Figure 4

Cigarette smoke extract inhibits CCR7 expression but does not suppress migration of dendritic cells towards a CCR7 ligand. A) Human dendritic cells were incubated for 18 hours in the presence or absence of 1% CSE. The NAC group was pretreated with 1 mM NAC for 60 minutes prior to the addition of 1% CSE and LPS. The nicotine group was pretreated with 1000 ng/ml nicotine for 60 minutes prior to the addition of LPS. Surface CCR7 expression was subsequently determined with flow cytometry. The numbers in the quadrants refer to the percentage of cells in that quadrant. Data shown is representative of 3 independent experiments. B) Human dendritic cells (1 × 10⁶/ml in 5 ml complete media) were incubated overnight with or without 2% CSE, or 1 mM NAC. Whole cellular CCR7 protein levels were determined following 18 hours using immunoblotting. β-Actin protein levels were determined as an internal control to ensure equal protein loading. Data shown is representative of 3 independent experiments. C) Half a million dendritic cells under different conditions suspended in 0.5 ml RPMI without serum, placed in cell culture inserts, and inserted into wells containing 0.5 ml of RPMI with 10% fetal calf serum and 10 ng/ml of CCL21. The number of migrated cells in the lower chamber was determined using flow cytometry. Data shown Data shown are means ± SEM. Data shown is representative of 4 independent experiments.