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Andrzej Sobota

Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

Recent publications
1.  Czerkies M., Borzęcka K., Zdioruk M.I., Płóciennikowska A., Sobota A., Kwiatkowska K., An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS, IMMUNOBIOLOGY, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2013.04.005, Vol.218, pp.1217-1226, 2013

Abstract:
Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000 ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

Keywords:
CD14, CTX-FITC, Endocytosis, HEPES-buffered saline, LBP, LPS conjugated with Alexa Fluor 488, LPS-AF488, LPS-binding protein, Lipopolysaccharide, PD buffer, SR-A, Scavenger receptor, TLR, poly(I:C), polyinosinic–polycytidylic acid, scavenger receptor A, subunit B of cholera toxin conjugated with FITC, transferrin conjugated with Alexa Fluor 647, transferrin-AF647

Affiliations:
Czerkies M. - other affiliation
Borzęcka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Zdioruk M.I. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Płóciennikowska A. - other affiliation
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
2.  Kleveta G., Borzęcka K., Zdioruk M., Czerkies M., Kuberczyk H., Sybirna N., Sobota A., Kwiatkowska K., LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility, JOURNAL OF CELLULAR BIOCHEMISTRY, ISSN: 0730-2312, DOI: 10.1002/jcb.23330, Vol.113, No.1, pp.80-92, 2012

Abstract:
Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

Keywords:
ACTIN CYTOSKELETON, CELL MOTILITY, LPS, N-WASP, PAXILLIN, SRC KINASES

Affiliations:
Kleveta G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Borzęcka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Zdioruk M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Czerkies M. - other affiliation
Kuberczyk H. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Sybirna N. - Ivan Franko Lviv National University (UA)
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
3.  Józefowski S., Czerkies M., Sobota A., Kwiatkowska K., Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling, ANALYTICAL BIOCHEMISTRY, ISSN: 0003-2697, DOI: 10.1016/j.ab.2011.02.031, Vol.413, No.2, pp.185-191, 2011

Abstract:
Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with 125I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.

Keywords:
Lipopolysaccharide, Toll-like receptor 4, ELISA, Radiolabeling, Flow cytometry

Affiliations:
Józefowski S. - other affiliation
Czerkies M. - other affiliation
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
4.  Józefowski S., Czerkies M., Łukasik A., Bielawska A., Bielawski J., Kwiatkowska K., Sobota A., Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-^5; Production Induced by Lipopolysaccharide, JOURNAL OF IMMUNOLOGY, ISSN: 0022-1767, DOI: 10.4049/jimmunol.0902926, Vol.185, No.11, pp.6960-6973, 2010

Abstract:
LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation

Keywords:
lipopolysaccharide, TNF alpha, MIP-2, ceramide. ceramide-1-phosphate, sphingomyelinase, J774, macrophages

Affiliations:
Józefowski S. - other affiliation
Czerkies M. - other affiliation
Łukasik A. - other affiliation
Bielawska A. - other affiliation
Bielawski J. - other affiliation
Kwiatkowska K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Sobota A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

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