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Increased activity of circulating polymorphonuclear leukocytes in acute myocardial infarction
392 International
Journal of Cardiology, 21 (1990) 392-393
Elsevier CARD10 10776
Increased activity of circulating polymorphonuclear in acute myocardial infarction M. Radomski *, K. Herbaczyhska-Cedro
‘v2and L. Ceremtiyiuki
leukocytes *
’ Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; ’ II Cardiac Department, Postgraduate School, Warsaw, Poland
(Received 18 October 1989; revision accepted 31 January 1990)
Acute myocardial infarction results in an increased sensitivity of circulating polymorphonuclear leukocytes to ex vivo aggregation. This increase was prevented by pretreatment of leukocytes with BW755, but not with aspirin, suggesting that the activation of blood leukocytes in infarction is due to a stimulation of cellular lipoxygenase.
Key words: Leukocyte aggregation; Lipoxygenase;
Acute myocardial infarction
Introduction Animal and human studies have shown the invading leukocytes to be involved in the pathogenesis of infarction [I]. Epidemiological observations suggest that the number of circulating peripheral leukocytes is a strong predictor of risk of infarction and stroke [2]. The func: tional status of blood leukocytes of patients with infarction, however, has not been clearly defined. The process of activation in vivo involves the adhesion of leukocytes to vascular endothelium and aggregation comparable with the formation of platelet thrombi. By monitoring the aggregation of leukocytes ex vivo as an index of their activity, we have examined the activity of these cells in patients with infarction (1-3 days). Materials and Methods The study was approved by the local ethical committee. Samples of peripheral venous blood (18 ml) were
Correspondence to: M. Radomski, Dept. of Pharmacology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. * On leave of absence at Wellcome Research Laboratories.
0167-5273/90/$03.50
obtained from patients (7 males and 5 females, age 48-64 years) with clinically and biochemically proven infarction. The control group consisted of healthy volunteers (5 males and 4 females, age 35-54 years) recruited from hospital and laboratory staff. A suspension of cells (> 95% viable leukocytes) was prepared from the blood by dextran sedimentation, differential centrifugation and hypotonic lysis. Leukocytes (2 x 106/ml) were preincubated for 3 minutes in a light aggregometer (Payton Ass.) in the presence or absence of aspirin (l-100 PM) or BW755 (0.1-10 PM, courtesy of Dr. S. Moncada, Wellcome Research Laboratories). After 3 minutes, aggregation was induced by addition of N-formyl-methionyl-leucyl-phenylalanine (N-formyl-Met-Leu-Phe, Sigma 0.03-10 PM) or arachidonic acid (Sigma, 0.1-100 PM) and the reaction was monitored for the next 10 minutes. The proaggregating activity of N-formyl-Met-Leu-Phe or arachidonic acid was expressed as a percentage of maximal transmission of light and was taken as an index of leukocytic activity. Results are the mean (plus or minus the standard error) calculated from 9 (control) and 12 (patients) separate experiments. Student’s t-test for unpaired data was used to assess the significance of any differences between groups.
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
393 10
A nz9-12
T
I
6
7 -Log
EMLPI]
T
1
5
6
5
Log
M
[AA]
4
M
Fig. 1. The sensitivity of human leukocytes to N-formyl-Met-Leu-Phe (FMLP, A) or arachidonic acid (AA, B)-induced aggregation by pretreatment 0). This sensitivity is decreased to the control range (0 ----0) increased in patients with infarction (0 0) but not with aspirin (100 PM, v -v). cells with BW755 (10 PM, q -
Results The incubation of control leukocytes with N-formylMet-Leu-Phe resulted concentration-dependent increase in transmission of light (Fig. 1A). A significant (P < 0.001) increase in sensitivity to this peptide was observed in leukocytes from patients with infarction. Aspirin ( < 100 PM) failed to affect (P > 0.05) the aggregation of leukocytes. In contrast, preincubation of these cells with BW755 reduced their sensitivity so that it was comparable with the control range (P > 0.05) (Fig. 1A). A similar pattern of aggregation and reactivity to aspirin and BW755 was observed with arachidonic acid (Fig. 1B). Neither compound affected aggregation of control leukocytes (P > 0.05; data not shown). Discussion These results clearly demonstrate that blood leukocytes are activated in patients with infarction. This activation does not seem to be dependent upon the formation of cyclooxygenase products, since aspirin failed to affect the aggregating properties of leukocytes. In contrast, a dual inhibitor of cyclooxygenase and lipoxygenase, BW755C [3] decreased the sensitivity of leukocytes from patients to aggregating agonists indicating a possible lipoxygenase activation in these cells. The precise mechanism of lipoxygenase activation in leukocytes from patients with infarction remains to be elucidated. Pontremoli et al. [4] found that blood leukocytes from patients with essential hypertension were activated
is of
and capable of producing enhanced amounts of superoxide anion via an NADPH oxidase-dependent pathway. If such an alteration in function of this enzyme existed in diseased leukocytes, it would provide free radicals necessary for activation of lipoxygenase. Our findings support the hypothesis that circulating leukocytes play an important role in the pathogenesis of infarction [5]. Studies are under way to investigate a correlation between aggregation of blood leukocytes and the course of uncomplicated and complicated infarction, with particular emphasis on the therapy and outcome of this disease. References Mehta JL, Nichols W, Mehta P. Neutrophils as potential participants in AMI. Relevance to reperfusion. J Am Co11 Cardiol 1988;11:1309-1316. Ernst E. Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA. Leukocytes and the risk of ischaemic diseases. J Am Med Assoc 1987;257:2318-2324. Higgs GA, Flower RJ, Vane JR. A new approach to anti-inflammatory drugs. Biochem Pharmacol 1979;28:1959-1961. PontremoIi S, Salamino F, Sparatore B, et al. Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients. Biochem Biophys Res Commun 1989;158:966-972. MuIIane K, Read N, Salmon JA, Moncada S. Role of leukocytes in acute myocardial infarction in anaesthetised dogs. Relationship to myocardial salvage by anti-inflammatory drugs. J Pharmacol Exp Ther 1984;228:510-522.
Journal of Cardiology, 21 (1990) 392-393
Elsevier CARD10 10776
Increased activity of circulating polymorphonuclear in acute myocardial infarction M. Radomski *, K. Herbaczyhska-Cedro
‘v2and L. Ceremtiyiuki
leukocytes *
’ Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland; ’ II Cardiac Department, Postgraduate School, Warsaw, Poland
(Received 18 October 1989; revision accepted 31 January 1990)
Acute myocardial infarction results in an increased sensitivity of circulating polymorphonuclear leukocytes to ex vivo aggregation. This increase was prevented by pretreatment of leukocytes with BW755, but not with aspirin, suggesting that the activation of blood leukocytes in infarction is due to a stimulation of cellular lipoxygenase.
Key words: Leukocyte aggregation; Lipoxygenase;
Acute myocardial infarction
Introduction Animal and human studies have shown the invading leukocytes to be involved in the pathogenesis of infarction [I]. Epidemiological observations suggest that the number of circulating peripheral leukocytes is a strong predictor of risk of infarction and stroke [2]. The func: tional status of blood leukocytes of patients with infarction, however, has not been clearly defined. The process of activation in vivo involves the adhesion of leukocytes to vascular endothelium and aggregation comparable with the formation of platelet thrombi. By monitoring the aggregation of leukocytes ex vivo as an index of their activity, we have examined the activity of these cells in patients with infarction (1-3 days). Materials and Methods The study was approved by the local ethical committee. Samples of peripheral venous blood (18 ml) were
Correspondence to: M. Radomski, Dept. of Pharmacology, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K. * On leave of absence at Wellcome Research Laboratories.
0167-5273/90/$03.50
obtained from patients (7 males and 5 females, age 48-64 years) with clinically and biochemically proven infarction. The control group consisted of healthy volunteers (5 males and 4 females, age 35-54 years) recruited from hospital and laboratory staff. A suspension of cells (> 95% viable leukocytes) was prepared from the blood by dextran sedimentation, differential centrifugation and hypotonic lysis. Leukocytes (2 x 106/ml) were preincubated for 3 minutes in a light aggregometer (Payton Ass.) in the presence or absence of aspirin (l-100 PM) or BW755 (0.1-10 PM, courtesy of Dr. S. Moncada, Wellcome Research Laboratories). After 3 minutes, aggregation was induced by addition of N-formyl-methionyl-leucyl-phenylalanine (N-formyl-Met-Leu-Phe, Sigma 0.03-10 PM) or arachidonic acid (Sigma, 0.1-100 PM) and the reaction was monitored for the next 10 minutes. The proaggregating activity of N-formyl-Met-Leu-Phe or arachidonic acid was expressed as a percentage of maximal transmission of light and was taken as an index of leukocytic activity. Results are the mean (plus or minus the standard error) calculated from 9 (control) and 12 (patients) separate experiments. Student’s t-test for unpaired data was used to assess the significance of any differences between groups.
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
393 10
A nz9-12
T
I
6
7 -Log
EMLPI]
T
1
5
6
5
Log
M
[AA]
4
M
Fig. 1. The sensitivity of human leukocytes to N-formyl-Met-Leu-Phe (FMLP, A) or arachidonic acid (AA, B)-induced aggregation by pretreatment 0). This sensitivity is decreased to the control range (0 ----0) increased in patients with infarction (0 0) but not with aspirin (100 PM, v -v). cells with BW755 (10 PM, q -
Results The incubation of control leukocytes with N-formylMet-Leu-Phe resulted concentration-dependent increase in transmission of light (Fig. 1A). A significant (P < 0.001) increase in sensitivity to this peptide was observed in leukocytes from patients with infarction. Aspirin ( < 100 PM) failed to affect (P > 0.05) the aggregation of leukocytes. In contrast, preincubation of these cells with BW755 reduced their sensitivity so that it was comparable with the control range (P > 0.05) (Fig. 1A). A similar pattern of aggregation and reactivity to aspirin and BW755 was observed with arachidonic acid (Fig. 1B). Neither compound affected aggregation of control leukocytes (P > 0.05; data not shown). Discussion These results clearly demonstrate that blood leukocytes are activated in patients with infarction. This activation does not seem to be dependent upon the formation of cyclooxygenase products, since aspirin failed to affect the aggregating properties of leukocytes. In contrast, a dual inhibitor of cyclooxygenase and lipoxygenase, BW755C [3] decreased the sensitivity of leukocytes from patients to aggregating agonists indicating a possible lipoxygenase activation in these cells. The precise mechanism of lipoxygenase activation in leukocytes from patients with infarction remains to be elucidated. Pontremoli et al. [4] found that blood leukocytes from patients with essential hypertension were activated
is of
and capable of producing enhanced amounts of superoxide anion via an NADPH oxidase-dependent pathway. If such an alteration in function of this enzyme existed in diseased leukocytes, it would provide free radicals necessary for activation of lipoxygenase. Our findings support the hypothesis that circulating leukocytes play an important role in the pathogenesis of infarction [5]. Studies are under way to investigate a correlation between aggregation of blood leukocytes and the course of uncomplicated and complicated infarction, with particular emphasis on the therapy and outcome of this disease. References Mehta JL, Nichols W, Mehta P. Neutrophils as potential participants in AMI. Relevance to reperfusion. J Am Co11 Cardiol 1988;11:1309-1316. Ernst E. Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA. Leukocytes and the risk of ischaemic diseases. J Am Med Assoc 1987;257:2318-2324. Higgs GA, Flower RJ, Vane JR. A new approach to anti-inflammatory drugs. Biochem Pharmacol 1979;28:1959-1961. PontremoIi S, Salamino F, Sparatore B, et al. Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients. Biochem Biophys Res Commun 1989;158:966-972. MuIIane K, Read N, Salmon JA, Moncada S. Role of leukocytes in acute myocardial infarction in anaesthetised dogs. Relationship to myocardial salvage by anti-inflammatory drugs. J Pharmacol Exp Ther 1984;228:510-522.