Polymorphonuclear leukocytes (PMN) which are """"""""primed"""""""" have enhanced responsiveness to stimulation in vitro. Priming of PMN responses (respiratory burst or degranulation) can be induced in vitro, and occurs in vivo in PMN obtained from patients with diseases associated with myeloid stimulation (e.g. bacteremia). Increases in the defensive or injurious potential of PMN exist in primed PMN. We have recently delineated a novel transductional sequence in human PMN which may be important in priming. It involves hydrolysis of choline-containing-phospholipids (PC) with accumulation of l-acyl-2-acylglycerol (AAG) and l-alkyl-2- acylglycerol (EAG) and their respective phosphatidic acids (AAPA & EAPA). We also have determined that AAG and EAG have distinct biological activities. Other preliminary studies in our laboratories show constitutive expression of specific genes of PMN in the basal state, and inducible regulation of certain genes during priming in vitro and in vivo (e.g. c-fos and IL-1 alpha). Our two aims pertain to these recent observations.
In Aim 1 we will test the hypothesis that diglycerides AAG and EAG or phosphatidic acids AAPA or EAPA which accumulate after PC hydrolysis play an important role in priming PMN. Under defined conditions of priming (immediate or delayed, transient or prolonged priming in vitro, and priming in vivo), we will analyze accumulation of radiolabelled AAG, EAG, AAPA, EAPA after hydrolysis of PC, as well as changes in mass of species and subspecies of these four products. Thereafter, we will test the ability of structurally-specific subspecies of AAG, EAG, AAPA, EAPA to prime PMN in vitro. Finally, we will elucidate in a cell-free system the enzymes required for production of diglycerides and phosphatidic acids from PC.
In Aim 2 we first will extend our studies on basal expression of PMN genes. Second we will test the hypothesis that delayed priming in vitro and priming in vivo are associated with specific alterations in transcriptional and translational events. Genes coding for three categories of proteins will be evaluated: known functional proteins (e.g. cytochrome B 559); putative regulatory proteins (e.g. protein kinases C); secreted proinflammatory proteins interleukin 1). Third, we will evaluate transductional stimuli which may modify gene expression in PMN, particularly AAG, EAG, AAPA, EAPA.
