Figure 8. LPS-regulation of DAT activity is CD14 dependent but TLR4 independent.

(A) Representative 40× images of macrophages that were unstimulated, LPS-stimulated, or cotreated with LPS and CLI095 (TLR4 antagonist), LPS and a neutralizing monoclonal antibody against CD14 (AbCD14), or LPS and Iaxo102 (a CD14 antagonist) and assayed for DAT-mediated IDT307. (B) DAT-dependent IDT307 uptake was calculated via blocker subtraction showing decreased DAT-dependent uptake in LPS-stimulated macrophages. Cotreatment with LPS and either AbCD14 or Iaxo102 prevented the LPS-induced reduction of DAT-dependent IDT307 uptake. (C and D) Quantification of rate (C, average slope) and magnitude (D, AUC) of DAT-dependent IDT307 uptake shown in B showed a significant decrease in the DAT-mediated IDT307 uptake following LPS stimulation (average slope, P = 0.005; AUC, P = 0.01). CLI095 did not prevent the LPS reduction of DAT-dependent uptake (AUC, P > 0.9), whereas cotreatment with either mAbCD14 or CD14 antagonist Iaxo102 rescued the DAT-dependent uptake back to unstimulated levels (average slope, P = 0.05; AUC, P = 0.06). Data are from n = 6–19 experiments/group, and statistical analysis was performed using Kruskal-Wallis tests with Dunn’s test for multiple comparisons. (E) Representative 60× images of cultured human macrophages that were either unstimulated, LPS-stimulated, or treated with LPS and mAbCD14 and labeled for CTxB-555 and DAT. (F) Quantifying colocalization between CTxB55 and DAT using Pearson’s correlation coefficient shows that AbCD14 reversed the increased DAT–CTxB-555 colocalization back to unstimulated levels (P = 0.03, Mann Whitney U test). Data in E and F are from n = 4–8 experiments/group. (G) The DAT-dependent dopamine efflux measured via simultaneous whole-cell patch-clamp and amperometry as in Figure 7 shows no significant increase in basal dopamine efflux between unstimulated macrophages and macrophages treated with LPS + mAbCD14 (P = 0.7, Welch’s 2-tailed t test, n = 5 experiments/group).