Etheno-NAD was obtained from Sigma-Aldrich Chemical, and 20 mM stock solution was prepared in 10 mM potassium phosphate buffer (pH 7.4). fMLP and retinoic acid were also from Sigma-Aldrich Chemical. WRW4 was from Tocris Bioscience (Bristol, UK).

Neutrophils were purified from venous blood of healthy volunteer. In brief, venous blood was collected with peripheral venous puncture and immediately anti-coagulated with 10 U/ml sodium heparin. Then, neutrophils were isolated by density gradient centrifugation in Histopaque-1077, followed by dextran sedimentation. Residual erythrocytes were eliminated with hypotonic lysis. The purity of neutrophils counted by Diff Quik staining was >95% average. Eosinophils were found to be <5%. The viability of neutrophils stained with tryphan blue was >99%.

Procedures for animal experiments were approved by the Animal Experimentation Committee at Hallym University. C57BL/6J female mice were sacrificed by cervical dislocation, and their femurs and tibiae were carefully cleaned from adherent tissues. After bone ends were cut off, the marrow was collected. Residual erythrocytes were eliminated with hypotonic lysis. The bone marrow neutrophils were then isolated by density gradient centrifugation in Percoll and suspended at a density of 1×10⁷ cells/ml in DMEM containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The purity of neutrophils counted by Giemsa staining was >90% average. Cultures were kept at 37℃ in a humidified atmosphere containing 95% air and 5% CO₂.

Mouse peripheral neutrophils were isolated according to the manufacturer's protocol (mouse neutrophil isolation kit, MACS Miltenyl Biotec, Germany). Briefly, peripheral blood was collected from C57BL/6J female mice and immediately anti-coagulated with 10 U/ml sodium heparin. Erythrocytes were eliminated from the whole blood cells by lysis with BD Pharm Lyse™ followed by hypotonic lysis. Then, leukocytes were treated with neutrophil biotin-antibody cocktail, and neutrophils were selectively isolated by magnetic separation with LS column.

Monocytes and lymphocytes were isolated as described previously [11]. For monocyte isolation, peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from buffy coats obtained by density sedimentation over Histopaque-1077. Residual erythrocytes were eliminated with hypotonic lysis, and then to separate monocytes from lymphocytes, PBMC suspension was carefully laid onto the hyper-osmotic Percoll solution in each tube and centrifuged with brake off. Monocytes layer at the interface was collected and suspended in RPMI-1640. To obtain lymphocytes, after collecting the monocyte fraction at the interface, the bottom layer was collected and after washing with PBS, the residual sediments were suspended in RPMI-1640 supplemented with 5% FBS.

HL-60 (a promyelocytic leukemic cell line), U937 (a promonocytic tumor cell line) and Jurkat (a T lymphocyte cell line) cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin and kept in a 5% CO₂ humidified chamber. To differentiate HL-60 cells into the neutrophil lineage by dimethyl sulphoxide (DMSO), cells at a density of 0.3×10⁶ cells/ml were grown in the presence of 1.3% DMSO for 3 days. Afterwards, the medium was replaced by fresh medium containing 0.65% DMSO and the culture continued for 3 more days. To differentiate HL-60 cells into the neutrophil lineage by retinoic acid, cells at a density of 0.2×10⁶ cells/ml were grown in the presence of 100 nM retinoic acid for 4 days as described previously [12].

Etheno-NAD cleaving activity was measured by a fluorimetric assay with etheno-NAD as the substrate [2]. Neutrophils or other immune cell types were suspended in Hank's balanced salt solution (HBSS) (in mM) (KCl 5.4, Na₂HPO₄ 0.338, KH₂PO₄ 0.44, NaHCO₃ 4.2, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.6, NaCl 137, D-glucose 5.6) 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, which causes a 10-fold fluorescence enhancement, was continuously followed at 37℃ using Spectramax M2/e fluorescence microplate reader (Molecular Devices) at an excitation of 300 nm and an emission wave length of 410 nm.

Human peripheral neutrophils at a density of 1×10⁷ cells/ml and mouse bone marrow neutrophils at a density of 3×10⁶ cells/ml 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) using a C18 column (4.6×250 mm, particle size 5 µm). Absorbance was measured at 254 nm using a UV detector (Jasco UV-2075 plus intelligent UV/VIS detector) and data were processed by the chromNAV data acquisition system from Jasco instruments. Peaks were identified by comparison to known standards.

Data were analyzed with Graphpad Prism 5.0 using ANOVA. Bonferroni test was used for post-hoc comparison. All data are presented as means±S.E.M from at least three independent experiments. p<0.05 was considered to indicate statistical significance.

Funaro et al. reported that human neutrophils are inactive in terms of extracellular ADP-ribosylcyclase activity [13]. However, there is no clear indication whether intact human neutrophils have any other extracellular NAD cleaving activities. To address this uncertainty, we undertook a simple fluorometric assay using etheno-NAD. As shown in Fig. 1A, intact human peripheral neutrophils showed scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse (either bone marrow or peripheral) neurophils. It is to be noted that mature mouse peripheral neutrophils have lower extracellular etheno-NAD cleaving activity than immature mouse bone marrow neutrophils (Fig. 1A). Also, there is no data until now comparing the extracellular NAD cleaving activity of intact human peripheral neutrophils with other immune cell types. Thus next, we compared extracellular etheno-NAD cleaving activity of human neutrophils with different human primary immune cells like monocytes and lymphocytes. As shown in Fig. 1B, human peripheral neutrophils showed a much less extracellular etheno-NAD cleaving activity compared to monocytes and lymphocytes. We also compared extracellular etheno-NAD cleaving activity of DMSO- and retinoic acid-differentiated neutrophillike HL-60 cells with U937 and Jurkat T cells to check whether the cell lines show the similar observation. As expected, DMSO-differentiated neutrophil-like HL-60 cells were devoid of extracellular etheno-NAD metabolizing activity, whilst U937 and Jurkat T cells, like primary human monocytes and lymphocytes, respectively, showed marked extracellular etheno-NAD metabolizing activity (Fig. 1B). However, retinoic acid-treated HL-60 cells showed a marked increase in extracellular etheno-NAD cleaving activity (Fig. 1B) as reported previously [14].

Next, we attempted to identify the extracellular metabolites generated from extracellular NAD degradation by human peripheral neutrophils and mouse bone marrow neutrophils with HPLC. As shown in Fig. 2, the major extracellular metabolite generated from extracellular NAD degradation by both human peripheral neutrophils and mouse bone marrow neutrophils was ADPR. Extracellular NAD degradation by human neutrophils, as expected, was scant following incubation of NAD with human neutrophils for 15 minutes or 1 hour (Fig. 2A). Though about three times less mouse bone marrow neutrophils (3×10⁶ cells/ml) than human neutrophils (1×10⁷ cells/ml) were used, degradation of extracellular NAD by mouse bone marrow neutrophils was remarkable (Fig. 2B).

Next, we investigated whether the scant extracellular etheno-NAD cleaving activity of human neutrophils is regulated following activation with fMLP, a chemoattractant for neutrophils. Since fMLP-like molecules could be abundant at bacterial infection sites, we investigated whether the extracellular etheno-NAD cleaving activity of intact human neutrophils are affected following fMLP stimulation. As shown in Fig. 3A, stimulation with fMLP induced a concentration-dependent decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils, which became significant at 100 nM, and was more marked at 1 µM concentration. In the next experiment, it was of interest that whether fMLP can affect extracellular etheno-NAD cleaving activity in other cells such as monocytes. Monocytes, however, was found to be unaffected by fMLP (Fig. 3B).

Human neutrophils express two types of fMLP receptors. The prototype receptor formyl peptide receptor (FPR) is activated by low concentration (in the picomolar to low nanomolar range) of fMLP and is considered a high affinity fMLP receptor. An FPR variant, FPR-like 1 (FPRL1), interacts with high concentrations (in the micromolar range) of fMLP and is defined as a low affinity fMLP receptor [15].

Since fMLP, at micromolar concentration showed the maximal inhibition in the extracellular etheno-NAD cleaving activity of intact human neutrophils (Fig. 3A), we hypothesized that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity might occur through FPRL1. To test this hypothesis, the effect of WRW4, a potent antagonist for FPRL1, on the fMLP-induced effect was examined. WRW4 at 10 µM but not at 1 µM reversed the fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutorphils (Fig. 4), suggesting that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils occur via FPRL1.