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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 - Korean J. Physiol. Pharmacol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon 200-702, Korea.

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,N(6)-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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: 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].


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 - Korean J. Physiol. Pharmacol. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 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.
Mentions: 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].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon 200-702, Korea.

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,N(6)-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.

No MeSH data available.


Related in: MedlinePlus