Hyperosmolarity Invokes Distinct Anti-Inflammatory Mechanisms in Pulmonary Epithelial Cells: Evidence from Signaling and Transcription Layers

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.


Introduction
Clinically, hypertonic saline (HTS) has been used to treat post-traumatic acute lung injury (ALI) [1,2,3] and the acute respiratory distress syndrome (ARDS) [4,5]. Following hemorrhagic shock, rats treated with nebulized HTS showed decreased lung leak [6]. HTS directly modulates human neutrophil [7], T-cell [2] and macrophage [8] responses, while HTS resuscitation decreases alveolar macrophage activation and neutrophil recruitment into the lung [1,2,8]. However, the effects of HTS on pro-inflammatory activation of lung epithelial cells have not been defined. Tumor necrosis factor-a (TNFa) and interleukin 1b (IL-1b) are pro-inflammatory cytokines [9,10] whose signaling pathways are expressed ubiquitously in human cells and induce expression of multiple proinflammatory cytokines and chemokines. Both TNFa and IL-1b play important roles in pulmonary inflammation and have been implicated in the development of ALI following hemorrhagic shock. Inhibition of these cytokines has been previously shown to diminish lung inflammation [11,12,13,14,15,16].
Nuclear factor-kB (NF-kB) represents a potential target for hyperosmolar (HOsm) modulation of inflammation [8,17]. Dimeric NF-kB, comprised of either p65, p50 or both, plays a central role in the transcription of several genes involved in inflammation in both animals and humans [18,19,20,21]. In animals, hemorrhagic shock induced NF-kB activation in the lung and increased TNFa and IL-1b production [13,22]. In humans, clinical studies of ARDS have documented increased NF-kB activation [22,23].
To investigate HTS inhibition of ALI and to investigate if the effects were Na + related or true HOsm effects, HTS was compared to a non-ionic, non toxic, clinically readily available substance at similar HOsm: sorbitol. Investigation into HOsm therapy for post-traumatic ALI may elucidate the mechanisms of HTS as a clinical therapy. Therefore we hypothesize that HOsm inhibits TNFa and IL-1b mediated signaling at multiple levels, decreasing cytokine/chemokine production in pulmonary epithelium.

Materials and Methods
Cell culture and treatment A549 pulmonary epithelial cells (CCL-185, ATCC, Manassas, VA, USA) were cultured in Ham's F12 medium with 10% fetal bovine serum and 100 IE/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown to 80-90% confluence for viability measurements (MTT, Cell Proliferation Kit, Roche Biochemicals, Indianapolis IN), cytokine, Western blotting, and phosphoprotein measurement and to 70-80% confluence for indirect immunofluorescent staining and DNA binding assays. A549 were cultured and treated in suspension, with end over end rotation, for cell size measurements by forward scatter (CyAn ADP analyzer,Beckman-Coulter, Indianapolis IN). Hyperosmolar treatments consisted of a 30 minute pre-treatment followed by continued immersion in media supplemented to a final osmolarity of 400¡5 mOsm with either sorbitol or NaCl, confirmed by a freezing point depression osmometer (Advanced Instruments, Inc, Norwood, MA, USA). For the study of the effects of osmolarity on cell size, in suspended cells, we performed a dose response to media ranging from iso-osmolar (290 mOsm) up to 600 mOsm with continuous immersion for 18 hours. HOsm concentrations were based upon previously measured concentrations following IV injection of HTS [27]. Cells were treated with isotonic (290 mOsm) medium (controls) or 400 mOsm (HTS, Sor) media, 10 ng/ml of TNFa or 10 ng/ml IL-1b (Sigma-Aldrich, St. Louis, MO, USA).

Cytokine and phosphoprotein analysis
Cytokine analyses on supernatants were completed with a multiplex bead-based assay (Bio-Rad Laboratories, Hercules, CA, USA) these data were confirmed via sandwich ELISAs (R&D Systems, Minneapolis, MN, USA). Whole cell lysates were generated using M-PER digestion buffer (Thermo Fisher Scientific Inc, Waltham, MA, USA) and were analyzed for cytokine production using identical ELISAs. Protein phosphorylation was measured using a cell-based colorimetric ELISA system which compared phosphorylated (Ser32) to total levels of I-kBa, phosphorylated (Ser73) to total levels of c-Jun and dual phosphorylated to total p38MAPK (SABiosciences, Frederick, MD, USA).

mRNA Expression
A549 cells grown in 12-well plates were harvested and total RNA extracted using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. mRNA levels were quantified by two-step real-time PCR. Briefly, 2.5 mg of total RNA per sample was incubated with 3 mg of random primers (Invitrogen Corp., Carslbad, CA, USA) at 70˚C for 2 minutes. The reaction was chilled and incubated with reverse transcriptase Superscript II (Invitrogen Corp.) and 1.25 mM dNTP (Applied Biosystems, Foster City, CA, USA) at 42˚C for 30 minutes and a third incubation at 70˚C. Equal amounts of cDNA were used for the real-time PCR. All reagents, primers and probes are validated Taqman expression assays purchased from Applied Biosystems (RANTES CCL5: Assay ID Hs00174575_m1 cat# 4453320, spans exons 2and 3 with an amplicon length of 63 bp; IP10 CXCL10: Assay ID Hs00171042_m1 cat # 4331182, spans exon 1-2 amplicon length 98 bp; MCP-1 CCL2: Assay ID Hs00234140_m1 Cat. # 433118, spans exon 1-2 amplicon length 99 bp; GAPDH: Assay ID Hs01922876_u1Cat. # 4331182, spans exon 7-7 amplicon length 139 bp). All reactions were performed and analyzed on an iCycler iQ5 multicolor real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). Levels of RANTES, IP-10 and MCP-1 in each sample were normalized and expressed as fold change over GAPDH.

Indirect immunofluorescent stain
A549 cells were grown and treated on glass slides then fixed and permeabilized with 70% acetone/30% methanol solution at 220˚C for 10 minutes. The slides were air-dried, blocked with 10% donkey serum in PBS (60 min) (Jackson Immuno Research Laboratories, West Grove, PA, USA), and incubated overnight incubation at 4˚C with (1:50) rabbit polyclonal anti-p65 (NF-kB) antibody in 1% BSA/PBS (Santa-Cruz Biotechnology Inc, Santa Cruz, CA, USA). After washing, the slides were incubated for 1 hour at room temperature with (1:100) Alexa fluor 488 conjugated donkey anti-rabbit antibody, and Alexa fluor 647 conjugated WGA to stain the membranes ((Molecular Probes Invitrogen Detection Technologies, Eugene, OR, USA). The chromatin stain DAPI was employed to demarcate the nuclei (Sigma-Aldrich, St. Louis, MO, USA). Images were acquired with a Leica DMRXA fitted with a Cooke CCD SensiCam using Chroma Sedat filters with single excitation and emission filter cubes. The three channel images were then digitally processed, for calculations of fluorescent mean intensities and for mean cell size (in voxels) measurements, using Intelligent Imaging Innovations Slidebook 4.1 software (Intelligent Imaging Innovations Inc, Denver, CO, USA).

Transcription factor DNA binding assay
Activity assays were performed on nuclear fractions of A549 cells and were added at 5 mg of nuclear protein per sample to plates containing transcription factor consensus sequences. Active nuclear forms of the transcription factor studied (NF-kB p65, NF-kB p50 or STAT-1) were detected per the manufacturer's protocol (Active Motif, Carlsbad, CA, USA)

Statistical Analysis
Data are represented as mean ¡ standard error of the mean. Comparisons between two groups were assessed by Student's t-test, and those between three or more groups were assessed by analysis of the variance (ANOVA) using JMP 5.0 software (SAS Institute, Inc. Cary, NC USA followed by Bonferreni Dunn correction for multiple comparisons. The comparisons depicted in Table 1 were used to select the candidates for the subsequent specific analysis. The false discovery rate (FDR) was set at 0.15; the q value (corrected level of significance by the Benjamini-Hochberg multiple comparison adjustment method [28]) was calculated and established at ,0.018. Variables associated with a significant pvalue (p,0.018) by a paired t-test were selected to be further analyzed.

Hyperosmolarity inhibits chemokine release and production
To investigate potential anti-inflammatory effects of HOsm on A549 cells, the stimulated production of inflammatory cytokines and chemokines were measured in response to TNFa and IL-1b. Table 1 shows a panel of 27 agents that have been identified in inflammatory lung injury [11,12,29,30,31], can be measured simultaneously by available multiplexed assay, and sorted by loci. This panel covers the production of various CC and CXC chemokines, growth factors and interleukins that could signal other cells in the pulmonary alveoli such as macrophages, neutrophils and T-cells, disposing toward TH1, 2 or 17 phenotypes. Previous time course experiments demonstrated significant cytokine and chemokine production at 18 hours (data not shown). TNFa induced synthesis and release of six pro-inflammatory cytokines/chemokines, of which three (IP10, MCP-1 and RANTES) were inhibited by HOsm pretreatment. Ten cytokines were stimulated by IL-1b, of which 4 were significantly inhibited by HOsm pretreatment: IP-10, RANTES, G-CSF and MIP-1. Specifically, IL-1b stimulation accounted for greater increases in chemokines and cytokines compared to TNFa except for IP-10 and MCP-1. Moreover, IL-1b stimulation caused increased release of CXCL, which resides on Chr 4, and 3/5 CCL which appear on Chr 17. VEGF was secreted constitutively (about 10 fold over baseline in 18 hrs). HTS and sorbitol were similar in their inhibitory effects on cytokine/chemokine synthesis and release.
ELISAs confirmed the four observed patterns of response to TNFa and IL-1b (  Fig. 1). The inhibitory effects of both HTS and SOR were similar on TNFa and Table 1. Hyperosmolarity inhibits secretion of archetypal cytokines induced by TNFa or IL-1b.

Common name
Other name Chromo-some   IL-1b stimulated increases in RANTES/CCL5 concentration, with 90-95% inhibition compared to isotonic media, p,0.005 [ Fig. 1a]. The 40-65% inhibition of IP-10/CXCL10 was proportionate for both stimuli despite different levels of expression (p,0.05) [ Figure 1b]. TNFa-induced MCP-1/CCL2 release was inhibited 40-50% by HOsm (p,0.05) [ Figure 1c], but IL-1b-induced MCP-1release remained was unaffected. IL-6 synthesis and release was induced by both stimuli and unaffected by HOsm [ Figure 1d]. IL-1b stimulation resulted in production of MIP-1b/CCL4 and G-CSF/CSF 3, which were inhibited by HOsm. Furthermore HOsm inhibited both IL-1b-induced IFN-c (but not TNFa) and TNFa production. Both stimuli increased production of IL-6, VEGF, IL-8, and IL-9 and were not affected by HOsm [ Table 1]. No significant differences were noted between HTS and SOR treatments; thus, HOsm appears to inhibit production of multiple cytokines/chemokines dependent upon the stimulus employed. Lastly, because decreased cytokine levels in the supernatant could be due to inhibition of release rather than inhibition of synthesis or increase in degradation, ELISAs for  (TABLE 2) demonstrated a similar pattern as the supernatants, indicating the observed inhibition is likely due to either to decreased synthesis or to increased degradation.

Hyperosmolar treatment affects mRNA translation
To determine if HOsm inhibition was due to decrease in mRNA expression, the intracellular mRNA levels of RANTES, IP-10, and MCP-1 were measured. These mRNA levels matched the released concentrations of these chemokines into the supernatant for both TNFa and IL-1b indicating that the inhibition may be upstream of mRNA [ Figure 2].

Hyperosmolarity decreased p65 translocation with TNFa but not IL-1b stimulation
HOsm treatment decreased p65 sub-unit of NF-kB translocation into the nucleus following TNFa but not IL-1b stimulation [ Figure 3].
Hyperosmolarity decreased NF-kB activation with TNFa but not IL-1b stimulation To quantify and further explore the effects of HOsm on NF-kB, a DNA binding assay to examine the activity of nuclear NF-kB was employed and, as an initial required preliminary, the effects of TNFa and IL-1b on NF-kB with respect to its functional differences in nuclear binding activity. NF-kB activation elicits a heterodimer between the p65 and p50 sub-units of NF-kB, according to the canonical pathway [20,32,33], and the described experiments focused on the p65 sub-unit, although samples run with the p50 sub-unit showed results similar to that of p65 (data not shown). At both the 30 and 60 minutes HOsm produced significant inhibition of TNFa-stimulated NF-kB activation (p,.05) [ Fig 4A]; however, IL-1b produced no significant differences at any of the time points studied [ Fig 4B]. TNFa stimulation of pulmonary epithelial cells in isotonic media produced an NF-kB peak activation equivalent to 60 ng of purified p65 before diminishing to a level of 30 ng, equivalent to the peak and sustained levels produced by HOsm treated cells. In contrast, IL-1b stimulated cells maintained an activation of around 60 ng across the entire time course, independent of HOsm treatment. Hyperosmolar treatment decreases I-kBa phosphorylation I-kBa phosphorylation represents a key regulatory step in NF-kB activation, leading to release of I-kBa from its inhibitory role on NF-kB. Western blot analysis of I-kBa with both stimuli in the presence of HOsm demonstrated a decrease in phosphorylation [ Fig 5]. This inhibition was noted most strongly at 5 minutes after stimulation. When quantified using a phosphoprotein ELISA, inhibition of I-kBa phosphorylation was noted at 10 minutes following stimulation with either TNFa or IL-1b (p,0.05) [ Figure 5B&C]. Interestingly, with either TNFa or IL-1b stimulation, I-kBa phosphorylation in the setting of HOsm treatment peaked at 30 minutes at or above the levels seen in isotonic media at the same time point. Thus, HOsm diminishes I-kBa phosphorylation similarly with either TNFa or IL-1b stimulation at the early time points but appears to only delay peak phosphorylation.

Transcription factor IRF-1 nuclear localization was inhibited by HOsm with both TNFa and IL-1b stimulation
Based on the RANTES promoter region [26,34], IRF-1 was identified as a proximal transcription factor and the increases in TNFa-or IL-1b-induced in nuclear IRF-1 levels were analyzed by western blotting [ Figure 6A]. In these experiments the IRF-1 levels on western blots were quantified by densitomery and IRF-1 levels were calculated as a fold change from nuclear IRF-1 densitometry from control cells. TNFa-and IL-1b-mediated increases in nuclear IRF-1 levels in A549 cells were inhibited by HOsm at 180 minutes [ Figure 6B] and at 90 and 180 minutes [ Figure 6C], respectively (p,0.05). The HTS and SOR groups were not statistically different.

Discussion
Of the pro-inflammatory mediators investigated in the preliminary screen, TNFa apparently increased the release of six cytokines/chemokines (.5-fold), of which 3 were inhibited by HOsm pretreatment: RANTES, MCP-1 and IP-10. In contrast, IL-1b induced 10 cytokines/chemokines of which HOsm attenuated 4: RANTES, IP-10, G-CSF and MIP1b. The 6 mediators that seemed increased in response to TNFa were also induced by IL-1b; but, IP-10 and MCP-1 were more responsive to TNFa. Thus, IL-1b apparently induced release of a larger array of cytokines/ chemokines than TNFa, at identical concentrations, suggesting different modes of transcriptional activation. Hyperosmolarity represents a promising potential therapy against inflammation. Both NaCl and sorbitol can be used to prepare hyperosmolar solutions and are clinically accessible. While NaCl is ionized in aqueous solutions, sorbitol is a C6 polyol (an open chain configuration of an hexose). We used these to determine whether the intracellular response to HOsm required ion flux to achieve anti-inflammatory benefit. We and others have used modest hyperosmolarity of about 400 mOsm against various cells [1,27,35,36,37]. These doses were derived from peak levels achievable in animal models and substantial cell shrinkage or death (Fig 7) are not evident. We have also used inhaled aerosolized hypertonic saline to protect animal lungs but the local dose is unknown [6]. The mechanism by which modest HOsm (,400 mOsm) reduces inflammatory signaling is unclear [1,6,35,37]. The cellular response to hyperosmolarity (HOsm) is an ancient pathway that led to the discovery of the modular p38MAPK (originally called hyperosmolar glycerol 1 HOG1 in yeast) pathways [38]. Some have suggested ionic mechanisms, others invoked cytostructural elements, but we have not found evidence for these in A549 and other mammalian cells [17,36].
Hyperosmolarity inhibited RANTES and IP-10 production, which requires NF-kB, whether induced by TNFa or IL-1b. Production of the NF-kB-dependent chemokine MCP-1 was inhibited by HOsm in response to TNFa but not IL-1b. But G-CSF and MIP-1b, both are NF-kB dependent, could only be induced by IL-1b, but not TNFa, and appeared inhibited by HOsm. Irrespective of the Figure 6. HOsm inhibits IRF-1 localization to the nucleus with either TNFa or IL-1b stimulation. IRF-1 in the nuclear extract of cells stimulated by TNFa or IL-1b 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 TNFa (A) or IL-1b stimulation (C); * p,0.05 for ISO vs. either HTS or SOR at matched time points; no significant differences were found between HTS and SOR groups. pro-inflammatory stimulus, A549 cells demonstrated similar HOsm inhibition, suggesting a similar non-ionic mechanism. In contrast, production of the ubiquitous, pro-inflammatory agents IL-6, IFNc and IL-8 appeared increased by both TNFa and IL-1b but not inhibited by HOsm. Similar to previous results [37,39], but here at more modest HOsm, TNFa-and IL-1b-elicited release of the neutrophil chemoattractant IL-8 was increased (see also below). The reasons for this increase are unclear, but again argues against cell dysfunction.
Due to their central role in inflammation, TNFa and IL-1b signal transduction pathways have been well delineated [9,10,40,41]. Following ligand binding, both receptors recruit distinct intracellular proteins that converge on the I-kB kinase (IKK) complex activating the NF-kB pathway [18,40,41]. NF-kB is a dimer held in the cytoplasm by its inhibitor, I-kBa, which masks its nuclear localization sequence. Upon stimulation through receptor signaling pathways (including TNFR, IL-1R or TLR) the IKK complex phosphorylates I-kBa at serine residues 32/36, leading to I-kBa degradation by the proteosome pathway. The NF-kB dimer translocates into the nucleus and begins transcription of many proinflammatory genes [40,41]. The TNFa and IL-1b pathways are also known to selectively activate other signal transduction processes, such as various MAPK modules [9,10,42,43] which could independently affect pro-inflammatory chemokine production.
Because multiple transcription factors have been implicated in pro-inflammatory activation of various cells [25,26] a number of potential transcription factors implicated in promoting cytokines that were inhibited by HOsm were investigated (Table 1). Firstly, HOsm did not affect TNFa-or IL-1b-elicited phosphorylation of Erk-1/2, JNK, or of c-Jun (a key component of AP-1) at 30 or 60 minutes by phosphoprotein analysis (data not shown). STAT-1 activation, as measured by a DNA binding assay, was also tested but no detectable STAT-1 binding was found in the nuclear fractions of any treatment groups (data not shown). Lastly, HOsm did not inhibit TNFa-or IL-1b-induced p38MAPK dual phosphorylation (data not shown).
IL-1b caused increased NF-kB nuclear translocation for both the p65 and p50 subunits of the dimer, which is an expected consequence of greater I-kBa activation in the first 30 minutes, similar to previous data, which demonstrated that either IKKa or IKKb were capable of phosphorylating I-kBa [18]. In contrast, TNFa requires both IKKa and IKKb for I-kBa phosphorylation. The phosphorylation of c-Jun and nuclear translocation of IRF-1 seem comparable with either stimulus; Erk-1/2, JNK, and STAT-1 do not seem to play a role in the A549 cellular response to HOsm inhibition of pro-inflammatory stimuli, contrary to their roles in other cell types [42,44,45]. Previously, in human vascular smooth muscle cells, we found that both TNFa and HOsm induced p38MAPK activation, additively [17]. Here, inhibiting p38MAPK reduced proinflammatory markers but not as much as HTS. Thus the mechanisms of HOsm inhibition is probably unrelated to p38MAPK [17,35].
At first glance, the early attenuation of TNFa-stimulated NF-kB translocation to the nucleus after HOsm pretreatment offers a satisfactory explanation for the attenuated expression of early NF-kB-driven genes such as RANTES or MCP-1. However, HOsm does not affect IL1b-driven NF-kB translocation, while suppressing cytokine expression. These data indicate that NF-kB promoter activity may be insufficient to explain the inhibition of cytokine/chemokine expression. To assess other HOsm suppression mechanisms, other TFs regulating RANTES-type promoter modules were examined [26]. After either stimulus, nuclear IRF-1 increased over 3 hours. Nuclear fractions showed that IRF-1 levels were elevated by 60 minutes and significantly reduced as early as 90 minutes for both. This decrease implies that HOsm may attenuate gene transcription by retarding the nuclear translocation of several, transcription factors.
Analysis of lavage fluids and injured lungs from animal studies and human clinical trials have found elevated levels of a variety of CXC (IL-8, IP-10) and CC chemokines (RANTES, MCP-1, MIP), which often correlate with leukocyte infiltration and outcomes [11,46,47]. Many of these (e.g. IL-8, RANTES/CCL5, MCP-1/CCL2, MIP1b/CCL4) are produced by alveolar Type II cells as part of their role in lung defense [48]. Although many of these gene products require NF-kB for expression, the actual promoter elements differ in their affinity [49]. These promoter regions also contain other transcription factor module binding elements such as for AP-1, C/EBP and IRF which interact with NF-kB to further regulate transcription [26,49].
The promoter region of RANTES has specifically been examined with bioinformatics [26]. In several cell types, the NF-kB promoter region of RANTES is neighbored by sites for other promoter modules, including IRF-1. However, there was no inhibition of c-jun phosphorylation (a component of AP-1) nor of STAT-1 DNA-binding (data not shown). This study in lung epithelial cells recapitulates previous work in endothelial cells stimulated by TNFa and IL-1b, where hyperosmolarity inhibited VCAM-1 production [50] via decreased IRF-1 activation. Further bioinformatics and TF translocation details are required to explain the selective stimulation and blockade of G-CSF and MIP-1b with IL-1b but not TNFa. Moreover, why does HOsm block MCP-1 with TNFa but not IL-1b? Lastly, the 25-30% suppression by HOsm of IL-6 IL-4 and IFNc when stimulated by IL-1b may deserve attention. The answers may involve the effects of HOsm on the IKK signalosome and gene specific transcription assembly.
While promoter organizations of IP-10, IL-6 and IL-1b genes resemble that of RANTES in some cell types [26], HOsm inhibited IP-10 production but did not suppress IL-6 release nor induce IL-1b production by TNFa. The striking lack of inhibition of IL-8 (a well-known NF-kB-regulated gene) by HOsm to either stimulus may be due to the tight binding of NF-kB to the consensus IL-8 promoter site [49], making this association impervious to the about 50% decrease in nuclear NF-kb levels. (Fig 4). The related, but weaker, NF-kB binding site in MCP-1 does not appear to contain a neighboring IRF-1 binding module, which may explain why HOsm treatment inhibits production of MCP-1 only with TNFa but not IL-1b (Table 1).
This study demonstrates that HOsm selectively inhibits the production of some pro-inflammatory mediators but not others; however, the synthesis and release of some specific agents involved in pulmonary epithelial inflammation (RANTES, IP-10, G-CSF, MIP-1b) are inhibited by HOsm. In A549 cells, HOsm produces effects on nuclear NF-kB, depending upon the stimulus. Controversy exists over the mechanism by which HTS acts in CF; clinical improvement may be due to mucolytic action, airway hydration or anti-inflammatory effects [51,52]. The effects of HOsm on inflammatory cell signaling could relate to the beneficial application of inhaled HTS in cystic fibrosis patients; furthermore, HTS inhalation may treat or prevent inflammation and/or ALI/ARDS following trauma in a broader patient population.
In summary, HOsm alters the TNFa-and IL-1b signaling in pulmonary epithelial cells thereby reducing the production of selected pro-inflammatory cytokines. The concordance between HTS and sorbitol suggests that the inhibitory mechanisms depend on hyperosmolarity and appear multi-pronged, affecting TNFa and IL-1b signaling cascades at distinct steps. Both HOsm conditions inhibit I-kBa degradation and p65 translocation when TNFa is stimulus, but not with IL-1b. The inhibition of IL-1b reveals another mechanism involving inhibition of IRF-1. IRF-1 is inhibited by both HOsm conditions for both stimuli. A broader analysis of how hyperosmolarity regulates transcription promoters of chemokine gene expression in different cellular contexts should lead to a better understanding of the anti-inflammatory properties of HOsm.