The heat sensitivity of cytokine-inducing effect of lipopolysaccharide

Journal of Leukocyte Biology

Yun Wang²  Baochong Gao¹  Min-Fu Tsan² 

[1] Research Service, VA Medical Center, Washington, DC; Department of Medicine, George Washington University, Washington, DC; and Research Service, VA Medical Center, Washington, DC; Research Service, VA Medical Center, Washington, DC; Department of Medicine, George Washington University, Washington, DC; and Department of Medicine, George Washington University, Washington, DC; and Research Service, VA Medical Center, Washington, DC; Department of Medicine, George Washington University, Washington, DC; and;Research Service, VA Medical Center, Washington, DC; Department of Medicine, Georgetown University, Washington, DC Research Service, VA Medical Center, Washington, DC; Research Service, VA Medical Center, Washington, DC; Department of Medicine, Georgetown University, Washington, DC Department of Medicine, Georgetown University, Washington, DC Research Service, VA Medical Center, Washington, DC; Department of Medicine, Georgetown University, Washington, DC

关键词: LPS;    endotoxin activity;    tumor necrosis factor α (TNF-α);    macrophages;   

DOI  :  10.1189/jlb.1205738

学科分类：生理学

来源: Federation of American Societies for Experimental Biology

【 摘 要 】

Heat inactivation by boiling has been widely used as a criterion to determine whether the observed effects of a protein preparation are a result of lipopolysaccharide (LPS) contamination. However, the heat sensitivity of LPS cytokine-inducing activity has not been characterized. In the current study, we demonstrated that the endotoxin activity, i.e., Limulus amebocyte lysate-gelating activity, and the tumor necrosis factor α (TNF-α)-inducing activity of LPS (Escherichia coli K-12 JM83, K-12 LCD25, and F583) were sensitive to boiling. Heat treatment by boiling for 15 min was sufficient to inactivate ∼90% of the LPS TNF-α-inducing activity. The heat-induced inactivation of LPS activities was not a result of adherence of boiled LPS to the wall of the container, i.e., polypropylene tubes, or aggregation of boiled LPS. In addition, boiled LPS retained its ability to bind polymyxin B. The presence of protein (ovalbumin) in LPS did not affect the heat sensitivity of LPS. Conversely, boiling reduced the size of LPS aggregates as determined by electrophoresis using native polyacrylamide gel. Likewise, the TNF-α-inducing activity of diphosphoryl lipid A (DPLA) was also sensitive to boiling. Thin-layer chromatographic analysis of boiled DPLA revealed that the heat-induced inactivation of DPLA TNF-α-inducing activity was not a result of its conversion to monophosphoryl lipid A. We conclude that the TNF-α-inducing activity of LPS and DPLA is sensitive to boiling and suggest that heat sensitivity as an indicator of whether the observed effects of a protein preparation are a result of LPS contamination should be used with caution.

【 授权许可】

Unknown