Lipopolysaccharide is a frequent and significant contaminant in microglia-activating factors.
Lipopolysaccharide (LPS/endotoxin) is a potent immunologic stimulant. Many commercial-grade reagents used in research are not screened for LPS contamination. LPS induces a wide spectrum of proinflammatory responses in microglia, the immune cells of the brain. Recent studies have demonstrated that a broad range of endogenous factors including plasma-derived proteins and bioactive phospholipids can also activate microglia. However, few of these studies have reported either the LPS levels found in the preparations used or the effect of LPS inhibitors such as polymyxin B (PMX) on factor-induced responses. Here, we used the Limulus amoebocyte lysate assay to screen a broad range of commercial- and pharmaceutical-grade proteins, peptides, lipids, and inhibitors commonly used in microglia research for contamination with LPS. We then characterized the ability of PMX to alter a representative set of factor-induced microglial activation parameters including surface antigen expression, metabolic activity/proliferation, and NO/cytokine/chemokine release in both the N9 microglial cell line and primary microglia. Significant levels of LPS contamination were detected in a number of commercial-grade plasma/serum- and nonplasma/serum-derived proteins, phospholipids, and synthetic peptide preparations, but not in pharmaceutical-grade recombinant proteins or pharmacological inhibitors. PMX had a significant inhibitory effect on the microglia-activating potential of a number of commercial-, but not pharmaceutical-grade, protein preparations. Novel PMX-resistant responses to alpha(2)-macroglobulin and albumin were incidentally observed. Our results indicate that LPS is a frequent and significant contaminant in commercial-grade preparations of previously reported microglia-activating factors. Careful attention to LPS levels and appropriate controls are necessary for future studies in the neuroinflammation field.
Weinstein
Jonathan R
JR
Department of Neurology, School of Medicine, University of Washington, Seattle, Washington 98195, USA. jweinste@u.washington.edu
Swarts
Sarah
S
Bishop
Caroline
C
Hanisch
Uwe-Karsten
UK
Möller
Thomas
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000954:Antigens, Surface / Q000032:analysis; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D018925:Chemokines / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004340:Drug Contamination; D004731:Endotoxins / Q000032:analysis / Q000494:pharmacology; D005434:Flow Cytometry; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000032:analysis* / Q000494:pharmacology*; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D011112:Polymyxin B / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D000954:Antigens, Surface; D018925:Chemokines; D016207:Cytokines; D004731:Endotoxins; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D011112:Polymyxin B