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Anti-inflammatory effects of N-acylethanolamines in rheumatoid arthritis synovial cells are mediated by TRPV1 and TRPA1 in a COX-2 dependent manner.

Lowin T, Apitz M, Anders S, Straub RH - Arthritis Res. Ther. (2015)

Bottom Line: The effects of OEA and PEA on SFs were diminished by FAAH inhibition.N-acylethanolamines exert anti-inflammatory effects in SFs.A dual FAAH/COX-2 inhibitor, increasing N-acylethanolamine levels with concomitant TRP channel desensitization, might be a good candidate to inhibit the production of proinflammatory mediators of synovial cells and to reduce erosions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Rheumatology and Neuroendocrine Immunology, Department of Internal Medicine I, University Hospital Regensburg, Franz Josef Strauss Allee 11, 93042, Regensburg, Germany. torsten.lowin@ukr.de.

ABSTRACT

Introduction: The endocannabinoid system modulates function of immune cells and mesenchymal cells such as fibroblasts, which contribute to cartilage destruction in rheumatoid arthritis (RA). The aim of the study was to determine the influence of N-acylethanolamines anandamide (AEA), palmitoylethanolamine (PEA) and oleylethanolamine (OEA) on several features of arthritic inflammation in vitro (human material) and in vivo (a mouse model).

Methods: Immunofluorescence and western blotting were used to detect cannabinoid receptors and related enzymes. Cytokines and MMP-3 were measured by ELISA. Intracellular signaling proteins were detected by proteome profiling. Proliferation was quantified by CTB reagent. Adhesion was assessed by the xCELLigence system. After onset of collagen type II arthritis, mice were treated daily with the FAAH inhibitor JNJ1661010 (20 mg/kg) or vehicle.

Results: IL-6, IL-8 and MMP-3 (determined only in synovial fibroblasts (SFs)) were downregulated in primary synoviocytes and SFs of RA and OA after AEA, PEA and OEA treatment. In SFs, this was due to activation of TRPV1 and TRPA1 in a COX-2-dependent fashion. FAAH inhibition increased the efficacy of AEA in primary synoviocytes but not in SFs. The effects of OEA and PEA on SFs were diminished by FAAH inhibition. Adhesion to fibronectin was increased in a CB1-dependent manner by AEA in OASFs. Furthermore, elevation of endocannabinoids ameliorated collagen-induced arthritis in mice.

Conclusions: N-acylethanolamines exert anti-inflammatory effects in SFs. A dual FAAH/COX-2 inhibitor, increasing N-acylethanolamine levels with concomitant TRP channel desensitization, might be a good candidate to inhibit the production of proinflammatory mediators of synovial cells and to reduce erosions.

No MeSH data available.


Related in: MedlinePlus

N-acylethanolamine binding receptors visualized by western blotting and immunofluorescence and quantification of CB1, CB2, TRPV1 and TRPA1 in response to hypoxia and TNF in RASF by cell-based ELISA. Immunofluorescent staining and western blotting revealed the cellular localization of CB1 (a), CB2 (b), TRPA1 (c), TRPV1 (d), FAAH (e) and COX-2 (f) protein. Original magnification is 400×. Quantification of CB1 (g), CB2 (h), TRPA1 (i) and TRPV1 (j) protein by cell-based ELISA after culture of RASF for 48 h under either hypoxia, TNF stimulation (10 ng/ml) or hypoxia combined with TNF (10 ng/ml) in RASF. g-j: Data are given as % of unstimulated control RASF. *p <0.05 vs. normoxic control (dotted line). Paired Wilcoxon test was used for comparisons. LY = complete cell lysate, CF = cytosolic fraction, NF = nuclear fraction, N = normoxia, H = hypoxia, PARP = poly(ADP-ribose)-polymerase 1 (control for nuclear localization), GAPDH = glycerinaldehyd-3-phosphat-dehydrogenase (control for cytosolic localization). CB1 cannabinoid receptor type 1, CB2 cannabinoid receptor type 2, COX-2 cyclooxygenase-2, ELISA enzyme-linked immunosorbent assay, FAAH fatty acid amide hydrolase, IL-6 interleukin 6, IL-8 interleukin 8, MMP-3 matrix metalloproteinase 3 (stromelysin), RA rheumatoid arthritis, SF synovial fibroblast(s), TNF tumor necrosis factor, TRPA1 transient receptor potential ankyrin 1, TRPV1 Transient receptor potential vanilloid channel
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Fig2: N-acylethanolamine binding receptors visualized by western blotting and immunofluorescence and quantification of CB1, CB2, TRPV1 and TRPA1 in response to hypoxia and TNF in RASF by cell-based ELISA. Immunofluorescent staining and western blotting revealed the cellular localization of CB1 (a), CB2 (b), TRPA1 (c), TRPV1 (d), FAAH (e) and COX-2 (f) protein. Original magnification is 400×. Quantification of CB1 (g), CB2 (h), TRPA1 (i) and TRPV1 (j) protein by cell-based ELISA after culture of RASF for 48 h under either hypoxia, TNF stimulation (10 ng/ml) or hypoxia combined with TNF (10 ng/ml) in RASF. g-j: Data are given as % of unstimulated control RASF. *p <0.05 vs. normoxic control (dotted line). Paired Wilcoxon test was used for comparisons. LY = complete cell lysate, CF = cytosolic fraction, NF = nuclear fraction, N = normoxia, H = hypoxia, PARP = poly(ADP-ribose)-polymerase 1 (control for nuclear localization), GAPDH = glycerinaldehyd-3-phosphat-dehydrogenase (control for cytosolic localization). CB1 cannabinoid receptor type 1, CB2 cannabinoid receptor type 2, COX-2 cyclooxygenase-2, ELISA enzyme-linked immunosorbent assay, FAAH fatty acid amide hydrolase, IL-6 interleukin 6, IL-8 interleukin 8, MMP-3 matrix metalloproteinase 3 (stromelysin), RA rheumatoid arthritis, SF synovial fibroblast(s), TNF tumor necrosis factor, TRPA1 transient receptor potential ankyrin 1, TRPV1 Transient receptor potential vanilloid channel

Mentions: In subsequent experiments, RASF generated from primary synovial cells were investigated, since effects of AEA were only augmented in mixed RA synoviocytes (see above). AEA, PEA and OEA bind a wide array of receptors and enzymes and, therefore, expression of possible target proteins was assessed in RASF (Fig. 2). Immunocytochemistry and western blotting (not for TRPA1, since antibodies were not suitable for western blotting) revealed that RASF not only express CB1, CB2, FAAH and COX-2 but also TRPV1 and TRPA1 (Fig. 2a-f) (see also Figure S1 in Additional file 1 for entire blots and blocking peptide control). Surprisingly, CB1 was found located exclusively in the nuclear membrane (Fig. 2a). Addition of TNF (10 ng/ml, 48 h) and hypoxia influenced protein levels of CB1, CB2, TRPV1 and TRPA1 (Fig. 2g-j). CB1 and CB2 protein were increased by TNF treatment (Fig. 2g, h). TRPV1 and TRPA1 were significantly increased in RASF when hypoxia was combined with TNF treatment (Fig. 2i, j).Fig. 2


Anti-inflammatory effects of N-acylethanolamines in rheumatoid arthritis synovial cells are mediated by TRPV1 and TRPA1 in a COX-2 dependent manner.

Lowin T, Apitz M, Anders S, Straub RH - Arthritis Res. Ther. (2015)

N-acylethanolamine binding receptors visualized by western blotting and immunofluorescence and quantification of CB1, CB2, TRPV1 and TRPA1 in response to hypoxia and TNF in RASF by cell-based ELISA. Immunofluorescent staining and western blotting revealed the cellular localization of CB1 (a), CB2 (b), TRPA1 (c), TRPV1 (d), FAAH (e) and COX-2 (f) protein. Original magnification is 400×. Quantification of CB1 (g), CB2 (h), TRPA1 (i) and TRPV1 (j) protein by cell-based ELISA after culture of RASF for 48 h under either hypoxia, TNF stimulation (10 ng/ml) or hypoxia combined with TNF (10 ng/ml) in RASF. g-j: Data are given as % of unstimulated control RASF. *p <0.05 vs. normoxic control (dotted line). Paired Wilcoxon test was used for comparisons. LY = complete cell lysate, CF = cytosolic fraction, NF = nuclear fraction, N = normoxia, H = hypoxia, PARP = poly(ADP-ribose)-polymerase 1 (control for nuclear localization), GAPDH = glycerinaldehyd-3-phosphat-dehydrogenase (control for cytosolic localization). CB1 cannabinoid receptor type 1, CB2 cannabinoid receptor type 2, COX-2 cyclooxygenase-2, ELISA enzyme-linked immunosorbent assay, FAAH fatty acid amide hydrolase, IL-6 interleukin 6, IL-8 interleukin 8, MMP-3 matrix metalloproteinase 3 (stromelysin), RA rheumatoid arthritis, SF synovial fibroblast(s), TNF tumor necrosis factor, TRPA1 transient receptor potential ankyrin 1, TRPV1 Transient receptor potential vanilloid channel
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: N-acylethanolamine binding receptors visualized by western blotting and immunofluorescence and quantification of CB1, CB2, TRPV1 and TRPA1 in response to hypoxia and TNF in RASF by cell-based ELISA. Immunofluorescent staining and western blotting revealed the cellular localization of CB1 (a), CB2 (b), TRPA1 (c), TRPV1 (d), FAAH (e) and COX-2 (f) protein. Original magnification is 400×. Quantification of CB1 (g), CB2 (h), TRPA1 (i) and TRPV1 (j) protein by cell-based ELISA after culture of RASF for 48 h under either hypoxia, TNF stimulation (10 ng/ml) or hypoxia combined with TNF (10 ng/ml) in RASF. g-j: Data are given as % of unstimulated control RASF. *p <0.05 vs. normoxic control (dotted line). Paired Wilcoxon test was used for comparisons. LY = complete cell lysate, CF = cytosolic fraction, NF = nuclear fraction, N = normoxia, H = hypoxia, PARP = poly(ADP-ribose)-polymerase 1 (control for nuclear localization), GAPDH = glycerinaldehyd-3-phosphat-dehydrogenase (control for cytosolic localization). CB1 cannabinoid receptor type 1, CB2 cannabinoid receptor type 2, COX-2 cyclooxygenase-2, ELISA enzyme-linked immunosorbent assay, FAAH fatty acid amide hydrolase, IL-6 interleukin 6, IL-8 interleukin 8, MMP-3 matrix metalloproteinase 3 (stromelysin), RA rheumatoid arthritis, SF synovial fibroblast(s), TNF tumor necrosis factor, TRPA1 transient receptor potential ankyrin 1, TRPV1 Transient receptor potential vanilloid channel
Mentions: In subsequent experiments, RASF generated from primary synovial cells were investigated, since effects of AEA were only augmented in mixed RA synoviocytes (see above). AEA, PEA and OEA bind a wide array of receptors and enzymes and, therefore, expression of possible target proteins was assessed in RASF (Fig. 2). Immunocytochemistry and western blotting (not for TRPA1, since antibodies were not suitable for western blotting) revealed that RASF not only express CB1, CB2, FAAH and COX-2 but also TRPV1 and TRPA1 (Fig. 2a-f) (see also Figure S1 in Additional file 1 for entire blots and blocking peptide control). Surprisingly, CB1 was found located exclusively in the nuclear membrane (Fig. 2a). Addition of TNF (10 ng/ml, 48 h) and hypoxia influenced protein levels of CB1, CB2, TRPV1 and TRPA1 (Fig. 2g-j). CB1 and CB2 protein were increased by TNF treatment (Fig. 2g, h). TRPV1 and TRPA1 were significantly increased in RASF when hypoxia was combined with TNF treatment (Fig. 2i, j).Fig. 2

Bottom Line: The effects of OEA and PEA on SFs were diminished by FAAH inhibition.N-acylethanolamines exert anti-inflammatory effects in SFs.A dual FAAH/COX-2 inhibitor, increasing N-acylethanolamine levels with concomitant TRP channel desensitization, might be a good candidate to inhibit the production of proinflammatory mediators of synovial cells and to reduce erosions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Rheumatology and Neuroendocrine Immunology, Department of Internal Medicine I, University Hospital Regensburg, Franz Josef Strauss Allee 11, 93042, Regensburg, Germany. torsten.lowin@ukr.de.

ABSTRACT

Introduction: The endocannabinoid system modulates function of immune cells and mesenchymal cells such as fibroblasts, which contribute to cartilage destruction in rheumatoid arthritis (RA). The aim of the study was to determine the influence of N-acylethanolamines anandamide (AEA), palmitoylethanolamine (PEA) and oleylethanolamine (OEA) on several features of arthritic inflammation in vitro (human material) and in vivo (a mouse model).

Methods: Immunofluorescence and western blotting were used to detect cannabinoid receptors and related enzymes. Cytokines and MMP-3 were measured by ELISA. Intracellular signaling proteins were detected by proteome profiling. Proliferation was quantified by CTB reagent. Adhesion was assessed by the xCELLigence system. After onset of collagen type II arthritis, mice were treated daily with the FAAH inhibitor JNJ1661010 (20 mg/kg) or vehicle.

Results: IL-6, IL-8 and MMP-3 (determined only in synovial fibroblasts (SFs)) were downregulated in primary synoviocytes and SFs of RA and OA after AEA, PEA and OEA treatment. In SFs, this was due to activation of TRPV1 and TRPA1 in a COX-2-dependent fashion. FAAH inhibition increased the efficacy of AEA in primary synoviocytes but not in SFs. The effects of OEA and PEA on SFs were diminished by FAAH inhibition. Adhesion to fibronectin was increased in a CB1-dependent manner by AEA in OASFs. Furthermore, elevation of endocannabinoids ameliorated collagen-induced arthritis in mice.

Conclusions: N-acylethanolamines exert anti-inflammatory effects in SFs. A dual FAAH/COX-2 inhibitor, increasing N-acylethanolamine levels with concomitant TRP channel desensitization, might be a good candidate to inhibit the production of proinflammatory mediators of synovial cells and to reduce erosions.

No MeSH data available.


Related in: MedlinePlus