Figure 1.
Effects of CSE on the clonal growth of normal human CFU-GM, BFU-E and CFU-E in BMCs.
Bone marrow mononuclear cells from healthy volunteers were treated with different concentrations of CSE for 24 h, then the BMCs were harvested and grown in methylcellulose medium with cytokines as described in Materials and methods. At the end of the 24 h, the number of colonies per well was determined. Each point represents the mean results of three normal individuals, and each experimental point represents duplicate plates. Results are expressed as mean value ± SD (* P<0.001).
Figure 2.
Cigarette smoke extract (CSE) induced the activation of NF-κB, AKT and ERK1/2 in BMCs.
(A) BMCs were incubated with media, 5% CSE, 10% CSE, 20% CSE or 1 µg/ml LPS for 24 h, then cells were harvested and the cytoplasmic and nuclear protein fractions were separated using Pierce Nuclear and Cytoplasmic Extraction Reagent Kit (NE-PER). 30 µg of each, cytoplasmic and nuclear extracts, was analyzed by 10% SDS-PAGE and Western blotted using NF-κB p65 antibody. For loading control of cytoplasmic protein, the membrane was stripped and immunoblotted for β-actin antibody; for loading control of nuclear protein, the membrane was blotted with anti-PARP antibody. Three independent experiments were done, but one representative experiment is shown. (B) BMCs from donor 1 and donor 2 were incubated with control media, 5% CSE or 10% CSE for 6 h at which point cells were harvested and lysed. Cell lysates were separated by SDS-PAGE and immunoblotted with an anti-p-ERK antibody or anti-p-AKT (ser473) antibody, and the membranes were subsequently stripped and reprobed with anti-ERK, anti-AKT or β-actin antibodies.
Figure 3.
CSE-induced NF-κB activation is independent of the activation of PI3K/AKT and ERK1/2 in BMCs.
BMCs were pretreated with PD98059 (10 µM), BAY11-7085 (5 µM), or Wortmannin (100 nM) for 60 min, and were subsequently exposed to 10% CSE for 6 h then cells were harvested and lysed. Cell lysates were separated by SDS-PAGE and immunoblotted with an anti-p-ERK antibody, anti-p-AKT (Ser473) or anti-p-IκBα (Ser32) antibody. The membranes were then stripped and reprobed with anti-ERK, anti-AKT or β-actin antibodies.
Figure 4.
CSE-induced TLR2 and TLR3 expression in BMCs may be in part mediated through activation of ERK1/2 and NF-κB signaling cascades.
BMCs were pretreated with PD98059 (10 µM), BAY11-7085 (5 µM), or Wortmannin (100 nM) for 60 min, and then exposed to 10% CSE for 24 h, after which the cells were harvested and used for flow cytometric analysis. (A) BMCs were gated according to forward scatter (FSC) and side scatter (SSC) profiles. For cell surface staining, 7-AAD negative cells (live cells) were gated for TLRs expression analysis. Histograms show representative fluorescence intensities of TLRs; BMCs were labeled with anti-TLR2-PE, anti-TLR3-PE or anti-TLR4-APC antibodies (blue line histogram) or appropriate isotype control antibodies (red line histogram). (B) For intracellular TLR2, TLR3 or TLR4 staining, cells were washed and then fixed and permeabilized. (C) For cell surface expression of TLR2, TLR3 or TLR4, the cells were incubated with anti-TLR2-PE, anti-TLR3-PE or anti-TLR4-APC antibodies for 30 min in the dark on ice, washed and resuspended in PBS. The experiment is a representation of three independent experiments.
Figure 5.
CSE had no effect on IL-10, TNF-α, and VEGF production.
BMCs were incubated with media, 5% CSE, 10% CSE or 1 µg/ml LPS for 24 h, and the supernants were harvested and analyzed for the presence of IL-10, TNF-α and VEGF by ELISA. Results are expressed as mean value ± SD of five different normal donors. (* P<0.001).
Figure 6.
Effects of ERK1/2, AKT and NF-κB signaling pathway on CSE-induced IL-8 release.
A, BMCs were incubated with media, 5% CSE, 10% CSE, or 1 µg/ml LPS for 24 h, and cell supernatants were tested for IL-8. B, BMCs were pretreated with PD98059 (10 µM), BAY11-7085 (5 µM), or Wortmannin (100 nM) for 60 min, and subsequently were exposed to 10% CSE for 1, 6, and 24 h. The levels of IL-8 were measured in cell supernatant by ELISA. Data are mean with SEM of five different normal donors. (* P<0.01; ** P<0.001).
Figure 7.
Effects of ERK1/2, AKT and NF-κB signaling pathway on CSE-induced TGF-β1 release.
BMCs were pretreated with PD98059 (10 µM), BAY11-7085 (5 µM), or Wortmannin (100 nM) for 60 min, and subsequently were exposed to 10% CSE for 24 h. The levels of TGF-β1 were measured in cell supernatant by ELISA. Data are mean with SEM of five different normal donors. (* P<0.05; ** P<0.01; *** P<0.001).
Figure 8.
Proposed Model for Overall effects of CSE on BMCs.
CSE can inhibit the growth of CFU-E, BFU-E and CFU-GM of human bone marrow cells and induce a significant increase in the expression of TLR2, TLR3 and TLR4. This effect correlates with the activation of ERK1/2 and NF-κB signaling cascades, but not AKT, as demonstrated by specific inhibition of these pathways. The activation of these two pathways by CSE induces the release of TGF-β1 and IL-8, and these cytokines may regulate the expression of Toll receptors. Our hypothesis is that these cytokines and TLR signaling pathways are tightly integrated.