Human eosinophils contain abundant amounts of 15-lipoxygenase (LO)-1. The biological role of 15-LO-1 in humans, however, is unclear. Incubation of eosinophils with arachidonic acid led to formation of a product with a UV absorbance maximum at 282 nm and shorter retention time than leukotriene (LT)C4 in reverse-phase HPLC. Analysis with positive-ion electrospray tandem MS identified this eosinophil metabolite as 14,15-LTC4. This metabolite could be metabolized to 14,15-LTD4 and 14,15-LTE4 in eosinophils. Because eosinophils are such an abundant source of these metabolites and to avoid confusion with 5-LO-derived LTs, we suggest the names eoxin (EX)C4, -D4, and -E4 instead of 14,15-LTC4, -D4, and -E4, respectively. Cord blood-derived mast cells and surgically removed nasal polyps from allergic subjects also produced EXC4. Incubation of eosinophils with arachidonic acid favored the production of EXC4, whereas challenge with calcium ionophore led to exclusive formation of LTC4. Eosinophils produced EXC4 after challenge with the proinflammatory agents LTC4, prostaglandin D2, and IL-5, demonstrating that EXC4 can be synthesized from the endogenous pool of arachidonic acid. EXs induced increased permeability of endothelial cell monolayer in vitro, indicating that EXs can modulate and enhance vascular permeability, a hallmark of inflammation. In this model system, EXs were 100 times more potent than histamine and almost as potent as LTC4 and LTD4. Taken together, this article describes the formation of proinflammatory EXs, in particular in human eosinophils but also in human mast cells and nasal polyps.
Abstract Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B 4 (LTB 4 ) in acute myeloid leukemia (AML) cells (16 clones) and G‐CSF mobilized peripheral blood CD34 + cells. CD34 + cells from patients with non‐myeloid malignancies expressed cytosolic phospholipase A 2 (cPLA 2 ), 5‐lipoxygenase activating protein (FLAP), and leukotriene A 4 (LTA 4 ) hydrolase but not 5‐lipoxygenase (5‐LO). The enzyme cPLA 2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5‐LO, FLAP, and LTA 4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187‐induced release of [ 14 C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA 2 protein. Biosynthesis of LTB 4 , upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB 4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1 − 14 C] AA release nor 5‐LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA 2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease.
Summary. Several lines of evidence suggest that phospho‐lipases A 2 , leukotrienes and prostaglandins play a role in the proliferation of haemopoietic cells. The expression of genes involved in the biosynthesis of leukotrienes and prostaglandins was investigated in peripheral B lymphoblasts, isolated from eight patients with acute pre‐B‐lymphocytic leukaemia (pre B‐ALL). RT‐PCR analysis demonstrated that four of the investigated pre‐B‐ALL clones expressed the gene coding for cytosolic phospholipase A 2 (cPLA 2 ), but not the gene coding for 5‐lipoxygenase. In contrast, the remaining four pre‐B‐ALL clones expressed 5‐lipoxygenase but not cPLA 2 , suggesting that the transcriptional regulation of these two genes are different and that their cellular functions are not linked to each other. The capacity of pre B‐ALL cells to produce LTB 4 and to express the 5‐lipoxygenase protein, correlated with the expression of 5‐lipoxygenase mRNA. All pre‐B‐ALL clones expressed genes coding for 5‐lipoxygenase activating protein (FLAP), leukotriene A 4 hydrolase and prostaglandin (PG)H synthase 1. Seven of the eight pre B‐ALL clones expressed PGH synthase 2. In comparison, normal tonsillar B cells did not express cPLA 2 or PGH synthase 2.
Classical Hodgkin lymphoma has unique clinical and pathological features and tumour tissue is characterized by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by inflammatory cells. In the present study, we report that the Hodgkin lymphoma-derived cell line L1236 has high expression of 15-lipoxygenase-1 and that these cells readily convert arachidonic acid to eoxin C(4), eoxin D(4) and eoxin E(4). These mediators were only recently discovered in human eosinophils and mast cells and found to be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15-lipoxygenase-1 was present mainly in the cytosol and that the enzyme translocated to the membrane upon calcium challenge. By immunohistochemistry of Hodgkin lymphoma tumour tissue, 15-lipoxygenase-1 was found to be expressed in primary Hodgkin Reed-Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15-lipoxygenase-1, however, was not expressed in any of 10 biopsies representing nine different subtypes of non-Hodgkin lymphoma. In essence, the expression of 15-lipoxygenase-1 and the putative formation of eoxins by Hodgkin Reed-Sternberg cells in vivo are likely to contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15-lipoxygenase-1 activity in L1236 cells demonstrates that this cell line comprises a useful model system to study the chemical and biological roles of 15-lipoxygenase-1.
