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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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Effect of exogenous  LTA4 hydrolase, Ado deaminase,  and RBCs on the biosynthesis of  LTB4 by PMNs in plasma. (A)  PMN (5 × 106) in suspension in  autologous plasma (0.5 ml) were  treated with 1.2 nM TNF-α and  1 μg/ml LPS for 30 min at  37°C. 10 μl of the preparation of  rLTA4 hydrolase (see Materials  and Methods) and/or 4 U of  Ado deaminase (or its diluent,  NaCl 0.9%) were added (per  milliliter of incubation media) 1  and 5 min, respectively, before  stimulation with 1 μM fMLP.  After 15 min of stimulation, the  incubations were stopped and  LTB4 production was measured  by RP-HPLC. (B) PMNs (5 ×  106) in suspension in autologous  plasma (0.5 ml) were treated with  1.2 nM TNF-α and 1 μg/ml LPS  for 30 min at 37°C. RBCs (0.5 ml of packed cells) treated or not with  captopril (see Materials and Methods) were next added to PMNs in  plasma; the incubation media were further treated or not with 30 μM  dipyridamole for 25 min at 37°C, and then stimulated with 1 μM fMLP  for 15 min. LTB4 biosynthesis was measured by RP-HPLC. Results  shown are the means ± SD of triplicate incubations from one experiment  representative of three. LTA4-H, LTA4-hydrolase; DIPY, dipyridamole;  CAPT-RBC, captopril-treated red blood cells; ADA, Ado deaminase.
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Figure 3: Effect of exogenous LTA4 hydrolase, Ado deaminase, and RBCs on the biosynthesis of LTB4 by PMNs in plasma. (A) PMN (5 × 106) in suspension in autologous plasma (0.5 ml) were treated with 1.2 nM TNF-α and 1 μg/ml LPS for 30 min at 37°C. 10 μl of the preparation of rLTA4 hydrolase (see Materials and Methods) and/or 4 U of Ado deaminase (or its diluent, NaCl 0.9%) were added (per milliliter of incubation media) 1 and 5 min, respectively, before stimulation with 1 μM fMLP. After 15 min of stimulation, the incubations were stopped and LTB4 production was measured by RP-HPLC. (B) PMNs (5 × 106) in suspension in autologous plasma (0.5 ml) were treated with 1.2 nM TNF-α and 1 μg/ml LPS for 30 min at 37°C. RBCs (0.5 ml of packed cells) treated or not with captopril (see Materials and Methods) were next added to PMNs in plasma; the incubation media were further treated or not with 30 μM dipyridamole for 25 min at 37°C, and then stimulated with 1 μM fMLP for 15 min. LTB4 biosynthesis was measured by RP-HPLC. Results shown are the means ± SD of triplicate incubations from one experiment representative of three. LTA4-H, LTA4-hydrolase; DIPY, dipyridamole; CAPT-RBC, captopril-treated red blood cells; ADA, Ado deaminase.

Mentions: We next examined whether endogenous Ado also suppressed LTB4 biosynthesis by PMNs resuspended in plasma. Stimulation of TNF-α/ LPS–primed PMNs with fMLP resulted in the biosynthesis of only minimal amounts of LTB4 (Fig. 3 A). The addition of Ado deaminase to the similarly primed PMN suspensions before fMLP stimulation failed to further increase the biosynthesis of LTB4. In contrast, the addition of both Ado deaminase and rLTA4 hydrolase resulted in a marked enhancement of LTB4 biosynthesis. As observed with PMNs treated with Ado deaminase alone, the addition of rLTA4 hydrolase alone failed to increase the synthesis of LTB4 upon fMLP stimulation. The effect of adding Ado deaminase and rLTA4 hydrolase to PMN suspensions in plasma on LTB4 biosynthesis was mimicked by the addition of RBCs, which are known to uptake extracellular Ado (18) and contain LTA4 hydrolase activity (7; Fig. 3 B). That the RBC LTA4 hydrolase was involved in the biosynthesis of LTB4 was confirmed by a pretreatment of RBCs with captopril, an inhibitor of LTA4 hydrolase (19), which resulted in a near complete inhibition of the effect of RBCs. Similarly, the involvement of RBCs in lowering extracellular Ado was demonstrated by adding dipyridamole, an inhibitor of Ado transport (18), to the incubation media 15 min before fMLP stimulation. Dipyridamole also caused a marked inhibition of LTB4 biosynthesis; that the inhibitory effect of dipyridamole was the consequence of an accumulation of Ado in the incubation media was assessed by the coaddition of Ado deaminase, which completely restored LTB4 biosynthesis to the level observed with RBCs (data not shown). The pretreatment of RBCs with captopril and addition of dipyridamole inhibited the biosynthesis of LTB4 to the level observed in the absence of RBCs.


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)

Effect of exogenous  LTA4 hydrolase, Ado deaminase,  and RBCs on the biosynthesis of  LTB4 by PMNs in plasma. (A)  PMN (5 × 106) in suspension in  autologous plasma (0.5 ml) were  treated with 1.2 nM TNF-α and  1 μg/ml LPS for 30 min at  37°C. 10 μl of the preparation of  rLTA4 hydrolase (see Materials  and Methods) and/or 4 U of  Ado deaminase (or its diluent,  NaCl 0.9%) were added (per  milliliter of incubation media) 1  and 5 min, respectively, before  stimulation with 1 μM fMLP.  After 15 min of stimulation, the  incubations were stopped and  LTB4 production was measured  by RP-HPLC. (B) PMNs (5 ×  106) in suspension in autologous  plasma (0.5 ml) were treated with  1.2 nM TNF-α and 1 μg/ml LPS  for 30 min at 37°C. RBCs (0.5 ml of packed cells) treated or not with  captopril (see Materials and Methods) were next added to PMNs in  plasma; the incubation media were further treated or not with 30 μM  dipyridamole for 25 min at 37°C, and then stimulated with 1 μM fMLP  for 15 min. LTB4 biosynthesis was measured by RP-HPLC. Results  shown are the means ± SD of triplicate incubations from one experiment  representative of three. LTA4-H, LTA4-hydrolase; DIPY, dipyridamole;  CAPT-RBC, captopril-treated red blood cells; ADA, Ado deaminase.
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Figure 3: Effect of exogenous LTA4 hydrolase, Ado deaminase, and RBCs on the biosynthesis of LTB4 by PMNs in plasma. (A) PMN (5 × 106) in suspension in autologous plasma (0.5 ml) were treated with 1.2 nM TNF-α and 1 μg/ml LPS for 30 min at 37°C. 10 μl of the preparation of rLTA4 hydrolase (see Materials and Methods) and/or 4 U of Ado deaminase (or its diluent, NaCl 0.9%) were added (per milliliter of incubation media) 1 and 5 min, respectively, before stimulation with 1 μM fMLP. After 15 min of stimulation, the incubations were stopped and LTB4 production was measured by RP-HPLC. (B) PMNs (5 × 106) in suspension in autologous plasma (0.5 ml) were treated with 1.2 nM TNF-α and 1 μg/ml LPS for 30 min at 37°C. RBCs (0.5 ml of packed cells) treated or not with captopril (see Materials and Methods) were next added to PMNs in plasma; the incubation media were further treated or not with 30 μM dipyridamole for 25 min at 37°C, and then stimulated with 1 μM fMLP for 15 min. LTB4 biosynthesis was measured by RP-HPLC. Results shown are the means ± SD of triplicate incubations from one experiment representative of three. LTA4-H, LTA4-hydrolase; DIPY, dipyridamole; CAPT-RBC, captopril-treated red blood cells; ADA, Ado deaminase.
Mentions: We next examined whether endogenous Ado also suppressed LTB4 biosynthesis by PMNs resuspended in plasma. Stimulation of TNF-α/ LPS–primed PMNs with fMLP resulted in the biosynthesis of only minimal amounts of LTB4 (Fig. 3 A). The addition of Ado deaminase to the similarly primed PMN suspensions before fMLP stimulation failed to further increase the biosynthesis of LTB4. In contrast, the addition of both Ado deaminase and rLTA4 hydrolase resulted in a marked enhancement of LTB4 biosynthesis. As observed with PMNs treated with Ado deaminase alone, the addition of rLTA4 hydrolase alone failed to increase the synthesis of LTB4 upon fMLP stimulation. The effect of adding Ado deaminase and rLTA4 hydrolase to PMN suspensions in plasma on LTB4 biosynthesis was mimicked by the addition of RBCs, which are known to uptake extracellular Ado (18) and contain LTA4 hydrolase activity (7; Fig. 3 B). That the RBC LTA4 hydrolase was involved in the biosynthesis of LTB4 was confirmed by a pretreatment of RBCs with captopril, an inhibitor of LTA4 hydrolase (19), which resulted in a near complete inhibition of the effect of RBCs. Similarly, the involvement of RBCs in lowering extracellular Ado was demonstrated by adding dipyridamole, an inhibitor of Ado transport (18), to the incubation media 15 min before fMLP stimulation. Dipyridamole also caused a marked inhibition of LTB4 biosynthesis; that the inhibitory effect of dipyridamole was the consequence of an accumulation of Ado in the incubation media was assessed by the coaddition of Ado deaminase, which completely restored LTB4 biosynthesis to the level observed with RBCs (data not shown). The pretreatment of RBCs with captopril and addition of dipyridamole inhibited the biosynthesis of LTB4 to the level observed in the absence of RBCs.

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