Intracellular LPS inhibits the activity of potassium channels and fails to activate NFκB in human macrophages

Abstract

Although much has been learned about signal transduction mechanisms and binding proteins involved in lipopolysaccharides (LPS)-induced activation of monocytes/macrophages, little is known about the ability of internalized LPS to activate cells. To approach this question we either exposed macrophages to LPS or microinjected the cells with LPS before studying early cellular events associated with LPS-mediated macrophage activation. We measured membrane currents and translocation of NFkappaB to the nucleus. Using the whole-cell patch clamp technique ion channels were analyzed and characterized as K+ sensitive inward and outward currents. Exogenous LPS was shown to increase the voltage-dependent outward current whereas the voltage-dependent inward current was unaffected. However when cells were microinjected with LPS both inward and outward current were completely abolished. The presence of LPS within the cells did not prevent them to perform phagocytosis or to respond to fMLP with an appropriate increase in [Ca2+]i. The immunocytological detection of NFkappaB p65 translocation revealed that exogenous LPS led to the nuclear localization of the p65 subunit of NFkappaB, whereas only the cytoplasmic localization of p65 was observed following microinjection of LPS. These data show that one major process in macrophage activation, the NFkappaB dependent transcription of a number of genes encoding for many inflammatory mediators cannot be induced by intracellular LPS but requires the interaction of LPS with external membrane components. However intracellular LPS causes a drastic decrease in potassium currents which by keeping the cell membrane at a depolarized potential may result in changed biological answers of these cells.

Polymyxin B-Conjugated α2-Macroglobulin as an Adjunctive Therapy to Sepsis: Modes of Action and Impact on Lethality

“…We could show that LPS is endocytosed via CD91 entering the lysosomal pathway for degradation. It is of interest to note that microinjected LPS did not induce nuclear factor-B translocation, indicating that intracellular LPS fails to induce an inflammatory response (Gerth et al, 2005). Therefore, internalization of LPS is an attractive way to abolish LPS toxicity in vivo.…”

“…Human monocytes were isolated by elutriation as described previously (Gerth et al, 2005). Isolated monocytes (purity of 96%) were cultivated in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 1% antibiotics (100 g/ml streptomycin and 100 U/ml penicillin) 1% glutamine, and 10% heat-inactivated FCS (Invitrogen).…”

Voltage-gated Potassium Channel Modulation of Neurotoxic Activity in Human Immunodeficiency Virus Type-1(HIV-1)-Infected Macrophages

“…LPS and phorbol myristate acetate (PMA) are capable of activating macrophages and modulating potassium channel expression (Qiu et al 2002). Exogenous LPS was shown to increase the voltage-dependent outward K current, whereas intracellular LPS inhibited the activity of potassium channels and failed to activate NFκB signaling in human macrophages (Gerth et al 2005). In addition, Kv channel activity can also be modulated by HIV-1 protein Tat (Visentin et al 2001).…”

A Cross-Disciplinary Perspective on the Innate Immune Responses to Bacterial Lipopolysaccharide

“…The study of intracellular LPS responses has been almost non-existent, as extensive characterization of cellular responses induced by extracellular LPS has dominated the past two decades. Nevertheless, scattered evidence suggested the existence of TLR4-independent host responses to LPS (Vogel et al 1980; Haziot et al, 2001; Gerth et al, 2005). Two recent studies revealed that intracellular LPS can trigger the Caspase-11-dependent formation of non-canonical inflammasomes by a process that does not require TLR4 (Hagar et al, 2013; Kayagaki et al, 2013).…”