The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osrKI) and wild-type mice (osrWT). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pHi) from 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na+/H+ exchanger activity from Na+-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osrKI DCs. Cell volume and pHi were similar in osrKI and osrWT DCs, but Na+/H+ exchanger activity and ROS production were higher in osrKI than in osrWT DCs. Before LPS treatment, migration was similar in osrKI and osrWT DCs. LPS (1 μg/ml), however, increased migration of osrWT DCs but not of osrKI DCs. Na+/H+ exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na+/H+ exchanger activity, and pHi to a greater extent in osrKI than in osrWT DCs. LPS increased cell volume, Na+/H+ exchanger activity, and ROS formation in osrWT DCs but not in osrKI DCs and blunted the difference between osrKI and osrWT DCs. Na+/H+ exchanger activity in osrWT DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osrKI DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na+/H+ exchanger activity in osrWT DCs but not in osrKI DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na+/H+ exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osrWT mice. In conclusion, partial OSR1 deficiency influences Na+/H+ exchanger activity, ROS formation, and migration of dendritic cells.
- intracellular pH
- oxidative stress-responsive kinase 1
- oxidative stress
- cell volume
the oxidative stress-responsive kinase 1 (OSR1) participates in the signaling of transport regulation during oxidative and osmotic stress (17, 37, 59, 66, 69, 83). The kinase is activated by the WNK (with no K) kinases WNK1 and WNK4, which are in turn activated by hyperosmotic stress (59, 79, 91). OSR1 stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC) and thus influences cell volume, transepithelial transport, renal salt excretion, and GABA neurotransmission (2, 15, 16, 27, 35–37, 59, 80). Mutations of WNK1 and WNK4 may lead to hypertension (20, 24, 37, 74, 84) and autonomic neuropathy (37). OSR1 is similarly considered to participate in the regulation of blood pressure (28, 77–79). Accordingly, OSR1 has been suggested as a potential drug target in the treatment of hypertension (28, 59, 77). The putative role of OSR1 in the regulation of immune cell function, remains, however, elusive.
The present study explored the role of OSR1 in the regulation of cell volume and formation of reactive oxygen species (ROS) in dendritic cells (DCs), antigen-presenting cells involved in the initiation of both innate and adaptive immunity and thus decisive for the regulation of the immune response (1, 3, 23, 58, 76). Exposure of DCs to bacterial wall components such as lipopolysaccharide (LPS) triggers generation of ROS (6, 49, 88), which contributes to pathogen defense (56). Similar to what has been shown in other cell types (25) including macrophages (13), ROS production is paralleled by H+ generation, which in turn inhibits ROS-generating NADPH oxidase (33). Regulation of cytosolic pH (pHi) involves the Na+/H+ exchanger in a wide variety of cells (13, 53, 55, 90) including macrophages (14, 32, 44, 71, 72) and monocytes (22, 38, 71). The Na+/H+ exchanger further participates in the regulation of cell volume (34, 42, 53).
The present study explored the impact of WNK-dependent OSR1 regulation on Na+/H+ exchanger activity, cell volume, and ROS formation in bone marrow-derived DCs.
MATERIALS AND METHODS
All animal experiments were conducted according to the German law for the welfare of animals and were approved by local authorities. Dendritic cells were cultured from bone marrow (87) of 7- to 11-wk-old heterozygous OSR1 knockin mice (osrKI) and respective wild-type mice (osrWT). The mice were kindly provided by D. Alessi (Univ. of Dundee, Dundee, UK). As described earlier (57), the knockin mice carry a mutation of the T-loop Thr residue in OSR1 (Thr185) to Ala preventing activation by WNK isoforms. Mice had free access to control diet (Ssniff, Soest, Germany) and tap drinking water.
Bone marrow-derived cells were flushed out of the cavities of the femur and tibia with PBS. Cells were then washed twice with RPMI 1640 medium and seeded out at a density of 2 × 106 cells per 60-mm dish (85). Cells were cultured for 6 days in RPMI 1640 (GIBCO, Darmstadt, Germany) containing 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% glutamine, 1% non-essential amino acids, and 0.05% β-mercaptoethanol. Cultures were supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF; 35 ng/ml, Preprotech, Hamburg, Germany) and fed with fresh medium containing GM-CSF on days 3 and 6. On day 7, >85% of the cells expressed CD11c, which is a marker for mouse DCs (85–87). Experiments were performed on days 7-8 of DC culture.
Immunostaining and flow cytometry.
Cells (4 × 105) were incubated in 100 μl FACS buffer [phosphate-buffered saline (PBS) plus 0.1% FCS] containing fluorochrome-conjugated antibodies at a concentration of 10 μg/ml (86). A total of 2 × 104 cells were analyzed. Staining with FITC-conjugated anti-mouse CD11c (BD Pharmingen, Heidelberg, Germany) was used as a positive marker for dendritic cells. After incubating with the antibody for 60 min at 4°C, the cells were washed twice and resuspended in FACS buffer for flow cytometric analysis.
To evaluate OSR1 transcript levels, mRNA abundance was determined by quantitative real-time PCR. To this end, total RNA was isolated using the Trifast Reagent (Peqlab, Erlangen, Germany). RNA (2 μg) was reverse-transcribed using oligo(dT)12–18 primers and SuperScript II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). Then, quantitative real-time PCR with the Bio-Rad iCycler iQ Real-Time PCR Detection System (Bio-Rad, Munich, Germany) was applied using the TaqMan Gene Expression Assay (Hs00362901 m1, Applied Biosystems) to determine OSR1 transcript levels. Reactions were performed using Universal TaqMan Master Mix (Applied Biosystems) in a final volume of 20 μl containing 2 μl of cDNA and the TaqMan Gene Expression Assay as recommended by the manufacturer. Following an initial incubation at 50°C for 2 min and 95°C for 10 min, 40 cycles of thermal cycling at 95°C for 15 s and 60°C for 60 s were performed. Calculated mRNA expression levels were normalized to the expression levels of TBP (Applied Biosystems) in the same cDNA sample. Relative quantification of gene expression was calculated according to the ΔΔCt method.
DCs (2 × 106 cells) were washed twice with PBS, then solubilized in lysis buffer (Pierce) containing protease inhibitor cocktail (Sigma-Aldrich, Schnelldorf, Germany). Samples were stored at −80°C until use for Western blotting. Cell lysates were separated by 7.5% SDS-PAGE and blotted on nitrocellulose membranes. The blots were blocked with 5% BSA in triethanolamine-buffered saline (TBS) and 0.1% Tween 20. Then the blots were probed overnight with anti p-NKCC1 (21) (1:5,000) and GAPDH (1:1,000, Cell Signaling) antibodies diluted in 5% milk in PBS and 0.1% Tween 20, washed 5 times, probed with secondary antibodies conjugated with horseradish peroxidase (1:2,000) for 1 h at room temperature, and washed final 5 times. Antibody binding was detected with the enhanced chemiluminescence (ECL) kit (Amersham, Freiburg, Germany). Densitometer scans of the blots were performed using Quantity One (Bio-Rad).
Cells were exposed either to bacterial LPS (1 μg/ml) or to t-butyl hydroperoxide (TBOOH, 10 μM) in the presence or absence of cariporide (10 μM). Stock solutions of LPS were prepared in culture medium whereas the rest of the substances were dissolved in sterile distilled water. The cells were treated by adding the substances to the cell suspension at the indicated final concentration and incubated at 37°C in a humidified 5% CO2 atmosphere.
Determination of cell volume.
Cell volume was estimated from forward scatter in flow cytometric analysis. Briefly, 8 × 105 cells were taken in a culture dish and treated with LPS in the presence and absence of cariporide (10 μM). After the treatment, cells were collected and centrifuged, the pellet was resuspended in FACS buffer, and the forward scatter was analyzed on a FACSCalibur (Becton-Dickinson, Heidelberg, Germany).
Determination of ROS production.
ROS production in DCs was determined utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) (68). Briefly, 8 × 105 cells were taken in a culture dish and treated with LPS in the presence or absence of cariporide (10 μM). After the treatment, cells were collected and DCFDA (Sigma, Schnelldorf, Germany) was added to the cell suspension at a final concentration of 10 μM. After 30 min of incubation in the dark at 37°C, cells were centrifuged and the pellet was washed twice with ice-cold PBS. The pellet was then resuspended in FACS buffer and the fluorescence was analyzed with flow cytometry (FACSCalibur). DCFDA fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
Measurement of intracellular pH.
For digital imaging of pHi, the cells were incubated in a HEPES-buffered Ringer solution containing 10 μM 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM, Molecular Probes, Leiden, The Netherlands) for 15 min at 37°C. After loading, the chamber was flushed for 5 min with Ringer solution to remove any deesterified dye (63). The perfusion chamber was mounted on the stage of an inverted microscope (Zeiss Axiovert 135), which was used in the epifluorescence mode with a ×40 oil immersion objective (Zeiss Neoplan). BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm using an intensified charge-coupled device camera (Proxitronic) and specialized computer software (Metafluor). Between 10 and 20 cells were outlined and monitored during the course of the measurements. The results from each cell were averaged and taken for final analysis. Intensity ratio (490/440) data were converted into pHi values using the high-K+/nigericin calibration technique (81). To this end, the cells were perfused at the end of each experiment for 5 min with standard high-K+/nigericin (10 μg/ml) solution (pH 7.0). The intensity ratio data thus obtained were converted into pH values using the rmax, rmin, pKa values previously generated from calibration experiments to obtain a standard nonlinear curve (pH range 5 to 8.5).
For acid loading, cells were transiently exposed to a solution containing 20 mM NH4Cl leading to initial alkalinization of pHi due to entry of NH3 and binding of H+ to form NH4+ (61). The acidification of pHi upon removal of ammonia allowed calculation of the mean intrinsic buffering power (β) of the cells (61). Assuming that NH4+ and NH3 are in equilibrium in cytosolic and extracellular fluid and that ammonia leaves the cells as NH3: where ΔpHi is the decrease of pHi following ammonia removal and Δ[NH4+]i is the decrease of cytosolic NH4+ concentration, which is identical to the concentration of [NH4+]i immediately before the removal of ammonia. The pK for NH4+/NH3 is 8.9 (9) and at an extracellular pH of 7.4 the NH4+ concentration in extracellular fluid ([NH4+]o) is 19.37 mM [20/(1 + 10pHo-pK)]. The intracellular NH4+ concentration ([NH4+]i) was calculated from:
To calculate the ΔpH/min during realkalinization, a manual linear fit was placed over a narrow pH range (pH 6.7 to 6.9) which could be applied to all measured cells.
The solutions were composed of the following (in mM): standard HEPES: 115 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4 10 glucose, and 32.2 HEPES; sodium-free HEPES: 132.8 NMDG, 3 KCl, 1 CaCl2, 1.2 MgSO4, 2 KH2PO4, 32.2 HEPES, 10 mannitol, and 10 glucose (for sodium-free ammonium chloride, 10 mM NMDG and mannitol were replaced with 20 mM NH4Cl); and high K+ for calibration: 105 KCl, 1 CaCl2, 1.2 MgSO4, 32.2 HEPES, 10 mannitol, and 5 μM nigericin. The pH of the solutions was titrated to 7.4 or 7.0 with HCl/NaOH, HCl/NMDG and HCl/KOH, respectively, at 37°C.
Determination of migration.
For migration assays, transwell inserts (BD Falcon 353097) and BD BioCoat Matrigel Invasion Chambers (354480, BD Biosciences) were used with a pore diameter size of 8 μm. The transwells were placed in a 24-well cell culture plate containing cell culture medium (750 μl) with or without chemokine ligand 21 (CCL21, 250 ng/ml, Peprotech) in the lower chamber. The upper chambers were filled with 500 μl cell culture medium containing DCs at a concentration of 50,000 cells/ml. The chamber was placed in a 5% CO2 37°C incubator for 4 h. In the following, the nonmigrated cells were removed by scrubbing with a cotton-tipped swab for two times and washing with PBS. The membrane was removed with a scalpel and fixed in 4% paraformaldehyde for 15 min. The migrated cells were then identified by staining with 4′,6-diamidino-2-phenylindole (DAPI).
Data are provided as means ± SE, and n represents the number of independent experiments. All data were tested for significance using Student's unpaired two-tailed t-test, and only results with P < 0.05 were considered statistically significant.
To determine the presence of a functional WNK signaling pathway in murine bone marrow-derived DCs, RT-PCR was performed. As shown in Fig. 1A, DCs express WNK1, WNK3, NCC, and NKCC1 but not SPAK. As illustrated in Fig. 1B, OSR1 mRNA is readily detectable in DCs and the transcript levels were similar in DCs from OSR1 knockin mice (osrKI) and DCs from wild-type mice (osrWT). Furthermore, the phosphorylation of NKCC1 was lower in osrKI DCs (Fig. 1C), indicative of reduced OSR1 activity in osrKI DCs.
As OSR1 is involved in regulatory cell volume increase, the present study determined the forward scatter of DCs in FACS analysis as a measure of cell volume. As illustrated in Fig. 2, no significant difference was observed in forward scatter between osrKI and osrWT. Treatment of the DCs with LPS was followed within 4 h by an increase in forward scatter of osrWT DCs, pointing to an increase in cell volume (Fig. 2). In contrast, the forward scatter of osrKI DCs was not significantly modified by LPS treatment. As a result, following LPS treatment, forward scatter was significantly higher in osrWT DCs than in osrKI DCs. To elucidate the contribution of the Na+/H+ exchanger, experiments were performed in the absence or presence of cariporide, a specific inhibitor of Na+/H+ exchanger 1 (NHE1) (48). As a result, in both genotypes, forward scatter was significantly decreased following treatment of DCs with cariporide (Fig. 2). The decrease of forward scatter tended to be higher in osrKI DCs than in osrWT DCs, a difference, however, not reaching statistical significance.
The lack of cell shrinkage in untreated osrKI DCs could have been due to lack of OSR1, NCC, and/or NKCC expression in osrWT DCs or due to a compensatory increase in Na+/H+ exchanger activity of osrKI DCs. To explore that possibility, cytosolic pH was determined in DCs utilizing BCECF fluorescence. The Na+/H+ exchanger activity was determined with the ammonium pulse technique (61). To this end, NH4Cl was added to the perfusate, leading to NH3 entry into the cells, binding of H+ to form NH4+ and thus to transient cytosolic alkalinization (Fig. 3). The subsequent removal of NH4Cl was followed by exit of NH3 leaving H+ behind. The H+ thus retained within the cell led to cytosolic acidification (Fig. 3). In the absence of Na+, the average cytosolic pH declined further after an ammonium pulse, indicating that the cells did not express significant Na+-independent H+ extrusion mechanisms to maintain or recover cytosolic pH (Table 1). Addition of Na+ was, however, followed by rapid pH recovery, pointing to the operation of the Na+/H+ exchanger. The Na+-dependent pH recovery was blunted in the presence of NHE1 inhibitor cariporide (Table 1). According to the ammonium pulse, the Na+/H+ exchanger activity was significantly higher in osrKI DCs than in osrWT DCs.
Treatment of osrWT DCs with LPS (1 μg/ml) was followed within 4 h by a significant increase in Na+-dependent realkalinization (Table 1 and Fig. 3). Again, in the absence of Na+, further acidification was observed, i.e., accelerated realkalinization following LPS treatment was due to Na+-dependent H+ extrusion, pointing to stimulation of Na+/H+ exchanger activity (Fig. 3). The increase in Na+/H+ exchanger activity was blunted in the presence of the NHE1 inhibitor cariporide (10 μM) (Table 1). In contrast to what was observed in osrWT DCs, treatment of osrKI DCs with LPS did not significantly modify Na+/H+ exchanger activity. Accordingly, following LPS treatment, Na+/H+ exchanger activity was significantly lower in osrKI DCs than in osrWT DCs (Fig. 3). Thus, LPS treatment reversed the difference between the genotypes (Fig. 3).
To determine whether inhibition of NKCC1 with furosemide in osrWT DCs mimics the effects seen in osrKI mice, osrWT DCs were treated with furosemide (100 nM) and Na+/H+ exchanger activity was studied. As seen in Fig. 4, treatment of DCs with furosemide led to a significant increase in basal Na+/H+ exchanger activity. In analogy to what had been observed in osrKI DCs, the LPS-induced stimulation of Na+/H+ exchanger activity was reversed in the presence of furosemide (Fig. 4).
LPS-induced ROS formation has previously been shown to account for the LPS-induced stimulation of Na+/H+ exchanger activity (62). Therefore, the effect of oxidative stress on Na+/H+ exchanger activity was studied. Treatment of osrWT DCs for 2 h with TBOOH (10 μM) to induce oxidative stress was followed by a significant increase in Na+-dependent realkalinization (Table 1 and Fig. 5). Again, in the absence of Na+, no acidification was observed, i.e., accelerated realkalinization following LPS treatment was due to Na+-dependent H+ extrusion pointing to stimulation of Na+/H+ exchanger activity (Fig. 5). As is apparent from Table 1, the increase in Na+/H+ exchanger activity was blunted in the presence of the NHE1 inhibitor cariporide (10 μM). In contrast to osrWT DCs, treatment of osrKI DCs with TBOOH did not significantly modify Na+/H+ exchanger activity. Following TBOOH treatment, Na+/H+ exchanger activity was significantly lower in osrKI DCs than in osrWT DCs (Fig. 5). Thus, similar to LPS, TBOOH reversed the difference between the genotypes (Fig. 5).
The buffer capacity of the cells was not significantly different between osrKI DCs and osrWT DCs and was not significantly modified by exposure to LPS or oxidative stress (Table 1).
Additional studies addressed the role of OSR1 and/or NHE1 in LPS-induced ROS formation. As illustrated in Fig. 6, before LPS treatment, ROS formation was significantly higher in osrKI DCs than in osrWT DCs. Cariporide treatment did not affect the intracellular ROS in osrWT DCs but significantly decreased ROS formation in osrKI DCs (Fig. 6). LPS enhanced the ROS formation in osrWT DCs but did not significantly alter ROS formation in osrKI DCs. Accordingly, following LPS treatment, ROS formation was not significantly different between osrKI DCs and osrWT DCs (Fig. 6).
The migratory potential of DCs is dependent on NHE1 activity. Thus, further experiments addressed the CCL21-induced migration of control- and LPS-(1 μg/ml) treated DCs. As presented in Fig. 7, migration of DCs from osrKI mice tended to be less pronounced than migration of DCs from wild-type mice under control conditions, a difference, however, not reaching statistical significance. LPS treatment markedly stimulated migration of osrWT DCs but did not significantly affect migration of osrKI DCs. Accordingly, upon LPS treatment, migration of osrKI DCs was significantly less pronounced than migration of osrWT DCs (Fig. 7).
The present study reveals that WNK resistance of OSR1 affects Na+/H+ exchanger activity and formation of ROS in DCs. In the absence of LPS, cell volume was similar in DCs from osrKI and osrWT, but Na+/H+ exchanger activity and formation of ROS were significantly higher in osrKI DCs than in osrWT DCs. Similar to what has been reported earlier (62), LPS triggered the formation of ROS, activated the Na+/H+ exchanger NHE1, and increased cell volume in osrWT DCs. All of those effects were blunted or even absent in osrKI DCs. Accordingly, LPS reversed the differences of ROS formation, Na+/H+ exchanger activity, and cell volume between osrKI DCs and osrWT DCs. The increase in Na+/H+ exchanger activity in osrWT DCs following LPS treatment was virtually abolished in the presence of cariporide, which inhibits both NHE1 and NHE2 (67). In DCs the NHE1, but not the NHE2, isoform is expressed (62).
As shown earlier (62), Na+/H+ exchanger activation by LPS depends on ROS activation. The signaling involves MAP kinases and phosphoinositide 3 kinase (82), well-known stimulators of Na+/H+ exchangers (8, 39, 43, 53, 73). Conversely, ROS production is sensitive to cytosolic pH (33). The higher cytosolic pH and Na+/H+ exchanger activity in osrKI DCs thus presumably contributes to the higher ROS production in those cells. ROS production is in turn important for antimicrobial activity (56).
Na+/H+ exchanger activity is enhanced in osrKI DCs despite the more alkaline pH, which should actually decrease Na+/H+ exchanger activity (29). At least in theory, the stimulation of the Na+/H+ exchanger in osrKI DCs could result from cell volume regulation. Cell shrinkage leads to stimulation of the Na+/H+ exchanger, which operates in parallel to the Cl−/HCO3− exchanger (34, 40). Collectively, the two carriers accomplish the entry of NaCl, followed by osmotically obliged water. The H+ and HCO3− extruded in exchange for Na+ (Na+/H+ exchanger) and Cl− (Cl−/HCO3− exchanger) are osmotically not relevant as they are replenished in the cell by cytosolic formation from CO2, which easily crosses the cell membrane (34, 40). Along those lines, LPS-induced cell swelling was abolished in the presence of cariporide, an observation highlighting the impact of the Na+/H+ exchanger in cell volume regulation.
The cell volume regulatory stimulation of the Na+/H+ exchanger may be required because of the lacking OSR1-dependent stimulation of NKCC in osrKI DCs. OSR1 is known to stimulate the NKCC and to participate in cell volume regulation (2, 15, 16, 27, 35–37, 59, 80). It is tempting to reason that OSR1 deficiency leads to decreased NKCC activity, requiring enhanced activity of the Na+/H+ exchanger for cell volume maintenance. We cannot rule out, however, the possibility that OSR1 regulates Na+/H+ exchanger directly, e.g., by phosphorylating the carrier protein. As furosemide treatment of osrWT DCs similarly increases Na+/H+ exchanger activity, a decreased NKCC activity at least contributes to the upregulation of Na+/H+ exchanger activity.
Cell volume may influence the formation of ROS and antioxidative defense (30, 31, 64, 65), and cell shrinkage interferes with ROS generation (62). Moreover, cytosolic pH modifies a variety of further functions, such as migration, cytokine release, adherence, nitric oxide formation, proliferation, and differentiation of macrophages and/or monocytes (5, 7, 10, 19, 44, 50–52, 54, 60, 71, 75). Na+/H+ exchanger activity further fosters (12, 26, 45, 46) or inhibits (4, 41, 47) apoptosis.
The present study did not address the functional significance of OSR1-sensitive DC function. DCs are antigen-presenting cells critically important in the regulation of innate and adaptive immunity (3, 18, 70, 89). To fulfill their diverse functions, DCs have to migrate into inflammatory tissues and return to secondary lymphoid sites following microbial challenge (11). The blunted stimulation of migration following LPS treatment is expected to compromise the function of osrKI DCs. On the other hand, augmented ROS formation of osrKI DCs is expected to foster the removal of pathogens (56). However, the difference of ROS formation between osrKI and osrWT DCs is lost following stimulation with LPS, and enhanced ROS production in immature DCs may not be relevant for the power of the immune response. Clearly, further studies will be required to elucidate the impact of reduced OSR1 activity on the immune system.
In conclusion, the present study disclosed that, in bone marrow-derived DCs, WNK resistance of OSR1 upregulates Na+/H+ exchanger activity, leading to cytosolic alkalinization and fostering formation of ROS. The study thus reveals a completely novel functional consequence of OSR1 inhibition.
This work was supported by the Deutsche Forschungsgemeinschaft DFG (SFB 766).
No conflicts of interest, financial or otherwise, are declared by the author(s).
V.P., A.R., W.Y., C.Z., and M.B. performed the experiments; V.P., A.R., W.Y., C.Z., M.B., M.F., and F.L. approved the final version of the manuscript; A.R. and F.L. conception and design of the research; A.R. and M.F. analyzed the data; A.R. and F.L. interpreted the results of the experiments; A.R. prepared the figures; M.F. and F.L. edited and revised the manuscript; F.L. drafted the manuscript.
Cariporide was a kind gift from Sanofi Aventis (Frankfurt, Germany), and the p-NKCC1 antibody (21) was a kind gift from Dr. Biff Forbush (Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT). The authors gratefully acknowledge the meticulous preparation of the manuscript by L. Subasic and S. Rübe.
- Copyright © 2012 the American Physiological Society