Objective: To determine the role played by mitogen-activated protein kinase (MAPK) signaling in the interactions between macrophages and intervertebral disc (IVD) cells, it was hypothesized that MAPK inhibition would modulate the production of the proinflammatory cytokines associated with inflammatory reaction in IVD cells.
Methods: Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were cocultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells, with and without SB202190 (a p38-α and -β inhibitor), SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor), and PD98059 (an extracellular signal-regulated kinase [ERK] 1/2 inhibitor). The cytokines in conditioned media from cocultured and macrophage-exposed (nemotic) cells were assayed using enzyme-linked immunosorbent assays (ELISAs).
Results: Interleukin (IL)-6 and IL-8 were secreted in greater quantities by the cocultured cells compared with naive IVD cells and macrophages (MΦ) cultured alone. The tumor necrosis factor (TNF)- α and IL-6 levels produced by the NP cells cocultured with MΦs (NP-MΦ) were significantly lower than those produced by AF cells cocultured with MΦs (AF-MΦ). SB202190 dose-dependently suppressed IL-6 secretion by AF-MΦ and NP-MΦ cocultures, and 10 μM SB202190 significantly decreased IL-6 and IL-8 production in nemotic AF and NP pellets. SP600125 at 10 μM significantly suppressed the production of TNF α IL-6. and IL-8 in AF-MΦ and NP-MΦ cocultures and significantly suppressed IL-1β production in the NP-MΦ coculture. Administration of 10 μM PD98059 significantly decreased IL-6 levels in the AF-MΦ coculture, and decreased the levels of TNF α and IL-8 in both the AF-MΦ and NP-MΦ cocultures.
Conclusions: The present study shows that inhibitors of p38 MAPK effectively controlled IL-6 production during inflammatory reactions and that JNK and ERK1/2 inhibitors successfully suppressed the production of major proinflammatory cytokines during interactions between macrophages and IVD cells. Therefore, selective blockade of these signals may serve as a therapeutic approach to symptomatic IVD degeneration.
Keywords: AF = annulus fibrosus; AF-MΦ = AF cells cocultured with MΦs; CCK-8 = Cell Counting Kit-8; CM = conditioned media; ELISA = enzyme-linked immunosorbent assay; ERK = extracellular signal-regulated kinase; FBS = fetal bovine serum; IL = interleukin; IVD = intervertebral disc; JNK = c-Jun N-terminal kinase; MAPK = mitogen-activated protein kinase; MΦ = macrophages; NP = nucleus pulposus; NP-MΦ = NP cells cocultured with MΦs; P/S = penicillin/streptomycin; PD98059; PMA = phorbol myristate acetate; SB202190; SP600125; TBS-T = Tris-buffered saline and Tween 20; TNF = tumor necrosis factor; cytokines; intervertebral disc degeneration; mitogen-activated protein kinases.