Natural killer cell mediated cytotoxicity of tumor cells initiated by neem leaf preparation is associated with CD40–CD40L–mediated endogenous production of interleukin-12

SummaryNeem leaf preparation (NLP) was found to activate natural killer (NK) cells (CD56+CD3−) to enhance their cytotoxic ability to tumor cells and stimulate the release of interleukin-12 (IL-12) from macrophages from healthy individuals and head-and-neck squamous cell carcionoma patients. NLP upregulated cytotoxic (CD16+ and CD56dim) NK cells, and the cytotoxicity of NK-sensitive K562 cells by NLP-stimulated peripheral blood mononuclear cells decreased significantly after IL-12 neutralization. This NK-mediated cytotoxicity was manifest by upregulation of IL-12–dependent intracellular expression of the perforin–granzyme B system. Moreover, NK cytotoxic function was abolished after use of concanamycin A, a perforin inhibitor, but not by brefeldin A, a Fas inhibitor, confirming the participation of the perforin–granzyme B system. In addition NLP upregulated the expression of CD40 in CD14+ monocytes and CD40L in CD56+ lymphocytes. Neutralization of CD40 and CD40L in NLP-stimulated peripheral blood mononuclear cells culture resulted in significant downregulation of IL-12 release and cytotoxicity of NK cells, demonstrating the role of a CD40–CD40L interaction in the observed functions. Signals involved in the NLP-induced release of IL-12, and thereby induction of NK cell cytotoxicity, are mediated by activating p38MAPK pathway, but not through the ERK1/2 signaling pathway. Overall the results suggest that NLP effects NK cellular cytotoxicity by CD40–CD40L–mediated endogenous production of IL-12, which critically controls perforin-dependent tumor cell cytotoxicity.