Aberrant expression of active leukotriene C4 synthase in CD16+ neutrophils from patients with chronic myeloid leukemia

Abstract

Elevated leukotriene (LT)C4 synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16+ neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase–polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16+ neutrophils from all tested patients transformed exogenous LTA4 to LTC4. These cells also produced LTC4 after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC4 synthase mRNA in CML CD16+neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC4 synthase at the protein level in CML CD16+ neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC4 synthase activity or expression of the protein could not be demonstrated in CD16+ neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16+neutrophils, transformed LTA4 to LTB4. The results indicate that aberrant expression of LTC4 synthase is a regular feature of morphologically mature CML CD16+neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC4 synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC4 has been demonstrated to stimulate proliferation of human bone marrow–derived myeloid progenitor cells.