Combination of High-Molecular-Weight Hyaluronic Acid and Cytokine Inhibitor Potently Inhibits Expression of Joint-Damage-Related Genes Induced By Synovial Fluid of RA Patients

Rheumatoid Arthritis (RA) is characterized by persistent inflammation of the synovial membranes, and many types of cells are activated by the inflammatory cytokines present in the synovial fluid. RA patients develop multiple systemic symptoms, particularly joint inflammation and destruction of bone and articular cartilage, with some patients suffering permanent disability [1]. The two components of joint damage appear to be consequences of pathogenic processes: (1) Bone erosion is mediated primarily by osteoclasts, which are activated by pro-inflammatory cytokines, and their destructive action is not counterbalanced by bone formation [2,3]; and (2) breakdown of the cartilage matrix is mediated by matrix degradation and inhibition of matrix synthesis induced by cytokines [4,5].


Introduction
Rheumatoid Arthritis (RA) is characterized by persistent inflammation of the synovial membranes, and many types of cells are activated by the inflammatory cytokines present in the synovial fluid. RA patients develop multiple systemic symptoms, particularly joint inflammation and destruction of bone and articular cartilage, with some patients suffering permanent disability [1]. The two components of joint damage appear to be consequences of pathogenic processes: (1) Bone erosion is mediated primarily by osteoclasts, which are activated by pro-inflammatory cytokines, and their destructive action is not counterbalanced by bone formation [2,3]; and (2) breakdown of the cartilage matrix is mediated by matrix degradation and inhibition of matrix synthesis induced by cytokines [4,5].
Pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6 play key roles in the pathogenesis of RA. These cytokines are abundant in the synovial fluid of affected joints, and anti-cytokine therapy greatly improves the signs and symptoms of RA [6][7][8] and prevents progression of joint destruction as assessed radio graphically [9][10][11]. Radiographic assessment of joint damage using the modified Sharp score is mainly restricted to small joints in the hands and feet; however, because radiographic damage in large weight-bearing joints, such as the hips, knees, and ankles, is strongly associated with walking disability and is an important determinant of functional capacity in patients with RA [6,7], assessment of the extent of radiographic damage in these large joints is essential. Seki et al. [12] reported that in all hip and knee joints with moderate to advanced pre-existing damage radiographic progression continued even if patients received TNF-α blockade therapy.
Intra-articular injection of Hyaluronate (HA) is now widely used in the treatment of Osteoarthritis (OA). The main mechanism through which HA prevents cartilage destruction and disease progression is thought to be its action in maintaining the viscoelastic properties of the synovial fluid [13]. In addition to this, we and others have reported that HA down-regulates the expressions of MMP (Matrix Metalloproteinase) and ADAMTS (A Disintegrin And Metalloproteinase With Thrombospondin Motifs), which are involved in the degradation of cartilage, and RANKL (Receptor Activator of NF-κBLigand), which is related to osteoclastogenesis, induced in synovial fibroblasts and chondrocytes by pro-inflammatory cytokines [14][15][16]. Moreover, intraarticular injection of HA is approved in Japan to reduce knee-joint pain in RA patients [17].
From these lines of evidence, we hypothesized that HA used concomitantly with a cytokine inhibitor may be beneficial for preventing joint destruction in weight-bearing joints of RA patients. In order to prove this hypothesis, we examined the combination effect of cytokine inhibitors and HA on expression of MMPs, ADAMTS, and RANKL induced in synovial fibroblasts and chondrocytes by synovial fluids of RA patients (RA-SF).

Synovial fluids of RA patients
Synovial fluids of RA patients (RA-SF) were obtained from ProteoGenex (Culver City, CA, USA). A total of 5 patients with RA were recruited for the study. Prior to participation, written informed consent was obtained from all subjects. All studies were performed in accordance with the Declaration of Helsinki.
Cytokine concentrations of RA-SF were measured by Procarta Cytokine Assay Kit (Affymetrix, Santa Clara, CA, USA). Soluble IL-6 receptor concentration was measured by human IL-6 receptor alpha Quantikine ELISA Kit (R&D Systems).

Cell culture
Human fibroblast-like synoviocytes from RA patients (RA-FLS) were purchased from Cell Applications (San Diego, CA, USA). RA-FLS were cultured using synoviocyte growth medium (Cell Applications). Human articular chondrocytes were purchased from Cell Applications and maintained in chondrocyte growth medium (Cell Applications).

Quantitative real-time PCR
Total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA, and USA). Synthesis of cDNA was performed using an OmniscriptRT kit (Qiagen) with random 9-mer primers (TaKaRa, Shiga, Japan) according to the manufacturer's protocol. Quantitative real-time PCR was performed by running a TaqMan gene expression assay (Applied Biosystems, Foster City, CA, USA), targeting human RANKL, MMP-3, ADAMTS-4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), on an ABI PRISM 7500 system (Applied Biosystems) according to the manufacturer's protocol.

Statistical analysis
Statistical significances were estimated by the unpaired t-test and Dunnett's multiple comparison test using a software package (SAS Institute Japan, Tokyo, Japan), with the significance level set to 5%.

Effect of cytokine inhibitors and HA
We examined the impact of the cytokine inhibitors on the RA-SFinduced RANKL, MMP-3, and ADAMTS-4 expressions in RA-FLS and chondrocytes. The inhibitors of IL-6 (TCZ), TNF-α (ETN), and IL-1 (IL-1Ra) suppressed RA-SF-induced RANKL and MMP-3 expressions in RA-FLS, and suppressed MMP-3 and ADAMTS-4 expressions in chondrocytes in a dose-dependent manner ( Figure 2). With each inhibitor, the inhibitory effect plateaued at 100 µg/mL. We then measured the concentrations of sIL-6R, IL-6, TNF-α, and IL-1β in RA-SF. We detected sIL-6R, IL-6, and TNF-α in all five samples, but IL-1β was below the limit of detection in two of the five samples (limit of detection: 15.8 pg/mL) ( Table 1)

Discussion
In the present study, we examined the effects of RA-SF on mRNA expression of MMP-3, ADAMTS-4, and RANKL in RA-FLS and μg/mL μg/mL inhibitor 0 10 100 300 Chondrocyte   TCZ  ETN  IL-1Ra   TCZ  ETN  IL-1Ra TCZ ETN IL-1Ra Joint damage such as bone destruction and cartilage degeneration is a major contributor to disability in RA patients [19]. In clinical trials, joint damage is routinely assessed using validated radiographic scores [20][21][22]. In the Sharp score and its modifications, bone destruction is reflected in the erosion score and cartilage degeneration is reflected in the joint-space narrowing score [22][23][24]. MMP and ADAMTS are enzymes which cleave cartilage matrix, so overproduction of these enzymes induces cartilage degeneration [25]. On the other hand, RANKL is an essential factor for osteoclastogenesis [26,27], and upregulation leads to abnormal bone resorption. Therefore, we chose to examine MMP-3, ADAMTS-4, and RANKL as joint-damage-related molecules. In this study, we measured mRNA expression of these molecules, rather than the proteins themselves, because large amounts of these proteins are already present in RA-SF.  cytokines exist in RA-SF, we focused on IL-1β, IL-6, and TNF-α because their inhibitors are widely used in the treatment of RA patients. As shown in Table 1, IL-6, TNF-α, and IL-1β (in part) are detectable in RA-SF. All cytokine inhibitors showed significant inhibitory effect on RA-SF-induced MMP-3, ADAMTS-4, and RANKL mRNA expression, but none achieved complete inhibition even if the concentration of inhibitors was increased. These results suggest that many cytokines play a role in the mRNA expression of MMP-3, ADAMTS-4, and RANKL.

RA-FLS
We also showed that HA suppressed RA-SF-induced MMP-3, ADAMTS-4, and RANKL expression in RA-FLS and chondrocytes. These are reasonable results, because we and others have reported that HA inhibits IL-1β-or IL-6-induced production of MMP and ADAMTS in RA-FLS and chondrocytes [15,16]. Although the inhibitory mechanism of HA is not fully understood, it is reported that the binding of HA to CD44 or ICAM-1 is essential for the induction of the inhibitory effect [28,29], and we and others have shown that HA induces MKP-1, a negative regulator of MAPK, and IRAK-M, a negative regulator of toll-like receptor signaling, and inhibited cytokine induced signal transductions [15,16]. HA is a major component of synovial fluid and cartilage matrix, and it is responsible for the viscoelastic qualities of synovial fluid, acting as both a lubricant and shock absorber. However, in joints affected by OA and RA, high-molecular-weight HA is cleaved into low-molecularweight HA which has low viscoelasticity [13]. Therefore, intra-articular injection of HA makes up for the loss of viscoelasticity of synovial fluid and protects against degradation of cartilage.
Each cytokine inhibitor partially suppressed RA-SF-induced expression of MMP-3, ADAMTS-4, and RANKL. These results lead to the idea that a combination of cytokine inhibitors might more potently inhibit the expression of these molecules. However, therapy with a combination of cytokine inhibitors is not recommended because of the strong immune-suppressing effect and high cost. HA on the other hand is very safe and low-cost, and is approved in Japan for therapy to reduce knee-joint pain in RA patients; therefore, we examined the combination effect of HA and a cytokine inhibitor.
To improve the quality of life of RA patients it is important to prevent joint destruction. Seki et al. [12] indicated the possibility that large joints are more resistant to the effects of cytokine inhibitors than are small joints. Reasons for this may be that larger amounts of drug are necessary to suppress joint destruction in large joints than are necessary in small joints or that weight bearing prevents the beneficial effects of the cytokine inhibitor. We demonstrated here that the combination of HA and a cytokine inhibitor more potently suppressed the expression of MMP-3, ADAMT-4, and RANKL than did cytokine inhibitor alone.
In conclusion, the combination of HA and cytokine inhibitor may be beneficial for the therapy of large joints of RA patients because the combination more effectively suppresses the expression of joint-damage-related molecules and HA makes up for the lost viscoelasticity of the synovial fluid.