Hyperosmolarity invokes distinct anti-inflammatory mechanisms in pulmonary epithelial cells: evidence from signaling and transcription layers

Abstract

Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. NaCl or sorbitol HOsmolarity (HOsm)inhibit chemokine secretion similarly, depending on stimulus.

RANTES (A) and IP-10 (B) release was inhibited by both HTS and SOR when stimulated by TNFα or IL-1β. HTS and SOR inhibited MCP-1 (C) release only when stimulated by TNFα but not by IL-1β. HOsm did not inhibit IL-6 (D) release with either stimulus. No significant differences were seen between HTS and SOR groups. Chemokine production was measured at 18 hours when treated with TNFα or IL-1β in the presence of 400 mOsm HTS or Sorbitol.

Figure 2. HOsm inhibits TNFα or IL-1β stimulated mRNA for RANTES (A), IP-10 (B) and MCP-1 (C) chemokines in parallel with overall chemokine release levels.

mRNA levels were measured following stimulation for 6 hours and normalized to constitutive GAPDH production after treatment with TNFα or IL-1β in the presence of 400 mOsm HTS or Sorbitol. No significant differences were seen between any of the HTS and SOR groups.

Figure 3. HOsm inhibits translocation of NF-κB into the nucleus induced by TNFα stimulation but not by IL-1β stimulation.

Indirect immunofluorescent staining was used to examine the intracellular location of the p65 subunit of NF-κB following 30 minutes of TNFα or IL-1β stimulation in the presence of isotonic (upper panels) or 400 mOsm HTS (SOR group not shown but appeared similar to HTS group). Cell structure is depicted in red (WGA glycoprotein stain), nuclei in blue (DAPI) and NF-κB in green. NF-kB translocation into the nucleus is seen in TNFα, IL-1β treated cells in isotonic medium. Under hyperosmolar conditions, translocation (thick arrows) stimulated by TNFα (but not IL-1β) is diminished (thin arrows). This figure is representative of three separate experiments. The bottom panel shows that HOsm does not significantly affect cell viability. Viabilty of pulmonary epithelial cells measured by MTT assay (absorbance) at 18 hours in the presence of 400 mOsm HTS or SOR. Cells were killed by heat treatment, at 90°Cfor 30 minutes prior to assay.

Figure 4. HOsm inhibits NF-κB activation by TNFα but not IL-1β stimulation.

The time course of NF-κB consensus binding of the p65 subunit in nuclear fraction of cells stimulated by TNFα (A) or IL-1β (B) in the presence of 400 mOsm HTS or SOR (n = 5). Activation assayed by DNA binding to consensus sequence; * p

Figure 5. HOsm inhibits phosphorylation of I-κBα induced by both TNFα and IL-1β.

Phosphorylation of I-κBα is inhibited at 10 minutes in cells stimulated with TNFα or IL-1β (10 ng/ml) by either 400 mOsm HTS or SOR treatment. The immunoblot is representative of five separate experiments. LC represent the loading control obtained by Ponceau-S stain of the nitro cellulose membranes. Phosphoprotein ELISA time course of cells stimulated by TNFα (B) or IL-1β (C) (both 10 ng/ml), in the presence of 400 mOsm HTS or Sorbitol; normalized for phosphoprotein/total protein levels. Phosphorylation of I-κBα initially diminished with either TNFα (B) or IL-1β (C) stimulation when treated with HOsm; however, I-κBα phosphorylation increases at 30 minutes with HOsm over isotonic treatment with either stimulus; * p

Figure 6. HOsm inhibits IRF-1 localization to the nucleus with either TNFα or IL-1β stimulation.

IRF-1 in the nuclear extract of cells stimulated by TNFα or IL-1β in the presence of 400 mOsm HTS or sorbitol was examined by immunoblotting (A), figure is representative of three separate experiments. The gels were digitized and quantitated for nuclear IRF-1 after TNFα (A) or IL-1β stimulation (C); * p

Figure 7. HOsm does not significantly affect cell volume.

Top left panel shows the mean total cell volume of pulmonary epithelial cells measured by immunofluorescent microscopy is not changed in the presence of 400 mOsm HTS or SOR after 18 hours of incubation (n = 3). The panel on top right shows mean (± sem) forward scatter of pulmonary epithelial cells measured by flow cytometry is also unchanged in the presence of isotonic and 400, 500, and 600 mOsm solutions of media plus HTS, after 18 hours of incubation (n = 3).