Study of the role of leukotriene B₄ in abnormal function of human subchondral osteoarthritis osteoblasts: Effects of cyclooxygenase and/or 5‐lipoxygenase inhibition

Abstract

Objective. To compare the effect of licofelone, NS-398 (an inhibitor of cyclooxygenase 2 [COX-2]), andBayX-1005 (an inhibitor of 5-lipoxygenase activating protein) on the production of leukotriene B 4 (LTB 4 ) and prostaglandin E 2 (PGE 2 ), and on cell biomarkers by human osteoarthritis (OA) subchondral osteoblasts.Methods. Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients and autopsy subjects. LTB 4 and PGE 2 levels were measured by enzyme-linked immunosorbent assay in conditioned media of osteoblasts incubated in the presence or absence of licofelone, NS-398, or BayX-1005. The effect of these drugs or of the addition of LTB 4 on alkaline phosphatase (AP) activity and osteocalcin release by OA and normal osteoblasts was determined. The presence of LTB 4 receptors in normal and OA osteoblasts was evaluated by Western blot analysis.Results. OA osteoblasts produced variable levels of PGE 2 and LTB 4 compared with normal osteoblasts. Licofelone, at the maximal dose used, inhibited production of PGE 2 and LTB 4 by OA osteoblasts by a mean ؎ SEM of 61.2 ؎ 6.4% and 67.0 ؎ 7.6%, respectively. NS-398 reduced PGE 2 production by 75.8 ؎ 5.3%. BayX-1005 inhibited LTB 4 production in OA osteoblasts by 38.7 ؎ 14.5% and marginally affected PGE 2 levels (reduction of 14.8 ؎ 5.3%). Licofelone dose-dependently stimulated 1,25-dihydroxyvitamin D-induced AP activity while inhibiting osteocalcin release. BayX-1005 partly reproduced these effects, but NS-398 failed to affect them. LTB 4 dose-dependently inhibited AP activity in OA osteoblasts, while its effect on osteocalcin depended on endogenous LTB 4 levels in these cells. In normal osteoblasts, LTB 4 dosedependently stimulated osteocalcin, whereas it failed to influence AP. LTB 4 receptors BLT1 and BLT2 were present in normal and OA osteoblasts.Conclusion.

Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production

“…Osteoblasts from the same patients showed elevated levels of transforming growth factor ␤1 (TGF␤1) (16), and the expression of TGF␤1 is increased in OA bone tissue compared with normal tissue (21). We also showed that OA osteoblasts produced variable levels of leukotriene B 4 (22), which could also differentiate the 2 subgroups of patients.…”

Licofelone Suppresses Prostaglandin E2 Formation by Interference with the Inducible Microsomal Prostaglandin E2 Synthase-1

“…It was previously found that licofelone suppresses cellular COX-1 in bovine platelets (IC 50 ϭ 0.21 M) (Laufer et al, 1994b) and thromboxane B 2 synthesis in ionophore-stimulated whole blood (IC 50 ϭ 3.9 M) (Tries et al, 2002b) or COX-2-mediated PGE 2 synthesis in ionomycin-stimulated human subchondral osteoblasts (IC 50 Ͻ 0.8 M) (Paredes et al, 2002). To investigate the direct interference of licofelone with isolated COX enzymes, purified ovine COX-1 and purified human recombinant COX-2 were preincubated with licofelone (or vehicle, DMSO) for 5 min; arachidonic acid (5 M for COX-1; 2 M for COX-2) was added and after 5 min, the formation of 12-HHT as the major COX-1/2-derived product under these experimental conditions (Capdevila et al, 1995) was determined.…”

“…Induction of COX-2 by proinflammatory stimuli coincides with the expression of mPGES-1, resulting in substantial PGE 2 formation (Murakami et al, 2000;Thoren and Jakobsson, 2000). Because licofelone potently reduced COX-2-coupled PGE 2 formation in intact cells (Paredes et al, 2002), but it caused no significant inhibition of isolated COX-2, we investigated whether licofelone could interfere with mPGES-1 activity. Expression of COX-2 and mPGES-1 in A549 cells was induced by treatment with 1 ng/ml interleukin-1␤ for 72 h. Microsomes were isolated, preincubated with or without licofelone, and PGE 2 formation was induced by addition of 20 M PGH 2 .…”

“…Although the effects of licofelone on general PGE 2 formation using a simple ELISA technique as read-out system in human osteoarthritis subchondral osteoblasts were shown by others previously (Paredes et al, 2002), we established a novel assay that allowed us to specifically assess COX-2-mediated PGE 2 synthesis in human whole blood (A. Koeberle, F. Pollastro, H. Northoff, and O. Werz, submitted for publication). Heparinized human whole blood was preincubated with licofelone for 10 min, before stimulation with 10 g/ml lipopolysaccharide for 5 h. To minimize the contribution of COX-1 in PGH 2 synthesis, and thus, to establish experimental settings where PGE 2 is primarily produced via the COX-2/mPGES-1 pathway, COX-1 was inactivated by the use of 50 M aspirin and thromboxane formation by platelets was blocked using the thromboxane synthase inhibitor CV4151 at 1 M. For measuring PGE 2 , we first separated PGE 2 from other arachidonic acid metabolites that may possibly interfere with commercially available PGE 2 ELISA detection systems.…”

5-Lipoxygenase Metabolic Contributions to NSAID-Induced Organ Toxicity

“…A greater induction of FLAP in cultured subchondral osteoblasts from patients with more severe OA was seen than from patients with moderate arthritis [128,129] RA, a true inflammatory arthritis, is also treated with NSAIDs along with disease modifying agents, biologics, and other therapies.…”