Figure 3.

CCL2 in neurons and CCR2/CCR4 in macrophages are required for in vitro neuron–macrophage interactions to produce proregenerative activity. A, C, E, G, I, Representative images of neurite outgrowth in DRG neuron cultures treated with CM obtained from neuron–macrophage cocultures using WT, CCL2-deficient (CCL2^−/−), or CCR2-deficient (CCR2^−/−) neurons (N) or macrophages (M). Genotype conditions for the cocultures to obtain CM were indicated at the left of the representative images. All cocultures were treated with either PBS or db-cAMP (cAMP), and the CMs were collected for 72 h. I, C 021, CCR4 antagonist, was added at a concentration of 0.1 μm during the coculture period. WT neurons were used for the neurite outgrowth assays for all conditions. DRG neurons and their neurites were visualized by immunofluorescence staining for β III tubulin. Scale bars, 100 μm. B, D, F, H, J, Quantification graphs of neurite outgrowth in the presence of CM from cultures of the different neuron–macrophage genotype combinations treated with PBS or cAMP. N = 4 independent cultures using independent CMs for each condition. ***p < 0.001 and **p < 0.01 compared with PBS values by unpaired t test.