MaxiK Blockade Selectively Inhibits the Lipopolysaccharide-Induced IκB-α/NF-κB Signaling Pathway in Macrophages

Abstract

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-α 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-α 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-κB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IκB-α is critically involved in the observed inhibition of NF-κB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-κB 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-κB-dependent inflammatory response of macrophages to bacterial LPS.

BK channels in innate immune functions of neutrophils and macrophages

“…Papavlassopoulos et al demonstrated that LPS-induced NF-B activation in human and rat macrophages was inhibited by BK channel blockade and proposed that BK channels might participate in TNF-␣ production by controlling NF-〉-dependent gene transcription. 17 However, similar NF-B activation occurred in BK ϩ/ϩ and BK Ϫ/Ϫ BMDMs ( Figure 5D). Moreover, no differences in Akt and ERK phosphorylation were observed between BK ϩ/ϩ and BK Ϫ/Ϫ BMDMs, indicating that BK channels do not control TNF-␣ release or signal transduction pathways that are implicated in TNF-␣ generation and release in BMDMs.…”

“…17 Using the BK channel inhibitors IbTX and paxilline, we observed that the dose-dependent LPS-induced TNF-␣ production in For personal use only. on May 11, 2018.…”

“…19 Prior studies using pharmacologic compounds implicated a BK channel dependence of LPS-induced macrophage activation, in particular with respect to NF-B activation and TNF-␣ production. 17,20 When we explored the potential role of BK channels using in vitro-differentiated BMDM from BK ϩ/ϩ and BK Ϫ/Ϫ mice, we found no biologic significance for this channel; neither TNF-␣ release nor the initiation of signaling pathways, such as NF-B, ERK, and phosphoinositide-3 kinase/Akt, was dependent on the presence of BK channels. Phosphoinositide-3 kinase has recently been implicated as a negative regulator of TLR4-mediated TNF-␣ release in human alveolar macrophages.…”

“…BK channels were observed earlier in human macrophages, and interestingly, the BK channel inhibitor effect on TNF-␣ and NF-B was also obtained by studying macrophages from either human blood or rat lungs. 17,22,23 It is therefore conceivable that differences between in vitrodifferentiated mouse BMDMs, and peripheral human and rat macrophage account for the observed differences. This assumption is further supported by the fact that mouse blood macrophages, but not BMDMs, initiated a respiratory burst to a strong activator such as PMA.…”

Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration

“…31 PGN is also a ligand of TLR2, suggesting that TLR2 or TLR4 might change intracellular K þ levels. Interestingly, TLRs and IL-1R were recently shown to associate and functionally depend on the large-conductance Ca 2 þ -activated K þ channel MaxiK 32,33 Since this K þ channel is abundantly expressed in macrophages, we could speculate that the activators MSU, PGN and probably imidazoquinoline activate the inflammasome via a MaxiK-mediated K þ efflux. However, the use of MaxiK inhibitor paxilline did not inhibit caspase-1 activation (data not shown), suggesting that different K þ channelforming proteins are involved.…”