Potential role of green tea amino acid l-theanine in the activation of innate immune response by enhancing expression of cytochrome b558 responsible for the reactive oxygen species-generating ability of leukocytes.

l-Theanine (N-ethyl- l-glutamine) is an analog of l-glutamine and l-glutamic acid, accounts for up to 50% of all free amino acids in green tea, and elicits an umami taste. As l-theanine also shows various physiological activities including immune response-modifying activities, it is expected to be an excellent health-promoting phytochemical agent. To know the influences of l-theanine on the human innate immune response, we investigated the effect of l-theanine on the superoxide anion (O2 - )-generating system of leukocytes using U937 cells. The O2 - -generating system in leukocytes consists of membrane cytochrome b558 protein (a complex of p22-phox and gp91-phox proteins) and cytosolic p40-phox, p47-phox, and p67-phox proteins. Addition of 500 µM l-theanine caused remarkable enhancement of the all-trans retinoic acid (ATRA)-induced O2 - -generating activity (to ~470% of ATRA-treated cells), but not l-glutamine and l-glutamic acid. Semiquantitative RT-PCR showed that the transcription level of gp91-phox is significantly increased in ATRA and l-theanine-co-treated cells. Chromatin immunoprecipitation revealed that l-theanine enhances acetylations of Lys-9 and Lys-14 residues of histone H3 within the chromatin surrounding the promoter region of the gp91-phox gene. Immunoblotting demonstrated that membrane cytochrome b558 proteins remarkably accumulate in ATRA + l-theanine-treated cells. These results suggested that l-theanine brings about a remarkable accumulation of cytochrome b558 protein via upregulating the transcription of the gp91-phox gene during leukocyte differentiation, resulting in marked augmentation of the O2 - -generating ability, which is one of the most important functions of leukocytes responsible for the innate immune system.

Curcumin dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells.

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide (O(2)(-))-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the O(2)(-)-generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the O(2)(-)-generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced O(2)(-)-generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator.