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 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.

Fig. 1

Comparison between effects of different commercial- and pharmaceutical-grade preparations in the absence or presence of PMX on a number of microglial activation parameters in the N9 cell line. N9 cells were either unstimulated or stimulated with 100 EU/mL of control standard endotoxin (CSE), 100-U/mL recombinant human α-thrombin (rh-thr 100), 10-U/mL recombinant mouse interferon-γ (rm-IFNγ), 100-U/mL plasma-derived bovine α-thrombin (pb-thr), 100-nM serum-derived human α₂-macroglobulin from supplier #5 (sh-α₂M-5), 100-nM serum-derived human α₂-macroglobulin from supplier #7 (sh-α₂M-7), 1-μM 2,4,6-trinitrophenol-conjugated egg-white-derived chicken ovalbumin (tnp-Oval), 15.4-μM (1 mg/mL) serum-derived rat serum albumin-essentially fatty acid free (sr-Alb-efaf), and/or 15.4-μM (1 mg/mL) serum-derived rat albumin-fraction V powder (sr-Alb-FV) as indicated in the absence or presence of PMX (10 μg/mL). Cell surface expression of CD95 (Fas) (A) and CD40 (B) was assessed 24 h later by flow cytometry. Values for the treatment groups that did and did not include PMX were normalized to their respective controls. Means ± S.E.M. for these values from at least three separate experiments are presented. *P < 0.05, **P < 0.01, ***P < 0.001 vs. corresponding preparation treatment without PMX. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. unstimulated control or PMX alone control (dotted line). Prenormalization mean absolute fluorescent intensity units ± S.E.M. for unstimulated cells were 15.9 ± 2.5 and 15.9 ± 2.8 in the absence or presence of PMX, respectively, for CD95(Fas) and 7.4 ± 1.2 and 5.5 ± 1.1 for CD40. Metabolic activity/proliferation (C), NO production (D), and TNF-α release (E) were also assessed 24 h following stimulation using the WST-1 assay, Griess reagent and cytokine ELISA, respectively. Values for the treatment groups that did and did not include PMX were normalized to their respective controls. Data are expressed as mean ± S.E.M. n ≥ 9, exp ≥ 3. Prenormalization mean absolute values for unstimulated cells were 0.32 ± 0.015 and 0.34 ± 0.032 absorbance units (C), 0.0 and 0.0 μM NO (D), and 0.0 and 1.33 ± 1.33 pg TNF-α/μg total protein (E) in the absence or presence of PMX, respectively.

Fig. 2

Comparison between effects of different commercial-grade preparations in the absence or presence of PMX on a number of microglial activation parameters in mouse primary microglia (pMG). Mouse pMG were either unstimulated or stimulated with 100 EU/mL of control standard endotoxin (CSE), 100-U/mL plasma-derived bovine α-thrombin (pb-thr), 100-nM serum-derived human α₂macroglobulin from supplier #5 (sh-α₂M-5), 100-nM serum-derived human α₂macroglobulin from supplier #7 (sh-α₂M-7), 1-μM 2,4,6-trinitrophenol-conjugated egg-white-derived chicken ovalbumin (tnp-Oval), 15.4-μM (1 mg/mL) serum-derived rat serum albumin-essentially fatty acid free (sr-Alb-efaf) and/or 15.4-μM (1 mg/mL) serum-derived rat albumin-fraction V powder (sr-Alb-FV) as indicated in the absence or presence of PMX (10 μg/mL). Cell surface expression of CD95 (Fas) (A) was assessed 24 h later by flow cytometry. Values for the treatment groups that did and did not include PMX were normalized to their respective controls. Means ± S.E.M. for these values from at least three separate experiments are presented. Statistical significance indicators are as in Figure 1. Prenormalization mean absolute fluorescent intensity units ± S.E.M. for unstimulated cells were 9.1 ± 1.8 and 8.8 ± 1.2 in the absence or presence of PMX, respectively. Metabolic activity/proliferation (B), TNF-α (C), MIP-1α (D), and KC (E) release were also assessed 24 h following stimulation using the WST-1 assay (B) and luminex system (C–E). Values for the treatment groups that did and did not include PMX were normalized to either their respective unstimulated (B) or CSE (C–E) controls. Data are expressed as mean ± S.E.M. n ≥ 4, exp ≥ 2. Prenormalization mean absolute values for unstimulated cells were 0.23 ± 0.008 and 0.22 ± 0.012 absorbance units (B), 5.27 ± 3.13 and 5.68 ± 3.38 pg TNF-α/μg total protein (C), 1.71 ± 0.50 and 2.02 ± 0.49 pg MIP-1α/μg total protein (D), 13.4 ± 7.19 and 20.7 ± 7.01 pg KC/μg total protein (E) in the absence or presence of PMX, respectively.