Phospholipase D (PLD) gene expression in human neutrophils and HL-60 differentiation

Abstract

Human neutrophils exhibit a regulated phospholipase D (PLD) activity that can be measured biochemically in vitro. However, the precise expression pattern of PLD isoforms and their specific biological role(s) are not well understood. Neutrophil mRNA is intrinsically difficult to isolate as a result of the extremely high content of lytic enzymes in the cell's lysosomal granules. Reverse transcription coupled to polymerase chain reaction indicated that pure populations of human neutrophils had the CD16b(+)/CD115(-)/CD20(-)/CD3zeta(-)/interleukin-5 receptor alpha(-) phenotype. These cells expressed the following splice variants of the PLD1 isoform: PLD1a, PLD1b, PLD1a2, and PLD1b2. As for the PLD2 isoform, neutrophils expressed the PLD2a but not the PLD2b mRNA variant. The relative amount of PLD1/PLD2 transcripts exists in an approximate 4:1 ratio. The expression of PLD isoforms varies during granulocytic differentiation, as demonstrated in the promyelocytic leukemia HL-60 cell line. Further, the pattern of mRNA expression is dependent on the differentiation-inducing agent, 1.25% dimethyl sulfoxide causes a dramatic increase in PLD2a and PLD1b transcripts, and 300 nM all-trans-retinoic acid induced PLD1a expression. These results demonstrate for the first time that human neutrophils express five PLD transcripts and that the PLD genes undergo qualitative changes in transcription regulation during granulocytic differentiation.