The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures

Pathobiology ◽  
2021 ◽  
pp. 1-9
Author(s):  
Hiroki Kohno ◽  
Kazuhisa Ouhara ◽  
Sho Mokuda ◽  
Tadahiro Tokunaga ◽  
Tomohiro Sugimoto ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Synovial Cell ◽  
Polymyxin B ◽  
Heat Inactivation ◽  
Synovial Cells ◽  
Toll Like Receptor ◽  
Blood Monocytes ◽  
Wild Type ◽  
Peripheral Blood Monocytes ◽  
Dose Dependent

<b><i>Introduction:</i></b> Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). <b><i>Methods:</i></b> RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. <b><i>Results:</i></b> cit-Fb induced expression of <i>CXCL10</i> and <i>IFNB</i> in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated <i>CXCL10</i> or <i>IL-6</i> induction; however, cit-Fb-mediated <i>CXCL10</i>expression was inhibited. Wild-type LPS from <i>Escherichia coli</i> (WT-LPS) strongly induces <i>CXCL10</i> expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated <i>CXCL10</i> induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated <i>CXCL10</i> induction in RSCs and PBMs. <b><i>Conclusion:</i></b> To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

scholarly journals Pertussis Toxin and Lipopolysaccharide Influence Phagocytosis of Bordetella pertussis by Human Monocytes

2001 ◽  
Vol 69 (12) ◽  
pp. 7635-7641 ◽  
Author(s):  
Lyndsay M. Schaeffer ◽  
Alison A. Weiss
Keyword(s):  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Fluorescent Protein ◽  
Polymyxin B ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Wild Type ◽  
Peripheral Blood Monocytes ◽  
Potential Mediator ◽  
Green Fluorescent

ABSTRACT The potential of human monocytes to mediate the clearance ofBordetella pertussis infection was examined. Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy. Monocytes internalized only a small percentage of the adherent bacteria. Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis. However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria. Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes. Opsonization with human serum played only a modest role in promoting phagocytosis. The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin. Less than 1% of internalized bacteria remained viable. These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance of B. pertussis infection.

scholarly journals Expression of sterile-α and armadillo motif in rheumatoid arthritis monocytes correlates with TLR2 induced IL-1β and disease activity

Rheumatology ◽  
2021 ◽  
Author(s):  
Ryan S Thwaites ◽  
Sarah Unterberger ◽  
Giselle Chamberlain ◽  
Henry Gray ◽  
Kelsey Jordan ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Disease Activity ◽  
Negative Regulator ◽  
Potential Contribution ◽  
Toll Like Receptor ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Pyrin Domain ◽  
Bone Damage ◽  
Receptor Family

Abstract Objective Cartilage and bone damage in rheumatoid arthritis (RA) are associated with elevated IL-1β. The effects of IL-1β can be reduced by biological therapies that target IL-1β or TNFα. However, the mechanisms responsible for increased IL-1β and the effect of anti-TNFα have not been fully elucidated. Recently, sterile-α and armadillo motif-containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1β secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR induced IL-1β secretion in RA peripheral blood monocytes and in patients commencing anti-TNFα treatment. Methods Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1β secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM, IL-1β and the components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. Results TLR1/2 activation induced elevated IL-1β in RA monocytes compared with heathy controls (p= 0.0009), which negatively correlated with SARM expression (p = 0.0086). Lower SARM expression also correlated with higher disease activity (p = 0.0246). Additionally, patients responding to anti-TNFα treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. Conclusion Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1β associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNFα blockade can modify IL-1β secretion.

scholarly journals Vitronectin (S protein) augments the functional activity of monocyte receptors for IgG and complement C3b

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 86-93
Author(s):  
CJ Parker ◽  
RN Frame ◽  
MR Elstad
Keyword(s):  
Cell Attachment ◽  
Attachment Site ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Tenfold Increase ◽  
Letter Code ◽  
Single Letter Code ◽  
Arginine Glycine Aspartic Acid ◽  
Dose Dependent Increase ◽  
Dose Dependent

An arginine-glycine-aspartic acid sequence (RGD in the single letter code for amino acids) is present in the cell attachment site of both vitronectin and fibronectin. Inasmuch as fibronectin and synthetic peptides containing RGD enhance ingestion of opsonized particles by monocytes, we investigated the effects of vitronectin on phagocytosis by monocytes of sheep erythrocytes bearing IgG (EA) or complement C3b (EC3b). Peripheral blood monocytes were isolated by countercurrent elutriation and allowed to adhere to slides that had been coated with either vitronectin or fibronectin. Next, EA or EC3b were incubated with the adherent monocytes, and phagocytosis was subsequently quantified. Vitronectin caused the same dose dependent increase in phagocytosis as fibronectin. The augmentation of phagocytosis of EA induced by vitronectin could be inhibited by the F(ab')2 fragments of anti- vitronectin IgG but not by preimmune F(ab')2. The maximum phagocytosis of EA induced by vitronectin could not be enhanced by the addition of fibronectin, suggesting that vitronectin and fibronectin act on the same population of monocytes and that the two proteins stimulate the same mechanism through which the enhanced phagocytosis is mediated. Fibronectin and vitronectin caused a tenfold increase in the attachment of EC3b to monocytes, but phagocytosis was augmented minimally. These studies demonstrate that vitronectin modulates interactions between monocytes and opsonized particles.

scholarly journals Characteristics of lipopolysaccharide interaction with human peripheral-blood monocytes

1985 ◽  
Vol 232 (2) ◽  
pp. 379-383 ◽  
Author(s):  
S J Warner ◽  
N Savage ◽  
D Mitchell
Keyword(s):  
Cell Membrane ◽  
Peripheral Blood ◽  
Lipid A ◽  
Human Peripheral Blood ◽  
Human Monocyte ◽  
Polymyxin B ◽  
Salmonella Typhi ◽  
Apparent Difference ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes

The interaction between radioiodinated lipopolysaccharide from Escherichia coli 0111:B4 (125I-LPS) and human peripheral-blood monocytes was studied. The association of 125I-LPS with monocytes at 37 degrees C appeared to depend on binding to the cell membrane with subsequent internalization of the molecule, and was not saturable with time (up to 2 h) or 125I-LPS concentration (up to 10 micrograms/ml). There was no apparent difference in the behaviour of unlabelled LPS and 125I-LPS with respect to monocyte association. 125I-LPS association with monocytes was inhibited by LPS and O-polysaccharide from E. coli 0111:B4 and Salmonella typhi 0901, but not by lipid A or polymyxin B. We propose that the mechanism of human monocyte stimulation by LPS involves polysaccharide-dependent binding to the cell membrane followed by internalization of the LPS molecule. We were unable to demonstrate a specific LPS receptor such as that found on murine B-lymphocytes.

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-κB activation in human peripheral blood monocytes

2003 ◽  
Vol 284 (4) ◽  
pp. L607-L613 ◽  
Author(s):  
Ling-Yu Chen ◽  
Bruce L. Zuraw ◽  
Ming Zhao ◽  
Fu-Tong Liu ◽  
Shuang Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Human Peripheral Blood ◽  
Toll Like Receptor ◽  
Blood Monocytes ◽  
Toll Like Receptor 4 ◽  
Peripheral Blood Monocytes ◽  
Protein Tyrosine

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-α, IL-1β, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-κB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-κB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.

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