Korean J Physiol Pharmacol.  2014 Dec;18(6):497-502. 10.4196/kjpp.2014.18.6.497.

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

• Hasan MA
• Sultan MT
• Ahn WG
• Kim YJ
• Jang JH
• Hong CW
• Song DK

• KMID: 2285552
• DOI: http://doi.org/10.4196/kjpp.2014.18.6.497

Abstract

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.

Keyword

Extracellular NAD cleaving activity; fMLP; FPRL1; Human neutrophils

MeSH Terms

Adenosine Diphosphate Ribose
Animals
Bone Marrow
Cell Membrane
Chromatography, Liquid
Humans
Lymphocytes
Mice
Monocytes
NAD*
Neutrophils*
Receptors, Formyl Peptide
Adenosine Diphosphate Ribose
NAD
Receptors, Formyl Peptide

Figure

• 

  Fig. 1 Intact human peripheral neutrophils show much less extracellular etheno-NAD cleaving activity than other immune cell types. (A) Comparison of extracellular etheno-NAD cleaving activity of human peripheral neutrophils with mouse bone marrow neutrophils and mouse peripheral neutrophils. (B) Comparison of extracellular etheno-NAD cleaving activity of human peripheral neutrophils with other primary immune cells and comparison of neutrophil-like cells with other immune cell lines. Briefly, each cell type was suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed for 15 min as described in Materials and Methods. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; ***p<0.0001.

• 

  Fig. 2 ADPR is the major extracellular metabolites generated from the degradation of NAD by both intact human peripheral neutrophils and mouse bone marrow neutrophil as determined by HPLC. Human peripheral neutrophils at a density of 1×107 cells/ml (A) and mouse bone marrow neutrophils at a density of 3×106 cells/ml (B) were incubated with or without NAD (1 µM) and incubated for 15 min or 1 hour at 37℃. Then the extracellular media were collected after centrifugation. Aliquots were analyzed by reverse-phase HPLC (Jasco Instruments) as described in Methods.

• 

  Fig. 3 fMLP induces a decrease in extracellular etheno-NAD cleaving activity of human neutrophils, but not of human monocytes. (A) Human neutrophils and (B) human monocytes were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml and incubated in the CO2 humidified chamber for 10 min. Then vehicle or fMLP of different concentration was treated. Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. **p<0.001; ***p<0.0001.

• 

  Fig. 4 fMLP-induced decrease in extracellular etheno-NAD cleaving activity of intact human neutrophils is reversed by WRW4, an FPRL1 antagonist. Human neutrophils were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Then, the cells were treated with vehicle or WRW4 (1 µM (A) or 10 µM (B)) for 5 min before the treatment of vehicle or fMLP (1 µM). Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; **p<0.001; ***p<0.0001.

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