Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N6-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

Lack of IL-10 Release from Human Polymorphonuclear Leukocytes

PURPOSE: Polymorphonuclear leukocytes (PMNs) are the first line of cellular response for host defense during acute inflammation. The ability of PMNs to produce and release numerous pro-inflammatory cytokines is now estabilished and plays an important role in triggering and maintaining the inflammatory response. We studied the autocrine downregulation of this process by invesgating the potential production by human PMNs of the major anti-inflammatory cytokine, interleukine 10 (IL-10). METHODS: Human PMNs were isolated from the peripheral blood of health volunteers by using the modified boyum method. Human PMNs were incubated at 37 degrees Cwith and without formyl Met-Leu-Phe (fMLP) for 30 minute, 2 hours, 4 hours, and 20 hours. The level of IL-10 was determined in each of the cell-free supernatants by using the enzyme linked immunosorbent assay (ELISA) method. RESULTS: Non-stimulated PMNs generated 1.40 +/- 1.791pg/mL to 3.46 +/- 1.607 pg/mL of IL-10 over the time course. Stimulation with fMLP resulted in an increase in the constitutive PMN-derived IL-10 by 2.74 +/- 0.762 pg/mL, 1.27 +/- 0.262 pg/mL, 1.19 +/- 0.364 pg/mL, and 2.44 +/- 1.317 pg/mL at 30 min, 2 hr, 4 hr, and 20 hr after stimulation, respectively, but these increases were not statiscally significant. CONCLUSION: Human PMNs seem unable to induce release of the most potent anti-inflammatory cytokine, IL-10, and down-regulate inflammatory response due to the autocrine mechanism. This could partly account for the persistence of local inflammation, when PMNs are the main infiltrating cells.