Lipopolysaccharide is a frequent and significant contaminant in microglia‐activating factors

Abstract

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 α 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 microgliaactivating factors. Careful attention to LPS levels and appropriate controls are necessary for future studies in the neuroinflammation field.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

“…Only trace amounts of LPS (ranging from 0.026 to 0.075 EU/nmol) were detected in the reagents. According to a previously study (Weinstein et al, 2008), these levels of LPS do not interfere with the results. Nevertheless, to assure that signal transduction through TLR2 and TLR4 would not be activated by these amounts of LPS, rats were intracerebroventricularly treated with 2.0 l solution containing 3.0 or 300 ng LPS (corresponding to the amounts of LPS equivalent to 0.075 and 7.5 EU/nmol, respectively), and signal transduction was determined by immunoblot, as described below and presented in Figure 2 A.…”

Critical Role of TLR4 Response in the Activation of Microglia Induced by Ethanol

“…The endotoxin content in the ethanol solution was Յ2.98 ϫ 10 Ϫ3 pg/ml, which is far below the concentration required to induce microglial activation under our assay conditions. Additionally, to further eliminate LPS-dependent effects in some experiments and as previously described (29,30), the LPS antagonist, polymyxin B sulfate (PMBS, 10 g/ml; Sigma-Aldrich), was added to the cell medium 15 min before and during ethanol or LPS treatment, or to supernatants from TLR4 Ϫ/Ϫ microglia treated with or without ethanol. PMBS is a cationic cyclic polypeptide that binds to the lipid A portion of the LPS in the cell membrane of Gramnegative bacteria, acting as a specific LPS antagonist.…”

Misfolded Truncated Protein τ Induces Innate Immune Response via MAPK Pathway

“…No other protein peaks were observed within the MS spectra demonstrating the absence of bacterial protein contaminants. The results presented earlier demonstrate that LPS is a frequent and functionally significant contaminant in many commercial-grade preparations of proteins and peptides used commonly in research on microglial activation (46). To exclude LPS contamination, we have tested truncated t in the absence and/or presence of the LPS inhibitor polymyxin B.…”