title: Annulus fibrosus cells express and utilize C-C chemokine receptor 5 (CCR5) for migration.
abstract: 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.
  
  
  
  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.
  
  
  
  This is an in vitro study.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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 to disc damage or inflammation.
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA; Department of Orthopedics, Wuhan Pu'Ai Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 473 Hanzheng St, Wuhan 430033, China.
authors.forename: Weijun
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA; College of Pharmacy, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd, North Chicago, IL 60064, USA.
authors.forename: David
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA; University of Illinois College of Medicine at Peoria, 1 Illini Dr, Peoria, IL 61605, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Shi
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA; Department of Orthopaedic and Traumatology, Taipei Veterans General Hospital, No. 201, Section 2, Shipai Rd, Beitou District, Taipei City 112, Taiwan; School of Medicine, National Yang-Ming University, No. 155, Section 2, Linong St, Taipei City 112, Taiwan.
authors.forename: Po-Hsin
authors.initials: PH
authors.lastname: Chou
authors.affiliation: Department of Biochemistry, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612, USA.
authors.forename: Chundo
authors.initials: C
authors.lastname: Oh
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA; Department of Biochemistry, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612, USA.
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA. Electronic address: howard.an@rushortho.com.
authors.forename: Howard S
authors.initials: HS
authors.lastname: An
authors.affiliation: Department of Orthopedic Surgery, Rush University Medical Center, 1611 W Harrison St, Suite 300, Chicago, IL 60612, USA.
authors.forename: Ana
authors.initials: A
authors.lastname: Chee
mesh_terms: D000070616:Annulus Fibrosus / Q000166:cytology* / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C584933:CCR5 protein, human; D016207:Cytokines; D019713:Receptors, CCR5
keywords: Cell migration; Cell signaling; Chemokine receptor; Chemokines; Flow cytometry; Intervertebral disc