Figure 2

TLR4 distribution and TLR4-mediated morphological alterations in purified microglia cultures. Purified microglial cultures were treated with 1 µg/mL LPS alone or with 20 µg/mL VB3323, for 6 or 18 h. Cells were then double-stained by CD11b and TLR4. Nuclei were revealed by Hoechst 33258 dye (blue). TLR4 was expressed in basal conditions (B) and appeared mostly unaltered during LPS treatment with (H, N) or without (E, K) TLR4 antagonist. After 18 h, LPS induced morphological alterations in CD11b-positive cells (J) that were prevented by cotreatment with VB3323 (M). (P) Morphometric parameters of CD11b-positive microglia were measured with Olympus DPSoft software. The LPS-induced increase (***p < 0.001; one-way ANOVA and Tukey test) in the mean microglial cell area and perimeter was counteracted by VB3323 cotreatment. At least 120 cells for each condition were analyzed from three independent experiments. (Q) Purified microglial cultures were harvested after treatment, and cell lysates were processed by Western blot. In the autoradiograms, the anti-CD68 antibody recognizes two protein bands at different molecular weights (87–115 kDa), which were used for quantification by optical densitometry. Expression data were normalized relative to actin and are expressed as the percentage of the mean CD68 levels in untreated cells (control). Data from three independent experiments were analyzed by one-way ANOVA and Tukey test. LPS doubled the CD68 level (***p < 0.001 versus control), and cotreatment with VB3323 prevented this. There was no stimulation by LPS in TLR4^LPS-del cultures. Scale bar, 20 µm.