Activated macrophage-like THP-1 cells modulate anulus fibrosus cell production of inflammatory mediators in response to cytokines.
METHODS
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.


OBJECTIVE
To investigate the role of macrophages in AF cell production of inflammatory mediators by cytokines stimulation.


BACKGROUND
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.


METHODS
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. RESULTS.: Tumor necrosis factor (TNF)-alpha, interleukin (IL)-8, IL-6, and NO (TNF-alpha: 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 micromol/L) were secreted in much greater amounts by cells maintained in coculture compared to macrophages (TNF-alpha: 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 micromol/L) or AF cells cultured alone. In addition, IL-6 secretion from AF cells in response to TNF-alpha was up-regulated by coculture, however, IL-6 secretion in response to IL-1 beta 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-alpha or IL-1beta stimulation.


CONCLUSIONS
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.
Kim
Joo Han
JH
Department of Orthopaedic Surgery, Ferguson Laboratory for Orthopaedic Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Studer
Rebecca K
RK
Sowa
Gwendolyn A
GA
Vo
Nam Viet
NV
Kang
James D
JD
D045744:Cell Line, Tumor; D018920:Coculture Techniques / Q000379:methods; D016207:Cytokines / Q000502:physiology*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D008262:Macrophage Activation / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D000093242:Interleukin-6 Inhibitors
D016207:Cytokines; D018836:Inflammation Mediators; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D000093242:Interleukin-6 Inhibitors