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Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells
Life Sciences 83 (2008) 666–670
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells Judith A. Stebulis a,⁎, David R. Johnson c, Ronald G. Rossetti b, Sumner H. Burstein b, Robert B. Zurier b a b c
UMass Memorial Medical Center, Rheumatology Division, 119 Belmont Street, Worcester, MA 01605, USA University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA Environmental Laboratory, U.S. Army Engineer Research and Development Center, 3909 Halls Ferry Rd., Vicksburg, MS 39180, USA
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Article history: Received 13 May 2008 Accepted 9 September 2008 Keywords: Inflammation Synovial cells Prostaglandins Cannabinoids Cyclooxygenase 2
a b s t r a c t Aims: To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). Main methods: FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10–30 μM), then stimulated with tumor necrosis factor α (TNFα). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with 14C-arachidonic acid for 20 h then treated with AjA (8– 32 μM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. Key findings: AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta12,14-PGJ2 (15d-PGJ2) production in a concentration dependent manner, but did not affect PGE2 production significantly. Significance: The capacity of AjA to increase selectively and markedly 15d-PGJ2, an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation. © 2008 Elsevier Inc. All rights reserved.
Introduction Ajulemic acid (AjA) (Fig. 1), a synthetic cannabinoid acid (Burstein et al., 1972), reduces significantly the severity of adjuvant arthritis in rats (Zurier et al., 1998). Manifestations (redness, swelling, pain) of joint inflammation in the rats were observed clinically, and histopathology indicated evidence of synovitis in AjA treated animals, but inflammation resolved without progression to cartilage degradation and bone erosion. Although some authors have questioned the lack of pyschoactivity of AjA (Dyson et al., 2005; Sumariwalla et al., 2004; Vann et al., 2007), the only published study in which AjA was given to humans showed that AjA did not alter cognition as assessed by validated instruments designed to screen for cognitive impairment in marijuana users (Karst et al., 2003). AjA also exhibits reduced CNS penetration and a superior therapeutic index compared with other cannabinoids (Dyson et al., 2005). Joint tissue injury in patients with RA is likely due to a multicellular assault on articular cartilage and bone. Nonetheless, studies in animals and humans suggest that joint damage can proceed with participation of synovial cells alone (Müller-Ladner et al., 1995, ⁎ Corresponding author. Tel.: +1 508 334 5224; fax: +1 508 334 5654. E-mail address: [email protected] (J.A. Stebulis). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.09.004
1996; O'Sullivan et al., 1985, 1992; Tanaka et al., 1988; Lehmann et al., 2000). Although not the sole mechanism of action of nonsteroidal antiinflammatory drugs (NSAIDs), suppression of COX2 has been considered important for treatment of inflammation (Abramson and Weissmann, 1989; Pillinger et al., 1998). In an effort to better understand mechanisms whereby AjA exerts antiinflammatory and joint protective activities, we investigated its influence on COX2 expression in and prostaglandin production by human FLS. Materials and methods Materials AjA was obtained from Organix (Woburn, MA). Its purity was monitored by high-pressure liquid chromatography and compared with material synthesized previously (Burstein et al., 1992). The sample was 97% chemically pure, and was 99% chirally pure in the R,R enantiomer. AjA was dissolved in DMSO, then diluted with MEM/2% FBS to achieve working concentrations of 10 to 30 μM. The concentration of DMSO was kept constant at 0.3%. Recombinant human TNFα (R&D Systems) was prepared in MEM/2% FBS. Vehicle control was MEM/2% FBS with 0.3% DMSO.
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DMSO treated FLS. Arachidonic acid release was measured by liquid scintillation counting of a 0.1 ml aliquot of medium. Prostaglandin measurement PGE2 and 15d-PGJ2 were measured in 4 and 24 h supernatants with commercially available ELISA kits (R&D systems). Cell viability
Fig. 1. Cannabinoid structures. (a) Δ9-THC is the principal component of Cannabis sativa responsible for the mood altering effects of the plant. (b) THC-11-oic acid is a major nonpsychoactive metabolite of THC in most species including humans. (c) AjA is a nonpsychoactive synthetic analog of THC-11-oic acid.
Cell viability was assessed by trypan blue uptake. FLS grown to confluence in 6-well culture plates were treated with AjA 30 μM or DMSO control for 1 h then stimulated with 1 ng/ml TNFα. 4 h after stimulation, cells were harvested by trypsinization, centrifuged at 250 g for 15 min, then resuspended in 1 ml PBS. 10 μl aliquots of cell suspension diluted 1:1 with trypan blue were transferred to a hemocytometer. Average cell counts of 3 aliquots from each specimen were determined. The number of cells with trypan blue uptake was compared with the total cell count. Statistical analysis
FLS isolation and stimulation FLS were isolated from synovial tissue or synovial fluid of patients with inflammatory arthritis (4 RA, 1 psoriatic arthritis) as we have described (Stebulis et al., 2005). Briefly, synovial tissue obtained at the time of joint replacement surgery was minced and plated in tissue culture dishes with growth medium (MEM with 15% FBS, 1% penicillin/ streptomycin solution, 1% nonessential amino acids). Synovial fluid was collected in heparinized syringes, then centrifuged at 250 g for 15 min. The cell pellet was resuspended in growth medium, then plated in tissue culture flasks. After 2–4 days primary cultures were washed with PBS to remove tissue fragments and nonadherent cells. FLS were incubated at 37 °C under 5% CO2 and passaged to 6-well tissue culture plates (split 1:3) when they reached confluence. Passages 2 through 8 were used for experiments. Experiments were done on FLS rested overnight in low serum medium (MEM/2% FBS). Medium was replaced before beginning the experiments. Cells were treated with 10 to 30 μM AjA for 1 h then stimulated or not with 1 ng/ml TNFα. Controls were vehicle treated cells. Whole cell lysates were collected in cell lysis buffer (Quantikine mRNA Cell Lysis Reagents—R&D Systems) 4 h after stimulation. Supernatants were collected at 4 and 24 h after stimulation. Samples were stored at −80 °C.
Except where otherwise indicated, data were analyzed using the Kruskal–Wallis rank sum analysis for nonparametric data. Dunn's test was used for all pair-wise comparisons against controls. Results COX2 expression Exposure of FLS to AjA increased COX2 expression in a concentration dependent manner in both unstimulated and TNFα-stimulated FLS (Fig. 2). Low level COX2 expression was detected in resting FLS. A 2-fold induction of COX2 was observed 4 h after stimulation of cells with TNFα. This increase was modest compared with the 5–8-fold induction of COX2 in FLS exposed to AjA. A further increase in the level of COX2 mRNA was observed in AjA treated FLS after stimulation with TNFα. Arachidonic acid release Because arachidonic acid is the major substrate for COX2, we examined the influence of AjA on arachidonate release from FLS. A significant increase (1.5–2 fold) in arachidonic acid release from FLS treated with AjA was observed compared with vehicle treated controls. The effect on arachidonic acid release was concentration dependent (Fig. 3).
Measurement of COX2 mRNA COX2 mRNA was measured by colorimetric assay per manufacturer's instructions (Quantikine mRNA Colorimetric Quantitation kit, R&D Systems). Cell lysates were hybridized with COX2 mRNA-specific biotin and digoxigenin labeled oligonucleotides. The mRNA–oligonucleotide hybrids were captured on streptavidin coated microtiter plates and detected with anti-digoxigenin alkaline phosphatase conjugate and appropriate substrate. Color intensity was measured on a standard plate reader at 490 nm with wave length correction at 650 nm. COX2 mRNA was quantified by comparison with a serially diluted COX2 mRNA standard. Measurement of arachidonic acid release FLS obtained and cultured as above were labeled (20 h) with 14Carachidonic acid, then medium (RPMI with 0.1% BSA) was replaced and cells were treated for 1 h with AjA (8–32 μM). Controls were
Fig. 2. AjA increases COX-2 expression in FLS in a concentration dependent manner. FLS cultured as described in Materials and methods are treated with AjA or vehicle control for 1 h then stimulated or not with 1 ng/ml TNFα. COX-2 mRNA was measured by hybridization/colorimetric assay of cell lysates collected 4 h after stimulation. Results represent mean ± SEM of 5 experiments with duplicate samples. ⁎p b 0.01 vs control. # p b 0.05 vs control.
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release significantly, although small increases in PGE2 were observed (Fig. 4b). Unstimulated FLS produced minimal amounts of both prostaglandins (mean 50–65 pg/ml) in the absence of AjA, and in most cultures resting 15d-PGJ2 levels were higher than PGE2. Production of PGE2, but not 15d-PGJ2, increased within 4 h after stimulation with TNFα, such that the relative secretion of 15d-PGJ2 to PGE2 declined. However, FLS treated with AjA before stimulation maintained a ratio of 15d-PGJ2 to PGE2 similar to or exceeding that of resting cells (Fig. 4c). The effect of AjA on prostaglandin production persisted for 24 h (Fig. 4d). FLS viability
Fig. 3. AjA induces release of free arachidonic acid from FLS. FLS cultured as described in Materials and methods are labeled (20 h) with 14C-arachidonic acid, then treated with AjA or vehicle control (DMSO 10 μL) for 1 h. Arachidonic acid release was measured by liquid scintillation counting. Results are representative of 2 experiments. Values shown are means ± SEM of 4 replicates of each sample. ⁎p b 0.02 vs control (statistical analysis performed using paired Student's t test).
We examined the effect of AjA on FLS viability under conditions identical to those used in experiments for COX2 and prostaglandin production. Trypan blue uptake was seen in 5 to 8% of cells, indicating 92 to 95% viability. There were no significant differences in percent viability or total cell counts between cells exposed to AjA or vehicle. Results were similar for stimulated and unstimulated cells. (Data not shown.)
Prostaglandin production by FLS
Discussion
Because AjA acts to suppress inflammation but increases expression of COX2 we considered that AjA might alter the eicosanoid profile in FLS. Addition of AjA to FLS increased 15d-PGJ2 production significantly in a concentration dependent manner in both unstimulated and TNFα-stimulated cells (Fig. 4a). AjA did not alter PGE2
Results of experiments presented in this paper indicate that addition to human FLS in vitro of an antiinflammatory cannabinoid acid increases release from cells of arachidonic acid, increases expression of COX2, and enhances production of 15d-PGJ2. Treatment of FLS with AjA promoted an antiinflammatory prostaglandin profile
Fig. 4. AjA alters prostaglandin production in FLS. (a) AjA increased production of 15d-PGJ2 in unstimulated and TNFα stimulated FLS, but (b) did not affect PGE2 production significantly. (c) AjA treatment promoted an antiinflammatory prostaglandin profile in FLS by preventing the post-stimulation reduction in the ratio of 15d-PGJ2 to PGE2 observed in stimulated cells not exposed to AjA. (d) The effect of AjA on prostaglandin production in stimulated FLS persisted for 24 h. FLS, cultured as described in Materials and methods, were exposed to AjA for 60 min then stimulated or not with 1 ng/ml TNFα for 4 h. Prostaglandins are assayed by ELISA of supernatants collected 4 and 24 h after cell stimulation. Results represent mean ± SEM of 5 experiments with duplicate samples. ⁎p b 0.01 vs control. #p b 0.05 vs control.
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in FLS by preventing the post-stimulation reduction in the ratio of 15d-PGJ2 to PGE2 observed in stimulated cells not exposed to AjA. Prostaglandin synthesis requires the coordinated activity of multiple enzymes. Arachidonic acid cleaved from cell membrane phospholipids by phospholipases is converted to the endoperoxide intermediate PGH2 by the constitutively expressed COX1 or the inducible COX2 enzyme. PGH2 is then converted to PGE2 and PGD2 by specific prostaglandin synthases, and PGD2 undergoes nonenzymatic dehydration to form the J series prostaglandins, including 15d-PGJ2. Our results demonstrate that AjA increases arachidonic acid release from and COX2 expression by FLS, thereby enabling the production of additional PGH2 necessary for the substantial increase in 15d-PGJ2 we observed. The complicated regulation of COX2 expression by overlapping signaling pathways and post-transcriptional modification underscores its biologic importance (Smith et al., 2000). Recent evidence supports a role for peroxisome proliferator-activated receptor (PPAR) mediated regulation of COX2 via a PPAR response element in the promoter region of the COX2 gene (Meade et al., 1999; Inoue et al., 2000). Both AjA and 15d-PGJ2 are PPARγ ligands (Liu et al., 2003; Forman et al., 1995). Thus, the AjA induced increase in 15d-PGJ2 production suggests that PPARγ activation might contribute to COX2 induction by AjA. However, 15d-PGJ2 can modify COX2 expression by PPARγ independent mechanisms (Kalajdzic et al., 2002; Tsubouchi et al., 2001), and we have observed that AjA can alter FLS activation in a PPARγ independent manner (Johnson et al., 2007). The regulatory effects of PPARγ and 15d-PGJ2 are far from straightforward, and appear to depend on cell type and physiologic conditions (Inoue et al., 2000; Tsubouchi et al., 2001; Chawla et al., 2001; Fahmi et al., 2002). The capacity of AjA to increase selectively and markedly 15d-PGJ2 release from cultured FLS is interesting in light of accumulating evidence that 15d-PGJ2 modulates inflammation. In animal models of acute and chronic inflammation (carrageenan induced pleuritis and collagen induced arthritis, respectively), COX2 inhibition suppresses 15d-PGJ2 production and prolongs the inflammatory phase, whereas administration of 15d-PGJ2 promotes resolution of inflammation (Gilroy et al.,1999; Cuzzocrea et al., 2002). Of particular interest in this regard is the demonstration that administration of 15d-PGJ2 suppresses inflammation and prevents joint tissue injury in rats with adjuvant induced arthritis (Kawahito et al., 2000), results which are similar to those observed when rats with adjuvant induced arthritis are treated with AjA (Zurier et al.,1998). Mechanisms whereby 15d-PGJ2 promotes resolution of inflammation are likely multifactorial. 15d-PGJ2 antagonizes inflammatory signaling pathways mediated by NFκB, AP-1, and STATs (Jiang et al., 1998; Ricote et al., 1998; Straus et al., 2000; Perez-Sala et al., 2003; Chen et al., 2003); suppresses iNOS, TNFα, IL-1β, and IL-12 production (Jiang et al., 1998; Maggi et al., 2000; Petrova et al., 1999); induces apoptosis of granulocytes, macrophages, lymphocytes, and synoviocytes (Kawahito et al., 2000; Gilroy et al., 2003; Ward et al., 2002; Nencioni et al., 2003); and regulates chemokine expression and cell trafficking (Zhang et al., 2001; Zernecke et al., 2003; Jackson et al., 1999; Pasceri et al., 2000). AjA has multiple effects on inflammatory processes that overlap with those of 15d-PGJ2: AjA suppresses IL-1β production by peripheral blood and synovial fluid monocytes (Zurier et al., 2003); induces apoptosis in human T lymphocytes (Bidinger et al., 2003); and results of preliminary experiments not reported here suggest that AjA suppresses NFκB activation in human FLS. It is likely that the antiinflammatory and joint protective activities of AjA involve mechanisms mediated by 15d-PGJ2 and those that are 15d-PGJ2 independent. In contrast to the substantial increase in the antiinflammatory 15dPGJ2 induced by AjA, release of PGE2, generally considered to promote inflammation and tissue damage in RA (Bingham, 2002), is not altered by AjA despite the marked increase in COX2 expression. Similarly, administration of 15d-PGJ2 to unstimulated rat and human chondrocytes in vitro also increases COX2 expression with no significant effect on PGE2 expression (Fahmi et al., 2002; Bianchi et al., 2005). However, in the absence of treatment with15d-PGJ2 or AjA, COX2 expression
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and PGE2 production are increased substantially in FLS in response to inflammatory stimuli (Tsubouchi et al., 2001; Bianchi et al., 2005; Stichtenoth et al., 2001; Cheng et al., 2004; Faour et al., 2001). In inflammatory and immune cells, PGH2 is converted to PGD2 by the catalytic action of hematopoietic PGD synthase (H-PGDS). H-PGDS has not been described in FLS, but studies in human megakaryoblastic cells demonstrate that H-PGDS expression is transcriptionally regulated (Fujimori et al., 2000), raising the possibility that AjA induces HPGDS gene expression. Alternatively, AjA could increase H-PGDS activity by impairing degradation of H-PGDS mRNA, stabilizing the enzyme, or altering enzyme kinetics. It is also possible that AjA increases 15d-PGJ2 secretion indirectly by suppressing the expected increase in PGE2, thus shunting terminal prostaglandin production to the PGD2 pathway. Precedent for such a mechanism exists with the observation that suppression of COX2 activity by NSAIDs leads to increased production of the lipoxygenase pathway product leukotriene B4 (Robinson et al., 1986). PGH2 is converted to PGE2 by microsomal PGE synthase-1 (mPGES-1) which is negatively regulated by PPARγ ligands, including 15d-PGJ2 (Bianchi et al., 2005; Cheng et al., 2004). Thus, AjA might shunt terminal prostaglandin production to the PGD pathway by inhibiting mPGES-1 gene expression. Conclusion In summary, we have shown that AjA alters prostaglandin production in human FLS such that 15d-PGJ2, an eicosanoid that facilitates resolution of inflammation, is expressed preferentially. Details of mechanisms whereby AjA increases 15d-PGJ2 production require further study. Although AjA and 15d-PGJ2 have similar effects in several in vitro systems and animal models, it is not clear that the joint protective actions of AjA are due to its capacity to increase 15d-PGJ2. It is clear that the design of agents with actions other than suppression of COX2 will be necessary for development of effective antiinflammatory drugs. Immune/inflammatory responses, designed to be protective, are extremely redundant. It follows that successful therapy of RA will require modification of several aspects of host defense responses with agents that can be given safely for long periods of time. Although in vitro/in vivo correlates and animal models must be interpreted with caution, the data presented here suggest that AjA or other cannabinoid acids, or particular nonpsychoactive constituents of Cannabis, may have value as therapeutic agents for the treatment of rheumatoid arthritis and other diseases characterized by acute and chronic inflammation. Acknowledgements We gratefully acknowledge Ann Skulas and Francisco J. Atez for their excellent technical assistance with FLS culture maintenance. This study was supported by NIH grants T32 AR07572 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (JS, Trainee), R01 AT000309 from the National Center for Complementary and Alternative Medicine (NCCAM), R01 DA13691 from the National Institute on Drug Abuse (NIDA), and AI056362 (NIAID). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the National Institutes of Health. References Abramson, S., Weissmann, G., 1989. The mechanisms of action of nonsteroidal antiinflammatory drugs. Clinical and Experimental Rheumatology 7 (Suppl 3), S163–S170. Bianchi, A., Moulin, D., Sebillaud, S., Koufany, M., Galteau, M.M., Netter, P., et al., 2005. Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxyΔ12,14prostaglandinJ2. Arthritis Research & Therapy 7 (6), R1325–R1337. Bidinger, B., Torres, R., Rossetti, R.G., Brown, L., Beltre, R., Burstein, S., et al., 2003. Ajulemic acid, a nonpsychoactive cannabinoid acid, induces apoptosis in human T lymphocytes. Clinical Immunology 108 (2), 95–102.
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells Judith A. Stebulis a,⁎, David R. Johnson c, Ronald G. Rossetti b, Sumner H. Burstein b, Robert B. Zurier b a b c
UMass Memorial Medical Center, Rheumatology Division, 119 Belmont Street, Worcester, MA 01605, USA University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA Environmental Laboratory, U.S. Army Engineer Research and Development Center, 3909 Halls Ferry Rd., Vicksburg, MS 39180, USA
a r t i c l e
i n f o
Article history: Received 13 May 2008 Accepted 9 September 2008 Keywords: Inflammation Synovial cells Prostaglandins Cannabinoids Cyclooxygenase 2
a b s t r a c t Aims: To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). Main methods: FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10–30 μM), then stimulated with tumor necrosis factor α (TNFα). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with 14C-arachidonic acid for 20 h then treated with AjA (8– 32 μM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. Key findings: AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta12,14-PGJ2 (15d-PGJ2) production in a concentration dependent manner, but did not affect PGE2 production significantly. Significance: The capacity of AjA to increase selectively and markedly 15d-PGJ2, an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation. © 2008 Elsevier Inc. All rights reserved.
Introduction Ajulemic acid (AjA) (Fig. 1), a synthetic cannabinoid acid (Burstein et al., 1972), reduces significantly the severity of adjuvant arthritis in rats (Zurier et al., 1998). Manifestations (redness, swelling, pain) of joint inflammation in the rats were observed clinically, and histopathology indicated evidence of synovitis in AjA treated animals, but inflammation resolved without progression to cartilage degradation and bone erosion. Although some authors have questioned the lack of pyschoactivity of AjA (Dyson et al., 2005; Sumariwalla et al., 2004; Vann et al., 2007), the only published study in which AjA was given to humans showed that AjA did not alter cognition as assessed by validated instruments designed to screen for cognitive impairment in marijuana users (Karst et al., 2003). AjA also exhibits reduced CNS penetration and a superior therapeutic index compared with other cannabinoids (Dyson et al., 2005). Joint tissue injury in patients with RA is likely due to a multicellular assault on articular cartilage and bone. Nonetheless, studies in animals and humans suggest that joint damage can proceed with participation of synovial cells alone (Müller-Ladner et al., 1995, ⁎ Corresponding author. Tel.: +1 508 334 5224; fax: +1 508 334 5654. E-mail address: [email protected] (J.A. Stebulis). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.09.004
1996; O'Sullivan et al., 1985, 1992; Tanaka et al., 1988; Lehmann et al., 2000). Although not the sole mechanism of action of nonsteroidal antiinflammatory drugs (NSAIDs), suppression of COX2 has been considered important for treatment of inflammation (Abramson and Weissmann, 1989; Pillinger et al., 1998). In an effort to better understand mechanisms whereby AjA exerts antiinflammatory and joint protective activities, we investigated its influence on COX2 expression in and prostaglandin production by human FLS. Materials and methods Materials AjA was obtained from Organix (Woburn, MA). Its purity was monitored by high-pressure liquid chromatography and compared with material synthesized previously (Burstein et al., 1992). The sample was 97% chemically pure, and was 99% chirally pure in the R,R enantiomer. AjA was dissolved in DMSO, then diluted with MEM/2% FBS to achieve working concentrations of 10 to 30 μM. The concentration of DMSO was kept constant at 0.3%. Recombinant human TNFα (R&D Systems) was prepared in MEM/2% FBS. Vehicle control was MEM/2% FBS with 0.3% DMSO.
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DMSO treated FLS. Arachidonic acid release was measured by liquid scintillation counting of a 0.1 ml aliquot of medium. Prostaglandin measurement PGE2 and 15d-PGJ2 were measured in 4 and 24 h supernatants with commercially available ELISA kits (R&D systems). Cell viability
Fig. 1. Cannabinoid structures. (a) Δ9-THC is the principal component of Cannabis sativa responsible for the mood altering effects of the plant. (b) THC-11-oic acid is a major nonpsychoactive metabolite of THC in most species including humans. (c) AjA is a nonpsychoactive synthetic analog of THC-11-oic acid.
Cell viability was assessed by trypan blue uptake. FLS grown to confluence in 6-well culture plates were treated with AjA 30 μM or DMSO control for 1 h then stimulated with 1 ng/ml TNFα. 4 h after stimulation, cells were harvested by trypsinization, centrifuged at 250 g for 15 min, then resuspended in 1 ml PBS. 10 μl aliquots of cell suspension diluted 1:1 with trypan blue were transferred to a hemocytometer. Average cell counts of 3 aliquots from each specimen were determined. The number of cells with trypan blue uptake was compared with the total cell count. Statistical analysis
FLS isolation and stimulation FLS were isolated from synovial tissue or synovial fluid of patients with inflammatory arthritis (4 RA, 1 psoriatic arthritis) as we have described (Stebulis et al., 2005). Briefly, synovial tissue obtained at the time of joint replacement surgery was minced and plated in tissue culture dishes with growth medium (MEM with 15% FBS, 1% penicillin/ streptomycin solution, 1% nonessential amino acids). Synovial fluid was collected in heparinized syringes, then centrifuged at 250 g for 15 min. The cell pellet was resuspended in growth medium, then plated in tissue culture flasks. After 2–4 days primary cultures were washed with PBS to remove tissue fragments and nonadherent cells. FLS were incubated at 37 °C under 5% CO2 and passaged to 6-well tissue culture plates (split 1:3) when they reached confluence. Passages 2 through 8 were used for experiments. Experiments were done on FLS rested overnight in low serum medium (MEM/2% FBS). Medium was replaced before beginning the experiments. Cells were treated with 10 to 30 μM AjA for 1 h then stimulated or not with 1 ng/ml TNFα. Controls were vehicle treated cells. Whole cell lysates were collected in cell lysis buffer (Quantikine mRNA Cell Lysis Reagents—R&D Systems) 4 h after stimulation. Supernatants were collected at 4 and 24 h after stimulation. Samples were stored at −80 °C.
Except where otherwise indicated, data were analyzed using the Kruskal–Wallis rank sum analysis for nonparametric data. Dunn's test was used for all pair-wise comparisons against controls. Results COX2 expression Exposure of FLS to AjA increased COX2 expression in a concentration dependent manner in both unstimulated and TNFα-stimulated FLS (Fig. 2). Low level COX2 expression was detected in resting FLS. A 2-fold induction of COX2 was observed 4 h after stimulation of cells with TNFα. This increase was modest compared with the 5–8-fold induction of COX2 in FLS exposed to AjA. A further increase in the level of COX2 mRNA was observed in AjA treated FLS after stimulation with TNFα. Arachidonic acid release Because arachidonic acid is the major substrate for COX2, we examined the influence of AjA on arachidonate release from FLS. A significant increase (1.5–2 fold) in arachidonic acid release from FLS treated with AjA was observed compared with vehicle treated controls. The effect on arachidonic acid release was concentration dependent (Fig. 3).
Measurement of COX2 mRNA COX2 mRNA was measured by colorimetric assay per manufacturer's instructions (Quantikine mRNA Colorimetric Quantitation kit, R&D Systems). Cell lysates were hybridized with COX2 mRNA-specific biotin and digoxigenin labeled oligonucleotides. The mRNA–oligonucleotide hybrids were captured on streptavidin coated microtiter plates and detected with anti-digoxigenin alkaline phosphatase conjugate and appropriate substrate. Color intensity was measured on a standard plate reader at 490 nm with wave length correction at 650 nm. COX2 mRNA was quantified by comparison with a serially diluted COX2 mRNA standard. Measurement of arachidonic acid release FLS obtained and cultured as above were labeled (20 h) with 14Carachidonic acid, then medium (RPMI with 0.1% BSA) was replaced and cells were treated for 1 h with AjA (8–32 μM). Controls were
Fig. 2. AjA increases COX-2 expression in FLS in a concentration dependent manner. FLS cultured as described in Materials and methods are treated with AjA or vehicle control for 1 h then stimulated or not with 1 ng/ml TNFα. COX-2 mRNA was measured by hybridization/colorimetric assay of cell lysates collected 4 h after stimulation. Results represent mean ± SEM of 5 experiments with duplicate samples. ⁎p b 0.01 vs control. # p b 0.05 vs control.
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release significantly, although small increases in PGE2 were observed (Fig. 4b). Unstimulated FLS produced minimal amounts of both prostaglandins (mean 50–65 pg/ml) in the absence of AjA, and in most cultures resting 15d-PGJ2 levels were higher than PGE2. Production of PGE2, but not 15d-PGJ2, increased within 4 h after stimulation with TNFα, such that the relative secretion of 15d-PGJ2 to PGE2 declined. However, FLS treated with AjA before stimulation maintained a ratio of 15d-PGJ2 to PGE2 similar to or exceeding that of resting cells (Fig. 4c). The effect of AjA on prostaglandin production persisted for 24 h (Fig. 4d). FLS viability
Fig. 3. AjA induces release of free arachidonic acid from FLS. FLS cultured as described in Materials and methods are labeled (20 h) with 14C-arachidonic acid, then treated with AjA or vehicle control (DMSO 10 μL) for 1 h. Arachidonic acid release was measured by liquid scintillation counting. Results are representative of 2 experiments. Values shown are means ± SEM of 4 replicates of each sample. ⁎p b 0.02 vs control (statistical analysis performed using paired Student's t test).
We examined the effect of AjA on FLS viability under conditions identical to those used in experiments for COX2 and prostaglandin production. Trypan blue uptake was seen in 5 to 8% of cells, indicating 92 to 95% viability. There were no significant differences in percent viability or total cell counts between cells exposed to AjA or vehicle. Results were similar for stimulated and unstimulated cells. (Data not shown.)
Prostaglandin production by FLS
Discussion
Because AjA acts to suppress inflammation but increases expression of COX2 we considered that AjA might alter the eicosanoid profile in FLS. Addition of AjA to FLS increased 15d-PGJ2 production significantly in a concentration dependent manner in both unstimulated and TNFα-stimulated cells (Fig. 4a). AjA did not alter PGE2
Results of experiments presented in this paper indicate that addition to human FLS in vitro of an antiinflammatory cannabinoid acid increases release from cells of arachidonic acid, increases expression of COX2, and enhances production of 15d-PGJ2. Treatment of FLS with AjA promoted an antiinflammatory prostaglandin profile
Fig. 4. AjA alters prostaglandin production in FLS. (a) AjA increased production of 15d-PGJ2 in unstimulated and TNFα stimulated FLS, but (b) did not affect PGE2 production significantly. (c) AjA treatment promoted an antiinflammatory prostaglandin profile in FLS by preventing the post-stimulation reduction in the ratio of 15d-PGJ2 to PGE2 observed in stimulated cells not exposed to AjA. (d) The effect of AjA on prostaglandin production in stimulated FLS persisted for 24 h. FLS, cultured as described in Materials and methods, were exposed to AjA for 60 min then stimulated or not with 1 ng/ml TNFα for 4 h. Prostaglandins are assayed by ELISA of supernatants collected 4 and 24 h after cell stimulation. Results represent mean ± SEM of 5 experiments with duplicate samples. ⁎p b 0.01 vs control. #p b 0.05 vs control.
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in FLS by preventing the post-stimulation reduction in the ratio of 15d-PGJ2 to PGE2 observed in stimulated cells not exposed to AjA. Prostaglandin synthesis requires the coordinated activity of multiple enzymes. Arachidonic acid cleaved from cell membrane phospholipids by phospholipases is converted to the endoperoxide intermediate PGH2 by the constitutively expressed COX1 or the inducible COX2 enzyme. PGH2 is then converted to PGE2 and PGD2 by specific prostaglandin synthases, and PGD2 undergoes nonenzymatic dehydration to form the J series prostaglandins, including 15d-PGJ2. Our results demonstrate that AjA increases arachidonic acid release from and COX2 expression by FLS, thereby enabling the production of additional PGH2 necessary for the substantial increase in 15d-PGJ2 we observed. The complicated regulation of COX2 expression by overlapping signaling pathways and post-transcriptional modification underscores its biologic importance (Smith et al., 2000). Recent evidence supports a role for peroxisome proliferator-activated receptor (PPAR) mediated regulation of COX2 via a PPAR response element in the promoter region of the COX2 gene (Meade et al., 1999; Inoue et al., 2000). Both AjA and 15d-PGJ2 are PPARγ ligands (Liu et al., 2003; Forman et al., 1995). Thus, the AjA induced increase in 15d-PGJ2 production suggests that PPARγ activation might contribute to COX2 induction by AjA. However, 15d-PGJ2 can modify COX2 expression by PPARγ independent mechanisms (Kalajdzic et al., 2002; Tsubouchi et al., 2001), and we have observed that AjA can alter FLS activation in a PPARγ independent manner (Johnson et al., 2007). The regulatory effects of PPARγ and 15d-PGJ2 are far from straightforward, and appear to depend on cell type and physiologic conditions (Inoue et al., 2000; Tsubouchi et al., 2001; Chawla et al., 2001; Fahmi et al., 2002). The capacity of AjA to increase selectively and markedly 15d-PGJ2 release from cultured FLS is interesting in light of accumulating evidence that 15d-PGJ2 modulates inflammation. In animal models of acute and chronic inflammation (carrageenan induced pleuritis and collagen induced arthritis, respectively), COX2 inhibition suppresses 15d-PGJ2 production and prolongs the inflammatory phase, whereas administration of 15d-PGJ2 promotes resolution of inflammation (Gilroy et al.,1999; Cuzzocrea et al., 2002). Of particular interest in this regard is the demonstration that administration of 15d-PGJ2 suppresses inflammation and prevents joint tissue injury in rats with adjuvant induced arthritis (Kawahito et al., 2000), results which are similar to those observed when rats with adjuvant induced arthritis are treated with AjA (Zurier et al.,1998). Mechanisms whereby 15d-PGJ2 promotes resolution of inflammation are likely multifactorial. 15d-PGJ2 antagonizes inflammatory signaling pathways mediated by NFκB, AP-1, and STATs (Jiang et al., 1998; Ricote et al., 1998; Straus et al., 2000; Perez-Sala et al., 2003; Chen et al., 2003); suppresses iNOS, TNFα, IL-1β, and IL-12 production (Jiang et al., 1998; Maggi et al., 2000; Petrova et al., 1999); induces apoptosis of granulocytes, macrophages, lymphocytes, and synoviocytes (Kawahito et al., 2000; Gilroy et al., 2003; Ward et al., 2002; Nencioni et al., 2003); and regulates chemokine expression and cell trafficking (Zhang et al., 2001; Zernecke et al., 2003; Jackson et al., 1999; Pasceri et al., 2000). AjA has multiple effects on inflammatory processes that overlap with those of 15d-PGJ2: AjA suppresses IL-1β production by peripheral blood and synovial fluid monocytes (Zurier et al., 2003); induces apoptosis in human T lymphocytes (Bidinger et al., 2003); and results of preliminary experiments not reported here suggest that AjA suppresses NFκB activation in human FLS. It is likely that the antiinflammatory and joint protective activities of AjA involve mechanisms mediated by 15d-PGJ2 and those that are 15d-PGJ2 independent. In contrast to the substantial increase in the antiinflammatory 15dPGJ2 induced by AjA, release of PGE2, generally considered to promote inflammation and tissue damage in RA (Bingham, 2002), is not altered by AjA despite the marked increase in COX2 expression. Similarly, administration of 15d-PGJ2 to unstimulated rat and human chondrocytes in vitro also increases COX2 expression with no significant effect on PGE2 expression (Fahmi et al., 2002; Bianchi et al., 2005). However, in the absence of treatment with15d-PGJ2 or AjA, COX2 expression
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and PGE2 production are increased substantially in FLS in response to inflammatory stimuli (Tsubouchi et al., 2001; Bianchi et al., 2005; Stichtenoth et al., 2001; Cheng et al., 2004; Faour et al., 2001). In inflammatory and immune cells, PGH2 is converted to PGD2 by the catalytic action of hematopoietic PGD synthase (H-PGDS). H-PGDS has not been described in FLS, but studies in human megakaryoblastic cells demonstrate that H-PGDS expression is transcriptionally regulated (Fujimori et al., 2000), raising the possibility that AjA induces HPGDS gene expression. Alternatively, AjA could increase H-PGDS activity by impairing degradation of H-PGDS mRNA, stabilizing the enzyme, or altering enzyme kinetics. It is also possible that AjA increases 15d-PGJ2 secretion indirectly by suppressing the expected increase in PGE2, thus shunting terminal prostaglandin production to the PGD2 pathway. Precedent for such a mechanism exists with the observation that suppression of COX2 activity by NSAIDs leads to increased production of the lipoxygenase pathway product leukotriene B4 (Robinson et al., 1986). PGH2 is converted to PGE2 by microsomal PGE synthase-1 (mPGES-1) which is negatively regulated by PPARγ ligands, including 15d-PGJ2 (Bianchi et al., 2005; Cheng et al., 2004). Thus, AjA might shunt terminal prostaglandin production to the PGD pathway by inhibiting mPGES-1 gene expression. Conclusion In summary, we have shown that AjA alters prostaglandin production in human FLS such that 15d-PGJ2, an eicosanoid that facilitates resolution of inflammation, is expressed preferentially. Details of mechanisms whereby AjA increases 15d-PGJ2 production require further study. Although AjA and 15d-PGJ2 have similar effects in several in vitro systems and animal models, it is not clear that the joint protective actions of AjA are due to its capacity to increase 15d-PGJ2. It is clear that the design of agents with actions other than suppression of COX2 will be necessary for development of effective antiinflammatory drugs. Immune/inflammatory responses, designed to be protective, are extremely redundant. It follows that successful therapy of RA will require modification of several aspects of host defense responses with agents that can be given safely for long periods of time. Although in vitro/in vivo correlates and animal models must be interpreted with caution, the data presented here suggest that AjA or other cannabinoid acids, or particular nonpsychoactive constituents of Cannabis, may have value as therapeutic agents for the treatment of rheumatoid arthritis and other diseases characterized by acute and chronic inflammation. Acknowledgements We gratefully acknowledge Ann Skulas and Francisco J. Atez for their excellent technical assistance with FLS culture maintenance. This study was supported by NIH grants T32 AR07572 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (JS, Trainee), R01 AT000309 from the National Center for Complementary and Alternative Medicine (NCCAM), R01 DA13691 from the National Institute on Drug Abuse (NIDA), and AI056362 (NIAID). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the National Institutes of Health. References Abramson, S., Weissmann, G., 1989. The mechanisms of action of nonsteroidal antiinflammatory drugs. Clinical and Experimental Rheumatology 7 (Suppl 3), S163–S170. Bianchi, A., Moulin, D., Sebillaud, S., Koufany, M., Galteau, M.M., Netter, P., et al., 2005. Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxyΔ12,14prostaglandinJ2. Arthritis Research & Therapy 7 (6), R1325–R1337. Bidinger, B., Torres, R., Rossetti, R.G., Brown, L., Beltre, R., Burstein, S., et al., 2003. Ajulemic acid, a nonpsychoactive cannabinoid acid, induces apoptosis in human T lymphocytes. Clinical Immunology 108 (2), 95–102.
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