Figure 2
Myelin potentiates pro-inflammatory macrophage activation in vitro. Bone marrow derived macrophages (BMDMs) were utilized to determine the function outcomes resulting from myelin debris application under three distinct activation states: M1 (IFN-γ and LPS), M2 (IL-4), or CTL (media, i.e. unstimulated). (A) Myelin stimulation significantly increased pro-inflammatory IL-12 cytokine levels relative to M1 alone (***p < 0.001), however myelin did not increase IL-12 in CTL or M2 treated cells. M1 stimulations had increased IL-12 relative to CTL or M2 (###p < 0.001). (B) Following the stimulation BMDMs were washed and treated with 2 µM H2DCFDA for 25 min. Myelin stimulation increased ROS relative to M1 alone (***p < 0.001), however myelin did not increase ROS in CTL or M2 treated cells. M1 stimulations had increased ROS relative to CTL or M2 (###p < 0.001). (C) Nitric oxide levels in treated supernatants were examined using the Griess assay of nitrite accumulation. Myelin stimulation increased nitric oxide relative to M1 alone (***p < 0.001), however myelin did not increase nitric oxide in CTL or M2 treated cells. M1 stimulations had increased nitric oxide relative to CTL or M2 (###p < 0.001). (D) Cell lysates were tested for arginase enzymatic activity. M2 macrophages had higher arginase activity relative to M1 and CTL (p < 0.001 and p < 0.0001, respectively). The addition of myelin however did not significantly alter arginase activity in any of stimulations tested. (E) Supernatants were applied to a neuron culture (N2A cells) for 24 h to determine supernatant toxicity. Myelin stimulation increased neurotoxicity relative to M1 alone (*p < 0.05), however myelin did not increase neurotoxicity in CTL or M2 treated cells. M1 stimulations had increased ROS relative to CTL or M2 (###p < 0.001). Representative of 3 independent biological replications of both BMDMs and myelin source. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001 mean ± SEM.
