Ethic Statement

All animal experiments were conducted in a licensed animal facility in accordance with the German law on the protection of experimental animals and were approved by local authorities of the state of Nordrhein-Westfalen (Landesamt für Natur, Umwelt und Verbraucherschutz NRW). Mice were sacrified by cervical dislocation.

Cell culture

The murine macrophage cell line RAW264.7 (#TIB-71) was purchased from ATTC. RAW264.7 macrophages with a stable TonEBP overexpression were generated as described earlier by Machnik et al [2]. Cells were cultured in Dulbeccós modified Eaglés Medium (DMEM) (PAA) supplemented with 10% Fetal Bovine Serum (Biochrom, Berlin, Germany) and 1% Pen-Strep (Biochrom) in a 5% CO₂ atmosphere with 95% humidity at 37°C.

Bone marrow-derived dendritic cells (BMDCs) were generated as described previously by Quast et al. [6]. In brief, bone marrow was harvested from the femurs and tibiae of seven week old C57BL/6 wildtype and filtered with 40 μm pore nylon cell strainers (BD Biosciences). Then, hematopoietic stem cells were plated into 10 cm non-treated petri dishes at a concentration of 5*10⁶ cells in 10 mL VLE-RPMI 1640 (Biochrom) supplemented with 10% vol/vol heat-inactivated FCS (Sigma-Aldrich), 100 u/mL Penicillin (PAA), 0.1 mg/mL Streptomycin (PAA) and 10 ng/mL recombinant murine GM-CSF (Peprotech). The culture medium was half-renewed every three days. At day 7–10 of culture, BMDCs were stimulated to mature by adding 200 ng/mL LPS (derived from E. coli, Sigma-Aldrich) for 48 h before they were used for functional assays.

For differentiation into bone marrow-derived macrophages (BMDMs), cells were plated into 10 cm non-treated petri dishes at a concentration of 5*10⁶ cells in IMDM medium (PAA) supplemented with 10% FCS (Sigma-Aldrich), 2 mM L-glutamine (PAA), 100 u/mL Penicillin (PAA), 0.1 mg/mL Streptomycin (PAA) and 10 ng/mL recombinant murine M-CSF (Peprotech).

Peritoneal macrophages were isolated via peritoneal lavage of C57BL/6 mice with 2 mM EDTA/PBS. After centrifugation at 1000 rpm for 8 min, remaining erythrocytes were lysed in erythrocyte lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃ in ddH₂O) for 5 min at 37°C. The reaction was stopped with 35 ml ice-cold PBS and washed twice with PBS following centrifugation at 1000 rpm for 5 min. Then, cells were plated into 10 cm non-treated petri dishes in IMDM supplemented with 1% Pen-Strep and 10% FCS. After adhesion of the macrophages, the cell culture medium was replaced in order to remove remaining B-cells in the supernatant.

In vitro migration assay

Chemotaxis of the macrophage cell line RAW264.7, bone marrow-derived macrophages and peritoneal macrophages was analyzed with a modified Boyden chamber (transwell) assay using cell culture membrane inserts with 8 µm pore size (BD Falcon #353097, Becton Dickinson). 2*10⁵cells were placed in serum-reduced (0.5% FCS) cell culture media (see cell culture) in the upper well while the culture medium of the lower compartment was supplemented with 25 nM CXCL12, 15 nM CCL2 (both from Peprotech), or NaCl (Merck) with concentrations between 10–100 mM (reaching a final concentration of 155 to 255 mM NaCl in the media), respectively. After 20 hours non-migratory cells on top of the membrane were removed with cotton swabs before the transmigrated cells on the bottom of the membrane were stained with 5 µM Vybrant CFDA-SE in PBS (Invitrogen) according to the manufacturer’s protocol. For each sample, 5–10 random fields were observed with an inverted Nikon Eclipse TE 2000-E fluorescence microscope (Nikon), equipped with a PlanFluor DL 10x/0.30 N.A. objective (Nikon). The number of migrated cells was counted using Cell Profiler software as described in [7].

For migration analysis of LPS-activated BMDCs, 20 nM CCL19 (Peprotech) and cell culture inserts with 5 µm pore size were used (Corning). Transmigrated BMDCs in the lower compartment were counted after incubation for 4 h at 37°C/5% CO₂.

Cell viability test

1*10⁴ cells per well were placed in a 96 well plate in serum-reduced media (0.5% FCS) to adhere. Subsequently, Na⁺ concentration of the media was increased by adding 10 to 100 mM NaCl. After 20 hours, the metabolic activity of the cells referring to cell viability was tested using a Cell Titer Blue Assay (Promega) according to manufacturer’s protocol. Fluorescence output of the assay was measured with a GloMax-Multi microplate reader (Promega).

Osmometer measurements

Osmolality of media solutions following different osmotic stimuli was determined with the freezing point micro-osmometer OM815 (Vogel Löser). A 300 mosm/kg H₂O standard solution was used to calibrate the osmometer and accuracy of measurements was ensured by using a sample of 0.9% NaCl with known osmolality of 186 mosm/kg H₂O.

Flow cytometry

Activation of BMDM macrophages was analyzed using flow cytometry (FACS CantoII device, BD Biosciences) following staining with an PE-conjugated anti-CD86 antibody (eBioscience).

Flame photometry

Media samples of both the upper and the lower chambers of the transwell system were collected at different time points after NaCl supplementation (1, 10, 30 minutes, and 1, 2, 4, 8, 12, 16 and 20 hours). Na⁺ content in the supernatants was determined via flame photometry in an EFOX 5053 device (Eppendorf) by comparison with an urine standard (EFK diagnostics). Liquichek Urine Chemistry Control (Biorad) containing known Na⁺ concentrations was used to ensure the accuracy of the photometer.

Phalloidin staining

5*10⁵ RAW264.7 cells were placed in DMEM with reduced FCS (0.5%) on fibronectin-coated (Bio-Harbor) glass coverslips. Cells were stimulated with 40 mM NaCl (Merck), 10 nM C5a (Peprotech), 15 nM CCL2 (Peprotech) or 25 nM CXCL12 (Peprotech) for 5 min. Subsequently, coverslips were washed twice with PBS followed by fixation with 4% PFA (Roth) in PBS for 20 minutes. Permeabilization of the cells was performed with 0.2% TritonX100 (Roth) in PBS for 5 minutes. Cells were stained with TRITC-Phalliodin (Sigma-Aldrich) and DAPI (Sigma-Aldrich) for 45 min. After washing three times with PBS and one time with bidest, coverslips were mounted in FluoroShield (ImmunoBioscience Corp.) containing 50 mg/ml DABCO anti-fade reagent (Sigma-Aldrich). Cell morphology and actin cytoskeleton organization was analyzed by the use of an inverted confocal laser scanning microscope (Fluoview 1000, Olympus) equipped with a Plan Apochromat 60x, NA 1.4 oil immersion objective (Olympus).

Analysis of lamellipodia dynamics

Motile RAW264.7 cells were placed in DMEM (PAA) with reduced FCS (0.5%) on a glass surface of a Lab-Tek Chamber Slide (Nunc) to adhere for 20 min in a 5% CO₂ atmosphere with 95% humidity at 37°C. Subsequently, cells were stimulated with 40 mM NaCl (Merck), 10 nM C5a (Peprotech), 15 nM CCL2 (Peprotech) or 25 nM CXCL12 (Peprotech), respectively. To analyze lamellipodia dynamics of macrophages, RAW264.7 cells were monitored over a period of 5 min by capturing phase contrast images every 2 s using an inverted Nikon Eclipse TE 2000-E microscope equipped with a climate chamber (37°C, 5% CO₂, humidity) and a PlanApo DM 100x/1.40 N.A. oil immersion phase contrast objective (Nikon). Subsequently, an area of interest was marked on each image of the time-series by lines (white line) that cross the motile lamellipodium of the polarized cell. The resulting 1-pixel-wide areas were cut out and lined up in time space plots. In these time space plots (so-called kymographs) lamellipodia protrusions appear as linear ascending contours (see yellow lines). The slope of these lines corresponds to the velocity of lamellipodia movement, v = dx (µm)/dt (min). Velocities of membrane protrusions were analyzed by kymograph and line-scan analysis following the walking average plug-in, using ImageJ1.38 (National Institutes of Health; http://imagej.nih.gov/ij/).

Western blot

For analysis of TonEBP protein expression, 2*10⁶ RAW264.7 cells were plated in cell culture dishes in serum reduced DMEM (0.5% FCS) and exposed to additional 40 mM NaCl for 20 hours. Afterwards, cells were washed with PBS and lysed in 8 M urea (Roth) with protease inhibitor (Roche Applied Science), followed by an incubation for 10 min on ice and centrifugation for 10 min at 13.000 rpm. The supernatant was used for immunoblot analysis and the protein concentration was measured by Bradford assay using Roti-Quant (Roth).

For immunoblotting, equal amounts of total protein were separated on 8% SDS-polyacrylamide gels under reducing conditions and electroblotted onto a polyvinylidene diflouride (PVDF) membrane (Biorad). The blots were blocked with 5% nonfat milk in phosphate buffered saline (PBS) and 0.1% Tween 20, pH 7.5 for 1 h at room temperature and then incubated overnight at 4°C with an anti-TonEBP antibody (ABR) and anti-β-actin (Sigma-Aldrich), respectively, both diluted 1:1.000 in 5% nonfat milk in PBST. After washing in PBST for three times, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Pierce) diluted 1:1000 in blocking solution for 90 minutes at room temperature. After washing in PBST for three times, chemiluminescent signals were detected using ECL Western Blotting Substrate (Pierce) on autoradiographic film (Fujifilm).

RNA interference of TonEBP

3*10⁵ RAW264.7 cells were placed into 6-well plates and transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen) on the next day according to the manufactureŕs protocol. Prior to transfection, we replaced the medium with serum-free medium without antibiotics and diluted 100 pmol of siRNA duplexes against the target sequence of TonEBP (Dharmacon; prevalidated siRNA database catalog L 058868) or 100 pmol of AllStars Negative siRNA (Qiagen catalog 1027281) in 250 µl Opti-MEM (Invitrogen), respectively. After incubation for 4 hours at 37°C/5% CO₂, cells were washed and the medium was replaced with normal DMEM. Three days after transfection cells were used for functional migration assays. Efficiency of the transfection and RNAi was evaluated by Western Blot analysis after stimulation with additional 40 mM NaCl for 20 hours.

Detection of CCL2 production

2*10⁵ RAW264.7 cells were placed in a 96 well plate in serum reduced DMEM (0.5% FCS) and stimulated with additional 40 mM NaCl. After 1, 2, 4, 8, 12, 16 and 20 hours, supernatants of NaCl stimulated cells and supernatants of cells cultivated in control medium (DMEM +0.5% FCS) were collected. Murine CCL2 in supernatants was quantified at the indicated timepoints by ELISA, according to the manufacturer`s instruction (R&D Duoset).

Statistical analysis

All experiments were repeated at least three times using duplicates. Data are expressed as mean ± SD. Two-tailed Student’s t test with values of p<0.05 considered significant was performed with IBM SPSS Statistics 19.0 (IBM) software.

NaCl induces chemotaxis of RAW264.7 cells

A recent study demonstrates that macrophages, which infiltrate the interstitium of the skin, are able to regulate salt-dependent extracellular volume and blood pressure homeostasis by a TonEBP-VEGF-C-dependent buffering mechanism [2]. However, it remained unclear whether hypertonic stress and NaCl act as a chemotactic stimulus per se. To investigate this issue we analyzed the ability of RAW264.7 macrophages to actively migrate toward excess NaCl by the use of modified Boyden chamber assays (so-called transwell migration assays).

The NaCl concentration in the lower well was increased by 40 mM (to a final NaCl concentration of 195 mM). This sodium concentration has been established previously by Machnik et al. to simulate the difference between the combined serum Na⁺ and K⁺ concentrations and the ratio of the Na⁺ and K⁺ content relative to water in skin observed in rats fed on a high salt diet [2]. Quantification of chemotaxis after 20 hours revealed a highly significant increase of transmigrated RAW264.7 cells toward excess NaCl as compared to unstimulated control cells (Fig. 1a). Migration capacity of NaCl-stimulated cells was about 60% as compared to cells stimulated with the chemokine CXCL12 (Fig. 1a). We found a lower but still significantly enhanced cell migration response toward 80 mM NaCl. However, the control osmolytes urea or mannitol were unable to elicit a significant chemotactic response of RAW264.7 cells (Fig. 1a), therefore illustrating that the observed cell migration was specific to increased hypertonicity by NaCl but not osmolality in general, or hypertonicity by mannitol. Indeed, cryoscopic (freezing point) measurements confirmed that the osmolality of the applied hypertonic solutions was equivalent and resulted in an increase from 320 mosm/kg H₂O in culture medium to approximately 400 mosm/kg H₂O following application of NaCl, urea or manntitol, respectively (Table S1).

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Figure 1. RAW264.7 cells recognize NaCl-induced hypertonicity as a chemoattractant stimulus.

Transwell migration assay of the murine macrophage cell line RAW264.7 toward the hypertonic stimuli NaCl, urea or mannitol, respectively (A). The difference in Na⁺ concentration measured in lower and upper wells by flame photometry is shown as Δ Na⁺ (B). Transwell migration assay of RAW264.7 cells in the presence or absence of Polymyxin B, respectively (C). Transwell migration assay of BMDMs activated with 200 ng/ml LPS (D). Dose-dependency of RAW264.7 cells toward different NaCl concentrations by the use of transwell migration assays (E). The migration index was determined by the number of transmigrated cells in relation to CXCL12-stimulated cells after 20 hours. [−/−] indicates that no hypertonic stimulus was added to the transwells. [−/+] indicates that the hypertonic stimulus was added to the lower well of the transwell. [+/−] indicates that the hypertonic stimulus was added to the upper well of the transwell. [+/+] indicates that the hypertonic stimulus was added to both wells of the transwell (A). “+”indicates that the stimulus was added to the lower well of the transwell (C, D and E). Error bars indicate ± SD. *p<0.05, **p<0.01, ***p<0.001 as compared to control of cells without stimulus. n.s. not significant. n>3 in duplicates.

https://doi.org/10.1371/journal.pone.0073439.g001

We then asked whether the observed induction of migration of RAW264.7 cells toward NaCl was due to a gradient-independent activation by a uniformly applied high salt concentration (so-called chemokinesis), and performed transwell migration assays with 40 mM excess NaCl in the upper well (+/−) or in both wells (+/+), respectively (Fig. 1a). In contrast to the setting in which NaCl was added to the lower well, we could not detect enhanced directional migration of RAW264.7 cells under these conditions. Taken together, these data provide strong evidence that a NaCl concentration gradient is a prerequisite for salt-induced chemotaxis of RAW264.7 cells.

Since our data suggested that concentration gradients of sodium and chloride ions are important for NaCl induced chemotaxis of RAW264.7 cells (see Fig. 1a), we assessed the stability of the NaCl gradient during the course of the transwell assay. For this reason we measured the Na⁺ concentration in the upper and lower wells of the transwell chambers with the help of a flame photometer. By comparing the Na⁺ content in the media with an urine standard (143.5 mmol/L), we found that the Na⁺ concentration in the lower well started to decrease rapidly from the 40 mM NaCl at t = 0, but a differential of at least 10 mM Na⁺ was still detectable for approximately 8 hours (Fig. 1c). We thus conclude that NaCl gradients can form and persist in this system and are likely responsible for the observed biological responses.

Previous reports have shown that motility of macrophages is enhanced when the cells are activated by endotoxins [8]. To exclude that migration of the cells toward high NaCl concentrations is due to a contamination by Lipopolysaccharide (LPS) followed by activation of macrophages, we incubated all media solutions with 50 µg/ml polymyxin B (PmxB). This concentration was able to attenuate nitric oxide production in RAW264.7 macrophages following stimulation with 1 µg/ml LPS without altering cell viability (data not shown). Analysis of the migration behavior revealed no significant difference between cells migrating in the presence or absence of PmxB (Fig. 1d). We conclude from these findings that NaCl-stimulated chemotaxis of RAW264.7 cells could not be attributed to a LPS-dependent inflammatory response. This is supported by the finding that LPS-activated, CD86- positive BMDMs (Fig. S1) show no random motility in the absence of a chemotactic stimulus (Fig. 1d). Furthermore, we found that the migration capacity of macrophages toward CCL2 or NaCl is unaltered following LPS-maturation as compared to unstimulated BMDMs.

Because we found a higher migration rate of RAW264.7 cells toward 40 mM excess NaCl as compared to 80 mM excess NaCl (see Fig. 1a) we sought to determine the maximum motility response toward NaCl by employing a dose-dependent transwell migration assay. Upon increasing excess NaCl concentrations (0 to 100 mM) in the lower well, we observed that the migratory responses of the cells toward NaCl were dose-dependent and showed a maximum at approximately 40 mM excess NaCl (Fig. 1e). The same NaCl concentrations were tested in a cell titer blue metabolic viability assay to investigate whether the decreased migratory response at concentrations higher than 40 mM excess NaCl is due to reduced cell viability. After 20 h exposure, cells showed more than 90% viability in added NaCl concentrations up to 40 mM. Nevertheless, cell viability slightly but significantly decreased from additional 60 mM NaCl onward (Fig. S2). We conclude from these data that decreased migration is likely caused by reduced viability of the cells at concentrations higher than 40 mM excess NaCl. Therefore, in all following experiments a stimulus of 40 mM excess NaCl was used, since this concentration elicited the maximum migration response, had no effect on cell viability and corresponded to in vivo data described previously [2].

Bone marrow derived macrophages, but not dendritic cells, migrate toward excess NaCl

In order to elucidate if primary macrophages also respond to excess NaCl as a chemoattractant, we investigated the migration behavior of murine bone marrow-derived macrophages (BMDMs) and macrophages isolated from the peritoneum of mice by the use of a transwell assay. We found in both BMDMs and peritoneal macrophages a significant migration response toward 40 mM excess NaCl. The migration capacity of BMDMs toward the NaCl stimulus was 75% as compared to the cells stimulated with the chemokine CCL2 (Fig. 2a), while peritoneal macrophages migrated about 50% as compared to CCL2-stimulated cells (Fig. 2b). Since the observed migration behavior was equivalent to the chemotactic response in RAW264.7 macrophages, we conclude that salt-dependent chemotaxis is not an isolated characteristic of a particular cell line.

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Figure 2. BMDMs and peritoneal macrophages, but no BMDCs show salt-dependent chemotaxis.

Transwell migration assays of BMDMs (A), peritoneal macrophages (B) and BMDCs (C). The migration index was determined by the number of transmigrated cells in relation to cells transmigrated toward CXCL12 (A), CCL2 (B) or CCL19 (C), respectively. “+”indicates that the stimulus was added to the lower well of the transwell. Error bars indicate ± SD, n = 3, ***p<0.001 as compared to cells without stimulus.

https://doi.org/10.1371/journal.pone.0073439.g002

We furthermore asked whether salt-dependent chemotaxis is a general feature of highly motile immune cells. Therefore, we analyzed the migration capacity of activated bone marrow-derived dendritic cells toward 40 mM excess NaCl. After stimulation with LPS dendritic cells mature into highly motile cells, which migrate toward the chemokine CCL19 in a transwell assay, but show no chemotactic response toward excess NaCl (Fig. 2c). Taken together, these results indicate that chemotaxis toward excess sodium is a macrophage specific process and not common in diverse motile myeloid cells.

The actin cytoskeleton is not involved directly in early processes of NaCl-induced macrophage chemotaxis

Cell migration to chemoattractants highly depends on the dynamics of the actin cytoskeleton and the formation of polarized membrane protrusions (lamellipodia). We therefore analyzed the effects of excess NaCl on cell morphology, polarization and actin cytoskeleton in RAW264.7 cells. By the use of confocal laser scanning microscopy of phalloidin-stained macrophages we could not detect striking changes in cell morphology and lamellipodia formation following stimulation with NaCl, CXCL12, CCL2 or C5a, respectively (Fig. 3a). To analyze putative cytoskeletal differences in more detail, we used a more sensitive assay system that allowed us to follow the morphologic response of RAW264.7 cells by time-lapse phase contrast video microscopy. Velocity of membrane protrusion formation was analyzed by computer-assisted kymograph and line-scan analysis, using ImageJ software (Fig. 3b, c). Figure 3d shows that C5a, CCL2 and CXCL12 significantly increase velocities of membrane formation. While stimulation with CCL2 and CXCL12 resulted in a lamellipodia formation speed of about 1.5 µm/min, the presence of C5a resulted in a slower response with about 1 µm/min. Nevertheless, no significant differences in lamellipodia dynamics could be detected between unstimulated RAW264.7 cells and those treated with 40 mM excess NaCl (Fig. 3d). These data indicate that excess sodium is not involved directly in early events that increase actin dynamics and cytoskeletal reorganization of cell morphology.

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Figure 3. Excess NaCl does not increase lamellipodia dynamics of motile RAW264.7 cells.

Microscopial analysis of TRITC-Phalloidin stained F-actin (red) in untreated RAW264.7 cells and cells stimulated with 40 mM NaCl, 10 nM C5a, 15 nM CCL2, or 25 nM CXCL12, respectively (A). Images were performed with an inverted confocal laser scanning microscope focussed to the basal plasma membrane of the cells. Nuclei were detected with DAPI staining (blue). Microscopical analysis of lamellipodia dynamics in RAW264.7 cells (B-D) on a glass surface stimulated with excess 40 mM NaCl, 10 nM C5a, 15 nM CCL2, 25 nM CXCL12, respectively. Membrane dynamics were visualized at the basal plasma membrane by the use of phase contrast over a period of 5 min at 2 sec per frame. Subsequently, an area of interest was marked on each image of the time-series by lines (white line in B) that cross the motile lamellipodium of the polarized cell. Velocities of lamellipodia protrusion formation were analyzed by kymograph analysis and line scan analysis of yellow lines (C) using ImageJ. Quantification of lamellipodia dynamics of motile RAW264.7 cells (D). Three kymographs per cell were analyzed; each dot represents the value of one single kymograph (C). Shown data are representative for one experiment out of three. Error bars indicate ± SD. ***p<0.001. Bars in microscopical images represent 10 µm (A, B).

https://doi.org/10.1371/journal.pone.0073439.g003

CCL2 and TonEBP are not involved in salt-dependent chemotaxis of macrophages

Since our results have shown that early migration-relevant processes like reorganization of the actin cytoskeleton were not altered under excess NaCl conditions in RAW264.7 cells, we investigated a possible induction of the chemokine CCL2 under hypertonic conditions. Studies by Kojima et al. had shown that NRK52E cells produced CCL2 in a time-dependent manner after exposure to hypertonicity by 100 mM NaCl and 200 mM mannitol [9]. For this reason, we measured CCL2 production in RAW 264.7 macrophages following stimulation with 40 mM excess NaCl over a time course of 20 hours. Enzyme linked immunosorbent assaying (ELISA) of the supernatants revealed significant CCL2 secretion of RAW264.7 macrophages, but no differences in CCL2 levels between unstimulated and NaCl-stimulated samples were observed at all time points investigated (Fig. 4). These results show that the secretion of CCL2 cannot be accounted for as a responsible indirect mechanism for salt-dependent chemotaxis in RAW264.7 cells.

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Figure 4. CCL2 production in RAW264.7 cells is not increased in excess NaCl.

RAW264.7 cells were stimulated with additional 40

https://doi.org/10.1371/journal.pone.0073439.g004

Since other chemokines or chemotactically active factors might play important roles in this system, we assessed the role of the transcription factor tonicity-responsive enhancer binding protein (TonEBP), which protects cells from osmotic stress [10], [11], [12], [13] and which previously had been shown to control the transcription of chemokine genes, too [9], [14]. We thus investigated a possible role of TonEBP in salt-dependent chemotaxis using RAW264.7 macrophages with a stable TonEBP overexpression as described previously [2]. These cells display as high protein expression levels of TonEPB under normal culture conditions as wildtype cells show following stimulation with 40 mM excess NaCl (Fig. 5a). However, TonEBP protein levels did not increase further after stimulation with 40 mM excess NaCl in TonEBP overexpressing cells.

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Figure 5. TonEBP is not involved in salt-dependent chemotaxis of macrophages.

Western blot analysis of TonEBP protein expression with or without excess 40(wt) and TonEBP overexpressing (TonEBP overexp) RAW264.7 cells, respectively (A). Kinetics of cell migration of TonEBP overexpressing and wildtype RAW264.7 macrophages toward 40 mM excess NaCl over 20 hours using transwell migration assays (B). Western blot analysis of TonEBP expression in RAW264.7 cells following RNAi of TonEBP using lipofection for 72 hours; mock, lipofection without siRNA; ns siRNA, nonspecific control siRNA (C). Transwell migration assay of RAW264.7 cells following RNAi of TonEBP toward 40 mM excess NaCl (D). For all western blot analysis (A, C), β-actin protein expression was used as a loading control. The migration index (B, D) was determined by the number of transmigrated cells in relation to CXCL12-stimulated cells. “+”indicates that the stimulus was added to the lower well of the transwell (D). All cell migration data shown represent mean ± SD from 3 experiments performed in duplicate. ***p<0.001 as compared to cells without stimulus. n.s. not significant. n = 3.

https://doi.org/10.1371/journal.pone.0073439.g005

We further hypothesized that if TonEBP were responsible for salt-dependent chemotaxis, TonEBP overexpressing cells should show an increased migration response toward excess NaCl as compared to wild-type macrophages. However, a comparison of cell migration of wildtype RAW264.7 macrophages with TonEBP overexpressing cells over 20 hours revealed no significant differences in migration behavior (Fig. 5b). This finding was further corroborated by showing that the migratory capacity toward excess NaCl in RAW264.7 cells following RNAi of TonEBP (Fig. 5c and 5d) was normal. Taken together, these results indicate that TonEBP is not involved in NaCl-induced chemotaxis of macrophages.