Cells of the synovium in rheumatoid arthritis. Macrophages
© BioMed Central Ltd 2007
Published: 21 December 2007
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© BioMed Central Ltd 2007
Published: 21 December 2007
The multitude and abundance of macrophage-derived mediators in rheumatoid arthritis and their paracrine/autocrine effects identify macrophages as local and systemic amplifiers of disease. Although uncovering the etiology of rheumatoid arthritis remains the ultimate means to silence the pathogenetic process, efforts in understanding how activated macrophages influence disease have led to optimization strategies to selectively target macrophages by agents tailored to specific features of macrophage activation. This approach has two advantages: (a) striking the cell population that mediates/amplifies most of the irreversible tissue destruction and (b) sparing other cells that have no (or only marginal) effects on joint damage.
Activation status of synovial macrophages and/or circulating monocytes in rheumatoid arthritis
Class of overexpressed molecules
Known or potential function
Class II major histocompatibility complex (overexpressed on Mφ)
Presentation of antigens relevant to disease initiation or severity  (Stuhlmuller B, et al., unpublished data) (reviewed in )
Cytokines and growth factors
For example, TNF-α, IL-1, IL-6, IL-10, IL-13, IL-15, IL-18, migration inhibitory factor, granulocyte macrophage colony-stimulating factor, and thrombospondin-1
Mediation and regulation of local and systemic inflammation and tissue remodelling (reviewed in [2,24,39,52])
Chemokines and chemoattractants
For example, IL-8, macrophage inflammatory protein-1, monocyte chemoattractant protein-1, and CXCL13
Mediation and regulation of monocyte migration Stimulation of angiogenesis (reviewed in )
MMP-9 and MMP-12
Tissue degradation and post-injury tissue remodelling [94,95]
Tissue inhibitors of MMP (TIMPs)
Attempt to control excessive tissue destruction 
For example, C-reactive protein and A-SAA (serum amyloid A)
Integrated hormone-like activation of hepatocytes by synovial Mφ and fibroblasts (mostly via IL-6)  (reviewed in )
Produced by interferon-gamma-stimulated monocytes/Mφ
Induces/enhances cytotoxicity and apoptosis Acts as antioxidant [98,99]
Produced by TNF-α-stimulated Mφ
Regulates nuclear factor-kappa-B and caspase-1 activation 
Thus, before a causal factor for RA is known, monocytes/Mφ remain an attractive research focus for the following reasons: (a) the radiological progression of joint destruction correlates with the degree of synovial Mφ infiltration , (b) the therapeutic efficacy of conventional anti-rheumatic therapy coincides with downregulation of MPS functions , (c) therapies directed at cytokines made predominantly by Mφ are effective in RA , (d) conventional or experimental drugs can be selectively targeted to Mφ or their different subcellular compartments (for example, [2, 8]), (e) differential activation of intracellular signal transduction pathways underlies different Mφ effector functions , and (f) more specific inhibitors of key metabolic enzymes or particular signal transduction pathways may become available as selective targets of anti-rheumatic therapy [9, 10]. In addition, the amplifying role of Mφ in RA has emerged so clearly that the effects of anti-rheumatic therapy (whether specific or conventional) on monocytes/Mφ may become an objective readout of the effectiveness of treatment [11–13] (Stuhlmuller B, Hernandez MM, Haeupl T, Kuban RJ, Gruetzkau A, Voss JW, Salfeld J, Kinne RW, Burmester GR, unpublished data).
Potential sites of myelomonocytic activation in rheumatoid arthritis and corresponding steps of macrophage intermediate or terminal (trans) differentiation
Joint or juxta-articular
• Recently immigrated monocytes
• Mφ (M1/M2? ; resident/inflammatory? )
• Dendritic cells
• Myelomonocytic precursors
• Endothelial cells
Epitheloid cells and multinucleated giant cells
Lung interstitial space
Cells of the MPS show clear signs of activation, not only in synovial and juxta-articular compartments such as the synovial membrane or the cartilage-pannus and bone-pannus junctions (including the subchondral bone), but also in extra-articular compartments (for example, peripheral blood and subendothelial space, the latter of which is the site of foam cell formation and development of atherosclerotic plaques in RA) (Table 2). This activation underlines the systemic inflammatory character of RA and may contribute to the occurrence of cardiovascular events and its increased mortality (reviewed in [2, 14, 15]).
Monocyte/macrophage functions and their (potential) role in rheumatoid arthritis
(Potential) role in rheumatoid arthritis
Clearance of immune complexes
Binding of immunoglobulins to Fc receptors (Fc-γ-R I, IIA, IIB, and IIIA)
Potential clearance of rheumatoid factor but further activation of monocytes/Mφ
Opsonization of complexes by complement, leading to binding to Mφ complement receptors and further cell activation [101,102] (reviewed in [2,103])
Notably, inhibition of monocyte activation by Fc-γ-R IIB 
Binding of complement factors to complement receptors 1 (CD35), 3 (CD11b), and 5a (CD88)
Recognition of activated complement (soluble phase or on immunoglobulin G-immune complexes)
Promotion of phagocytosis and activation of monocytes/Mφ 
Phagocytosis of particulate antigens
Conventional (Fc-mediated) → lysosomal degradation and MHC-II antigen processing
Scavenging of debris but potential import of arthritogenic molecules 
Antigen presentation and activation of CD4+ and CD8+ T cells, possibly relevant to disease initiation or perpetuation (spreading of autoimmunity) (reviewed in )
Coiling phagocytosis → lysosomal degradation and MHC-I antigen processing
Involved in phagocytosis of Borrelia burgdorferi, active agent of Lyme arthritis (reviewed in )
Clearance of intracellular pathogens and apoptotic cells
Removal of pathogens and recognition of apoptotic cells via exposed intracellular membrane components
Induction of Mφ-derived cytokines by bacterial toxins or superantigens [26,28,103]
Modulation of Mφ responses by mycobacterial lipoarabinomannan [104,105] or Toll-like receptors [29,106]
Persistence of obligate/facultative intracellular pathogens with arthritogenic potential [107,108]
Antigen processing and presentation
Enzymatic degradation of antigens and binding of antigenic peptides to MHC molecules and transport to the cell surface
Important cognate functions upon antigen recognition via presentation of antigen on MHC-II molecules  and expression of membrane second signal molecules adjacent to T cells (reviewed in )
Chemotaxis and angiogenesis
Attraction of other inflammatory cells and induction of neo-vascularization
Positive feedback between Mφ-derived cytokines and chemotactic factors (for example, IL-8 and monocyte chemoattractant protein-1) Promotion of angiogenesis by IL-8 and soluble forms of adhesion molecules (for example, vascular cell adhesion molecule-1 and endothelial-leukocyte adhesion molecule-1) 
Remodelling of tissue via interaction with fibroblasts
Sustained monocyte recruitment at wound injury sites via monocyte chemoattractant macrophage inflammatory protein-1α Phagocytosis of matrix debris and endogenous production of IL-1, TNF-α, and so on as well as post-injury tissue remodelling (reviewed in )
Mφ synthesis of prostaglandins (PGs) E2 and I2 Expression of scavenger receptor A (uptake of oxidized low-density lipoprotein)
Pro-inflammatory activity of PGE2 and PGI2 and leukotrienes in rheumatoid arthritis, but also autocrine negative feedback through peroxisome proliferator-activated receptors α and γ (reviewed in ) Fish-based diets are associated with clinical improvement of human and experimental arthritis (reviewed in )
Modulation of T cell-contact-induced production of IL-1β and TNF-α in Mφ by apolipoprotein A-I 
A significant part of Mφ effector responses is mediated by cell contact-dependent signalling with different inflammatory or mesenchymal cells (as exemplified in the lower left quadrant of Figure 2).
Because of the prominent numbers of Mφ and fibroblasts and their activated status in RA synovial tissue, the interaction of these cells is critical for the resulting inflammation and tissue damage. Indeed, the mere contact of these cells elicits the production of interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-8. The cytokine output can be enhanced or down-modulated not only by addition of pro-inflammatory or regulatory cytokines (for example, IL-4, IL-10, IL-13, or IL-1 receptor antagonist [IL-1RA]), but also by neutralization of the CD14 molecule . Also, in vitro, significant cartilage degradation occurs in co-cultures of mouse fibroblasts and Mφ, a response markedly exceeding that observed with each culture alone (reviewed in ). Furthermore, purified human synovial fibroblasts co-cultured with myelomonocytic cells induce cartilage degradation in vitro, but with a strong contribution of soluble IL-1 and tumor necrosis factor (TNF)-α .
Accessory, inflammatory, effector, and inhibitory Mφ functions can be stimulated by fixed T cells or their plasma membranes if T cells are pre-activated and express activation surface molecules. In response to such interaction, monocytes produce metalloprotease (MMP), IL-1α, and IL-1β [19, 20]. Also, T cells pre-stimulated in an antigen-mimicking fashion stimulate TNF-α and IL-10 production once in contact with monocytes . Conversely, fixed T cells stimulated in an antigen-independent fashion (that is, with IL-15, IL-2, or a combination of IL-6 and TNF-α, the so-called Tck cells) induce monocyte production of TNF-α but not the anti-inflammatory IL-10 [20, 21]. These findings suggest that early RA may reflect antigen-specific T cell-Mφ interactions . Conversely, chronic RA may be associated with antigen-independent interactions dominated by an exuberant cytokine milieu and Tck cells. This may also explain the relative paucity of IL-10 in the synovial membrane in chronic RA, as discussed below.
Several ligand pairs on T cells and monocytes/Mφ have been implicated in this interaction , although the importance of individual ligand pairs, as well as the influence of soluble mediators, remains unclear. Interestingly, T cells isolated from RA synovial tissue show phenotypical and functional features similar to Tck cells and the above-mentioned signal transduction pathways differentially contribute to the induction of TNF-α and IL-10 production in monocytes/Mφ by co-culture with Tck cells. If applicable in vivo in RA, this would allow selective therapeutic targeting of pro-inflammatory TNF-α and sparing of anti-inflammatory IL-10.
The interaction between monocytes and endothelial cells in RA (Figure 2), critical for the sustained influx of activated monocytes in the synovial membrane, relies on the altered expression of integrin/selectin pairs on the surface of the two cell types (reviewed in ). Because the synovial cytokine milieu (including the Mφ-derived TNF-α) upregulates the expression of these ligand pairs, a self-perpetuating cycle ensues by which sustained Mφ-derived mechanisms lead to further influx and activation of circulating monocytes. Upon cell contact, monokine-activated CD56bright natural killer cells induce monocytes to the production of TNF-α, thus representing another possible reciprocal loop of activation in RA .
Overview of pro-inflammatory interleukins relevant to macrophage (dys)function in rheumatoid arthritis
Main pathogenetic features
Predominantly produced by Mφ
Critical mediator of tissue damage
Possesses autocrine features [43,51-53]
Predominantly produced by Mφ
Critical pleiotropic mediator of disease
Possesses autocrine features [59-61]
Produced by endothelial cells
Important Th2-inducing component in allergy/autoimmunity
Signals via IL-1 receptor-related protein (ST2)
Nuclear factor with transcriptional repressor properties (≈ nuclear factor from high endothelial venules) [111-113]
Pro-inflammatory effects on both myeloid and non-myeloid cells [114,115]
Elevated in RA, although a relative paucity is also possible [116,117]
Induces osteoclastic bone loss in mice 
Produced by Mφ
Important autocrine mediator of disease processes [21,56-58]
Only IL-21R is expressed by synovial Mφ and fibroblasts 
Predominantly produced by fibroblasts under the influence of Mφ
Most strikingly elevated cytokine in acute RA, with phase-dependent differential effects [17,75,76] (reviewed in [2,77])
Induces experimental dermatitis 
Stimulates proteoglycan resorption in cartilage 
Recruits leukocytes to inflammatory sites and stimulates production of metalloprotease (MMP) and tissue inhibitor of MMP 
IFN type I/IL-10
Involved in both Th1 and Th2 inflammatory disorders [122,123]
Possesses autocrine features [124,125]
Overexpressed in psoriasis
Possesses autocrine features 
Relevant to innate immunity and acute-phase response 
Possible antagonism with regulatory IL-10 
Polymorphism possibly contributes to RA sex-bias susceptibility 
Involved in microbial recognition by upregulation of Toll-like receptors
Possesses autocrine features [30,129,130]
Predominantly produced by synovial Mφ and dendritic cells
Promotes Th1 responses (reviewed in )
Predominantly produced by synovial Mφ and dendritic cells
Shares p40 subunit with IL-12 and possibly antagonizes IL-12  (reviewed in )
Produced by Mφ and its neutralization has anti-arthritic effects
Possesses autocrine features 
Pro-inflammatory role 
Th0-Th1 lymphokine with pleiotropic, amplifying effects on Mφ arthritis (reviewed in [24,25])
The ability of bacterial toxins or superantigens to initiate the secretion of Mφ-derived cytokines is relevant in view of a possible microorganism etiology of RA and in view of side effects of anti-TNF-α therapy, particularly mycobacterial infections [26, 27]. Lipopolysaccharide (LPS), for example, binds to Mφ through the CD14/LPS-binding protein receptor complex and, in vitro, stimulates the production of IL-1β, TNF-α, and macrophage inflammatory protein-1α. Staphylococcal enterotoxin B (SEB), a potent Mφ activator, enhances arthritis in MRL-lpr/lpr mice. Anti-TNF-α therapy, in this case, reverses both the severe wasting effects of SEB and the incidence of arthritis, indicating that TNF-α is central in this system. Finally, the staphylococcal enterotoxin A increases the expression of the Toll-like receptor (TLR)-4 in human monocytes by ligation of major histocompatibility complex-II, with subsequent enhancement of pro-inflammatory cytokines by known TLR-4 ligands (for example, LPS ).
TLRs are part of the recently discovered cellular pattern-recognition receptors (PPRs) involved in first-line defense of the innate immune system against microbial infections. In addition to bacterial or viral components, some PPRs recognize host-derived molecules, such as the glycoprotein gp96, nucleic acids, hyaluronic acid oligosaccharides, heparan sulfate, fibronectin fragments, and surfactant protein A (reviewed in ). In RA, notably, functional TLR-2 and TLR-4 are expressed on CD16+ synovial Mφ, peripheral blood mononuclear cells, and synovial fibroblasts . Also, their expression can be upregulated by cytokines present in the inflamed RA joint (for example, IL-1β, TNF-α, macrophage colony-stimulating factor, and IL-10); this suggests that activation of synovial cells via TLRs may contribute to disease processes , as supported by findings in experimental arthritis . On the other hand, the chronic polyarthritis observed in mice with deletion of the DNase II gene, whose Mφ are incapable of degrading mammalian DNA, appears to occur independently of the nucleic acid-specific TLR-9 .
Females are affected by RA at a ratio of approximately 3:1 compared with males and experience clinical fluctuations during the menstrual cycle and pregnancy, indicating a major modulating role for sex hormones. Due to their expression of sex-hormone receptors and their cytokine response upon exposure to estrogens, monocytes/Mφ are strongly involved in hormone modulation of RA . Indeed, physiological levels of estrogens stimulate RA Mφ to the production of the pro-inflammatory cytokine IL-1, whereas higher levels inhibit IL-1 production, conceivably mimicking the clinical improvement during pregnancy. Interestingly, selective estrogen receptor ligands inhibiting nuclear factor (NF)-κB transcriptional activity (but lacking estrogenic activity) can markedly inhibit joint swelling and destruction in experimental arthritis .
Overview of anti-inflammatory cytokines relevant to macrophage (dys)function in rheumatoid arthritis
Main pathogenetic features
Produced by differentiated Mφ and upregulated by pro-inflammatory mediators, including IL-1 itself or granulocyte macrophage colony-stimulating factor
Autocrine contribution to the termination of inflammatory reactions [54,55] (reviewed in [53,56])
Strong regulator of Mφ functions but virtually absent in synovial tissue [73,131-133]
Produced by synovial Mφ
Strong regulator of Mφ functions but relatively deficient in RA
Possesses autocrine features [73,74]
Regulator of Mφ functions in a paracrine regulatory loop with synovial fibroblasts [36,134]
Selective regulator of Mφ functions
Improves experimental arthritis (reviewed in [2,91])
Known as an anti-inflammatory molecule [135,136], IL-16 also has pro-inflammatory properties (that is, correlates with metalloprotease-3 levels, progression of joint destruction, and levels of other pro-inflammatory cytokines) [137,138].
Clear anti-inflammatory and anti-destructive effects in experimental arthritides
Therapy attempts in human RA thus far have been unsuccessful .
Produced by Mφ [78-80]
Main regulator of connective tissue remodelling
Potent inducer of hyaluronan synthase 1
Induces synovial inflammation (reviewed in ) but also suppresses acute and chronic arthritis [81,82]
Induces inflammation and cartilage degradation in a rabbit model 
Possesses autocrine features
MMP can affect TGF-β via shedding of latent TGF-β attached to decorin (disease-enhancing loop).
TNF-α is a pleiotropic cytokine that increases the expression of cytokines, adhesion molecules, prostaglandin E2, collage-nase, and collagen by synovial cells. TNF-α exists in membrane-bound and soluble forms, both acting as pro-inflammatory mediators. Transmembrane TNF-α is involved in local, cell contact-mediated processes and appears to be the prime stimulator of the R75 receptor . Interestingly, the transgenic expression of this form is alone sufficient to induce chronic arthritis ; likewise, a mutant membrane TNF-α, which uses both R55 and R75 receptors, can cause arthritis. Conversely, the soluble form of TNF-α, shed via MMP cleavage from the membrane-bound form, primarily stimulates the R55 receptor, acting transiently and at a distance .
In RA, TNF-α is mostly produced by Mφ in the synovial membrane and at the cartilage-pannus junction and possibly occupies a proximal position in the RA inflammatory cascade . While an average of approximately 5% of synovial cells express TNF-α mRNA/protein in situ , the degree of TNF-α expression in the synovial tissue depends upon the prevailing histological configuration, resulting in different clinical variants . Different disease stages and clinical variants are also reflected in serum and synovial fluid levels of TNF-α .
The critical importance of TNF-α in RA is supported by several experimental observations: (a) TNF-α in combination with IL-1 is a potent inducer of synovitis , (b) transgenic, deregulated expression of TNF-α causes the development of chronic arthritis , (c) TNF-α is produced in synovial membrane and extra-articular/lymphoid organs in experimental arthritides, mimicking the systemic character of RA , (d) neutralization of TNF-α suppresses experimental arthritides [39, 43], and (e) administration of chimeric/humanized anti-TNF-α monoclonal antibodies or TNF-α receptor constructs has shown remarkable efficacy in acute disease and retardation of radiographic progression [3, 7, 11].
As an interesting development, the analysis of gene expression in monocytes of anti-TNF-α-treated patients with RA may represent a powerful tool to identify regulation patterns applicable for diagnosis and therapy stratification or monitoring [45, 46] (Stuhlmuller B, Hernandez MM, Haeupl T, Kuban RJ, Gruetzkau A, Voss JW, Salfeld J, Kinne RW, Burmester GR, unpublished data). A reasonable expectation is that gene analyses also provide means to predict which patients are future responders to anti-TNF-α therapy.
TNF receptors are found in synovial tissue and fluid of patients with RA, especially in cases of severe disease . There are two known TNF receptors, the R55 (TNF-R1) (high-affinity receptor) and the R75 (TNF-R2) (low-affinity receptor), which are expressed by both synovial Mφ and fibroblasts [47, 48]. The two TNF receptors can operate independently of one another, cooperatively, or by 'passing' TNF-α to one another , a complexity that may explain the tremendous sensitivity of target cells (such as Mφ) to minute concentrations of TNF-α. TNF receptors can also be shed, binding to soluble TNF-α and hence acting as natural inhibitors in disease. Recent studies have demonstrated that TNF-R1 may be primarily responsible for pro-inflammatory effects of TNF-α, whereas TNF-R2 may predominantly mediate anti-inflammatory effects of TNF-α  (reviewed in ). Thus, selective blockade of TNF-R1, instead of broad blockade of all effects of TNF-α, may become an attractive therapeutic approach [48, 50].
In the RA synovial membrane, IL-1 is found predominantly in CD14+Mφ ; also, IL-1 levels in the synovial fluid significantly correlate with joint inflammation . The two existing forms of IL-1 (IL-1α and IL-1β) show some differences (for example, low protein homology, stronger pro-inflammatory regulation of the IL-1β promoter, and secretion of inactive pro-IL-1β versus expression of membrane-bound IL-1α activity) but also strong similarities (that is, three-dimensional structures of the essential domains, molecular masses of pro-peptides, and mature-form processing proteases), resulting in almost identical binding capacity to the IL-1 receptors and comparable function. In arthritis, IL-1 appears to mediate a large part of the articular damage, as it profoundly influences proteoglycan synthesis and degradation [43, 53]. At the same time, IL-1 induces the production of MMP-1 and MMP-3 and enhances bone resorption; this is compatible with recent evidence from arthritis models and human RA suggesting that the tissue-destruction capacities of IL-1β may outweigh its genuine role in joint inflammation .
The IL-1 type I receptor (IL-1R1), which mediates cell activation via IL-1R accessory protein and IL-1 receptor-associated kinase (IRAK), is found on numerous cells in the synovial tissue of patients with RA . In contrast, the type II receptor (IL-1R2) (also found in soluble form in serum), which lacks cell-activating properties and acts exclusively as a decoy receptor, is low in synovial tissue . Similarly, IL-1RA, a soluble protein that blocks the action of IL-1 by binding to the type I receptor without receptor activation, has been detected only sporadically in RA synovial samples. In RA, the balance between IL-1 and its physiological inhibitor IL-1RA is therefore shifted in favor of IL-1, indicating a dysregulation crucial in promoting chronicity . However, therapeutic application of IL-1RA (anakinra) appears to be only modestly effective in RA (reviewed in ). Therefore, it remains to be clarified whether the IL-1 pathway is a less suitable therapeutic target than TNF-α (for example, due to functional redundancy in the IL-1 receptor superfamily) or whether the biological molecule IL-1RA is suboptimal for therapy.
IL-15, a cytokine of the IL-2 family with chemoattractant properties for memory T cells, is produced by lining layer cells (including Mφ) and is increased in RA synovial fluid . Notably, peripheral or synovial T cells stimulated with IL-15 induce Mφ to produce IL-1β, TNF-α, IL-8, and monocyte chemotactic protein-1 [21, 57] but not the regulatory IL-10. Because IL-15 is also produced by Mφ themselves, this cytokine may (re)stimulate T cells, possibly self-perpetuating a pro-inflammatory loop . The expression of IL-15 in the RA synovial membrane, its biological function, and its successful targeting in experimental arthritis have generated large expectations on the use of a fully humanized anti-IL-15 antibody in clinical trials [56–58].
In the RA synovial membrane, this cytokine of the IL-1 family is expressed in CD68+ Mφ contained in lymphoid aggregates. CD14+ Mφ of the RA synovial fluid also express the IL-18 receptor . The pro-inflammatory role of IL-18 in arthritis (and its potential suitability as a therapeutic target in RA) is indicated by the following findings: (a) IL-18 treatment markedly aggravates experimental arthritis , (b) intra-articular overexpression of IL-18 induces experimental arthritis, (c) IL-18 is involved in the development of experimental streptococcal arthritis (a strongly Mφ-dependent model), (d) IL-18 is selectively overexpressed in the bone marrow of patients with juvenile idiopathic arthritis and Mφ activation syndrome , (e) IL-18 can stimulate osteoclast formation through upregulation of RANKL (receptor activator of NF-κB ligand) production by T cells in RA synovitis, and (f) IL-18 mediates its action via classic induction of TNF-α, GM-CSF, and interferon (IFN)-γ  or functional Toll-like receptors TLR-2 and TLR-4 in synovial cells  or else through the induction of synovial acute-phase serum amyloid proteins. The clinical relevance of synovial IL-18 is emphasised by its correlation with the systemic levels of C-reactive protein (CRP); also, IL-18 and CRP decrease in parallel in synovial tissue and serum following effective treatment with disease-modifying anti-rheumatic drugs . In addition, peripheral blood mononuclear cells of RA patients show low levels of the IL-18 binding protein (a natural inhibitor of IL-18) and reduced sensitivity to stimulation with IL-12/IL-18, indicating profound dysregulation of the IL-18 system .
The genuine role of IL-23, a cytokine of the IL-12 family predominantly produced by Mφ or dendritic cells, is unclear due to the sharing of the p40 subunit with IL-12 . IL-23 has prominent pro-inflammatory functions, since transgenic expression in mice leads to multi-organ inflammation and premature death. IL-23 promotes various T-cell responses potentially relevant to RA . Recent studies in experimental arthritis have demonstrated that mice lacking only IL-12 (p35-/-) show exacerbated arthritis, whereas mice lacking only IL-23 (p19-/-) are completely protected from arthritis . In addition, activation of Mφ derived from arthritis-susceptible rats is paradoxically associated with reduced levels of pro-inflammatory mediators but high expression of IL-23 (p19), whereas non-susceptible rats show the inverse phenotype. If these findings were transferable to human RA, IL-23 would have a pro-inflammatory role and IL-12 a protective one. At the present time, it is unclear whether these findings fit into the recently introduced M1/M2 paradigm of differential Mφ activation [64, 65] and especially whether this paradigm can be exploited for a better understanding of the role of Mφ in RA.
IL-27, another cytokine of the IL-12 family, is expressed by monocytes/Mφ following common inflammatory stimuli and displays a variety of pro- and anti-inflammatory properties . In support of a pro-inflammatory role in arthritis, neutralizing antibodies against IL-27p28 suppress experimental arthritis .
Chemokines (subdivided into the CXC, CC, C, and CX3C families) are small proteins specialized in differential recruitment of leukocyte populations via a number of transmembrane receptors. Chemokines not only favor monocyte influx into inflamed tissue, but also play a key role in activation, functional polarization, and homing of patrolling monocytes/Mφ . Notably, monocytes/Mφ express only select types of the numerous chemokine receptors (for example, CCR1, 2, 5, 7, and 8 as well as CX3CR1), representing a partially specific basis for prominent trafficking of monocyte/Mφ in arthritis. In RA, synovial Mφ produce several chemokines (for example, CCL3 [or Mφ inflammatory protein 1α], CCL5 [or RANTES], and CX3CL1 [or fractalkine]) and at the same time carry chemokine receptors, indicating the presence of autocrine loops in disease (reviewed in ). At the same time, chemokines are upregulated by the Mφ-derived TNF-α and IL-1. Significantly, some chemokines expressed in synovial Mφ (for example, IL-8 and fractalkine) are powerful promoters of angiogenesis, thus providing a link between Mφ activation and the prominent neo-vascularization of the RA synovium . In RA, angiogenesis may be further promoted via activation of Mφ by advanced glycation end products, whereas thrombospondin-2 seems to downregulate angiogenesis. Because the enlargement of the vascular bed potentiates the influx of activated monocytes, down-modulation of the chemokine system represents a multi-potential target of anti-rheumatic therapy, as indicated by the promising results of treatment with a CCR1 antagonist in RA .
One of the first ILs ever discovered, migration inhibitory factor (MIF), is an early-response cytokine abundantly released by Mφ. MIF stimulates a number of Mφ functions in an autocrine fashion (for example, secretion of TNF-α, phagocytosis, and generation of reactive oxygen species [ROS]). In addition, MIF confers resistance to apoptosis in Mφ and synovial fibroblasts, thus prolonging the survival of activated, disease-relevant cells. In RA, MIF is overexpressed in serum and synovial tissue in correlation with disease activity. Also, polymorphisms in the promoter or coding region of the human MIF gene are associated with features of juvenile idiopathic arthritis or adult RA .
Differentiated Mφ constitutively express IL-1RA, which is upregulated by pro-inflammatory mediators, including IL-1 itself or GM-CSF, and induces strong anti-inflammatory effects. By means of this feedback mechanism, Mφ therefore contribute to the termination of inflammatory reactions (reviewed in [71, 72]) (see above).
IL-10, a Th2- and Mφ-derived cytokine with clear autocrine functions, reduces HLA-DR expression and antigen presentation in monocytes and inhibits the production of pro-inflammatory cytokines, GM-CSF, and Fc-γ receptors by synovial Mφ. Consistently with cytokine and chemokine downregulation, IL-10 clearly suppresses experimental arthritis. In spite of IL-10 elevation in serum and synovial compartments of patients with RA , some studies suggest a relative deficiency of IL-10 . A combined IL-4/IL-10 deficiency probably tilts the cytokine balance to a pro-inflammatory predominance. In addition, the ex vivo production of IL-10 by RA peripheral blood mononuclear cells is negatively correlated with radiographic joint damage and progression of joint damage, suggesting that high IL-10 production is protective in RA. Similarly to IL-4, however, treatment with recombinant IL-10 does not improve RA. This may be partially explained by upregulation of the pro-inflammatory Fc-γ receptors I and IIA on monocytes/Mφ (reviewed in ).
IL-6 is the most strikingly elevated cytokine in RA, especially in the synovial fluid during acute disease . The acute rise is consistent with the role of IL-6 in acute-phase responses (Table 1). However, while IL-6 levels in the synovial fluid correlate with the degree of radiological joint damage, and IL-6 and soluble IL-6 receptors promote the generation of osteoclasts, this cytokine has phase-dependent effects; for example, it protects cartilage in acute disease but promotes excessive bone formation in chronic disease. While IL-6 is mostly produced by synovial fibroblasts and only partially by Mφ, two findings suggest that the striking IL-6 rise is a prominent outcome of Mφ activation: (a) the morphological vicinity of IL-6-expressing fibroblasts with CD14+ Mφ in the RA synovial tissue (reviewed in ) and (b) co-culture studies showing that IL-1 stimulates IL-6 production . The role of IL-6 in experimental arthritis and the anti-arthritic effects of anti-IL-6 receptor antibodies suggest a role for anti-IL-6 therapy in RA  (reviewed in ).
In RA, Mφ express different transforming growth factor-beta (TGF-β) molecules and TGF-β receptors in the lining and sublining layers, at the cartilage-pannus junction, and in the synovial fluid [78–80]. The pro-inflammatory effects of TGF-β are substantiated by induction of Mφ expression of Fc-γ receptor III (which elicits the release of tissue-damaging ROS) and promotion of monocyte adhesion and infiltration during chronic disease (reviewed in ). At the same time, TGF-β has anti-inflammatory properties; for example, it counteracts some IL-1 effects, including phagocytosis of collagen and possibly MMP production. A protective role of TGF-β in RA is also suggested by the association between TGF-β polymorphism and disease severity; that is, alleles associated with low TGF-β expression are correlated with stronger inflammation and poorer outcome . Likewise, experimental arthritis is significantly ameliorated by activation of TGF-β via adenoviral expression of thrombospondin-1 . The effects of TGF-β on tissue inhibitor of MMP (TIMP) are also unclear, as the regulation of MMP and TIMP may depend on different tissue domains (superficial versus deep cartilage layers) and may vary for intra- or extracellular digestion of collagen (reviewed in ).
The role of Mφ-derived cytokines in the perpetuation of RA, the pathophysiological dichotomy between joint inflammation and cartilage destruction, and the crucial significance of activated synovial Mφ in relation to permanent joint damage  have led to a radical re-evaluation of the conventional anti-inflammatory and disease-modifying treatments in relation to Mφ parameters in order to potentiate therapeutic effects (for example, via combination approaches ) and reduce side effects. For anti-Mφ effects of conventional anti-rheumatic therapy in RA (including methotrexate, leflunomide, anti-malarials, gold compounds, corticosteroids, and non-steroidal anti-inflammatory drugs), the reader is referred to a recent comprehensive review . Recent findings show that conventional and specific anti-rheumatic treatments predominantly target sublining rather than lining Mφ; also, different therapeutic approaches seem to result in similar histological changes in the inflamed synovial membrane, including significant reduction of sublining Mφ. This, in turn, is significantly correlated with the degree of clinical improvement [11, 12]. Thus, different pathogenetic mechanisms may funnel into similar disease pathway(s), leading to massive activation of Mφ and providing the rationale for targeted anti-Mφ therapy.
The transcription of most cytokine genes in monocytes/Mφ depends on the activation of NF-κB and NF-κM transcription factors or that of the activator protein-1 (AP-1) complex. In RA synovial Mφ, the expression of NF-κB is more pronounced than that of AP-1, a selectivity that may bear important therapeutic implications . Accordingly, the anti-arthritic effects of IL-4 may be based on the selective suppression of NF-κB in Mφ. IL-10 also downregulates the production of pro-inflammatory monokines, inhibiting the nuclear factors NF-κB, AP-1, or NF-IL-6. Unlike IL-4, IL-10 can also enhance degradation of the mRNA for IL-1 and TNF-α (reviewed in ). In general, therefore, targeted inhibition of 'pro-inflammatory' signal transduction pathways in Mφ represents an attractive therapeutic approach .
Gene therapy has been applied in experimental arthritis models to counteract Mφ-derived IL-1 and TNF-α or to deliver/overexpress protective IL-1RA, soluble IL-1 type I receptor-IgG fusion protein, and type I soluble TNF-α receptor-IgG fusion protein. This has been extended to (Mφ-derived) anti-inflammatory cytokines (that is, IL-4, IL-10, IL-13, IFN-β, or TGF-β) and to 'molecular synovectomy' (either by expression of herpes simplex virus-thymidine kinase with subsequent administration of ganciclovir or by overexpression of Fas-ligand/inhibitors of nuclear translocation of NF-κB, resulting in synovial cell apoptosis [88, 91, 92]). Therefore, gene therapy aimed at neutralizing pro-inflammatory Mφ products, overexpressing Mφ-regulating mediators, or simply eliminating overly activated Mφ remains promising for the treatment of arthritis.
The multitude and abundance of Mφ-derived mediators in RA and their paracrine and autocrine effects (including those directed to other cells of the myeloid lineage) indicate that Mφ are local and systemic amplifiers of disease severity and perpetuation. The main local mechanisms include (a) self-perpetuating chemokine-mediated recruitment of inflammatory cells, (b) cytokine-mediated activation of newly immigrated inflammatory cells, (c) cell contact-mediated activation of neighboring inflammatory cells, (d) cytokine- and cell contact-mediated secretion of matrix-degrading enzymes, (e) activation of mature dendritic cells and cytokine-mediated differentiation of Mφ (and possibly B cells, T cells, and mesenchymal cells) into antigen-presenting cells, with possible effects on spreading of autoimmunity to cryptic epitopes, (f) neo-vascularization, with potentiation of cellular and exudatory mechanisms, and (g) (trans)differentiation of Mφ into osteoclasts involved in subchondral bone damage. At a systemic level, amplification of disease can proceed at least through the following mechanisms: (a) acute-phase response network, (b) systemic production of TNF-α, (c) anomalies in bone marrow differentiation, and (d) chronic activation of circulating monocytes.
Although uncovering the etiology of disease remains the ultimate goal of research, the efforts in understanding how activated Mφ influence disease have led to optimization strategies to selectively target activated Mφ in RA (Figure 3). This approach has at least two advantages: (a) striking the very cell population that mediates/amplifies most of the irreversible cartilage destruction and (b) minimizing adverse effects on other cells that may have no (or marginal) effects on joint damage.
granulocyte macrophage colony-stimulating factor
interleukin-1 receptor antagonist
migration inhibitory factor
mononuclear phagocyte system
reactive oxygen species
staphylococcal enterotoxin B
transforming growth factor-beta
tissue inhibitor of metalloprotease
tumor necrosis factor
tumor necrosis factor receptor 1
tumor necrosis factor receptor 2
Ernesta Palombo-Kinne is gratefully acknowledged for critical revision of the manuscript. This study was supported by the German Federal Ministry of Education and Research (BMBF) (grants FKZ 01ZZ9602, 01ZZ0105, and 010405 to RWK), the Interdisciplinary Center for Clinical Research (IZKF) Jena, including grants for junior researchers, (grants FKZ 0312704B and 0313652B to RWK), the Jena Centre for Bioinformatics; (grant 01GS0413, NGFN-2 to RWK); the German Research Foundation (DFG) (grants KI 439/7-1/3 and KI 439/6-1/3 to RWK); the Thuringian Ministry of Science, Research, and Art (FKZ B 311-00026); and a grant for the advancement of female scientists (LUBOM Thuringia).