*Article* **Impact of Δ9-Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells**

**Torsten Lowin \*, Christina Kok, Sophie Smutny and Georg Pongratz**

> Poliklinik, Funktionsbereich & Hiller Forschungszentrum für Rheumatologie, University Hospital Duesseldorf, 40225 Duesseldorf, Germany; christina.Kok@uni-duesseldorf.de (C.K.); sophie.Smutny@hhu.de (S.S.); georg.pongratz@hhu.de (G.P.)

**\*** Correspondence: torsten.lowin@med.uni-duesseldorf.de; Tel.: +49-211-8106150

**Abstract:** δ9-Tetrahydrocannabinol (THC) has demonstrated anti-inflammatory effects in animal models of arthritis, but its mechanism of action and cellular targets are still unclear. The purpose of this study is to elucidate the effects of THC (0.1–25 μM) on synovial fibroblasts from patients with rheumatoid arthritis (RASF) and peripheral blood mononuclear cells (PBMC) from healthy donors in respect to proliferation, calcium mobilization, drug uptake, cytokine and immunoglobulin production. Intracellular calcium and drug uptake were determined by fluorescent dyes Cal-520 and PoPo3, respectively. Cytokine and immunoglobulin production were evaluated by ELISA. Cannabinoid receptors 1 and 2 (CB1 and CB2) were detected by flow cytometry. RASF express CB1 and CB2 and the latter was increased by tumor necrosis factor (TNF). In RASF, THC (≥5 μM) increased intracellular calcium levels/PoPo3 uptake in a TRPA1-dependent manner and reduced interleukin-8 (IL-8) and matrix metalloprotease 3 (MMP-3) production at high concentrations (25 μM). Proliferation was slightly enhanced at intermediate THC concentrations (1–10 μM) but was completely abrogated at 25 μM. In PBMC alone, THC decreased interleukin-10 (IL-10) production and increased immunoglobulin G (IgG). In PBMC/RASF co-culture, THC decreased TNF production when cells were stimulated with interferon-γ (IFN-γ) or CpG. THC provides pro- and anti-inflammatory effects in RASF and PBMC. This is dependent on the activating stimulus and concentration of THC. Therefore, THC might be used to treat inflammation in RA but it might need titrating to determine the effective concentration.

**Keywords:** synovial fibroblast; rheumatoid arthritis; cannabis; tetrahydrocannabinol; cytokines; calcium; PBMC; cannabinoid receptors; TRPA1
