Figure 3.

LR integrity is required for LTB₄ enhancement of AM signaling and antimicrobial functions. (A) AMs were pretreated for 5 minutes with vehicle control or 5 mM MβCD, 2-βHPCD, or MαCD, followed by 5-minute incubation with or without 10 nM LTB₄. They were then incubated with IgG-RBCs (50:1) and phagocytic indices, determined as described in “Phagocytosis and bacterial killing.” Phagocytosis is expressed as the percentage of the control value in which no drugs were added, and each bar represents the mean ± SEM from 3 individual experiments, each performed in quintuplicate. *P < .05 compared with control; #P < .001 vs LTB₄-treated cells. (B) AMs were preincubated with cyclodextrin compounds as in panel A before the addition of 50:1 serum-opsonized K pneumoniae. Thirty minutes after infection, cells were incubated with 10 nM LTB₄ or vehicle. Microbicidal activity was assessed and expressed as the mean ± SEM percentage of survival of ingested bacteria from 3 individual experiments, each performed in triplicate. *P < .05 compared with control; #P < .001 vs LTB₄-treated cells. (C) AMs were pretreated for 5 minutes with vehicle control or cyclodextrins, followed by 5-minute treatment with or without 10 nM LTB₄, after which they were incubated with 30:1 IgG-RBCs for 15 minutes. Immunoblots depict phosphorylated forms of PKC-α, PKC-δ, ανδ Syk, as well as β-actin as a loading control. Results shown are representative of 3 independent experiments. (D) Relative PKC-α, PKC-δ, and Syk phosphorylation was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel C, and is expressed as a percentage of control. The intensity of phosphorylation was quantitated as the density of the phosphorylated protein band divided by that of the actin band, and this ratio was then expressed relative to that of the untreated control, which was set at 100%. The dashed line in the figure indicates lanes in the membrane that contained experimental conditions that were not pertinent and were omitted. *P < .05 vs IgG-RBC; #P < .001 vs IgG-RBC plus LTB₄.