Figure 6

Macrophage M1 polarization in degenerated NP tissue.

(A) Immunofluorescence double-labeling of iNOS or CD86 (green) and CD11b (red) in the degenerated NP tissue. Macrophage in degenerated NP tissue displayed positive CD86 and iNOS staining, which were M1 marker. Moreover, M2 marker (Arginase-1 and CD206) were negative. Bar = 50 μm. (B,G) Western blot analysis of macrophage treated with CM of primary NP cells from degenerated nucleus pulposus samples of MRI grades II (n = 3), III (n = 3), IV (n = 3) and V (n = 3). The M1 marker iNOS and M2 marker Arginase-1 were analyzed. Blots were was analysed by densitometry (*P < 0.05). (C,H) Macrophage treated with CM of primary human NP cell (grade III) was transfected with lentivirus expressing sh-p38α or β or γ or δ or sh-C immediately. The M1 marker iNOS and M2 marker Arginase-1were analyzed. Blots were was analysed by densitometry (n = 3, respectively, *P < 0.05). (D) flow cytometric analysis of cytokines in CM from primary human NP cells (grade III) which were transfected with lentivirus expressing sh-p38α or β or γ or δ or sh-C immediately. The expression of GM-CSF, IFNγ, MCP-1, IL-1β and TNF-α were analyzed (n = 3, respectively, *P < 0.05). (E,I) macrophage were treated with CM including neutralization antibodies against GM-CSF or IFNγ. The M1 marker iNOS and M2 marker Arginase-1were analyzed. Grade II sample served as a normal control. Blots were was analysed by densitometry (n = 3, respectively, *P < 0.05). (F) Quantification of the percent of CD11b positive cells expressing M1 marker(iNOS and CD86) or M2 marker(Arginase-1 and CD206) (n = 150, *P < 0.05).