stimulus present in RA synovial fluid causes the
osteoclasts to stay in a more macrophage like phenotype than in the cultures with
OA synovial fluid, which seemed to create a very good environment for the
osteoclastogenesis. Interestingly, when comparing the volume of bone resorbed by
a single osteoclast the change in morphology did not affect it. The change in
morphology may, however, affect the osteoclasts’ interaction with the other bone
cells, and this may partly explain why the changes in the bone metabolism between
RA and OA happen in the different areas of the joint.
RANKL is the main factor of osteoclastogenesis, along with M-CSF, which
first activates the production of the RANK cell surface receptor, as explained in
chapter 2.1.3 (Boyce, 2013). Our data are in accordance with RANKL signaling
being the most crucial for osteoclastogenesis, as deprivation of it in negative control
samples inhibited the osteoclast differentiation. To evaluate the effect of other
factors affecting osteoclastogenesis, the effect of RANKL already present in the
samples was nullified by using supraphysiological RANKL concentrations, and
synovial fluid RANKL and OPG levels were measured to avoid confounding by
76
the natural RANKL and OPG in them. Earlier studies have assessed various
cytokines that promote osteoclastogenesis via RANKL or other pathways. These
cytokines include TNF-α, IL-1, IL-6, IL-7, IL-8, IL-11, IL-15, IL-17, IL-23, and
IL-34 (Amarasekara et al., 2018). Of the cytokines associated with increased
osteoclastogenesis, we found increased levels of TNF-α, IL-1, IL-6, IL-7, IL-8, IL-
15, IL-17 in our RA patient samples, IL-11 and IL-23 were not tested. Also, various
other disease and inflammation related molecules and proteins, such as, ACPAs,
CRP and VEGF in RA, are known to increase osteoclast differentiation
independently of RANKL (Harre et al., 2015; H. Kim et al., 2015; K. Kim et al.,
2015). An increase of VEGF was seen in the RA synovial fluid samples. CRP was
not tested. All the RA patients selected for the study were ACPA positive.
However, all cytokines are not pro-inflammatory and increase
osteoclastogenesis: various anti-inflammatory cytokines are known to be increased
in both RA and OA. Cytokines that are associated with decreased osteoclast
differentiation include IFN-α, IFN-β, IFN-γ, IL-3, IL-4, IL-10, IL-12, IL-27 and
IL-33 (Amarasekara et al., 2018). Of the above we found IFN-γ, IL-4 and IL-10 to
be increased in the tested RA samples. Due to limitations in the number of cytokines
tested in the multiplex assay, the other anti-osteoclastic cytokines were not tested.
However, previous studies have found increased concentrations of IFN-α, IL-3, IL-
27 and IL-33 in RA patient samples (Ali Khan, 2018; Lai et al., 2016; Pavlovic et
al., 2016; Sellam et al., 2016).
It is evident that the combined cytokine effect in our cell culture experiment
favours osteoclastogenesis, as could be seen from the osteoclast cultures with RA
serum or either synovial fluid compared to healthy control serum. The
inflammatory stimulus present in both synovial fluid and serum increased the
osteoclastogenesis significantly, but apparently the effect is limited, as OA synovial
fluid, which contained less pro-inflammatory factors increased the osteoclast
differentiation significantly more than RA. Since the study samples are from real
patients, we believe that this depicts rather well the complex situation in the
diseased joint where both pro- and anti-inflammatory factors work at the same time.
As discussed earlier, the unexpected result seen between the synovial fluids is likely
caused by the inflammatory osteoclastogenesis, which drives the osteoclasts
towards a more macrophage-like phenotype (Suzuki et al., 2001; Tsuboi et al.,
2003). It is notable that the RA serum samples were collected from untreated RA
patients at the time of diagnosis and the synovial fluid samples during knee
prothesis operation from RA patients suffering from secondary arthrosis of the knee.
This limitation is due to the availability of patient material. However, the data
77
shows, that a significant inflammatotry factors exist in the synovial fluid of RA
patients with secondary arthrosis of the knee, which etiology differs from that of
primary OA.
RA and OA are both diseases which are associated with both local and systemic
changes in the bone metabolism specific for the disease (Greisen et al., 2015;
Prieto-Potin, Largo, Roman-Blas, Herrero-Beaumont, & Walsh, 2015; Suzuki et al.,
2001). In RA, the inflamed synovial tissue is the main source of inflammatory
cytokines, including RANKL (Tsuboi et al., 2003), and the bone erosions start from
the perichondral areas where the synovial tissue is in contact with the bone, which
is not covered by the articular cartilage. In OA, the mechanical wear of the cartilage
and the subchondral bone along with the synovial inflammation cause changes in
local bone metabolism, which results in multiple pathological changes, such as
sclerosis, cysts and osteophytes, all of which are also associated with increased
osteoclastic activity (Barr et al., 2015; Favero et al., 2015).
The bone erosions in RA are caused by osteoclasts (Cappariello et al., 2014).
According to our data we suggest that the monocytes or macrophages in the
inflamed synovial tissue differentiate into osteoclasts due to inflammatory
osteoclastogenesis and cause the inflammatory bone erosions in RA. According to
the data in study I, the synovial cells are unlikely to resorb bone themselves as they
present a more macrophage than osteoclastic phenotype, as they are TRAcP 5A,
not 5B positive. However, it has been suggested that under the right conditions it
may be possible for them to undergo changes that would allow them to fuse into
bone resorbing cells (Suzuki et al., 2001). RA is also associated with an increased
risk of osteoporosis, which is thought to be a result of both medication and
circulating inflammatory cytokines filtered into the serum from the inflamed
synovial tissue (Clayton & Hochberg, 2013). OA is not associated with a risk of
increased osteoporosis (Clayton & Hochberg, 2013). Our osteoclast culture
experiment with RA serum shows how the circulating inflammatory cytokines in
RA increase the osteoclastic activity, and hence the risk of general osteoporosis.
Our cytokine data is in accordance with the previous literature of cytokines in
RA and OA, where the severity of both diseases has been shown to correlate with
the increased cytokine levels (Barra et al., 2014; Hampel, Sesselmann, Iserovich,
Sel, Paulsen, & Sack, 2013; Vangsness, Burke, Narvy, MacPhee, & Fedenko, 2011).
In the synovial fluid and serum, the level of the major pro-inflammatory cytokines
associated with RA, such as Il-1, Il-6, IL-8, VEGF etc., were increased as expected.
In general, the measured cytokines were ten times more concentrated in the
synovial fluids than in the serums, which was also seen in the fold changes. The
78
changes in the cytokines, which reached statistically significant differences,
between serum and synovial fluid experiments are explained by the low number of
study subjects and measurements of certain serum cytokines falling under the
detection level in the multicytokine assay.
In conclusion, OA and RA fluids affect the osteoclastogenesis and the bone
resorption by osteoclasts in vitro. Both RA synovial fluid and serum increased the
osteoclastogenesis and the bone resorption. Surprisingly, the osteoclastogenesis
and the bone resorption increased the most in the presence of OA patient synovial
fluid. The bone changes happening in the diseases can better understood with these
data. However, further studies of inflammatory osteoclastogenesis are required to
obtain better understanding for optimal therapeutic interventions for the
inflammatory diseases affecting joints and bone. This in vitro disease cell model
could be used to evaluate the effect of new potential drugs on osteoclast function.
6.5 Future aspects
The observation that the TRAcP 5B/5A ratio differs and affects OPN’s
phosphorylation in RA raises questions about the mechanism behind this. TRAcP
positive cells were abundant in the synovial tissue. In future studies it would be
interesting to study the expression of TRAcP in the synovial cells in detail to
distinguish the cell type that produces it. The mechanism behind the change in the
TRAcP 5B/5A ratio between RA and OA synovial fluids would also be interesting
to study further, as discussed earlier in chapter 6.1. It seemed possible, that the
proteolytic mechanism behind TRAcP 5A being processed to 5B does not work in
RA, as the total TRAcP levels did not differ between RA and OA.
As the other factor that affects OPN’s phosphorylation, the in vivo relevance
of extracellular Fam20C kinase on OPN’s phosphorylation should also be studied
from real patient samples, along with its relation to TRAcP. Which affects OPN’s
phosphorylation more?
The effect of extracellular OPN and its phosphorylation on osteoblasts should
be studied more, to gain further understanding of the osteoclast-osteoblast
connection. Better understanding of the basic functions of the bone cells may reveal
new therapeutical prospectives in diseases related to changes in bone metabolism,
such as osteoporosis.
To possibly reveal new insights in the pathogenesis of OA, it would be
interesting to measure OPN’s phosphorylation in the calcified cartilage of healthy
79
and OA patients. The change in OPN’s phosphorylation may be an important factor
in the changes seen in the OA cartilage and the subchondral bone.
Inflammatory osteoclastogenesis is an interesting phenomenon and according
to our results the osteoclasts may differ from the normal osteoclasts. The
inflammatory osteoclastogenesis is not yet well understood. In future studies, the
inflammatory osteoclasts should be studied to reveal further differences, which
could possibly be influenced with targeted therapies. The therapies against
inflammatory osteoclastogenesis would allow us to affect only the pathological
osteoclasts, without affecting the normal bone metabolism.
80
81
7 Conclusions
The main goal of this study was to investigate the osteoclast function and the factors
that affect it in normal or pathological conditions of RA and OA. Based on the
study’s results, the following major conclusions were made.
1. The phosphorylation of OPN is increased in RA synovial fluid, which is the
result of the decreased TRAcP 5B/5A ratio.
2. Human osteoclasts secrete OPN into the resorption pit during the resorption of
human bone or a protein free resorbable material. OPN is the most abundant
protein secreted during the bone resorption into the resorption area.
3. RA serum and synovial fluid increased the osteoclastogenesis and the bone
resorption when compared to the healthy control serum. However, OA synovial
fluid increased the osteoclastogenesis and activity even more. RA synovial
fluid contains high concentrations of inflammatory cytokines. These cause the
inflammatory osteoclastogenesis, which generates osteoclasts of a more
macrophage like phenotype.
These interesting new discoveries will reveal their clinical significance in future
studies.
82
83
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Original publications
I. Luukkonen, J., Pascual, L. M., Patlaka, C., Lång, P., Turunen, S., Halleen, J., Nousiainen, T., Valkealahti, M., Tuukkanen, J., Andersson, G., Lehenkari, P. (2017). Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio. Plos One, 12(8). doi:10.1371/journal.pone.0182904.
II. Luukkonen, J., Hilli, M., Nakamura, M., Ritamo, I., Valmu, L., Kauppinen, K., Tuukkanen J., Lehenkari, P. (2019). Osteoclasts secrete osteopontin into resorption lacunae during bone resorption. Histochemistry and Cell Biology. doi:10.1007/s00418-019-01770-y.
III. Luukkonen J., Huhtakangas J., Turunen S., Tuukkanen J., Vuolteenaho O., Lehenkari P. (2019). Bone resorption and osteoclastogenesis are stimulated by synovial fluid from osteoarthritis and rheumatoid arthritis patients in vitro. Manuscript.
I and II are published under Creative Commons Attribution 4.0 International
License.
Original publications are not included in the electronic version of the dissertation.
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U N I V E R S I TAT I S O U L U E N S I S
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TAJani Luukkonen
OULU 2019
D 1532
Jani Luukkonen
OSTEOPONTIN AND OSTEOCLASTSIN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU