Background and Role in RA

Macrophages, together with osteoclasts and myeloid DCs, are derived from myelomonocytic origins and are key cellular components of the innate immune system. Macrophages differentiate from circulating monocytes and have primary roles in tissues as phagocytes of invading pathogens and as scavengers of apoptotic debris. In addition, macrophage activation results in the expression of chemokines and cytokines, such as TNF-α and IL-1β, that helps to attract other cells and proteins to the sites of inflammation. The central role of macrophages in RA pathogenesis is supported by the fact that conventional therapies, including methotrexate and cytokine inhibitors, act to decrease the production of cytokines that are produced primarily by macrophages. Indeed, a correlation has been found between synovial macrophage infiltration and subsequent radiographic joint destruction. A remarkable fact is that a reduction in the number of sublining macrophages in RA synovial tissue has been shown to strongly correlate with the degree of clinical improvement, regardless of the type of therapy chosen. In addition to local effects of macrophages in the synovial tissue, systemic consequences of macrophage-mediated inflammation in RA may be manifested by damage to other areas such as the subendothelial space where macrophages become foam cells contributing to atherosclerotic plaques.

Mechanisms for Increased Macrophage Number in RA Tissue

Possible mechanisms for the increased number of macrophages in diseased tissue include increased chemotaxis and reduced emigration. Some studies also suggest local proliferation of macrophages in areas of inflammation. Decreased apoptosis may also contribute to the accumulation of macrophages in the RA joint. Several studies have shown that induction of synoviocyte apoptosis in animal models of inflammatory arthritis ameliorates joint inflammation and joint destruction. In both experimental arthritis and synovial tissue from patients with RA, reduced expression of the proapoptotic Bcl-2 family member, Bim, was seen in macrophages and corresponded to the increased expression of IL-1β by macrophages. Furthermore, administration of a Bim mimetic dramatically reduced the incidence of arthritis and successfully ameliorated established arthritis in mice. This result suggests that therapies that restore the homeostasis between survival and cell death of RA macrophages may be successful in ameliorating arthritis in patients.

Heterogeneity of Monocyte and Macrophage Populations

Within monocyte and macrophage populations, there is a great deal of heterogeneity. For example, 2 human monocyte populations have been defined based on their surface marker expression: the CD14 ⁺ CD16 ⁻ and the CD14 ^low CD16 ⁺ subsets. CD16 is a receptor for immunoglobulin (Ig) G, FcγIIIA, which binds to IgG-containing immune complexes (see later discussion). The number of CD14 ^low CD16 ⁺ monocytes is elevated in RA peripheral blood, and CD14 ^low CD16 ⁺ macrophages are enriched in RA synovial tissue. CD14 ^low CD16 ⁺ monocytes produce more TNF-α in response to the microbial toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) compared with the CD14 ⁺ CD16 ⁻ subset. These observations suggest that the proinflammatory CD14 ^low CD16 ⁺ monocytes migrate to the RA joint and become highly responsive macrophages. However, CD14 ⁺ CD16 ⁻ monocytes also express the chemokine receptor (CCR) 2, which binds monocyte chemotactic protein 1, and thereby promotes monocyte migration to the site of inflammation. Because the RA joint is rich in this chemokine, it is possible that CD14 ⁺ CD16 ⁻ and CCR2 ⁺ monocytes are recruited to the joint where CD16 expression is then induced. Nonetheless, because monocytes migrate from the peripheral blood into RA synovial tissue, identification of circulating monocyte subpopulations may be an extremely useful clinical tool for tracking disease activity and for identifying additional therapeutic targets.

In addition, diversity in activation states of macrophages has been found. In general, macrophages exhibiting a more inflammatory phenotype have been named M1, or classically activated macrophages, whereas those that trend toward a more antiinflammatory and repair role are known as M2, or alternatively activated macrophages. Most macrophages in the RA joint express proinflammatory cytokines and are thus most consistent with classically activated macrophages. Therapies that promote the balance in favor of an M2 phenotype may be useful in RA.

Therapies Targeting Macrophages

Conventional therapies such as prednisone, methotrexate, leflunomide, sulfasalazine, and TNF-α inhibitors have been shown to decrease the number of CD68 ⁺ macrophages in the synovial sublining. Another study of synovial tissue response to rituximab found a significant reduction in the number of sublining macrophages at 16 weeks, providing evidence for synovial tissue sublining macrophage reduction after B-cell depletion therapy in RA as well. Furthermore, a reduction in the number of synovial sublining macrophages correlated clinically with the improvement of the values of disease activity score (DAS) 28, suggesting an association between sublining CD68 ⁺ macrophages and therapeutic efficacy. The positive correlation between the change in RA clinical activity and CD68 expression in the synovial sublining has been independently confirmed.

Specifically, targeting activated macrophages at sites of inflammation would be a way of circumventing the potential untoward effects of systemic macrophage depletion. The bisphosphonate clodronate, encapsulated within liposomes, has been used to specifically deplete macrophages. After injecting rats intraperitoneally with streptococcal cell-wall (SCW) fragments to induce arthritis, intravenous (IV) liposomal clodronate suppressed the development of chronic arthritis for up to 26 days after treatment. Treatment was also associated with the depletion of synovial and hepatic, but not splenic, macrophages, as well as a reduction in articular IL-1β, IL-6, TNF-α, and matrix metallopeptidase (MMP) 9 levels. Similarly, in the K/B × N serum transfer model of arthritis, where spontaneously produced anti–glucose phosphate isomerase (GPI) antibodies from a K/B × N mouse are injected into a naive host, treatment with liposomal clodronate before serum transfer caused depletion of macrophages in the bone marrow and liver, and the treated mice were completely resistant to arthritis. Resistance to arthritis was reversed when the macrophage-depleted mice were reconstituted with macrophages from naive animals and immediately injected with K/B × N serum. In rabbits with established antigen-induced arthritis (AIA), repeated intraarticular administrations of low, noncytotoxic doses of liposomal clodronate led to an early reduction of joint swelling, delay in radiographic progression, and decrease in the number of synovial lining macrophages. However, no difference was seen in pannus formation or radiographic erosions at 8 weeks. In patients with RA undergoing knee replacement surgery, a single intraarticular dose of clodronate liposomes significantly reduced the number of CD68 ⁺ cells and the expression of adhesion molecules in the synovial lining. In contrast, no immunohistologic difference was observed in the control group. These observations suggest that depletion of synovial tissue macrophages may be an important therapeutic goal in RA.

Systemic depletion of all macrophages could have serious consequences in patients, and this may be avoided by specifically targeting receptors present on activated macrophages. Folate receptor β (FRβ) has been described on both activated macrophages from RA synovial fluid and animal models of arthritis but not on resting or quiescent macrophages or normal cells of the body except for the proximal tubule cells of the kidneys. The FRβ has been used to deliver folate-conjugated imaging agents to inflamed joints in patients with RA and is a target for novel therapeutic agents. Several new-generation folate antagonists are selectively taken up by the FRβ and show growth inhibition capabilities against FRβ-expressing cells, thus circumventing the systemic effects. In addition, antibodies or fragments of antibodies against FRβ linked with immunotoxins have been developed and have been shown to reduce the number of macrophages and levels of IL-6 and to increase the number of apoptotic cells in RA synovial tissue engrafted into severe combined immunodeficient mice. Another approach involved the conjugation of folate to superoxide dismutase and catalase, 2 enzymes that scavenge the damaging reactive oxygen species secreted by activated macrophages. Folate conjugation dramatically enhanced the ability of catalase and superoxide dismutase to scavenge reactive oxygen species produced by activated macrophages in cell culture experiments and the uptake of the enzyme catalase into activated macrophages. Folate has also been conjugated to small molecules, or haptens such as fluorescein, and given to rodents previously immunized to the hapten after the onset of experimental arthritis. The folate-hapten conjugate selectively “decorated” and promoted immune-mediated elimination of activated macrophages and decreased paw swelling, spleen size, systemic inflammation, arthritis score, and bone erosion. Thus, the presence of select folate receptors on activated macrophages offers exciting potential to target activated macrophages in the RA joint.

Background

DCs, along with macrophages and B cells, have the ability to present antigen to T cells, and therefore play a central role in the development of innate and adaptive immune responses. In the periphery, immature DCs are stimulated to undergo differentiation by an array of pathogens, mainly via the activation of TLRs by exogenous or endogenous stimuli, and also in response to cytokines or immune complexes produced during the inflammatory response. TLR signaling results in a significant change in chemokine receptors expressed by DCs, allowing for maturation of DCs and migration to the lymphoid tissue, where mature DCs display antigen on MHC molecules to naive T cells. DCs also express the critical costimulatory molecules, CD80 and CD86, which interact with CD28 on T cells, completing the necessary signal for antigen-specific effector T-cell maturation to occur. In addition to stimulation of naive T cells, DCs can process and display antigen in local tissues and contribute to the inflammatory response by the production of cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, DCs can direct the formation of distinct T helper (T _h ) cells by producing key cytokines, such as IL-12 and IL-18 for T _h 1 cells and IL-23 for T _h 17 cells. Finally, DCs are important in the development of both central and peripheral tolerance, and their depletion in animal models is associated with the onset of fatal autoimmune-type disease. In the thymus, DCs present endogenous self-antigens to T cells and delete those that are strongly reactive, whereas in the periphery, interaction between autoreactive T cells and immature DCs bearing self-antigen may result in anergy, apoptosis, or differentiation into regulatory T cells. Deviations in this pathway, either failed clearance of dead cells or exposure of DCs bearing self-antigens to maturation signals, can abrogate their tolerogenic ability and are implicated in the development of autoimmunity.

DCs may be categorized into subtypes based on the expression of various cell-surface markers. Functionally, however, DCs may be separated into 2 main classes: classical or conventional DCs (cDCs), which are resident in lymphoid tissues or migratory in nonlymphoid tissues, and plasmacytoid DCs (pDCs). Both types may be activated by particular TLRs that induce the molecules necessary to promote antigen presentation, T-cell stimulation, and cytokine production. cDCs express CD11c:CD18, also known as complement receptor (CR) 4, and all known TLRs, except for TLR9. cDCs are the main participants in antigen presentation and activation of naive T cells as well as the mediators of peripheral tolerance. pDCs, on the other hand, are particularly important in modifying the immune response toward viruses. They do not express high levels of CD11c and have been identified by the expression of specific markers, such as blood dendritic cell antigen 2. In addition, pDCs express TLRs 1, 7, and 9 and other TLRs to a lesser degree. In response to stimuli such as viruses, pDCs are able to generate abundant amounts of type I interferons (IFNs) (IFN-α and IFN-β) and other cytokines such as TNF-α and IL-12. These cytokines increase the production of inflammatory mediators by macrophages, and in the case of IL-12, they can direct a potent T _h 1 response.

DCs and RA

The vast majority of studies that have examined the role of DCs in RA have relied on immunohistochemical techniques or have isolated DCs from peripheral blood and characterized their phenotype and function. In RA synovial tissue, the number of pDCs that are localized to perivascular lymphocytic infiltrates correlates with anti–cyclic citrullinated peptide (anti-CCP) antibodies. These pDCs produced B-cell activating factor, IL-18, and IFN-α/-β, whereas the cDCs secreted IL-12 and IL-23. The total numbers of cDCs or pDCs or the number of mature DCs in RA synovial tissue were not significantly different from patients with osteoarthritis (OA) or psoriatic arthritis, although there was a statistical increase in the ratio of pDC/cDC in RA synovial tissue. These data suggest that the number of DCs in synovial tissue may not reflect their true contribution to RA pathogenesis.

Few cDCs or pDCs are detected in peripheral blood of patients with RA, and the numbers are lower compared with healthy controls. The expression of the inhibitory FcγRIIB on DCs derived from peripheral blood monocytes of patients with RA correlated with disease activity. In addition, DCs expressing higher levels of FcγRIIB inhibited T-cell proliferation and promoted the T-regulatory phenotype after TLR and Fc receptor (FcR) stimulation in coculture studies. Treatment with methotrexate or infliximab dramatically affects the number, maturation, and function of the DC. DCs derived from monocytes of patients treated with infliximab displayed an antiinflammatory phenotype and increased numbers of cDCs in the circulation. In addition, the numbers of pDCs were increased in the peripheral blood in patients in clinical remission induced by either methotrexate or infliximab. Further, isolation of these pDCs and coculture with naive T cells led to an induction of the T-regulatory phenotype, which was capable of inhibiting autologous T-cell proliferation. In contrast, there was a reduction in both circulating cDC and pDC numbers in anti–IL-6 receptor–treated patients with RA, which was not observed in anti-TNF or cytotoxic T-lymphocyte antigen (CTLA) 4 immunoglobulin–treated patients with RA. These data suggest that clinically relevant information may be gleaned from examining circulating DCs in patients with RA and that there are differences related to the mode of therapy.

DCs and Murine Models of RA

The use of murine models of inflammatory arthritis has supported the human studies on the roles of DCs in RA. Follicular DCs are required for the development of the K/B × N mouse model of arthritis. In contrast, selective depletion of pDCs enhanced the severity and pathology of collagen-induced arthritis (CIA). These data suggest that pDCs may prevent the break of tolerance and that the follicular DC or cDC may be the central culprit that leads to the activation of autoreactive lymphocytes. Adoptive transfer of DCs from CTLA4 immunoglobulin–treated mice was sufficient to inhibit arthritis in CIA-recipient mice. In addition, adoptive transfer of TLR-stimulated DCs after immunization reduced CIA. Thus, similar to patients with RA, modification of the DC function by biologic therapies may lead to a skewing of T-cell development toward a T-regulatory phenotype mediated by DCs.

Background

DCs, along with macrophages and B cells, have the ability to present antigen to T cells, and therefore play a central role in the development of innate and adaptive immune responses. In the periphery, immature DCs are stimulated to undergo differentiation by an array of pathogens, mainly via the activation of TLRs by exogenous or endogenous stimuli, and also in response to cytokines or immune complexes produced during the inflammatory response. TLR signaling results in a significant change in chemokine receptors expressed by DCs, allowing for maturation of DCs and migration to the lymphoid tissue, where mature DCs display antigen on MHC molecules to naive T cells. DCs also express the critical costimulatory molecules, CD80 and CD86, which interact with CD28 on T cells, completing the necessary signal for antigen-specific effector T-cell maturation to occur. In addition to stimulation of naive T cells, DCs can process and display antigen in local tissues and contribute to the inflammatory response by the production of cytokines, such as TNF-α, IL-1β, and IL-6. Furthermore, DCs can direct the formation of distinct T helper (T _h ) cells by producing key cytokines, such as IL-12 and IL-18 for T _h 1 cells and IL-23 for T _h 17 cells. Finally, DCs are important in the development of both central and peripheral tolerance, and their depletion in animal models is associated with the onset of fatal autoimmune-type disease. In the thymus, DCs present endogenous self-antigens to T cells and delete those that are strongly reactive, whereas in the periphery, interaction between autoreactive T cells and immature DCs bearing self-antigen may result in anergy, apoptosis, or differentiation into regulatory T cells. Deviations in this pathway, either failed clearance of dead cells or exposure of DCs bearing self-antigens to maturation signals, can abrogate their tolerogenic ability and are implicated in the development of autoimmunity.

DCs may be categorized into subtypes based on the expression of various cell-surface markers. Functionally, however, DCs may be separated into 2 main classes: classical or conventional DCs (cDCs), which are resident in lymphoid tissues or migratory in nonlymphoid tissues, and plasmacytoid DCs (pDCs). Both types may be activated by particular TLRs that induce the molecules necessary to promote antigen presentation, T-cell stimulation, and cytokine production. cDCs express CD11c:CD18, also known as complement receptor (CR) 4, and all known TLRs, except for TLR9. cDCs are the main participants in antigen presentation and activation of naive T cells as well as the mediators of peripheral tolerance. pDCs, on the other hand, are particularly important in modifying the immune response toward viruses. They do not express high levels of CD11c and have been identified by the expression of specific markers, such as blood dendritic cell antigen 2. In addition, pDCs express TLRs 1, 7, and 9 and other TLRs to a lesser degree. In response to stimuli such as viruses, pDCs are able to generate abundant amounts of type I interferons (IFNs) (IFN-α and IFN-β) and other cytokines such as TNF-α and IL-12. These cytokines increase the production of inflammatory mediators by macrophages, and in the case of IL-12, they can direct a potent T _h 1 response.

DCs and RA

The vast majority of studies that have examined the role of DCs in RA have relied on immunohistochemical techniques or have isolated DCs from peripheral blood and characterized their phenotype and function. In RA synovial tissue, the number of pDCs that are localized to perivascular lymphocytic infiltrates correlates with anti–cyclic citrullinated peptide (anti-CCP) antibodies. These pDCs produced B-cell activating factor, IL-18, and IFN-α/-β, whereas the cDCs secreted IL-12 and IL-23. The total numbers of cDCs or pDCs or the number of mature DCs in RA synovial tissue were not significantly different from patients with osteoarthritis (OA) or psoriatic arthritis, although there was a statistical increase in the ratio of pDC/cDC in RA synovial tissue. These data suggest that the number of DCs in synovial tissue may not reflect their true contribution to RA pathogenesis.

Few cDCs or pDCs are detected in peripheral blood of patients with RA, and the numbers are lower compared with healthy controls. The expression of the inhibitory FcγRIIB on DCs derived from peripheral blood monocytes of patients with RA correlated with disease activity. In addition, DCs expressing higher levels of FcγRIIB inhibited T-cell proliferation and promoted the T-regulatory phenotype after TLR and Fc receptor (FcR) stimulation in coculture studies. Treatment with methotrexate or infliximab dramatically affects the number, maturation, and function of the DC. DCs derived from monocytes of patients treated with infliximab displayed an antiinflammatory phenotype and increased numbers of cDCs in the circulation. In addition, the numbers of pDCs were increased in the peripheral blood in patients in clinical remission induced by either methotrexate or infliximab. Further, isolation of these pDCs and coculture with naive T cells led to an induction of the T-regulatory phenotype, which was capable of inhibiting autologous T-cell proliferation. In contrast, there was a reduction in both circulating cDC and pDC numbers in anti–IL-6 receptor–treated patients with RA, which was not observed in anti-TNF or cytotoxic T-lymphocyte antigen (CTLA) 4 immunoglobulin–treated patients with RA. These data suggest that clinically relevant information may be gleaned from examining circulating DCs in patients with RA and that there are differences related to the mode of therapy.

DCs and Murine Models of RA

The use of murine models of inflammatory arthritis has supported the human studies on the roles of DCs in RA. Follicular DCs are required for the development of the K/B × N mouse model of arthritis. In contrast, selective depletion of pDCs enhanced the severity and pathology of collagen-induced arthritis (CIA). These data suggest that pDCs may prevent the break of tolerance and that the follicular DC or cDC may be the central culprit that leads to the activation of autoreactive lymphocytes. Adoptive transfer of DCs from CTLA4 immunoglobulin–treated mice was sufficient to inhibit arthritis in CIA-recipient mice. In addition, adoptive transfer of TLR-stimulated DCs after immunization reduced CIA. Thus, similar to patients with RA, modification of the DC function by biologic therapies may lead to a skewing of T-cell development toward a T-regulatory phenotype mediated by DCs.

Background

There are several mechanisms by which macrophages and other innate immune cells become activated. One way is via PRRs that are designed to recognize simple and regular nonself patterns of molecular structure, conserved during evolution, called pathogen-associated molecular patterns (PAMPs). Furthermore, when cells are under duress, such as in chronic inflammation, they may express danger-associated molecular patterns (DAMPs), such as uric acid, adenosine triphosphate, heat shock proteins (HSPs), or glycoprotein 96 (gp96), that may also be recognized by PRRs. PRRs may be membrane bound or soluble plasma proteins. Examples include mannose-binding lectin (MBL) that is important in the lectin pathway of complement activation, the transmembrane PRRs composed of 10 known human TLRs that may be activated on cell surfaces or within endosomal compartments, and the cytosolic PRRs that include the nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) and the retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs).

Toll-like Receptors

The cell-surface TLRs include TLR1, 2, 4, 5, and 6, with the recognition motifs outside the cell, whereas TLR3, 7, 8, and 9 are on the endosomal membrane, with the PRR recognition motifs within the endosomal compartment. The TLR system recognizes PAMPs, including LPS (TLR4), peptidoglycans (PGNs) (TLR2 together with TLR1 or 6), unmethylated CpG DNA motifs (TLR9) from bacteria, and single-stranded RNA (TLR7) and double-stranded RNA (TLR3) from viruses. The cytoplasmic domain of the TLR is called the Toll–IL-1 receptor (TIR) motif because it is also present in the IL-1 receptor. TLR signals are mediated through the TIR, which interacts with adapter molecules. All TLRs except TLR3 signal through the adapter molecule myeloid differentiation factor 88 (MyD88), whereas TLR3 signals only through the adapter molecule TIR domain-containing adapter-inducing IFN-β (TRIF), and TLR4 signals through both MyD88 and TRIF. Signaling through the MyD88 leads to the activation of nuclear factor κB (NF-κB) and the mitogen-activated protein (MAP) kinases, c-Jun N-terminal kinase and p38. Activation of NF-κB and the MAP kinases leads to the transcription of genes involved in inflammation, proliferation, and protection against apoptosis, whereas activation through TRIF results in the expression of type I IFNs, IFN-α and -β. Clinically, further elucidation of TLR signaling cascades is important because they offer attractive targets for intervention.

TLR Expression in RA

TLRs are expressed in the RA joint. In RA synovial tissue, CD16 ⁺ synovial lining macrophages also expressed TLR2, and the expression of both TLR2 and TLR4 was significantly higher in RA tissue than in samples from patients with OA. RA synovial tissues have a pronounced expression of TLR2 messenger RNA (mRNA) in the synovial lining and at sites of attachment and invasion into cartilage or bone tissue. In addition, TLR3 and TLR7 were also found to be highly expressed in RA synovium, and samples of tissue from patients with either early or long-standing RA showed similar levels of TLR3 and TLR4, both of which were significantly higher than those in patients with OA.

In RA synovial fibroblasts, levels of baseline mRNA for TLR2 and TLR4 did not differ compared with OA tissue fibroblasts. However, compared with OA fibroblasts, RA synovial fibroblasts demonstrated a significantly increased expression of TLR2 after treatment with IL-1β, TNF-α, LPS, or synthetic bacterial lipopeptide, leading to a strong increase of NF-κB translocation into the nucleus. In contrast, another study found that levels of both TLR2 and TLR4 mRNA were significantly higher in RA synovial fibroblasts compared with those from patients with OA and normal skin fibroblasts. In contrast to whole synovial tissue, TLR7 mRNA expression by synovial fibroblasts was not seen, suggesting that previous TLR7 staining may have been reflecting expression by macrophages or DCs.

Peripheral blood monocytes also express TLRs. Both TLR2 and TLR4 were increased on CD16 ⁺ and CD16 ⁻ peripheral blood monocytes from patients with RA versus healthy controls, and the TLR2 expression on the CD16 ⁺ subset was higher than that on the CD16 ⁻ subset. Furthermore, IFN-γ increased the expression of TLR2 and TLR4 on RA peripheral blood monocytes. In RA synovial fluid, CD14 ⁺ macrophages demonstrated increased expression of TLR2 and TLR4 compared with peripheral blood monocytes or control macrophages differentiated in vitro from normal monocytes.

Activation of Cells from the RA Joint by Microbial TLR Ligands

Isolated RA synovial fibroblasts treated with microbial TLR2 and TLR4 ligands demonstrated a marked increase in the osteoclast activator RANKL, at both the mRNA and protein levels. Inflammatory cytokines IL-6 and IL-8, as well as MMP-1 and -3, were induced by stimulation of RA synovial fibroblasts with bacterial PGN. Furthermore, RA synovial fibroblasts demonstrated increased expression of vascular endothelial growth factor after stimulation with bacterial PGN and increased IL-15 after stimulation by TLR2 and TLR4 ligands. Stimulation of RA synovial fibroblasts with the synthetic TLR3 ligand, poly I:C, led to the production of IL-6 and TNF-α, which was significantly enhanced when the cells were preincubated with IFN-α compared with cells stimulated with poly I:C alone. These observations suggest that in RA, synovial fibroblasts may be activated through the TLR pathway.

RA synovial fluid macrophages demonstrate an increased response to microbial TLR2 and TLR4 ligands compared with control macrophages differentiated in vitro from normal monocytes, macrophages from the joints of patients with other forms of inflammatory arthritis, or RA peripheral blood monocytes. In addition, treatment of RA synovial fluid macrophages with a microbial TLR2 ligand significantly increased the levels of IFN-γ and IL-23 mRNA compared with in vitro–differentiated control macrophages. It is possible that alterations of intracellular signaling pathways, such as those regulated by IFN-γ or IL-10, might be responsible.

Endogenous TLR Ligands in RA

Because microbial ligands are not likely the cause of TLR signaling in the RA joint, studies have examined RA synovial fluids and tissues for the presence of potential endogenous TLR ligands. RA synovial fluid activated human embryonic kidney 293 cells only when these cells expressed TLR4, suggesting the presence of endogenous TLR4 ligands in RA synovial fluid. In addition, the authors have demonstrated that RA synovial fluid is capable of activating normal macrophages and that this activation was mediated through TLR2 and TLR4 (Pope and colleagues, unpublished data, 2010). Together, these observations suggest that endogenous TLR ligands or DAMPs may be released in response to inflammation in early RA and result in continuous persistence of inflammatory mediators through activation of cells of the innate immune system.

Several potentially relevant endogenous TLR ligands or DAMPs have been identified in the RA joint. Ligands such as HSP22, tenascin-C, high-mobility group box chromosomal protein 1, serum amyloid A protein, and fragments of hyaluronic acid are highly expressed in the RA joint and are capable of activating monocytic cells through TLR2, or TLR4, or both. Another DAMP, gp96, was also highly expressed in RA synovial fluid and synovial tissue. The addition of gp96 in vitro to RA synovial fluid macrophages induced significantly higher levels of TNF-α, IL-8, and TLR2 compared with control macrophages. Although gp96 bound to both TLR2 and TLR4, macrophage activation was mediated primarily through TLR2. The quantity of TLR2 expression on synovial fluid macrophages strongly correlated with the level of gp96 in the same synovial fluids. Further studies, using a murine model of arthritis mediated by immune complexes, demonstrated that in the normal joint, an extracellular matrix (ECM) glycoprotein, tenascin-C, was not expressed. However, tenascin-C was induced during the early phase of the arthritis, and it contributed to the progression of the arthritis, mediated through TLR4 activation of macrophages and synovial fibroblasts. These observations support a potentially important role of endogenous TLR ligands in the persistent activation of macrophages and synovial fibroblasts that is observed in the joints of patients with RA.

TLR Signaling as a Target in RA

Understanding the potential role of TLRs in inflammation has led to its therapeutic exploitation. In mice with CIA, in which intradermal injections of type II collagen leads to the priming and the effector phases of inflammatory arthritis, treatment with a TLR4 antagonist both before the onset of disease and during established arthritis led to a significant reduction of arthritis. Furthermore, there was decreased histologic destruction of the cartilage matrix and infiltration of inflammatory cells into the joint space. In addition, IL-1 receptor antagonist–deficient mice developed a spontaneous chronic arthritis, which was ameliorated when treated with a TLR4 antagonist. The results of these animal models support the potential benefits of suppressing TLR signaling as a therapeutic approach in RA.

In ex vivo synovial tissue cultures from patients with RA, the addition of a TLR4 antagonist suppressed the spontaneous secretion of IL-1β and TNF-α, thus supporting the role of TLR4 in the production of inflammatory cytokines. Currently available antirheumatic therapies that are known to have a suppressive effect on the TLR signaling pathway include hydroxychloroquine (TLR7, 8, and 9) and auranofin (TLR4). Antagonists of TLR4 are being studied for possible use in sepsis and endotoxemia. Lipid A, the innermost of the 3 regions of LPS, was created in a stable, synthetic form called E5564 (eritoran). It is currently undergoing clinical trials and may become a viable agent in RA. Another TLR4 receptor antagonist, chaperonin 10 (HSP10), has been studied in a randomized, double-blind, multicenter study of 23 patients with moderate to severe active RA who received twice weekly IV therapy at different concentrations for 12 weeks. All 3 treatment groups tolerated the therapy well and had significant improvement in the primary endpoint of clinical improvement as measured by the DAS28 score. The effect seemed to be dose dependent, with 4 of 7 patients in the highest group achieving an American College of Rheumatology (ACR) 50 response and 2 of 7 achieving an ACR 70 response. The highest treatment group also had significant improvement in all secondary endpoint measures, including swollen and tender joint count, patient’s assessment of pain on the visual analog scale, disability index on the health assessment questionnaire, and morning stiffness. In summary, the TLR signaling pathway, which may be activated by endogenous TLR ligands or DAMPs, is a novel target for therapeutic intervention in patients with RA.

Nucleotide-binding Oligomerization Domain–like Receptors

Similar to TLRs, the NLRs are intracellular, cytosolic receptors that sense PAMPs or DAMPS and mediate an inflammatory response. Thus far, there are 22 proteins in the NLR family, including the NOD and NTPases implicated in apoptosis and multihistocompatability complex transcription leucine rich repeat protein (NALP) subfamilies, with the 14 NALPs characterizing the largest subfamily. Common features of the NLRs include a central nucleotide-binding domain, a C-terminal leucine-rich repeat domain, and N-terminal caspase-recruitment and pyrin domains. The NOD proteins recognize fragments of bacterial cell-wall proteoglycans and activate the transcription factor NF-κB. Although NOD proteins are expressed in phagocytes along with TLRs, they are especially important activators of the innate response in epithelial cells, where expression of TLRs is weak or absent. Members of the NALP family, including NALP1, 3, and 12, are capable of forming functional caspase-1 activation complexes, or inflammasomes, that are important in the processing and release of IL-1β and IL-18 in response to PAMPs or DAMPs.

It has been shown that the induction of SCW-driven arthritis in NOD2 gene–deficient mice results in reduced joint swelling and decreased levels of TNF-α and IL-1β, whereas the opposite effect was seen in NOD1 –deficient mice, suggesting an antiinflammatory role for NOD1. Moreover, the microbial ligand for NOD2, muramyl dipeptide (MDP), has been detected in RA synovium, whereas none was found in synovium from patients with OA. NOD2 was expressed by both macrophages and synovial fibroblasts, as detected by immunohistochemistry, but not by lymphocytes or blood vessels. Transcription of NOD2 mRNA by RA synovial fibroblasts was induced by TNF-α, poly I:C, and LPS. Adding MDP to other inflammatory stimuli augmented RA synovial fibroblast production of inflammatory cytokines and matrix-degrading enzymes compared with the stimuli alone. These observations suggest that TLR activation may induce NOD2 expression and that TLRs and NOD2 may act synergistically in promoting inflammation and matrix destruction in the RA joint. Further studies will be required to determine whether NODs may potentially become effective therapeutic targets in RA.

The Inflammasome and RA

In macrophages, the essential link between pro-IL-1β and pro-IL-18 and their bioactive counterparts is the protease caspase 1, which cleaves these molecules and generates the active cytokines that are then released from the cell. Activation of caspase 1 depends on the formation of inflammasomes, which are multiprotein complexes consisting of an NLR protein such as an NALP, the adapter molecule apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase 1, and which are assembled in response to cellular recognition of DAMPs or PAMPs. Inflammasomes are implicated in diseases such as systemic onset juvenile idiopathic arthritis and familial Mediterranean fever. In addition, mutations in NALP3 are responsible for cryopyrin-associated periodic syndromes, and NALP3-containing inflammasomes are activated by monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals in gout and pseudogout, respectively. In addition to MSU and CPPD crystals, other ligands of the NALP3 inflammasome include Alzheimer disease–associated amyloid deposits (amyloid-β) and the ECM components, biglycan and hyaluronan.

NALP3 is expressed in the RA joint. Using real-time PCR, it has been shown that NALP3 mRNA levels were increased in RA synovium compared with OA and that monocyte-derived macrophages from healthy donors differentiated in vitro increased NALP3 expression when stimulated by TNF-α. Another study did not find any differences between RA and OA synovial expression of NALP1, NALP3, NALP12, or ASC using densitometric analysis of Western blots. However, using enzyme-linked immunosorbent assay, caspase-1 levels were significantly enhanced in RA synovial tissues, even though there was no difference in concentrations of IL-1β. Thus, further studies are needed to clarify the role of the inflammasome in RA pathogenesis.

Caspase 1 is not the only IL-1β converting enzyme (ICE) involved in IL-1β processing, which is likely the reason why inhibition of caspase 1 only partially inhibits experimental models of RA, whereas deficiency of IL-1β completely ameliorates the arthritis. Alternative ICEs, such as elastase or proteinase 3 in neutrophils or chymase in mast cells, may be involved in the early stages of inflammatory arthritis by contributing to the processing of IL-1, whereas caspase 1 may play more of a role in the chronic stage of the arthritis.

Retinoic Acid–inducible Gene I–like Receptors

Three genes encode RLRs in human and mouse genomes. One of these genes, RIG-I , encodes an RNA helicase protein whose expression is induced by IFN-γ and which is found in cells such as endothelial cells, bronchial epithelial cells, smooth muscle cells, and macrophages. It is a sensor of viral RNAs and activates cells of the innate immune system. It is also associated with various chronic inflammatory diseases, including lupus nephritis, and is considered important in mediating reactions induced by IFN-γ. Recently, high levels of RIG-I expression have been found in RA synovial tissues compared with OA controls. Treatment of RA synovial fibroblasts with IFN-γ significantly induced the expression of RIG-I, and knockdown of RIG-I in RA synovial fibroblasts with small interfering RNA (siRNA) resulted in the inhibition of the expression of the chemokine CXCL10. In addition, RIG-I was expressed in RA synovial fibroblasts stimulated with TNF-α and knockdown of RIG-I with siRNA suppressed TNF-α–induced RANTES (chemokine (C-C motif) ligand 5 [CCL5]) expression, suggesting a possible role for the TNF-α/RIG-I/CCL5 pathway in RA pathogenesis. Further studies are needed to show whether RIG-I is a plausible target for therapy in RA.