REVIEW

Regulation of TNF-a with a focus on rheumatoidarthritis

Eva AV Moelants, Anneleen Mortier, Jo Van Damme and Paul Proost

Cytokines and chemokines represent two important groups of proteins that control the human immune system. Dysregulation

of the network in which these immunomodulators function can result in uncontrolled inflammation, leading to various diseases

including rheumatoid arthritis (RA), characterized by chronic inflammation and bone erosion. Potential triggers of RA include

autoantibodies, cytokines and chemokines. The tight regulation of cytokine and chemokine production, and biological activity is

important. Tumor necrosis factor-a (TNF-a) is abundantly present in RA patients’ serum and the arthritic synovium. This review,

therefore, discusses first the role and regulation of the major proinflammatory cytokine TNF-a, in particular the regulation

of TNF-a production, post-translational processing and signaling of TNF-a and its receptors. Owing to the important role of

TNF-a in RA, the TNF-a-producing cells and the dynamics of its expression, the direct and indirect action of this cytokine and

possible biological therapy for RA are described.

Immunology and Cell Biology advance online publication, 30 April 2013; doi:10.1038/icb.2013.15

Keywords: TNF-a; post-translational modification; autoantibodies; rheumatoid arthritis; cytokine

Chemokines and cytokines represent two important groups ofproteins that control the human immune system. A tight regulationof the cytokine and chemokine network is essential for an optimalfunctioning of the human immunological system. Uncontrolledcytokine production has been associated with many diseases includingrheumatoid arthritis (RA). Chronic inflammation and bone erosionare two central features of RA.1 Potential triggers of RA includeautoantibodies such as rheumatoid factor and anticitrullinatedpeptides antibodies (ACPAs), proinflammatory cytokines like tumornecrosis factor-a (TNF-a), interleukin (IL)-1b and receptor activatorof nuclear factor kB ligand (RANKL), and chemokines such asCXCL8/IL-8. TNF-a is abundantly present in RA patients’ serum andthe arthritic synovium as a primary and major cytokine disturbing thecontrolled balance between pro- and anti-inflammatory cytokines.2

Indeed, TNF-a is known to be a potent inducer of otherproinflammatory mediators including IL-1b, chemokines andproteases. Since a number of years, neutralization of TNF-a activityis used to treat RA patients.3,4

PATHOGENESIS OF RA

RA is a systemic chronic inflammatory disease, with a prevalencebetween 0.5 and 1%. Women are affected about three times morethan men.5 Its etiology is still unknown, but genetic factors,environmental influences, as well as life style (for example,smoking) all determine disease susceptibility.6,7 Pain, stiffness andsynovitis (inflammation of the synovial membrane) of joints aretypical symptoms of patients with RA.5

Bone resorptionUnder normal physiological conditions, a balance between boneformation and bone resorption exists. In patients with RA, thisbalance is disturbed in favor of bone resorption. RA is primarilyassociated with inflammation within the peripheral joints, and thedestruction of joint tissues and structures.8 Bone resorption dependson osteoclasts that are multinucleated giant cells developing fromhematopoietic cells of the monocyte/macrophage lineage at theinterface between the synovial tissue and articular bone. Boneerosion allows invasion of cells of the synovial membrane andresults in pannus formation.9 The pannus is an abnormal layer ofgranulation tissue composed of immune cells, blood vessels andfibrous cells. It starts growing from the synovial membrane andultimately invades the joint in RA, causing irreversible cartilagedestruction and bone erosion.10 The influx of osteoclast precursorsinto inflamed synovial tissue and their differentiation into matureosteoclasts is controlled by cytokines. Macrophage-colony-stimulatingfactor (M-CSF) and RANKL are essential for the maturation ofosteoclasts. M-CSF is mainly produced by synovial fibroblastsand endothelial cells.11–13 Although M-CSF is essential forosteoclastogenesis, on its own it is insufficient to induce finaldifferentiation of osteoclasts. RANKL is necessary to induce thefinal maturation steps of osteoclasts and their bone-resorbing activity.RANKL is predominantly expressed by synovial mesenchymal cellsincluding fibroblasts and activated synovial T cells.9,14

Osteoprotegerin is a soluble decoy receptor that, by bindingRANKL, prevents RANKL-dependent osteoclastogenesis.15

Laboratory of Molecular Immunology, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven, BelgiumCorrespondence: Professor P Proost, Laboratory of Molecular Immunology, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven,Minderbroedersstraat 10, Leuven 3000, Belgium.E-mail: paul.proost@rega.kuleuven.be

Received 23 January 2013; revised 21 March 2013; accepted 23 March 2013

Immunology and Cell Biology (2013), 1–9& 2013 Australasian Society for Immunology Inc. All rights reserved 0818-9641/13

www.nature.com/icb

Autoantibodies and cytokines in RARA belongs to the family of autoimmune diseases. Autoimmunity inRA refers to the production of antibodies specific for immunoglo-bulin G, called rheumatoid factor, or ACPAs, chronic inflammationand joint damage (including the synovial membrane, cartilage andbone). A spectrum of autoantibodies is known to be specificallyassociated with RA.16 These antibodies that bind the citrullinatedisoform of proteins are collectively named ACPAs. Recently, it wasshown that ACPAs can recognize citrullinated vimentin expressed onthe surface of osteoclast precursor cells. Binding of ACPAs to the cellsurface induces osteoclast differentiation through the autocrinestimulation of TNF-a production leading to bone loss.17 Duringosteoclast differentiation, the production of peptidylargininedeiminase (PAD)2, an enzyme responsible for protein citrullination,increases.17 A calcium flux will activate PAD2, and activated PAD2subsequently citrullinates vimentin, which is abundantly expressed onthe surface of osteoclast-lineage cells.

Chronic inflammation in RA is supported by the induction ofautoimmunity and the imbalance between pro- and anti-inflamma-tory cytokines. Cytokines regulate a broad range of inflammatoryprocesses associated with the pathogenesis of RA, and are abundantlypresent in RA patients’ serum and the arthritic synovium.2 Oncesynovial inflammation or synovitis develops from autoimmunity inRA, additional triggers such as the production of proinflammatorycytokines, for example, TNF-a, IL-1b and IL-6, further stimulateosteoclastogenesis (development of bone-resorbing osteoclasts) andbone erosion. Cytokine inhibition is one of the most effectiveapproaches to slow or even arrest bone erosion and preventprogression of systemic bone loss. Blocking of TNF-a is particularlyimportant in the pathogenesis of inflammatory osteolysis becauseTNF-a induces the expression of RANKL and synergizes with RANKLto directly promote osteoclast differentiation.18,19 TNF-a alsostimulates bone loss by mobilizing CD11bþ osteoclast precursorsfrom the bone marrow and by reducing bone formation by inhibitingosteoblast differentiation and function.20,21 In addition, TNF-ainduces the production of inflammatory chemokines resulting inthe accumulation of proinflammatory leukocytes, includingneutrophils, monocytes and activated T cells.22 However, inhibitionof TNF-a may result in unwanted side effects (for example,reactivation of tuberculosis in RA patients treated with TNF-ablockers) due to compromised host defense functions.23 Despite thepotential unwanted side effects,23 TNF-a blockers (infliximab,etanercept, adalimumab, certolizumab and golimumab) arenowadays a standard treatment for patients with RA.3,4 Therefore,the following discussion will focus on TNF-a as a major cytokineimplicated in RA. TNF-a was discovered in 1975 as an endotoxin-induced protein, which caused necrosis of sarcomas transplanted intomice.24 The importance of TNF-a in inflammation and disease hasbeen demonstrated by the success of anti-TNF-a antibodies oradministration of soluble TNF-a receptor (TNFR) as treatment forRA and other inflammatory diseases.

REGULATION OF THE PRODUCTION, PROCESSING AND

SIGNALING OF TNF-a AND ITS RECEPTORS

Regulation of the production of TNF-a and its receptorsOwing to its pleiotropic actions, the biosynthesis of TNF-a is underthe control of multiple and complex regulatory mechanisms. Theseregulatory mechanisms control different stages in the TNF-a pathwayincluding gene transcription, mRNA turnover, translation and intra-cellular signaling of the protein.

Regulation of TNF-a gene transcription. The induction of inflamma-tion in response to Toll-like receptor ligands is essential for hostdefense during infection. Expression of TNF-a is highly regulated atthe transcriptional level by a network of transcription factors, co-regulators and chromatin modifications.25 The TNF-a gene promoter,third intron and enhancer located immediately after the gene containmultiple transcription-factor-binding sites. These binding sitesregulate transcription through binding with members oftranscription factor families as nuclear factor of activated T cells(NFAT), nuclear factor-kB (NF-kB), Ets, interferon-regulating factorand CCAAT/enhancer-binding protein a.26 Co-regulators aretranscriptional regulators that, in contrast to transcription factors,lack DNA-binding properties. They modulate gene transcription inseveral ways for example, by histone-modifying activities andpromoting the assembly of a transactivating complex.25 DNAmethylation and covalent modification of histones (for example,phosphorylation, ubiquitination and methylation) influence thechromatin conformation, representing the epigenetic level of TNF-alocus regulation.27 Besides a proper induction of inflammation uponinfection, also the timely resolution is crucial to avoid harmful effectsof prolonged inflammation. Therefore, inflammatory genes andpathways are also negatively regulated. Negative feedbackmechanisms control both gene expression of TNF-a, as well as theintracellular signal transduction pathways of TNF-a. Members of theinhibitor of kB (IkB) family are examples of negative regulators ofinflammation that inhibit transcription of different genes includingTNF-a in a gene-specific manner.28

Regulation of TNF-a mRNA transport, stability and translation. Reg-ulatory mechanisms that operate after transcription also have a keyrole in balancing the TNF-a production. Short-lived RNA transcripts,such as several cytokine-encoding mRNAs, contain RNA-destabilizingelements that may in concert contribute to stringent regulation ofcytokine production. AU-rich elements (AREs), which are adenosine-and uridine-rich sequences in the 30 untranslated region of genes, arethe best-known destabilizing elements.29,30 AREs in the TNF-amRNA control its transport, stability and translation initiation.29,31

Mutant mice expressing a TNF-a gene with deletion of the TNF-aARE region showed higher levels of circulating TNF-a and evendeveloped two specific pathologies, that is, chronic inflammatoryarthritis and Crohn’s-like inflammatory bowel disease.31 Theinstability of ARE-containing mRNAs is regulated by ARE-bindingproteins. Tristetraprolin is an important ARE-binding protein thatinduces mRNA destabilization and has been found to associate withdifferent RNA degradation enzymes. Tristetraprolin was found todirectly bind the TNF-a mRNA ARE motif and in this way inhibitsthe production of TNF-a in macrophages by destabilizing itsmRNA.32 The importance of tristetraprolin in regulating TNF-alevels is also illustrated by tristetraprolin-knockout mice, whichdevelop a complex inflammatory phenotype and displayinflammatory arthritis, dermatitis, conjunctivitis and myeloidhyperplasia caused by chronic effective TNF-a excess.33

Another post-transcriptional mechanism for regulating the TNF-aactivity is by targeting of cytokine-encoding mRNA by microRNAs(miRNAs).30 Cytokine-encoding mRNAs contain regulatory miRNA-binding motifs, which can bind small 20–24nt noncoding RNAspecies called miRNAs transcribed from noncoding intronic andintergenic regions across the mammalian genome. miRNAs interactwith mRNAs and trigger translational suppression/activation ormRNA degradation.30 By computational analysis, 10 cytokine-encoding genes were predicted to harbor miRNA target sites.34

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miR-125b was reported to directly target TNF-a mRNA, therebynegatively influencing TNF-a production.35,36 In contrast, miR-369-3p was found to mediate translational activation of TNF-a.37 LikemiR-369-3p, miR-16 also showed sequence complementarity withTNF-a ARE.30 However, miR-16 inhibition led to a marked increasein TNF-a mRNA stability.38

Regulation of TNFRs. Another way of regulating the action of TNF-a is by controlling its receptor expression and activation. TNFRs aredifferently expressed as TNFR1 is found on the membrane of a widevariety of cells, whereas TNFR2 is mainly expressed by immune andendothelial cells.39,40 Receptor expression (mRNA and protein level) isinfluenced by a number of stimuli including TNF-a andIL-1.41 Other factors, as lipopolysaccharide, regulate receptor levelsby stimulating rapid internalization of TNFRs.42 Differentialexpression of TNFR1 and TNFR2 is caused by the structure of theirpromoter region. The promoter of TNFR1 possesses features of atypical housekeeping gene promoter, whereas the inducibility ofTNFR2 is obtained by a cAMP-response element and consensuselements for multiple transcription factors, like NF-kB, activatingprotein-1 (AP-1), interferon regulatory factor-1 and g-interferonactivation sequence.43,44 The presence of TNFRs at the cellsurface is also influenced by shedding of the extracellular domainby proteolytic cleavage. Soluble TNFRs attenuate the action ofTNF-a by competing with the membrane-bound receptors forTNF-a.45 Shedding of TNFR1 is induced by various inflammatorymediators as phorbol myristate acetate and reactive oxygen species.Nitric oxide and hydrogen peroxide are responsible for the activationof matrix metalloproteinases (MMPs) involved in sheddingof TNFR1.46,47

The importance of a proper regulation of expression and activationof TNFR is underscored by the detection of raised levels of mRNA,and surface protein of TNFR1 and TNFR2 in mononuclear cellsisolated from RA synovial joints.48 Furthermore, an association is seenbetween a single-nucleotide polymorphism in the TNFR2 gene andRA, and higher sTNFR2 levels in serum and synovial fluid were foundfor patients with familial RA.49–51

Regulation by the interaction with cytokines or other molecules. Theinduction of inflammation by inflammatory cytokines and chemo-kines is essential for host defense during infection. However,prolonged inflammation causes irreversible damage and even disease.Therefore, the inflammatory effect of TNF-a is also regulated at thelevel of intracellular signaling by anti-inflammatory cytokines such asIL-4 and IL-10. Immunization of mice with anti-IL-10 resulted in agreater and more sustained increase in plasma TNF-a levels, anindication for the importance of IL-10 for the downregulation ofTNF-a.52 Inhibitors binding directly with TNF-a such as solubleTNFR1 and TNFR2 were also reported.53

Processing of TNF-a and its receptorsBesides controlling the production of TNF-a and its receptors, theaction of TNF-a and its receptors can be regulated by post-translational modification performed by enzymes.

Proteolytic cleavage of TNF-a and its receptor. The proteolysis ofcytokines by proteases can lead to altered bioavailability and activity.Shedding of the major proinflammatory cytokine TNF-a representsone example of how proteolytic cleavage of cytokines can modulatethe inflammatory reaction. TNF-a is produced as a 26 kDa propro-tein, mainly by activated monocytes and macrophages, but also by awide range of other cells like T cells, B cells, endothelial cells and

fibroblasts.40 Pro-TNF-a is expressed on the plasma membrane,where it can be cleaved by proteases such as MMPs and othermetalloproteases. Cleavage by the metalloprotease TACE/adisintegrin-like metalloproteinase (ADAM)-17 releases the soluble17 kDa form of TNF-a from the cell surface.54 In vivo studies haveshown that most of the biological functions of TNF-a require itsshedding and release as a soluble mediator. Mice carrying amutation in their pro-TNF-a sequence, that blocks effective releaseof the membrane-anchored protein, suboptimally developedinflammation.55 Moreover, MMP-1, MMP-2, MMP-3, MMP-7,MMP-9, MMP-12, MMP-14 and MMP-17 release active TNF-afrom the cell surface by a similar mechanism as ADAM-17-mediated shedding.56 On the other hand, ADAM-17 can also actanti-inflammatory as it is also involved in the processing of TNFRinto its soluble variant, which can neutralize the action of TNF-a.40

Citrullination of TNF-a. Higher levels of TNF-a can be measured insynovial fluid of RA patients compared with healthy controls.57,58

PAD2 mRNA or protein have been detected in lymphocytic andmonocytic cells.59 PAD4 is widely expressed in T cells, B cells,macrophages, neutrophils and fibroblast-like cells. Both isotypes arefound in the synovium of RA patients.59,60 Furthermore, a haplotypeof PADI4 associated with susceptibility to RA and with increasedmRNA stability was identified.61 This haplotype is associated withincreased levels of autoantibodies to citrullinated peptides in serafrom individuals with RA. The colocalization of the cytokine TNF-aand PAD4 combined with the higher stability of PAD4 mRNA in RAmake this cytokine a potential target for citrullination. Ascitrullination is known to influence the biological activity ofchemokines, citrullination of TNF-a might represent an additionalway of affecting the role of this cytokine in inflammatory processes.Recently, it was shown that citrullination of TNF-a reduced itspotency to stimulate chemokine production in vitro on humanprimary fibroblasts.62

Signaling of TNF-aTNF-a executes its function through binding to two structurallydistinct membrane receptors, TNFR1 (also known as p55 orTNFRSF1A) or TNFR2 (also known as p75 or TNFRSF1B).40,63–65

Binding will activate two separate intracellular signaling pathwaysleading to gene transcription. TNFRs are expressed by a wide varietyof cells. No cell type of the body has yet been found that does notexpress TNFR1, whereas TNFR2 is selectively expressed by immuneand endothelial cells.39,40 In general, binding of TNF-a to TNFR1promotes cell survival and proinflammatory functions through NF-kB and AP-1 signaling, and apoptosis. The regulation of T cellmigration, proliferation, activation and antigen presentation, tissuerepair and angiogenesis mediated by TNF-a, in contrast, relies onsignaling through TNFR2.66,67 However, some degree of receptorcrosstalk and overlapping functioning exists.67,68 In contrast to theextracellular-ligand-binding domains of these TNFRs, which bothcontain similar cysteine-rich subdomains, the intracellular domainsshow no sequence homology. Both receptors activate signaltransduction by recruitment of different proteins from the cytosolthrough specific protein–protein interaction domains.69 TNFR1 isactivated by both the 26 kDa membrane-bound and 17 kDa solubleform of TNF-a, whereas TNFR2 only signals after binding ofmembrane-bound TNF-a.70 The difference in principal ligands forTNFR1 and TNFR2, soluble and membrane-bound TNF-a,respectively, also contributes to the various signaling effects ofTNFR, as both forms of TNF-a have opposite biological effects, for

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example, only soluble TNF-a is able to induce the formation ofsignificant levels of osteoclasts.71 Signaling by TNFR1 starts with thetrimerization of the receptor, bringing together the death domains(DDs) and initiating the recruitment of TNFR-associated DD protein(TRADD) (Figure 1). Under unstimulated conditions, the cytoplas-matic DD of TNFR1 will bind the DD of silencer of DD protein(SODD).18,72 TNF-a binding to TNFR1 leads to the release of SODD,allowing the binding of TRADD.72 TRADD initiates signaling byrecruiting two additional molecules, receptor interacting protein-1(RIP-1) and TNFR-associated factor (TRAF)2.66,73–75 Furthersignaling of the TRADD–RIP-1–TRAF2 complex stimulates AP-1and NF-kB activation. NF-kB is activated in two ways.66 First, RIP-1can recruit kinases like mitogen-activated protein (MAP) kinasekinase kinase (MAP3K or MEKK)3, which activates the b-subunitof the inhibitor of kB kinase (IKK) complex.76 This activation leads tophosphorylation, ubiquitination and finally degradation of thecytosolic IkB protein, which forms a complex with NF-kB.77

Unbound NF-kB can enter the nucleus and initiates genetranscription. NF-kB controls the expression of multiple genesencoding proteins that control inflammatory processes includingproinflammatory cytokines (for example IL-1, IL-6 andTNF-a).10,78 In their turn, these cytokines induce the production ofchemokines (for example CXCL8, CC chemokine ligand (CCL)3/macrophage inflammatory protein-1a, CCL2/monocyte chemotacticprotein-1 and CCL5/RANTES (which stands for regulated onactivation, normal T cell expressed and secreted)) and theirreceptors.22 Secondly, TRAF2 can also activate NF-kB signalingthrough direct binding to the IKK complex and the subsequent

recruitment of inhibitor of cellular apoptosis proteins (cIAPs)-1and -2 that participate in IkB degradation.79 TRAF2 can alsoassociate with MAP3K that activates MAP kinase kinases (MAP2Kor MEK), including MEK-6, which in turn phosphorylate and activatec-Jun NH2-terminal kinases (JNKs) and p38 MAP kinase.80 ActivatedJNKs phosphorylate a subunit of the transcription factorAP-1, named c-Jun, which is essential for the initiation of genetranscription by AP-1.81 Apart from promoting cell survival andproinflammatory functions through NF-kB and AP-1 signalingleading to cytokine and chemokine induction, binding of TNF-a toTNFR1 can also initiate apoptosis. Therefore, Fas-associated DDprotein has to interact with TRADD to initiate the recruitment ofprocaspase-8.75,82 Autocatalytic activation of procaspase-8 releasesactive caspase-8, which induces the activation of other caspases andfinally leads to apoptosis.82 The cell death signaling pathway isinhibited by NF-kB, which induces cFLIP, an inhibitor of therecruitment and activation of procaspase-8.83

The signaling pathways activated by TNFR2 are still poorlyunderstood (Figure 1). TNFR2 lacks an intracellular DD, but bindingof TNF-a causes trimerization of TNFR2, enabling its direct interac-tion with TRAF2. TRAF2 interacts with TRAF1, TRAF3, cIAP-1 andcIAP-2, resulting in the activation of transcription factors NF-kB orAP-1, through, respectively, the activation of IKK complex anddegradation of IkB, or the activation of MAP3K and JNK.84–86

Binding of TNF-a to TNFR2 can also initiate the noncanonial NF-kB pathway by first activating NF-kB inducing kinase, whichsubsequently interacts with IKKa and IKKb. This interaction resultsin NF-kB signaling.87 Signaling through TNFR2 is autoregulated by

Figure 1 TNF-a-mediated signaling through TNFR1 and TNFR2. Signaling by TNFR1 starts with the trimerization of the receptor, bringing together the DDsand initiating the recruitment of TRADD. TRADD initiates signaling by recruiting two additional molecules, RIP-1 and TRAF2. Further signaling of the

TRADD-RIP-1-TRAF2 complex stimulates AP-1 and NF-kB activation, and apoptosis. Firstly, NF-kB is activated through the activation of the IKK complex.

IKK activation leads to phosphorylation, ubiquitination and finally degradation of cytosolic IkB protein, which forms a complex with NF-kB. Unbound NF-kB

can enter the nucleus and initiates gene transcription. Secondly, TRAF2 can also associate with MAP3K/MEKK3, which activates MAP2K or MEK, which in

turn phosphorylates and activates JNKs and p38 MAP kinase (MAPK). Activated JNKs phosphorylate a subunit of the transcription factor AP-1, named

c-Jun, which is essential for the initiation of gene transcription by AP-1. Finally, binding of TNF-a to TNFR1 can also initiate apoptosis. Therefore, Fas-

associated DD protein (FADD) has to interact with TRADD to initiate the recruitment of procaspase-8. Autocatalytic activation of procaspase-8 releases

active caspase-8, which induces the activation of other caspases and finally leads to apoptosis. The signaling pathways activated by TNFR2 are still poorly

understood. TNFR2 lacks an intracellular DD, but binding of TNF-a causes trimerization of TNFR2, enabling its direct interaction with TRAF2. TRAF2

interacts with TRAF1, TRAF3, cIAP-1 and cIAP-2, resulting in the activation of transcription factors NF-kB or AP-1, through the activation of IKK complex

and degradation of IkB, respectively, or the activation of MAP3K and JNK. Binding of TNF-a to TNFR2 can also initiate the noncanonial NF-kB pathway by

first activating NF-kB-inducing kinase (NIK), which subsequently interacts with IKKa and IKKb. This interaction results in NF-kB signaling. Furthermore,

TNFR2 can also activate endothelial/epithelial tyrosine kinase (Etk) independently of TRAF2. Active Etk mediates crosstalk with vascular endothelial growth

factor receptor 2 (VEGFR2) through reciprocal phosphorylation, resulting in activation of the phosphatidylinositol 3-kinase (PI3K)-Akt angiogenic pathway in

endothelial cells.

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cIAP-1- and cIAP-2-dependent ubiquitination and proteasomaldegradation of TRAF2.88 TNFR2 can also activate endothelial/epithelial tyrosine kinase independently of TRAF2. Binding ofTNF-a induces a conformational change of TNFR2 that causesunfolding of the bound inactive endothelial/epithelial tyrosinekinase into its active form. Active endothelial/epithelial tyrosinekinase mediates crosstalk with vascular endothelial growth factorreceptor 2 through reciprocal phosphorylation, resulting in activationof the phosphatidylinositol 3-kinase-Akt angiogenic pathway inendothelial cells.89

TNF-a EXPRESSION AND ACTION IN RA

TNF-a has a pivotal role in the pathophysiology of RA. TNF-a acts ona range of cell types in the joint space to amplify and perpetuate theinflammatory process.90 Levels of TNF-a are elevated in synovial fluidof RA patients compared with healthy controls.57,58

TNF-a expression in RANeither the initiating nor the perpetuating events in the pathogenesisof RA are well understood. However, the role of TNF-a herein isevident. It is hypothesized that initially the binding of ACPAs toosteoclast precursor cells stimulates the release of TNF-a. TNF-a thanpromotes the differentiation of these cells into mature osteoclasts,leading to synovitis and initial bone loss.1 Moreover, Toll-likereceptors are hypothesized to have a key function at an initial stageof synovial activation by binding microbial components orendogenous ligands, thereby activating synovial fibroblasts leadingto the expression of proinflammatory cytokines and chemokines, andsubsequent attraction and accumulation of immune cells in thesynovium.91 In the inflamed joint, TNF-a can be produced by variouscells (for example activated macrophages, synovial fibroblasts, T cellsand B cells, neutrophils, natural killer cells, osteoblasts andosteoclasts) causing chronic inflammation and bone loss.18,92–96

Synovial T cells can contribute directly to synovitis through theproduction of TNF-a.92–94 However, T cells primarily indirectlyinfluence TNF-a levels by the production of cytokines that willstimulate other cells to produce TNF-a.10 Moreover, the contact-dependent interaction of T cells with neighboring macrophages andsynovial fibroblasts activates these cells, inducing TNF-aproduction.97 Macrophages are a second important source ofcytokines in the synovium.92,94 Activation likely occurs throughpattern-recognition receptors such as Toll-like receptors, as Toll-likereceptor2-deficient mice with streptococcal cell wall-induced arthritiswere unable either to develop joint swelling or to inhibit cartilagematrix synthesis.98 Neutrophils are present in high numbers insynovial fluid. Once activated, they synthesize and release a varietyof cytokines including TNF-a and contribute significantly to thehypoxic environment in inflamed joints.94,99 Natural killer cells arewidely distributed within RA synovial tissue and release significantlevels of TNF-a in response to other cytokines.95 Natural killer cellsmay also contribute indirectly to high TNF-a concentrations found inRA by stimulating monocytes to produce TNF-a in a contact-dependent manner.100 Human osteoblasts release TNF-a that canact as an endogenous mitogenic factor stimulating osteoblasticproliferation.96

The dynamics of TNF-a expression in early stages of RA are notwell understood because sampling and subsequent examination hasonly been performed on patients with well-established disease.However, by utilizing animal collagen-induced arthritis models forRA, the kinetics of proinflammatory expression before and afterdisease onset were studied. Interestingly, TNF-a was detected

preceding disease onset, inflammatory cell infiltration and thedevelopment of destructive changes in the joint. Initially, TNF-a ismainly produced by synoviocytes within the synovial-lining layer,causing chemotaxis of monocytes and neutrophils into the syno-vium.101 TNF-a expression progressively increases from onset ofarthritis in the collagen-induced arthritis model until a maximum isreached on day 10 corresponding to the peak of paw swelling and tomanifestation of erosive changes in cartilage and bone.102

Indirect action of TNF-a by the induction of mediatorsTNF-a indirectly exerts different functions in the pathogenesis of RAby the induction of multiple mediators. First, TNF-a stimulatesendothelial cells to express integrins and adhesion molecules at theirsurface to allow leukocyte accumulation. Intracellular adhesionmolecule-1, which is required for the extravasation of neutrophilsfrom the bloodstream into the inflamed joint tissue, is upregulated onendothelial cells by TNF-a.103 Secondly, TNF-a induces theproduction and release of chemokines as CXCL8, CCL2 and CCL5,which are also important for leukocyte recruitment andangiogenesis.22 For example, CXCL8 is known to be abundantlypresent in RA synovial fluid, synovial tissue and serum.104,105 TNF-aactivates neutrophils and fibroblasts in the inflamed synovial tissue tosecrete large amounts of CXCL8.105–107 In turn, CXCL8 mainlyactivates and attracts neutrophils during inflammatory processes.TNF-a also stimulates leukotriene B4 production, which attractsmyeloid cells and induces neutrophils to synthesize and secretebiologically active CXCL8.108 The attracted inflammatory cells willproduce additional TNF-a, resulting in a positive feedback andenhanced inflammation. A third function of TNF-a is theinduction of other proinflammatory cytokines like IL-1, IL-6 andgranulocyte macrophage-colony-stimulating factor.2,109–111 Inaddition, TNF-a also stimulates the production of osteoclastogeniccytokines such as M-CSF by endothelial cells.12,13 Furthermore,TNF-a stimulates RA pathogenesis by promoting dendritic celldifferentiation leading to autoantigen presentation to T cells in thesynovium of RA patients.112,113 The action of TNF-a was alsoperceived to be important for angiogenesis typical for RA, and aprerequisite for inflammation and destruction. For instance,inhibition of TNF-a is associated with the downregulation ofvascular endothelial growth factor and the suppression ofneovascularization.114 Another important role of TNF-a in thedevelopment of RA is to enhance the development, activation andrecruitment of bone-resorbing osteoclasts by inducing the expressionof the cytokine RANKL by bone marrow stromal cells through theinduction of IL-1b and IL-1bR, and by synergizing with RANKL(Figure 2).19,109 Binding of RANKL to its receptor RANK causesactivation of nuclear factor of activated T cells, cytoplasmic 1(NFATc1) through two pathways.115 The NF-kB/AP-1/c-fos pathwayis triggered through TRAF6, which is the main adapter molecule ofRANK. On the other hand, activation of NFATc1 is dependent oncalcium signaling. The tyrosine kinases Tec and Btk are first activatedby RANKL–RANK interaction and cause phosphorylation of PLCg.116

Phosphorylated PLCg consequently mediates calcium release in thecytoplasm that in turn activates the phosphatase calcineurin.115,117

Calcineurin finally activates NFATc1 by dephosphorylating its NH2-terminal regulatory domain.117 Intracellular calcium can also activateCa2þ /calmodulin-dependent kinases, which then induce theexpression of NFATc1 and facilitate NFATc1-dependent generegulation.118 During osteoclast differentiation, the production ofPAD2 is known to increase, therefore, elevated Ca2þ might alsoactivate PAD2 and cause citrullination of different target proteins.17

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TNF-a-induced prostaglandin E2 stimulates osteoblasts to releasefactors that stimulate bone resorption by osteoclasts.119 Finally,TNF-a also stimulates cartilage destruction by accelerating thechondrocyte switch from an anabolic matrix-synthesizing state to acatabolic state, the production of matrix-degrading enzymes andMMPs, and the secretion of Dickkopf homolog-1, which acts as aWnt inhibitor and blocks bone and cartilage formation.10,120

Direct action of TNF-aFurthermore, TNF-a may also directly act on osteoclast precursorsand stimulate osteoclast differentiation through a mechanism inde-pendent of the RANKL/RANK system (Figure 2).121

Osteoclastogenesis is then promoted through TNFR1 signaling,whereas TNFR2 acts inhibitory.71,122 Moreover, their principalligands, soluble and membrane-bound TNF-a, respectively, haveopposite effects, as only soluble TNF-a is able to induce theformation of significant levels of osteoclasts.71 Under unstimulatedconditions, SODD is bound to the cytoplasmatic DD of TNFR1.18

SODD detaches upon binding of TNF-a to TNFR1, allowing thebinding of TRADD.72 TRADD attracts RIP-1 and TRAF2.74 TRAF2, 5and 6 mediate further signaling through NF-kB, JNK and

p38.18,66,73,123 This will lead to the activation of NFATc1.115,123

NFATc1 is a key regulator of osteoclast differentiation, as it inducesosteoclast-specific genes (for example, TNFR-associated protein,calcitonin receptor, cathepsin K and MMP-9) and positivelyregulates its own promoter.115

In conclusion, elevated levels of TNF-a detected in RA patients cancause osteoclastogenesis directly by binding to TNFR1 on osteoclastprecursors and indirectly by the production of RANKL. TNF-afurther influences bone resorption by mobilizing CD11bþ osteoclastprecursors from the bone marrow and reducing bone formation byinhibiting osteoblast differentiation and function.20,21

Therapy used for RA patientsTraditionally, the treatment of RA was based upon disease-modifyinganti-rheumatic drugs like methotrexate, which slow down jointdamage and reduce joint swelling and pain.124 However, disease-modifying anti-rheumatic drugs are associated with problems due tolack of efficacy and side effects.3 More recently, biologic therapeuticstargeting molecules involved in the inflammatory response such asproinflammatory cytokines have been launched. TNF-a blockers(infliximab, etanercept, adalimumab, certolizumab and golimumab)are nowadays regularly used for treating RA patients.3,4 Infliximab,adalimumab and golimumab are monoclonal anti-TNF-a antibodies.Certolizumab is a humanized Fab fragment conjugated topolyethylene glycol. Etanercept is a fusion protein of twoextracellular domains of the TNFR2 receptor and the Fc region ofhuman immunoglobulin G1. However, because of the high non-responder rate to anti-TNF-a therapy (approximately one-third of thepatients) drugs are being developed targeting either other cytokines,chemokine or chemokine receptors.125 Besides TNF-a, also IL-1brepresents a potential trigger for inflammation and bone erosion inRA. Drugs mimicking IL-1 receptor antagonist, like anakinra,competitively bind with high affinity to IL-1 receptors, therebyreducing disease progression of RA.126 Canakinumab, a fully humananti-IL-1b monoclonal antibody, represents a second drug thatsuppresses the action of this cytokine.127 Another fully humanmonoclonal antibody targeting RANKL, denosumab, was shown tobe able to induce a rapid and sustained inhibition of boneresorption.128 Current data suggest that also the inhibition of othercytokines like IL-6, IL-12, IL-15, IL-17, IL-18 and IL-23 can beconsidered as novel therapeutic strategies.1,10 Targeting chemokinesand their receptors also seems promising as future therapy for RA.Recently, a CCR1 antagonist was examined in a phase Ib clinical trial.Treatment with this inhibitor resulted in a decrease in macrophages inthe synovium and clinical improvement compared with the placebogroup.129 An overview of other small molecule antagonists of CCR1developed by pharmaceutical companies was published.130 Thechemokine receptor CCR5 is important for chemotaxis of T cells byCCL3 and CCL5, which are found in high levels in the synovial fluidof RA patients.131 Patients with the non-functional CCR5 D32 alleleshow a decreased severity and incidence of RA.132 However, blockageof CCR5 did not seem to cause improvement of RA symptoms.133

Moreover, other CC chemokine receptors (for example, CCR2 onmonocytes and CCR6 on immature dendritic cells) are recognized asattractive drug targets for small molecule antagonists in the treatmentof RA.130

CONCLUSION

Uncontrolled cytokine production can cause many diseases. Themajor proinflammatory cytokine TNF-a is abundantly present in RApatients’ serum and the arthritic synovium. TNF-a stimulates

Figure 2 TNF-a-mediated signaling in osteoclasts. Elevated levels of TNF-adetected in RA patients can cause osteoclastogenesis directly by binding to

TNFR1 and indirectly by the production of RANKL. Binding of TNF-a to

TNFR1 leads to the release of SODD from the cytoplasmatic DD of the

receptor. This allows the binding of TRADD to TNFR1. TRADD recruits

RIP-1 and TRAF2. TRAF2, 5 and 6 mediate further signaling through

NF-kB, JNK and p38. This will lead to the potent activation of nuclear

factor of activated T cells, cytoplasmic 1 (NFATc1). NFATc1 is a key

regulator of osteoclast differentiation, as it induces osteoclast-specific genes

and positively regulates its own promoter. TNF-a also promotes

osteoclastogenesis by inducing the expression of RANKL by bone marrowstromal cells. Binding of RANKL to its receptor RANK causes activation of

NFATc1 in two ways. The NF-kB/AP-1/c-fos pathway is triggered through

TRAF6, which is the main adapter molecule of RANK. On the other hand,

activation of NFATc1 is dependent on calcium signaling. The tyrosine

kinases Tec and Btk are first activated by RANKL–RANK interaction, and

cause phosphorylation of PLCg. Phosphorylated PLCg consequently

mediates calcium release in the cytoplasm, which in turn activates

the phosphatase calcineurin. Calcineurin finally activates NFATc1 by

dephosphorylating its NH2-terminal regulatory domain. Intracellular calcium

can also activates Ca2þ /calmodulin-dependent kinases (CaMKs), which then

induce the expression of NFATc1 and facilitate NFATc1-dependent gene

regulation. During osteoclast differentiation, the production of PAD2 is

known to increase. Thus, elevated Ca2þ might also activate PAD2 and

cause citrullination of different target proteins.

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Immunology and Cell Biology

inflammation, osteoclastogenesis, and subsequent destruction of jointtissues and bone erosion within the peripheral joints, known to bemain features associated with RA. Therefore, the role and regulationof TNF-a in RA, in particular, (1) the regulation of TNF-aproduction, (2) post-translational processing, and (3) signaling ofTNF-a and its receptors were unraveled. The biosynthesis of TNF-a isunder the control of multiple and complex regulatory mechanisms.These regulatory mechanisms control different stages in the TNF-apathway including gene transcription, mRNA turnover, translationand intracellular signaling of the protein. The activity of TNF-a is alsoregulated by post-translational processing of the cytokine and itsreceptor (for example, proteolytic cleavage and citrullination). Finally,TNF-a performs its pleiotropic functions by signaling through tworeceptors, TNFR1 and TNFR2. Owing to its pivotal role in thepathophysiology of RA, TNF-a production and action in RA werefurther explained. TNF-a can be produced by different cells in theinflamed joint. This cytokine has various direct and indirect actionsby the binding to TNFR or the induction of inflammatory mediators,respectively, (for example integrins, adhesion molecules and chemo-kines). A successful therapy for RA is to block the action of TNF-a bydrugs such as infliximab, etanercept, adalimumab, certolizumab andgolimumab. However, a significant part of RA patients do notrespond to anti-TNF-a therapy. Therefore, the inhibition of othercytokines like IL-1 and RANKL, or chemokine receptors like CCR1,are alternative approaches to effectively slow down or even arrestbone erosion and prevent progression of systemic bone loss in theseRA patients.

ACKNOWLEDGEMENTSThis work was supported by the Fund for Scientific Research of Flanders

(FWO-Vlaanderen) and the Concerted Research Actions (GOA) of the

Regional Government of Flanders. Mortier A holds a postdoctoral fellowship

of the FWO-Vlaanderen.

1 Schett G, Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosisand treatment. Nat Rev Rheumatol 2012; 8: 656–664.

2 Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis.J Clin Invest 2008; 118: 3537–3545.

3 Edwards CJ. Immunological therapies for rheumatoid arthritis. Br Med Bull 2005;73-74: 71–82.

4 Thalayasingam N, Isaacs JD. Anti-TNF therapy. Best Pract Res Clin Rheumatol 2011;25: 549–567.

5 Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001; 358: 903–911.6 Lundstrom E, Kallberg H, Alfredsson L, Klareskog L, Padyukov L. Gene-environment

interaction between the DRB1 shared epitope and smoking in the risk of anti-citrullinated protein antibody-positive rheumatoid arthritis: all alleles are important.Arthritis Rheum 2009; 60: 1597–1603.

7 Kallberg H, Padyukov L, Plenge RM, Ronnelid J, Gregersen PK, van der Helm-van MilAH et al. Gene-gene and gene-environment interactions involving HLA-DRB1,PTPN22, and smoking in two subsets of rheumatoid arthritis. Am J Hum Genet2007; 80: 867–875.

8 Goldring SR, Gravallese EM. Pathogenesis of bone erosions in rheumatoid arthritis.Curr Opin Rheumatol 2000; 12: 195–199.

9 Gravallese EM, Manning C, Tsay A, Naito A, Pan C, Amento E et al. Synovial tissue inrheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum2000; 43: 250–258.

10 McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat RevImmunol 2007; 7: 429–442.

11 Seitz M, Loetscher P, Fey MF, Tobler A. Constitutive mRNA and protein production ofmacrophage colony-stimulating factor but not of other cytokines by synovialfibroblasts from rheumatoid arthritis and osteoarthritis patients. Br J Rheumatol1994; 33: 613–619.

12 Clinton SK, Underwood R, Hayes L, Sherman ML, Kufe DW, Libby P. Macrophagecolony-stimulating factor gene expression in vascular cells and in experimental andhuman atherosclerosis. Am J Pathol 1992; 140: 301–316.

13 Nakano K, Okada Y, Saito K, Tanikawa R, Sawamukai N, Sasaguri Y et al. Rheumatoidsynovial endothelial cells produce macrophage colony-stimulating factor leadingto osteoclastogenesis in rheumatoid arthritis. Rheumatology (Oxford) 2007; 46:597–603.

14 Crotti TN, Smith MD, Weedon H, Ahern MJ, Findlay DM, Kraan M et al. Receptoractivator NF-kappaB ligand (RANKL) expression in synovial tissue from patients withrheumatoid arthritis, spondyloarthropathy, osteoarthritis, and from normal patients:semiquantitative and quantitative analysis. Ann Rheum Dis 2002; 61: 1047–1054.

15 Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R et al.Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell 1997; 89: 309–319.

16 Goldbach-Mansky R, Lee J, McCoy A, Hoxworth J, Yarboro C, Smolen JS et al.Rheumatoid arthritis associated autoantibodies in patients with synovitis of recentonset. Arthritis Res 2000; 2: 236–243.

17 Harre U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E et al. Induction ofosteoclastogenesis and bone loss by human autoantibodies against citrullinatedvimentin. J Clin Invest 2012; 122: 1791–1802.

18 Braun T, Zwerina J. Positive regulators of osteoclastogenesis and bone resorption inrheumatoid arthritis. Arthritis Res Ther 2011; 13: 235.

19 Zhang YH, Heulsmann A, Tondravi MM, Mukherjee A, Abu-Amer Y. Tumor necrosisfactor-alpha (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNFtype 1 receptor and RANK signaling pathways. J Biol Chem 2001; 276: 563–568.

20 Li P, Schwarz EM, O’Keefe RJ, Ma L, Boyce BF, Xing L. RANK signaling is notrequired for TNFalpha-mediated increase in CD11(hi) osteoclast precursors but isessential for mature osteoclast formation in TNFalpha-mediated inflammatoryarthritis. J Bone Miner Res 2004; 19: 207–213.

21 Gilbert L, He X, Farmer P, Boden S, Kozlowski M, Rubin J et al. Inhibition ofosteoblast differentiation by tumor necrosis factor-alpha. Endocrinology 2000; 141:3956–3964.

22 Koch AE. Chemokines and their receptors in rheumatoid arthritis: future targets?Arthritis Rheum 2005; 52: 710–721.

23 Mohan AK, Cote TR, Siegel JN, Braun MM. Infectious complications of biologictreatments of rheumatoid arthritis. Curr Opin Rheumatol 2003; 15: 179–184.

24 Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 1975;72: 3666–3670.

25 Medzhitov R, Horng T. Transcriptional control of the inflammatory response. Nat RevImmunol 2009; 9: 692–703.

26 Shebzukhov YV, Kuprash DV. Transcriptional regulation of TNF/LT locus in immunecells. Mol Biol 2011; 45: 56–67.

27 Murr R. Interplay between different epigenetic modifications and mechanisms. AdvGenet 2010; 70: 101–141.

28 Bates PW, Miyamoto S. Expanded nuclear roles for IkappaBs. Sci STKE 2004; 2004:e48.

29 Espel E. The role of the AU-rich elements of mRNAs in controlling translation. SeminCell Dev Biol 2005; 16: 59–67.

30 Bak RO, Mikkelsen JG. Regulation of cytokines by small RNAs during skininflammation. J Biomed Sci 2010; 17: 53.

31 Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G. Impairedon/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements:implications for joint and gut-associated immunopathologies. Immunity 1999; 10:387–398.

32 Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of macrophage tumornecrosis factor-alpha production by tristetraprolin. Science 1998; 281: 1001–1005.

33 Taylor GA, Carballo E, Lee DM, Lai WS, Thompson MJ, Patel DD et al. A pathogeneticrole for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resultingfrom tristetraprolin (TTP) deficiency. Immunity 1996; 4: 445–454.

34 Asirvatham AJ, Gregorie CJ, Hu Z, Magner WJ, Tomasi TB. MicroRNA targets inimmune genes and the Dicer/Argonaute and ARE machinery components. MolImmunol 2008; 45: 1995–2006.

35 Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B et al. Modulation ofmiR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulationand their possible roles in regulating the response to endotoxin shock. J Immunol2007; 179: 5082–5089.

36 Huang HC, Yu HR, Huang LT, Huang HC, Chen RF, Lin IC et al. miRNA-125bregulates TNF-alpha production in CD14þ neonatal monocytes via post-transcrip-tional regulation. J Leukoc Biol 2012; 92: 171–182.

37 Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAscan up-regulate translation. Science 2007; 318: 1931–1934.

38 Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J et al. Involvementof microRNA in AU-rich element-mediated mRNA instability. Cell 2005; 120:623–634.

39 Aggarwal BB. Signaling pathways of the TNF superfamily: a double-edged sword. NatRev Immunol 2003; 3: 745–756.

40 Callard R, Gearing A. The Cytokine FactsBook. Academic Press, London, 1994.41 Winzen R, Wallach D, Kemper O, Resch K, Holtmann H. Selective up-regulation

of the 75-kDa tumor necrosis factor (TNF) receptor and its mRNA by TNF and IL-1.J Immunol 1993; 150: 4346–4353.

42 Ding AH, Sanchez E, Srimal S, Nathan CF. Macrophages rapidly internalize theirtumor necrosis factor receptors in response to bacterial lipopolysaccharide. J BiolChem 1989; 264: 3924–3929.

43 Kemper O, Wallach D. Cloning and partial characterization of the promoterfor the human p55 tumor necrosis factor (TNF) receptor. Gene 1993; 134:209–216.

44 Santee SM, Owen-Schaub LB. Human tumor necrosis factor receptor p75/80(CD120b) gene structure and promoter characterization. J Biol Chem 1996; 271:21151–21159.

Regulation of TNF-aEAV Moelants et al

7

Immunology and Cell Biology

45 Engelmann H, Aderka D, Rubinstein M, Rotman D, Wallach D. A tumor necrosisfactor-binding protein purified to homogeneity from human urine protects cells fromtumor necrosis factor toxicity. J Biol Chem 1989; 264: 11974–11980.

46 Hino T, Nakamura H, Abe S, Saito H, Inage M, Terashita K et al. Hydrogen peroxideenhances shedding of type I soluble tumor necrosis factor receptor from pulmonaryepithelial cells. Am J Respir Cell Mol Biol 1999; 20: 122–128.

47 Okuyama M, Yamaguchi S, Yamaoka M, Nitobe J, Fujii S, Yoshimura T et al. Nitricoxide enhances expression and shedding of tumor necrosis factor receptor I (p55) inendothelial cells. Arterioscler Thromb Vasc Biol 2000; 20: 1506–1511.

48 Brennan FM, Gibbons DL, Mitchell T, Cope AP, Maini RN, Feldmann M. Enhancedexpression of tumor necrosis factor receptor mRNA and protein in mononuclearcells isolated from rheumatoid arthritis synovial joints. Eur J Immunol 1992; 22:1907–1912.

49 Faustman D, Davis M. TNF receptor 2 pathway: drug target for autoimmune diseases.Nat Rev Drug Discov 2010; 9: 482–493.

50 Cope AP, Aderka D, Doherty M, Engelmann H, Gibbons D, Jones AC et al. Increasedlevels of soluble tumor necrosis factor receptors in the sera and synovial fluid ofpatients with rheumatic diseases. Arthritis Rheum 1992; 35: 1160–1169.

51 Barton A, John S, Ollier WE, Silman A, Worthington J. Association betweenrheumatoid arthritis and polymorphism of tumor necrosis factor receptor II, but nottumor necrosis factor receptor I, in Caucasians. Arthritis Rheum 2001; 44: 61–65.

52 Standiford TJ, Strieter RM, Lukacs NW, Kunkel SL. Neutralization of IL-10 increaseslethality in endotoxemia. Cooperative effects of macrophage inflammatory protein-2and tumor necrosis factor. J Immunol 1995; 155: 2222–2229.

53 Bemelmans MH, van Tits LJ, Buurman WA. Tumor necrosis factor: function, releaseand clearance. Crit Rev Immunol 1996; 16: 1–11.

54 Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF et al. Ametalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature 1997; 385: 729–733.

55 Ruuls SR, Hoek RM, Ngo VN, McNeil T, Lucian LA, Janatpour MJ et al. Membrane-bound TNF supports secondary lymphoid organ structure but is subservient tosecreted TNF in driving autoimmune inflammation. Immunity 2001; 15: 533–543.

56 Van Lint P, Libert C. Chemokine and cytokine processing by matrix metalloproteinasesand its effect on leukocyte migration and inflammation. J Leukoc Biol 2007; 82:1375–1381.

57 Larche MJ, Sacre SM, Foxwell BM. Pathogenic role of TNF [alpha] in rheumatoidarthritis. Drug Discovery Today: Disease Mechanisms 2005; 2: 367–375.

58 Schiff MH. Role of interleukin 1 and interleukin 1 receptor antagonist in themediation of rheumatoid arthritis. Ann Rheum Dis 2000; 59 (Suppl 1), i103–i108.

59 Foulquier C, Sebbag M, Clavel C, Chapuy-Regaud S, Al Badine R, Mechin MC et al.Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, andPAD-6 are expressed in rheumatoid arthritis synovium in close association with tissueinflammation. Arthritis Rheum 2007; 56: 3541–3553.

60 Chang X, Yamada R, Suzuki A, Sawada T, Yoshino S, Tokuhiro S et al. Localization ofpeptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue ofrheumatoid arthritis. Rheumatology (Oxford) 2005; 44: 40–50.

61 Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M et al.Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylargininedeiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003; 34:395–402.

62 Moelants EAV, Mortier A, Grauwen K, Ronsse I, Van Damme J, Proost P. Citrullinationof TNF-alpha by peptidylarginine deiminases reduces its capacity to stimulate theproduction of inflammatory chemokines. Cytokine 2012; 61: 161–167.

63 Mukai Y, Shibata H, Nakamura T, Yoshioka Y, Abe Y, Nomura T et al. Structure-function relationship of tumor necrosis factor (TNF) and its receptor interaction basedon 3D structural analysis of a fully active TNFR1-selective TNF mutant. J Mol Biol2009; 385: 1221–1229.

64 Mukai Y, Nakamura T, Yoshioka Y, Tsunoda S, Kamada H, Nakagawa S et al.Crystallization and preliminary X-ray analysis of the tumour necrosis factor alpha-tumour necrosis factor receptor type 2 complex. Acta Crystallogr Sect F Struct BiolCryst Commun 2009; 65: 295–298.

65 Mukai Y, Nakamura T, Yoshikawa M, Yoshioka Y, Tsunoda S, Nakagawa S et al.Solution of the structure of the TNF-TNFR2 complex. Sci Signal 2010; 3: ra83.

66 Bradley JR. TNF-mediated inflammatory disease. J Pathol 2008; 214: 149–160.67 Naude PJ, den Boer JA, Luiten PG, Eisel UL. Tumor necrosis factor receptor

cross-talk. FEBS J 2011; 278: 888–898.68 Pimentel-Muinos FX, Seed B. Regulated commitment of TNF receptor signaling: a

molecular switch for death or activation. Immunity 1999; 11: 783–793.69 Ledgerwood EC, Pober JS, Bradley JR. Recent advances in the molecular basis of

TNF signal transduction. Lab Invest 1999; 79: 1041–1050.70 Grell M, Douni E, Wajant H, Lohden M, Clauss M, Maxeiner B et al. The

transmembrane form of tumor necrosis factor is the prime activating ligand of the80 kDa tumor necrosis factor receptor. Cell 1995; 83: 793–802.

71 Abu-Amer Y, Erdmann J, Alexopoulou L, Kollias G, Ross FP, Teitelbaum SL. Tumornecrosis factor receptors types 1 and 2 differentially regulate osteoclastogenesis.J Biol Chem 2000; 275: 27307–27310.

72 Jiang Y, Woronicz JD, Liu W, Goeddel DV. Prevention of constitutive TNF receptor 1signaling by silencer of death domains. Science 1999; 283: 543–546.

73 Kanazawa K, Kudo A. TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.J Bone Miner Res 2005; 20: 840–847.

74 Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV. TNF-dependent recruitment of theprotein kinase RIP to the TNF receptor-1 signaling complex. Immunity 1996; 4:387–396.

75 Hsu H, Shu HB, Pan MG, TRADD-TRAF2 GoeddelDV. and TRADD-FADD interactionsdefine two distinct TNF receptor 1 signal transduction pathways. Cell 1996; 84:299–308.

76 Yang J, Lin Y, Guo Z, Cheng J, Huang J, Deng L et al. The essential role of MEKK3 inTNF-induced NF-kappaB activation. Nat Immunol 2001; 2: 620–624.

77 Israel A. The IKK complex: an integrator of all signals that activate NF-kappaB?Trends Cell Biol 2000; 10: 129–133.

78 Akira S, Kishimoto T. IL-6 and NF-IL6 in acute-phase response and viral infection.Immunol Rev 1992; 127: 25–50.

79 Devin A, Lin Y, Yamaoka S, Li Z, Karin M, Liu Z. The alpha and beta subunits ofIkappaB kinase (IKK) mediate TRAF2-dependent IKK recruitment to tumor necrosisfactor (TNF) receptor 1 in response to TNF. Mol Cell Biol 2001; 21: 3986–3994.

80 Deacon K, Blank JL. MEK kinase 3 directly activates MKK6 and MKK7, specificactivators of the p38 and c-Jun NH2-terminal kinases. J Biol Chem 1999; 274:16604–16610.

81 Karin M, Gallagher E. From JNK to pay dirt: jun kinases, their biochemistry,physiology and clinical importance. IUBMB Life 2005; 57: 283–295.

82 Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281: 1305–1308.

83 Micheau O, Lens S, Gaide O, Alevizopoulos K, Tschopp J. NF-kappaB signals inducethe expression of c-FLIP. Mol Cell Biol 2001; 21: 5299–5305.

84 Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV. The TNFR2-TRAF signalingcomplex contains two novel proteins related to baculoviral inhibitor of apoptosisproteins. Cell 1995; 83: 1243–1252.

85 Rothe M, Wong SC, Henzel WJ, Goeddel DV. A novel family of putative signaltransducers associated with the cytoplasmic domain of the 75 kDa tumor necrosisfactor receptor. Cell 1994; 78: 681–692.

86 Cabal-Hierro L, Lazo PS. Signal transduction by tumor necrosis factor receptors. CellSignal 2012; 24: 1297–1305.

87 Malinin NL, Boldin MP, Kovalenko AV, Wallach D. MAP3K-related kinase involved inNF-kappaB induction by TNF, CD95 and IL-1. Nature 1997; 385: 540–544.

88 Li X, Yang Y, JD Ashwell. TNF-RII and c-IAP1 mediate ubiquitination and degradationof TRAF2. Nature 2002; 416: 345–347.

89 Zhang R, Xu Y, Ekman N, Wu Z, Wu J, Alitalo K et al. Etk/Bmx transactivates vascularendothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediatethe tumor necrosis factor-induced angiogenic pathway. J Biol Chem 2003; 278:51267–51276.

90 Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. AnnuRev Immunol 1996; 14: 397–440.

91 Pierer M, Rethage J, Seibl R, Lauener R, Brentano F, Wagner U et al. Chemokinesecretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2ligands. J Immunol 2004; 172: 1256–1265.

92 MacNaul KL, Hutchinson NI, Parsons JN, Bayne EK, Tocci MJ. Analysis of IL-1 andTNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytesby in situ hybridization. J Immunol 1990; 145: 4154–4166.

93 Millet I, Ruddle NH. Differential regulation of lymphotoxin (LT), lymphotoxin-beta(LT-beta), and TNF-alpha in murine T cell clones activated through the TCR.J Immunol 1994; 152: 4336–4346.

94 Grivennikov SI, Tumanov AV, Liepinsh DJ, Kruglov AA, Marakusha BI,Shakhov AN et al. Distinct and nonredundant in vivo functions of TNF produced byT cells and macrophages/neutrophils: protective and deleterious effects. Immunity2005; 22: 93–104.

95 Ahern DJ, Brennan FM. The role of natural killer cells in the pathogenesis ofrheumatoid arthritis: major contributors or essential homeostatic modulators?Immunol Lett 2011; 136: 115–121.

96 Modrowski D, Godet D, Marie PJ. Involvement of interleukin 1 and tumour necrosisfactor alpha as endogenous growth factors in human osteoblastic cells. Cytokine1995; 7: 720–726.

97 McInnes IB, Leung BP, Sturrock RD, Field M, Liew FY. Interleukin-15 mediatesT cell-dependent regulation of tumor necrosis factor-alpha production in rheumatoidarthritis. Nat Med 1997; 3: 189–195.

98 Joosten LA, Koenders MI, Smeets RL, Heuvelmans-Jacobs M, Helsen MM, Takeda Ket al. Toll-like receptor 2 pathway drives streptococcal cell wall-induced jointinflammation: critical role of myeloid differentiation factor 88. J Immunol 2003;171: 6145–6153.

99 Edwards SW, Hallett MB. Seeing the wood for the trees: the forgotten role ofneutrophils in rheumatoid arthritis. Immunol Today 1997; 18: 320–324.

100 Dalbeth N, Gundle R, Davies RJ, Lee YC, McMichael AJ, Callan MF et al. CD56brightNK cells are enriched at inflammatory sites and can engage with monocytes in areciprocal program of activation. J Immunol 2004; 173: 6418–6426.

101 Palmblad K, Erlandsson-Harris H, Tracey KJ, Andersson U. Dynamics of earlysynovial cytokine expression in rodent collagen-induced arthritis: a therapeuticstudy using a macrophage-deactivating compound. Am J Pathol 2001; 158:491–500.

102 Marinova-Mutafchieva L, Williams RO, Mason LJ, Mauri C, Feldmann M, Maini RN.Dynamics of proinflammatory cytokine expression in the joints of mice withcollagen-induced arthritis (CIA). Clin Exp Immunol 1997; 107: 507–512.

103 Bevilacqua MP. Endothelial-leukocyte adhesion molecules. Annu Rev Immunol 1993;11: 767–804.

104 Brennan FM, Zachariae CO, Chantry D, Larsen CG, Turner M, Maini RN et al.Detection of interleukin 8 biological activity in synovial fluids from patients withrheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells.Eur J Immunol 1990; 20: 2141–2144.

Regulation of TNF-aEAV Moelants et al

8

Immunology and Cell Biology

105 Koch AE, Kunkel SL, Burrows JC, Evanoff HL, Haines GK, Pope RM et al. Synovialtissue macrophage as a source of the chemotactic cytokine IL-8. J Immunol 1991;147: 2187–2195.

106 Strieter RM, Kasahara K, Allen RM, Standiford TJ, Rolfe MW, Becker FS et al.Cytokine-induced neutrophil-derived interleukin-8. Am J Pathol 1992; 141:397–407.

107 Van Damme J, Decock B, Conings R, Lenaerts JP, Opdenakker G, Billiau A. Thechemotactic activity for granulocytes produced by virally infected fibroblasts isidentical to monocyte-derived interleukin 8. Eur J Immunol 1989; 19: 1189–1194.

108 McCain RW, Holden EP, Blackwell TR, Christman JW. Leukotriene B4 stimulateshuman polymorphonuclear leukocytes to synthesize and release interleukin-8 in vitro.Am J Respir Cell Mol Biol 1994; 10: 651–657.

109 Wei S, Kitaura H, Zhou P, Ross FP, Teitelbaum SL. IL-1 mediates TNF-inducedosteoclastogenesis. J Clin Invest 2005; 115: 282–290.

110 Munker R, Gasson J, Ogawa M, Koeffler HP. Recombinant human TNF inducesproduction of granulocyte-monocyte colony-stimulating factor. Nature 1986; 323:79–82.

111 Van Damme J, Cayphas S, Van Snick J, Conings R, Put W, Lenaerts JP et al.Purification and characterization of human fibroblast-derived hybridoma growth factoridentical to T-cell-derived B-cell stimulatory factor-2 (interleukin-6). Eur J Biochem1987; 168: 543–550.

112 Sarkar S, Fox DA. Dendritic cells in rheumatoid arthritis. Front Biosci 2005; 10:656–665.

113 Zou GM, Tam YK. Cytokines in the generation and maturation of dendritic cells:recent advances. Eur Cytokine Netw 2002; 13: 186–199.

114 Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res 2002; 4 (Suppl 3),S81–S90.

115 Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H et al. Induction andactivation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling interminal differentiation of osteoclasts. Dev Cell 2002; 3: 889–901.

116 Shinohara M, Koga T, Okamoto K, Sakaguchi S, Arai K, Yasuda H et al. Tyrosinekinases Btk and Tec regulate osteoclast differentiation by linking RANK and ITAMsignals. Cell 2008; 132: 794–806.

117 Hwang SY, Putney JW Jr. Calcium signaling in osteoclasts. Biochim Biophys Acta2011; 1813: 979–983.

118 Sato K, Suematsu A, Nakashima T, Takemoto-Kimura S, Aoki K, Morishita Y et al.Regulation of osteoclast differentiation and function by the CaMK-CREB pathway. NatMed 2006; 12: 1410–1416.

119 Nakao S, Ogtata Y, Shimizu E, Yamazaki M, Furuyama S, Sugiya H. Tumor necrosisfactor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by theactivation of cyclooxygenase-2 (COX-2) transcription via NFkappaB in human gingivalfibroblasts. Mol Cell Biochem 2002; 238: 11–18.

120 Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D et al. Dickkopf-1 is amaster regulator of joint remodeling. Nat Med 2007; 13: 156–163.

121 Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S et al.Tumor necrosis factor alpha stimulates osteoclast differentiation by amechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 2000;191: 275–286.

122 Abu-Amer Y, Ross FP, Edwards J, Teitelbaum SL. Lipopolysaccharide-stimulatedosteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor. J ClinInvest 1997; 100: 1557–1565.

123 Kanazawa K, Azuma Y, Nakano H, Kudo A. TRAF5 functions in both RANKL- andTNFalpha-induced osteoclastogenesis. J Bone Miner Res 2003; 18: 443–450.

124 Herold M. Rheumatoid arthritis. In: Shoenfeld Y and Meroni PL (eds). The GeneralPractice Guide to Autoimmune Disease. Pabst Science Publisher, Lengerich, 2012:63–71.

125 Verweij CL. Predicting the future of anti-tumor necrosis factor therapy. Arthritis ResTher 2009; 11: 115.

126 Jiang Y, Genant HK, Watt I, Cobby M, Bresnihan B, Aitchison R et al. A multicenter,double-blind, dose-ranging, randomized, placebo-controlled study of recombinanthuman interleukin-1 receptor antagonist in patients with rheumatoid arthritis:radiologic progression and correlation of Genant and Larsen scores. Arthritis Rheum2000; 43: 1001–1009.

127 Alten R, Gomez-Reino J, Durez P, Beaulieu A, Sebba A, Krammer G et al. Efficacy andsafety of the human anti-IL-1beta monoclonal antibody canakinumab in rheumatoidarthritis: results of a 12-week, Phase II, dose-finding study. BMC MusculoskeletDisord 2011; 12: 153.

128 Hamdy NA. Denosumab: RANKL inhibition in the management of bone loss. DrugsToday (Barc) 2008; 44: 7–21.

129 Haringman JJ, Kraan MC, Smeets TJ, Zwinderman KH, Tak PP. Chemokine blockadeand chronic inflammatory disease: proof of concept in patients with rheumatoidarthritis. Ann Rheum Dis 2003; 62: 715–721.

130 Liu J, Merritt JR. CC chemokine receptor small molecule antagonists in the treatmentof rheumatoid arthritis and other diseases: a current view. Curr Top Med Chem 2010;10: 1250–1267.

131 Suzuki N, Nakajima A, Yoshino S, Matsushima K, Yagita H, Okumura K. Selectiveaccumulation of CCR5þ T lymphocytes into inflamed joints of rheumatoid arthritis.Int Immunol 1999; 11: 553–559.

132 Zapico I, Coto E, Rodriguez A, Alvarez C, Torre JC, Alvarez V. CCR5 (chemokinereceptor-5) DNA-polymorphism influences the severity of rheumatoid arthritis.Genes Immun 2000; 1: 288–289.

133 van Kuijk AW, Vergunst CE, Gerlag DM, Bresnihan B, Gomez-Reino JJ,Rouzier R et al. CCR5 blockade in rheumatoid arthritis: a randomised, double-blind,placebo-controlled clinical trial. Ann Rheum Dis 2010; 69: 2013–2016.

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