353
Fundam Clin Phannacol(1992) 6,353-358 0 Elsevier, Paris
Activation of guinea pig eosinophil respiratory burst by leukotriene B,: role of protein kinase C KF Rabe*, MA Giembycz, G Dent
Departmnr of Thoracic Medicine, National Heart and h
I*,
PJ Barnes
g Imtitute. Dovehouse Street, London SW3 6LY,VK
(Received 9 June 1992; accepted 9 October 1992)
Summary - Leukotriene B, (LTB,) and the protein kina= C activator, 4-/%phorbol dibutyrate (PDBu), both induced a pronounced and concentrationdependent stimulation of hydrogen peroxide (%OJ generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM - 1 pM. The LTB, response was inhibited competitively by the specific LTB, receptor antagonist, U-75302, with a KBof 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB, and PDBu, respectively) by the competitive protein .kinase C inhibitor, I-0-hexadecyl-2-0-methylglycerolat a concentration of 300 pM. LTB, appears. therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C. leukotriene B, / eosinophils / respiratory burst / protein kinase C / protein kinase C inhibitors / LTB, antagonists / U-75302
Introduction
Leukouiene B, (LTB,; 5(S)-12(R)dihydroxy-6,14cis-8,10-tra~-eicosatetraenoicacid) is a product of the 5-lipoxygenation of arachidonic acid in a variety of cell types, including human neuuophils (BorSeat and Samuelsson. 1979), blood monocytes (Czop and Austen, 1985). alveolar macrophages (Fels et af, 1982) and lung mast cells (Freeland et al, 1988). LTB, is a potent chemoattractant for neuuophils and monocytes (Nagy et af. 1982). stimulates aggregation and degranulation of human
neuuophils (Ford-Hutchinson et af, 1980;O’Flaherty el af, 1981), promotes the cyclooxygenation of arachidonic acid in eosinophils (Rabe el al, 1991) and is both chemokinetic and chemotactic for guinea pig eosinophils (Maghni el af, 1991). Recent evidence suggests that these functional responses are mediated by specific cell surface receptors. Thus, on human neutrophils (Goldman and Goetzl, 1982;Kriesle and Parker, 1983). guinea pig lung macrophages (Cristol et af, 1988) and, more recently, on guinea pig alveolar (Maghni el af. 1991) and peritoneal eosinophils (Ng el af, 1991)
* Present address: Krankenhaus Grosshansdorf, LVA Hamburg, 2070 Grosshansdorf, Germany.
354
KFRabertd
specific binding sites have been identified for [3H]LTB, with strict stereospecificity for the 5(S),12(R) isomer. Moreover, radioligand binding studies have shown two prototype LTB, receptor antagonists, U-75302 and SC-41930, to compete effectively with [3H]LTB, for binding to intact eosinophils (Maghni et al, 1991; Ng et al, 1991). LTB, is only generated in very small quantities by human eosinophils (Weller et al, 1983) and has only slight chemotactic activity for these cells (Wardlaw et al, 1986). In contrast, guinea pig eosinophils preferentially generate LTB, (Sun el al, 1989) and this mediator has been shown to be chemotactic for guinea pig eosinophils in vitro as well as inducing bronchopulmonary eosinophilia after inhalation in vivo (Richards et al, 1991). Here we report the stimulation of respiratory burst in guinea pig eosinophils by LTB, and the involvement of the Ca++/phospholipid-dependent protein kinase (protein kinase C) in this response. A preliminary account of some of these data has been presented to the British Pharmacological Society (Rabe et al, 1990).
Materials and methods Eosinophils were isolated from peritoneal exudates of human serumtreated male Dunkin-Hartley guinea pigs by differential centrifugation over Percoll density gradients, as described previously (Yukawa et al. 1989). Briefly, guinea pigs (700-1 300 g body weight), treated by weekly intraperitoneal injection of 1 ml human serum for at least 3 weeks prior to experimentation, were anaesthetised with ketamine (25 mgkg) and xylazine (5 mg/kg) and their peritoneal cavities lavaged with 50 ml sterile 5% glucose solution. Cells were precipitated by centrifugation of the lavage fluid, resuspended in 1.070 g/ml Percoll, supplemented with FCS 10% and DNase 4.5 pg/ml. and layered onto 4-step density gradients (1.080/1 .085/1.090/1.100 g/ml Percoll in Pipes buffer). After centrifugation at 1600 g for 20 min at 18OC. cells were removed from the interfaces between gradient fractions, washed twice in HBSS and resuspended in Hepes-buffered, CaZ+/Mg2+-freeKrebs Ringer bicarbonate solution (KRB). Cell number and eosinophil purity were assessed by counting Kimura stained cells in
an improved Neubauer haemocytometer. Fractions containing > 90% eosinophils were pooled and stored on ice until required. Contaminating cells were mainly macrophages and, occasionally, small numbers of lymphocytes and mast cells. The purity of eosinophil preparations were 95.7 f 0.35% (mean f sem, n = 12). Respiratory burst activity was measured as the generation of hydrogen peroxide (H202)in a superoxide dismutase-saturated system; H,O, was assayed by the horseradish peroxidase-catalysed oxidation of the fluorescent dye, scopoletin (Sigma Chemical Co. Poole, Dorset, UK). as described (Dent et al, 1991). Eosinophils (1 x l@/ml) were incubated at 37OC for 5 min, in the absence or presence of the LTB, receptor antagonist, 6-(6-(3-hydrox y- 1E. 52-undecadien- 1-yl)-2-pyridinyl)1.5-hexanediol) (U-75302. The Upjohn Company, Kalamazoo, Michigan, USA), or the protein kinase C inhibitor, 1-0-hexadecyl-2-0-methylgycerol (AMG-C,,. B achem Feinchemi kalien, B ubendorf. Switzerland). prior to the addition of 4-Fphorbol dibutyrate (PDBu, Sigma) or LTB, (a gift from Bayer UK, Stoke Poges. Bucks, UK) and the loss of scopoletin fluorescence was monitored continuously for 15-30 min (fig 1. insets). Inverted first derivative traces of scopoletin fluorescence were generated (fig 1. main panels) and the rate of generation of H,O, was calculated by comparison with a standard curve for known H20, concentrations. Results are expressed as quantities of H,O, generated after stimulation with LTB, but, owing to the prolonged response to PDBu leading to an exhaustion of scopoletin in the assay system and thus precluding the calculation of quantities of H,O,. PDBu responses are reported as the maximal rate of generation of H,O, as calculated from the first derivative traces. Data are expressed as arithmetic mean f sem and comparisons were made using Student's t-test; a probability of less than 0.05 was defined as statistically significant.
Results Both PDBu (1 nM-1 pM) and LTB, (10 nM100 pM) caused a pronounced, concentrationdependent stimulation of H,O, generation by guinea pig peritoneal eosinophils (fig 1). 'Ihe response to LTB, was rapid and transient. with rates of generation being maximal within 45 s of exposure of the cells to the agent and returning to baseline levels within 2 min (fig 1 lower panel). In
LTB, and guinea pig eosinophils
contrast, PDBu caused a prolonged respiratory burst response whose time of onset became shorter with increasing concentrations of the stimulus (fig 1 upper panel). In all cases the basal generation of H,O, was negligible.
E
.-
I-
a
0
Y
5
10
15
Time
0
3
6
IS
12
9
20
25
30
(mid
18
Time (minl
355
Pre-incubation of eosinophils with U-75302,a specific LTB, receptor antagonist (Lin et al, 1988). caused a 5-fold rightward-shift in the LTB, concentration-response curve (fig 2). The mean EC,, value for stimulation of respiratory burst was increased from 42.9 f 9.35 nM in the absence to 236.7 f 79.5 nM in the presence of 100 nM U75302 ( n = 3, P < 0.05). From this shift an antagonist affinity constant (K,) of 25.3 k 6.56 nM was calculated. Treatment of eosinophils with the protein kinase C inhibitor AMG-C,, led to rightward shifts in the concentration-response curves for both PDBu and LTB, (fig 3);the EC50 for induction of H,O, generation by PDBu was increased 2.8-fold, from 10.8 f 3.0 nM ( n = 9) to 30.4 f 13.8 nM ( n = 3, P < 0.05), while the EC,, for LTB, was increased 3.8-fold, from 70.2 k 5.1 nM (n = 9)to 266 f 12.1 nM ( n = 5, P < 0.05) after treatment with 300 p M AMG-C,, - a concentration which causes total inhibition of PKC in human neutrophils (Kramer et al, 1989).
:
.I-
.
a m
l2
0
m
1
U
0 L
0
Y
a 0
3
6
Tim.
9
1
2
1
5
(minl
Fig 1. Original traces of respiratory burst responses of e o s i n e phils to increasing concentrations of PDBu (upper panel) and LTB, (lower panel). T h e insets s h o w a series of realtime recordings of $0, generation measured as the loss of scopoletin fluorescence over time which was monitored continuously for 15-30 min. The main panels show the kinetics of loss of fluorescence for both stimuli by means of generated inverted first derivative traces of the respective realtime recordings. The response to LTB, was rapid and transient, with rates of generation being maximal within 45 s of exposure of the cells to the agent and returning to baseline levels within 2 min (lower panel). In contrast, PDBu caused a prolonged respiratory burst response whose time of onset became shorter with increasing concentrations of the stimulus. The rate of generation of $0, was calculated by comparison with a standard curve for known H,O, concentrations.
O J I lo-’
I
1
10-a
10-7
I
10-6
[LTBJ (MI Fig 2. Pre-incubation of eosinophils with the specific LTB,, receptor antagonist U-75302 (100 nM) caused a S-fold rightward-shift in the LTB, concentration-response curve. The mean EC, value for stimulation of respiratory burst was increased from 42.9 f 9.35 nM in the absence (open squares) to 236.7 f 79.5 nM in the presence (closed squares) of U-75302. This antagonism appeared competitive in nature and an antagonist affinity constant (K,)of 25.3 f 6 3 6 nM was calculated.
356
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a A
v)
= 9)
6 -
0 0
z
5 -
1 0
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4 -
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2
3 -
9)
s
2-
Fundam Clin Phannacol(1992) 6,353-358 0 Elsevier, Paris
Activation of guinea pig eosinophil respiratory burst by leukotriene B,: role of protein kinase C KF Rabe*, MA Giembycz, G Dent
Departmnr of Thoracic Medicine, National Heart and h
I*,
PJ Barnes
g Imtitute. Dovehouse Street, London SW3 6LY,VK
(Received 9 June 1992; accepted 9 October 1992)
Summary - Leukotriene B, (LTB,) and the protein kina= C activator, 4-/%phorbol dibutyrate (PDBu), both induced a pronounced and concentrationdependent stimulation of hydrogen peroxide (%OJ generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM - 1 pM. The LTB, response was inhibited competitively by the specific LTB, receptor antagonist, U-75302, with a KBof 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB, and PDBu, respectively) by the competitive protein .kinase C inhibitor, I-0-hexadecyl-2-0-methylglycerolat a concentration of 300 pM. LTB, appears. therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C. leukotriene B, / eosinophils / respiratory burst / protein kinase C / protein kinase C inhibitors / LTB, antagonists / U-75302
Introduction
Leukouiene B, (LTB,; 5(S)-12(R)dihydroxy-6,14cis-8,10-tra~-eicosatetraenoicacid) is a product of the 5-lipoxygenation of arachidonic acid in a variety of cell types, including human neuuophils (BorSeat and Samuelsson. 1979), blood monocytes (Czop and Austen, 1985). alveolar macrophages (Fels et af, 1982) and lung mast cells (Freeland et al, 1988). LTB, is a potent chemoattractant for neuuophils and monocytes (Nagy et af. 1982). stimulates aggregation and degranulation of human
neuuophils (Ford-Hutchinson et af, 1980;O’Flaherty el af, 1981), promotes the cyclooxygenation of arachidonic acid in eosinophils (Rabe el al, 1991) and is both chemokinetic and chemotactic for guinea pig eosinophils (Maghni el af, 1991). Recent evidence suggests that these functional responses are mediated by specific cell surface receptors. Thus, on human neutrophils (Goldman and Goetzl, 1982;Kriesle and Parker, 1983). guinea pig lung macrophages (Cristol et af, 1988) and, more recently, on guinea pig alveolar (Maghni el af. 1991) and peritoneal eosinophils (Ng el af, 1991)
* Present address: Krankenhaus Grosshansdorf, LVA Hamburg, 2070 Grosshansdorf, Germany.
354
KFRabertd
specific binding sites have been identified for [3H]LTB, with strict stereospecificity for the 5(S),12(R) isomer. Moreover, radioligand binding studies have shown two prototype LTB, receptor antagonists, U-75302 and SC-41930, to compete effectively with [3H]LTB, for binding to intact eosinophils (Maghni et al, 1991; Ng et al, 1991). LTB, is only generated in very small quantities by human eosinophils (Weller et al, 1983) and has only slight chemotactic activity for these cells (Wardlaw et al, 1986). In contrast, guinea pig eosinophils preferentially generate LTB, (Sun el al, 1989) and this mediator has been shown to be chemotactic for guinea pig eosinophils in vitro as well as inducing bronchopulmonary eosinophilia after inhalation in vivo (Richards et al, 1991). Here we report the stimulation of respiratory burst in guinea pig eosinophils by LTB, and the involvement of the Ca++/phospholipid-dependent protein kinase (protein kinase C) in this response. A preliminary account of some of these data has been presented to the British Pharmacological Society (Rabe et al, 1990).
Materials and methods Eosinophils were isolated from peritoneal exudates of human serumtreated male Dunkin-Hartley guinea pigs by differential centrifugation over Percoll density gradients, as described previously (Yukawa et al. 1989). Briefly, guinea pigs (700-1 300 g body weight), treated by weekly intraperitoneal injection of 1 ml human serum for at least 3 weeks prior to experimentation, were anaesthetised with ketamine (25 mgkg) and xylazine (5 mg/kg) and their peritoneal cavities lavaged with 50 ml sterile 5% glucose solution. Cells were precipitated by centrifugation of the lavage fluid, resuspended in 1.070 g/ml Percoll, supplemented with FCS 10% and DNase 4.5 pg/ml. and layered onto 4-step density gradients (1.080/1 .085/1.090/1.100 g/ml Percoll in Pipes buffer). After centrifugation at 1600 g for 20 min at 18OC. cells were removed from the interfaces between gradient fractions, washed twice in HBSS and resuspended in Hepes-buffered, CaZ+/Mg2+-freeKrebs Ringer bicarbonate solution (KRB). Cell number and eosinophil purity were assessed by counting Kimura stained cells in
an improved Neubauer haemocytometer. Fractions containing > 90% eosinophils were pooled and stored on ice until required. Contaminating cells were mainly macrophages and, occasionally, small numbers of lymphocytes and mast cells. The purity of eosinophil preparations were 95.7 f 0.35% (mean f sem, n = 12). Respiratory burst activity was measured as the generation of hydrogen peroxide (H202)in a superoxide dismutase-saturated system; H,O, was assayed by the horseradish peroxidase-catalysed oxidation of the fluorescent dye, scopoletin (Sigma Chemical Co. Poole, Dorset, UK). as described (Dent et al, 1991). Eosinophils (1 x l@/ml) were incubated at 37OC for 5 min, in the absence or presence of the LTB, receptor antagonist, 6-(6-(3-hydrox y- 1E. 52-undecadien- 1-yl)-2-pyridinyl)1.5-hexanediol) (U-75302. The Upjohn Company, Kalamazoo, Michigan, USA), or the protein kinase C inhibitor, 1-0-hexadecyl-2-0-methylgycerol (AMG-C,,. B achem Feinchemi kalien, B ubendorf. Switzerland). prior to the addition of 4-Fphorbol dibutyrate (PDBu, Sigma) or LTB, (a gift from Bayer UK, Stoke Poges. Bucks, UK) and the loss of scopoletin fluorescence was monitored continuously for 15-30 min (fig 1. insets). Inverted first derivative traces of scopoletin fluorescence were generated (fig 1. main panels) and the rate of generation of H,O, was calculated by comparison with a standard curve for known H20, concentrations. Results are expressed as quantities of H,O, generated after stimulation with LTB, but, owing to the prolonged response to PDBu leading to an exhaustion of scopoletin in the assay system and thus precluding the calculation of quantities of H,O,. PDBu responses are reported as the maximal rate of generation of H,O, as calculated from the first derivative traces. Data are expressed as arithmetic mean f sem and comparisons were made using Student's t-test; a probability of less than 0.05 was defined as statistically significant.
Results Both PDBu (1 nM-1 pM) and LTB, (10 nM100 pM) caused a pronounced, concentrationdependent stimulation of H,O, generation by guinea pig peritoneal eosinophils (fig 1). 'Ihe response to LTB, was rapid and transient. with rates of generation being maximal within 45 s of exposure of the cells to the agent and returning to baseline levels within 2 min (fig 1 lower panel). In
LTB, and guinea pig eosinophils
contrast, PDBu caused a prolonged respiratory burst response whose time of onset became shorter with increasing concentrations of the stimulus (fig 1 upper panel). In all cases the basal generation of H,O, was negligible.
E
.-
I-
a
0
Y
5
10
15
Time
0
3
6
IS
12
9
20
25
30
(mid
18
Time (minl
355
Pre-incubation of eosinophils with U-75302,a specific LTB, receptor antagonist (Lin et al, 1988). caused a 5-fold rightward-shift in the LTB, concentration-response curve (fig 2). The mean EC,, value for stimulation of respiratory burst was increased from 42.9 f 9.35 nM in the absence to 236.7 f 79.5 nM in the presence of 100 nM U75302 ( n = 3, P < 0.05). From this shift an antagonist affinity constant (K,) of 25.3 k 6.56 nM was calculated. Treatment of eosinophils with the protein kinase C inhibitor AMG-C,, led to rightward shifts in the concentration-response curves for both PDBu and LTB, (fig 3);the EC50 for induction of H,O, generation by PDBu was increased 2.8-fold, from 10.8 f 3.0 nM ( n = 9) to 30.4 f 13.8 nM ( n = 3, P < 0.05), while the EC,, for LTB, was increased 3.8-fold, from 70.2 k 5.1 nM (n = 9)to 266 f 12.1 nM ( n = 5, P < 0.05) after treatment with 300 p M AMG-C,, - a concentration which causes total inhibition of PKC in human neutrophils (Kramer et al, 1989).
:
.I-
.
a m
l2
0
m
1
U
0 L
0
Y
a 0
3
6
Tim.
9
1
2
1
5
(minl
Fig 1. Original traces of respiratory burst responses of e o s i n e phils to increasing concentrations of PDBu (upper panel) and LTB, (lower panel). T h e insets s h o w a series of realtime recordings of $0, generation measured as the loss of scopoletin fluorescence over time which was monitored continuously for 15-30 min. The main panels show the kinetics of loss of fluorescence for both stimuli by means of generated inverted first derivative traces of the respective realtime recordings. The response to LTB, was rapid and transient, with rates of generation being maximal within 45 s of exposure of the cells to the agent and returning to baseline levels within 2 min (lower panel). In contrast, PDBu caused a prolonged respiratory burst response whose time of onset became shorter with increasing concentrations of the stimulus. The rate of generation of $0, was calculated by comparison with a standard curve for known H,O, concentrations.
O J I lo-’
I
1
10-a
10-7
I
10-6
[LTBJ (MI Fig 2. Pre-incubation of eosinophils with the specific LTB,, receptor antagonist U-75302 (100 nM) caused a S-fold rightward-shift in the LTB, concentration-response curve. The mean EC, value for stimulation of respiratory burst was increased from 42.9 f 9.35 nM in the absence (open squares) to 236.7 f 79.5 nM in the presence (closed squares) of U-75302. This antagonism appeared competitive in nature and an antagonist affinity constant (K,)of 25.3 f 6 3 6 nM was calculated.
356
KFRabeCtal
a A
v)
= 9)
6 -
0 0
z
5 -
1 0
-E
4 -
c 0 ’i;
2
3 -
9)
s
2-
