Sequential CCL2 Expression Profile After Disc Injury in Mice.

Macrophages produce proinflammatory cytokines in injured intervertebral discs (IVDs). We recently showed that macrophage-derived inflammatory cytokines contribute to the production of pain-related factors. However, the mechanism by which macrophages are recruited to injured IVDs has not been fully clarified. Here, we examined the expression dynamics of the chemokine CCL2 in a mouse IVD injury model and the mechanisms of its regulation. The percentage of macrophages increased from day 1 after injury and persisted up until day 28. At 1 and 3 days after injury, the expression of both Ccl2 messenger RNA (mRNA) and CCL2 protein was elevated in the IVD injury group, after which expression decreased to basal levels. Consistent with the increase in CCL2 expression, Ccr2 and Tnfa expression and various types of macrophages were also immediately elevated following disc injury. Further, tumor necrosis factor-α (TNF-α) stimulated Ccl2 mRNA and CCL2 protein expression in IVD cells in vitro. The expressions of M1 (Cd86 and Nos2) and M2a (Ym1) macrophage markers were all significantly elevated from  day 1 following injury in injured compared with control mice. Meanwhile, the expression of Cd206 (M2a and M2c marker) was significantly elevated on days 3, 7, 14, and 28 following injury. These results suggest that in IVD injury, TNF-α stimulates CCL2, which, in turn, mediates the recruitment of macrophages with the recruited macrophages subsequently differentiating into M1 and M2 subtypes. CCL2 signaling may, therefore, play an important role in IVD pathology via macrophage recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:895-901, 2020.