Hypertonic saline attenuates TNF-alpha-induced NF-kappaB activation in pulmonary epithelial cells

Abstract

Resuscitation with hypertonic saline (HTS) attenuates acute lung injury (ALI) and modulates postinjury hyperinflammation. TNF-alpha-stimulated pulmonary epithelium is a major contributor to hemorrhage-induced ALI. We hypothesized that HTS would inhibit TNF-alpha-induced nuclear factor (NF)-kappaB proinflammatory signaling in pulmonary epithelial cells. Therefore, we pretreated human pulmonary epithelial cells (A549) with hypertonic medium (180 mM NaCl) for 30 min, followed by TNF-alpha stimulation (10 ng/mL). Key regulatory steps and protein concentrations in this pathway were assessed for significant alterations. Hypertonic saline significantly reduced TNF-alpha-induced intercellular adhesion molecule 1 levels and NF-kappaB nuclear localization. The mechanism is attenuated phosphorylation and delayed degradation of IkappaB alpha. Hypertonic saline did not alter TNF-alpha-induced p38 mitogen-activated protein kinase phosphorylation or constitutive vascular endothelial growth factor expression, suggesting that the observed inhibition is not a generalized suppression of protein phosphorylation or cellular function. These results show that HTS inhibits TNF-alpha-induced NF-kappaB activation in the pulmonary epithelium and, further, our understanding of its beneficial effects in hemorrhage-induced ALI.

Figures

FIG. 1. Pulmonary epithelial cell viability and constitutive VEGF expression are unchanged by HTS

A, Metabolism of tetrasodium salt to colored formazan salt crystal is quantified by solubilization and spectrophotometry. After 18 h of incubation, with and without HTS, the measured absorbance values are not significantly different. Data from three separate experiments are averaged and presented as SEM. B, Supernatant concentrations of VEGF were measured by flow cytometry. Resting cells produce VEGF at a steady rate over 12 h of incubation. Hypertonic saline does not alter the rate of expression or the amount produced, indicating that protein synthesis and cellular function are intact. Data from three separate experiments are averaged and presented as SEM.

FIG. 2. Hypertonic saline decreases TNF-α–induced ICAM expression

A, Western blot of whole cell lysate does not find any ICAM-1 in unstimulated cells. TNF-α markedly increases ICAM-1 amounts. Hypertonic saline pretreatment alone does not induce ICAM-1 production. Hypertonic saline largely decreases the amount of TNF-α–induced ICAM-1 production. Vascular cell adhesive molecule 1 amounts are unchanged by TNF-α or HTS. B, Hypertonic saline significantly decreases TNF-α–induced ICAM-1 production. Densitometry data from four separate experiments are presented as SEM. Both the TNF-α–only group and the HTS/TNF-α group are significantly different from all other groups (*#P < 0.001).

FIG. 3. Hypertonic saline significantly attenuates TNF-α–induced NF-κB nuclear localization

A, Immunofluorescent images show the intracellular localization of NF-κB. The nuclear stains (blue) are omitted from the bottom row. In unstimulated cells, most of the NF-κB p65 subunit (green) is sequestered in the cytoplasm (first column). Hypertonic saline pretreatment does not significantly change the intracellular location of the p65 subunit (second column). TNF-α causes the p65 subunit to accumulate in the nucleus at 30 min (third column). Hypertonic saline attenuates TNF-α– induced NF-κB nuclear translocation (fourth column) with more of the p65 subunit left within the cytoplasm. The orange bar equals 10 μm. All images acquired at 40 magnification. B, Hypertonic saline reduces nuclear NF-κB MFI. Nuclear staining was used to produce masks to measure the relative MFI of the p65 subunit within the nuclei. The MFI of HTS pretreatment–only cells are not different from controls. TNF-α causes a 4-fold increase that HTS reduces by more than 40%. Data from four separate experiments are presented as SEM. Both the TNF-α–only group and the HTS/TNF-α group are significantly different from all other groups (*#P < 0.001).

FIG. 4. Hypertonic saline alters IκBα phosphorylation and degradation dynamics

A, Western blot of whole cell lysate does not find phosphorylated IκBα in unstimulated cells. TNF-α provokes a marked increase in phosphorylated IκBα at 5 min that degrades by 10 min. Hypertonic saline significantly decreases the amount of TNF-α–induced phosphorylation that degrades by 10 min as in controls. Hypertonic saline does not change total IκBα concentration in unstimulated cells. TNF-α stimulates IκBα degradation at 5 min that is complete by 10 min. Hypertonic saline inhibits degradation with a significant amount remaining at 10 min and a small but detectable amount remaining at 30 min. B, Hypertonic saline significantly decreases the amount of phosphorylated IκBα at 5 min of TNF-α stimulation. Densitometry data from four separate experiments are presented as SEM (*P < 0.001). C, TNF-α stimulates near complete total IκBα degradation at 10 min. Hypertonic saline delays the same level of degradation to approximately 30 minutes. Hypertonic saline significantly decreases total IκBα degradation at 10 min (*P < 0.001). Densitometry data of four separate experiments are presented as SEM.

FIG. 5. Hypertonic saline does not alter cell surface localization or whole cell expression of TNF R1

A, Flow cytometry detection of cell surface TNF R1 finds the MFI of cells incubated in HTS for 30 min unchanged from controls. Data from three separate experiments are averaged and presented as SEM. B, Flow cytometry detection of cell surface TNF R1 produces similar histograms from both controls and HTS. This similarity is confirmed by an overlay of the histograms. C, Western blot of whole cell lysate finds TNF R1 levels of cells incubated in HTS for 30 min unchanged from controls.

FIG. 6. TNF-α–induced p38 MAPK phosphorylation is unchanged by HTS

A, Western blot of whole cell lysate does not find phosphorylated p38 MAPK in unstimulated cells. TNF-α provokes a marked increase in phosphorylated p38 levels at 5 min that continues to increase and plateau at 10 and 30 min. Hypertonic saline pretreatment alone before TNF-α stimulation causes a low level of p38 phosphorylation. TNF-α provokes proportional increases in phosphorylated p38 levels at 5, 10, and 30 min while in the presence of HTS. B, Densitometry data confirm that HTS alone causes a measurable increase in phosphorylated p38. At each time point, the paired groups are significantly different. However, the similarly shaped density curves show that TNF-α induces the same relative increase in both the control and HTS groups. Densitometry data of four separate experiments are averaged and presented as SEM (*P < 0.01). C, Subtracting the average densitometry value of the initial HTS-only group from the remaining HTS groups yields a curve that is identical to that of TNF-α only. The paired time points from each curve are not significantly different with overlapping error bars. Densitometry data of four separate experiments are presented as SEM.