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. 2013 Mar 15;288(11):7875-7884.
doi: 10.1074/jbc.M112.403220. Epub 2013 Jan 29.

Mechanical stretch inhibits lipopolysaccharide-induced keratinocyte-derived chemokine and tissue factor expression while increasing procoagulant activity in murine lung epithelial cells

Sara C Sebag  1 , Julie A Bastarache  1 , Lorraine B Ware  2
Affiliations

Mechanical stretch inhibits lipopolysaccharide-induced keratinocyte-derived chemokine and tissue factor expression while increasing procoagulant activity in murine lung epithelial cells

Sara C Sebag et al. J Biol Chem. .

Abstract

Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.

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Figures

FIGURE 1.
FIGURE 1.
LPS-induced total and cell surface TF expression and mRNA levels are reduced by cell stretch. MLE-12 cells were treated with 10 μg/ml LPS for 4 h prior to stimulation with equibiaxial strain (20%) for 2 h. Cells were lysed, and proteins were resolved by SDS-PAGE. Blots were probed with anti-TF antibody (A), and then quantitative analysis of total cell-associated TF was performed as described under “Experimental Procedures” (B). In other experiments, surface expression of TF was measured by ELISA in non-permeabilized cells (C), whereas detection of TF mRNA from mRNA extracts was analyzed by RT-PCR (D). Results are means ± S.E. (n = three to four individual experiments (three wells/n)). *, p < 0.05 (versus control medium-treated cells); #, p < 0.05 (LPS-treated versus (LPS + stretch)-treated cells).
FIGURE 2.
FIGURE 2.
Cell stretch decreases LPS-mediated KC secretion and mRNA levels. MLE-12 cells were treated with 10 μg/ml LPS for 4 h prior to stimulation with equibiaxial strain (20%) for 2 h (black bars). A, cell culture supernatants were then assayed for KC by sandwich ELISA. B, RNA isolation from cell lysates was also analyzed for KC as described under “Experimental Procedures.” Results are means ± S.E. (n = three to four individual experiments (three wells/n)). *, p < 0.05 (compared with control medium-treated cells); #, p < 0.05 (LPS-treated versus (LPS + stretch)-treated cells).
FIGURE 3.
FIGURE 3.
Effects of LPS and MS on TLR4 and NF-κB activation. MLE-12 cells were treated with 10 μg/ml LPS for 4 h prior to stimulation with equibiaxial strain (20%) for 30 min (dark gray bars) or 2 h (light gray bars) before cell lysates were analyzed for TLR4 mRNA expression (A) or total cell-associated expression by ELISA (B). C, in other experiments, cells were fixed, and then surface TLR4 expression was measured. For NF-κB measurements, cell lysates were collected after 4.5 h. D, nuclear proteins were isolated and analyzed for NF-κB activation by activity ELISA. Results are means ± S.E. (n = three to four individual experiments (three wells/n)). *, p < 0.05 (compared with control medium-treated cells); #, p < 0.05 (stretch-treated versus (LPS + stretch)-treated cells).
FIGURE 4.
FIGURE 4.
Role of NF-κB activation in LPS and stretch-mediated LPS-induced TF and KC expression. MLE-12 cells were transiently transfected with either the plasmid encoding the IκBα-DN cDNA or the empty vector (pBSII-KS/Asc) 48 h prior to exposure for 4 h to 10 μg/ml LPS. Cells were then exposed to equibiaxial strain (20%) for 2 h (black bars). RNA isolated from cell lysates was analyzed for TF (A) or KC (B) as described under “Experimental Procedures.” In addition, cell culture supernatants were assayed for KC by sandwich ELISA (C). Results are means ± S.E. (n = two to four individual experiments (three wells/n)). *, p < 0.05 (compared with control cells or LPS-treated cells (bars)).
FIGURE 5.
FIGURE 5.
Mechanotransduction effects on F-actin microfilament organization in unstretched and stretched MLE-12 cells. Cells were grown on flexible silicone membranes, fixed, and stained with fluorescein isothiocyanate-phalloidin. Arrows point to linear densely labeled fibers in unstretched control MLE-12 cells (A), linear densely labeled fibers in cells exposed to 10 μg/ml LPS for 6 h (B), short bundled fibers in cells subjected to 20% MS for 2 h (C), and linear less defined fibers in cells exposed to 10 μg/ml LPS for 4 h prior to 20% MS for 2 h (D) (magnification ×40).
FIGURE 6.
FIGURE 6.
EtOH inhibits LPS-mediated surface TLR4 and KC release. MLE-12 cells were pretreated for 10 min with 0.4% (w/v) EtOH before exposure to 10 μg/ml LPS for 4.5 h. A, surface expression of TLR4 was examined on fixed non-permeabilized cells. B, cell culture supernatants were assayed for KC by sandwich ELISA. *, p < 0.05 (compared with control medium-treated cells or LPS-treated cells (brackets)).
FIGURE 7.
FIGURE 7.
Cell stretch augments surface TF activity without modifying LPS-mediated clot time. MLE-12 cells were treated with 10 μg/ml LPS for 4 h prior to stimulation with equibiaxial strain (20%) for 2 h (black bars). Cell lysates were then assayed for surface TF activity (A), whereas culture supernatants were assayed for clot time (B) as described under “Experimental Procedures.” Results are means ± S.E. (n = three to four individual experiments (three wells/n)). *, p < 0.05 (compared with control medium-treated cells); #, p < 0.05 (LPS-treated versus (LPS + stretch)-treated cells).
FIGURE 8.
FIGURE 8.
MS-induced increase in LPS-induced cell surface TF activity is not mediated through calcium signaling. MLE-12 cells were pretreated with 20 μm BAPTA-AM for 30 min prior to stimulation with 10 μg/ml LPS for 4 h. Cells were then stretched at 20% for 2 h. As a vehicle control, cells were incubated with 0.01% Me2SO. Cells were then assayed for TF activity. Results are means ± S.E. (n = three individual experiments (three wells/n)).
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