THROMBOSIS RESEARCH 59; 669-674,199O 00493648/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
BRIEF
COMMUNICATION
~DU~ONINHuMANNEuTRopHlLcHEMoTAxIsBYTHEpR~AcYcLIN ANAmGUElLOPROsT
F. A. Nicolini, P. Mehta, D. Lawson, J.L. Mehta Departments of Medicine and Pediatrics, University of Florida College of Medicine and the Veterans Affairs Medical Center, Gainesville, FL 32608-l 197 USA
(Received
23.4.1990;
accepted
in revised form 6.6.1990
by Editor N.U. Bang)
ODUCTION Platelets, endothelial cells and neutrophils interact together in the maintenance of vascular tone, the development of thrombosis and the induction of’reperfusion-related tissue injury (l-3). Platelet activation is a key initial event in the formation of an occlusive thrombus in an atherosclerotic vessel with damaged or dysfunctional endothelium (4). Neutrophils perpetuate the ischemic injury by release of superoxide radicals, arachidonate metabolites and proteolytic enzymes (4). Prostacyclin has been shown to exert an inhibitory effect on platelet and neutrophil functions (5,6), and these properties may relate to its beneficial effects (7). In this study, we describe the effects of a stable prostacyclin analogue iloprost on function of neutrophils taken from healthy human volunteers. ODS p Peripheral venous blood was obtained from 10 normal subjects in heparin (10 units/ml), layered over Mono-Poly Resolving Medium (Flow Labs, Maclean, VA) and centrifuged at 300g for 30 minutes at 24’ C. Red blood cells in the neutrophil-rich layer were removed by hypotonic lysis. Subsequent cell preparation contained greater than 95% neutrophils. Platelets were isolated by centrifugation of blood collected in 3.8% sodium citrate at 800g for 15 minutes at 4’ C as described previously (8). Neutrophils were incubated with washed platelets (WP) in a ratio of 1:lO in Hank’s Balanced Salt Solution (I-IBSS) with Ca++ and Mg++ for 10 minutes at 37” C. As controls, neutmphils alone and platelets alone were also incubated in I-IBSS. Oxygen Neutrophil-derived free oxygen radical production was determined by luminol-dependent chemiluminescence using formyl-methionyl-leucylphenylalanine (f-MLP) lo-7M as the stimulus as described previously (8). Briefly, neutrophils (with or without WP) suspended at a concentration of 1 x 106 cells/ml in HBSS were incubated with f-MLP for 10 minutes at 37’ C. Iloprost in different concentrations (0,O. 1, 1.0, and 10 @I) was added to the suspension. Luminol (initial concentration 5 pM) was added to amplify chemiluminescence. f-MLP (10 pM) was added and chemiluminescence measured for 30 seconds from time zero and then every 10 minutes for 2 hours in a liquid scintillation counter. Counts per minute over time were plotted on a semilog paper and the area under the curve calculated as an arbitrary index of free oxygen radical release by f-MLP-stimulated neutrophils. Key words: Chemotaxis, Iloprost, Neutrophils,6~~tacyclin
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Chemotaxis: Directed neutrophil chemotaxis in response to f-MLP was measured in a chemotaxis chamber (8). The number of neutrophils in the upper chamber was kept constant at 1 x 105 cells/ml while concentration of iloprost was varied from 0, 0.1, 1.0, and 10 PM. The lower chamber contained f-MLP (0, 10q9, 10e8, and lo-TM). A polycarbonate filter (5.0 p.M pore) separated the upper and lower compartments, which were incubated in humidified air with 5% Co;! for 60 minutes at 37’ C. In each experiment, all measurements were made in triplicate and the number of cells were counted in five separate high-power fields (x 400) at each concentration of f-MLP. The data were averaged and expressed as cells per 5 high-power fields. Neutronhil LTl34 generation: To determine LTB4 generation, neutrophils (1 x 107 cells/ml) were incubated with calcium ionophore A 23187 (10 PM) at 37’ C for 15 minutes in a shaking water bath. Iloprost was added in increasing concentrations (0, 0.1, 1.0, and 10 PM). The reaction was terminated by placing the tubes in ice for 5 minutes and subsequently adding cold methanol (0.5 ml). Suspensions were centrifuged at 25,000g for 5 minutes at 4’ C and the supematants analyzed for LTB4 by radioimmunossay (RIA) in duplicate using supplies from New England Nuclear, Boston, MA. Our previous studies (9) have shown that a specific 5-lipoxygenase inhibitor, piriprost (10 PM), decreases neutrophil LTB4 formation by over 70% as determined by this RIA. Furthermore, the values obtained from RIA correlated (r-0.91) with those obtained from HPLC. Statistics: The data from multiple observations were averaged and expressed as a mean f standard deviation. Data were analyzed by analysis of variance and Student’s t-test for paired observations, as appropriate. A p value of less than 0.05 was considered significant. RESULTS Neutronhi free oxygen radical production; Iloprost at all concentrations exerted a slight inhibitory effect on neutrophil free oxygen radical production as indicated by a change in chemiluminescence. However, there was no dose-dependent effect (Table 1). The presence of WP increased the chemiluminescence (177.3 + 37.7 vs 153.8 f 69.2 cm2) as noted previously (8). Addition of iloprost had only a very minimal effect on the free oxygen radical produced by neutrophils in the presence of WP (Table 1). TABLE 1: Effect of Iloprost on Neutrophil Chemiluminescence Chemiluminescence Area (cm2) Neutrophils (1 x 1061ml) alone + Iloprost 0.1 m + Iloprost 1.0 pM + Iloprost 10 PM
153.8
Neutrophils (1 x lO%nl) + WP (1 x 107/ml) + Iloprost 0.1 PM + Iloprost 1.0 ~_LM + Iloprost 10 PM Data from 5 experiments expressed as mean f SD; WP -
136.3 rt 58.1 134.8 + 36.5 135.0 f 65.9 177.3 f37.7 176.8 f 67.0 170.0 f 62.0 169.7 + 57.6 washed platelets
Chemotaxis: In all experiments, migration of neutrophils was markedly increased as the concentration of f-MLP was increased from 10-g to 10-T M (Table 2). The presence of platelets enhanced neutrophil chemotaxis, as described previously (8). Although iloprost decreased neutrophil chemotaxis (spontaneous as well as f-MLP stimulated) in the neutrophil suspension (with or without WP), the reduction was significant at the 10 pM concentration. Furthermore, the
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degree of inhibition of neutrophil chemotaxis was similar in the neutrophil suspension with or without platelets. TABLE 2: Effect of Iloprost on f-MLP-Induced Neutrophil Chemotaxis
0 Neutrophils + Iloprost + Iloprost + Iloprost
alone 0.1 pM 1.0 pM 10 pM
244 f 53 222f60 194 * 55 167 + 47*
Neutrophils (per 5 high-power fields) f-MLP concentration (M) 10-9 10-a 10-7 294 + 63 272 -t 62 247 f 61 221 f 57*
362 + 56 330 * 49 307 + 49 282 f 46*
480 432 412 378
f f f +
7.5 76 81 66*
Neutrophils + WP 268 f 44 345 f 74 431 f 83 508 * 73 + Iloprost 0.1 pM 244+36 315 f68 394 * 79 470 f74 + Iloprost 1.0 pM 218 + 32 286 f 70 374 f 73 449 f 76 198+34** ~257f56** + Iloprost 10 pM 325 f 107** 411+64** Data expressed as mean f SD; concentration of neutrophils and WP are the same as in Table 1. * p < 0.02 vs neutrophils alone ** p c 0.05 vs neutrophils + WP Neutrouhil LTB3 generation: LTB4 generation by neutrophils ranged from 17.6 to 24.6 q/107 cells, and was unaffected by iloprost. Addition of platelets to neutrophils increased LTB4 generation, but iloprost did not affect the enhanced LTB4 generation (Table 3). TABLE 3: Effect of Iloprost in Neutrophil LTB4 Generation
Neutrophils (107/ml) + Iloprost 0.1 pM + Iloprost 1.0 pM + Iloprost 10 pM Neutrophils (107/ml) + WP (107/ml) + Iloprost 0.1 pM + Iloprost 1.0 pM + Iloprost 10 pM Data from 5 experiments expressed as mean f SD
LTB4 (ng/107 cells) 21.1 f 6.9 29.7 + 7.2 34.2 f 6.4 33.6 + 12.7 30.7 f 1.5 22.5 f 4.9 22.5 f 4.9 27.8 rt 2.5
DISCUSSION Previous studies (10) showed that iloprost reduces myocardial infarct size in dogs subjected to total coronary occulsion for 90 minutes and reperfusion for 6 hours. In these studies, myocardial myeloperoxidase activity, an index of neutrophil accumulation, was reduced at the interface between the infarcted and the noninfarcted region. The authors postulated that the protective effect of iloprost was related to its potential to decrease migration of neutrophils in response to ischemic myocardial injury. The authors also demonstrated an iloprost-induced decrease in canine neutrophil superoxide radical generation in response to opsonized zymosan. It is also possible that, besides the inhibitory effect on platelets, iloprost contributed to myocardial protection by its neutrophil-inhibitory effects. Iloprost was also found to be beneficial in the treatment of patients with critical limb ischemia by virtue of its modulating effects on platelet-neutrophil-endothelial cells interactions (11). However, iloprost was not very effective in patients with stable angina pectoris and severe coronary artery disease in whom &hernia was provoked by exercise testing and anial pacing (12).
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That iloprost was not efficacious in treating this condition is probably related to the fact that neutrophils as well as platelets are not activated in stable angina, unlike in acute mocardial ischemic syndromes (e.g. unstable angina and acute myocardial infarction) when neutrophil activity as measured by their chemotactic response, elastase release, superoxide radical production and LTB4 generation is markedly enhanced (13). Platelet activity is also enhanced in acute myocardial ischemia (14). Because iloprost may be clinically useful, we examined the effects of iloprost on human neutrophil functions in vitro. Our observations indicate that iloprost decreases the neutrophil chemotactic response to f-MLP, while free oxygen radical generation is only slightly decreased. Neutrophil LTB4 generation was not affected by iloprost. A decrease in directed chemotaxis is consistent with the observations of Simpson et al (10) in dogs and also lends support to the results of clinical trials of iloprost in critical limb ischemia (11). PGI2 also decreases neutrophil chemotactic activity (7). Reduction in neutrophil chemotaxis could be a rationale for the potential use of iloprost in acute ischemic syndromes when complement fragments appear early in injured tissues (14). However, the inhibitory effects of iloprost were observed in our experiments in high concentrations. A possible explanation for the apparent discrepancy between our in vitro results and the in vivo studies wherein the inhibition of neutrophil accumulation is seen with therapeutic use of iloprost may be that other prostaglandins, such as PGEl and PGI2, which are released during ischemic injury, may exert a synergistic effect with iloprost in vivo. Furthermore, a reduction in afterload with iloprost may also contribute to its tissue-protective effects (10). Neutrophils when activated produce different free oxygen radical species, such as superoxide anion and hydrogen peroxide. Chemiluminescence measures total oxidative burst but cannot discriminate between superoxide anions and hydrogen peroxide (15). It is noteworthy that the method we used to quantitate oxidative burst employs measurements of the area under the curve over 2h and the vertical axis is plotted (in counts per minute) in log scale (8, 9). As such, only major reductions in chemiluminescence are identified. Furthermore, superoxide radical release occurs within the fiit few seconds, whereas most of the remaining chemiluminescence is due to the release of hydrogen peroxide. It is likely that the measurement of early superoxide radical release was missed in our experiments, and iloprost does not affect hydrogen peroxide release. Nonetheless, superoxide radical generation measured by reduction of ferricyochrome C is reduced by iloprost in very high concentrations (lo), which are unlikely to be achieved in vivo. Lastly; arachidonate metabolism in neutrophils does not seem to be decreased by iloprost in concentrations up to 10 @VI. Thus the major inhibitory effect of iloprost appears to be limited to a reduction in neutrophil chemotaxis. Iloprost is a very potent inhibitor of platelet aggregation, with an IQ0 of 0.1 to 0.6 nM. In previous studies platelets were shown to enhance neutrophil activity (8), and we hypothesized that iloprost may exert a more pronounced inhibition of neutrophil function in the presence of platelets. In this study we demonstrated potentiation of neutrophil activity in the presence of platelets. However, iloprost affected neutrophil function by a similar magnitude whether platelets were present or not. This indicates that the effects of iloprost on neutrophil activity are independent of its effects on platelets, In other studies (16), a selective TxA2 synthetase inhibitor was shown to decrease neutrophil function only in the presence of platelets by redirecting cyclic endoperoxides towards PGI2 formation in the neutrophils. Obviously, this mechanism would not be operative in the present experiments. In summary, this study shows that iloprost exerts inhibitory effects on neutrophil chemotaxis without significantly affecting their oxidative burst. Reduction in neutrophil migration into the ischemic area may result in protection from tissue injury induced by subsequent neutrophil activation.
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The authors thank Ms. Janet Wootten for editorial assistance. This study was supported by a Department of Veterans Affairs Merit Review Award and a grant-in-aid from the American Heart Association, Florida Affiliate. JLM is a Clinical Investigator of the Department of Veterans Affairs, Central Office. _S R 1. VANHOUTTE, P.M., and HOUSTON, D.S. Circulation 72,728-734, 1985.
Platelets, endothelium and vasospasm.
2.
MEHTA,J.L., NICHOLS,W.W., and MEHTA,P. Neutrophils as potential participants in acute myocardial &hernia: Relevance to reperfusion. J.A.C.C. 11, 1309-1316, 1988.
3.
NICHOLS,W.W., MEHTA,J.L., DONNELLY,W.H., LAWSON,D., THOMPSON,L., and TERRET, M. Reduction in coronary vasodilator reserve following coronary occlusion and reperfusion in anesthetized dog: role of endothelium-derived relaxing factor, myocardial neuuophil infiltration and prostaglandins. J. Mol. Cell. Cardiol. 20,943-954, 1988.
4.
FALK,E. Thrombosis in Unstable Angina. In: Thrombosis and Platelets in Myocardial ~;~.h;miz, Cardiovascular Clinics 18. J.L. Mehta (Ed.) Philadelphia: F.A. Davis, 1987, pp.
5.
BAYER, B.L., BLASS, K.E., and FORSTER,W. Antiaggregatory effect of prostacyclin (PGI2) in vivo. Br. J. Pharmacol. 66, 10-12, 1979.
6.
FANTONE,J. and KINNES,D.A. Prostaglandin El and prostaglandin 12 modulation of superoxide production by human neutrophils. Biochem. Biophys. Res. Commun. 113,506512, 1983.
7.
SIMPSON, P.J., MITSOS, S.E., GALLAGHER,K.P., FANTONE, J.C., ABRAMS, G.D., SCHORK, M.A., and LUCCHESI, B.R. Prostacyclin protects ischemic-reperfused myocardium in the dog by inhibition of neutrophil activation. Am. Heart J. 113, 129-137, 1987.
8. DINERMAN, J., MEHTA, J., LAWSON, D., and MEHTA, P. Enhancement of human neutrophil function by platelets: effects of indomethacin. Thromb. Res. 49,509-517, 1988. 9.
MEHTA,J.L., MEHTA,P., WARD,M.B., and LAWSON,D. Inhibition of human platelets and neutrophil functions by piriprost (U60257). ProstaglandinsLeuhotrienes Med. 29,259267, 1987.
10.
SIMPSON, P.J., MICKELSON,J., FANTONE,J.C., GALLAGHER,K.P., and LUCCHESI, B.R. Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart. Circ. Res. 60,666-673, 1987.
11.
MULLER, B., STURZEBECHER,S., and KRAIS, R. The experimental and clinical pharmacology of iloprost. In: The Pathophysiology of Critical Limb Ischemia and
Pharmacological Interventionwitha Stable ProstacyclinAnalogue, Iloprost. J.A. Dormandy (Ed.) London: Royal Society of Medicine Services Ltd., 1989, pp. 33-49.
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12.
BUGIARDINI, R., GALVANI, M., PERRINI, D., GRIDELLI, C., TOLLEMETO, D., MARI, L., PUDDU, P., and LENZI, S. Myocardial ischemia induced by prostacyclin and iloprost. Clin. Phatmacol. Ther. 38, 101-108, 1985.
13.
MEHTA, J.L., DINERMAN, J., MEHTA, P., SALDEEN, T.G.P., LAWSON, D., DONNELLY, W.H., and WALLIN, R. Neutrophil function in ischemic heart disease. Circulation 79,549556, 1989.
14.
ROSSEN, R.D., SWAIN, J.L., MICHAEL, L.H., WEAKLEY, S., GIEUUINI, E., and ENIMAN, N.L. Selective accumulation of the first component of complement and leukocytes in ischemic canine heart muscle: a possible initiator of an extra myocardial metabolism of ischemic injury. Circ. Res. 57, 119-130, 1985.
15.
MILES, E.L., RHOLL, K.S., and QUIE, P.G. Luminol-amplified chemiluminescence: a sensitive method for detecting the carrier state in chronic granulomatous disease. J. Clin. Microbial. 12,52-56, 1980.
16.
MEFITA,J.L., LAWSON,D.L., and MEHTA, P. Modulation of human neutrophil superoxide production by sekective thromboxane synthetase inhibitor U63,557A. Life Sciences 43,923928, 1988.
Address for reprints: J.L. Mehta, M.D., Ph.D., Box J-277, JHMHC, University of Florida, Gainesville, PL 326 10
BRIEF
COMMUNICATION
~DU~ONINHuMANNEuTRopHlLcHEMoTAxIsBYTHEpR~AcYcLIN ANAmGUElLOPROsT
F. A. Nicolini, P. Mehta, D. Lawson, J.L. Mehta Departments of Medicine and Pediatrics, University of Florida College of Medicine and the Veterans Affairs Medical Center, Gainesville, FL 32608-l 197 USA
(Received
23.4.1990;
accepted
in revised form 6.6.1990
by Editor N.U. Bang)
ODUCTION Platelets, endothelial cells and neutrophils interact together in the maintenance of vascular tone, the development of thrombosis and the induction of’reperfusion-related tissue injury (l-3). Platelet activation is a key initial event in the formation of an occlusive thrombus in an atherosclerotic vessel with damaged or dysfunctional endothelium (4). Neutrophils perpetuate the ischemic injury by release of superoxide radicals, arachidonate metabolites and proteolytic enzymes (4). Prostacyclin has been shown to exert an inhibitory effect on platelet and neutrophil functions (5,6), and these properties may relate to its beneficial effects (7). In this study, we describe the effects of a stable prostacyclin analogue iloprost on function of neutrophils taken from healthy human volunteers. ODS p Peripheral venous blood was obtained from 10 normal subjects in heparin (10 units/ml), layered over Mono-Poly Resolving Medium (Flow Labs, Maclean, VA) and centrifuged at 300g for 30 minutes at 24’ C. Red blood cells in the neutrophil-rich layer were removed by hypotonic lysis. Subsequent cell preparation contained greater than 95% neutrophils. Platelets were isolated by centrifugation of blood collected in 3.8% sodium citrate at 800g for 15 minutes at 4’ C as described previously (8). Neutrophils were incubated with washed platelets (WP) in a ratio of 1:lO in Hank’s Balanced Salt Solution (I-IBSS) with Ca++ and Mg++ for 10 minutes at 37” C. As controls, neutmphils alone and platelets alone were also incubated in I-IBSS. Oxygen Neutrophil-derived free oxygen radical production was determined by luminol-dependent chemiluminescence using formyl-methionyl-leucylphenylalanine (f-MLP) lo-7M as the stimulus as described previously (8). Briefly, neutrophils (with or without WP) suspended at a concentration of 1 x 106 cells/ml in HBSS were incubated with f-MLP for 10 minutes at 37’ C. Iloprost in different concentrations (0,O. 1, 1.0, and 10 @I) was added to the suspension. Luminol (initial concentration 5 pM) was added to amplify chemiluminescence. f-MLP (10 pM) was added and chemiluminescence measured for 30 seconds from time zero and then every 10 minutes for 2 hours in a liquid scintillation counter. Counts per minute over time were plotted on a semilog paper and the area under the curve calculated as an arbitrary index of free oxygen radical release by f-MLP-stimulated neutrophils. Key words: Chemotaxis, Iloprost, Neutrophils,6~~tacyclin
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Chemotaxis: Directed neutrophil chemotaxis in response to f-MLP was measured in a chemotaxis chamber (8). The number of neutrophils in the upper chamber was kept constant at 1 x 105 cells/ml while concentration of iloprost was varied from 0, 0.1, 1.0, and 10 PM. The lower chamber contained f-MLP (0, 10q9, 10e8, and lo-TM). A polycarbonate filter (5.0 p.M pore) separated the upper and lower compartments, which were incubated in humidified air with 5% Co;! for 60 minutes at 37’ C. In each experiment, all measurements were made in triplicate and the number of cells were counted in five separate high-power fields (x 400) at each concentration of f-MLP. The data were averaged and expressed as cells per 5 high-power fields. Neutronhil LTl34 generation: To determine LTB4 generation, neutrophils (1 x 107 cells/ml) were incubated with calcium ionophore A 23187 (10 PM) at 37’ C for 15 minutes in a shaking water bath. Iloprost was added in increasing concentrations (0, 0.1, 1.0, and 10 PM). The reaction was terminated by placing the tubes in ice for 5 minutes and subsequently adding cold methanol (0.5 ml). Suspensions were centrifuged at 25,000g for 5 minutes at 4’ C and the supematants analyzed for LTB4 by radioimmunossay (RIA) in duplicate using supplies from New England Nuclear, Boston, MA. Our previous studies (9) have shown that a specific 5-lipoxygenase inhibitor, piriprost (10 PM), decreases neutrophil LTB4 formation by over 70% as determined by this RIA. Furthermore, the values obtained from RIA correlated (r-0.91) with those obtained from HPLC. Statistics: The data from multiple observations were averaged and expressed as a mean f standard deviation. Data were analyzed by analysis of variance and Student’s t-test for paired observations, as appropriate. A p value of less than 0.05 was considered significant. RESULTS Neutronhi free oxygen radical production; Iloprost at all concentrations exerted a slight inhibitory effect on neutrophil free oxygen radical production as indicated by a change in chemiluminescence. However, there was no dose-dependent effect (Table 1). The presence of WP increased the chemiluminescence (177.3 + 37.7 vs 153.8 f 69.2 cm2) as noted previously (8). Addition of iloprost had only a very minimal effect on the free oxygen radical produced by neutrophils in the presence of WP (Table 1). TABLE 1: Effect of Iloprost on Neutrophil Chemiluminescence Chemiluminescence Area (cm2) Neutrophils (1 x 1061ml) alone + Iloprost 0.1 m + Iloprost 1.0 pM + Iloprost 10 PM
153.8
Neutrophils (1 x lO%nl) + WP (1 x 107/ml) + Iloprost 0.1 PM + Iloprost 1.0 ~_LM + Iloprost 10 PM Data from 5 experiments expressed as mean f SD; WP -
136.3 rt 58.1 134.8 + 36.5 135.0 f 65.9 177.3 f37.7 176.8 f 67.0 170.0 f 62.0 169.7 + 57.6 washed platelets
Chemotaxis: In all experiments, migration of neutrophils was markedly increased as the concentration of f-MLP was increased from 10-g to 10-T M (Table 2). The presence of platelets enhanced neutrophil chemotaxis, as described previously (8). Although iloprost decreased neutrophil chemotaxis (spontaneous as well as f-MLP stimulated) in the neutrophil suspension (with or without WP), the reduction was significant at the 10 pM concentration. Furthermore, the
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degree of inhibition of neutrophil chemotaxis was similar in the neutrophil suspension with or without platelets. TABLE 2: Effect of Iloprost on f-MLP-Induced Neutrophil Chemotaxis
0 Neutrophils + Iloprost + Iloprost + Iloprost
alone 0.1 pM 1.0 pM 10 pM
244 f 53 222f60 194 * 55 167 + 47*
Neutrophils (per 5 high-power fields) f-MLP concentration (M) 10-9 10-a 10-7 294 + 63 272 -t 62 247 f 61 221 f 57*
362 + 56 330 * 49 307 + 49 282 f 46*
480 432 412 378
f f f +
7.5 76 81 66*
Neutrophils + WP 268 f 44 345 f 74 431 f 83 508 * 73 + Iloprost 0.1 pM 244+36 315 f68 394 * 79 470 f74 + Iloprost 1.0 pM 218 + 32 286 f 70 374 f 73 449 f 76 198+34** ~257f56** + Iloprost 10 pM 325 f 107** 411+64** Data expressed as mean f SD; concentration of neutrophils and WP are the same as in Table 1. * p < 0.02 vs neutrophils alone ** p c 0.05 vs neutrophils + WP Neutrouhil LTB3 generation: LTB4 generation by neutrophils ranged from 17.6 to 24.6 q/107 cells, and was unaffected by iloprost. Addition of platelets to neutrophils increased LTB4 generation, but iloprost did not affect the enhanced LTB4 generation (Table 3). TABLE 3: Effect of Iloprost in Neutrophil LTB4 Generation
Neutrophils (107/ml) + Iloprost 0.1 pM + Iloprost 1.0 pM + Iloprost 10 pM Neutrophils (107/ml) + WP (107/ml) + Iloprost 0.1 pM + Iloprost 1.0 pM + Iloprost 10 pM Data from 5 experiments expressed as mean f SD
LTB4 (ng/107 cells) 21.1 f 6.9 29.7 + 7.2 34.2 f 6.4 33.6 + 12.7 30.7 f 1.5 22.5 f 4.9 22.5 f 4.9 27.8 rt 2.5
DISCUSSION Previous studies (10) showed that iloprost reduces myocardial infarct size in dogs subjected to total coronary occulsion for 90 minutes and reperfusion for 6 hours. In these studies, myocardial myeloperoxidase activity, an index of neutrophil accumulation, was reduced at the interface between the infarcted and the noninfarcted region. The authors postulated that the protective effect of iloprost was related to its potential to decrease migration of neutrophils in response to ischemic myocardial injury. The authors also demonstrated an iloprost-induced decrease in canine neutrophil superoxide radical generation in response to opsonized zymosan. It is also possible that, besides the inhibitory effect on platelets, iloprost contributed to myocardial protection by its neutrophil-inhibitory effects. Iloprost was also found to be beneficial in the treatment of patients with critical limb ischemia by virtue of its modulating effects on platelet-neutrophil-endothelial cells interactions (11). However, iloprost was not very effective in patients with stable angina pectoris and severe coronary artery disease in whom &hernia was provoked by exercise testing and anial pacing (12).
672
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That iloprost was not efficacious in treating this condition is probably related to the fact that neutrophils as well as platelets are not activated in stable angina, unlike in acute mocardial ischemic syndromes (e.g. unstable angina and acute myocardial infarction) when neutrophil activity as measured by their chemotactic response, elastase release, superoxide radical production and LTB4 generation is markedly enhanced (13). Platelet activity is also enhanced in acute myocardial ischemia (14). Because iloprost may be clinically useful, we examined the effects of iloprost on human neutrophil functions in vitro. Our observations indicate that iloprost decreases the neutrophil chemotactic response to f-MLP, while free oxygen radical generation is only slightly decreased. Neutrophil LTB4 generation was not affected by iloprost. A decrease in directed chemotaxis is consistent with the observations of Simpson et al (10) in dogs and also lends support to the results of clinical trials of iloprost in critical limb ischemia (11). PGI2 also decreases neutrophil chemotactic activity (7). Reduction in neutrophil chemotaxis could be a rationale for the potential use of iloprost in acute ischemic syndromes when complement fragments appear early in injured tissues (14). However, the inhibitory effects of iloprost were observed in our experiments in high concentrations. A possible explanation for the apparent discrepancy between our in vitro results and the in vivo studies wherein the inhibition of neutrophil accumulation is seen with therapeutic use of iloprost may be that other prostaglandins, such as PGEl and PGI2, which are released during ischemic injury, may exert a synergistic effect with iloprost in vivo. Furthermore, a reduction in afterload with iloprost may also contribute to its tissue-protective effects (10). Neutrophils when activated produce different free oxygen radical species, such as superoxide anion and hydrogen peroxide. Chemiluminescence measures total oxidative burst but cannot discriminate between superoxide anions and hydrogen peroxide (15). It is noteworthy that the method we used to quantitate oxidative burst employs measurements of the area under the curve over 2h and the vertical axis is plotted (in counts per minute) in log scale (8, 9). As such, only major reductions in chemiluminescence are identified. Furthermore, superoxide radical release occurs within the fiit few seconds, whereas most of the remaining chemiluminescence is due to the release of hydrogen peroxide. It is likely that the measurement of early superoxide radical release was missed in our experiments, and iloprost does not affect hydrogen peroxide release. Nonetheless, superoxide radical generation measured by reduction of ferricyochrome C is reduced by iloprost in very high concentrations (lo), which are unlikely to be achieved in vivo. Lastly; arachidonate metabolism in neutrophils does not seem to be decreased by iloprost in concentrations up to 10 @VI. Thus the major inhibitory effect of iloprost appears to be limited to a reduction in neutrophil chemotaxis. Iloprost is a very potent inhibitor of platelet aggregation, with an IQ0 of 0.1 to 0.6 nM. In previous studies platelets were shown to enhance neutrophil activity (8), and we hypothesized that iloprost may exert a more pronounced inhibition of neutrophil function in the presence of platelets. In this study we demonstrated potentiation of neutrophil activity in the presence of platelets. However, iloprost affected neutrophil function by a similar magnitude whether platelets were present or not. This indicates that the effects of iloprost on neutrophil activity are independent of its effects on platelets, In other studies (16), a selective TxA2 synthetase inhibitor was shown to decrease neutrophil function only in the presence of platelets by redirecting cyclic endoperoxides towards PGI2 formation in the neutrophils. Obviously, this mechanism would not be operative in the present experiments. In summary, this study shows that iloprost exerts inhibitory effects on neutrophil chemotaxis without significantly affecting their oxidative burst. Reduction in neutrophil migration into the ischemic area may result in protection from tissue injury induced by subsequent neutrophil activation.
Vol59, No. 3
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The authors thank Ms. Janet Wootten for editorial assistance. This study was supported by a Department of Veterans Affairs Merit Review Award and a grant-in-aid from the American Heart Association, Florida Affiliate. JLM is a Clinical Investigator of the Department of Veterans Affairs, Central Office. _S R 1. VANHOUTTE, P.M., and HOUSTON, D.S. Circulation 72,728-734, 1985.
Platelets, endothelium and vasospasm.
2.
MEHTA,J.L., NICHOLS,W.W., and MEHTA,P. Neutrophils as potential participants in acute myocardial &hernia: Relevance to reperfusion. J.A.C.C. 11, 1309-1316, 1988.
3.
NICHOLS,W.W., MEHTA,J.L., DONNELLY,W.H., LAWSON,D., THOMPSON,L., and TERRET, M. Reduction in coronary vasodilator reserve following coronary occlusion and reperfusion in anesthetized dog: role of endothelium-derived relaxing factor, myocardial neuuophil infiltration and prostaglandins. J. Mol. Cell. Cardiol. 20,943-954, 1988.
4.
FALK,E. Thrombosis in Unstable Angina. In: Thrombosis and Platelets in Myocardial ~;~.h;miz, Cardiovascular Clinics 18. J.L. Mehta (Ed.) Philadelphia: F.A. Davis, 1987, pp.
5.
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Address for reprints: J.L. Mehta, M.D., Ph.D., Box J-277, JHMHC, University of Florida, Gainesville, PL 326 10
