PMC

SP-A is a key regulator of TLR expression and signaling in macrophages. SPA binds to its receptor(s) leading to increased expression of TLR2 but not TLR4 (this report), the MR *() and SR-A ** (). Simultaneously, SP-A regulates TLR activity in response to agonists by decreasing the phosphorylation of key TLR signaling proteins, including Akt and MAP kinases. Finally, SP-A decreases the phosphorylation of IκBα and nuclear translocation of p65 which results in diminished pro-inflammatory cytokine production.

SP-A selectively regulates the phosphorylation of MAP kinases in macrophages following the addition of TLR ligands. Following the same protocol as in , treated MDMs were lysed and SDS-PAGE and Western blots were performed using pErk (A,D), pp38 (B,E), pJNK (C,F) or actin (control) Abs. Representative Western blots are shown and bar graphs generated by densitometric analysis, represent cumulative data (duplicate samples in each experiment; mean ± SEM; n = 4 for pErk, LPS and Pam₃Cys 15min incubation; n = 4 for pp38, LPS and Pam₃Cys 15min incubation; and n = 3 for pJNK, LPS and Pam₃Cys 10min incubation). A Student t-test was performed. * p < 0.05 compared to LPS and Pam₃Cys.

SP-A regulates the phosphorylation of Akt in human macrophages following the addition of TLR ligands. 5-day old MDMs were adhered to a 12-well tissue culture plate, washed, and incubated overnight in autologous serum at 37°C. Cells were incubated with or without SP-A (10 μg/ml) for 10 min. After washing, cells were incubated for an additional 10 min at 37°C to internalize any bound SP-A. LPS (30 min) or Pam₃Cys (15 min) was added to appropriate wells. MDMs were lysed and SDS-PAGE and Western blots were performed with lysates using pAkt Ab or actin Ab (control). Representative Western blots are shown (A, C) and bar graphs (B, D), generated by densitometric analysis, represent cumulative data (duplicate samples in each experiment; mean ± SEM; n = 3 LPS; n = 4 Pam₃Cys). A Student t-test was performed. * p < 0.05 compared to LPS or Pam3Cys.

Human monocytes and macrophages express TLR2 and TLR4. Peripheral blood mononuclear cells (PBMCs) were isolated from human blood and incubated in Teflon wells with autologous serum. The monocytes differentiate into macrophages (MDM) by day 5. 1-(A,E), 3-(B,F), or 5- (C,G) day old PBMCs were harvested and incubated with either an APC-conjugated human TLR2 mAb (or an APC-conjugated subtype control mAb) or a PE-conjugated human TLR4 mAb (or a PE-conjugated subtype control mAb). The stained cells were analyzed using flow cytometry by gating on the monocytes or macrophages (), and a representative experiment is shown. The number in the top right corner represents the specific mean fluorescence intensity (MFI; TLR2 or TLR4 MFI minus subtype control MFI). (D, H) Bar graph with cumulative data (triplicate samples in each experiment; n = 5 for TLR2; n = 4 for TLR4). One-way ANOVA with post Tukey test *** p < 0.005 compared to day 1 cells (± SEM).

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 , 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.

SP-A regulates steady state mRNA expression of TLR2, but not TLR4, during monocyte differentiation into macrophages. SP-A (10 μg/ml) was added to 1-, 3-, and 5-day old PBMCs in selected Teflon wells for 1, 2, 6, or 20h. PBMCs were harvested and monocytes/macrophages adhered to a 12-well tissue culture plate in autologous serum. After washing away lymphocytes, the monocytes or MDMs were lysed in Trizol and total RNA was isolated. mRNA was converted to cDNA and real time PCR was performed. TLR2 and TLR4 mRNA amplification was normalized to the β actin housekeeping gene. The fold change was determined by comparing SP-A-treated samples to samples with no SP-A. Cumulative data are shown (± SEM) [TLR2: n = 6 (d5 20h); n = 5 (d5 2h); n = 4 (d1 1h, 2h, 6h, 20h; d3 1, 2h; d5 1h, 6h); n = 2 (d3 6h, 20h)] [TLR4: n = 6 (d1 2h); n = 5 (d1 1h, 20h); n = 4 (d1 6h); n = 3 (d3 1h, 2h; d5 2h, 20h); n = 2 (d3 6h, d5 6h); n = 1 (d3 20h; d5 20h)]. Student t-test was performed. * p < 0.05 compared to day 1 monocytes

SP-A regulates the phosphorylation of IκBα in macrophages following the addition of TLR ligands. 5 day old MDMs were adhered to a 12-well tissue culture plate, washed, and incubated overnight in autologous serum at 37°C. Cells were incubated with or without SP-A (10 μg/ml) for 10 min. After washing, cells were incubated for an additional 10 min at 37°C to internalize any bound SP-A. LPS (10 min) or Pam₃Cys (5 min) was added to appropriate wells. MDMs were lysed and SDS-PAGE and Western blots were performed using pIκBα or actin (control) Abs. Representative Western blots are shown (A, D) and bar graphs (B, E), generated by densitometric analysis, represent cumulative data (duplicate samples in each experiment; mean ± SEM; n = 4, LPS 10 min incubation or n = 4 Pam₃Cys 5 min incubation). (C, F) 5 day old MDMs were adhered to a 12-well tissue culture plate, washed, and incubated overnight in autologous serum at 37°C. Cells were incubated with or without SP-A (10 μg/ml) for 10 min. After washing, cells were incubated for an additional 10 min at 37°C to internalize any bound SP-A. LPS (1 h) or Pam₃Cys (1 h) was added to appropriate wells, media was removed, and nuclear extracts were prepared. p65 nuclear translocation was measured according to the manufacturers of the colorimetric TransFactor kit. (mean ± SEM; n = 3) A Student t-test was performed. * p < 0.05 compared to LPS or Pam₃Cys

TLR2 steady state mRNA levels decrease, while TLR4 levels vary to only a small extent, as monocytes differentiate into macrophages. 1-, 3-, and 5- day old PBMCs in Teflon wells were harvested and adhered to a 12-well tissue culture plate in RPMI containing 10% autologous serum. After washing away lymphocytes, the monocytes or MDMs were lysed in Trizol and total RNA was isolated. mRNA was converted to cDNA and real time PCR was performed. TLR2 (A), TLR4 (B), and MR (C) amplification was normalized to the β actin housekeeping gene and the relative copy number was determined. A and B are representative experiments (mean ± SD, triplicate samples) and C are cumulative data (mean ± SEM) [n = 6 (TLR2, TLR4); n = 4 (MR)]. The MR was used as a positive control as a macrophage marker. One-way ANOVA with post-Tukey test. * p < 0.05 compared to day 1 monocytes. ***p < 0.005 compared to day 1 monocytes.