Purification and characterization of cytokine-inducing protein of seed extract from Aeginetia indica L., a parasitic plant

We have isolated 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by an affinity chromatography on N-hydroxysuccinimide (NHS)-activated Sepharose High Performance column bound F3 monoclonal antibody which neutralizes cytokine-inducing and antitumor effect of AIL. In in vitro model using human peripheral blood mononuclear cells (PBMC), the 55 kDa protein (AILb-A) induced multiple cytokines, such as IFN-γ, tumor necrosis factor (TNF)-α, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-2, IL-6, IL-10, IL-12 and IL-18, and also accelerated killer cell activities of PBMC. When compared with a commonly used immunotherapeutic agent OK-432, AILb-A induced Th1 cytokines are greater than OK-432. Of the Th2 cytokines, the amounts of IL-6 and IL-10 induced by AILb-A were lower than those by OK-432. No significant induction of IL-4 and IL-13 was observed in AILb-A-stimulated PBMC. TNF family including TNF-α, TNF-β, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) were suggested to be important for AILb-A-induced killing activity of PBMC by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, the neutralizing test using cytokine-specific antibodies demonstrated that IL-18 plays a most significant role for IFN-γ- and killer cell-inducing ability of AILb-A among the cytokines tested. These findings clearly indicated that AILb-A, a 55 kDa protein of AIL, is a potent Th1 cytokine inducer and may be a useful immunotherapeutic agent for the patients with malignancies.