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Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils.

Krump E, Picard S, Mancini J, Borgeat P - J. Exp. Med. (1997)

Bottom Line: Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs).The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs.This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie, Centre de recherche du CHUL, Université Laval, Québec, Canada.

ABSTRACT
Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.

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Time course of Ado accumulation in PMN suspensions.  Freshly isolated PMNs were resuspended in HBSS (A) or autologous  plasma (B) at concentrations of 5 × 106/ml (squares) or 20 × 106/ml (circles). At various time points, 1-ml aliquots of the cell suspensions were denatured with TCA and the Ado content was measured by liquid chromatography–mass spectrometry. Results shown are the means ± SD of  triplicate incubations from one experiment representative of three. Error  bars are not shown when smaller than symbols.
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Figure 1: Time course of Ado accumulation in PMN suspensions. Freshly isolated PMNs were resuspended in HBSS (A) or autologous plasma (B) at concentrations of 5 × 106/ml (squares) or 20 × 106/ml (circles). At various time points, 1-ml aliquots of the cell suspensions were denatured with TCA and the Ado content was measured by liquid chromatography–mass spectrometry. Results shown are the means ± SD of triplicate incubations from one experiment representative of three. Error bars are not shown when smaller than symbols.

Mentions: Isolated PMNs in suspension in salt buffers have been reported to release Ado at levels that influence their functions (12). We thus performed measurements of endogenous Ado concentrations in PMN suspensions under conditions used for the assessment of leukotriene biosynthesis. After the centrifugation and resuspension of PMNs in fresh buffers, aliquots were removed for up to 60 min and the concentration of Ado was measured. PMNs resuspended in HBSS or autologous plasma released Ado in a time– and cell concentration– dependent manner (Fig. 1). Cell-depleted plasma was found to contain 30 ± 3 nM (mean ± SE, n = 6) of Ado. The addition of 0.1 U Ado deaminase after the incubation of 2.0 × 107 PMN/ml for 15 min in HBSS reduced the concentration of Ado within seconds to <4 nM, and remained below this level for up to 30 min. Stimulation of PMNs with 0.6 μM platelet-activating factor (PAF) did not have any effect on the levels of endogenous Ado (not shown).


Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils.

Krump E, Picard S, Mancini J, Borgeat P - J. Exp. Med. (1997)

Time course of Ado accumulation in PMN suspensions.  Freshly isolated PMNs were resuspended in HBSS (A) or autologous  plasma (B) at concentrations of 5 × 106/ml (squares) or 20 × 106/ml (circles). At various time points, 1-ml aliquots of the cell suspensions were denatured with TCA and the Ado content was measured by liquid chromatography–mass spectrometry. Results shown are the means ± SD of  triplicate incubations from one experiment representative of three. Error  bars are not shown when smaller than symbols.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199092&req=5

Figure 1: Time course of Ado accumulation in PMN suspensions. Freshly isolated PMNs were resuspended in HBSS (A) or autologous plasma (B) at concentrations of 5 × 106/ml (squares) or 20 × 106/ml (circles). At various time points, 1-ml aliquots of the cell suspensions were denatured with TCA and the Ado content was measured by liquid chromatography–mass spectrometry. Results shown are the means ± SD of triplicate incubations from one experiment representative of three. Error bars are not shown when smaller than symbols.
Mentions: Isolated PMNs in suspension in salt buffers have been reported to release Ado at levels that influence their functions (12). We thus performed measurements of endogenous Ado concentrations in PMN suspensions under conditions used for the assessment of leukotriene biosynthesis. After the centrifugation and resuspension of PMNs in fresh buffers, aliquots were removed for up to 60 min and the concentration of Ado was measured. PMNs resuspended in HBSS or autologous plasma released Ado in a time– and cell concentration– dependent manner (Fig. 1). Cell-depleted plasma was found to contain 30 ± 3 nM (mean ± SE, n = 6) of Ado. The addition of 0.1 U Ado deaminase after the incubation of 2.0 × 107 PMN/ml for 15 min in HBSS reduced the concentration of Ado within seconds to <4 nM, and remained below this level for up to 30 min. Stimulation of PMNs with 0.6 μM platelet-activating factor (PAF) did not have any effect on the levels of endogenous Ado (not shown).

Bottom Line: Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs).The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs.This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie, Centre de recherche du CHUL, Université Laval, Québec, Canada.

ABSTRACT
Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.

Show MeSH
Related in: MedlinePlus