Fig. 5: CCL2 is released by macrophages and neutrophils in vivo and in vitro with IL-33 stimulation.
From: IL-33/ST2 drives inflammatory pain via CCL2 signaling and activation of TRPV1 and TRPM8
A Co-localization of CCL2 with neutrophil marker (MPO) in the hindpaw skin at 4 h after IL-33 injection (n = 3 mice, scale bar: 50 μm). Quantification of (A) for neutrophil infiltration (B) and percentage of neutrophils expressing CCL2 (C) (n = 3 mice). D Co-localization of CCL2 with macrophage marker(F4/80) in the hindpaw skin at 4 h after IL-33 injection (n = 3 mice, scale bar: 50 μm). Quantification of (D) for macrophage infiltration (E) and percentage of macrophages expressing CCL2 (F) (n = 3 mice). Depletion of macrophages by intraperitoneal injection of Clodronate liposome (i.p: 200 μl, once a day for 2 days) attenuates IL33-induced mechanical (G) and thermal (H) hypersensitivity (n = 5-6 mice). Depletion of macrophages by intraplantar injection of Clodronate liposome (i.p: 200 μl) reverses gene expression of CCL2 (I) and ST2 (J) (n = 5-6 mice). K Infiltration of macrophages and neutrophils after CCR2 blocking (n = 3 mice, scale bar: 100  μm). Quantification for infiltration of macrophages (L) and neutrophils (M) after CCR2 blocking in the IL-33 model. N Western blots and quantification of protein levels of MPO in the hindpaw of INCB3344-treated mice at 4 h after IL-33 injection. (n = 6 mice). O IL-33 stimulation increases CCL2 secretion from the Raw264.7 cells in 24 h (n = 3 trials). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05. #represents vehicle vs drug treatment. Unpaired t-test for (B, C, E, F, O), One-way ANOVA with Bonferroni’s post-hoc test for (I, J, L–N), Repeated measures for two-way ANOVA with Bonferroni’s post-hoc test for (G–H). The skin layers are stratum corneum (SC), epidermis (Epi) and Dermis.
