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

Abstract

<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.