Modulation of the response of rheumatoid arthritis synovial fibroblasts to proinflammatory stimulants with cyclic tensile strain

Cytokine 51 (2010) 35–41

Contents lists available at ScienceDirect

Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

Modulation of the response of rheumatoid arthritis synovial fibroblasts to proinflammatory stimulants with cyclic tensile strain R.A. Bader *, K.L. Wagoner Department of Biomedical and Chemical Engineering, 121 Link Hall, Syracuse University, Syracuse, NY 13244, USA Syracuse Biomaterials Institute, Syracuse University, Syracuse, NY, USA

a r t i c l e

i n f o

Article history: Received 7 August 2009 Received in revised form 5 February 2010 Accepted 30 March 2010

Keywords: Rheumatoid arthritis Synovial fibroblasts MH7A Mechanical stimulation Tensile strain

a b s t r a c t Although physical therapy has been shown to be an effective method for treatment of rheumatoid arthritis, a thorough investigation on the impact of mechanical signals upon the complex cytokine network associated with pathogenesis has not yet been conducted. In the current study, our research group investigated the effect of mechanical stimulation on primary and immortalized rheumatoid arthritis synovial fibroblasts (RASFs) through analysis of secreted proteins using multiplex immunoassay. Equibiaxial tensile strain was applied to 2D cultures grown on collagen-coated, flexible silicone membranes at a magnitude of 10% and a frequency of 0.5 Hz using the Flexcell System. After 24 h, supernatant was removed and assayed for the following cytokines: IL-1b, IL-6, IL-8, VEGF, FGF-2, GM-CSF, MCP-1, RANTES, TNF-a. The results were compared to unstimulated control groups. Mechanical stimulation alone only impacted secretion of IL-8 by primary RASFs. However, in the presence of proinflammatory mediators (TNF-a or IL-17), application of cyclic tensile strain increased secretion of a number of proteins by both primary and immortalized RASFs, although the responses were not analogous. In contrast, MCP-1 secretion was decreased when mechanical stimulation was applied in combination with IL-17 to primary cultures. In general, the study suggests that cyclic tensile strain can be used to modulate the effects of proinflammatory stimulants on RASFs; however, given the highly variable results, more research will be necessary to identify the pathways that are implicated in mechanotransduction. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction An imbalance in pro- and anti-inflammatory cytokines released by synovial fibroblasts, mononuclear leukocytes, and lymphocytes has been implicated in the joint inflammation and cartilage degradation that characterizes rheumatoid arthritis (Fig. 1) [1–3]. Although a number of cell types are active participants in the pathogenesis of rheumatoid arthritis, activated rheumatoid arthritis synovial fibroblasts (RASFs) have emerged as the conductors of joint destruction. Even in the absence of inflammatory cells and

Abbreviations: ADR, above detectable range; BDR, below detectable range; FGF2, basic fibroblast growth factor; GM-CSF, granulocyte–macrophage colonystimulation factor; IL-1b, interleukin-1 beta; IL-6, interleukin-6; IL-8, interleukin8; MCP-1, monocyte chemotactic protein-1; MMP, matrix metalloproteinase; NFjB, nuclear factor-jB; NSF, normal synovial fibroblast; RANTES, regulated upon activation, normal T-cell expressed, and secreted; RASF, rheumatoid arthritis synovial fibroblast; TIMP, tissue inhibitor of metalloproteinase; TNF-a, tissue necrosis factor-alpha; VEGF, vascular endothelial growth factor. * Corresponding author at: Department of Biomedical and Chemical Engineering, 121 Link Hall, Syracuse University, Syracuse, NY 13244, USA. Tel.: +1 (315) 443 9709; fax: +1 (315) 443 9175. E-mail address: [email protected] (R.A. Bader). 1043-4666/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2010.03.015

mediators, RASFs retain the ability to aggressively invade cartilage, seemingly through the continued upregulation and expression of adhesion molecules and matrix degradative enzymes, particularly matrix metalloproteinases (MMPs) and cysteine proteases [4–7]. Additionally, RASFs release proinflammatory cytokines, such as interleukin-1b (IL-b), tissue necrosis factor-a (TNF-a), and interleuking-6 (IL-6) that promote the immune response; growth factors, such as transforming growth factor (TGF-b), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2), and granulocyte macrophage colony stimulating factor (GM-CSF) that support angiogenesis; and chemokines, for example interleukin-8 (IL-8), macrophage inflammatory protein-1 (MIP-1), monocyte chemoattractant protein-1 (MCP-1), and regulated upon activation, normal T-cell expressed, and secreted (RANTES) that attract leukocytes to the inflamed joint [8]. Despite the prominent role played by RASFs in joint inflammation, few studies have been conducted thus far to elucidate the mechanosensitive nature of the cells and, consequently, the contribution of biomechanical signals to pathogenesis remains largely unknown. Clinical and experimental evidence points towards the importance of the biomechanical environment in rheumatoid arthritis pathogenesis. Physical therapy is often used to reduce inflamma-

36

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

Fig. 1. Representation of a rheumatic joint. An imbalance in the cytokines secreted by inflammatory effector cells is associated with the symptoms that characterize rheumatoid arthritis.

tion and improve mobility in patients with rheumatoid arthritis [9]. The duration of manual treatment ranges from several days for continuous passive motion to several hours for assisted or voluntary exercise [10]. In concurrence with the results from clinical studies, continuous passive motion has been correlated with significantly less articular degradation relative to immobilization in animal models of arthritis [3]. Accordingly, the latter treatment has been shown to reduce glycosaminoglycan degradation, suppress expression of proinflammatory mediators, and induce expression of anti-inflammatory interleukin-10 (IL-10) within the meniscal fibrocartilage of rabbits with antigen induced arthritis [11]. Despite the evidence that the progression of rheumatoid arthritis can be altered by mechanical stimuli, a thorough investigation on the response of rheumatoid arthritis synovial fibroblasts to stress and strain has not yet been conducted. In contrast, the affect of various loading modes upon chondrocytes, another cell type of importance associated with rheumatoid arthritis, has been widely studied [12–15]. Most notably, IL-1b activated chondrocytes subjected to cyclic tensile strain at a low physiological frequency (3% strain at 0.25 Hz) showed a sustained reduction in the expression of inflammatory genes, including cyclooxygenase, inducible nitric oxide synthase, and MMPs [14]. Further study demonstrated that cyclic tensile strain regulated multiple points within the NF-jB (nuclear factor kappa-lightchain-enhancer of activated B cells) signaling cascade [16–19]. NF-jB transcription factors play an integral role in immune response through the regulation of genes for chemokines/cytokines, adhesion molecules, and cell proliferation. Upon treatment with cyclic tensile strain, nuclear translocation of NF-kB was reduced and degradation of the inhibitors of NF-kB was abrogated [16– 19]. Dynamic compression of chondrocytes at 15% strain, 1 Hz embedded within an agarose construct, with or without an anti-

inflammatory additive, also was shown to produce an anti-inflammatory response [12]. Given the knowledge that chondrocytes are strongly impacted by biomechanical factors, the influence of mechanical stimuli on other key cell types involved in rheumatoid arthritis needs to be researched. Although an in depth investigation has not yet been conducted, several studies have implied that gene expression and protein secretion by synovial fibroblasts are impacted by mechanical stimulation in a manner analogous to that of chondrocytes. Transcription of MMPs by SV40 T antigen immortalized RASFs has been shown to be controlled by shear stress in a magnitude dependent manner. Whereas gentle shear stress resulted in a reduction of mRNA levels, intermediate shear stress stimulated MMP transcription. The latter study also demonstrated that transcription of tissue inhibitors of metalloproteinases (TIMPs), inhibitors of MMPs, is elevated in response to increased shear stress [20]. Similar results were obtained for immortalized synovial fibroblasts exposed to proinflammatory cytokines and subjected to a 2% compressive strain at 0.7 Hz when cultured on collagen-coated agar [20]. In contrast to the above results which imply that the proinflammatory response of synovial fibroblasts can be reduced through the appropriate application of mechanical strain, one study found that NF-kB was activated, rather than inhibited, in response to tensile strain [21]. However, the meaningfulness of the results is questionable given that the strain level was set at 120%. A vast amount of research remains to be conducted on the response of synovial fibroblasts to mechanical stimulation, particularly as pertains to the impact upon the cytokine network. In this study, the influence of cyclic tensile strain on the secretion of cytokines, growth factors, and chemokines by rheumatoid arthritis synovial fibroblast, with and without the addition of proinflammatory co-stimulants (TNF-a and IL-17), was evaluated by

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

multiplex immunoassay. For comparison, the response of both primary and immortalized RASFs was determined. TNF-a and IL-17 were chosen as co-stimulants in an effort to mimic paracrine effects mediated by macrophages and T-cells, respectively. The cells were exposed to dynamic equibiaxial tensile strain at a magnitude of 10% and a physiologically relevant frequency of 0.5 Hz. The chosen frequency is consistent with jogging or fast walking [22]. The results provide new insight into the mechanism by which biomechanical signals modulate the symptoms of rheumatoid arthritis. 2. Materials and methods

37

Stop™ rubber stopper (Flexcell International, Hillsborough, NC) was inserted into three of the wells of each plate to prevent deformation of the elastic membrane. After 24 h, the program was halted, and supernatant was removed and stored in aliquots at 80 °C until analysis. RASFs are intrinsically activated at low passage numbers [24]; however, in an effort to mimic the paracrine effects of nearby cells, experiments were also conducted whereby the cells were subjected to stimulation by either TNF-a or IL-17 (R&D Systems, Minneapolis, MN) at a concentration of 1 ng/ml. A literature survey indicated that the chosen concentration induces a significant response from synovial fibroblasts for both proinflammatory cytokines [25,26].

2.1. Isolation of synovial fibroblasts 2.4. Live/Dead cell viability assay Three different lots of primary rheumatoid arthritis synovial fibroblasts (RASFs) were isolated from the synovial tissue of Caucasian females between the ages of 60 and 68 receiving total hip or knee replacement for rheumatoid arthritis. The tissue samples were collected by Dr. Michael Clarke at Community General Hospital (Syracuse, NY) following a protocol approved by the Institutional Review Board. Cells were isolated following the procedure outlined by Zimmermann et al. [23]. In brief, the synovial tissue was diced into small pieces and digested in 1X phosphate-buffered-saline (PBS) containing 0.1% trypsin (Invitrogen, Carlsbad, CA) for 30 min at 37 °C, 5% CO2. The PBS solution was removed and digestion was continued overnight at 37 °C, 5% CO2 with 0.1% collagenase P (Roche, Indianapolis, IN) in RPMI 1640 supplemented with 10% fetal bovine serum (Lonza, Inc., Allendale, NJ). The digested tissue was filtered through a cell strainer with a 100 lm nylon mesh to obtain a cell suspension. The cells were collected by centrifugation, seeded into T-75 plates, and cultured in RPMI 1640 supplemented with 10% FBS at 37 °C, 5% CO2. After three passages, isolation of synovial fibroblasts was confirmed using direct fluorescent antibody staining with CD44-FITC mAB and CD14-Alexa FluorÒ 647 mAb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Both synovial fibroblasts and macrophages exhibit positive CD44 staining; however, in regards to CD14, synovial fibroblasts are negative, while synovial macrophages are positive. 2.2. Synovial fibroblast cell cultures The primary and immortalized RASFs were cultured in RPMI 1640 supplemented with HEPES, L-glutamine, penicillin–streptomycin, and 10% fetal bovine serum (Lonza, Inc., Allendale, NJ) at 37 °C, 5% CO2. Immortalized RASFs, the MH7A cell line, were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. 2.3. Mechanical stimulation of synovial fibroblasts At passage 3 or 4, primary and immortalized RASFs were seeded onto collagen-coated 6-well BioFlexÒ culture plates (Flexcell International, Hillsborough, NC), which possess flexible silicone elastomer bottoms, at a concentration of 7  104 cells/well. Cells were grown for 4–5 days such that the cells were 70–80% confluent. The media was replaced, and the cells were subjected to dynamic equibiaxial tensile strain at a magnitude of 10% and a frequency of 0.5 Hz (sinusoidal waveform) in the FlexCellÒ Tension Plus™ System (Flexcell International, Hillsborough, NC). Within the latter system, the flexible membranes of the culture plates are stretched over plastic loading posts when negative pressure is applied. The average strain value is calculated from the applied pressure and a manufacturer supplied calibration factor. Stretch and release is controlled by a computer program. For unloaded controls, a Flex-

A LIVE/DEADÒ viability/cytotoxicity kit (Invitrogen, Carlsbad, CA) was used after the application of mechanical stimulation and removal of supernatant to confirm that the primary and immortalized RASFs remained viable and adherent. Live cells fluoresce green as a result of the conversion of calcein-AM to calcein upon uptake, while dead cells fluoresce red after ethidium homodimer enters the nucleus and binds to nucleic acid. The kit was used following manufacturer specifications. 2.5. Multiplex immunoassay Cytokine profiles were obtained using a Milliplex Map human cytokine panel (Millipore, Billerica, MA) and a Luminex 200 system (Luminex, Austin, TX) following a manufacturer provided protocol. With the latter bead-based assay, capture antibodies are conjugated to beads that have been dyed with red and infrared fluorophores. Through variation of fluorophore intensity, 100 separate microspheres are generated with unique identities. Following cytokine capture, a fluorescently tagged detection antibody is added. Each bead is separately identified and quantified within the Luminex platform using a flow cytometry system with a dual laser detector. Concentrations were determined based upon a standard curve generated from dilution of a recombinant cytokine standard. The cytokines investigated, as well as the detectable ranges, were as follows: IL-1b (6.1–10,000 pg/ml), IL-6 (8.1–10,000 pg/ml), IL-8 (7.1– 10,000 pg/ml), MCP-1 (6.1–10,000 pg/ml), RANTES (6.2–10,000 pg/ ml), GM-CSF (10.4–10,000 pg/ml), TNF-a (10.5–10,000 pg/ml), VEGF (5.8–10,000 pg/ml), and FGF-2 (7.5–10,000 pg/ml). 2.6. Statistical analysis In general, cytokine concentrations were expressed relative to those obtained from unstimulated control cells. Absolute values were given when the control cells secreted cytokines at concentrations below the detectable range. All data is presented as mean plus or minus standard error (N = 3). Unpaired t-tests were used to compare cytokine concentrations from cell cultures stimulated with TNF-a or IL-17 to cells not stimulated with a proinflammatory mediator subjected to the same loading regime, as well as mechanically stimulated versus stationary cells for cells exposed to the same co-stimulant. 3. Results 3.1. Live/Dead viability assay Live/Dead assay verified that primary and immortalized RASFs remained adherent and viable following the application of cyclic tensile strain. Staining of live cells with calcein-AM also provided a method to observe morphological differences between the pri-

38

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

Fig. 2. After the application of cyclic tensile strain, Live/Dead assay was used to ensure the viability of (A) primary RASFs, and (B) immortalized RASFs. The primary and immortalized RASFs are morphologically distinct.

mary and immortalized cells. The immortalized cells were smaller in size and less organized relative to the primary cells (Fig. 2). The morphological observations are consistent with previous studies on immortalized RASFs [27,28]. 3.2. Effect of cyclic tensile strain and proinflammatory mediators on protein secretion by primary RASFs Cyclic tensile strain alone at a frequency of 0.5 Hz and a magnitude of 10% significantly increased the secretion of only IL-8 by primary RASFs. The addition of TNF-a, in the absence of mechanical stimulation, significantly increased secretion of MCP-1 and IL-6. Application of cyclic tensile strain had no further impact on secretion of the latter cytokines (Fig. 3). GM-CSF secretion was also increased to detectable levels following TNF-a stimulation (not shown). In contrast, VEGF secretion was only significantly increased by the combination of cyclic tensile strain and TNF-a. Analogously, although IL-17 alone elicited no change in the secretion of any of the cytokines investigated, IL-17 in conjunction with mechanical stimulation led to a significant increase in IL-8 and a decrease in MCP-1 secretion. The impact of stimulants on RANTES secretion was highly variable and no significant differences between stimulated and unstimulated cells were found. The effect of TNF-a on IL-8 secretion, both with and without cyclic tensile strain, could not be assessed because the values were above the detectable range (Fig. 3). IL-1b was not detected under any culture conditions, and TNF-a levels were only within the detectable range when TNF-a was incorporated into the media (not shown). 3.3. Effect of cyclic tensile strain and proinflammatory mediators on protein secretion by immortalized RASFs The effect of cyclic tensile strain and proinflammatory mediators, both alone and together, on immortalized RASFs, the MH7A cell line, differed from that of primary RASFs. Cyclic tensile strain alone did not affect the secretion of any of the cytokines. IL-8 concentrations remained within the detectable range and secretion was significantly increased upon the addition of TNF-a. Mechanical stimulation with TNF-a significantly increased IL-8 secretion relative to TNF-a alone. TNF-a also significantly increased secretion of IL-6 and GM-CSF, but cyclic tensile strain had no further impact (Fig. 4). TNF-a induced the secretion of VEGF, as indicated by a significant increase in concentration from below the detectable range to over 100 pg/ml upon stimulation. VEGF secretion was also significantly increased by the combination of IL-17 with mechanical stimulation (Fig. 5). With the latter exception, IL-17 did not elicit a cellular response from the immortalized cells regardless of mechanical stimulation (Fig. 4). There was also a complete lack

of detectable MCP-1, regardless of the culture conditions (not shown), in addition to a significant rise in FGF-2 secretion relative to primary cultures (Fig. 4). As with primary cultures, immortalized RASFs did not secrete detectable levels of IL-1b, and TNF-a was only observed when included in the media. 4. Discussion The cytokines observed in the current study, IL-1b, TNF-a, and IL-6, are known to be paramount to the pathogenesis of rheumatoid arthritis. IL-1b and TNF-a induce lymphocytes, macrophages, and synovial fibroblasts to release additional proinflammatory cytokines, as well as MMPs that degrade underlying cartilage [2]. TNF-a is the focus of many of the ‘‘biologic” therapies currently in use due to an ability to affect the secretion of other cytokines, including IL-1b [2]. In this study, the lack of detectable amounts of IL-1b and TNF-a is consistent with their production primarily by synovial macrophages, rather than synovial fibroblast. IL-6 is also an effector cytokine for cellular activation, particularly for T and B cells [2]. Additionally, IL-6 has been shown to induce expression of VEGF, thereby facilitating angiogenesis, another critical component of rheumatoid arthritis pathogenesis [29]. In the current study, although IL-6 secretion was increased upon the addition of TNF-a to both primary and immortalized RASF cultures, application of cyclic tensile strain had no effect. Whether mechanical stimulation has an impact upon secretion of IL-6 has been shown to be cell type dependent. However, based upon the increased secretion of IL-6 by osteoarthritic chondrocytes subjected to shear [30] and by human tendon fibroblasts exposed to biaxial stretch [31], an effect on the secretion of IL-6 by RASFs subjected to cyclic tensile strain was anticipated. A more pronounced response in regards to cytokine secretion may be observed at a different magnitude and/or frequency of tensile loading. Growth factors VEGF, FGF-2, and GM-CSF play a role in neovascularization of the synovial tissue [32]. Proinflammatory cytokines, particularly TNF-a and IL-1b, in conjunction with the hypoxic environment of the synovium, stimulate the release of VEGF, which acts as a mitogen for endothelial cells and increases microvascular permeability [33]. GM-CSF and FGF-2 also act upon endothelial cells to trigger angiogenesis [32]. GM-CSF plays an additional role in chemoattracting neutrophils and regulating the growth and differentiation of granulocyte/macrophage precursors [33]. In the present study, GM-CSF was only detected upon stimulation of primary and immortalized synovial fibroblasts with TNF-a, but mechanical stimulation had no additional effect (results not shown). FGF-2 secretion by primary cells was also below the detectable range, regardless of stimulation. Immortalized cells, however, secreted FGF-2 under all culture conditions. TNF-a in-

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

39

Fig. 4. Effect of cyclic tensile strain and proinflammatory co-stimulants on protein secretion by immortalized RASFs. Cytokine concentrations are expressed relative to those obtained from unstimulated control cells. All data is presented as mean ± SE (N = 3). *Indicates a significant difference of p < 0.05 versus stationary cell cultures that received the same proinflammatory co-stimulant.  Indicates a significant difference of p < 0.05 versus cells subjected to the same loading condition that did not receive a proinflammatory co-stimulant.

Fig. 3. Effect of cyclic tensile strain and proinflammatory co-stimulants on protein secretion by primary RASFs. Cytokine concentrations are expressed relative to those obtained from unstimulated control cells. All data is presented as mean ± SE (N = 3). ADR indicates that the concentrations were above the detectable range. *Indicates a significant difference of p < 0.05 versus stationary cell cultures that received the same proinflammatory co-stimulant. **Indicates a significant difference of p < 0.1 versus stationary cell cultures that received the same proinflammatory costimulant.  Indicates a significant difference of p < 0.05 versus cells subjected to the same loading condition that did not receive a proinflammatory co-stimulant. à Indicates a significant difference of p < 0.1 versus cells subjected to the same loading condition that did not receive a proinflammatory co-stimulant.

creased FGF-2 secretion by the immortalized RASFs and there was a trend towards increased FGF-2 secretion upon co-stimulation with mechanical strain and TNF-a or IL-17, but the differences

were not significant. VEGF secretion by primary cells was not altered by proinflammatory mediators or cyclic tensile strain alone, but a significant rise was detected when cells were stimulated with TNF-a in combination with strain. Therefore, mechanical stimulation at the frequency and magnitude chosen may augment the angiogenesis that partially characterizes rheumatoid arthritis. The response of the immortalized RASFs was not analogous. TNF-a significantly increased VEGF secretion; however, mechanical stimulation only had an effect in combination with IL17. The different response of the immortalized cells in regards to growth factor secretion as compared to primary cells suggest that the MH7A cell line may not be appropriate for studying the role of mechanotransduction in rheumatoid arthritis pathogenesis. IL-8, RANTES, and MCP-1 are all involved with recruiting lymphocytes that further promote the inflammatory response. RANTES

40

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

Fig. 5. Effect of cyclic tensile strain and proinflammatory co-stimulants on VEGF secretion by immortalized RASFs. Relative concentrations could not be determined; therefore, absolute concentrations are given. All data is presented as mean ± SE (N = 3). BDR indicates that the concentrations were below the detectable range. *Indicates a significant difference of p < 0.05 versus stationary cell cultures that received the same proinflammatory co-stimulant.  Indicates a significant difference of p < 0.05 versus cells subjected to the same loading condition that did not receive a proinflammatory co-stimulant.

and MCP-1 both belong to the CC subfamily of chemokines and stimulate chemotaxis of a variety of cell types involved with chronic inflammation, including monocytes and T-cells [34]. In clinical studies, elevated RANTES and MCP-1 levels have been found in both the serum and synovial fluid of patients with rheumatoid arthritis [35]. CXC chemokines, such as IL-8, stimulate chemotaxis of cells associated with acute inflammation, particularly neutrophils. IL-8 has also been found to play a role in the promotion of angiogenesis and the activation of immune cells. Both types of chemokines, CC, and CXC, have been shown to activate synovial fibroblasts in an autocrine manner to increase production of IL-6 and IL-8 [34]. RANTES secretion was highly variable between primary cells isolated from different patients and, consequently, significant differences could not be found between unstimulated and stimulated cells. IL-8 was the only cytokine whose secretion from primary cells was directly altered by the application of cyclic tensile strain, without the addition of proinflammatory mediators. The result lends support to the notion that mechanical stimulation activates the NF-jB pathway in synovial fibroblasts. NF-jB binding to the IL-8 promoter has been found to be essential for IL-8 gene activation [36]. In the current study, the connection between NF-kB activation and IL-8 expression was also observed through stimulation of the cells with TNF-a, which resulted in an increase in IL-8 secretion by primary cells to levels that were above the detectable range. In immortalized cells, mechanical stimulation alone did not significantly alter the secretion of IL-8, and TNF-a stimulation increased IL-8 secretion to a lesser degree than in primary cells. Previous studies have shown that immortalized synovial fibroblasts possess an altered phenotype whereby NF-jB is constitutively activated [27]. Therefore, responses that are dependent upon the NF-kB pathway may be less apparent in immortalized than primary cells. MCP-1 secretion by primary cells was increased by TNF-a, but the addition of tensile strain did not further alter secretion. Stimulation with IL-17 did not change the secretion of MCP-1 by primary cells; however, MCP-1 secretion was reduced by the combination of IL-17 and cyclic tensile strain. The MCP-1 reduction suggests that a level of cyclic tensile strain may be found that can lead to a general reduction in inflammation. Given that MCP-1 levels are decreased when cyclic tensile strain is applied in combination with IL-17, while the levels of other inflammatory proteins are increased, indicates that mechanical stimulation may be acting upon multiple signaling pathways. The immortalized RASFs did not secrete MCP-1, providing further evidence that their use in the study of rheumatoid arthritis pathogenesis may not be appropriate.

In general, the study presented herein indicates that cyclic tensile strain can be used to modulate the effects of proinflammatory stimulants on RASFs or vice versa. Similar phenomena have been observed with other cell types. For example, cyclic tensile strain was found to abrogate the inflammatory effects of IL-1b and TNF-a on chondrocytes, while cyclic tensile strain alone had little impact [15,37]. Likewise, mechanical stimulation was found to mitigate the effect of TNF-a on myoblast-like cells; however, these cells also responded to strain alone in a magnitude dependent manner [38]. The impact of cyclic tensile strain on RASFs was highly dependent upon the presence of a proinflammatory mediator and upon the cytokine, growth factor, or chemokine observed. The variable results suggest that multiple pathways are implicated in mechanotransduction in RASFs. Future studies on the role of biomechanical signals in rheumatoid arthritis pathogenesis will focus on identifying changes in associated transcription factors, including NF-kB, NF-AT, STAT, and AP-1 [39,40]. This study also demonstrated that, despite previous publications asserting that the MH7A cell line thoroughly mimics primary RASFs [28,41,42], immortalized RASFs may not be appropriate for the investigation of rheumatoid arthritis pathogenesis, particularly in regards to the effects of mechanical stimulation. Acknowledgements The authors would like to thank Andrew Boucher for assistance in cell culture and for helping to set up our lab. References [1] Firestein GS. Evolving concepts of rheumatoid arthritis. Nature 2003;423(6937):356–61. [2] McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007;7(6):429–42. [3] Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned, future directions. Nat Rev Drug Discov 2007;6(1):17. [4] Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T. Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford) 2006;45(6):669–75. [5] Kontoyiannis D, Kollias G. Fibroblast biology. Synovial fibroblasts in rheumatoid arthritis: leading role or chorus line? Arthritis Res 2000;2(5): 342–3. [6] Muller-Ladner U, Ospelt C, Gay S, Distler O, Pap T. Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts. Arthritis Res Ther 2007;9:223 (6). [7] Pap T, Muller-Ladner U, Gay RE, Gay S. Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res 2000;2(5):361–7. [8] Ritchlin C. Fibroblast biology. Effector signals released by the synovial fibroblast in arthritis. Arthritis Res 2000;2(5):356–60. [9] Lundberg IE, Nader GA. Molecular effects of exercise in patients with inflammatory rheumatic disease. Nat Clin Pract Rheumatol 2008;4(11): 597–604. [10] Rannou F, Poiraudeau S, Revel M. Cartilage: from biomechanics to physical therapy. Ann Readapt Med Phys 2001;44(5):259–67. [11] Ferretti M, Srinivasan A, Deschner J, Gassner R, Baliko F, Piesco N, et al. Antiinflammatory effects of continuous passive motion on meniscal fibrocartilage. J Orthop Res 2005;23(5):1165–71. [12] Chowdhury TT, Bader DL, Lee DA. Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes. Biochem Biophys Res Commun 2006;339(1):241–7. [13] Fermor B, Jeffcoat D, Hennerbichler A, Pisetsky DS, Weinberg JB, Guilak F. The effects of cyclic mechanical strain and tumor necrosis factor alpha on the response of cells of the meniscus. Osteoarthr Cartilage 2004;12(12):956–62. [14] Madhavan S, Anghelina M, Rath-Deschner B, Wypasek E, John A, Deschner J, et al. Biomechanical signals exert sustained attenuation of proinflammatory gene induction in articular chondrocytes. Osteoarthr Cartilage 2006;14(10): 1023–32. [15] Xu Z, Buckley MJ, Evans CH, Agarwal S. Cyclic tensile strain acts as an antagonist of IL-1 beta actions in chondrocytes. J Immunol 2000;165(1): 453–60. [16] Agarwal S, Deschner J, Long P, Verma A, Hofman C, Evans CH, et al. Role of NFkappaB transcription factors in antiinflammatory and proinflammatory actions of mechanical signals. Arthritis Rheum 2004;50(11):3541–8. [17] Agarwal S, Long P, Seyedain A, Piesco N, Shree A, Gassner R. A central role for the nuclear factor-kappaB pathway in anti-inflammatory and proinflammatory actions of mechanical strain. FASEB J 2003;17(8):899–901. [18] Dossumbekova A, Anghelina M, Madhavan S, He L, Quan N, Knobloch T, et al. Biomechanical signals inhibit IKK activity to attenuate NF-kappaB

R.A. Bader, K.L. Wagoner / Cytokine 51 (2010) 35–41

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

transcription activity in inflamed chondrocytes. Arthritis Rheum 2007;56(10):3284–96. Chowdhury TT, Salter DM, Bader DL, Lee DA. Signal transduction pathways involving p38 MAPK, JNK, NFkappaB and AP-1 influences the response of chondrocytes cultured in agarose constructs to IL-1beta and dynamic compression. Inflamm Res 2008;57(7):306–13. Sun HB, Yokota H. Messenger-RNA expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and transcription factors in rheumatic synovial cells under mechanical stimuli. Bone 2001;28(3):303–9. Inoh H, Ishiguro N, Sawazaki S, Amma H, Miyazu M, Iwata H, et al. Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor kappaB in human fibroblast cells. FASEB J 2002;16(3):405–7. Huang J, Ballou LR, Hasty KA. Cyclic equibiaxial tensile strain induces both anabolic and catabolic responses in articular chondrocytes. Gene 2007; 404(1–2):101–9. Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, et al. Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture–primary culture cells markedly differ from fourth-passage cells. Arthritis Res 2001;3(1):72–6. Hirth A, Skapenko A, Kinne RW, Emmrich F, Schulze-Koops H, Sack U. Cytokine mRNA and protein expression in primary-culture and repeated-passage synovial fibroblasts from patients with rheumatoid arthritis. Arthritis Res 2002;4(2):117–25. Cho ML, Jung YO, Kim KW, Park MK, Oh HJ, Ju JH, et al. IL-17 induces the production of IL-16 in rheumatoid arthritis. Exp Mol Med 2008;40(2): 237–45. Scaife S, Brown R, Kellie S, Filer A, Martin S, Thomas AM, et al. Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display. Rheumatology (Oxford) 2004;43(11):1346–52. Asamitsu K, Sakurada S, Mashiba K, Nakagawa K, Torikai K, Onozaki K, et al. Alteration of the cellular response to interleukin-1 beta by SV40 large T antigen in rheumatoid synovial fibroblasts. Arch Virol 1999;144(2):317–27. Haas C, Aicher WK, Dinkel A, Peter HH, Eibel H. Characterization of SV40T antigen immortalized human synovial fibroblasts: maintained expression patterns of EGR-1, HLA-DR and some surface receptors. Rheumatol Int 1997;16(6):241–7. Nakahara H, Song J, Sugimoto M, Hagihara K, Kishimoto T, Yoshizaki K, et al. Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial

[30]

[31]

[32] [33] [34] [35]

[36] [37]

[38]

[39] [40]

[41]

[42]

41

growth factor production in rheumatoid arthritis. Arthritis Rheum 2003;48(6):1521–9. Mohtai M, Gupta MK, Donlon B, Ellison B, Cooke J, Gibbons G, et al. Expression of interleukin-6 in osteoarthritic chondrocytes and effects of fluid-induced shear on this expression in normal human chondrocytes in vitro. J Orthop Res 1996;14(1):67–73. Skutek M, van Griensven M, Zeichen J, Brauer N, Bosch U. Cyclic mechanical stretching enhances secretion of Interleukin 6 in human tendon fibroblasts. Knee Surg Sports Traumatol Arthrosc 2001;9(5):322–6. Szekanecz Z, Koch AE. Angiogenesis and its targeting in rheumatoid arthritis. Vascul Pharmacol 2009;51(1):1–7. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440. Koch AE. Chemokines and their receptors in rheumatoid arthritis: future targets? Arthritis Rheum 2005;52(3):710–21. Yao TC, Kuo ML, See LC, Ou LS, Lee WI, Chan CK, et al. RANTES and monocyte chemoattractant protein 1 as sensitive markers of disease activity in patients with juvenile rheumatoid arthritis: a six-year longitudinal study. Arthritis Rheum 2006;54(8):2585–93. Kunsch C, Rosen CA. NF-kappa B subunit-specific regulation of the interleukin8 promoter. Mol Cell Biol 1993;13(10):6137–46. Long P, Gassner R, Agarwal S. Tumor necrosis factor alpha-dependent proinflammatory gene induction is inhibited by cyclic tensile strain in articular chondrocytes in vitro. Arthritis Rheum 2001;44(10):2311–9. Chandran R, Knobloch TJ, Anghelina M, Agarwal S. Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation. Am J Physiol Cell Physiol 2007;293(1):C267–76. Iqbal J, Zaidi M. Molecular regulation of mechanotransduction. Biochem Biophys Res Commun 2005;328(3):751–5. Okamoto H, Cujec TP, Yamanaka H, Kamatani N. Molecular aspects of rheumatoid arthritis: role of transcription factors. FEBS J 2008;275(18): 4463–70. Miyazawa K, Mori A, Okudaira H. Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen. J Biochem 1998;124(6):1153–62. Shimozato O, Watanabe N, Goto M, Kobayashi Y. Cytokine production by SV40immortalized adherent synovial cells from rheumatoid arthritis patients. Cytokine 1996;8(1):99–105.