Endotoxin contamination in commercially available pokeweed mitogen contributes to the activation of murine macrophages and human dendritic cell maturation
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Published on: May 31, 2018
Jae Seung Yang1, Hye Jin Kim, Young Hee Ryu
1International Vaccine Institute, SNU Research Park, San 4-8 Bongcheon-7 dong, Kwanak-gu, Seoul 151-818, Republic of Korea.
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.
Insights
Commercial pokeweed mitogen (PWM) may have misleading immunological effects due to endotoxin contamination. Repurified PWM, free of endotoxin, still stimulates lymphocyte proliferation, suggesting endotoxin, not PWM, drives other immune responses.
Area of Science:
- Immunology
- Biochemistry
Background:
- Pokeweed mitogen (PWM) is commercially available and known to activate macrophages, inducing proinflammatory cytokines and nitric oxide (NO).
- Previous studies attributed these immune responses to PWM itself.
Purpose of the Study:
- To investigate the role of endotoxin contamination in the reported immunological effects of commercial pokeweed mitogen (PWM).
- To determine if PWM possesses inherent properties beyond lymphocyte proliferation that activate immune cells.
Main Methods:
- Commercial PWM was tested for endotoxin contamination using a Limulus amebocyte lysate assay.
- Polymyxin B (PMB), an endotoxin inhibitor, was used to assess its effect on PWM-induced immune responses.
- PWM was repurified using PMB-coupled beads and its immunological properties were re-evaluated.
- Immune cell activation, cytokine production, nitric oxide (NO) release, Toll-like receptor 4 (TLR4) activation, dendritic cell maturation, and lymphocyte proliferation were measured.
Main Results:
- Commercial PWM contained high levels of endotoxin (over 10(4) endotoxin units/mg).
- PMB significantly inhibited PWM-induced proinflammatory cytokine and NO production, as well as TLR4 activation.
- Repurified PWM, devoid of endotoxin, did not induce proinflammatory cytokine expression, TLR4 activation, or dendritic cell maturation.
- Repurified PWM retained its ability to induce human lymphocyte proliferation.
Conclusions:
- Commercial PWM is heavily contaminated with endotoxin, which is responsible for inducing proinflammatory cytokine production, NO release, and TLR4 activation.
- The mitogenic property of PWM, specifically its ability to induce lymphocyte proliferation, is independent of endotoxin contamination.
- Previous attributions of certain immunological properties to PWM may be inaccurate due to endotoxin contamination.
