Osteoarthritis (OA) is a painful, chronic degenerative disease which affects the quality of life in both man and animals  . OA is characterized by cartilage breakdown, bone remodeling, osteophyte formation and joint inflammation. Humans and animals such as horses afflicted with OA suffer from debilitating morbidity  . OA in horses accounts for substantial monetary loss in the equine industry. It is estimated that 60% of lameness problems in horses are due to OA  . Approximately one third of 2 and 3-year old thoroughbred racehorses have macroscopic and histologic changes of the metacarpophalangeal joint associated with OA  . Humans and horses have similar joint volume, structure and biomechanical function   . Their articular cartilage also exhibits similar cellular and molecular characteristics   . Because of their common structural, functional and cellular characteristics, horses have been used as an in vivo model for studying human OA. Equine joint tissues such as cartilage and their constituent chondrocytes have also been beneficial as models for in vitro studies      .
Pro-inflammatory mediators including prostaglandins (PG), cytokines, and chemokines play important roles in the pathogenesis of OA  -  . PGE2, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α are well documented mediators of chronic pain and cartilage degradation in OA  -  . More recently, chemokines including MCP-1 (CCL2) and their receptors have been recognized as important players in OA pathogenesis  -  . Stimulation of human chondrocytes with IL-1β or TNF-α results in upregulation of MCP-1 gene expression compared to untreated controls  . In addition, there is evidence that MCP-1 contributes to cartilage matrix degeneration by increasing secretion of matrix metalloproteinase (MMP)-3 by chondrocytes  . MCP-1 as a key mediator of chronic pain has also been reported. Knockout mice with deleted MCP-1 receptor have been used to define this chemokine as a factor in nociception. While acute pain behavior is relatively unchanged in these animals, hyperalgesia is inhibited  . Administration of exogenous MCP-1 is seen to increase firing of dorsal root ganglia neurons, leading to neuronal hyperexcitability and neuropathic pain  . Studies also reinforced the role of MCP-1 as a mediator of osteoarthritic pain  -  . In addition, increased levels of MCP-1 in situ appear to correlate with the severity of the disease. These observations strengthen the possibility that MCP-1 plays a role in chronic pain and hyperalgesia.
Evidence supporting the role of pro-inflammatory molecules such as prostaglandins, cytokines and chemokines in the pathogenesis of OA led to the use of non-steroidal anti-inflammatory drugs (NSAIDs). These drugs help ease the pain and alleviate symptoms in OA patients but do not modify disease progression. Moreover, NSAIDs are associated with deleterious side effects affecting cardiovascular, gastrointestinal, renal and hepatic systems  . NSAIDs that ablate PGE2 production also disrupt bone healing and fracture repair    . Awareness of the adverse effects of NSAIDs has prompted the need to evaluate complementary and alternative agents for the safe management of OA. Non-pharmacologic agents that have been evaluated include glucosamine (GLU), chondroitin sulfate (CS) and avocado/soybean unsaponifiables (ASU) alone or in combination. ASU, GLU and CS have been documented to reduce inflammation in vitro    ,  -  , and in vivo  -  . These agents have been reported to ameliorate OA in man and animals with minimal adverse side effects  -  . The molecular mechanism behind the beneficial effects of ASU, GLU and CS has been attributed to their ability to inhibit activation of NF-κB transcription factor  -  . When activated, NF-κB translocates from the cytoplasm to the nucleus which induces the expression of pro-inflammatory genes and catabolic enzymes that break down cartilage  -  .
To address the need for safe alternative anti-inflammatory agents, this study was conducted to determine whether the combination of ASU, GLU and CS will inhibit production of key inflammatory mediators: MCP-1 and PGE2. We also determined whether production of these pro-inflammatory mediators will similarly be inhibited by the NSAID, phenylbutazone (PBZ). Using an in vitro equine chondrocyte culture model, we observed that the [ASU + GLU + CS] combination inhibited production of both MCP-1 and PGE2 while PBZ suppressed PGE2 but not MCP-1. Identifying compounds that inhibit both the chemokine MCP-1 and PGE2 in the osteoarthritic joint may offer a more effective therapeutic approach.
2. Materials and Methods
2.1. Isolation of Equine Chondrocytes
Articular cartilage was harvested from the radiocarpal and tibiotarsal joints of apparently healthy horses of unknown age, sex, or breed. The horses used in this study were obtained from commercial sources (Celsis In Vitro Technologies, Baltimore, MD) and one of the authors (ARR). For animals obtained by ARR, animals were euthanized for reasons unrelated to this study in compliance with Michigan State University’s Institutional Animal Care and Use Committee. All harvested articular cartilage appeared smooth, glassy, and without macroscopic abnormalities. Cartilage was aseptically harvested, cut into 1 - 5 mm2 pieces, and digested with type II collagenase medium (10,000 U/L, Gibco, Invitrogen, Carlsbad, CA, USA) at 37˚C, 5% CO2 for 12 - 18 hours and chondrocytes were isolated as previously described  .
2.2. Treatment Design
Cells were seeded in flasks, and allowed to grow to confluency in control media consisting of Dulbeccos’ Modified Eagle’s basal medium (Sigma; St. Louis, MO, USA) supplemented with 10% v/v fetal bovine serum (Gemini Bio-Products; Woodland, CA, USA), 300 mg/ml L-glutamine (Sigma), 30 mg/ml antibiotic/antimycotic (Sigma), and 3.7 g/L sodium bicarbonate (Sigma), pH 7.4at 37°C, 5% CO2  . Chondrocytes were used at passage 2 - 4. Chondrocytes were seeded in 6-well plates at 5 × 106 cells/well, and incubated at 37˚C, 5% CO2 for 24 hours. Chondrocytes were then pre-treated with 1) control media, 2) [ASU (NMX1000®, 8.3 µg/mL) + GLU (FCHG49®, 11 µg/mL) + CS (TRH122®, 20 µg/mL)] (Nutramax Laboratories, Inc., Edgewood, MD, USA), or 3) PBZ (4 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) for an additional 24 hours. The concentrations of ASU, GLU, CS and PBZ used in the present study were previously reported to have significant anti-inflammatory effects and are clinically relevant  -  . Cells were then activated for 24 hours with IL-1β (10 ng/mL, R&D Systems, Minneapolis, MN, USA) or lipopolysaccharide (LPS, 1 ng/mL, Sigma-Aldrich). Supernatant was collected and stored at −80˚C until assays were performed. Nine chondrocyte lines at passage 2 - 4 from different horses were used and experiments were performed in triplicate.
2.3. Collagen and Aggrecan Immunofluorescence
Chondrocyte phenotype was characterized by immunofluorescence staining for collagen and aggrecan  . Chondrocytes were seeded at 1 × 104 cells/well on 8-well chambered slides (Fisher Scientific, Waltham, MA, USA) and incubated for 24 hours at 37˚C, 5% CO2. Cells were fixed with 10% v/v formalin (Thermo, Fisher Scientific) for 20 minutes and washed four times with 1x phosphate buffered saline (PBS, Gibco, Invitrogen). To prevent non-specific binding, a blocking solution (Gibco, Invitrogen) was applied and incubated for 30 minutes, followed by a 5 minute wash using PBS. Fixed cells were double stained for type I and type II collagen using a cocktail containing a goat anti-type I antibody (Southern Biotechnology Associates, Birmingham, AL, USA) diluted 1:500 and a mouse monoclonal anti-type II (Calbiochem, La Jolla, CA, USA) diluted 1:50 in PBS containing 0.05% Triton X-100. Cells were incubated overnight at 4˚C and then washed 3 times with PBS for 5 minutes each.
For aggrecan immunostaining, fixed chondrocyte-seeded 8-well chambered cover glass slides were incubated with a blocker diluent solution at room temperature for 30 minutes (Gibco Invitrogen) to prevent non-specific binding. Slides were washed for 5 minutes with PBS and then incubated overnight at 4˚C with mouse monoclonal anti-aggrecan (U.S. Biological, Swampscott, MA, USA) diluted 1:20 in PBS. Cells were washed 3 times with PBS then incubated with Alexa Fluor 488 donkey anti-mouse IgG (Gibco Invitrogen) and Alexa Fluor 594 donkey anti-goat IgG for 2 hours at room temperature with gentle agitation. Slides were washed 3 times with PBS and nuclei were stained with DAPI (Gibco Invitrogen) for 30 minutes as described above. Staining was analyzed using a Nikon epifluorescence TE 200 microscope and digital images were captured with a digital camera (Nikon Spot Camera, USA).
2.4. Determination of NF-κB Nuclear Translocation Using Immunofluorescence
Chondrocytes were seeded on 8-well chamber slides as described above. Cells were incubated at 37˚C, 5% CO2 overnight with 1) control media, 2) [ASU (NMX1000®, 8.3 µg/mL) + GLU (FCHG49®, 11 µg/mL) + CS (TRH122®, 20 µg/mL)], or 3) PBZ (4 µg/mL). Cells were activated for 1 hour with IL-1β (10 ng/mL). Rabbit anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied to the cells and incubated overnight at 4˚C. Slides were washed with 1x PBS, and a secondary donkey anti-rabbit IgG antibody labeled with Alexa Fluor-488 (Invitrogen) was applied for 2 hours. Cells were washed and analyzed using a Nikon epifluorescence TE 200 microscope and digital images were captured with a digital camera (Nikon Spot Camera, USA).
2.5. MCP-1 and PGE2 Immunoassay (ELISA)
MCP-1 and PGE2 concentrations were determined in cell culture supernatant using commercially available ELISA kits (Equine CCL2 (MCP-1) VetSet, Kingfisher Biotech Inc., St. Paul, MN, USA and Prostaglandin E2 Parameter Assay Kit, R&D Systems). All assays were run according to manufacturers’ instructions and a standard was run in parallel to samples during each assay. Optical density was measured immediately using the SpectraMAX 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm with wavelength correction at 540 nm.
2.6. Statistical Analysis
Pair-wise multiple comparisons were performed using one-way ANOVA, Tukey post-hoc using SigmaStat statistical software (Windows Version 3.11), where p < 0.05 was considered statistically significant.
3.1. Chondrocyte Phenotype Characterization
Chondrocytes in monolayer culture showed immunostaining for collagen (Figure 1, top image) and aggrecan (Figure 1, bottom image). At passage 2 and 3, some chondrocytes showed only type II collagen immunostaining. A few cells
Figure 1. Representative immunofluorescent staining of collagen and aggrecan in equine chondrocytes at passage 3 monolayer culture. Equine chondrocytes stained green for type II collagen, red for type I collagen (top image) and green for aggrecan (bottom image). DAPI nuclear staining is blue (top and bottom image). Magnification indicated by bar = 20µ.
stained for type I collagen alone and some stained for both (Figure 1, top image). Collagen types I and II (Figure 1, top image) were colocalized in the peri-nuclear cytoplasm. In comparison, all chondrocytes immunostained for aggrecan (Figure 1, bottom image). Chondrocyte morphology varied from the more rounded to spindle shape.
3.2. Stimulation of Chondrocyte MCP-1 and PGE2 Production by IL-1β and LPS
Stimulation of MCP-1 and PGE2 production by IL-1β was confirmed in chondrocyte cell lines 1 - 3 (Figure 2) and in chondrocytes cell lines 4 - 6 by LPS (Figure 3). Untreated control chondrocytes produced MCP-1 concentrations ranging from 10 - 25 × 103 pg/ml which significantly increased 5 - 7 times with IL-1β stimulation, p < 0.001 (Figure 2, left panel). Lines 1 - 3 also produced variable baseline levels of PGE2 ranging from 1 - 16 × 102 pg/ml which significantly increased 8 - 28 times with IL-1β stimulation, p < 0.001 (Figure 2, right panel).
Figure 2. MCP-1 and PGE2 production by chondrocyte lines 1 - 3 following stimulation with IL-1β. MCP-1 and PGE2 were measured in cell culture supernatant following incubation with IL-1β for 24 hours. Concentrations of both MCP-1 (left panel) and PGE2 (right panel) were significantly increased following IL-1β stimulation compared to control cells. Results are presented as means ± 1SD, n = 3. *indicates p < 0.001.
LPS stimulation significantly increased MCP-1 production in chondrocyte cell lines 4 - 6, p < 0.001 (Figure 3 left panel). Control, non-stimulated chondrocytes produced MCP-1 concentrations ranging from 4 to 35 × 103 pg/ml which significantly increased 3 to 6 times after LPS stimulation, p < 0.001. In comparison, PGE2 production in control non-stimulated cells ranged from 4 to 35 × 103 pg/ml (Figure 3, right panel). Production increased 2 to 6 times following LPS stimulation (Figure 3, right panel).
3.3. Effect of PBZ or [ASU + GLU + CS] on MCP-1 and PGE2 Production
Pre-treatment with [ASU + GLU + CS] or PBZ significantly inhibited PGE2 production in chondrocyte cell lines 7 and 8 stimulated with IL-1β, and in line 9 stimulated with LPS (Figure 4). Treatment with [ASU + GLU + CS] significantly reduced PGE2 production by 46% - 60%, p < 0.001). In contrast, PBZ ablated PGE2 production by 97% - 99%, p < 0.001 (Figure 4). MCP-1 production was significantly inhibited by pre-treatment with [ASU + GLU + CS] in chondrocyte cell lines 7 and 8 stimulated with IL-1β, and in cell line 9 stimulated with LPS (Figure 5). It is striking that PBZ did not suppress MCP-1 production whereas [ASU + GLU + CS] reduced MCP-1 levels by 30% - 41% in stimulated cells, p < 0.001
Figure 3. MCP-1 and PGE2 production by chondrocytes lines 4 - 6 following stimulation with LPS. MCP-1 and PGE2 were measured in cell culture supernatant following incubation with LPS for 24 hours. Concentrations of both MCP-1 (left panel) and PGE2 (right panel) were significantly increased following LPS stimulation compared to control cells. Results are presented as means ± 1SD, n =3. *indicates p < 0.001.
3.4. Effect of PBZ or [ASU + GLU + CS] on NF-κB Nuclear Translocation
The association between inhibition of MCP-1 and PGE2 production and suppression of NF-κB activation was analyzed using immunostaining. Chondrocytes seeded on 8-well chamber slides were pre-treated with [ASU + GLU + CS] or PBZ and then stimulated with IL-1β and then immunostained for NF-κB. Non-stimulated, control cells showed cytoplasmic fluorescence, and no nuclear fluorescence with NF-κB immunostaining. IL-1β stimulated chondrocytes showed increased nuclear fluorescence, indicative of translocation of NF-κB from the cytoplasm to the nucleus. Pre-treatment with [ASU + GLU + CS] or PBZ diminished nuclear fluorescence in IL-1β stimulated cells (Figure 6).
The principal finding of the present study is that [ASU + GLU + CS] inhibits MCP-1 and PGE2 production whereas PBZ ablates PGE2 levels but has minimal effect on MCP-1 levels. We used equine chondrocytes in monolayer culture since they display phenotypic changes similar to those of osteoarthritic cartilage.
Figure 4. PGE2 production following pre-treatment with [ASU + GLU + CS] or PBZ and stimulation with IL-1β by chondrocytes lines 7 - 8, or LPS by chondrocytes line 9. Pre-treatment of cells with [ASU + GLU + CS] or PBZ significantly reduced PGE2 production in stimulated cells. Results are presented as means ± 1SD, n = 3. *indicates p < 0.001.
Chondrocytes shift from producing the articular cartilage phenotype marker type II collagen to the fibroblastic-osteoblastic type I collagen observed in osteoarthritic cartilage. At passage 2 - 4, some chondrocytes produce type II while some produce either type I collagen alone or both (Figure 1). These chondrocyte cultures also showed robust response to IL-1β and LPS stimulation indicated by significant increase in MCP-1 andPGE2 production confirming earlier reports (Figures 2, Figure 3).
The finding that [ASU + GLU + CS] significantly suppressed but did not totally ablate MCP-1 and PGE2 production suggests that this compound could minimize disruption of physiologic functions of these molecules (Figure 4, Figure 5). It has been reported that NSAIDs that totally block PGE2 production interfere with bone healing and fracture repair    . Although PGE2 is well-characterized for its key role in the pathogenesis of OA, PGE2 is also critical for healing and tissue repair    . While sparing the physiologic functions of PGE2, [ASU + GLU + CS] could effectively inhibit the ability of PGE2 to 1) stimulate the production of degradative enzymes and 2) suppress the synthesis
Figure 5. MCP-1 production following pre-treatment with [ASU + GLU + CS] or PBZ and stimulation with IL-1β by chondrocytes lines 7 - 8, or LPS by chondrocytes line 9. Pre-treatment of cells with [ASU + GLU + CS] significantly reduced MCP-1 production but not with PBZ in stimulated cells (p > 0.05). Results are presented as means ±1SD, n = 3. * indicates p < 0.001), ▲indicates p = 0.011.
of cartilage extracellular matrix components, as well as attenuate induction of other pro-inflammatory mediators  . More importantly [ASU + GLU + CS] could minimize PGE2 mediated sensitization of pain nociceptors  -  .
Similarly, [ASU + GLU + CS] suppresses but does not ablate MCP-1 production in stimulated chondrocytes. This compound could thus help preserve physiologic biologic functions of MCP-1 while modifying its deleterious participation in OA pathogenesis. MCP-1 expression which was reported to induce MMPs in osteoarthritic patients has been correlated with severity of the disease    . In vitro treatment of human chondrocytes with exogenous MCP-1 results in significant MMP-3 production. MMP-3 is an important catabolic enzyme that has been linked to pain induction in OA. This conclusion is supported by a study which showed reduction in movement-related pain behavior in CCR2 knockout mice compared to wild-type mice with experimentally induced OA  . However, osteoarthritic pain attributed to MCP-1 may not involve degradation of cartilage matrix and tissue destruction.
PBZ profoundly suppressed PGE2 production in chondrocytes stimulated with
Figure 6. NF-κB immunofluorescence in stimulated and pre-treated cells. Control cells (A, D) show cytoplasmic fluorescence, and no nuclear fluorescence with NF-κB. Stimulation of cells with IL-1β (B, E) increases nuclear fluorescence, indicative of nuclear translocation of NF-κB. Pre-treatment of cells with [ASU + GLU + CS] (C) or PBZ (F) diminished nuclear fluorescence following IL-1β stimulation. Arrows point to nuclei. Magnification indicated by bar = 25µ.
IL-β or LPS (Figure 4). Notably, PBZ did not inhibit MCP-1 production in equine chondrocytes compared to [ASU + GLU + CS] (Figure 5). This observation suggests that the inhibitory effect on MCP-1 by [ASU + GLU + CS] could be regulated along different pathways. Further support of this possibility is the observation that both PBZ and [ASU + GLU + CS] inhibited nuclear translocation of NF-κB (Figure 6), but did not result in concomitant suppression of MCP-1 production by PBZ. Previous studies have shown that ASU, GLU and CS individually or in combination with other agents inhibit NF-κB translocation   -  . NF-κB is well known as a critical regulator of the inflammatory response in chondrocytes including PGE2 production and expression of enzymes that degrade cartilage matrix       .
It is now acknowledged that the pathogenesis of OA involves a wide variety of inflammatory mediators. Among the mediators receiving increasing attention are chemokines such as MCP-1 that are suspected to induce pain in the joint. Understanding of how production of these mediators can be suppressed and how their inhibition may affect OA pathology is still limited. As demonstrated in this study, commonly used therapeutics, such as non-steroidal anti-inflammatory drugs (NSAIDs), may not be able to attenuate production of all relevant inflammatory mediators. The observation that [ASU + GLU + CS] and PBZ differ in their effect on MCP-1 in stimulated horse chondrocytes may be of practical significance since MCP-1and its receptors have been implicated in the pathogenesis of OA. The data from this study suggest that the combination of [ASU + GLU + CS] may have broader effects than PBZ alone.
This study provides evidence that the inhibition of MCP-1 can be a specific target for therapeutic intervention. Targeting multiple pathways leading to inflammation and joint destruction may offer a more effective treatment. Further studies using OA cartilage and in vivo techniques would be useful to determine the full extent of MCP-1 production in osteoarthritic joints. Additionally, studies looking at combination treatment with [ASU + GLU + CS] and PBZ are warranted following the results from this study. Combination treatment may result in greater reduction of the inflammatory response. Alternatively, combination treatment may allow for the use of lower doses of PBZ or other NSAIDs clinically, therefore reducing the risk of adverse effects associated with NSAID use in horses and man, such as gastrointestinal ulceration and renal damage. Further work to elucidate the downstream effects of MCP-1 would be useful to determine the need for additional treatment options.
This study was supported by Nutramax Laboratories, Inc. We would like to thank Dr. Reinhard Grzanna for designing the immunohistochemistry and Western blot experiments as well as for constructive review of the manuscript and Lowella Fortuno for technical assistance.
Carmelita G. Frondoza, Ph. D. and Mark Grzanna, MS are former employees of Nutramax Laboratories, Inc. but do not hold stocks or royalties. Erica J Secor, DVM, MS and Ann M. Rashmir-Raven, DVM do not have competing interests.
List of Abbreviations
MCP: monocyte chemotactic protein
ASU: avocado/soybean unsaponifiables
CS: chondroitin sulfate
NSAID: non-steroidal anti-inflammatory drugs
PBS: phosphate buffered saline
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