Neutrophil migration and activation declined in response to CLEC9A-knocked down macrophages that had undergone H37Ra stimulation.

<p>The neutrophil migration levels of (A) HL-60D cells and (B) human primary PMNs were assayed using a multiwell chamber chemotaxis assay. Neutrophils were resuspended in the upper chamber of a Transwell plate and conditioned medium was added to the lower chamber. Conditioned medium was obtained from control or CLEC9A-knocked down THP-1 cells that had been treated with heat-killed H37Ra for the indicated times. Only after removal of H37Ra by centrifugation was the conditioned medium used for the migration assays. Following incubation for 90 min, the neutrophils that had migrated through Transwell membrane were counted. (C) HL-60D cells were incubated with conditioned medium from knock-down control or CLEC9A-silencing THP-1 cells with or without H37Ra treatment for the indicated times. Total RNA was extracted, reverse transcribed, and the expression of the MMP9 mRNA was measured by Q-PCR and normalized against GAPDH mRNA. The relative mRNA level in the cells incubated with conditioned medium from knock-down control THP-1 cells without H37Ra treatment (shluc CM) at 0 hour was set as 1.0. Data are mean ± SD of three separate experiments. Two-tailed multiple t-tests were performed (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01).