Bone Marrow Mononuclear Cells Up-Regulate Toll-Like Receptor Expression and Produce Inflammatory Mediators in Response to Cigarette Smoke Extract

Several reports link cigarette smoking with leukemia. However, the effects of cigarette smoke extract (CSE) on bone marrow hematopoiesis remain unknown. The objective of this study was to elucidate the direct effects of cigarette smoke on human bone marrow hematopoiesis and characterize the inflammatory process known to result from cigarette smoking. Bone marrow mononuclear cells (BMCs) from healthy individuals when exposed to CSE had significantly diminished CFU-E, BFU-E and CFU-GM. We found increased nuclear translocation of the NF-κB p65 subunit and, independently, enhanced activation of AKT and ERK1/2. Exposure of BMCs to CSE induced IL-8 and TGF-β1 production, which was dependent on NF-κB and ERK1/2, but not on AKT. CSE treatment had no effect on the release of TNF-α, IL-10, or VEGF. Finally, CSE also had a significant induction of TLR2, TLR3 and TLR4, out of which, the up-regulation of TLR2 and TLR3 was found to be dependent on ERK1/2 and NF-κB activation, but not AKT. These results indicate that CSE profoundly inhibits the growth of erythroid and granulocyte-macrophage progenitors in the bone marrow. Further, CSE modulates NF-κB- and ERK1/2-dependent responses, suggesting that cigarette smoking may impair bone marrow hematopoiesis in vivo as well as induce inflammation, two processes that proceed malignant transformation

Effects of CSE on the clonal growth of normal human CFU-GM, BFU-E and CFU-E in BMCs.

<p>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).</p

CSE-induced NF-κB activation is independent of the activation of PI3K/AKT and ERK1/2 in BMCs.

<p>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.</p

CSE had no effect on IL-10, TNF-α, and VEGF production.

<p>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).</p

Effects of ERK1/2, AKT and NF-κB signaling pathway on CSE-induced TGF-β1 release.

<p>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).</p

CSE-induced TLR2 and TLR3 expression in BMCs may be in part mediated through activation of ERK1/2 and NF-κB signaling cascades.

<p>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.</p

Proposed Model for Overall effects of CSE on BMCs.

<p>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.</p