Abstract
Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/or 1-14C-labeled or unlabeled arachidonic acid. Using reverse phase high pressure liquid chromatography with simultaneous monitoring of ultraviolet light absorption at 229 and 280 nm and, where appropriate, of radioactivity, our studies reveal that human peripheral blood mononuclear cells generate leukotrienes C4 and B4 (LTC4 and LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) following stimulation with A23187. The ratio of LTC4 to LTB4 was approximately 10-fold greater among the mononuclear cells than among similar incubations of polymorphonuclear leukocytes. Furthermore, the mononuclear cells failed to metabolize LTB4 into the omega-hydroxy or omega-carboxy derivatives that were always present in, and very characteristic of incubations of polymorphonuclear leukocytes. Depletion of monocytes from the mononuclear cells by double adherence resulted in virtual loss of the generation of 5-lipoxygenase-derived products by the remaining nonadherent cells, supporting the conclusion that the monocytes and not the lymphocytes were the source of LTC4, LTB4, and 5-HETE. The presence of both 12-HETE and the cyclooxygenase-derived 12-hydroxyheptadecatrienoic acid correlated with the degree of platelet contamination, suggesting that the platelets account for the presence of these compounds.
Highlights
Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets
We decided to evaluate the lipoxygenasederived metabolites of arachidonic acid generated by human peripheral blood mononuclear cellsusing a high pressure liquid chromatography system that permitted identification and B4 (LTC4and LTB4)and 5-hydroxyeicosatetrae- of the individual leukotrienes and the hydroxyeicosatetraenoic acid(5-HETE)followingstimulationwith noic acids.The results demonstrate that PBMC virtually free
The ratio of LTC4to LTB4 was approximately of PMNL (1%or less) generate lipoxygenase-derived metab10-fold greater among the mononuclear cells than olites of arachidonic acid in a profile that differs both qualiamong similar incubations of polymorphonuclear leu- tatively and quantitatively from that generated by PMNL. kocytes
Summary
From the $Department of Dermatology and the Rosalind Russell Arthritis Research Laboratory, University of California at San Francisco, San Francisco General Hospital, SanFrancisco, California, 94110 and the lIGroupe de Recherche sur ks Lewotrienes, Luboratoire d’Endocrinologie Moleculaire, Centre Hospitalier del’hiversite Laval, Quebec GIV4G2, Canada. Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/ or l-14C-labeledor unlabeled arachidonic acid. Generation of 5-lipoxygensse-derived products by the remaining nonadherent cells, supporting the conclusion that themonocytes and not the lymphocytes were the source of LTC4,LTB,, and 5-HETE The presence of both 12-HETE and thecyclooxygenase-derived 12hydroxyheptadecatrienoic acid correlated withthe degree of platelet contamination, suggesting thatthe platelets account for the presence of these compounds. Total amounts of compounds in the sample were as follows: w-OH-LTB,, 180 pmol; LTB4, 565 pmol; 12-HETE, 235 pmol;5-HETE, 750 pmol; and LTC,, 35 pmol.C~(LWar,achidonic acid
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