BACKGROUND CONTEXT Disc degeneration is associated with the progressive loss of the proteoglycan content of the intervertebral disc, decreased matrix synthesis, higher concentrations of proteolytic enzymes, and increased levels of proinflammatory cytokines. In previous studies, we have shown that C-C chemokine ligand (CCL)2, CCL3, and CCL5 are highly expressed by cultured nucleus pulposus (NP) and annulus fibrosus (AF) cells that have been treated by interleukin-1. The major function of these chemokines is to recruit immune cells into the disc. It is unclear if disc cells can respond to these chemokines. Recent studies by Phillips et al. (2015) showed that NP cells express a number of cytokines and chemokine receptors. PURPOSE The purpose of this study is to determine the gene and protein expression of C-C chemokine receptor (CCR)1, CCR2, and CCR5 in NP and AF cells, and to test if these receptors can respond to their ligands in these cells by cell signaling and migration. STUDY DESIGN/SETTING This is an in vitro study. METHODS For RNA, surface expression, and cell signaling studies, human cells were isolated from the NP and AF tissues collected after spine surgery or from donated spine segments (Gift of Hope Human Donor & Tissue Network of Illinois) and cultured in monolayer. The gene expression of human CCR1, CCR2, and CCR5 was analyzed using real-time polymerase chain reaction. The surface expression of CCR1, CCR2, and CCR5 was analyzed using flow cytometry and fluorescently tagged antibodies specific for these proteins. Extracellular signal-regulated kinase (ERK) phosphorylation was analyzed from the cell lysates of NP and AF cells treated with CCL2 and CCL5 for 1 hour using enzyme-linked immunosorbent assay. Migration of primary rabbit AF cells was assayed using 8-μm Corning Transwell inserts in the presence or absence of CCL5. This study was partially funded by a North American Spine Society 2014 Basic Research Grant Award ($50,000). RESULTS RNA analysis showed that gene expression of CCR1, CCR2, and CCR5 was evident in human NP and AF cells (n=6). Only a small population of NP and AF cells expressed CCR1 (1.9% and 1.2%, respectively) and CCR2 (0.8% and 1.4%, respectively) on the cell surface, whereas a larger percentage expressed CCR5 (12.7% and 11.6%, respectively). Significantly higher levels of ERK phosphorylation were detected in AF cells after treatment with CCL5 and not CCL2. Treatment with either chemokine did not cause significantly higher ERK phosphorylation in NP cells. There was an increase in average AF cell migration in the presence of CCL5. The increase was significant when the migration was induced with CCL5 (500 ng/mL) at both 2- and 6-hour time points. CONCLUSIONS CCR5 is expressed at the RNA level and on the cell surface of NP and AF cells. In the presence of CCL5, we detected increased levels of ERK phosphorylation and AF cell migration, suggesting that the CCR5 receptors in AF cells are functional. These data suggest that AF cells may have the ability to migrate in response ...

Short chain fatty acids (SCFAs) are products of anaerobic bacteria at mucosal sites, such as the female genital tract in bacterial vaginosis (BV). IL-1β, a potent proinflammatory cytokine, is found at elevated levels in the vaginal fluids of BV+ women. We recently found that SCFAs induce IL-1β in monocytes. Here, we tested whether SCFAs could control IL-1β production by either: 1) inducing pro IL-1β in the cytoplasm, and/or 2) causing inflammasome activation and release of IL-1β by acting as a “second” danger signal. We found that exposing peripheral blood mononuclear cells (PBMCs) for 24-hours to physiological levels of either acetic or butyric acid resulted in a modest increase in IL-1β levels in vitro. The release of IL-1β was more robust and occurred more rapidly when PBMCs were treated with SCFAs first, followed by exogenous ATP as a “danger” signal. SCFAs themselves were not potent danger signals as they failed to increase the release of LPS-induced pro IL-1β. Since SCFAs can enter cells via MCTs, we blocked these transporters using pharmacological agents and found that MCTs were necessary for SCFA-mediated IL-1β production. These findings show that SCFAs can induce cytoplasmic pro IL-1β production and that their effects are MCT-dependent. Because IL-1β is an important proinflammatory mediator, these results enhance our understanding of how SCFAs may contribute to the inflammatory environment seen in BV.

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