Immunocytochemical Localization of Bacterial Lipopolysaccharide with Colloidal-Gold Probes in Different Target Cells

Municio, A.M.; Abarca, S; Carrascosa, José L.; Garcı́a, R.; Díaz‐Laviada, Inés; Ainaga, M. J.; Portolés, Manuel; Pagani, Raffaella; Risco, Cristina; Bosch, María Asunción

doi:10.1007/978-1-4757-5140-6_17

Cited by 8 publications

(4 citation statements)

References 6 publications

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“…Although microinjection of LPS circumvents the contact between LPS and the cell membrane and thus does not exactly mimic the fate of externally LPS it may serve as a valuable tool to gain more insight into the mechanisms by which intracellular LPS may induce biological responses. The fact that internalized LPS has been found not only in cellular compartments but also in the cytoplasm (Municio et al, 1990;Kang et al, 1992;Kutuzova et al, 2001) underlines the suitability of this approach. Using the whole-cell patch clamp technique which will give a direct measurement of the net current flow through the whole cell membrane, we could show that LPS when applied extracellularly induces a depolarization-evoked outwardly rectifying K þ current.…”

Section: Discussionmentioning

confidence: 93%

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

Gerth

Grosche

Nieber

et al. 2004

Journal Cellular Physiology

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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.

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Section: Discussionmentioning

confidence: 93%

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

Gerth

Grosche

Nieber

et al. 2004

Journal Cellular Physiology

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“…Exactly how internalization processes involving lipid trafficking and vesicular transport could lead to an interaction with postulated intracellular LPS recognition molecules such as the cytosolic nod proteins remains speculative. In this context, microinjection of LPS into the cytoplasm does not mimick the exact pathways of LPS internalization, however, since earlier experiments have detected internalized LPS also in the cytoplasm (Kang et al, 1990;Municio et al, 1990;Diaz-Laviada et al, 1991;Kutuzova et al, 2001), the microinjection procedure for the detection of intracellular activity of LPS disassociated from extracellular engagement by the LPS-recognition complex has validity. It can thus be concluded that microinjected LPS leading to cytoplasmic localization does not lead to NFkB translocation and NFkB dependent gene transcription of the investigated inflammatory mediators, suggesting that an intracellular cytoplasmic LPS receptor leading to activation of this pathway is not present or functional in the cellular systems used.…”

Section: Discussionmentioning

confidence: 96%

“…Besides stimulating cells by engagement of the above mentioned activation and signaling cascade, LPS is also internalized, probably by a number of mechanisms (micropinocytosis, macropinocytosis, receptor mediated and penetration of the plasma membrane) leading to different subcellular localizations of endotoxin including the cytoplasm (Kang et al, 1990;Municio et al, 1990;Diaz-Laviada et al, 1991;Kutuzova et al, 2001). This internalization process is discussed as representing a mechanism for clearance of this toxic compound (Munford and Hall, 1986) which, although mediated partly by the same molecule (CD14 (Poussin et al, 1998)), does not appear to activate cells (Kitchens et al, 1992;Gegner et al, 1995;Lentschat et al, 1999).…”

mentioning

confidence: 99%

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

Seitzer

Gerdes

2002

Journal Cellular Physiology

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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.

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“…LPS triggers a wide range of cellular responses, including the synthesis and release of a variety of inflammatory mediators (e.g., tumor necrosis factor alpha [TNF-ot], interleukin 1 [IL-1], IL-6, IL-8, and prostaglandins) which contribute to host defense mechanisms but may induce major immunopathological disorders when produced in excess. The triggering of cellular responses is initiated by the binding of LPS to the cell surface which precedes its internalization (2,18,19,27). Over the last few years, major efforts have been devoted to the characterization of cell membrane structures which may function as LPS receptors.…”

mentioning

confidence: 99%

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae

et al. 1994

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A water-soluble acylpolygalactosyl (APG) of 34 kDa was obtained from the Klebsiella pneumoniae membrane by alkaline hydrolysis and delipidation. APG comprises a poly(1,3)galactose chain, a core, and a lipid moiety made of a glucosamine disaccharide with two N-linked ,OH-myristates. The monocyte binding sites for APG were investigated by flow cytometry. Biotin-labelled APG (Biot-APG) bound to monocytes at 4°C in the absence of serum, calcium, and magnesium. The binding was dose dependent, saturable, and displaced by unlabelled APG. Neither the polysaccharide chain present in APG-related molecules nor the PP; group or additional ester-linked myristates and palmitates were required for APG binding. The role of CD11b and CD14 was demonstrated by competitive inhibition with monoclonal antibodies and by the uptake of APG by these solubilized proteins. APG was rapidly internalized into monocytes at 37°C while CD14 and CD11b/CD18 molecules were partially down-modulated. Lipopolysaccharides (LPS) from the same K. pneumoniae strain and

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Immunocytochemical Localization of Bacterial Lipopolysaccharide with Colloidal-Gold Probes in Different Target Cells

Cited by 8 publications

References 6 publications

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

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

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

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae

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