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The Journal of Immunology, 2006, 177: 4086-4093.
Copyright © 2006 by The American Association of Immunologists, Inc.

MaxiK Blockade Selectively Inhibits the Lipopolysaccharide-Induced I{kappa}B-{alpha}/NF-{kappa}B Signaling Pathway in Macrophages1

Martin Papavlassopoulos*,{dagger}, Cordula Stamme*,{ddagger}, Lutz Thon||, Dieter Adam||, Doris Hillemann*,§, Ulrich Seydel*,¶ and Andra B. Schromm2,*,{dagger}

* Research Center Borstel, Center for Medicine and Biosciences, Department of Immunochemistry and Biochemical Microbiology, {dagger} Emmy-Noether Group Immunobiophysics, {ddagger} Division of Cellular Pneumology, § Division of Mycobacteriology, and Division of Biophysics, Borstel, Germany; and || Institute of Immunology, University Clinic of Schleswig-Holstein Campus Kiel, Kiel, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-{alpha} and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+-activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-{alpha} nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-{kappa}B and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor I{kappa}B-{alpha} is critically involved in the observed inhibition of NF-{kappa}B translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-{kappa}B and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-{kappa}B-dependent inflammatory response of macrophages to bacterial LPS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system responds to bacterial infection with the activation of several types of immune-competent cells. Among these, the macrophage plays a pivotal role. Macrophages are present in all body compartments and elicit a fast and strong response to bacterial virulence factors, producing huge amounts of inflammatory mediators such as TNF-{alpha} and ILs (IL-1, IL-6, IL-8, IL-12, IL-23), lipid mediators like leukotrienes, PGs, and platelet-activating factor, as well as NO and other reactive oxygen compounds, essential for the recruitment of phagocytes and lymphocytes to the place of infection and the initiation of bacterial killing. One of the most potent stimuli for the macrophage is endotoxin (LPS), the main component of the outer leaflet of the outer membrane of Gram-negative bacteria (1). The receptor complex responsible for the recognition of LPS is built by TLR4 and the extracellular protein MD-2 (2, 3, 4). The detailed mechanism of how LPS activates this complex receptor cluster is still not very well understood. Different models have been proposed and a still growing number of signaling molecules and adapters are discussed. The basic model is that soluble CD14 and the acute phase protein LPS-binding protein (LBP),3 in a soluble (sLBP) or in a membrane-associated form (mLBP), bind LPS and transport it to the cell surface (5, 6, 7, 8). The GPI-anchored mCD14 is also thought to act as a scavenger, concentrating LPS on the cell surface and thus enhancing the sensitivity toward low amounts of LPS (9). Then MD-2 enables the recognition of LPS by TLR4 homodimers (10). The intracellular portion (Toll/IL-1R (TIR) domain) of the TLR4 receptor recruits several adapter proteins. The first TIR domain-containing adapter that was found was MyD88 (11, 12). The so-called MyD88-dependent pathway is triggered by the TIR domain-containing adaptor protein (Mal) (13, 14) that binds to MyD88 followed by IL-1R-associated kinase-4, IL-1R-associated kinase-1, and TNFR-associated factor-6 (15, 16). The MyD88-dependent pathway leads to the nuclear translocation of NF-{kappa}B (early NF-{kappa}B), a transcription factor that plays an essential role in the regulation of a broad variety of genes coding for inflammatory mediators (17). The translocation of NF-{kappa}B is regulated by a family of inhibitory proteins, termed I{kappa}B, which sequester NF-{kappa}B in the cytoplasm. The best-characterized proximal regulator of NF-{kappa}B activation is the I{kappa}B-{alpha} isoform. Upon phosphorylation, I{kappa}B-{alpha} undergoes ubiquitin-dependent degradation in the 26S proteasome, allowing the nuclear translocation of NF-{kappa}B.

A second TLR4-dependent signaling pathway is the MyD88-independent pathway. MyD88-knockout mice revealed a delayed activation of signaling molecules (late NF-{kappa}B) and an abolished production of various mediators, such as TNF-{alpha} and IL-6 (12). In contrast to this, these mice showed an unaffected activation of the transcription factor IFN regulatory factor-3 and subsequent release of IFN-beta. The MyD88-independent pathway is triggered by two TIR domain-containing adapters, TIR domain-containing adaptor inducing IFN-beta (TIR-containing adapter molecule-1) and TIR domain-containing adaptor inducing IFN-beta-related adaptor molecule (TIR-containing adapter molecule-2) (18, 19, 20).

Intriguingly, TLR4 appears to recruit numerous other surface proteins of immune cells to the process of cell activation assisting the described core receptor. This behavior is unique among all known members of the TLR family, and the structural basis underlying the ability of TLR4 to engage all these proteins is not understood. Only TLR2 has been shown to associate with other TLRs to build heteromeric receptors (10). TLR4/MD-2 as been found to associate in complexes that include CD11/CD18, CD55, CD81, hsp70, hsp90, GDF5, and CXCR4 (21, 22, 23). However, for most of these proteins, a functional participation in signal transduction is not clear, yet. For other molecules, such as MOESIN (24, 25) and mLBP (26), a functional participation in cell activation by LPS has been shown. Several publications including our own have reported that potassium channels are involved in the activation of monocytes, macrophages, and endothelial cells by LPS (27, 28, 29, 30, 31, 32, 33). In previous work, we have shown that in human macrophages the large-conductance Ca2+-activated potassium channel MaxiK (BK) is activated by stimulation of cells with LPS (33, 34). To get further insights into the role of MaxiK in the activation process of macrophages by LPS, we investigated the effect of channel blockade on intracellular signaling cascades. We present data supporting an essential and specific role of MaxiK channel function in activation of the signal transduction pathway leading to activation of NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Paxilline was obtained from Alomone Labs. Deep rough mutant LPS (Re LPS) was extracted from Escherichia coli strain F515 according to the phenol/chloroform/petrol ether procedure (35). The LPS preparation was lyophilized and used in the triethylammonium salt form. The chemical purity of the LPS preparation was confirmed by mass spectrometry. Human recombinant TNF-{alpha} was obtained from R&D Systems. The monoclonal anti-CD14 Ab biG14 was purchased from Biometec.

Isolation of monocytes and macrophages and incubation conditions

Monocytes were isolated from human peripheral blood of healthy donors by the Hypaque-Ficoll gradient method and cultivated in Teflon bags in RPMI 1640 medium (endotoxin, ≤0.01 EU/ml; Biochrom) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 4% heat-inactivated human serum type AB from healthy donors at 37°C and 6% CO2. Cells were cultured in the presence of 2 ng/ml M-CSF (R&D Systems) for 7 days to differentiate monocytes to macrophages. To determine cytokine induction after cell stimulation, cells were seeded at 200-µl aliquots of a suspension of 1 x 106 cells/ml in 96-well tissue culture dishes (Nunc). The MaxiK blocker paxilline was added 10 min before cell stimulation with LPS or TNF-{alpha}. Cell-free supernatants of duplicate samples were collected 4 h after stimulation for TNF-{alpha} determination or 24 h after stimulation for IL-8 determination, respectively, pooled, and stored at –20°C until determination of cytokine content. The presence of paxilline did not affect cell viability as verified by trypan blue staining. Data shown are representative of at least three independent experiments.

To determine p38 and ERK phosphorylation and I{kappa}B-{alpha} expression in human blood macrophages, cells were seeded at 200-µl aliquots of a suspension of 1 x 105 cells/ml in 96-well tissue culture dishes and stimulated with LPS in the absence and presence of MaxiK blocker paxilline or anti-CD14 Ab (biG14).

Rat alveolar macrophages were isolated by lung lavage of male Sprague Dawley rats (Charles River) as described previously (36). Cell viability was routinely checked by erythrosine B exclusion and averaged 94–98%. Cells were resuspended in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 0.2% heat-inactivated human serum type AB from healthy donors.

To determine nuclear translocation of NF-{kappa}B in alveolar macrophages and human blood macrophages, cells were seeded at 200-µl aliquots of a suspension of 1 x 105 cells/ml in 96-well tissue culture dishes and stimulated with LPS or TNF-{alpha} in the absence and presence of MaxiK blocker paxilline for 60 min.

Cytokine determination

TNF-{alpha} was determined in pooled cell-free supernatants of stimulated cells by sandwich ELISA using monoclonal mouse Ab against human TNF-{alpha} and HRP-conjugated rabbit anti-human TNF-{alpha} Ab (Intex) as stated in detail elsewhere (37). IL-8 was determined in pooled cell-free supernatants of stimulated cells by sandwich ELISA using IL-8 cytoset from Biosource exactly according to the manufacturers’ protocol. Data shown are mean and SD (±SD) of triplicate samples of one representative experiment.

RT-PCR analysis

Total cellular RNA was isolated from the cells using the RNeasy Mini kit from Qiagen, and 0.2–0.5 µg of RNA was reverse transcribed with oligo(dT) primers.

For analysis of MaxiK mRNA during macrophage maturation, PCR was performed with 1% (v/v) of Taq polymerase (Eppendorf) in a total volume of 10.3 µl. The primers used were 5'-GCT ACA GCA CCC CGC AGA CA-3' and 5'-GGG GGA CTA CAG GGG AAA ACA GG-3', which yield a 560-bp product. Primers used for the amplification of beta-actin mRNA were 5'-AGC GGG AAA TCG TGC GTG-3' and 5'-CAG GGT ACA TTG TTG TGC-3', which yield a 309-bp product. PCR conditions for MaxiK PCR were 30 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 40 s, and extension at 72°C for 60 s in a Mastercycler Gradient (Eppendorf). PCR conditions for the amplification of beta-actin were 30 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 40 s, and extension at 72°C for 60 s. PCR products were separated by electrophoresis in 1% agarose gels containing 0.1 µg/ml ethidium bromide and visualized under UV light.

For the analysis of cytokine mRNA, real-time PCR was performed on a Rotor-Gene 2000 (Corbett Research) using QuantitTect gene expression assays for human beta-actin, IL-1beta, IL-6, IL-8, and TNF-{alpha} and the QuantiTect probe PCR kit from Qiagen according to the manufacturers’ protocol. Before PCR, a preincubation step (95°C for 15 min) was performed to activate the HotStart TaqDNA polymerase (Qiagen). The PCR was conducted for 40 cycles of denaturation at 94°C for 15 s, annealing at 56°C for 30 s, and extension at 76°C for 30 s. Product quantification was performed by fluorescence detection of fluorescein reporter dye emission at 510 nm. The threshold values (CT values) were determined with the Rotor Gene software 4.6. For the relative quantification normalized CT values of the target genes were estimated using CT values of the standard curves of the beta-actin PCR as control target.

Western blot analysis

To determine whether paxilline affects p38 and ERK phosphorylation and I{kappa}B-{alpha} protein expression in human macrophages, Western blot analysis was performed. After exposing cells to the experimental conditions, whole-cell lysates were produced by the application of 20 µl of lysis buffer (pH 6.8) containing 2% (v/v) SDS (Bio-Rad), 50 mM DTT (Boehringer-Mannheim), 62.5 mM Tris, and 10% (v/v) glycerol. Protein content was determined with Bradford reagent. Aliquots of lysates containing equal amounts of protein (12–15 µg) were electrophoretically separated on a 12% SDS gel and transferred onto nitrocellulose by blotting at 100 mV for 1 h. Nonspecific binding sites were blocked with 5% nonfat dry milk in 10 mM Tris, 100 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature. The membranes were then incubated in 0.1% Tween 20 with 5% nonfat dry milk with rabbit Abs against p38 (1:1,000), phospho-p38 (1:1,000), phospho-ERK (1:1,000) from Cell Signaling Technology or I{kappa}B-{alpha} (1:2,000) from Santa Cruz and detected by goat anti-rabbit IgG-HRP (1:10,000) from Jackson ImmunoResearch. Immunoreactive proteins were visualized by ECL using the ECL Western blotting detection system (Amersham Biosciences). Data shown are representative of at least three independent experiments.

Protein extraction and NF-{kappa}B activation assay

After exposing cells to the experimental conditions, nuclear extracts were prepared and analyzed as described previously (38). The cells were scraped off the plates, centrifuged, and resuspended in 400 µl of ice-cold buffer A (10 mM Tris, 5 mM MgCl2, 10 mM KCl, 1 mM EGTA, 0.3 M sucrose, 1 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 10 mM beta-glycerol phosphate, and 1.5 µl of protease inhibitor mixture (Complete; Roche). After 15 min on ice, 25 µl of 10% NP-P40 was added. The solution was vortexed and nuclei were pelleted by centrifugation. The supernatants (cytoplasmic extracts) were collected and frozen at –80°C. The nuclear pellet was resuspended in 30 µl of buffer B (20 mM Tris, 5 mM MgCl2, 320 mM KCl, 0.2 mM EGTA, 1 mM DTT, 25% glycerol, and protease inhibitor mixture, and incubated for 15 min on ice. Lysates were cleared by centrifugation at 14,000 x g for 15 min. The activity of NF-{kappa}B in the nuclear extracts was determined by EMSA. NF-{kappa}B oligonucleotides were end-labeled with [{gamma}-32P]ATP using T4 kinase. A 2-µg sample of crude nuclear extract was incubated for 20 min in binding buffer containing 50 µg/ml poly(dI/dC) with 7.5 fmol of the 32P-labeled oligonucleotides encoding the consensus NF-{kappa}B site 5'-AGCTCAGAGGGGACTTTCCGAGAGAGC-3' (MWG-Biotech). Samples were separated by electrophoresis in 5% polyacrylamide gels for 2 h at 150 V, and gels were analyzed with a PhosphorImager (Molecular Dynamics). In competition experiments, 100x unlabeled NF-{kappa}B probe was added along with radiolabeled NF-{kappa}B probe. For supershift analysis, nuclear extracts were incubated with anti-p65 and anti-p50 Abs (Santa Cruz) for 20 min before addition of radiolabeled probe.

Caspase activation assay

For analysis of caspase-1 activation, 4.5 x 105 human macrophages were seeded on cover glasses (18-mm diameter; Carl Roth) and stimulated with LPS in the absence and presence of paxilline at the indicated concentrations for 4 h. During the last hour of stimulation, the caspase-1 pseudosubstrate FAM-YVAD-FMK (BIOCARTA) was added to cells as recommended by the manufacturer. Cells were then fixed in 4% w/v paraformaldehyde, mounted with VectaShield containing 4',6'-diamidino-2-phenylindole (Vector Laboratories), and visualized using a Zeiss LSM 510 confocal laser-scanning microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dose-response and specificity of the inhibitory effect of paxilline

To elucidate the role of MaxiK channel in LPS-induced cell activation and signal transduction of human macrophages, we used the specific MaxiK blocker paxilline (39) to inhibit the activation of the channel. Blockade of MaxiK by the application of paxilline led to a decrease in secretion of the proinflammatory cytokine TNF-{alpha} and the chemokine IL-8 after stimulation of cells with LPS (Fig. 1A). In contrast, secretion of IL-8 after stimulation of cells with TNF-{alpha} was not affected in the presence of paxilline (Fig. 1B). To elucidate whether the function of MaxiK is required for the release of cytokines after cell stimulation or is involved in the intracellular signaling process leading to the transcription of cytokine genes, we isolated RNA from macrophages stimulated with either LPS or TNF-{alpha} in the absence and presence of paxilline. Relative transcription of the genes coding for IL-1beta, IL-6, IL-8, and TNF-{alpha} was determined in a quantitative real-time PCR. Gene transcription was enhanced upon stimulation of cells with LPS compared with controls for all cytokines measured (Fig. 1C). In the presence of paxilline, significant reduction in gene transcription was observed. In contrast, the induction of gene transcription induced by TNF-{alpha} was not reduced in the presence of paxilline. These data indicate that inhibition of MaxiK activation by LPS interferes with cell activation at the level of gene transcription or before gene transcription, whereas transcription downstream of TNF-{alpha}-signaling cascades is not inhibited.


Figure 1
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  		FIGURE 1. The MaxiK blocker paxilline inhibits LPS-induced cytokine transcription and release. Influence of prior application of paxilline on LPS-induced TNF-{alpha} and IL-8 production (A) and TNF-{alpha}-induced IL-8 production (B). Human blood macrophages were incubated in the absence and presence of 20 µM paxilline for 10 min and subsequently stimulated with the indicated concentrations of LPS or TNF-{alpha}. Cell-free supernatants were harvested after 4 h for the determination of TNF-{alpha} and after 24 h for the determination of IL-8. Cytokine concentrations are mean ± SD of triplicates. Data shown is representative of three independent experiments. C, Real-time RT-PCR of cytokine mRNA from human blood macrophages. Cells were incubated in the absence and presence of 20 µM paxilline for 10 min and subsequently stimulated with either 5 ng/ml LPS or 50 ng/ml TNF-{alpha}. After 30 min of stimulation, the mRNA was harvested, reverse-transcribed, and analyzed by real-time PCR for the relative transcription levels of the indicated cytokines.

 
The expression of MaxiK in cells of myelo-monocytic origin is restricted to macrophages. In monocytes, expression of the protein cannot be detected as shown earlier by the absence of channel activity in monocytes measured by patch-clamp technique (33). We confirmed these results by analyzing MaxiK mRNA in freshly isolated human monocytes and cells differentiated in vitro for increasing times in the presence of M-CSF. The expression of MaxiK was determined by PCR amplification of reverse-transcribed RNA using gene-specific primers for the pore-forming {alpha} subunit of MaxiK. In human monocytes freshly isolated from peripheral blood, MaxiK was not detected. Differentiation of monocytes in the presence of M-CSF induced transcription of MaxiK, leading to a positive signal on day 3, which enhances over time until day 7 (Fig. 2A). Analyzing primary human alveolar macrophages from various donors, we could show that also in vivo-differentiated macrophages transcribe MaxiK mRNA (shown exemplarily in Fig. 2B). Blockade of MaxiK function by paxilline led to a reduction in cytokine production already at a concentration of 2 µM paxilline as shown for TNF-{alpha} release after LPS stimulation of in vitro-differentiated human macrophages (Fig. 2C, right panel). A sensitive dose-response was also observed for the inhibition of TNF-{alpha} mRNA transcription in in vitro-differentiated human macrophages (Fig. 2D). In contrast, application of paxilline did not attenuate LPS-induced TNF-{alpha} release from human monocytes even at the highest concentration of 20 µM (Fig. 2C, left panel), supporting that the inhibitory effects of paxilline depend on the presence of MaxiK channel and excluding effects on cell viability or unspecific inhibition of intracellular signaling events independent of the ion channel. Analysis of the effect of paxilline on cell activation of monocytes and macrophages from data of various independent experiments (Table I) supports that paxilline does not have inhibitory effects on the activation of monocytes by LPS. Surprisingly, cytokine production in monocytes appears to be even enhanced at the lowest concentration of paxilline (2 µM), an observation we cannot explain yet. The collective data also revealed that, in contrast to the individual experiment shown in Fig. 2C, where the inhibitory effect of paxilline was less pronounced at the highest dose, paxilline inhibits the activation of macrophages by LPS in a dose-dependent manner at the concentrations tested.


Figure 2
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  		FIGURE 2. Dose-response and specificity of the inhibitory effect of paxilline. A, Expression of MaxiK mRNA detected by PCR of reverse-transcribed RNA from human blood monocytes directly after isolation and after increasing time of differentiation in the presence of M-CSF. B, Expression of MaxiK mRNA detected by PCR of reverse-transcribed RNA from alveolar lavages of two donors. The controls included were cDNA from blood-derived human monocytes (–) and from corresponding macrophages (+). C, Influence of prior application of paxilline on LPS-induced TNF-{alpha}-production of human blood monocytes (left) and macrophages (right) of the same donor. Cytokine concentrations are mean ± SD of triplicates. Data shown are representative of three independent experiments. D, Real-time PCR of corresponding production of TNF-{alpha} mRNA in human macrophages.

 

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  		Table I. Influence of prior application of paxilline on LPS-induced TNF{alpha} production of human blood monocytes and macrophagesa

 
MAPK pathway of signal transduction is partially affected by blockade of MaxiK

Cell activation by LPS initiates several lines of intracellular signaling cascades cumulating in the transcription of proinflammatory genes. Among the major signaling cascades are the MAPK pathways that are initiated by stimulation of TLR4 with LPS. Thus, we investigated the effects of the MaxiK blocker paxilline on the activation of MAPK proteins. Stimulation of human macrophages by LPS lead to a phosphorylation of the ERK already after 15 min (Fig. 3A). This phosphorylation was neither affected by the presence of paxilline at 15 min nor at later time points until 90 min poststimulation. In contrast, the presence of an anti-CD14 Ab completely abolished the phosphorylation of ERK proteins after stimulation with LPS. Similar results were obtained for the phosphorylation of JNK (data not shown). In a time course of p38 phosphorylation after stimulation of human macrophages with LPS, a slight reduction of p38 phosphorylation was observed 15 and 30 min after stimulation of cells in the presence of paxilline (Fig. 3B, upper panel). To confirm the inhibitory function of paxilline in these experiments, cell supernatants of the same setup were also analyzed for TNF-{alpha} production after 4 h. Cytokine determination showed a strong inhibition of TNF-{alpha} production after stimulation with LPS in the presence of paxilline (Fig. 3B, lower panel).


Figure 3
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  		FIGURE 3. MAPK pathway of signal transduction is partially affected by blockade of MaxiK. Western blot analysis of ERK phosphorylation after 15 min (A) and p38 phosphorylation after the indicated time points (B). Human blood macrophages were incubated in the absence and presence of 20 µM paxilline or 10 µg/ml inhibitory CD14 Ab for 10 min and subsequently stimulated with 10 ng/ml LPS. Cytosolic extracts were separated by gel electrophoresis, and proteins were blotted on nitrocellulose and detected by Abs specific for phosphorylated ERK and phosphorylated p38. B, Lower panel, TNF-{alpha} production of the experiment shown in A (upper panel) after 4 h of stimulation in the presence and absence of paxilline. Western blots shown are representative of five experiments. Analysis of band intensities was performed by densitometry using Optimas software (Media Cybernetics). Densitometric analysis summarizing the data from independent experiments is depicted below the respective representative experiment.

 
Blockade of MaxiK inhibits the nuclear translocation of transcription factor NF-{kappa}B

Another major signaling pathway downstream of cell activation by LPS is the NF-{kappa}B pathway. NF-{kappa}B is an important transcription factor regulating the activation of a broad range of proinflammatory genes upon translocation into the nucleus. In contrast to the slight effects observed on MAPK pathways, we found that the presence of paxilline completely abolished translocation of NF-{kappa}B into the nucleus after stimulation of human macrophages with LPS (Fig. 4A). In accordance with the data on cell activation by the cytokine TNF-{alpha} presented above, translocation of NF-{kappa}B after stimulation with TNF-{alpha} was not affected by paxilline (Fig. 4A). The identity of the protein determined in nuclear extracts by radiolabeled probes was verified using a panel of control probes and could be confirmed to be NF-{kappa}B p65 (Fig. 4B). Using alveolar macrophages from rats, we could show that the translocation of NF-{kappa}B after stimulation with LPS is also completely abrogated in these primary macrophages (Fig. 4C), confirming that MaxiK function appears to play a pivotal role in the induction of NF-{kappa}B pathway of signal transduction.


Figure 4
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  		FIGURE 4. Blockade of MaxiK inhibits the nuclear translocation of transcription factor NF-{kappa}B. EMSA of nuclear NF-{kappa}B in human blood macrophages (A and B) and alveolar macrophages of the rat (C). Macrophages were incubated in the absence and presence of 20 µM paxilline for 10 min and subsequently stimulated with 10 ng/ml LPS or 50 ng/ml TNF-{alpha}. After 60 min of stimulation, nuclear extracts were prepared, separated by gel electrophoresis, and probed with 32P-labeled oligonucleotide specific for NF-{kappa}B. B, To test for the specificity of the oligonucleotide bands detected, nuclear extracts from LPS-stimulated cells was preincubated with buffer or Abs specific for p65, p50 subunits of NF-{kappa}B, unlabeled oligos for AP-1 or unlabeled oligos for NF-{kappa}B, and probed with 32P-labeled oligonucleotide specific for NF-{kappa}B. Analysis of band intensities was performed by densitometry. Data shown are representative of three (A and B) and two (C) independent experiments, respectively.

 
Blockade of MaxiK inhibits degradation of I{kappa}B-{alpha}

The translocation of NF-{kappa}B into the nucleus is tightly regulated. In resting cells, NF-{kappa}B is retained in the cytoplasm by the family of inhibitory proteins (I{kappa}B). The I{kappa}B-{alpha} isoform represents the major proximal regulator of NF-{kappa}B. Upon stimulation, I{kappa}B-{alpha} is phosphorylated at specific serine residues and ubiquitinylated, and subsequently undergoes degradation in the proteasome, releasing NF-{kappa}B for nuclear translocation. We investigated the degradation of I{kappa}B-{alpha} in human macrophages and found degradation of I{kappa}B-{alpha} strongest 20 min after stimulation of cells by LPS and reappearing 40 min after stimulation (Fig. 5). The observed kinetics of I{kappa}B-degradation matches the kinetics of NF-{kappa}B translocation, with a maximum observed 40–60 min after cell activation. The degradation of I{kappa}B-{alpha} at 20 min after stimulation by LPS was completely abolished in the presence of paxilline, confirming that this pathway of signal transduction critically depends on the function of the MaxiK channel.


Figure 5
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  		FIGURE 5. Blockade of MaxiK inhibits degradation of I{kappa}B-{alpha}. Western blot analysis of I{kappa}B-{alpha} protein expression in LPS-stimulated cells. Macrophages were incubated in the absence and presence of 20 µM paxilline for 10 min and subsequently stimulated with 5 ng/ml LPS. At time points 0, 20, and 40 min after stimulation, cytosolic extracts were prepared and separated by gel electrophoresis, and proteins were blotted on nitrocellulose and probed with an Ab specific for I{kappa}B-{alpha}. Analysis of band intensities was performed by densitometry. Data shown are representative of three independent experiments.

 
Blockade of MaxiK inhibits caspase-1 activity

The activation of MaxiK is accompanied by an efflux of potassium ions from the cell. To identify a possible intracellular target that is regulated by this potassium efflux, we have investigated the activation of caspases, i.e., potassium-regulated proteases involved in apoptosis and inflammatory processes. Staining for caspase activity with the carboxyfluorescein-labeled caspase-1 pseudosubstrate FAM-YVAD-FMK showed that stimulation of human macrophages with LPS induced an activation of caspase-1 (Fig. 6). In contrast, no caspase-1 activity was observed in unstimulated cells or cells treated with the MaxiK blocker paxilline alone. However, the LPS-induced caspase-1 activity was strongly inhibited in the presence of paxilline, indicating that MaxiK function is critically involved in this caspase activity in human macrophages.


Figure 6
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  		FIGURE 6. LPS-induced activation of caspase-1 is inhibited by paxilline. Macrophages were pretreated as indicated with 20 µM paxilline for 20 min and then stimulated with 5 ng/ml LPS for 4 h or were left untreated. Caspase-1-like activity was analyzed by staining with a FAM-labeled pseudosubstrate for caspase-1 (green) and subsequent visualization by confocal microscopy. Chromatin was stained with 4',6'-diamidino-2-phenylindole (blue). Figure shows representative pictures from three independent experiments with cells from different donors. Bar, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cell activation by bacterial LPS appears to be a highly complex process that is still not understood in detail. Signaling proteins at the cell surface, TLR4 and MD-2, which are involved in the perception of the stimulus, connect to several intracellular signaling cascades orchestrating the process of the cellular response. Recent investigations have revealed that the receptor complex is not a static structure but assembles upon stimulation with LPS by movement of the proteins into lipid raft domains of the cytoplasmic membrane, resulting in the formation of an active signaling complex (21, 22, 23). However, the molecular mechanisms of this receptor recruitment to specific membrane domains are not understood. In addition, the signaling complex appears to recruit several accessory proteins with yet mostly unknown functions for the process of stimulus recognition and signal transduction. One of these proteins is the large-conductance Ca2+-dependent potassium channel MaxiK. This ion channel is expressed in macrophages and has been shown in a previous work to be activated by LPS (33).

The data presented here provide evidence for a specific role of MaxiK in initiating an NF-{kappa}B-dependent signaling cascade. We could show that the blockade of MaxiK abolishes the degradation of the inhibitory factor I{kappa}B-{alpha} (Fig. 5) and the subsequent translocation of NF-{kappa}B (Fig. 4A) into the nucleus in LPS-stimulated cells, assigning the ion channel a central role in the signal generation process. Also, the MAPK pathway of p38 revealed a sensitivity for the blockade of MaxiK (Fig. 3B), whereas ERK was found to be unaffected by channel blockade during cell activation by LPS (Fig. 3A). LPS-induced activation of NF-{kappa}B via TLR4 is accomplished via two different pathways, the MyD88-dependent pathway and the MyD88-independent pathway. Because the kinetics of NF-{kappa}B translocation is different in both pathways, different mechanisms of proximal regulation can be assumed. The observed dependence of nuclear translocation of NF-{kappa}B on the function of MaxiK can be interpreted in a way that the ion channel represents a general principle in both pathways of TLR4 activation.

In monocytes, which do not express the MaxiK channel, the channel blocker paxilline had no effect on LPS-induced cell activation, excluding unspecific inhibitory effects of paxilline on other cellular targets. Also, activation of macrophages by the cytokine TNF-{alpha} was not affected by the blockade of MaxiK. In summary, the inhibitory effects of MaxiK blockade appeared to be specific for MaxiK-expressing cells and revealed a role for this ion channel in a central pathway of the signaling process initiated by LPS.

Patch-clamp experiments on excised membrane patches from human macrophages have shown that the application of LPS to the outer leaflet of the membrane leads to an activation of the MaxiK channel. This observation indicates that MaxiK activation is a very early process in cell activation taking place in the cytoplasmic membrane (33). The role of MaxiK activation in the initiation of the signaling process is not understood yet. MaxiK activation leads to an opening of the ion channel with subsequent potassium efflux. Thus, MaxiK activation could participate in the signaling process by either of two possible mechanisms: regulation of a potassium-sensitive protein in the signaling cascade or regulation of a membrane potential-sensitive process.

Caspases are an evolutionarily conserved group of proteases involved in apoptosis and inflammation (40). Caspase-1 exhibits upon activation an enzymatic activity that cleaves pro-IL-1beta and pro-IL-18 to form the active secreted cytokines (41, 42, 43). The maturation of these cytokines is attributed to large caspase-1-containing protein complexes termed "inflammasome" (44). Several reports have described the activation of caspases by decreasing intracellular potassium concentrations (45, 46). Caspase-1 has been found to be activated by K+ efflux via P2X7 channels in mouse macrophages, which were activated by the application of LPS and ATP (46). In colon epithelial cells stimulated with LPS, caspase-1 is activated by a MyD88-independent mechanism (47). Similar results have been obtained for the role of caspase-3 in the activation of various cell types by LPS (48, 49). The LPS-induced production of IL-1 by human macrophages has been shown to be dependent on the activation of caspase-1 (44, 50). Recently, a novel function of caspase-1 in the activation of NF-{kappa}B and p38 MAPK has been described, which is independent of its enzymatic activity (51). The authors showed that, in HEK293T cells, caspase-1 activation is necessary for the LPS-induced activation of p38 and NF-{kappa}B. Caspase-3 has been found to affect activation of the MAPK p38 but does not appear to be involved in the activation of NF-{kappa}B (48). In this study, we have investigated a functional connection between MaxiK and the activation of caspase-1. Our results clearly demonstrate that caspase-1 is activated in human macrophages by LPS, but not by the MaxiK blocker paxilline (Fig. 6). However, the LPS-induced activation of caspase-1 is inhibited in the presence of paxilline. These data show for the first time that the MaxiK channel has a regulatory function for caspase-1 activation in human macrophages. Thus, caspase-1 is an attractive candidate that could link MaxiK activation to the NF-{kappa}B-dependent signaling pathway.

The data presented provide evidence that the initiation and regulation of NF-{kappa}B-dependent signaling cascades obviously differ in monocytes and macrophages. In the latter, a strong dependency of the NF-{kappa}B pathway of the activation of MaxiK could be shown for in vitro-generated macrophages from peripheral blood monocytes as well as for in vivo-differentiated primary pulmonary macrophages. This pathway obviously represents a general principle in different types of macrophages. At present, we can only speculate about the reasons for this difference between monocytes and macrophages. A possible explanation might be that in monocytes simply other potassium channels fulfill the function of MaxiK in regulation of the signaling process. But it is also conceivable that the macrophage elicits a more complex pattern of signal regulation to allow this cell a much tighter control of regulating activation processes.

We have shown that the potassium channel MaxiK is critically involved in the very early steps of the proinflammatory response of macrophages to bacterial LPS. For the first time we could show that the central I{kappa}B-{alpha}/NF-{kappa}B-dependent proinflammatory pathway depends on the function of MaxiK channel in macrophages. This adds a new level of regulation in the complex signal transduction process elicited by macrophages in response to bacterial infection.


   Acknowledgments
 
We thank C. Hamann, S. Groth, and S. Adam for excellent technical assistance and Prof. Dalhoff (University of Lübeck) for kindly providing cells from human lung lavage.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Deutsche Forschungsgemeinschaft (SCHR 621/2-1, SFB 367 Project B8, and DFG STA 609/1-3). Back

2 Address correspondence and reprint requests to Dr. Andra Schromm, Research Center Borstel, Department of Immunochemistry and Biochemical Microbiology, Emmy-Noether Group Immunobiophysics, Parkallee 10, 23845 Borstel, Germany. E-mail address: aschromm{at}fz-borstel.de Back

3 Abbreviations used in this paper: LBP, LPS-binding protein; TIR, Toll/IL-1R. Back

Received for publication September 16, 2005. Accepted for publication June 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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