Cytoplasmic bacterial lipopolysaccharide does not induce NFκB activation or NFκB mediated activation signals in human macrophages and an LPS reporter cell line

Abstract

Although many membrane components have been described to be involved in the activation of cells by bacterial lipopolysaccharide (LPS), the question remains whether LPS, once internalized by target cells, is also capable of interacting with cytoplasmic elements in such a way that activation of cells results independently of receptor engagement. This is an important aspect to consider with respect to the development of strategies aimed at attenuating adverse effects of LPS in the framework of bacterial infections. In this study, human monocyte derived macrophages as representatives of one of the primary target cells activated by LPS, were microinjected with LPS to circumvent exogenous LPS stimulation. Parameters correlating to cytoplasmic activation of the nuclear transcription factor NFkappaB (intracellular calcium mobilization), to nuclear translocation of the NFkappaB p65 subunit and to mRNA-transcription of inflammatory cytokines known to be expressed upon exogenous LPS-stimulation and to require NFkappaB activation (interleukin-1beta, interleukin-6, tumor necrosis factor alpha) were investigated. In addition, the LPS-reporter cell line 3E10, which contains a reporter gene under the control of an NFkappaB-inducible promoter was analyzed with respect to NFkappaB nuclear translocation and reporter gene expression. None of the cellular systems used and none of the parameters investigated led to the observation that intracellular LPS leads to activation of the cells in comparison to external LPS stimulation. These experiments allow the conclusion that LPS in the cytoplasmic compartment does not lead to NFkappaB translocation, cytokine mRNA transcription, and NFkappaB dependent protein expression and suggest that these activation parameters require the interaction of LPS with external membrane components.

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

“…In search of another LPS-induced short time activation signal that allows to be detected in microinjected cells we studied the effect of LPS on the activation of NFkB. In accordance with Seitzer and Gerdes (2003) who injected LPS into an LPS reporter cell line, we did not observe any translocation of NFkB p65 into the nucleus. The viability of the cells was verified by showing that NFkB p65 was translocated into the nucleus of cells that had been microinjected with LPS (10 mg/ml) prior to the addition of exogenous LPS (10 mg/ml) (data not shown).…”

“…As Seitzer and Gerdes (2003) neither observed an effect of intracellular LPS on NFkB dependent gene transcription of IL-1 b and IL-6 in human macrophages they conclude that the mere presence of LPS in the cytoplasmic compartment is not sufficient to activate downstream signaling. According to Hornef et al (2002), however, in epithelial cells LPS-mediated cellular activation requires ligand internalization indicating that cells might differ in their intracellular means to recognize LPS.…”

MicroRNA-145-5p and microRNA-320a encapsulated in endothelial microparticles contribute to the progression of vasculitis in acute Kawasaki Disease

“…Validation studies showed that in monocytes transfected with hsa-miR-1, PTK9 mRNA expression was significantly reduced (87%) at 3 hours post-transfections, and this suppression continued for 24 hours (Supplementary Figure 4 ). Since LPS efficiently induces inflammation response and is commonly used many experiments for studying the cellular inflammation signaling 7 , 22 – 24 , we conducted the experiments in the presence or absence of LPS. Three hours post-transfection cells were harvested and mRNAs of inflammatory cytokines ( TNF , IL1B , IL6 , IL10 and IL18 ) and the target mRNAs ( BMPR1A and TMEM9B ) were measured by real-time PCR.…”