Anulus fibrosus (AF) cells obtained from patients undergoing surgery were cocultured with macrophage-like cells and production of inflammatory mediators was analyzed by quantitative assay.
To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation.
Discogenic pain caused by anular disruption is an important cause of low back pain and recent studies show the presence of macrophages in symptomatic discs but not in normal and aging discs. We hypothesize that macrophages play a major role in development of symptomatic disc.
Human AF cells were cocultured with phorbol myristate acetate stimulated macrophage-like THP-1 cells. The conditioned medium from cells cultured alone or in coculture was assayed for cytokines by Enzyme-linked immunosorbent assay and nitric oxide (NO) by the Greiss method. Using the same outcome measures, comparisons of cell response to cytokines were made among macrophage-like cells, naïve AF cells, and macrophage exposed AF cells.
Tumor necrosis factor (TNF)-α, interleukin (IL)-8, IL-6, and NO (TNF-α: 1.45 ± 0.29 ng/mL, IL-8: 97.02 ± 7.94 ng/mL, IL-6: 33.40 ± 3.55 ng/mL, NO: 8.42 ± 0.78 μmol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-α: 0.78 ± 0.12 ng/mL, IL-8: 58.04 ± 4.44 ng/mL, IL-6: 0.14 ± 0.03 ng/mL, NO: 0.30 ± 0.08 μmol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-α was up-regulated by coculture, however, IL-6 secretion in response to IL-1 β was downregulated in a dose-dependent manner. Coculture with macrophages also up-regulated AF cell secretion of IL-8 dose-dependently and downregulated NO to TNF-α or IL-1β stimulation.
We conclude that exposure to macrophages, as can be expected after anular injury, can result in enhanced response to local inflammation. Although changes were observed in all inflammatory mediators after macrophage exposure, the most significant change was observed in IL-6 and IL-8, implicating these mediators in development of symptomatic disc.
To evaluate macrophage’s role in symptomatic discs, we analyzed AF cell cytokine production after macrophage exposure. AF cytokine secretion was increased by coculture. Prior macrophages exposure upregulated IL-8, downregulated NO, and alternatively regulated IL-6 by TNF-α/IL-1β. We conclude that exposure to macrophages will result in enhanced AF response to inflammation.
From the *Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA; †Department of Neurosurgery, College of Medicine, Korea University, Seoul, Korea; ‡VA Pittsburgh Healthcare System; and §Department of Physical Medicine and Rehabilitation, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Acknowledgment date: February 19, 2008; Revision date: May 6, 2008; Acceptance date: May 8, 2008.
The manuscript submitted does not contain information about medical device(s)/drug(s).
Foundation funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.
Supported by the Pittsburgh Foundation at Department of Orthopedic Surgery, University of Pittsburgh.
Address correspondence and reprint requests to James D. Kang, MD, Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street, E1641 BST, Pittsburgh, PA 15261. E-mail: firstname.lastname@example.org