The spine journal : official journal of the North American Spine Society 2017 01 17() pii 10.1016/j.spinee.2017.01.010
Disc degeneration is associated with the progressive loss of the proteoglycan content of the intervertebral disc (IVD), decreased matrix synthesis, higher concentrations of proteolytic enzymes and increased levels of pro-inflammatory 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 (IL)-1. The major function for 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.
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.
In vitro studies 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 and Tissue Network of Illinois) and cultured in monolayer. Gene expression of human CCR1, CCR2 and CCR5 was analyzed using real-time PCR. 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).
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 while 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.
CCR5 is expressed at the RNA level and at 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 on AF cells are functional. These data suggest that AF cells may have the ability to migrate in response to disc damage or inflammation.