Detection and cellular localization of 12R-lipoxygenase in human tonsils

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin ct al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearance of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal center B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-C-14]arachidonic acid with homogenates of human tonsillar tissue yielded mixtures of radiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying ratios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configuration. Using stereospecifically labeled [10S-H-3]- and [10R-H-3]arachidonic acid substrates we detected pro-R hydrogen abstraction at carbon 10 associated with formation of 12R-HETE. This mechanist ic evidence implicates a 12R-lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was identified in tonsils by RT-PCR and Northern analysis. The cellular distribution was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific reaction was restricted to squamous epithelial cells, including the epithelium lining the tonsillar crypts. Ln this location the 12R-lipoxygenase might help regulate differentiation of the epithelium or participate in lymphocyte-epithelial cell interactions. (C) 2001 Academic Press.