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. 2013 Dec 10;141(1):39–51. doi: 10.1111/imm.12164

Figure 7.

Figure 7

Mycobacterium tuberculosis infected dendritic cells (DCs) exhibit a reduced ability to adhere to lung endothelial cells and migrate toward lymphatic chemokines. (a) Human microvascular lung endothelial cells (HMVLEC) were seeded in 96-well plates and activated to express adhesion molecules with tumour necrosis factor-α (TNF-α; 20 ng/ml). DCs were cultured in medium alone, infected with M. tuberculosis, treated with lipopolysaccharide (LPS; 200 ng/ml) or latex beads for 48 hr. The DCs were then harvested, syto-61-stained and placed over the HMVLEC for 30 min. Fluorescence indicative of DC adherence was measured by spectrophotometry. Data are expressed as average per cent decrease in relative fluorescent units (RFU) ± standard error relative to medium alone. Data represent three separate donors for LPS and six for all other DC treatments. Each treatment contained a replicate. Statistical significance for the 95% confidence interval was determined by a Student's t-test. (b) CCR7 on resting (medium), LPS-stimulated (200 ng/ml) and M. tuberculosis-infected (multiplicity of infection ∼ 2) DCs was measured by flow cytometry. Per cent CCR7-positive cells over four independent blood donors is shown. (c) HMVLEC were seeded on porous inserts and cultured for 48 hr. TNF-α (20 ng/ml) was added to induce expression of adhesion molecules overnight. DCs (1 × 105) that were cultured in medium alone, LPS-treated (200 ng/ml), or infected with M. tuberculosis (multiplicity of infection ∼ 2) were fluorescently stained and overlaid. The lymphatic chemokines CCL19 and CCL21 (100 ng/ml) were added to the lower chamber of the well. The cultures were incubated for 24 hr. Replicate well-readings were taken for each DC treatment and the data are expressed as the mean per cent change in relative fluorescent units (RFU) ± standard error relative to medium alone. Data represent three experiments carried out with separate donors. Statistical significance in the 95% confidence interval was determined by a Student's t-test; n.s. = not significant.