Endotoxin contamination in commercially available pokeweed mitogen contributes to the activation of murine macrophages and human dendritic cell maturation

Abstract

Commercially available pokeweed mitogen (PWM) has been reported to activate macrophages, leading to production of proinflammatory cytokines and nitric oxide (NO). However, we found that polymyxin B (PMB), a specific inhibitor of endotoxin activity, inhibited the PWM-induced expression of proinflammatory cytokines and NO and the activation of Toll-like receptor 4 (TLR4). A kinetic-turbidimetric Limulus amebocyte lysate assay demonstrated that commercial PWM contained substantial endotoxin, over 10(4) endotoxin units/mg of the PWM. A PWM repurified by PMB-coupled beads no longer induced the expression of proinflammatory cytokines, TLR4 activation, or dendritic cell maturation. However, the repurified PWM remained able to induce proliferation of human lymphocytes, which is a representative characteristic of PWM. These results suggest that commercial PWM might be contaminated with a large amount of endotoxin, resulting in the attribution of misleading immunological properties to PWM.

FIG. 1.

Induction of proinflammatory cytokines TNF-α and IL-6 by PWM-activated RAW 264.7 cells is inhibited by PMB treatment. (A) RAW 264.7 cells were stimulated with the indicated concentrations of PWM for 15 h, and supernatants were collected to analyze TNF-α and IL-6 concentrations. (B) RAW 264.7 cells were treated with 50 μg/ml PMB for 1 h and then further incubated with 5 or 20 μg/ml PWM for 15 h to quantitatively examine the concentrations of TNF-α and IL-6. LPS was used as a control to confirm the inhibitory effect of PMB. Error bars indicate standard deviations.

FIG. 2.

The mode of action of PWM mimics LPS. CHO/CD14/TLR4 cells were incubated for 16 h with (A) PWM (0, 0.01, 0.1, or 1 μg/ml), (B) 0, 0.1, 1 or 10 μg/ml PWM in the presence of 50 μg/ml PMB, (C) PWM or heat-treated PWM (1 μg/ml), and (D) PWM or proteinase K-treated PWM (1 μg/ml). TLR4-dependent NF-κB activation was determined by flow-cytometric analysis of CD25.

FIG. 3.

RPWM induces neither the expression of proinflammatory cytokines TNF-α and IL-6 nor TLR4 activation. RPWM was prepared by eliminating endotoxin with repeated application of polymyxin B-coupled beads. (A) RAW 264.7 cells were stimulated with 0, 5, or 20 μg/ml of PWM or RPWM for 15 h, and supernatants were analyzed for TNF-α and IL-6. (B) CHO/CD14/TLR4 cells were stimulated with 0, 1, 5, or 25 μg/ml of PWM or RPWM for 16 h. TLR4-dependent NF-κB activation was determined by flow-cytometric analysis of CD25.

FIG. 4.

PWM, but not RPWM, induces human DC maturation. Immature DC derived from CD14⁺ monocytes of human PBMC were incubated with the indicated concentrations of (A) PWM or (B) RPWM for 48 h. Then, the expression of CD80, CD83, and CD86 was determined by flow cytometry. Data represent the means ± standard deviations of the geometric mean fluorescence intensity.

FIG. 5.

RPWM does not lose the ability to induce proliferation of human lymphocytes. Human PBMC were labeled with 2 μM of CFSE fluorescence dye for 10 min and then stimulated with PWM (5 μg/ml), RPWM (5 μg/ml), proteinase K (control), proteinase K-treated RPWM (5 μg/ml), or PHA (0.5 μg/ml) for 6 days. At the end of incubation, the cells were stained with APC-labeled anti-CD3 antibody and cell division was examined by measuring FL-1 fluorescence intensity of CD3-positive cells with flow-cytometric analysis.