review article osteoimmunology: major and costimulatory

Review ArticleOsteoimmunology Major and CostimulatoryPathway Expression Associated with ChronicInflammatory Induced Bone Loss

Tania N Crotti1 Anak A S S K Dharmapatni1 Ekram Alias12 and David R Haynes1

1Discipline of Anatomy and Pathology School of Medical Sciences The University of Adelaide Adelaide SA 5005 Australia2Department of Biochemistry Faculty of Medicine Universiti Kebangsaan Malaysia Jalan Yaacob Latif Bandar Tun Razak56000 Kuala Lumpur Malaysia

Correspondence should be addressed to Tania N Crotti taniacrottiadelaideeduau

Received 17 October 2014 Accepted 10 December 2014

Academic Editor Giorgio Mori

Copyright copy 2015 Tania N Crotti et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship betweenthe immune system and bone metabolism This is pertinent to immune-mediated diseases such as rheumatoid arthritis andperiodontal disease where there are chronic inflammation and local bone erosion Periprosthetic osteolysis is another exampleof chronic inflammation with associated osteolysis This may also involve immune mediation when occurring in a patient withrheumatoid arthritis (RA) Similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatorycytokines expressed in these diseases This review highlights the role of immune-related factors influencing bone loss particularlyin diseases of chronic inflammation where there is associated localized bone loss The importance of the balance of the RANKL-RANK-OPG axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in theimmunoreceptor tyrosine-based activation motif (ITAM) signaling pathway play Although animal models are briefly discussedthe focus of this review is on the expression of ITAM associated molecules in relation to inflammation induced localized bone lossin RA chronic periodontitis and periprosthetic osteolysis with an emphasis on the soluble andmembrane bound factor osteoclast-associated receptor (OSCAR)

1 Introduction

The term osteoimmunology and the study of osteoimmunol-ogy have developed due to the close interrelationship betweenthe immune system and bone metabolism [1] This is evidentin immune-mediated diseases such as rheumatoid arthritisand periodontal disease (periodontitis) where there are localbone erosion and inflammation as reviewed in detail inmultiple publications [2ndash4] Similarities in the mechanismsof bone loss in disease are likely related to the inflammatorycytokines expressed in a number of bone loss diseases Thesecytokines are known to upregulate osteoclast activity viaincreased expression levels of receptor activator NF kappaB ligand (RANKL) relative to osteoprotegerin (OPG) (asexplored below) and increase localized bone loss in diseases

such as RA periodontal disease and periprosthetic osteolysis[5ndash10]

This review highlights the role of immune-related cellsand factors in modulating bone loss particularly in thesediseases While the importance of the RANKL-RANK-OPGaxis has been appreciated for nearly two decades [11ndash13]morerecent studies have highlighted the importance of factorsassociated with immunoreceptor tyrosine-based activationmotif (ITAM) signalling This review will briefly discussthe RANKL-RANK-OPG axis but its major focus will beon the role of ITAM-associated factors the more recentlyinvestigated pathway and how it relates to inflammatory boneloss diseases in particular osteoclast-associated receptor(OSCAR) [14]

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 281287 13 pageshttpdxdoiorg1011552015281287

2 Journal of Immunology Research

2 Chronic Inflammation-Mediated Bone Loss

21 Rheumatoid Arthritis Rheumatoid arthritis (RA) affects1-2 of the population and involves an autoimmune reactionwith an autoantibody response to citrullinated proteins (andothers such as rheumatoid factor and collagen type II) [15]RA is characterized by synovitis involving angiogenesissynovial proliferation increased infiltration survival anddecreased apoptosis of inflammatory cells [16] Further to thisthere is an increase in osteoclast number and activity leadingto focal bone erosions juxta-articular osteopenia and jointdestruction [17ndash20] Animal models suggest that there mayalso be suppression of localised osteoblast formation of bone[21]

22 Periodontitis and Similarities to RA Periodontitis is achronic inflammatory disease of the gingival tissues withan associated loss of the supporting structures includingthe periodontal ligament and alveolar bone The aetiologyinvolves an inflammatory response to bacterial infectionsuch as P gingivalis and possibly an autoimmune reactionas reviewed [22] Periodontitis is the most common andwidespread bone loss pathology in humans with 64 ofthe US population aged 65 years and older reported ashaving moderate or severe periodontitis [23] Despite theprevalence of this disease the most common treatment iseither mechanical subgingival plaque removal or surgicaldebridement Inevitably in the absence of effective treatmentsupport structures (periodontium) are compromised and theaffected teeth will loosen and fall out

RA and periodontitis have a similar pathophysiologycharacterized by destructive inflammation that culminatesin localized bone loss The citrullination of proteins by Pgingivalis and the subsequent generation of autoantigens thatdrive autoimmunity in RA have been proposed as a possiblemechanism linking these two diseases [24] Similarities in RAand periodontitis may relate to citrullinated enolase as thespecific antigen involved aswell as cross-reaction between theantibodies directed towards the immunodominant epitope ofhuman citrullinated alpha-enolase and a conserved sequenceon citrullinated P gingivalis enolase [25]

New evidence suggests a relationship between the extentand severity of chronic periodontitis and RA [24 25]Individuals with advanced RA are more likely to experiencemore significant periodontal problems compared to theirnon-RA counterparts and vice versa This is supported byfindings in a study using a combined animalmodel of RA andperiodontitis [26] which demonstratedmore severe develop-ment of arthritis in mice with periodontitis Further to thismice in which periodontitis alone was induced had evidenceof radiocarpal bone loss in the absence of arthritic disease[26] Additionally mice in which inflammatory arthritis wasinduced also had evidence of periodontitis [26]This suggestspresence of either RA or periodontitis places the individualat risk of developing the other disease Both conditionsinvolve an imbalance between proinflammatory and anti-inflammatory cytokines and increased bone-resorbing activ-ity Cantley et al (2011) thus proposed that these two diseasesare related through a common underlying dysfunction of

fundamental inflammatorymechanisms [26] New treatmentstrategies are needed for both diseases that target the inhi-bition of proinflammatory cytokines destructive enzymesand bone-resorbing activity The clinical implications ofthe current research strongly suggest that patients with RAshould be carefully screened for their periodontal status asreviewed [27]

23 Peri-Prosthetic Osteolysis Joint replacement surgery isused as a last resort in osteoarthritis (OA) and RA patientsand is a relatively successful operation however a largeproportion of implants fail within 10ndash20 years as a result ofbone loss and implant loosening [4 10 28]The pathogenesisbehind prosthetic implant failure involves wear of prostheticalloys such as polyethylene (PE) cobalt chrome and tita-nium liberated from the implant surface [29ndash31] These par-ticles stimulate a chronic inflammatory response [29] whichincreases bone-resorbing activity of the osteoclasts [32] andsuppresses bone formation by the osteoblast [33 34] resultingin bone loss [25] Periprosthetic tissues contain granuloma-tous lesions dominated by inflammatory cells particularlymacrophages and foreign-body giant cells [29ndash31 35 36] It isbelieved that an inflammatory reaction is initiated within thetissues in an attempt at particle clearance This then becomesa chronic reaction resulting in a granulomatous lesion Thisgranulomatous lesion in periprosthetic osteolysis often leadsto the formation of pseudosynovium-like structure in whichcells are organized into lining layer in the membranoustissues adjacent to the failed implant surface [35] Juxtaposedto this pseudosynovium are fibrous and collagenous regionspossibly scar tissues which could be indicative of late stageperiprosthetic osteolysis The plethora of factors release inthis inflammatory reaction within the tissues contributestowards the promotion of osteoclast formation [9 37]

Higher numbers of T lymphocytes have been observedin the periprosthetic tissues of human and mouse modelscontaining polyethylene and metal particles compared withnormal tissues [38ndash40] and osteoarthritic tissues [41] Sandhuet al (1998) proposed that T cells are indirectly affected bythe inflammatory cascade induced by wear particles [40] It ishowever important to note that T cells make up less than 10of total cell population [42] in periprosthetic tissues Giventhe low levels of T lymphocytes the general belief held isthat lymphocyte infiltrates are not normally associated withwear-particle induced periprosthetic osteolysis in particularin the granulomatous region [29 32 42 43]The role of T cellsmay however bemore pertinent in RApatients with implantsThe prevalence of foreign-body giant cells in response toimplant-derived wear debris in RA patients and non-RApatients does not differ but appears to be linked to the amountof polyethylene wear debris [36] As would be expectedthis study reported a high prevalence of plasma cells inlymphocytic infiltrates in untreated RA patients comparedwith non-RA patients with a different distribution [36]Whether implant wear is inducing a different reaction in RAversus non-RA patients may potentially have implications forcombination and immune targeted treatment of inflamma-tion and osteolysis in these patients

Journal of Immunology Research 3

3 RANKL-RANK-OPG Axis

Receptor activator NF kappa B ligand- (RANKL-) RANKsignalling has several important roles in the immune systemand bone [13 44ndash46] Physiologically RANKL is requiredfor normal development of lymph nodes [13] as evident inknockoutmice In bone RANKL interactionwith its receptorRANK expressed by the osteoclast induces the transcriptionfactor nuclear factor of activated T cells-1 (NFATc1) [47ndash49] NFATc1 is an essential factor for differentiation multi-nucleation and activation [47ndash49] NFATc1 binds directly toand regulates osteoclast differentiation genes such as tartrateresistant acid phosphatase (TRAP) [50] cathepsin K (CathK) [51] osteoclast-associated receptor (OSCAR) [52] 1205733-integrin [53 54] and calcitonin receptor (CTR) [50] NFATc1is also involved in autoregulation of itself further enhancinggene expression and osteoclast differentiation [55]

RANK-RANKL interaction is inhibited by the decoyreceptor OPG [12] and thus the ratio of RANKL to OPGhas a crucial influence on bone resorption [56] Interactionsbetween RANK expressing cells of the lamina propria andT cells expressing RANKL also play a role in intestinalinflammation [57] In the vasculature RANKL interactswith RANK to promote survival of endothelial cells [58]Additionally RANKL is upregulated in the keratinocytes ofinflamed skin [59] Further to this in an inflammatory arthri-tismodel reminiscent of RA activated T cells exacerbate jointdestruction via RANKL upregulation [46]

31 RANKL-RANK-OPG in Bone Pathologies The RANK-RANKL-OPG axis is known to regulate not only normalbone physiology but also alterations in RANK-RANKL-OPGinteractions that play a role in bone disease Uncoupling inthe balance between the level and activity of these moleculesculminates in osteoporotic or osteopetrotic phenotypes dueto an increase or decrease in osteoclast formation and activityThis is particularly evident in focal bone loss associatedwith chronic inflammatory diseases such as rheumatoidarthritis periodontal disease and periprosthetic bone lossIn active RA periodontal disease and prosthetic looseningelevated levels of RANKL relative to OPG are observed inthe synovial-like soft tissue gingival tissue and soft tissuesadjacent to sites of osteolysis [5ndash10] Further to this theelevated ratio of RANKL OPG expression is associated withincreased differentiation and activity of the bone-resorbingosteoclasts [5 7ndash9 32 60] suggesting RANKL OPG ratio asan important indicator for bone erosion

As RANKL and OPG are key molecules regulating boneloss in diseases therapeutic interventions targeting thesemolecules and their signaling cascades are being investigatedto treat a wide range of diseases

32 RANKL-RANK-OPG in RA Osteoclasts are the promi-nent cell eroding bone in inflammatory arthritis [20] Aseminal paper in the field of osteoimmunology used aRANKL knockout background to demonstrate that animalsdeveloped an osteopetrotic phenotype and a reduction inbone erosion characterized by the absence of osteoclasts

whilst inflammation did not differ between wild-type andRANKL knockout mice [61] In contrast cartilage erosionwas present in both control littermate and RANKL knockoutmice [61] suggesting that the RANK-RANKL-OPG axis doesnot directly affect cartilage metabolism

In human studies the levels of soluble RANKL have beenfound to be higher than OPG in synovial fluids from patientswith RA compared with osteoarthritis (OA) patients [62]suggesting a role in increased bone resorption In supportof this a more recent large center study has reported theratio of RANKL OPG and markers of bone and cartilagedegradation (such as collagen terminal 1 (CTX-1)) to bepredictive of progression of radiological bone damage in RA[60]

In synovial tissues from patients with active RA RANKLexpression is predominantly located in sublining regions [6263] concentrated at focal sites of osteoclastic bone erosion inthe pannus- bone interface [64] In contrast OPG has beendescribed as being in regions of synovium some distancefrom the sites of bone erosion in RA [64] We reportedOPG is associated with endothelial cells and macrophagesin the synovial lining layer of OA and normal tissues whilstabsent in patients with active RA [63] RANKL expressingcells have been detected in a subset of fibroblast-like syn-oviocytes and infiltrating mononuclear cells [62] Further tothis activated synoviocytes from RA tissue express RANKLand have decreased OPG and are capable of supportingosteoclastogenesis in vitro [65] RANKL expressing cells arealso seen within areas of lymphocyte infiltration and dualimmunostaining by ourselves and others have shown thatmany of the RANKL-positive cells are a subset of CD3+ andCD4+ cells [6 62 63] ActivatedT cells fromRApatients haveincreased RANKL are able to induce osteoclast formation invitro [62] This study also reported a higher ratio of solubleRANKL relative to OPG suggesting T cells as a source ofsoluble RANKL in RA [62]

NFATc1 is a transcription factor crucial to RANKL-RANK signaling in the osteoclasts [47] and is initially identi-fied as being expressed by T cells and involved in regulationof cytokine transcription [66] We observed NFATc1 positivecells in lymphocyte aggregates in RA tissues [67] Manyof the NFATc1-immunostained mononuclear cells observedwere single nucleated and thus could be either precursor cellsof osteoclasts such as macrophages or lymphocytes Thosewith lymphocyte morphology are most likely activated Tcells as most of them demonstrated NFATc1 positive staininglocalized mainly in nucleus [67] These cells may promoteosteoclastogenesis through the RANKRANKL pathway [39]because as already mentioned as activated T-cells demon-strate elevated expression of membrane-bound RANKL withthe ability to support osteoclastogenesis in vitro [62]

33 RANKL-RANK-OPG in Periodontal Disease The relativeratio of RANKL to OPG is also a significant indicator inbone loss associated with periodontal disease [5 69] SolubleRANKL is significantly higher in gingival crevicular fluid(GCF) of periodontitis patients than in healthy GCF whileOPG is not [69] Similar to RA B and T lymphocytes express


RANKL in gingival tissues associated with periodontitis [569] with expression of more than 50 and 90 of T cellsand B cells respectively Consistent with a role in osteoclastregulation lymphocytes isolated from gingival tissues ofpatients induced differentiation of mature osteoclast cellsin a RANKL-dependent manner in vitro [69] These resultssuggest that activated T and B cells can be the cellular sourceof RANKL and an inducer of bone resorption in periodontaldisease

In a crude mRNA analysis of tissue from dentalpatients those with periodontitis exhibited significantlyhigher NFATc1 gene expression compared with healthy sub-jects Interestingly NFATc1 and RANKL expression levelsstrongly correlated with each other and with clinical peri-odontal parameters [70]

34 RANK-RANKL-OPG in Peri-Prosthetic Osteolysis Asep-tic bone loss adjacent to orthopedic joint implants is acommon cause of joint implant failure in humans RANKRANKL and tumour necrosis factor (TNF-120572) are key mod-ulators of bone turnover and their expression has beenreported by ourselves and others in the tissues near peripros-thetic osteolysis in patients undergoing revision of total hipprostheses [9 71 72] These factors were strongly expressedby large multinucleated cells containing polyethylene weardebris in revision tissues [9] More interestingly a strongstatistical correlationwas found between volume of bone losspolyethylene wear debris and RANK RANKL and TNF-120572expression [9]This was consistent with the earlier findings ofStea et al (2000) [72] where immunohistochemical detectionof TNF-120572 positively correlated with radiographic scores ofosteolysis

In periprosthetic osteolysis elevated levels of RANKLrelative to its competitor OPG are associated with increaseddifferentiation and activity of the bone-resorbing osteoclasts[8 32 73] An earlier study had shown that cells isolated fromperiprosthetic tissues containing wear particles expressedmRNA encoding for the proosteoclastogenic moleculesRANKL its receptor RANK monocyte colony-stimulatingfactor (M-CSF) interleukin- (IL-) 1 beta TNF-120572 IL-6 andsoluble IL-6 receptor as well as OPG [8] Other studiesshowed that osteoclasts formed from cells isolated fromperiprosthetic tissues in the presence and absence of humanosteoblastic cells in vitro [8 74] When osteoclasts formedin the absence of osteoblastic cells markedly higher levels ofRANKL mRNA relative to OPG mRNA were expressed Par-ticles of prosthetic materials also stimulated human mono-cytes to express both osteoclast-associated genes and osteo-clastmediating factors in vitro [8]These findings suggest thatingestion of prosthetic wear particles by macrophages resultsin expression of osteoclast-differentiating molecules andstimulation of macrophage differentiation into osteoclasts[8] Subsequent immunohistochemical studies demonstratesignificantly higher levels of RANKL in the periprosthetictissues of patients with implant failure than in similar tissuesfrom osteoarthritic and healthy subjects [32] In contrastOPG protein levels were similar in all tissues with the netresult of higher RANKL OPG ratio [32] Of interest RANKL

protein and mRNA were predominantly associated withmacrophage cells containing wear particles in the peripros-thetic tissues [32]These findings support the contention thathigh levels of RANKL in periprosthetic tissues of patientswith prosthetic loosening may significantly contribute toaseptic implant loosening [32]

We observed both mRNA and protein expressions ofNFATc1 to be higher in periprosthetic osteolysis than in OAtissues although levels did not reach significance [75] Thisis consistent with low T lymphocyte numbers observed inthese tissues This may provide an explanation why lowerthan expected NFATc1 protein and mRNA levels are foundin periprosthetic osteolytic tissues

4 The ITAM Pathway in Osteoimmunology

The ITAM pathway regulates proliferation survival and dif-ferentiation of effector immune cells and provides osteoclastswith costimulatory signals [14 76ndash80] In preosteoclasts andosteoclasts innate immune receptors TREM2 and OSCARassociate with the ITAM adaptor proteins DAP12 and Fcreceptor gamma-chain (FcR120574) respectively [80 81] DAP12and TREM2 are required for differentiation into multinucle-ated bone-resorbing osteoclasts in vitro via phosphorylationof the Syk tyrosine kinase [79] OSCAR on the cell surfacemediates signal transduction via FcR120574 [82 83] (Figure 1)Theinduction of intracellular calcium via this pathway is requiredin conjunction with RANKL-RANK interaction for NFATc1induction [84]

41Mutations of ITAM-AssociatedMolecules and Bone Pheno-types In Vivo In the context of human pathology the roles ofTREM2 or DAP12 have only begun to be recognized Studiesin diseased tissues particularly in Nasu-Hakola disease [85ndash87] and very recently in Alzheimerrsquos disease [88] have shownthat these molecules may be involved Mutations in TREM2or DAP12 have been associated with bone pathologies such asbone cysts and increased fractures (in addition to preseniledementia) in Nasu-Hakola disease [87 89] These studiessupport a role for DAP12 and a relationship between theskeletal and psychotic characteristics observed in Nasu-Hakola disease and for schizophrenia and presenile dementia[90] In TREM2 deficient individuals the osteoclast precur-sors failed to differentiate into effective bone-resorbing cells[91] Consistent with this Paloneva et al [87] demonstratedthat function mutations in DAP12 and TREM2 result in aninefficient and delayed differentiation of osteoclasts in vitroIn postmenopausal osteoporosis a rare allele (G allele) ofOSCAR-2322Aampgt G (SNP in the 51015840 flanking region) hasbeen associated with lower bone mineral density [92]

Although animal models are not the focus of this reviewit is interesting to note the phenotypes of ITAM relatedmolecules in single and combination knockouts TREMminusminusmice have an osteopenic phenotype similar to Nasu-Hakoladisease In vitro lack of TREM2 impairs proliferation osteo-clast precursors and affects the rate of osteoclastogenesis byaccelerating differentiation into mature osteoclasts [93] sug-gesting different effects of knocking out TREM2 in vivo and


RANKL

RANKL

OSCARL OSCARL

OSCARL

RANKL

RANK

TREM2

DAP12

OSCAR

OSCAR

OSCAR

OPGsOSCAR

Nucleus

FcRy

ITAM

NFATc1

NFATc1

Osteoblast

Osteocyte

Figure 1 RANGL-RANK-OPG axis and ITAM the costimulatory pathway in inflammation induced localised bone loss pathologies

in vitro In DAP12 deficient mice (minusminus) there are an increasedbone mass (osteopetrosis) and impeded development ofosteoclasts Mice that are double knockout for the adaptorsDAP12minusminus and FcR120574minusminus are severely osteopetrotic and bonemarrow derived osteoclast precursors from these mice areunable to differentiate into mature osteoclasts in a RANKL-andM-CSF-mediated culture system [79 82] Although thereis some redundancy findings from these studies suggest thatDAP12 is the predominant factor responsible for optimalosteoclast differentiation

42 OSCAR Signalling and Function in Bone RegulationOSCAR is an IgG-like receptor expressed by monocytesmacrophages monocyte-derived dendritic cells in humansand is involved in antigen presentation as well as survivalmaturation and activation of dendritic cells [14 76 77 8394 95] Ligation of human OSCAR on monocytes and neu-trophils results in the induction of a proinflammatory cascadeand the initiation of downstream immune responses [95]Importantly cell bound OSCAR on osteoclast precursors isan essential costimulatory factor in osteoclast formation butdoes not bypass the requirement of RANKL RANKL-RANKinduction of NFATc1 expression precedes that of OSCAR

[96] and is crucial for induction of OSCAR gene expression[52] In addition ligand-activated OSCAR interacts withFcR120574 to produce an increase in intracellular calcium [95]that augments NFATc1 expression [96] This establishes apositive feedback loop that results in marked elevation ofboth OSCAR and NFATc1 expressions in terminal stages ofosteoclast formation [52 96] These findings demonstrate asignificant role for OSCAR in immune modulation as well asosteoclastogenesis

In vitro studies demonstrate that addition of OSCAR-Fcto osteoblast-osteoclast cocultures results in the inhibition ofosteoclast differentiation with Kim et al (2002) suggestingthat this was due to OSCAR-Fc blocking an osteoblastderived OSCAR ligand binding to OSCAR [14] This maybe in addition to the recent identification of the motifswithin fibrillar collagens in the extracellular matrix (ECM)as OSCAR ligands [97]The importance of OSCAR is furtherhighlighted by the fact that soluble OSCAR (s)OSCAR in theform of OSCAR-Fc has also been shown to inhibit osteoclastdifferentiation from PBMCs in the presence of RANKL M-CSF and TGF-120573 [94]

Costimulatory immune pathways may further increaseosteoclast differentiation and activity [81 82] particularly in


chronic inflammatory diseases with an immune componentsuch as in RA periprosthetic osteolysis and periodontaldisease In fact our studies [67 75] and those of otherssuggest that deregulation of ITAM-associated moleculescontributes to the pathogenesis and severity of rheumatoidarthritis periodontal disease periprosthetic osteolysis andosteoporosis [10 67 75 92 94 98 99]

43 Expression of ITAM-Associated Molecules in ChronicInflammation Induced Localised Osteolysis We and othershave demonstrated increased levels of ITAM-related factorsincluding TREM2 DAP12 OSCAR and FcR120574 in humanperiprosthetic tissues adjacent to sites of osteolysis [75]and in RA synovial tissues [67 94] Additionally we haveobserved ITAM-related factors expressed in periodontitistissue adjacent to bone loss (unpublished observations) Ofthese factors soluble and membrane-bound OSCAR havebeen more extensively assessed in the context of RA andvascular disease (expanded on below)

44 Expression of ITAM-Associated Molecules in RheumatoidArthritis We observed markedly higher levels of TREM2DAP12 OSCAR and FcR120574 in active RA patients com-pared to synovial tissues from inactive RA OA or controlhealthy joint Multiple cell types expressed TREM2 includingmononuclear cells in lymphoid aggregates and fibroblasts[67] In OA tissues TREM2 immunostaining was noticed inmonocytemacrophage-like cells mainly around perivascularareas and on blood vessels (unpublished observations) Thepositive TREM2 immunostaining on the vasculature wasconsistent with the finding on expression of TREM2 inendothelial cells that has been documented earlier [100]TREM2 immunostaining was also occasionally spotted onlymphocyte-like cells in some OA tissues however to datethere has been no study indicating the expression of TREM2in lymphocytes but further investigation is needed for confir-mation

Interestingly DAP12 appeared predominantly associatedwith macrophage-like cells in the sublining of the synovialtissue particularly in the macrophage-like cells in the liningof the OA group [67] More recently a study by Chen etal (2014) [101] reported that mRNA expression levels ofDAP12 in the peripheral blood mononuclear cells of activeRA patients were significantly higher in active RA patientsthan in inactive RAorOApatientsThis is consistent with ourobservations [67] of higher levels of DAP12 protein expressedin the synovium in active RA patients than in inactive RAor OA patients They also noted that the levels significantlydecreased after effective therapy [101]

FcR120574 protein associates with fibroblasts and monocytesof the synovial sublining whilst lymphoid aggregates and thevasculature do not express FcR120574 [67] Of note similar toDAP12 FcR120574 was associated with macrophage-like synovio-cytes in the synovial lining with some scattered monocytesin the sublining of the OA tissue This increased DAP12 andFcR120574 expression might indicate a role in the pathogenesis ofOA but this is yet to be determined [67]

45 Soluble and Synovial Tissue Levels of OSCAR in RAAnalysis of human synovial tissue serum plasma andsynovial fluid suggests that OSCAR expression is associatedwith disease activity in RA [67 94 98 99] Recent studiesshow that OSCAR protein expression is increased in mono-cytes from RA patients compared with healthy individualscorrelating with inflammatory disease activity (DAS28) [94]OSCAR has also been noted to be expressed by mononuclearcells adjacent to synovial microvessels in RA tissues [94]Consistent with these findings our immunohistochemicalstudies show that high levels of OSCAR are associated withmono- and multinuclear cells in active RA tissues comparedto tissues from OA and normal patients [67] (Figure 2)Furthermore semiquantitative analysis confirmed that thereis a significant elevation of OSCAR (119875 lt 005) in active RAsynovial tissues compared to osteoarthritis synovial tissuesThis increased expression of OSCAR in the synovial tissueof active RA suggests OSCAR regulation by inflammatorycytokines and supports a role for OSCAR in the pathogenesisof RA

A study investigating the clinical radiological andsynovial immunopathological responses following anti-rheumatic treatment in RA proposed that high synovialtissue vascularity predicted favorable clinical and radiologicalresponses to treatment [102] Similar to this we previouslyreported the increased OPG staining associated with thevasculature in synovial tissues retrieved from the patientin remission OA and normal compared with active RA[63] Furthermore we have reported increased levels ofOPG associated with vasculature following treatment withDMARDS [7]

More recently we have detected increased expressionof OSCAR protein associated with the microvasculatureof synovial tissue from all inactive and active RA patienttissues (99) compared to none in the normal synovial tissuegroup (09) [67] Importantly in diseased tissues OSCARwasexpressedmostly on the luminal side of themicrovasculatureconsistent with OSCAR expression by endothelial cells [67103] Our findings suggest that OSCAR is associated withthe endothelial cells of the microvasculature and is eitherproduced by endothelial cells or secreted by other cells andbound by the endothelial cells in inflammatory states Themarked reduction in the OSCAR associated with endothelialcells observed in OA and healthy synovial tissues comparedwith active RA indicates an immune modulatory mechanism[67] which may also signal back to the osteoclasts andregulate bone resorption

Following observations ofOSCAR associationwith bloodvessels the expression ofOSCARwas investigated in endothe-lial cell lines in vitro Our analysis on bone marrow-derivedendothelial cells (BMECs) challenged with IL-1120573 and TNF-120572 in vitro demonstrated that the inflammatory cytokinesincreased OSCAR expressed as both mRNA and secretedand membrane-bound proteins [67] Together with in vivoobservations on synovial tissues and serum levels thesestudies suggest that inflammatory cytokines in RA regulatecleavage or secretion of sOSCAR from preosteoclasts or themicrovasculature


(a) (b)

Figure 2 OSCAR positive cells (red) in synovial tissue (a) OSCAR immunostaining in the lining cells of OA tissues (b) MononuclearOSCAR positive cells as indicated by arrow The magnification was 400x

(a) (b) (c)

Figure 3 Expression of osteoclast and vascular-associated molecules in mildly inflamed gingival tissues (a) TRAP (osteoclast marker) (b)OSCAR C Von Willebrand factor to identify the microvasculature The magnification was 200x

The regulation by inflammatory cytokines such as TNF-120572 of OSCAR messenger RNA expression has also beenobserved in monocytes [94] Interestingly levels of OSCARwere found to increase in serum from RA patients followinganti-TNF treatment [94] Of note these studies did notinvestigate gene or the release of protein OSCAR by humanperipheral blood derived osteoclasts in response to TNF-120572 inconjunction with RANKL

46 Expression of ITAM-Associated Molecules in PeriodontalDisease To our knowledge very limited descriptive or func-tional studies have investigated ITAM factors in periodontitisand normal gingival tissues An early study however reportedthat isolated polymorphonuclear neutrophils from GCF ofadult periodontitis patients exhibited higher Fc alpha RI andFc gamma RI levels and lower Fc gamma RIIa and Fc gammaRIIIb levels than peripheral blood polymorphonuclear neu-trophils They found that GCF derived polymorphonuclearneutrophils had a reduced ability to phagocytose and killIgG1-opsonized P gingivalis compared to peripheral bloodpolymorphonuclear neutrophils [104]

Our recent unpublished observations have identifiedOSCAR colocalizing with TRAP in cells in serial sectionsof mildly inflamed gingival tissue (Figure 3) Of note theseosteoclast markers are highly expressed in themultinucleatedcells on the bone Similar to the previous observationsof expression of ITAM-associated molecules in active andinactive RA patients OSCAR expression was also noted in themicrovasculature Given the similarities in pathogenesis ofRA and periodontitis [27] it is worth investigating expressionof ITAM factors in periodontitis gingivitis and normalgingival tissues

47 Expression of ITAM-Associated Molecules in Peri-Prosthetic Osteolysis We have reported a marked increasein the levels of TREM2 and DAP12 OSCAR and FcR120574 intissues containing PE particles compared with OA synovialcontrol tissue when assessed by a semiquantitative scoringsystem [75] Furthermore the observed increased levels ofthese proteins in peri-prosthetic tissues were consistent withthe finding that the corresponding mRNA levels were alsoincreased [75] Of interest PE-containing osteoclast-like


(a) (b)

(c) (d)

Figure 4 Expression of ITAM-associated molecules in PE-containing tissues from sites of aseptic loosening due to osteolysis (a) TREM2(b) DAP12 (c) OSCAR and (d) FcR120574 immunostaining The magnification was 400x

cells in these tissues were associated with high levels ofTREM2 and OSCAR protein and their respective adaptormolecules DAP12 and FcR120574 [75] (Figure 4) Consistent withthese in vivo observations PE particles added to humanperipheral blood derived osteoclast cells in vitro upregulatedITAM expression [75]

It is important to also understand the role synovialfluid may play in modulating regulators of cartilage andbone destruction in the joint Andersson et al (2007) foundthat synovial fluid from patients with OA stimulated themRNA expression of OSCAR and NFATc1 in mouse calvarialimplants in vitro while mRNA expressions of DAP12 andFcR120574 were not affected by synovial fluid from either revisionor OA patient groups The authors suggested that perhapsOSCAR and NFATc1 mRNA might be regulated by solublefactors that are present in OA synovial fluid Howeverexpression of DAP12 and FcR120574 was not regulated in the sameway [105] This is an area that could further be explored

Considering that inflammation recruits osteoclast pre-cursors and can induce the differentiation and activationof osteoclasts the enhanced expression of ITAM-relatedmolecules in revision tissues could exacerbate bone loss inthis disease

48 Potential Role for OSCAR in the Clinic Previous studieshave demonstrated that soluble fusion (OSCAR-fc) proteincomprising the extracellular domain ofOSCAR could inhibitosteoclastogenesis in murine preosteoclastosteoblast cocul-tures [14] and PBMCs cultured in the presence of RANKLM-CSF and TGF-120573 [94] In this situation sOSCAR couldcompete with OSCAR ligand and reduce OSCAR signalling

The ability of soluble sOSCAR in vitro to impede osteoclastformation may prove useful in inhibiting osteoclast differen-tiation and may thus prevent bone damage in diseases suchas RA

There is conflicting data as to whether sOSCAR increasesin healthy individuals or it increases as a result of erosiveactivity in RA Soluble OSCAR has been detected in serumand reported to be higher in healthy compared to RA patients[94 99] Serum levels of sOSCAR were shown to inverselycorrelate with erosion and disease activity [99] A recentstudy however reported higher levels of sOSCAR in theplasma of RA patients rather than healthy individuals [98]We have also detected sOSCAR in the synovial fluid of OAand active RA patients with no significant difference betweenthese diseases [67]We believe that sOSCAR has the potentialto act as decoy receptor for OSCAR ligand within the jointand affect osteoclast development in RA It is possible thatsuccessful treatment results in increased cleavage of cellassociated OSCAR resulting in increased sOSCAR levels inthe joint In this way sOSCAR regulates osteoclastic boneresorption and is an early marker that predicts joint damageThe biological effect of serum and synovial fluid-derivedOSCAR on osteoclastogenesis is yet to be investigated

Synovitis and erosion are not always linked with somepatients having progressive erosive disease despite being inremission and it is unclear what factors drive this [106] Thediscordance between clinical inflammatory disease activityand radiological outcomes emphasizes the need for a vali-dated marker of bone damage in conjunction with currentclinical parameters that are routinely assessed [107 108] Theability to monitor bone erosion will allow the clinician to


make important decisions on therapy earlier [109] and reducestructural joint damage [110] Currently unaddressed RA-induced joint damage affects mobility of patients later in lifeand predisposes to secondary osteoarthritis [110] In additionwhile we have numerous markers that reflect inflammationand related disease activity there are none that monitor jointerosion other than X-rays which only indicate damage afterit has occurred [111] Therefore it is essential that an accurateearly marker of joint erosion is identified in order to guideeffective treatment modalities in order to protect the joint ofarthritic patients

5 Conclusion

While the significance of ITAM-associated molecules hasbeen largely established in the context of bone biology andan immunological point of view limited studies have beencarried out on osteoclast ITAM-related molecules in humanbone pathologies The increased levels of ITAM factors ininflamed tissues adjacent to sites of localized bone loss inRA periodontal disease and periprosthetic osteolysis mayprove indicative of the disease progression Further to thislevels of the soluble factor OSCAR in serum or local fluidmay provide us with a potential bone destructive marker andpotential target for modulation of bone erosion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgements

This work was funded by the National Health and MedicalResearch Council of Australia the Allan and Beryl Stephensgrant from Arthritis Australia and an ASBMR GAP grantE Alias was supported by Adelaide Fee Scholarship Inter-national of the University of Adelaide and the MalaysianMinistry of Higher Education The authors would like tothank Tavik Morgenstern for designing the schematic usedin Figure one

References

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[2] S R Goldring P E Purdue T N Crotti et al ldquoBone remod-elling in inflammatory arthritisrdquo Annals of the RheumaticDiseases vol 72 no 2 pp ii52ndashii55 2013

[3] K Sato and H Takayanagi ldquoOsteoclasts rheumatoid arthritisand osteoimmunologyrdquo Current Opinion in Rheumatology vol18 no 4 pp 419ndash426 2006

[4] R A Kayal ldquoThe role of osteoimmunology in periodontaldiseaserdquo BioMed Research International vol 2013 Article ID639368 12 pages 2013

[5] T Crotti M D Smith R Hirsch et al ldquoReceptor activatorNF 120581B ligand (RANKL) and osteoprotegerin (OPG) protein

expression in periodontitisrdquo Journal of Periodontal Researchvol 38 no 4 pp 380ndash387 2003

[6] T N Crotti M D Smith H Weedon et al ldquoReceptor acti-vator NF-120581B ligand (RANKL) expression in synovial tissuefrom patients with rheumatoid arthritis spondyloarthropathyosteoarthritis and from normal patients semiquantitative andquantitative analysisrdquo Annals of the Rheumatic Diseases vol 61no 12 pp 1047ndash1054 2002

[7] D Haynes T Crotti H Weedon et al ldquoModulation of RANKLand osteoprotegerin expression in synovial tissue from patientswith rheumatoid arthritis in response to disease-modifyingantirheumatic drug treatment and correlation with radiologicoutcomerdquoArthritis Care and Research vol 59 no 7 pp 911ndash9202008

[8] D R Haynes T N Crotti A E Potter et al ldquoThe osteo-clastogenic molecules RANKL and RANK are associated withperiprosthetic osteolysisrdquo Journal of Bone and Joint Surgery vol83 no 6 pp 902ndash911 2001

[9] C A Holding D M Findlay R Stamenkov et al ldquoThecorrelation of RANK RANKL and TNF120572 expression with boneloss volume and polyethylene wear debris around hip implantsrdquoBiomaterials vol 27 no 30 pp 5212ndash5219 2006

[10] C Jiang Z Li H Quan et al ldquoOsteoimmunology in orthodon-tic tooth movementrdquo Oral Diseases 2014

[11] W S Simonet D L Lacey C R Dunstan et al ldquoOsteoprote-gerin a novel secreted protein involved in the regulation of bonedensityrdquo Cell vol 89 no 2 pp 309ndash319 1997

[12] D L Lacey E Timms H-L Tan et al ldquoOsteoprotegerinligand is a cytokine that regulates osteoclast differentiation andactivationrdquo Cell vol 93 no 2 pp 165ndash176 1998

[13] Y-Y Kong H Yoshida I Sarosi et al ldquoOPGL is a key regulatorof osteoclastogenesis lymphocyte development and lymph-node organogenesisrdquo Nature vol 397 no 6717 pp 315ndash3231999

[14] N Kim M Takami J Rho R Josien and Y Choi ldquoA novelmember of the leukocyte receptor complex regulates osteoclastdifferentiationrdquoThe Journal of Experimental Medicine vol 195no 2 pp 201ndash209 2002

[15] D Aletaha T Neogi A J Silman et al ldquo2010 rheumatoidarthritis classification criteria an American college of rheuma-tologyEuropean League against rheumatism collaborative ini-tiativerdquo Arthritis amp Rheumatism vol 62 no 9 pp 2569ndash25812010

[16] M G H van de Sande M J H de Hair C van der Leij etal ldquoDifferent stages of rheumatoid arthritis features of thesynovium in the preclinical phaserdquo Annals of the RheumaticDiseases vol 70 no 5 pp 772ndash777 2011

[17] M-M Tamas E Filippucci A Becciolini et al ldquoBone erosionsin rheumatoid arthritis ultrasound findings in the early stage ofthe diseaserdquo Rheumatology vol 53 no 6 Article ID ket484 pp1100ndash1107 2014

[18] R J Black L Spargo C Schultz et al ldquoDecline in hand bonemineral density indicates increased risk of erosive change inearly rheumatoid arthritisrdquo Arthritis Care and Research vol 66no 4 pp 515ndash522 2014

[19] M Bromley and D E Woolley ldquoChondroclasts and osteoclastsat subchondrial sites of erosion in the rheumatoid jointrdquoArthritis and Rheumatism vol 27 no 9 pp 968ndash975 1984

[20] E M Gravallese Y Harada J-T Wang A H Gorn T SThornhill and S R Goldring ldquoIdentification of cell types


responsible for bone resorption in rheumatoid arthritis andjuvenile rheumatoid arthritisrdquoThe American Journal of Pathol-ogy vol 152 no 4 pp 943ndash951 1998

[21] N C Walsh S Reinwald C A Manning et al ldquoOsteoblastfunction is compromised at sites of focal bone erosion ininflammatory arthritisrdquo Journal of Bone and Mineral Researchvol 24 no 9 pp 1572ndash1585 2009

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[23] P I Eke B A Dye L Wei G O Thornton-Evans and RJ Genco ldquoPrevalence of periodontitis in adults in the unitedstates 2009 and 2010rdquo Journal of Dental Research vol 91 no10 pp 914ndash920 2012

[24] G P Harvey T R Fitzsimmons A A S S K DhamarpatniC Marchant D R Haynes and P M Bartold ldquoExpression ofpeptidylarginine deiminase-2 and -4 citrullinated proteins andanti-citrullinated protein antibodies in human gingivardquo Journalof Periodontal Research vol 48 no 2 pp 252ndash261 2013

[25] K Lundberg N Wegner T Yucel-Lindberg and P J VenablesldquoPeriodontitis in RA-the citrullinated enolase connectionrdquoNature Reviews Rheumatology vol 6 no 12 pp 727ndash730 2010

[26] M D Cantley D R Haynes VMarino and PM Bartold ldquoPre-existing periodontitis exacerbates experimental arthritis in amouse modelrdquo Journal of Clinical Periodontology vol 38 no 6pp 532ndash541 2011

[27] P M Bartold R I Marshall and D R Haynes ldquoPeriodontitisand rheumatoid arthritis a reviewrdquo Journal of Periodontologyvol 76 no 11 supplement pp 2066ndash2074 2005

[28] D T Graves T Oates and G P Garlet ldquoReview of osteoim-munology and the host response in endodontic and periodontallesionsrdquo Journal of Oral Microbiology vol 3 article 5304 2011

[29] D W Howie ldquoTissue response in relation to type of wearparticles around failed hip arthroplastiesrdquoThe Journal of Arthro-plasty vol 5 no 4 pp 337ndash348 1990

[30] H-G Willert H Bertram and G Hans Buchhorn ldquoOsteolysisin alloarthroplasty of the hip the role of bone cement fragmen-tationrdquo Clinical Orthopaedics and Related Research no 258 pp108ndash121 1990

[31] H-G Willert and G H Buchhorn ldquoThe significance of wearandmaterial fatigue in loosening of hip prosthesesrdquoOrthopadevol 18 no 5 pp 350ndash369 1989

[32] T N Crotti M D Smith D M Findlay et al ldquoFactorsregulating osteoclast formation in human tissues adjacent toperi-implant bone loss Expression of receptor activator NF120581BRANK ligand and osteoprotegerinrdquo Biomaterials vol 25 no 4pp 565ndash573 2004

[33] G J Atkins K J Welldon C A Holding D R Haynes DW Howie and D M Findlay ldquoThe induction of a catabolicphenotype in human primary osteoblasts and osteocytes bypolyethylene particlesrdquo Biomaterials vol 30 no 22 pp 3672ndash3681 2009

[34] C Vermes K A Roebuck R Chandrasekaran J G Dobai J JJacobs andT TGlant ldquoParticulatewear debris activates proteintyrosine kinases and nuclear factor 120581B which down-regulatestype I collagen synthesis in human osteoblastsrdquo Journal of Boneand Mineral Research vol 15 no 9 pp 1756ndash1765 2000

[35] S R Goldring A L Schiller M Roelke C M Rourke D AOrsquoNeil and W H Harris ldquoThe synovial-like membrane at thebone-cement interference in loose total hip replacements and its

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[36] A Vasudevan E F DiCarlo T Wright et al ldquoCellular responseto prosthetic wear debris differs in patients with and withoutrheumatoid arthritisrdquo Arthritis and rheumatism vol 64 no 4pp 1005ndash1014 2012

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[38] B Hercus and P A Revell ldquoPhenotypic characteristics of Tlymphocytes in the interfacial tissue of aseptically loosenedprosthetic jointsrdquo Journal of Materials Science Materials inMedicine vol 12 no 10ndash12 pp 1063ndash1067 2001

[39] I Roato D Caldo L DrsquoAmico et al ldquoOsteoclastogenesis inperipheral blood mononuclear cell cultures of periprostheticosteolysis patients and the phenotype of T cells localized inperiprosthetic tissuesrdquo Biomaterials vol 31 no 29 pp 7519ndash7525 2010

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[41] S Landgraeber M Toetsch C Wedemeyer et al ldquoOver-expression of p53BAK in aseptic loosening after total hipreplacementrdquo Biomaterials vol 27 no 15 pp 3010ndash3020 2006

[42] W A Jiranek M Machado M Jasty et al ldquoProduction ofcytokines around loosened cemented acetabular componentsanalysis with immunohistochemical techniques and in situhybridizationrdquo Journal of Bone and Joint SurgerymdashSeries A vol75 no 6 pp 863ndash879 1993

[43] F F Buechel D Drucker M Jasty W Jiranek and W HHarris ldquoOsteolysis around uncemented acetabular componentsof cobalt-chrome surface replacement hip arthroplastyrdquoClinicalOrthopaedics and Related Research no 298 pp 202ndash211 1994

[44] T Nakashima and H Takayanagi ldquoOsteoimmunologycrosstalk between the immune and bone systemsrdquo Journal ofClinical Immunology vol 29 no 5 pp 555ndash567 2009

[45] W C Dougall M Glaccum K Charrier et al ldquoRANK isessential for osteoclast and lymph node developmentrdquo Genes ampDevelopment vol 13 no 18 pp 2412ndash2424 1999

[46] Y-Y Kung U Felge I Sarosi et al ldquoActivated T cells regulatebone loss and joint destruction in adjuvant arthritis throughosteoprotegerin ligandrdquoNature vol 402 no 6759 pp 304ndash3091999

[47] H Takayanagi S Kim T Koga et al ldquoInduction and activationof the transcription factor NFATc1 (NFAT2) integrate RANKLsignaling in terminal differentiation of osteoclastsrdquo Develop-mental Cell vol 3 no 6 pp 889ndash901 2002

[48] H Hirotani N A Tuohy J-T Woo P H Stern and N AClipstone ldquo The calcineurinnuclear factor of activated T cellssignaling pathway regulates osteoclastogenesis in RAW2647cellsrdquo The Journal of Biological Chemistry vol 279 no 14 pp13984ndash13992 2004

[49] N Ishida K Hayashi M Hoshijima et al ldquoLarge scale geneexpression analysis of osteoclastogenesis in vitro and elucida-tion of NFAT2 as a key regulatorrdquo The Journal of BiologicalChemistry vol 277 no 43 pp 41147ndash41156 2002

[50] K Matsuo D L Galson C Zhao et al ldquoNuclear factor of acti-vated T-cells (NFAT) rescues osteoclastogenesis in precursorslacking c-Fosrdquo Journal of Biological Chemistry vol 279 no 25pp 26475ndash26480 2004


[51] M Matsumoto M Kogawa S Wada et al ldquoEssential roleof p38 mitogen-activated protein kinase in cathepsin K geneexpression during osteoclastogenesis through association ofNFATc1 and PU1rdquoThe Journal of Biological Chemistry vol 279no 44 pp 45969ndash45979 2004

[52] Y Kim K Sato M Asagiri I Morita K Soma and HTakayanagi ldquoContribution of nuclear factor of activated T cellsc1 to the transcriptional control of immunoreceptor osteoclast-associated receptor but not triggering receptor expressed bymyeloid cells-2 during osteoclastogenesisrdquo The Journal of Bio-logical Chemistry vol 280 no 38 pp 32905ndash32913 2005

[53] T N Crotti M Flannery N C Walsh J D Fleming S RGoldring and K P McHugh ldquoNFATc1 regulation of the human1205733 integrin promoter in osteoclast differentiationrdquo Gene vol372 no 1-2 pp 92ndash102 2006

[54] T N Crotti S M Sharma J D Fleming et al ldquoPU1 andNFATc1mediate osteoclastic induction of themouse120573

3integrin

promoterrdquo Journal of Cellular Physiology vol 215 no 3 pp 636ndash644 2008

[55] M Asagiri K Sato T Usami et al ldquoAutoamplification ofNFATc1 expression determines its essential role in bone home-ostasisrdquo The Journal of Experimental Medicine vol 202 no 9pp 1261ndash1269 2005

[56] T-Y Ho K Santora J C Chen A-L Frankshun and C ABagnell ldquoEffects of relaxin and estrogens on bone remodelingmarkers receptor activator of NF-kB ligand (RANKL) andosteoprotegerin (OPG) in rat adjuvant-induced arthritisrdquoBonevol 48 no 6 pp 1346ndash1353 2011

[57] T Totsuka T Kanai Y Nemoto et al ldquoRank-rankl signalingpathway is critically involved in the function of CD4+CD25+regulatory T cells in chronic colitisrdquoThe Journal of Immunologyvol 182 no 10 pp 6079ndash6087 2009

[58] H-H Kim H S Shin H J Kwak et al ldquoRANKL regulatesendothelial cell survival through the phosphatidylinositol 3rsquo-kinaseAkt signal transduction pathwayrdquo The FASEB Journalvol 17 no 14 pp 2163ndash2165 2003

[59] K Loser A Mehling S Loeser et al ldquoEpidermal RANKLcontrols regulatory T-cell numbers via activation of dendriticcellsrdquo Nature Medicine vol 12 no 12 pp 1372ndash1379 2006

[60] L H D van Tuyl A E Voskuyl M Boers et al ldquoBaselineRANKLOPG ratio and markers of bone and cartilage degra-dation predict annual radiological progression over 11 years inrheumatoid arthritisrdquoAnnals of the Rheumatic Diseases vol 69no 9 pp 1623ndash1628 2010

[61] A R Pettit H Ji D Von Stechow et al ldquoTRANCERANKLknockout mice are protected from bone erosion in a serumtransfer model of arthritisrdquoThe American Journal of Pathologyvol 159 no 5 pp 1689ndash1699 2001

[62] S Kotake N Udagawa M Hakoda et al ldquoActivated humanT cells directly induce osteoclastogenesis from human mono-cytes possible role of T cells in bone destruction in rheumatoidarthritis patientsrdquo Arthritis amp Rheumatism vol 44 no 5 pp1003ndash1012 2001

[63] D R Haynes E Barg T N Crotti et al ldquoOsteoprotegerinexpression in synovial tissue from patients with rheumatoidarthritis spondyloarthropathies and osteoarthritis and normalcontrolsrdquo Rheumatology vol 42 no 1 pp 123ndash134 2003

[64] A R Pettit N C Walsh C Manning S R Goldring and EM Gravallese ldquoRANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoidarthritisrdquo Rheumatology vol 45 no 9 pp 1068ndash1076 2006

[65] H Takayanagi H Iizuka T Juji et al ldquoInvolvement ofreceptor activator of nuclear factor kappaB ligandosteoclastdifferentiation factor in osteoclastogenesis from synoviocytes inrheumatoid arthritisrdquoArthritis ampRheumatism vol 43 no 2 pp259ndash269 2000

[66] S Monticelli and A Rao ldquoNFAT1 and NFAT2 are positiveregulators of IL-4 gene transcriptionrdquo European Journal ofImmunology vol 32 no 10 pp 2971ndash2978 2002

[67] T N Crotti A A S S K Dharmapatni E Alias A C WZannettino M D Smith and D R Haynes ldquoThe immunore-ceptor tyrosine-based activation motif (ITAM)-related factorsare increased in synovial tissue and vasculature of rheumatoidarthritic jointsrdquo Arthritis Research amp Therapy vol 14 no 6article R245 2012

[68] N J Horwood V Kartsogiannis J MW Quinn E Romas T JMartin and M T Gillespie ldquoActivated T lymphocytes supportosteoclast formation in vitrordquo Biochemical and BiophysicalResearch Communications vol 265 no 1 pp 144ndash150 1999

[69] T Kawai T Matsuyama Y Hosokawa et al ldquoB and T lym-phocytes are the primary sources of RANKL in the boneresorptive lesion of periodontal diseaserdquoThe American Journalof Pathology vol 169 no 3 pp 987ndash998 2006

[70] G N Belibasakis G Emingil B Saygan O Turkoglu GAtilla andN Bostanci ldquoGene expression of transcription factorNFATc1 in periodontal diseasesrdquoAPMIS vol 119 no 3 pp 167ndash172 2011

[71] J Chiba H E Rubash and Y Iwaki ldquoThe characterization ofcytokines in the interface tissue obtained from failed cementlesstotal hip arthroplasty with and without femoral osteolysisrdquoClinical Orthopaedics and Related Research no 300 pp 304ndash312 1994

[72] S Stea M Visentin D Granchi et al ldquoCytokines and osteolysisaround total hip prosthesesrdquo Cytokine vol 12 no 10 pp 1575ndash1579 2000

[73] D R Haynes T N Crotti and H Zreiqat ldquoRegulation ofosteoclast activity in peri-implant tissuesrdquo Biomaterials vol 25no 20 pp 4877ndash4885 2004

[74] A SabokbarOKudo andNAAthanasou ldquoTwodistinct cellu-lar mechanisms of osteoclast formation and bone resorption inperiprosthetic osteolysisrdquo Journal of Orthopaedic Research vol21 no 1 pp 73ndash80 2003

[75] E Alias A S S KDharmapatni A CHolding et al ldquoPolyethy-lene particles stimulate expression of ITAM-related moleculesin peri-implant tissues andwhen stimulating osteoclastogenesisin vitrordquo Acta Biomaterialia vol 8 no 8 pp 3104ndash3112 2012

[76] E Merck B De Saint-Vis M Scuiller et al ldquoFc receptor 120574-chain activation via hOSCAR induces survival and maturationof dendritic cells and modulates Toll-like receptor responsesrdquoBlood vol 105 no 9 pp 3623ndash3632 2005

[77] E Merck C Gaillard D M Gorman et al ldquoOSCAR is anFcR120574-associated receptor that is expressed by myeloid cells andis involved in antigen presentation and activation of humandendritic cellsrdquo Blood vol 104 no 5 pp 1386ndash1395 2004

[78] K Nemeth M Schoppet N Al-Fakhri et al ldquoThe role ofosteoclast-associated receptor in osteoimmunologyrdquo The Jour-nal of Immunology vol 186 no 1 pp 13ndash18 2011

[79] A Mocsai M B Humphrey J A G van Ziffle et al ldquoTheimmunomodulatory adapter proteins DAP12 and Fc receptor120574-chain (FcR120574) regulate development of functional osteoclaststhrough the Syk tyrosine kinaserdquo Proceedings of the National


Academy of Sciences of the United States of America vol 101 no16 pp 6158ndash6163 2004

[80] M B Humphrey L L Lanier and M C Nakamura ldquoRole ofITAM-containing adapter proteins and their receptors in theimmune system and bonerdquo Immunological Reviews vol 208 no1 pp 50ndash65 2005

[81] H Takayanagi ldquoMechanistic insight into osteoclast differentia-tion in osteoimmunologyrdquo Journal of Molecular Medicine vol83 no 3 pp 170ndash179 2005

[82] T Koga M Inui K Inoue et al ldquoCostimulatory signalsmediated by the ITAM motif cooperate with RANKL for bonehomeostasisrdquo Nature vol 428 no 6984 pp 758ndash763 2004

[83] S Ishikawa N Arase T Suenaga et al ldquoInvolvement ofFcR120574 in signal transduction of osteoclast-associated receptor(OSCAR)rdquo International Immunology vol 16 no 7 pp 1019ndash1025 2004

[84] K Kim J H Kim J Lee et al ldquoNuclear factor of activatedT cells c1 induces osteoclast-associated receptor gene expres-sion during tumor necrosis factor-related activation-inducedcytokine-mediated osteoclastogenesisrdquoThe Journal of BiologicalChemistry vol 280 no 42 pp 35209ndash35216 2005

[85] Y Numasawa C Yamaura S Ishihara et al ldquoNasu-Hakoladiseasewith a splicingmutation of TREM2 in a Japanese familyrdquoEuropean Journal of Neurology vol 18 no 9 pp 1179ndash1183 2011

[86] J Paloneva T Manninen G Christman et al ldquoMutations intwo genes encoding different subunits of a receptor signalingcomplex result in an identical disease phenotyperdquo AmericanJournal of Human Genetics vol 71 no 3 pp 656ndash662 2002

[87] J Paloneva J Mandelin A Kiialainen et al ldquoDAP12TREM2deficiency results in impaired osteoclast differentiation andosteoporotic featuresrdquo The Journal of Experimental Medicinevol 198 no 4 pp 669ndash675 2003

[88] B Melchior A E Garcia B-K Hsiung et al ldquoDual inductionof TREM2 and tolerance-related transcript Tmem176b in amy-loid transgenic mice implications for vaccine-based therapiesfor Alzheimerrsquos diseaserdquo ASN Neuro vol 2 no 3 Article IDe00037 2010

[89] J Paloneva M Kestila JWu et al ldquoLoss-of-functionmutationsin TYROBP (DAP12) result in a presenile dementia with bonecystsrdquo Nature Genetics vol 25 no 3 pp 357ndash361 2000

[90] T Kaifu J NakaharaM Inui et al ldquoOsteopetrosis and thalamichypomyelinosis with synaptic degeneration in DAP12-deficientmicerdquo The Journal of Clinical Investigation vol 111 no 3 pp323ndash332 2003

[91] M Cella C Buonsanti C Strader T Kondo A Salmaggi andM Colonna ldquoImpaired differentiation of osteoclasts in TREM-2-deficient individualsrdquo The Journal of Experimental Medicinevol 198 no 4 pp 645ndash651 2003

[92] G S Kim J-M Koh J S Chang et al ldquoAssociation ofthe OSCAR promoter polymorphism with BMD in post-menopausal womenrdquo Journal of Bone andMineral Research vol20 no 8 pp 1342ndash1348 2005

[93] K Otero M Shinohara H Zhao et al ldquoTREM2 and beta-catenin regulate bone homeostasis by controlling the rate ofosteoclastogenesisrdquo Journal of Immunology vol 188 no 6 pp2612ndash2621 2012

[94] S Herman R B Muller G Kronke et al ldquoInduction ofosteoclast-associated receptor a key osteoclast costimulationmolecule in rheumatoid arthritisrdquo Arthritis and Rheumatismvol 58 no 10 pp 3041ndash3050 2008

[95] E Merck C Gaillard M Scuiller et al ldquoLigation of theFcR120574 chain-associated human osteoclast-associated receptorenhances the proinflammatory responses of human monocytesand neutrophilsrdquo Journal of Immunology vol 176 no 5 pp3149ndash3156 2006


[97] A D Barrow N Raynal T L Andersen et al ldquoOSCAR is a col-lagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and micerdquo Journal of Clinical Investigationvol 121 no 9 pp 3505ndash3516 2011

[98] N Ndongo-Thiam G de Sallmard J Kastrup and P MiossecldquoLevels of soluble osteoclast-associated receptor (sOSCAR) inrheumatoid arthritis Link to disease severity and cardiovascu-lar riskrdquo Annals of the Rheumatic Diseases vol 73 no 6 pp1276ndash1277 2014

[99] S Zhao Y-Y GuoNDing L-L Yang andN Zhang ldquoChangesin serum levels of soluble osteoclast-associated receptor inhuman rheumatoid arthritisrdquo Chinese Medical Journal vol 124no 19 pp 3058ndash3060 2011

[100] L C Chen J D Laskin M K Gordon and D L LaskinldquoRegulation of TREM expression in hepatic macrophages andendothelial cells during acute endotoxemiardquo Experimental andMolecular Pathology vol 84 no 2 pp 145ndash155 2008

[101] D-Y Chen L Yao Y-M Chen et al ldquoA potential role ofmyeloid DAP12-associating lectin (MDL)-1 in the regulation ofinflammation in rheumatoid arthritis patientsrdquo PLoS ONE vol9 no 1 Article ID e86105 2014

[102] A R Pettit HWeedonM Ahern et al ldquoAssociation of clinicalradiological and synovial immunopathological responses toanti-rheumatic treatment in rheumatoid arthritisrdquo Rheumatol-ogy vol 40 no 11 pp 1243ndash1255 2001

[103] C Goettsch M Rauner K Sinningen et al ldquoThe Osteoclast-Associated Receptor (OSCAR) is a novel receptor regulated byoxidized low-density lipoprotein in human endothelial cellsrdquoEndocrinology vol 152 no 12 pp 4915ndash4926 2011

[104] T Kobayashi K Yamamoto N Sugita et al ldquoEffective in vitroclearance of Porphyromonas gingivalis by Fc120572 receptor I (CD89)on gingival crevicular neutrophilsrdquo Infection and Immunity vol69 no 5 pp 2935ndash2942 2001

[105] M K Andersson P Lundberg A Ohlin et al ldquoEffects onosteoclast and osteoblast activities in cultured mouse calvarialbones by synovial fluids from patients with a loose jointprosthesis and from osteoarthritis patientsrdquo Arthritis ResearchandTherapy vol 9 no 1 article R18 2007

[106] F McQueen and E Naredo ldquoThe ldquodisconnectrdquo between synovi-tis and erosion in rheumatoid arthritis a result of treatment orintrinsic to the disease process itselfrdquo Annals of the RheumaticDiseases vol 70 no 2 pp 241ndash244 2011

[107] E Villeneuve and B Haraoui ldquoUncoupling of disease activityand structural damage Does it matter clinicallyrdquo Annals of theRheumatic Diseases vol 72 no 1 pp 1ndash2 2013

[108] E M Ruderman ldquoDefining the value of structural inhibition islow-dose etanercept inferior to the standard doserdquo Rheumatol-ogy vol 51 no 12 Article ID kes263 pp 2118ndash2119 2012

[109] E M Shanahan M Smith L Roberts-Thomson A Estermanand M Ahern ldquoInfluence of rheumatoid arthritis on work


participation inAustraliardquo InternalMedicine Journal vol 38 no3 pp 166ndash173 2008

[110] A Finckh N Bansback C A Marra et al ldquoTreatment of veryearly rheumatoid arthritis with symptomatic therapy disease-modifying antirheumatic drugs or biologic agents a cost-effectiveness analysisrdquo Annals of Internal Medicine vol 151 no9 pp 612ndash621 2009

[111] J S Smolen and D Aletaha ldquoForget personalised medicineand focus on abating disease activityrdquo Annals of the RheumaticDiseases vol 72 no 1 pp 3ndash6 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Chronic Inflammation-Mediated Bone Loss

21 Rheumatoid Arthritis Rheumatoid arthritis (RA) affects1-2 of the population and involves an autoimmune reactionwith an autoantibody response to citrullinated proteins (andothers such as rheumatoid factor and collagen type II) [15]RA is characterized by synovitis involving angiogenesissynovial proliferation increased infiltration survival anddecreased apoptosis of inflammatory cells [16] Further to thisthere is an increase in osteoclast number and activity leadingto focal bone erosions juxta-articular osteopenia and jointdestruction [17ndash20] Animal models suggest that there mayalso be suppression of localised osteoblast formation of bone[21]

22 Periodontitis and Similarities to RA Periodontitis is achronic inflammatory disease of the gingival tissues withan associated loss of the supporting structures includingthe periodontal ligament and alveolar bone The aetiologyinvolves an inflammatory response to bacterial infectionsuch as P gingivalis and possibly an autoimmune reactionas reviewed [22] Periodontitis is the most common andwidespread bone loss pathology in humans with 64 ofthe US population aged 65 years and older reported ashaving moderate or severe periodontitis [23] Despite theprevalence of this disease the most common treatment iseither mechanical subgingival plaque removal or surgicaldebridement Inevitably in the absence of effective treatmentsupport structures (periodontium) are compromised and theaffected teeth will loosen and fall out

RA and periodontitis have a similar pathophysiologycharacterized by destructive inflammation that culminatesin localized bone loss The citrullination of proteins by Pgingivalis and the subsequent generation of autoantigens thatdrive autoimmunity in RA have been proposed as a possiblemechanism linking these two diseases [24] Similarities in RAand periodontitis may relate to citrullinated enolase as thespecific antigen involved aswell as cross-reaction between theantibodies directed towards the immunodominant epitope ofhuman citrullinated alpha-enolase and a conserved sequenceon citrullinated P gingivalis enolase [25]

New evidence suggests a relationship between the extentand severity of chronic periodontitis and RA [24 25]Individuals with advanced RA are more likely to experiencemore significant periodontal problems compared to theirnon-RA counterparts and vice versa This is supported byfindings in a study using a combined animalmodel of RA andperiodontitis [26] which demonstratedmore severe develop-ment of arthritis in mice with periodontitis Further to thismice in which periodontitis alone was induced had evidenceof radiocarpal bone loss in the absence of arthritic disease[26] Additionally mice in which inflammatory arthritis wasinduced also had evidence of periodontitis [26]This suggestspresence of either RA or periodontitis places the individualat risk of developing the other disease Both conditionsinvolve an imbalance between proinflammatory and anti-inflammatory cytokines and increased bone-resorbing activ-ity Cantley et al (2011) thus proposed that these two diseasesare related through a common underlying dysfunction of

fundamental inflammatorymechanisms [26] New treatmentstrategies are needed for both diseases that target the inhi-bition of proinflammatory cytokines destructive enzymesand bone-resorbing activity The clinical implications ofthe current research strongly suggest that patients with RAshould be carefully screened for their periodontal status asreviewed [27]

23 Peri-Prosthetic Osteolysis Joint replacement surgery isused as a last resort in osteoarthritis (OA) and RA patientsand is a relatively successful operation however a largeproportion of implants fail within 10ndash20 years as a result ofbone loss and implant loosening [4 10 28]The pathogenesisbehind prosthetic implant failure involves wear of prostheticalloys such as polyethylene (PE) cobalt chrome and tita-nium liberated from the implant surface [29ndash31] These par-ticles stimulate a chronic inflammatory response [29] whichincreases bone-resorbing activity of the osteoclasts [32] andsuppresses bone formation by the osteoblast [33 34] resultingin bone loss [25] Periprosthetic tissues contain granuloma-tous lesions dominated by inflammatory cells particularlymacrophages and foreign-body giant cells [29ndash31 35 36] It isbelieved that an inflammatory reaction is initiated within thetissues in an attempt at particle clearance This then becomesa chronic reaction resulting in a granulomatous lesion Thisgranulomatous lesion in periprosthetic osteolysis often leadsto the formation of pseudosynovium-like structure in whichcells are organized into lining layer in the membranoustissues adjacent to the failed implant surface [35] Juxtaposedto this pseudosynovium are fibrous and collagenous regionspossibly scar tissues which could be indicative of late stageperiprosthetic osteolysis The plethora of factors release inthis inflammatory reaction within the tissues contributestowards the promotion of osteoclast formation [9 37]

Higher numbers of T lymphocytes have been observedin the periprosthetic tissues of human and mouse modelscontaining polyethylene and metal particles compared withnormal tissues [38ndash40] and osteoarthritic tissues [41] Sandhuet al (1998) proposed that T cells are indirectly affected bythe inflammatory cascade induced by wear particles [40] It ishowever important to note that T cells make up less than 10of total cell population [42] in periprosthetic tissues Giventhe low levels of T lymphocytes the general belief held isthat lymphocyte infiltrates are not normally associated withwear-particle induced periprosthetic osteolysis in particularin the granulomatous region [29 32 42 43]The role of T cellsmay however bemore pertinent in RApatients with implantsThe prevalence of foreign-body giant cells in response toimplant-derived wear debris in RA patients and non-RApatients does not differ but appears to be linked to the amountof polyethylene wear debris [36] As would be expectedthis study reported a high prevalence of plasma cells inlymphocytic infiltrates in untreated RA patients comparedwith non-RA patients with a different distribution [36]Whether implant wear is inducing a different reaction in RAversus non-RA patients may potentially have implications forcombination and immune targeted treatment of inflamma-tion and osteolysis in these patients

























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