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

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• The enzyme 12R-lipoxygenase and its product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), were previously identified exclusively in human skin.
• An EST sequence suggested the presence of 12R-lipoxygenase in human tonsillar germinal center B lymphocytes.

Purpose of the Study:

• To investigate the presence and function of 12R-lipoxygenase in human tonsils.
• To determine the cellular localization and biosynthetic pathway of 12R-HETE in tonsillar tissue.

Main Methods:

• Incubation of human tonsillar homogenates with radiolabeled arachidonic acid.
• Stereochemical analysis of lipoxygenase products (12-HETE and 15-HETE).
• RT-PCR, Northern analysis, and in situ hybridization to detect mRNA and localize the enzyme.

Main Results:

• 12R-lipoxygenase activity was detected in human tonsillar homogenates, producing 12R-HETE.
• Mechanistic studies indicated pro-R hydrogen abstraction at carbon 10 during 12R-HETE biosynthesis.
• mRNA for 12R-lipoxygenase was found in tonsils but localized to squamous epithelial cells, not germinal center lymphocytes.

Conclusions:

• Human tonsils contain 12R-lipoxygenase activity, primarily in squamous epithelial cells.
• The enzyme's localization suggests roles in epithelial cell differentiation and/or lymphocyte-epithelial cell interactions.
• The initial EST finding in lymphocytes was misleading regarding the enzyme's cellular distribution in tonsils.