International Jownal of Cardiology, 37 (1992) 301-307 0 1992 Elsevier Science Publishers B.V. All rights reserved

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Spontaneous superoxide generation by polymorphonuclear leukocytes isolated from patients with stable angina after physical exercise Carlo Guarnieri, Giovanni Melandri, Ilaria Caldarera, Vittorio Cervi, Franc0 Semprini and Angelo Branzi DepartmerIt of Biochemistry, Centre of Research on Cardiac Metabolism and Institute of Cardiology, Unirrr.sity of Bolopu, Bologna. Itab (Received

11 April 1992; revision

accepted

76 May lYY2)

Guarnieri C, Melandri G, Caldarera I, Cervi V, Semprini F, Branzi A. Spontaneous superoxide generation by polymorphonuclear leukocytes isolated from patients with stable angina after physical exercise. Int J Cardiol 1992;37:301-307. The activation of circulating polymorphonuclear leukocytes was determined in terms of 0, generation and elastase release in patients with stable angina (n = 12) and in control subjects (n = 81 after maximal physical exercise and after a 15min recovery. There was no spontaneous 0; formation under basal conditions in both groups of patients. On the contrary, there was significant formation of 0; (p < 0.001) from patients with stable angina measured directly after exercise, along with a slight spontaneous 0; formati,on in control subjects (p < 0.05). After recovery, the spontaneous polymorphonuclear leukocyte-O; formation decreased but was still present in the patients with stable angina, while in the healthy subjects these values returned to resting levels. The activation of polymorphonuclear leukocytes with phorbol 12-myristate 13-acetate enhanced 0, formation both in healthy subjects and in patients with stable angina, with a lesser effect in the latter. Moreover, no differences were observed in polymorphonuclear leukocyte-stimulated 0, formation during the protocol, both in the angina stable patients and healthy subjects. No changes were found in plasma elastase levels among stable angina patients nor in control subjects as a consequence of exercise or recovery. This study indicates there is an early activation of circulating polymorphonuclear leukocytes in terms of 0; production in stable angina patients during maximal exercise, which is still present after a 15min recovery. Such activation occurs without elastase release. However, in healthy subjects maximal exercise resulted in very little increase in neutrophil activation. Key words: Polymorphonuclear

leukocyte; Stable angina; Exercise; Superoxide; Elastase

Correspondence to: Prof. C. Guarnieri, Dept. istry, Via lrnerio 48. 40126 Bologna. Italy.

of Biochem-

This research was supported in part by research grants from CNR and the Minister0 dell’llniversita‘ e della Ricerca Scientifica e Tecnologica. Italy.

302

Introduction Activated polymorphonuclear leukocytes have been shown to have a detrimental role in ischemic myocardial damage because of their cytotoxic production of free oxygen radicals [l], and their release of both lysosomal enzymes [21 and arachidonate metabolites [3]. Activation of polymorphonuclear leukocytes initiates with a sudden increase in 0; radical formation catalyzed by membrane-bound NADPH oxidase which is quiescent in the resting state [4]. This stimulation is triggered by phagocytosable particles (bacteria, viruses, aggregate materials, debris, etc.) and by a number of soluble factors (chemotactic peptides, lectins, Ca2+ ionophores, phorbol esters, cytochalasin, detergents). In the course of myocardial ischemia, the most potent stimuli are probably factors generated through protein cascades such as complement [5] and coagulation pathways [6], or factors released from endothelial or circulating cells [7]. On the other hand, some endogenous factors released in response to ischemic conditions, such as PGI? [8], adenosine [9], and C-reactive protein [lo] are able to inhibit PMN superoxide generation. Therefore, the activation of NADPH oxidase probably occurs when the intensity of the stimulating signals overcomes that of the inhibitors. Some recent reports describe that circulating polymorphonuclear leukocytes can be activated in different cardiovascular pathologies [ 11,121, and they can also be exhausted as in acute myocardial infarction [13]. Until now the exact means of this early activation was not clear. It was also unclear whether activation intensity of polymorphonuclear leukocytes is characteristic of particular cardiovascular disorders. To answer these questions it is necessary to monitor several pathological cardiovascular conditions. In this research we have studied the behavior of circulating polymorphonuclear leukocytes in terms of ability to generate 0; radicals in patients with stable angina undergoing physical exercise. In addition, we have studied the levels of plasma elastase which provide information on

the polymorphonuclear process.

leukocyte

degranulation

Materials and Methods Patient population The study was conducted in 12 patients with stable angina and 8 healthy male volunteers. In the stable angina group, age was 55 f 8 yr; 11 patients were male and a previous myocardial infarction had occurred in 5 patients. Patients with recent myocardial infarction (< 3 months), hypertension (> 170/100 mmHg undergoing treatment), or those with angina due to valvular disease were excluded. Symptoms were present for a minimum of 6 months and were associated with a minimum of 1 mm ST-segment depression measured 80 ms after the J point during treadmill exercise (on average the ST-segment depression was 1.9 + 1.1 mm>. Coronary angiography disclosed single-vessel disease in 3 patients, two-vessel disease in 6 and three-vessel disease in 3. Normal healthy volunteers (age = 48 + 10 yr) did not have a family history of heart disease and were judged normal according to physical examination, two-dimensional echocardiography, and an exercise test. Most patients with stable angina (n = 9) were not taking any drugs and none of the patients or control subjects had smoked. Study protocol After a 12-h fast, all study subjects were sampled for peripheral venous blood collection at 8.00 a.m. (basal sample). At 9.00 a.m. a symptomlimited exercise test was performed (Bruce protocol). Blood samples were obtained before exercising, at the end of the test, and 15 min into the recovery period. A total of four blood samples were collected. Exercise duration was significantly longer in control subjects as compared with patients with stable angina (12.5 f 1.6 vs 6.4 + 3.0 min; p < 0.01). Likewise the peak double product achieved was higher in control individuals (28.6 + 2.8 vs 22.0 * 5.8 x 103; p < 0.01).

303

Separation

of polymorphonuclear

leukocytes

Peripheral venous blood was collected in 10 mM EDTA and immediately layered over MonoPoly Resolving Medium (Flow Laboratories). After centrifugation at 500 X g for 30 min at 24°C the polymorphonuclear leukocyte-rich layer was suspended in Dulbecco buffered salt solution without Ca’+ and MgZc as described previously [13]. The pellet collected after centrifugation at 50 x g for 10 min was treated briefly with 0.8% (w/v) NH,Cl to lyse the red blood cell and then the pelleted polymorphonuclear leukocytes were washed twice in Dulbecco buffer containing 11 mM glucose (buffer A). The final pellet suspended in this medium at a concentration of l- 1.5 x 10’ cells/ml, contained more than 9698% polymorphonuclear leukocytes and their viability, as determined by Trypan Blue exclusion, was more than 96%. Polymorphonuclear generation

leukocyte

zymatic method [15] (Granulocyte Elastase kit from Merck Immuno assay, Darmstadt, Germany). The elastase and elastase-alpha r-proteinase inhibitor complex present in the plasma was bound to the antibodies provided in the kit. Alkaline phosphatase-labeled antibodies specific to the alpha proteinase inhibitor were then added. The hydrolysis of the substrate of the labeled enzyme 4-nitrophenylphosphate was stopped by adding 2 M NaOH and the concentration of polymorphonuclear leukocyte elastase was determined spectrophotometrically from a calibration curve. Statistical

analysis

Values are expressed as mean f SEM. Comparison between groups was made by analysis of variance. The Bonferroni test [16] was used to determine differences between groups. A p value of less than 0.05 was considered significant.

superoxide

Results

Superoxide (01’) was measured by following the superoxide dismutase inhibitable reduction of ferricytochrome C at 550 nm in a double beam spectrophotometer. The assay mixture contained: 0.1 mM ferricytochrome C and 0.5-l x lo6 polymorphonuclear leukocytes suspended in buffer A. The cells were incubated for 3 min at 37°C then the reaction was started by adding 0.1 pg/ml phorbol 12-myristate 13-acetate (PMA). The reference cuvette contained all the components listed above plus JO pg of superoxide dismutase. The release of 0; was calculated from the linear portion of cytochrome C reduction plot, using a molar absorption coefficient of 21.1 x lo3 M-’ cm-‘. The proteins were determined according to Bradford [14], employing bovine serum albumin as a standard. The results are expressed in nanomoles of superoxide/min . mg polymorphonuclear leukocyte protein. Each determination was performed in triplicate.

Peripheral blood polymorphonuclear leukocyte counts in patients with stable angina (n = 12) were 4150 ? 500/mm” (mean &-SEM), in control subjects tn = 8) 3800 k 531/mm3 tp < 0.05). Number of polymorphonuclear leukocytes as percent of total leukocytes in peripheral blood was 51.2 k 3% and 55.5 + 4%, respectively, in each group. The yield of polymorphonuclear leukocytes from peripheral blood in the patients with stable angina and in control subjects was 11.8 x lo6 and 11.2 x lo6 cells/ml, respectively (p = NS). At the peak of maximal exercise, the level of polymorphonuclear leukocytes increased both in stable angina patients and control subjects (5170 I 315/mm” and 5200 k 350/mm3, respectively). The content of polymorphonuclear leukocytes returned to basal values in patients with stable angina at the end of a 15-min post-exercise recovery period (4730 _t 588/mm”). However, in control subjects polymorphonuclear leukocyte counts were higher than basal values after the recovery period (4400 _t 354/mm3; JJ < 0.03).

Assay of elastase

Neutrophil

Plasma levels of polymorphonuclear leukocyte elastase were measured with an immuno-en-

In control subjects no spontaneous 0; radicals were found in polymorphonuclear leukocytes

0,

generation

Control

Subiects

Stable

Angina

Patients

. . a

.

.

t i . 0

L

. Basal

.a+i Basal before exercise

Maximal exercise

0

0 Recovery

Basal

+t

+I Basal before exercise

Maximal exercise

Fig. 1. Spontaneous 0, formation from circulating polymorphonuclear leukocytes in control subjects and in patients angina after physical execise and following recovery. a, p < 0.05 significantly different from basal.

under basal conditions (Fig. 1). Similar results were found in most patients with stable angina, even if some patients in the basal condition before exercise showed a slight spontaneous polymorphonuclear leukocyte Ozl formation. At the time of maximal exercise, polymorphonuclear leukocytes from patients with stable angina exhibited a marked increase in 0; formation which diminished after the recovery period (p < 0.001). Also, in control subjects there was a slight but significant elevation in spontaneous 0; generation after maximal exercise (p < 0.03, which returned to basal levels at the end of the recovery period. Fig. 2 shows the rate of 0; formation by polymorphonuclear leukocytes after stimulation with PMA. There was no significant difference in control subjects during the protocol. Maximally stimulated 0; generation was close to 25 nmol/min * mg prot. Likewise in the patients with stable angina, the rate of 0; formation in the polymorphonuclear leukocytes stimulated with PMA was similar at all time periods and was about 16 nmol/min mg prot. However, this mean value is significantly lower than the control subjects ( p < 0.001). isolated

;

a

Recovery

with stable

Plasma elastase level The levels of plasma elastase did not change in response to physical exercise and recovery in either patients with stable angina or control subjects (Table 1). Moreover, no significant differences in plasma elastase levels were found between patients with stable angina and control subjects at all times considered. Discussion This study confirms previous research that describes polymorphonuclear leukocytes activation as an early event involved in the pathogenesis of myocardial ischemia [12,17-201. We show that in patients with documented stable angina, circulating polymorphonuclear leukocytes isolated in concomitance with the maximal physical exercise able to induce ST-segment depression have increased spontaneous 02 formation. Moreover, in the same patients polymorphonuclear leukocytes are still active at the end of the 15-min recovery, when ST-segment depression had nor-

305 TABLE

cytes more in patients with stable angina than in control subjects. In this regard, according to animal experiments [21], it may be suggested that the drop of poststenotic arterial pressure in the ischemic myocardium increases the intravascular cardiac pool of polymorphonuclear leukocytes in patients with stable angina during exercise-induced ischemia. Moreover, because of slower passage through the microvascular network, exposure of polymorphonuclear leukocytes to factors released by ischemia may in turn activate polymorphonuclear leukocytes [5,22-241. At the end of exercise, when perfusion tends to normalize, part of the increased intravascular pool of activated polymorphonuclear leukocytes may be washed out, and found in peripheral blood because of recirculation.

1

Plasma elastase levels in patients with stable angina (n = 12) and control subjects (n = 8) after physical exercise and following recovery. Groups

Control

subjects

Stable angina

(ILg/l) Basal Basal before exercise Maximal exercise Recovery

76.Jk

The results

are expressed

patients

(Kg/l) 1.8

62.3 + 5.3

60.2 f 18.1

61.1*8.8

69.3+ 65.5+

60.2 f 9.4 55.J+ 9.5

8.51 9.5 as mean+

SEM.

malized. These results clearly indicate that stimuli released in consequence of the stress exercise activate circulating polymorphonuclear leukoControl

30

Stable

Subjects

_

.

I 25

* 2 P

F ._

20

15

_

t + : .

. i

: . t : .

t

.

.

_

t 8 . t : . . .

_

E \ .I N O

10

Patients

Angina

. .: . . . t ..

8 . . .

t i

0

t :

: .

.

.

_

. .

5

E I

c 5

0

t Basal

Basal before exercise

Maximal exercise

Recover

v

Basal

Basal before exercise

Fig. 2. Oi formation from circulating polymorphonuclear leukocytes stimulated with phorbol patients with stable angina after physical exercise and following recovery. The values in stable different (p < 0.05) from control subjects.

Maximal exercise

Recovery

ester in control angina patients

subjects and in are significantly

306

Therefore, we suggest that some of the compounds originating from the vasculature or platelets in response to exercise-induced ischemia [71 could elicit the spontaneous polymorphonuclear leukocyte-O; formation, or induce a priming effect on polymorphonuclear leukocyte activation. In this regard, it should be noted that proteolytic enzymes, ionophores, and lipopolysaccharides are able to prime isolated. polymorphonuclear leukocytes to produce 0; radicals [25] by enhancing the intracellular Ca2+ movements, even in the presence of very low levels of stimuli. However, it is noteworthy that the eventual stimuli should be present after isolation of polymorphonuclear leukocytes since the interaction between ligand and receptor is required for maintaining NADPH oxidase in the activated state [4]. We exclude that the spontaneous activation of polymorphonuclear leukocytes occurs as a consequence of the cell isolation, because we used a procedure that did not employ dextran which could be a potential activator. Moreover, we measured 0,’ formation in the absence of possible interferences because the rate of superoxide generated was measured in the initial 3 min rather than after a prolonged period of incubation. It is also possible that in the patients with stable angina the modifications in activation of polymorphonuclear leukocytes following physical exercise and recovery are in part caused by the presence of a plasmatic pool of more functionally competent polymorphonuclear leukocytes, since their number increases in the circulation in response to exercise and then decreases at the end of recovery. In fact, this study confirms that physical exercise is able to increase the number of circulating leukocytes [26] and to cause leukocyte activation [27]. Moreover, according to Schaefer’s study we were unable to find an increased plasma level of elastase after short-time exercise, probably because the 0, formation by polymorphonuclear leukocytes is an earlier activating event preceding the phase of degranulation [4]. Interestingly, in the patients with stable angina the stimulation of polymorphonuclear leukocytes with the potent NADPH-oxidase activator phorbol ester leads to a lower 0; generation in

comparison to the control subjects. These results suggest that polymorphonuclear leukocytes in the patients with stable angina, already in the basal condition, are in a condition of partial “exhaustion” or “desensitization” and the biochemical machinery leading to 0,: generation is unable to reach its maximal activation. However, this situation does not prevent patients with stable angina from spontaneously releasing 0; radicals by polymorphonuclear leukocytes. The increase in the function of polymorphonuclear leukocytes documented in this research in terms of spontaneous 0; formation in the patients with stable angina may be one of the earliests events able to influence the complex interactions among endothelial cells, platelets, and leukocytes, all maintaining a hemodynamic equilibrium and appropriate vascular tone. In fact, Dinerman and Mehta [7] clearly described in their review that a flux of 0; causes vascular smooth muscle contraction, in vivo thrombosis and a reduction in blood flow. Again, the Oi radicals can produce lipid peroxides that increase membrane permeability with loss of membrane function 1281.We suggest that polymorphonuclear leukocyte activation through the generation of 0; may account for the still unexplained alterations occurring in patients with stable angina undergoing physical exercise. Further studies are necessary to clarify if the early activation of polymorphonuclear leukocytes induced by stressed exercise has detrimental implications in the clinical evaluation of angina patients. Acknowledgements The authors thank Daniela Donadio and Giovanna Grandi for their assistance in the preparation of this manuscript and Dr. Jane Davis for revising the manuscript. References Jolly SR, Kane WJ, Bailie MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-285.

307 2 Mullane KM, Read N, Salmon JA, Moncada S. Role of leukocytes in acute myocardial infarction in anesthetized dogs: relationship to myocardial salvage by anti-inflammatory drugs. J Pharmacol Exp Ther 1984;228:510-522. 3 Zurier RB, Weissmann G, Hoffstein S, Kammerman S, Tai HH. Mechanisms of lysosomal enzyme release from human leukocytes. J Clin Invest 1974;53:297-309. 4 Rossi F. The 0; forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 1986;853:65-89. 5 Crawford MH, Grover FL, Kolb WP et al. Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury. Circulation 1988;78:1449-1458. 6 Wactfogel YT, Pixley RA, Kucich U et al. Purified plasma factor XII a aggregates human neutrophils and causes of degranulation. Blood 1986;67:1731-1737. 7 Dinerman JL. Mehta JL. Endothelial, platelet and leukocyte interactions in ischemic heart disease: insights into potential mechanisms and their clinical relevance. J Am Coil Cardiol 1990;16:207-222. 8 Simpson PJ, Mitsos SE, Ventura A et al. Prostacycline protects ischemic reperfused myocardium in the dog by inhibition of neutrophil activation. Am Heart J 1987;113: 129-137. 9 Cronstein BN, Kramer SB. Weissman G, Hirsch-Horn R. Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J Exp Med 1983;158: 1160-l 177. 10 Tatsumi N, Ashimoto K, Okuda K, Kyougoku T. Neutrophil chemiluminescence induced by platelet activating factor and suppressed by C-reactive protein, Clin Chim Acta 1988;171:85-92. 11 Mehta J, Dinerman J, Metha P, Saldeen TGP, Lawson D, Donnelly WH, Wallin R. Neutrophil function in ischemic heart disease. Circulation 1989:79:549-556. 12 Berliner S, Sclarovsky S, Lavie G, Pinkhas J, Aronson M, Agmon J. The ieukergy test in patients with ischemic heart disease. Am Heart J 1986;111:19-22. 13 Guarnieri C, Melandri G, Caldarera I et al. Reduced oxidative activity of circulating neutrophils patients after myocardial infarction. Cell Biochem Funct 1990;8:157-162. 14 Bradford MA. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 2482.54.

1.5 Neumann S, Gunzer G, Henrich M. Lang H. PMN elastase assay: enzyme immunoassay for human polymorphonuclear elastase complexed with alfa-1-proteinase inhibitor. J Clin Chem Clin Biochem 1984;22:693-697. 16 Wallestein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res 1980;47:1-9. 17 Ciuffetti Cl, Bellomo G, Mercuri M, Lombardini R, Savino K, Corea L. Leucocyte rheology in controlled coronary ischaemia. Int J Cardiol 1989;25:193-198. 18 Dinerman JL, Mehta JL, Saldeen TGP et al. Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction. J Am Coil Cardiol 199fk15: 1559-1563. 19 Radomski M, Herbaczynskacedro K. Ceremuzynski L. Increased activity of circulating PMN in acute myocardial infarction. Int J Cardiol 1990;27:392-398. 20 Bell D, Jackson M. Nicoll JJ, Millar A, Dawes J, Muir AL, Inflammatory response, neutrophil activation, and free radical production after acute myocardial infarction: effect of thrombolytic treatment. Br Heart J 1990;63:82-87. 21 Ley K, Meyer JU, Intagliette M, Arfors KE. Shunting of leukocytes in the rabbit tenuissimus muscle. Am J Physiol 1989;256:H85-H93. 22 Hill JH, Waer PA. The phlogistic role of C, leukotactic fragments in myocardial infarctions in rats. J Exp Med 1971;133:885-9~. 23 Rossen RD, Swain JP, Michael LM, Weakly S, Giannini E, Entman ML. Selective accumulation of the first component of complement and leukocytes in ischemic canine heart muscle: a possible initiator of an extra myocardial mechanism of ischemic injuries. Circ Res 1985:57:119-130. 24 Petrone WF, English DK, Wong K, MC Cord JM. Free radicals and inflammation: superoxide dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 1980;77:1159-1163. 25 Finkel TH, Pabst MJ, Suzuki H et al. Priming of neutrophils and macrophages for enhanced release of superoxide anion by the calcium ionophore ionomycin. J Biol Chem 198~262:12589-12596. 26 Schaefer RM, Kokot K, Meidland A, Plass R. Jogger-s leukocytes. N Engl J Med 1987;316:223-224. 27 Cannon JG, Kluger MJ. Endogenous pyrogen activity in human plasma after exercise. Science 1983;220:617-619. 28 Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-376.

Spontaneous superoxide generation by polymorphonuclear leukocytes isolated from patients with stable angina after physical exercise.

The activation of circulating polymorphonuclear leukocytes was determined in terms of O2.- generation and elastase release in patients with stable ang...
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