3H arachidonic acid incorporation and metabolism in purified human basophils

A central feature of the allergic response is the generation of arachidonic acid (AA) metabolites by basophils and mast cells. In addition, AA metabolism may play a role in regulating the anti-IgE mediated degranulation of human basophils. To study this biochemistry, purified human basophils (>80%) were labeled with 3H-AA (0.3 μM, 25 μCi/ml, 2 hours at 370C) and subsequently challenged with anti-IgE. Basophils were found to incorporate 45 +/- 3% of the exogenous AA which distributed into phospholipids (PL) (77.1 +/- 3.5%) and neutral lipids (19.7 +/- 3.3%) with only 5.3 +/- 2.7% remaining as the free acid (n = 7). Phosphatidylcholine (23.9 +/- 1.7%), phosphatidylinositol (22.0 +/- 1.4%) and phosphatidylethanolamine (14.5 +/- 2.7%) accounted for the majority of the AA with the remaining PL containing <3%. Anti-IgE (0.1 μg/ml) challenge led to the release of histamine (23.8 +/- 4.7%) and 3H-AA (8.1 +/- 1.7%) (n = 5). HPLC analysis revealed unmetabolized 3H-AA, 3H-LTC4, 3H-HETE and an unidentified peak which migrated in the prostaglandin region of the elution profile. The same metabolites were released when the basophils were challenged with antigen. The calcium ionophore A23187 (1μg/ml) also caused the release of histamine (37.4 +/- 4.1%) and 3H-AA (17.0 +/- 2.9%), while the phorbol ester, TPA caused HR (19.7 +/- 5.8%) but no increase in 3H AA turnover. Because of limited cell numbers this is the first time the authors have been able to study AA metabolism in human basophils