Nerve growth factor (NGF) is increased in intervertebral discs (IVDs) after disc injury and anti-NGF therapy improves low back pain in humans. Furthermore, M1 and M2 macrophage subtypes play a role in degenerative IVD injury. We examined M1 and M2 macrophage markers and NGF and cytokine expression in IVD-derived cells from control and IVD-injured mice for 28 days following injury. Ngf messenger RNA (mRNA) expression was increased 1 day after injury in injured compared with control mice, and persisted for up to 28 days. Flow cytometric analysis demonstrated that the proportion of F4/80+ CD11b+ cells was significantly increased from 1 day after injury for up to 28 days in injured compared to control mice. mRNA expression of M1 macrophage markers Tnfa, Il1b, and Nos2 was significantly increased 1 day after injury in injured compared to control mice, before gradually decreasing. At 28 days, no significant difference was observed in M1 markers. The M2a marker, Ym1, was significantly increased 1 day after injury in injured compared with control mice, while M2a and M2c markers Tgfb and Cd206 were significantly increased 7, 14, and 28 days after injury. Tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) stimulated Ngf mRNA and NGF protein expression in IVD cells. Our results suggest that TNF-α and TGF-β may stimulate NGF production under inflammatory and non-inflammatory conditions following IVD injury. As TNF-α and TGF-β are produced by M1 and M2 macrophages, further investigations are needed to reveal the role of macrophages in NGF expression following IVD injury. Our results may aid in developing treatments for IVD-related LBP pathology.

Macrophages, particularly M1 macrophages, produce proinflammatory cytokines and contribute to the degenerative process in injured intervertebral discs (IVDs). We previously showed that macrophages in both intact and injured IVDs increased following IVD injury. Resident macrophages and macrophages recruited from the peripheral blood have distinct roles in tissue. However, it remains to be determined whether increased macrophages derive from resident or recruited macrophages. We investigated the origin of M1 macrophages in injured IVDs using green fluorescent protein (GFP) transgenic bone marrow chimeric mice. The M1 macrophage marker, CD86, increased in both disc‐derived resident macrophages and bone marrow‐derived macrophages (BMMs) after lipopolysaccharide/interferon γ stimulation in vitro. Following IVD injury, the proportion of cells positive for the CD86 ligand, the F4/80 antigen, and the surface glycoprotein CD11b (CD86+ CD11b+ F4/80+) significantly increased in GFP+ populations at days 3, 7, and 14. In contrast, CD86+ CD11b+ F4/80+ cells in GFP− populations significantly increased on day 3, and thereafter decreased on days 7 and 14. The proportion of CD86+ CD11b+ F4/80+ cells in the GFP+ populations was significantly higher than that in the GFP− populations at days 1, 3, 7, and 14. Monocyte chemoattractant protein‐1 expression in disc‐derived macrophages, but not in BMMs, increased following interleukin‐1β stimulation. Our results suggest M1 macrophages following IVD injury originate from recruited macrophages. Resident macrophages may behave differently in IVD injury. The role of resident macrophages needs to be clarified. Further investigation is needed.

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

Multiple human and animal studies suggest that the upregulation of inflammatory cytokines and other pain‐related molecules in degenerated or injured intervertebral discs (IVDs) may cause discogenic low back pain (LBP). We previously reported that macrophages in injured IVD in mice produced inflammatory cytokines, but not other pain‐related molecules. CD14 is a monocyte marker expressed mainly by macrophages. The aim of the current study was to evaluate the role of CD14‐positive cells in inflammatory cytokine and pain‐related molecule expression in human degenerated IVD. IVD samples were harvested from 14 patients, including 10 with lumbar spinal stenosis, four with adult spinal deformity, and one with lumbar disc herniation during spinal interbody fusion surgery. Harvested IVD‐derived mononuclear cells were obtained and CD14‐positive (+) and CD14‐negative (−) cells were separated using CD14 antibody and streptavidin‐labeled magnetic beads. Inflammatory cytokines messenger RNA (mRNA) in the CD14(+) and CD14(−) cells, including tumor necrosis factor ɑ (TNFA), in, terleukin‐1β (IL1B) and IL6, were determined using quantitative polymerase chain reaction (qPCR) and their expression levels were compared. To evaluate factors controlling the regulation of pain‐related molecules mRNA expression, cultured CD14(−) and CD14(+) cells from IVDs were stimulated with recombinant human TNF‐ɑ and IL‐1β and levels of pain‐related molecules, including calcitonin gene‐related peptide (CGRP) and nerve growth factor (NGF) were determined using qPCR. Levels of TNFA, IL1B, IL6, and NGF in CD14(+) cells were significantly increased compared with those in CD14(−) cells (TNFA, p = 0.006; IL1B, p = .017; IL6, p = .010; NGF, p = .027). Following TNFA stimulation, NGF levels were significantly increased in CD14(−) and CD14(+) cells (CD14(−), p = .003; CD14(+), p < .001) and CGRP was significantly increased in CD14(−) IVD cells (p = .040). Following IL1B stimulation, NGF levels were significantly increased in CD14(−) cells (p = .004). CD14(+) cells had higher TNFA, IL1B, IL6, and NGF expressions than CD14(−) cells in human degenerated IVDs. Additionally, TNFA stimulation promoted the upregulation of NGF and CGRP in CD14(−) cells. These findings suggested that CD14(+) cells directly and indirectly contributed to inflammatory cytokine and pain‐related molecule expression in human degenerated IVD. CD14(+) cells might be important in the pathological mechanism of chronic discogenic LBP in humans.

BackgroundThe purpose of this study to compare glenohumeral joint motion during active shoulder axial rotation between subacromial impingement syndrome (SIS) shoulders and asymptomatic shoulders using cine-magnetic resonance imaging (cine-MRI). Measurement of glenohumeral joint motion via manual intervention does not assess the usual glenohumeral joint motion, and the glenoid surface cannot be confirmed manually. However, cine-MRI can produce clear images of glenohumeral joint rotation. Therefore, we sought to measure the active ROM of the glenohumeral rotation using cine-MRI.MethodsSeventy-three shoulders in 42 asymptomatic volunteers and 110 SIS shoulders in 103 consecutive patients were included in this study. We evaluated 36 matched pairs (72 shoulders in total) adjusting for baseline characteristics with propensity score matching method. The patients underwent cine-MRI during axial rotation of the adducted arm. During imaging, participants rotated their shoulder from the maximum internal rotation to the maximum external rotation over the first 10 s and then back to the maximum internal rotation over the subsequent 10 s. We assessed internal/external rotation, and compared the asymptomatic and SIS shoulders in this regard. Evaluation of rotation angles was performed on a series of axial images through the humeral head center.ResultsThe mean internal rotation angles of the asymptomatic and patient groups were 55° ± 10° and 41° ± 23°, respectively, (P = .002; 95% Confidence Interval [CI], 51–58 vs 33–49); the mean external rotation angles were 47° ± 15° and 21° ± 25°, respectively, (P < .001; CI, 42–52 vs 13–29).ConclusionsCompared to asymptomatic shoulders, SIS shoulders showed significantly restricted glenohumeral rotation as determined by cine-MRI. Our results suggested that the significant limitation of active glenohumeral rotation might be associated with rotator cuff dysfunction.