Figure 3.

SP-A increases TLR2, but not TLR4, surface expression on MDMs. 5-day old PBMCs in Teflon wells were incubated with or without SP-A (10 μg/ml) for 2 h (A,B). Following the same protocol as in figure 1, SP-A-treated and untreated MDMs were incubated with either a human APC-conjugated TLR2 mAb or APC-conjugated subtype control mAb (A) or human PE-conjugated TLR4 mAb or PE-conjugated subtype control mAb (B). The stained cells were analyzed using flow cytometry by gating on the MDMs and the average of triplicate samples is shown in this experiment which is representative of n = 4 (A) and n = 5 (B). 5-day old MDMs in monolayer culture on glass coverslips were incubated with or without SP-A (10 μg/ml) for 2h. After washing, cells were fixed in paraformaldehyde (no permeabilization) and stained with mouse anti-human TLR2 mAb, mouse anti-human TLR4 mAb, mouse anti-human MR mAb, or subtype control mAb followed by a secondary Alexa-488-conjugated anti-mouse Ab. Glass coverslips were mounted and visualized by confocal microscopy. A representative experiment is shown in (C) [n = 3 (TLR2); n = 2 (TLR4, MR)]. Cumulative data are shown in (D). A Student t-test was performed. * p < 0.05 relative to TLR2 expression on untreated MDMs. The MR was used as a positive control.