Leukotriene C4 synthesis and export in human platelets and leukocytes : LTC4 synthase expression in normal and leukemic myeloid cells

Sammanfattning: Leukotriene (LT)C4 synthase catalyses conjugation of the unstable epoxide LTA4 with reduced glutathione and is the rate-limiting enzyme in the formation of cysteinyl leukotrienes, powerful mediators of asthma and inflammation. The presence of LTC4 synthase in platelets was demonstrated by purification and characterization of bovine platelet LTC4 synthase. Screening of platelets from six mammalian species for LTC4 synthase activity revealed that bovine, equine, ovine, rabbit and rat platelets produced LTC4 with variable efficiency after incubation with LTA4. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced LTB4, indicating the presence of LTA4 hydrolase in these cells. Partial information concerning the primary structure of bovine and equine LTC4 synthase was obtained by protein sequencing and cloning by PCR. The capacity of human platelets to actively release LTC4 upon incubation with LTA4 was investigated. The presence of a mechanism for rapid and carrier-mediated active export of LTC4 into the extracellular space was observed and suggested the involvement of an energydependent and probenecide-sensitive transporter, possibly MRP1. Furthermore, the presence of MRP1 mRNA and protein in human platelets was demonstrated. The effect of the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on the metabolism of exogenous LTA4 in human granulocytes was investigated. A PMA-provoked suppression of the conversion of LTA4 to LTC4 was observed, indicating PKC-dependent downregulation of LTC4 synthase activity. The effects of PMA could be prevented by specific protein kinase C inhibitors, confirming the involvement of PKC. The leukotriene-producing capacity of peripheral blood leukocyte suspensions from patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) in blast crisis (CMLbc) was investigated. Leukocytes from 19 of 29 AML patients, but only from 2 of 12 CMLbc patients demonstrated a deficiency at the 5-LO/FLAP level as judged by absent or very low leukotriene-production from endogenous substrate after ionophore A23187 stimulation. However, regardless of the 5-LO/FLAP deficiency, leukocyte suspensions from AML as well as CMLbc patients consistently displayed significantly increased LTC4 synthase activity as demonstrated by incubation with ionophore A23187 or LTA4. Peripheral blood CD16(+) neutrophils were purified from CML patients and found to consistently transform exogenous LTA4 to LTC4. These cells also produced LTC4 after activation with ionophore A23187 or the chemotactic peptide f-Met-Leu-Phe. Expression of LTC4 synthase mRNA and protein in CML neutrophils was confirmed by RT-PCR and immunoblot analysis. Expectedly, LTC4 synthase activity or expression of the protein could not be demonstrated in neutrophil suspensions from any of the healthy individuals. The leukotriene-forming capacity of immature myeloid cells from human bone marrow was investigated. Highest LTC4 synthase activity was observed in CD34-expressing progenitors and the presence of LTC4 synthase in CD34(+) myeloid progenitors was confirmed by immunoblot analysis. In addition, LTC4 synthase activity was observed to be gradually downregulated with myeloid maturation. A mechanism mediating LTC4 synthase downregulation by post-transcriptional events could be identified in mature peripheral neutrophils.