Clinical and Experimental Allergy, 1992, Volume 22, pages 91 97
IgE binding inhibits arachidonic acid-induced chemiluminescence of human platelets p. RIO*, J. M. TUNON DE LARA*, R. MARTHANf atid A. TAYTARD* *Scrvicc des Maladies Re.spiratoires. Hopital du Haut Li-veque, CHR de Bordeaux, Pe.ssac and ^Laboratoire de Physiologie. Universite de Bordeaux II. Bordeaux. France Summary
Arachidonie acid (AA)-induced chemiluminescence (CHL) was studied in vitro by means of a luminometer in platelets from nine healthy volunteers and six allergie patients. The amplitude ofthe CHL signal increased with AA concentration from 250 /(M to 7 mM. At a low A A concentration (250 /m). the CHL signal consisted of two peaks. The first one occurred at 6 + 3 sec and the second one at 90± 15 sec {n = 9). The mean amplitude of these peaks was I-95+ 0-61 mV/sec and 0-82 ± 0 22 mV/sec for normal subjects, and 2-35 + 0-62 mV/sec and 0 78 + 0 26 mV/sec for allergic patients, respectively. Aspirin (a cycloxygenase inhibitor) and baiealein (a lipoxygenase inhibitor) reduced in a concentration-dependent manner, the first and second peak, respectively. The binding of immunoglobulin F (IgE) alone to platelets from both normal and allergic subjeets inhibited both ihe first and seeond peak of AA-induced CHL. This inhibitory effect was specifically due to the action of IgF as it was (i) concentration-dependent and (ii)not observed when immunoglobulin G (IgG) was substituted for IgF. It is concluded that in normal subjects, as well as in allergie patients, the binding of IgE alone to its speeific receptor on human platelets eould alter arachidonate metabolism that probably involves cycloxygenase and lipoxygenase pathways. Clinica! and E.xperimental Allergy. Vol. 21. pp. 91 97. Submitted 19 June 1991; revised 21 August 1991, accepted 30 August 1991 Introduction Platelets may be involved in the pathophysiology of asthma [1]. In a previous study, we demonstrated that the life span of platelets was reduced in atopie asthmatic patients [2]. In addition, there is evidence that the function of platelets from subjects suffering from allergic diseases is altered [3]. Platelets from both normal and allergie subjects bear the low affinity receptor for IgE (Fc-epsilon-RII) [4 6]. The binding of IgE to its receptor activates, in the presence of either the specific antigen or anti-IgE. the platelet whieh then releases eytotoxie mediators sueh as oxygen metabolites. In allergic patients, there are extended periods of time during which IgF is bound to its receptor in Ihe absence of the specific antigen. Nevertheless, the efTeet ofthe binding of IgF alone on platelets has nol been extensively studied, although it has been shown Corrcspmidcncf: Dr P. Rio. Scrviuc des Muhdics Rcspiraloircs, Hopital iju Haul Levcquc. CHR dc Bordeaux, F-33604 Pessae eedex, France.
that this binding does not induce a release of mediators [1,3,7]. In a recent study, we have shown that the binding of IgF alone to the Fc-epsilon-RlI alters monoamine uptake in platelets [8]. Since metabolism of arachidonic acid (AA) is an important function of platelets [9]. we examined the eflect of the binding of IgF alone on this metabolism in platelets from both normal subjects and allergic patients. For this purpose, we studied A A metabolism in both normal and allergic platelets by means of chcmiluminescenee (CHL) as it has been previously shown that this teehnique is well suited to study AA-activated platelets [10,1 1,12]. Materials and methods Subjecf.s A lotal of nine healthy volunteers, five men and four women aged 23 to 40 years, participated in the study. Their serum IgE eoncentration was 42 3+18-5 IU/ml (normal values+ 2 s.d. = 100+IU/ml). All the subiects 91
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gave their written consent and the protocol was approved by the Local Ethics Committee of the Hospital. None of the subjects had taken any medication, including oral contraceptives or non-steroidal anti-infiammatory drugs, for at least 15 days. Also included in the study were six allergic patients, aged from 22 to 35 years. They were suffering from either allergic rhinitis or allergie asthma. However, they were symptom-free at the moment of the study. The clinical diagnosis of allergy was confirmed by skin tests and radioallergosorbent tests. Their IgE concentration was 596 + 317 IU/ml. They did not take any medication at the moment ofthe study.
Isolation of hlood platelets Blood samples were withdrawn between 08.00 and 10.UO a.m. Venous blood (40 ml) was collected without a tourniquet in a syringe containing 5 ml of ACD anticoagulant (citric aeid: I 4 g; sodium: 2 5 g; dextrose: 2 g; in 100 ml of distilled water). A first centrifugation at 200 g for 25 min separated the platelet-rich plasma (PRP) which was then acidified with 3 ml of ACD. A second eentrifugation at 1500 g for 10 min enabled the separation of the platelet-poor plasma (PPP) from the pellet which was then washed three times in phosphate-buffered saline (PBS) at pH 6 5 . and resuspended in Hcpes-KrebsRinger-Glucosc bulTer (HKRG) (Hepes: 25 niM: NaCi: 125 mM; KCI: 4-8 niM; glucose: 5 6 mM; ascorbic acid: 1 niM) to a final concentration of 5 x 10*^ platelets/ml. Platelets, leucocytes and red cells were counted in a counter (S-Plus IV Coulter. Paris). The platelet suspensions used in this study contained less than 1% red cells and 0 03% leucocytes per 5 x 10** platelets.
Chemilumine.scence The chemiluminescencc experiments were carried out with a LKB-Wallac luminometer 1251 with a mixing device and temperature control. The experiments were performed at 37 C. After incubation for 45 min in HKRG, 100 fi\ of luminol (3-aminophthalhydrazide; 200 fiM) were added to the platelet suspension which was then activated with AA. The concentration-dependent effeet was analysed using increasing concentrations of AA ranging from 0 25 to 7 mM. In order to characterize the chemiluminescence response, cycloxygenase and lipoxygenase pathways were inhibited by a previous incubation for 20 min with acetylsalicylie acid and baicalein. respectively.
The effect of immunoglobulins was studied by incubating 400/tl of the platelet suspension for 45 min with 100 /il of HKRG. or with 100 /i\ of IgE solution at various concentrations ranging from 30 to 500 IU/ml, or with 100/il ofthe TgG solution at a concentration of 12 mg/ ml. In addition, we also studied the effect of IgG at a concentration of 9 mg/ml, this concentration being the normal plasma one for that class of immunoglobulin. AA-induced chemiluminescence was then measured during the 3 min following the AA addition. Drugs used Sodium araehidonate (Sigma. St Louis. Missouri, U.S.A.); protein A purified plasma IgG (a gift from V. Jallu. Section de pathologie de hemostase. Pessac, France); polyclonal purified human IgE and baicaiein (5,6,7-trihydroxyfiavone) (Calbiochem, La Jolla, California, U.S.A.); acetylsalicylic acid (Sigma). Statistical analysis Data are given as means ± s.d. and compared between the different series (e.g. IgE. HKRG. IgG) with two-tailed Student's /-tests for unpaired samples. Comparison of concentration-response curves was carried out using a computerized ANOVA procedure. Results AA-induccd chemiluminescence of platelets In normal subjects, addition of AA to a suspension of washed platelets (200 /il) preincubated with luminol (200 /iM) produced a signal that reached a maximum in about 6 sec. The amplitude of this signal was concentration-dependent up to 29 + 6 mV/sec (/? —9) for an A A concentration of 7 niM (Fig. I). In both normal and allergic subjects, addition of AA at a low concentration (i.e. 250 fiwi) to 400 ft\ of the platelet suspension in the presence of luminol led to two luminescence signals. The mean amplitudes ofthe first peak were 1 95 + 061 mV/sec and 2-35±062 mV/sec (NS), and those of the second peak were 0 8 2 + 0 22 mV/sec and 0 78 + 0 26mV/sec(NS). respectively. In the both groups, the maximal amplitude ofthe first peak occurred about 6-9 sec after the addition of AA. This first signal then decreased within 20 sec to the background level. The onset of the second peak occurred at 35 sec. reached a maximum in about 90 sec and returned to background level within 150 sec (Fig. 2). This amplitude of the second signal was 25 + 8 7% that ofthe first peak (« = 9) for the normal subjects and 22 7+11"^) (« = 6) for the allergic subjects (Fig. 3).
IgE and platelet chemilumlnescence
50 r
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3r
40
30
20
10
0 T 0
1
2
3
4
5
6
7
8
AA (nM)
Fig, 1, Concentration-dependent effect of AA on platelet CHL in hciillhy siibjecls. Mean values of CHL (/f = 9) are plotted vs AA conL-cnlrations ranging from 025 lo 7 mM. Vertical bars indicate s.d.
Characterization of the two peaks Pretreatment of platelets with acetylsalicylic acid (ASA) inhibited the first signal of AA-induced luminescence without aitering the second signai (Eig. 2). This efl'ect was concentration-dependent from iO"^to iO ^' M. At iO"''M concentration of ASA, the ampiitudeofthe first peak was not significantly different from that of the background noise. Baicalein reduced the second signal of AA promoted chemilumineseence. Indeed, the second peak was completely abolished by baicalein at 10"^ M. However, the first peak was aiso decreased by a concentration as iow as iO-^Mofbaicalein(Flg. 2).
0
15 30 45 60
75
90 105 120 135 150 165 180
Time ( s ) Kig, 2, AA-induced CHL of platelets vs lime. Open circles represent CHL of platelets from one healthy suhjecl induced by a low concentration of AA (250 /(M). Closed circles and closed squares represent AA-induced CHL of platelets from the same subject after incuhalion with ASA (10 "^ M) and baicalein (10 * M), respectively.
In allergic patients, IgE inhibited AA induced CHL in a concentration-dependent manner. The potency of IgE was similar to that of the normal subjects (i.e. IC 5 0 - 9 5 iU/ml; NS). At low concentrations of AA (i.e. 0 25 mM), no difference was observed between normal and atopic subjects for the IgE dependent inhibition of the two peaks of CHL (Fig. 3). Preincubation with IgG soiution under conditions similar to that with IgE did not aiter the AA-induced ehemilumineseence of piatelets (Fig. 4).
Effect of immunoglobulins Incubation of washed human plateiets from normal subjects with a high concentration of IgE (500 IU/ml) suppressed the signals of luminescence induced by AA ranging from 0 25 to 7 niM (Eigs 4 & 5). IgE aitered the chemilumineseence response in a concentration-dependent manner. Both luminescence signals (i.e. first and second peak) decreased when the concentration of IgE increased (Fig. 5). Regarding the first peak, the concentration of IgE causing a 5Q"A, decrease in amplitude (i.e. IC 50) was equivalent to liO IU/ml (H = 7) (Fig. 6).
Diseussion These results show that AA-stimulation of piatelets from both normai and ailergic subjects induces a concentration-dependent iuminescence response that consists of two components. The first and the second peaks may depend on the cycio.Kygenase and iipoxygenase pathways, respectiveiy. The binding of IgE alone inhibits both peaks in a concentration-dependent manner. The chemilumineseence enables the measurement of reactive oxygen species production such as singlet oxy-
94
P. Rio et al.
NS
1 „
2
T
T
1st peok
Q
2na peak
Normol subjects
1st peak
2nd peak
Allergic subiects
Kig. 3. Mean values of AA-induced peaks of CH L in normal and allergic subjects. White columns represent mean values of the maximal CHL responses to AA at 9 and 90 sec (i.e. first and second peak) in normal subjects (« = 9) and allergic subJeL-ls (« = 6) in the absence of IgE. Vertical bars indicate s.d. Grey columns represent the similar values in the presence oflgE (500 IU/ml).
gen, superoxide and hydroxyl radicals and hydrogen peroxide. These oxygen metabohtes emit light either directly or upon their interaction with luminol [13]. Activation of platelets by AA releases reactive oxygen species that can be recorded by means o[ CHL as previously shown by Worncr et al. [11], Melsa-Ketela et
al. [12] and MiWs ct ai [10]. It has been demonstrated that the luminescence signal in the platelet suspension is not due to leucocyte contamination [11]. At low concentrations, the AA-induced luminescence response consisted of two peaks. This result is in agreement with that of Worner et al. [11] who also described two peaks, the time-course of which was close to that reported in the present study. In contrast. Mills et al. [ 10] described only one peak of CHL induced by AA whereas Metsa-Ketela ct al. [12] found either one or two peaks. It is likely that a higher concentration of AA and a lower concentration of platelets are responsible for the difference in the results of these studies, with respect to the number of peaks observed. The two successive peaks of CHL could be due to the two pathways of AA metabolism in platelets. Indeed, lhe first peak was inhibited after incubating with ASA, a specific inhibitor of cycloxygenase and the second peak was inhibited by baicalein. a specific inhibitor of 12iipoxygenase pathway [14] in platelets [15]. These results confirm those of Mills et al. [10] and Melsa-Ketela et al. [ 12] regarding lhe pharmacological sensitivity of the first peakand those of Worner ('/«/. [I I] regarding that of both peaks. The fact that ASA {as well as indomethacin [11]) reduced in a concentration-dependent manner the amplitude of the first peak strongly suggests that il involves the cycloxygenase pathway. Moreover, Worner demonstrated that incubation of platelets with ASA decreased the first peak and concurrently increased the amplitude of the second peak.
35 -
I
30
25
20
15
NS
10
3-5 AA {mM)
Fig. 4. Eflect of IgE and IgG on phtlelet CHL response to AA at high concentrations in healthy subjects. Columns rcprcsenl mean valties (« = 9) of CHL response lo AA at 3 high concentrations (i.e. 2. 3'5. and 7 mM). White, grey, and black columns represent CHL afler inctibalion with a bufTer, IgG solution or IgE solution, respeclively (*/'
IgE binding inhibits arachidonic acid-induced chemiluminescence of human platelets p. RIO*, J. M. TUNON DE LARA*, R. MARTHANf atid A. TAYTARD* *Scrvicc des Maladies Re.spiratoires. Hopital du Haut Li-veque, CHR de Bordeaux, Pe.ssac and ^Laboratoire de Physiologie. Universite de Bordeaux II. Bordeaux. France Summary
Arachidonie acid (AA)-induced chemiluminescence (CHL) was studied in vitro by means of a luminometer in platelets from nine healthy volunteers and six allergie patients. The amplitude ofthe CHL signal increased with AA concentration from 250 /(M to 7 mM. At a low A A concentration (250 /m). the CHL signal consisted of two peaks. The first one occurred at 6 + 3 sec and the second one at 90± 15 sec {n = 9). The mean amplitude of these peaks was I-95+ 0-61 mV/sec and 0-82 ± 0 22 mV/sec for normal subjects, and 2-35 + 0-62 mV/sec and 0 78 + 0 26 mV/sec for allergic patients, respectively. Aspirin (a cycloxygenase inhibitor) and baiealein (a lipoxygenase inhibitor) reduced in a concentration-dependent manner, the first and second peak, respectively. The binding of immunoglobulin F (IgE) alone to platelets from both normal and allergic subjeets inhibited both ihe first and seeond peak of AA-induced CHL. This inhibitory effect was specifically due to the action of IgF as it was (i) concentration-dependent and (ii)not observed when immunoglobulin G (IgG) was substituted for IgF. It is concluded that in normal subjects, as well as in allergie patients, the binding of IgE alone to its speeific receptor on human platelets eould alter arachidonate metabolism that probably involves cycloxygenase and lipoxygenase pathways. Clinica! and E.xperimental Allergy. Vol. 21. pp. 91 97. Submitted 19 June 1991; revised 21 August 1991, accepted 30 August 1991 Introduction Platelets may be involved in the pathophysiology of asthma [1]. In a previous study, we demonstrated that the life span of platelets was reduced in atopie asthmatic patients [2]. In addition, there is evidence that the function of platelets from subjects suffering from allergic diseases is altered [3]. Platelets from both normal and allergie subjects bear the low affinity receptor for IgE (Fc-epsilon-RII) [4 6]. The binding of IgE to its receptor activates, in the presence of either the specific antigen or anti-IgE. the platelet whieh then releases eytotoxie mediators sueh as oxygen metabolites. In allergic patients, there are extended periods of time during which IgF is bound to its receptor in Ihe absence of the specific antigen. Nevertheless, the efTeet ofthe binding of IgF alone on platelets has nol been extensively studied, although it has been shown Corrcspmidcncf: Dr P. Rio. Scrviuc des Muhdics Rcspiraloircs, Hopital iju Haul Levcquc. CHR dc Bordeaux, F-33604 Pessae eedex, France.
that this binding does not induce a release of mediators [1,3,7]. In a recent study, we have shown that the binding of IgF alone to the Fc-epsilon-RlI alters monoamine uptake in platelets [8]. Since metabolism of arachidonic acid (AA) is an important function of platelets [9]. we examined the eflect of the binding of IgF alone on this metabolism in platelets from both normal subjects and allergic patients. For this purpose, we studied A A metabolism in both normal and allergic platelets by means of chcmiluminescenee (CHL) as it has been previously shown that this teehnique is well suited to study AA-activated platelets [10,1 1,12]. Materials and methods Subjecf.s A lotal of nine healthy volunteers, five men and four women aged 23 to 40 years, participated in the study. Their serum IgE eoncentration was 42 3+18-5 IU/ml (normal values+ 2 s.d. = 100+IU/ml). All the subiects 91
92
P. Rio et al.
gave their written consent and the protocol was approved by the Local Ethics Committee of the Hospital. None of the subjects had taken any medication, including oral contraceptives or non-steroidal anti-infiammatory drugs, for at least 15 days. Also included in the study were six allergic patients, aged from 22 to 35 years. They were suffering from either allergic rhinitis or allergie asthma. However, they were symptom-free at the moment of the study. The clinical diagnosis of allergy was confirmed by skin tests and radioallergosorbent tests. Their IgE concentration was 596 + 317 IU/ml. They did not take any medication at the moment ofthe study.
Isolation of hlood platelets Blood samples were withdrawn between 08.00 and 10.UO a.m. Venous blood (40 ml) was collected without a tourniquet in a syringe containing 5 ml of ACD anticoagulant (citric aeid: I 4 g; sodium: 2 5 g; dextrose: 2 g; in 100 ml of distilled water). A first centrifugation at 200 g for 25 min separated the platelet-rich plasma (PRP) which was then acidified with 3 ml of ACD. A second eentrifugation at 1500 g for 10 min enabled the separation of the platelet-poor plasma (PPP) from the pellet which was then washed three times in phosphate-buffered saline (PBS) at pH 6 5 . and resuspended in Hcpes-KrebsRinger-Glucosc bulTer (HKRG) (Hepes: 25 niM: NaCi: 125 mM; KCI: 4-8 niM; glucose: 5 6 mM; ascorbic acid: 1 niM) to a final concentration of 5 x 10*^ platelets/ml. Platelets, leucocytes and red cells were counted in a counter (S-Plus IV Coulter. Paris). The platelet suspensions used in this study contained less than 1% red cells and 0 03% leucocytes per 5 x 10** platelets.
Chemilumine.scence The chemiluminescencc experiments were carried out with a LKB-Wallac luminometer 1251 with a mixing device and temperature control. The experiments were performed at 37 C. After incubation for 45 min in HKRG, 100 fi\ of luminol (3-aminophthalhydrazide; 200 fiM) were added to the platelet suspension which was then activated with AA. The concentration-dependent effeet was analysed using increasing concentrations of AA ranging from 0 25 to 7 mM. In order to characterize the chemiluminescence response, cycloxygenase and lipoxygenase pathways were inhibited by a previous incubation for 20 min with acetylsalicylie acid and baicalein. respectively.
The effect of immunoglobulins was studied by incubating 400/tl of the platelet suspension for 45 min with 100 /il of HKRG. or with 100 /i\ of IgE solution at various concentrations ranging from 30 to 500 IU/ml, or with 100/il ofthe TgG solution at a concentration of 12 mg/ ml. In addition, we also studied the effect of IgG at a concentration of 9 mg/ml, this concentration being the normal plasma one for that class of immunoglobulin. AA-induced chemiluminescence was then measured during the 3 min following the AA addition. Drugs used Sodium araehidonate (Sigma. St Louis. Missouri, U.S.A.); protein A purified plasma IgG (a gift from V. Jallu. Section de pathologie de hemostase. Pessac, France); polyclonal purified human IgE and baicaiein (5,6,7-trihydroxyfiavone) (Calbiochem, La Jolla, California, U.S.A.); acetylsalicylic acid (Sigma). Statistical analysis Data are given as means ± s.d. and compared between the different series (e.g. IgE. HKRG. IgG) with two-tailed Student's /-tests for unpaired samples. Comparison of concentration-response curves was carried out using a computerized ANOVA procedure. Results AA-induccd chemiluminescence of platelets In normal subjects, addition of AA to a suspension of washed platelets (200 /il) preincubated with luminol (200 /iM) produced a signal that reached a maximum in about 6 sec. The amplitude of this signal was concentration-dependent up to 29 + 6 mV/sec (/? —9) for an A A concentration of 7 niM (Fig. I). In both normal and allergic subjects, addition of AA at a low concentration (i.e. 250 fiwi) to 400 ft\ of the platelet suspension in the presence of luminol led to two luminescence signals. The mean amplitudes ofthe first peak were 1 95 + 061 mV/sec and 2-35±062 mV/sec (NS), and those of the second peak were 0 8 2 + 0 22 mV/sec and 0 78 + 0 26mV/sec(NS). respectively. In the both groups, the maximal amplitude ofthe first peak occurred about 6-9 sec after the addition of AA. This first signal then decreased within 20 sec to the background level. The onset of the second peak occurred at 35 sec. reached a maximum in about 90 sec and returned to background level within 150 sec (Fig. 2). This amplitude of the second signal was 25 + 8 7% that ofthe first peak (« = 9) for the normal subjects and 22 7+11"^) (« = 6) for the allergic subjects (Fig. 3).
IgE and platelet chemilumlnescence
50 r
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3r
40
30
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0 T 0
1
2
3
4
5
6
7
8
AA (nM)
Fig, 1, Concentration-dependent effect of AA on platelet CHL in hciillhy siibjecls. Mean values of CHL (/f = 9) are plotted vs AA conL-cnlrations ranging from 025 lo 7 mM. Vertical bars indicate s.d.
Characterization of the two peaks Pretreatment of platelets with acetylsalicylic acid (ASA) inhibited the first signal of AA-induced luminescence without aitering the second signai (Eig. 2). This efl'ect was concentration-dependent from iO"^to iO ^' M. At iO"''M concentration of ASA, the ampiitudeofthe first peak was not significantly different from that of the background noise. Baicalein reduced the second signal of AA promoted chemilumineseence. Indeed, the second peak was completely abolished by baicalein at 10"^ M. However, the first peak was aiso decreased by a concentration as iow as iO-^Mofbaicalein(Flg. 2).
0
15 30 45 60
75
90 105 120 135 150 165 180
Time ( s ) Kig, 2, AA-induced CHL of platelets vs lime. Open circles represent CHL of platelets from one healthy suhjecl induced by a low concentration of AA (250 /(M). Closed circles and closed squares represent AA-induced CHL of platelets from the same subject after incuhalion with ASA (10 "^ M) and baicalein (10 * M), respectively.
In allergic patients, IgE inhibited AA induced CHL in a concentration-dependent manner. The potency of IgE was similar to that of the normal subjects (i.e. IC 5 0 - 9 5 iU/ml; NS). At low concentrations of AA (i.e. 0 25 mM), no difference was observed between normal and atopic subjects for the IgE dependent inhibition of the two peaks of CHL (Fig. 3). Preincubation with IgG soiution under conditions similar to that with IgE did not aiter the AA-induced ehemilumineseence of piatelets (Fig. 4).
Effect of immunoglobulins Incubation of washed human plateiets from normal subjects with a high concentration of IgE (500 IU/ml) suppressed the signals of luminescence induced by AA ranging from 0 25 to 7 niM (Eigs 4 & 5). IgE aitered the chemilumineseence response in a concentration-dependent manner. Both luminescence signals (i.e. first and second peak) decreased when the concentration of IgE increased (Fig. 5). Regarding the first peak, the concentration of IgE causing a 5Q"A, decrease in amplitude (i.e. IC 50) was equivalent to liO IU/ml (H = 7) (Fig. 6).
Diseussion These results show that AA-stimulation of piatelets from both normai and ailergic subjects induces a concentration-dependent iuminescence response that consists of two components. The first and the second peaks may depend on the cycio.Kygenase and iipoxygenase pathways, respectiveiy. The binding of IgE alone inhibits both peaks in a concentration-dependent manner. The chemilumineseence enables the measurement of reactive oxygen species production such as singlet oxy-
94
P. Rio et al.
NS
1 „
2
T
T
1st peok
Q
2na peak
Normol subjects
1st peak
2nd peak
Allergic subiects
Kig. 3. Mean values of AA-induced peaks of CH L in normal and allergic subjects. White columns represent mean values of the maximal CHL responses to AA at 9 and 90 sec (i.e. first and second peak) in normal subjects (« = 9) and allergic subJeL-ls (« = 6) in the absence of IgE. Vertical bars indicate s.d. Grey columns represent the similar values in the presence oflgE (500 IU/ml).
gen, superoxide and hydroxyl radicals and hydrogen peroxide. These oxygen metabohtes emit light either directly or upon their interaction with luminol [13]. Activation of platelets by AA releases reactive oxygen species that can be recorded by means o[ CHL as previously shown by Worncr et al. [11], Melsa-Ketela et
al. [12] and MiWs ct ai [10]. It has been demonstrated that the luminescence signal in the platelet suspension is not due to leucocyte contamination [11]. At low concentrations, the AA-induced luminescence response consisted of two peaks. This result is in agreement with that of Worner et al. [11] who also described two peaks, the time-course of which was close to that reported in the present study. In contrast. Mills et al. [ 10] described only one peak of CHL induced by AA whereas Metsa-Ketela ct al. [12] found either one or two peaks. It is likely that a higher concentration of AA and a lower concentration of platelets are responsible for the difference in the results of these studies, with respect to the number of peaks observed. The two successive peaks of CHL could be due to the two pathways of AA metabolism in platelets. Indeed, lhe first peak was inhibited after incubating with ASA, a specific inhibitor of cycloxygenase and the second peak was inhibited by baicalein. a specific inhibitor of 12iipoxygenase pathway [14] in platelets [15]. These results confirm those of Mills et al. [10] and Melsa-Ketela et al. [ 12] regarding lhe pharmacological sensitivity of the first peakand those of Worner ('/«/. [I I] regarding that of both peaks. The fact that ASA {as well as indomethacin [11]) reduced in a concentration-dependent manner the amplitude of the first peak strongly suggests that il involves the cycloxygenase pathway. Moreover, Worner demonstrated that incubation of platelets with ASA decreased the first peak and concurrently increased the amplitude of the second peak.
35 -
I
30
25
20
15
NS
10
3-5 AA {mM)
Fig. 4. Eflect of IgE and IgG on phtlelet CHL response to AA at high concentrations in healthy subjects. Columns rcprcsenl mean valties (« = 9) of CHL response lo AA at 3 high concentrations (i.e. 2. 3'5. and 7 mM). White, grey, and black columns represent CHL afler inctibalion with a bufTer, IgG solution or IgE solution, respeclively (*/'
