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Unstimulated polymorphonuclear neutrophils regulate proximal coronary arterial tone
International
Journal
of
cardiology ELSEVIER
International Journal of Cardiology 55 (1996) 15-27
Unstimulated polymorphonuclear neutrophils regulate proximal coronary arterial tone Yukihiko Abe”, Tomiyoshi Saito”, Satoshi Kurodaa, Toshiyuki Ishibashi”, Mitsumasa Keitokub, Yukio Maruyamaa’* “First Department of Internal Medicine, Fukushima Medical College, 1 Hikarigaoka, Fukushima, 960-12, Japan bFirst Department of Internal Medicine, Tohoku University, Sendai, Miyagi, Japan
Received 26 September 1995; revised 16 January 1996; accepted 5 March 1996
Abstract Our objective is to clarify, by measuring the superoxide production as a marker of active state of polymorphonuclear neutrophils, whether unstimulated polymorphonuclear neutrophils would influence coronary arterial tone. We recorded the isometric tension of the porcine coronary arterial ring in a bath of oxygenated Krebs Ringer solution. Unstimulated porcine polymorphonuclear neutrophils that contained little superoxide were added to the bath. We also analyzed the prostaglandins produced in the bath. The isometric tension of arterial rings increased dose-dependently when polymorphonuclear neutrophils were added to the bath. The vasoconstriction induced by unstimulated polymorphonuclear neutrophils was inhibited by endothelial denudation, indomethacin, anti-CD1 la/l&like antibody. Thromboxane A, synthetase inhibitor and superoxide dismutase did not affect the vasoconstriction. Prostaglandin E, predominated among the prostaglandins produced in the bath; its production was significantly inhibited by indomethacin (without vs. with indomethacin; 3898 + 1704 vs 1956 + 715 pg/ml, P < 0.05, n = 6). Pretreatment of vascular rings with indomethacin blocked the interaction of the coronary artery with polymorphonuclear neutrophils. Results suggested that unstimulated polymorphonuclear neutrophils constrict the proximal coronary artery. Such vasoconstriction may be produced by cyclooxygenase products, especially prostaglandin E, produced in the vascular wall via the interaction between the polymorphonuclear neutrophils and the endothelium. Polymorphonuclear neutrophils may regulate coronary arterial tone. Keywords:
Unstimulated polymorphonuclear
neutrophils; Endothelium; Prostaglandin E,; Coronary arterial tone; Pig
1. Introduction Interaction between the polymorphonuclear neutrophils and the blood vessels releases a variety of substances that affect vascular tone. However, results of studies of polymorphonuclear neutrophils isolated *Corresponding author. Tel.: +0245 48 2111; fax: +0245 48 1821.
from various species do not necessarily coincide [l-lo]. Studies have reported vasoaction that was endothelium-dependent or -independent, i.e. the endothelium-dependent vasoconstrictor responses in rabbit and dog polymorphonuclear neutrophils [2,3,5], the endothelium-independent vasoconstrictor responses by rat and rabbit polymorphonuclear neutrophils [4,8]. Endothelium-dependent vasodilation has been observed in human polymorphonuclear
0167-5273/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO167-5273(96)02624-l
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neutrophils [9], while endothelium-independent vasodilation has been reported in rat and rabbit polymorphonuclear neutrophils [3,10]. The effects of polymorphonuclear neutrophils on vessel tone seem to be influenced by their condition. The functional status of polymorphonuclear neutrophils is thought to influence their interaction with the vessels. According to Sessa and Mullane [4], polymorphonuclear neutrophils activated by the calcium ionophore A23187 constricts aortic rings via the release of a protein-like contractile factor. Polymorphonuclear neutrophils activated by phorbol myristate acetate induce the contraction of rabbit coronary arteries via the generation of hydroxyl radical [l]. Ohlstein and Nichols reported that the contraction of rabbit aortic rings induced by fMLPactivated polymorphonuclear neutrophils was blocked by superoxide dismutase 121.In contrast, rat polymorphonuclear neutrophils elicited by the intra peritoneal injection of oyster glycogen relaxes aortic rings via the release of a substance resembling endothelium-derived relaxing factor [ 111. Unstimulated, spontaneous polymorphonuclear neutrophils reportedly produce differing changes in vascular tone; i.e. contraction and relaxation by polymorphonuclear neutrophils of the rabbit [3], contraction by these of the dog [5] and of the rabbit [4], and vasorelaxation by the supernatants of human polymorphonuclear neutrophils [7]. Thus, polymorphonuclear neutrophils may produce a variety of vasoactive effects in the unstimulated as well as the stimulated condition. No report has described whether the polymorphonuclear neutrophils used were determined to be unstimulated, especially during the interaction between polymorphonuclear neutrophils and blood vessels. Polymorphonuclear neutrophils may be stimulated by mechanical manipulation during isolation [3] or are upon addition to the organ bath [4]. Differing stimuli may then produce differing profiles of the polymorphonuclear neutrophils and blood vessel interactions. Thus, our objective was to clarify, by measuring the superoxide production as a marker of active state of polymorphonuclear neutrophils, whether unstimulated polymorphonuclear neutrophils would influence coronary arterial tone and regulate the basal tone. We conducted in vitro experiments to exclude the role of various factors which alter vascular tension, and
investigated the mechanism(s) of the vascular response produced between unstimulated polymorphonuclear neutrophils and the vascular endothelium.
2. Materials
and methods
2.1. Preparation of polymorphonuclear
neutrophils
Polymorphonuclear neutrophils were freshly prepared as follows [ 121. Thirty milliliters of porcine blood anti-coagulated with 17% volume of acidcitrate-dextrose (ACD) solution was mixed with 20ml of 6% dextran 70 in normal saline in a 60-ml syringe. The syringe was maintained upright for 1 h. Following sedimentation, the straw-colored upper layer was carefully removed and centrifuged at 150 g for 10 min. The pellet was washed with phosphatebuffered saline. After centrifugation, the cells were overlaid on Mono-poly-resolving-medium (Flow Laboratories Inc., Costa Mesa, CA.) and centrifuged at 300g for 20 min. The cells of the fraction below the interface were washed with phosphate-buffered saline and centrifuged at 150g for 10 min. To exclude mononuclear cells, the pellet was overlaid on Lymphoprep (NYCOMED AS, Oslo, Norway) and centrifuged at 300g for 20 min. The bottom pellet was washed with phosphate-buffered saline and adjusted to 10’ cells/ml. All procedures were carried out at room temperature. In this procedure, the purity of polymorphonuclear neutrophils, as determined by May-Giemsa staining, exceeded 98%. Cellular viability was determined by the trypan blue exclusion method and exceeded 95% in the final fraction, The prepared polymorphonuclear neutrophils did not contain measurable levels of either hemoglobin (
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NaHCO, 2.1, Ca-N, 0.00973, CaCl, 0.166, and glucose 2.0. The right coronary artery was dissected free and cleaned of adherent connective tissue, then cut into ring preparations 2Smm in length. Care was taken to avoid stretching and to keep endothelial cells intact and unrubbed. The endothelial cells of some vascular rings were mechanically damaged by gently rubbing the intimal surface with a wooden stick to create de-endothelialized rings. 2.3. Tension measurement
We used 115 coronary arterial rings from 36 pigs in the experiments on tension measurement. Coronary arterial rings were suspended in water-jacketed organ chambers filled with 10 ml Krebs Ringer bicarbonate solution at 37°C and continuously gassed with 95% 02-5%CO,. Each ring was suspended on a pair of stainless steel hooks, one of which was fixed to the side wall of the chamber, while the other was connected to a force-displacement transducer (Model T7-15-240, Orientec Corporation, Tokyo, Japan). Changes in isometric tension were recorded on a polygraph system (Model 1363, NEC-SANEI, Tokyo, Japan). The arterial rings were gradually stretched until the resting force reached 3g. The solution in the organ chamber was replaced every 20 min to wash out blood cells adherent to the vascular wall. After equilibration for 180 min, the arterial rings were contracted with 35 mM KCl, followed by the addition of 0.01 PM substance P to the organ chamber for the estimation of endothelial integrity. Endothelium-unrubbed rings relaxed in response to substance P, whereas the de-endothelialized rings did not. After washing the arterial rings and observing a steady resting state, we recorded changes in tension following the addition of polymorphonuclear neutrophils. The change in tension was expressed as a percentage of the contraction induced by 35 mM KCl. No polymorphonuclear neutrophils stimulants were used during the measurement of tension.
(2) Indomethacin was used to block cyclooxygenase. Since various concentrations of indomethacin (1 PM, 10 ,LLM, or 100 PM) [2,5,13] had been used, to determine the concentration appropriate for blocking the action of cyclooxygenase in porcine coronary arterial rings, we studied the concentration-dependency of indomethacin required to inhibit the vasoconstriction induced by arachidonic acid. Arachidonic acid at concentration of 0.1, 1 and 10 ,uM, respectively, was added cumulatively to the organ chamber. Changes in isometric vascular tension were recorded before and after the addition of indomethacin, 2 or 20 ,uM. (3) To detect the vasoactive substances that altered the isometric vascular developed tension, we added the antagonist to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml. Seven antagonists were used: 1 ,uM ON03708 (a TXA,/PGH, receptor antagonist, n=6) [14-161, 20 PM indomethacin (a cyclonordioxygenase inhibitor, n=6), 20 PM hydroguaiaretic acid (a lipoxygenase inhibitor, n = 6) [17-201, 100 PM DP-1904 (a TXA, synthetase inhibitor, n=5) [21,22], 1 ,uM ketanserin (an S,-blocker, n=5), 1 ,uM pyrilamine (an H,blocker, n=5) or 2OOU/ml superoxide dismutase (n=5). The concentrations of ON03708 and nordihydroguaiaretic acid used were obtained from previous reports [14-201. We confirmed that the
lo5
2.4. Experimental
protocol
(1) A cumulative volume of polymorphonuclear neutrophils, lo’--5 x lo6 cells/ml, was added to the organ chamber, and the changes in isometric vascular tension were recorded (n = 7).
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5x105 Concentration
105 of
5X106ceIIs,mI PYNs
Fig. 1. Relation between polymorphonuclear neutrophil concentration and polymorphonuclear neutrophils-induced developed tension in porcine coronary arterial rings. Polymorphonuclear neutrophils-induced vasoconstriction (mean ? S.D.) was expressed as % of the contraction induced by 35mM KC1 (n=7).
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Y. Abe et al. I International
.
.. ..
. . .. . .
PMNs
b -te..f . PMNs
IM
wo
wo
PMNs
.. . . wo
ON0
PMNs
c . PMNs
. . . wo
. NDGA
. .
.
wo
PMNs I 1 Omin
Fig. 2. Typical traces of polymorphonuclear neutrophils-induced vasoconstriction suppressed by several blockers; a, IM; b, ONO; c, NDGA. PMNs, 106/ml polymorphonuclear neutrophils; wo, washout; IM, 20 PM indomethacin; ONO, 1 PM ONO-3708; NDGA, 20 PM nordihydroguaiaretic acid.
concentrations of the antagonists used in this study did not affect the vasoconstriction induced by 35 mM KCl. (4) To detect the main site of production of vasoactive substances, we pretreated the vascular rings or the polymorphonuclear neutrophils with 20pM indomethacin, then observed the interaction between the vascular rings and polymorphonuclear neutrophils as follows. Vascular rings were pretreated with indomethacin for 15 min followed by repeated washings. Polymorphonuclear neutrophils were then added to the vascular rings (n = 5), or a suspension of polymorphonuclear neutrophils pretreated with indomethacin for 15 min was washed and added to the vascular rings not exposed to indomethacin (n = 5). (5) Rings denuded of endothelium were used to study the role of the vascular endothelium in the polymorphonuclear neutrophils-induced alteration of vascular tone. Following denudation as described previously, the vascular ring was suspended. Polymorphonuclear neutrophils, lo6 cells/ml, were then added to the chamber, with the vascular tension
recorded by the procedure used without denudation (n = 6). (6) To examine the effect of NO on the poly-
morphonuclear neutrophils-induced alteration of vascular tone, we added 100 ,uM Nw-nitro-L-arginine methyl ester (NO blocker) [23] to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml (n = 5). (7) To examine the effect of stimulated polymorphonuclear neutrophils with a stimulant on the polymorphonuclear neutrophils-induced alteration of vascular tone, we added 2 ,uM A23187 to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml (n = 5). (8) To examine the role of receptors on the polymorphonuclear neutrophils and vascular endothelium on the polymorphonuclear neutrophils-induced alteration of vascular tone, 35 mM KC1 was first added to the organ chamber. After vascular tension had reached a plateau, a concentration of 1 or long/ml anti-CD 1la/ 18-like antibody was added to the organ chamber, followed by the addition of polymorphonuclear neutrophils, lo6 cells/ml, 15-20
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19
%
% 40
35-i
35
T
pco.01 l-r
30
25
25 20 15 10 5 0 1 control
control
IM
control
ON03708
NDGA
%
% 30
40 -
N.S.
pco.01
35 -
contol
DPI 904
control
SOD
control
denuded
Fig. 3. Bar graphs showing the effects of several antagonists or de-endothelialization on polymorphonuclear neutrophils-induced vasoconsttiction. IM, ON03708, NDGA, DP1904, SOD; 20 PM indomethacin, 1 PM ONO-3708, 20 PM nordihydroguaiaretic acid, 100 PM DP-1904 or 2OOU/ml superoxide dismutase was introduced into the chamber, respectively, and polymorphonuclear neutrophils-induced vasoconsttiction was compared with that without intervention (‘control’); denuded, contraction of ring denuded of endothelium, control; vascular ring and polymorphonuclear neutrophils were untreated.
min later (II = 6). Anti-CD 1la/ 18-&e antibody was selected because CD lla/18 is present on the surface of polymorphonuclear neutrophils, even spontaneous polymorphonuclear neutrophils [24], and only this antibody was commercially available for use in pigs. This antibody is active against porcine as well as equine leukocytes [25]. We conducted this experiment at the concentrations of
anti-CD lla/l8-like antibody that did not affect the vasoconstriction induced by 35 mM KCl. 2.5. Determination of superoxide production We used 28 coronary arterial rings from 21 pigs to evaluate the production of superoxide from polymorphonuclear neutrophils, which was measured
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Fig. 4. Bar graphs showing the effects of indomethacin pretreatment of vascular ring (n = 5) or polymorphonuclear neutrophils (n = 5) alone on polymorphonuclear neutrophils-induced vasoconstriction. IM-vascular, IM-PMNs; vascular ring or polymorphonuclear neutrophils alone was pretreated with 20 PM indomethacin respectively. Control; neither the vascular ring nor the polymorphonuclear neutrophils were treated with indomethacin.
spectrophotometrically using ferricytochrome c [26]. A porcine coronary arterial ring was mounted in a 20-ml chamber containing Krebs-Ringer bicarbonate buffer. The solution in that chamber was maintained at 37°C and continuously aerated with 95% O,-5% CO, under the conditions described earlier. Polymorphonuclear neutrophils, lo6 cells/ml, and ferricytochrome c, 50 PM, were added to the chamber, which was composed of a couple of small chambers connected to each other. Vascular tension was con-
Table 1 Superoxide production and vasoconstriction Min.
0 5 10 15 20 30 40 50 60
observed following
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tinuously measured in one of chambers. At the same time, 3 ml of the solution was taken from another chamber, decanted into a small tube, and the absorbance was measured at 550 nm every 5 min until 60 min after administration of polymorphonuclear neutrophils. In 14 cases, polymorphonuclear neutrophils were stimulated with 2 PM A23187. In 7 of those cases, 200U/ml superoxide dismutase was added to the chambers. Thereafter, superoxide production was measured in the same way. Data were calculated as nanomoles of superoxide released per lo6 polymorphonuclear neutrophils, based on an extinction coefficient of 15.5mM-‘cm-’ for cytochrome c reduction [26]. 2.6. Microscopic observation of polymorphonuclear neutrophils In 21 preparations from 21 pigs, we microscopically observed the polymorphonuclear neutrophils in a smear from each chamber, which was stained with May-Giemsa solution, before and after the experiment on tension measurement. The number of degranulated polymorphonuclear neutrophils per 1000 cells was counted. 2.7. Measurement of prostaglandins We analyzed the prostaglandins produced by the 67 coronary arterial rings from 14 pigs in the organ chamber using radioimmunoassay. A volume of 106/
administration of polymorphonuclear
Production of superoxide (n mol/ lo6 PMNs) VandPandCa (n=7)
VandPandCaandS (n=7)
VandP (n=7)
0+-o 14.6 t 9.2 16 5 9.2 17 _t 8.6 18.3 I 8.4 19.6 2 10.5 19.6 +- 10.7 19.6 ? 10.7 19.4 k 11.1
o-t-0 020 020 o-co 020 Ok0 020 oio oio
Ok0 0.1 t 0.3 0.6 -c 0.8 0.7 lb 1.0 0.7 2 1.5 0.7 i 1.1 0.5 t 1.2 0.5 i 1.2 0.6 k 1.2
V, coronary arterial ring; P or PMNs, polymorphonuclear
neutrophils Vasoconstriction (%) produced by PMNs without stimulant (n = 7) Ok0 0.6 IT 0.8 6.4 i 1.4 8.4 i- 1.8 10.0 ? 1.9 20.2 2 2.2 22.9 k 34 24.8 i 4.6 25.1 i 4.6
neutrophils; Ca, Ca ionophor A23187; S, superoxide dismutase.
Journal of Cardiology
Y. Abe et al. I International p
n
p<0.05.n=4
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p
n
n
N.S.
PGE: wlml
PGhs n=5 wlm n
pco.01 n=5
N.S. n=4
n
n
I,
T
200
cant
IM
100 I L, pco.05 n=4 i---7
T
cant
IM
cant
CA
cant
PMNs
cant
IM
cant
CA
cant
p
T
cant
PMNs
p
T
CA
cant
PMNs
Fig. 5. Production of prostaglandin E, (PGE,, left panel), prostaglandin F,, (PGF,,, right upper panel), and tbromboxane B, (TXB,, right lower panel). cant, produced in the solution with lo6 polymorphonuclear neutrophils and coronary arterial ring; TM, produced in the solution with polymorphonuclear neutrophils, coronary arterial ring and 20 PM indomethacin; CA, produced in the solution with coronary arterial ring alone; PMNs, produced in the solution with polymorphonuclear neutrophils alone.
ml polymorphonuclear neutrophils was added to the organ chamber in which the porcine coronary artery ring was mounted. When the vascular tension reached a maximum, the solution was removed to a tube containing indomethacin at -20°C. The concentrations of thromboxane B, and prostaglandin E, in the solution were then measured with a lz51 assay system (Amersham, Buckinghamshire, England). ProstaglandinF,, was measured with a 3H radioimmunoassay kit (Baxter Travenol Diagnostics, Inc., Cambridge, MA). The prostaglandins released during the interaction between the arterial ring and the polymorphonuclear neutrophils were measured both without and with the addition of 20 ,uM indomethacin. We also determined the concentration of
prostaglandins in the chamber containing only the vascular ring or the polymorphonuclear neutrophils. 2.8. Agents
The following agents were purchased from Sigma Chemical, St. Louis, MO: calcium ionophore A23 187 (free acid), indomethacin, nordihydroguaiaretic acid (NDGA), pyrilamine maleate salt, ketanserin, prostaglandin Fza, prostaglandin E,, ferricytochrome c (equine heart type VI), Nw-nitro-Larginine methyl ester. Recombinant human superoxide dismutase (4000 units/mg) was a gift from Ube IL, Tokyo, Japan. ONO-3708 was kindly supplied by the OPCL, Osaka, Japan. DP-1904 was a gift from
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DPCL, Tokyo, Japan. Anti-CD 1la/ lx-like antibody was purchased from VMRD, Inc., Pullman, WA. 2.9. Analysis of data Results are expressed as mean + S.D. The vascular contraction induced by polymorphonuclear neutrophils was compared with that induced by 35mM KCl. We compared the tension developed in porcine coronary arterial rings at differing concentrations of polymorphonuclear neutrophils, or the polymorphonuclear neutrophils-induced vasoconstriction with differing concentrations of anti-CD1 la/ 18-like antibody, using one-way analysis of variance followed by Fisher’s PLSD. Other results were analyzed by the paired Student’s t-test. A P value less than 0.05 was considered statistically significant.
3. Results
3.1. Tension induced by polymorphonuclear neutrophils in porcine coronary arterial rings The isometric tension of porcine arterial rings increased dose-dependently when porcine polymorphonuclear neutrophils were added to the organ chamber at concentrations of lo5 to 5 x 106/ml (Fig. 1, n =7). The vasoconstriction induced by polymorphonuclear neutrophils achieved a maximum tension in approximately 60 min, and persisted for at least 30 min. The sustained contraction returned to basal tension following repeated washing. To examine the reproducibility of the polymorphonuclear neutrophils-induced vasoconstriction, the arterial rings were repeatedly exposed to polymorphbnuclear neutrophils. The arteries after the exposure to polymorphonuclear neutrophils produced the contraction with same magnitude as observed before the exposure (data not shown). 3.2. Concentration of indomethacin required to inhibit cyclooxygenase in porcine coronary arterial rings The contraction of porcine coronary arterial rings was dependent on the concentration of arachidonic acid; that is, it was 10.1 +- 8.8, 24.9 ? 12.4 and 52.9 + 8.7% (n=7) at doses of 0.1, 1.0 and 10 PM,
of Cardiology 55 (1996) 15-27 respectively. A concentration of 20 PM indomethacin suppressed the vasoconstriction induced by arachidonic acid (0.0 t 0.0, 1.0 ? 0.97, 1.7 +1.25%, n=7, P < 0.01 vs without indomethacin). However, 2 PM indomethacin did not significantly suppress vasoconstriction (4.0 ‘-t 1.0, 13.9 2 4.8, 36.4 + 17.0%, n=7, N.S.). The vasoconstriction induced by 35mM KC1 was not affected by exposure to 20 PM indomethacin (data not shown). From these results, we chose a concentration of 20 PM indomethacin for study. 3.3. Effect of vasoactive substances on vasoconstriction induced by polymorphonuclear neutrophils The tracing in Fig. 2 shows the effects of indomethacin (IM in Fig. 2a), ONO-3708 (ON0 in Fig. 2b) and nordihydroguaiaretic acid (NDGA in Fig. 2c) on the vasoconstriction induced by polymorphonuclear neutrophils. Fig. 3 shows the mean values of the effects of the various blockers on the polymorphonuclear neutrophils-induced vasoconstriction. It was significantly suppressed by indomethacin (IM in Fig. 3, n=6), ONO-3708 (ON03708 in Fig. 3, n=6), and nordihydroguaiaretic acid (NDGA in Fig. 3, n=6). By contrast, the polymorphonuclear neutrophils-induced vasoconstriction was unaffected by DP-1904 (DP1904 in Fig. 3, n=5), ketanserin (data not shown), pyrilamine (data not shown) and superoxide dismutase (SOD in Fig. 3, n=5). 3.4. Detection of site of production of vasoactive substances When a coronary ring alone was pretreated with indomethacin (‘IM-vascular’, in Fig. 4), the vasoconstriction induced by polymorphonuclear neutrophils was suppressed. However, pretreatment of the polymorphonuclear neutrophils suspension alone (‘IMPMNs’ in Fig. 4) did not alter the development of isometric tension. 3.5. Role of endothelium in polymorphonuclear neutrophils-induced vasoconstriction When polymorphonuclear neutrophils were added to the chamber in which a vascular ring denuded of
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endothelium was suspended (‘denuded’ in Fig. 3), the vascular ring contracted only slightly (P < 0.01, n=6). 3.6. Effect of NO in polymorphouclear induced vasoconstriction
neutrophils-
When the Nw-nitro-L-arginine methyl ester was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was not affected (control vs Nw-nitro-L-arginine methyl ester; 20.7 5 5.8 vs 21.8 2 6.5%, n = 5, NS). 3.7. Comparison between unstimulated and stimulated polymorphonuclear neutrophils-induced vasoconstriction When A23187 was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was increased (unstimulated vs A23187-stimulated; 20.5 + 8.2 vs 52.2 2 18.5%, n = 5, P < 0.01). 3.8. Role of receptors on polymorphonuclear neutrophils and vascular endothelium in vasoconstriction induced by polymorphonuclear neutrophils When the anti-CD lla/l8-like antibody was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was suppressed dose-dependently in the range of 1 to 10 rig/ml of the antibody (control, 1 rig/ml, long/ml; 16.7 i. 4.8, 11.8 -+ 6.4, -3.2 + 5.4%*, n = 6, *P < 0.01 vs control).
55 (1996) 15-27
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3.10. Microscopic observation of polymorphonuclear neutrophils Degranulated polymorphonuclear neutrophils comprised less than 1% of the sample as observed on microscopic examination before and after the experiments without and with polymorphonuclear neutrophils-stimulant (without and with stimulant; n = 10, n = 11). Thus, their cellular structure of polymorphonuclear neutrophils remained virtually intact during these experiments. 3.11. Measurement of prostaglandins Fig. 5 shows the production of prostaglandins. Prostaglandin E, was produced most prominently in the chamber that contained a vascular ring and polymorphonuclear neutrophils (‘cant’ in the left panel). However, the amount of prostaglandin E, produced by either the vascular ring or the polymorphonuclear neutrophils alone was significantly less than that containing a vascular ring and polymorphonuclear neutrophils. Indomethacin pretreatment (IM in the left panel) inhibited the production of prostaglandin E,. With the vascular ring and polymorphonuclear neutrophils (right upper panel), prostaglandin F,, was produced, but to a lesser extent than prostaglandin E,. Production of prostaglandin F,, was not inhibited by indomethacin for an unknown reason. Little prostaglandin F,, was produced by either the vascular ring or the polymorphonuclear neutrophils alone. With the vascular ring and polymorphonuclear neutrophils, thromboxane B, was produced (right lower panel), but to a lesser extent than prostaglandin E,. Thromboxane B, production was inhibited by indomethacin. Little thromboxane B, was produced by either the vascular ring or the polymorphonuclear neutrophils alone.
3.9. Measurement of superoxide anion
4. Discussion
In this experiment conducted without the polymorphonuclear neutrophils-stimulant, the calcium ionophore A23 187, the vascular tension increased, but only a trace of superoxide was detected. Use of A23187 led to the marked production of superoxide which was suppressed by 2OOU/ml superoxide dismutase (Table 1).
The effects of polymorphonuclear neutrophils on vessel tone seem to be influenced by their condition. Polymorphonuclear neutrophils in the present study were considered to be unactivated from (a) evidence of only a trace of superoxide production, even after tension development, and (b) microscopic observation of small number (i.e. less than 1%) of degranu-
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lated polymorphonuclear neutrophils after tension development due to administration of polymorphonuclear neutrophils to the bath containing a vessel. These unactivated polymorphonuclear neutrophils elicited contraction of the large epicardial coronary artery. The magnitude of the endothelium-dependent vasoconstriction depended upon the number of polymorphonuclear neutrophils administered. The vasoconstriction induced by polymorphonuclear neutrophils was inhibited by a cyclooxgenase inhibitor, a thromboxane A,/prostaglandin H, blocker or an anti-CD 1la/ 1&-like antibody, which showed that cyclooxygenase products, which are produced in the vascular wall via the interaction of polymorphonuclear neutrophils to the endothelium of the coronary artery, may be responsible for the vasoconstriction. From the results of the reduction in arterial tension induced by esculetin and the AA861, Nishida et al. considered that the polymorphonuclear neutrophilsinduced vasoconstriction in isolated canine coronary arterial rings was produced by the release of vasoconstrictive substances generated by the polymorphonuclear neutrophils-endothelial system, such as the leukotriene A, steal mechanism, in which leukotriene A, synthesized by polymorphonuclear neutrophils was transferred intercellularly to endothelial cells and served as the substrate of the leukotriene C, synthetase system that exists in endothelial cells [5]. As the vasoconstriction induced by polymorphonuclear neutrophils was also suppressed in our study by the lipoxygenase inhibitor, nordihydroguaiaretic acid, we considered that this mechanism plays a partial role. In Nishida’s report, indomethacin did not reduce the vasoconstriction induced by polymorphonuclear neutrophils. However, in our study, the vasoconstriction induced by polymorphonuclear neutrophils was suppressed by indomethacin and by the thromboxane A, /prostaglandin H, receptor antagonist, ONO-3708. Since Toda et al. previously reported that the coronary arterial contractions induced by prostaglandin F 2a, prostaglandin E, and prostaglandin D, were also suppressed by ONO-3708 u51, our findings indicated that cyclooxygenase products, as well as leukotriene, participate in the vasoconstriction induced by polymorphonuclear neutrophils. When only the coronary ring was pretreated with indomethacin, the polymorphonuclear neutrophils-induced vasocohstriction was suppressed. In
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contrast, pretreatment of the polymorphonuclear neutrophils suspension with indomethacin did not alter the development of isometric tension. These results strongly suggest that the constriction of the coronary artery induced by polymorphonuclear neutrophils is brought about by the cyclooxygenase products generated in the vascular wall. The reason for the discrepancy between our findings and those of Nishida regarding the reduction in polymorphonuclear neutrophils-induced vasoconstriction by indomethacin is not clear. A difference in methodology, such as species differences and differing doses of indomethacin, may be responsible. Before using indomethacin in our protocol, we evaluated the concentration of this agent that would inhibit the vasoconstriction induced by arachidonic acid. An indomethacin concentration of 20 ,uM, but not of 2 ,uM, suppressed the vasoconstriction induced by arachidonic acid. Accordingly, we used 20 PM of indomethacin. However, a lower dose of 1 PM indomethacin was used in Nishida’s study [5]. The functional status of the polymorphonuclear neutrophils used in their study was also not defined. Our differing results may be related to the differing characteristics of the polymorphonuclear neutrophils evaluated. To study the effect of NO in the unstimulated polymorphonuclear neutrophils-induced vasoconstriction, we added 100 PM Nw-nitro-L-arginine methyl ester to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils. The polymorphonuclear neutrophils-induced vasoconstriction was unnaffected by Nw-nitro-L-arginine methyl ester. We considered that NO did not participate in the unstimulated polymorphonuclear neutrophils-induced vasoconstriction. By contrast, Ohlstein and Nichols reported that activated polymorphonuclear neutrophils can release superoxide anion and produce endothelium-dependent contraction. They considered that the endothelium-dependent contraction may be the result of superxide anion inactivation of endothelium-derived relaxing factor [2]. From our study, A23187 (polymorphonuclear neutrophils-stimulant) increased polymorphonuclear neutrophils-induced vasoconstriction. We speculate that the mechanism of unstimulated polymorphonuclear neutrophils-induced vasoconstriction may differ from that of stimulated polymorphonuclear neutrophils.
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Prostaglandin E, produces vasodilation in the artery of the hind leg of the rabbit [27] and the dog [28]. This substance is a potent vasoconstrictor in the coronary arteries of the cow, dog and human [29]. Lopez et al. reported that fMLP contracts the atherosclerotic artery of the monkey in vivo and that the prostaglandin E, released from the leukocytes participates in that phenomenon [30]. Prostaglandin E, and thromboxane B, were the predominant prostaglandins detected in our study; such production was inhibited by indomethacin. We also confirmed that lo-‘M prostaglandin E, contracted the porcine coronary arterial ring (46.8 t 16.8%, n = 4). However, doses of prostaglandin E, below 10M6 M had no effect. The concentrations of prostaglandin E, (left panel in Fig. 5, 3.9 + 1.7 x 1O-6 mg/ml = 1.1 + 0.5 x lo-‘M) were well below that value. We suggest that the tissue concentration of prostaglandin E, in the vascular wall produced by the interaction between polymorphonuclear neutrophils and the coronary arterial ring (not measured in our study) may be sufficient to induce vasoconstriction, and that the concentration of prostaglandin E, measured in the bath may have been diluted. As the polymorphonuclear neutrophils-induced vasoconstriction was not inhibited by DP-1904, a selective thromboxane A, synthetase inhibitor, we consider cyclooxygenase products, especially prostaglandin E,, to be major participants in the vasoconstriction induced by polymorphonuclear neutrophils. In the present experiment, anti CD lla/l&like antibody reduced the vasoconstriction induced by polymorphonuclear neutrophils. Our data showed that the receptor mechanism on the polymorphonuclear neutrophils and the vascular endothelium may participate in the polymorphonuclear neutrophils-induced vasoconstriction. It has been considered that CD 1la/ 18 emerges when the polymorphonuclear neutrophils are stimulated. However, another report holds that the intracellular adhesion molecules, i.e. CDlla, CDllb, CDllc and CDl8, are present on the surface of polymorphonuclear neutrophils without stimulation [24], and that ICAMis generally present on the vascular endothelium [31]. We suggest that the CDlla/CDl& which is present on the surface of unstimulated polymorphonuclear neutrophils, and the ligand on the vascular endothelium may participate in the vasoconstriction induced by
55 (1996) 15-27
25
unstimulated polymorphonuclear neutrophils. That is, if polymorphonuclear neutrophils adherence to the endothelium is considered to be the first step in the polymorphonuclear neutrophils/blood vessel interaction, the present data appear to be reasonable. Several methodological problems should be pointed out. First, it is difficult to assess how much the polymorphonuclear neutrophils were activated. In the present experiment, polymorphonuclear neutrophils were isolated under germ-free conditions. No polymorphonuclear neutrophils stimulants were used in the measurement of vascular contraction, resulting in the generation of very little superoxide. Further, A23 187 (polymorphonuclear neutrophils-stimulant) increased polymorphonuclear neutrophils-induced vasoconstriction, that was useful to provide additional evidence that mechanical stimuli during isolation process did not stimulate polymorphonuclear neutrophils in the present study. The reason of microscopic observation of small number (i.e. less than 1%) of degranulated polymorphonuclear neutrophils with Ca-ionophore A23 187 is unclear. We speculate that polymorphonuclear neutrophils have various stimulated stages and polymorphonuclear neutrophils used in our experiments might not reach the degranulated stage, even if A23187 was administered. We thus consider that unstimulated polymorphonuclear neutrophils can change vascular tone. There are several reports which described that polymorphonuclear neutrophils migrate to sites of tissue damage and release oxygen free radicals, arachidonic acid metabolites and lysosomal proteases, which exacerbate tissue injury during coronary artery occlusion [32,33]. Thus, we compared the difference of stimulated and unstimulated polymorphonucIear neutrophils. As a result, developed vascular tension due to stimulated polymorphonuclear neutrophils was clearly different, as mentioned above. Although we studied mainly unstimulated polymorphonuclear neutrophils-induced vasoconstriction, it is necessary to further investigate the difference between stimulated and unstimulated polymorphonuclear neutrophils-induced alteration of vascular tone in more detail. Second, concerning the interaction between the vessel and the polymorphonuclear neutrophils, it is considered that, in small veins, the rolling and sliding of polymorphonuclear neutrophils occur initially along the-endothelial surface during inflamma-
26
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tion which is then followed by their extravascular migration [34]. Velocity of flow in the coronary artery is rapid relative to that of venous flow; therefore, it is not known whether an interaction between polymorphonuclear neutrophils and the arterial vascular wall is normally induced in vivo. Kloner et al. showed in vivo that the neutrophils migrated into the large epicardial coronary arteries after ischaemia and reperfusion, and that this migration occurred after reperfusion and not during ischaemia [35]. Monocytes migrate into the vascular wall during formation of the atherosclerotic plaque [13,36-381, suggesting that polymorphonuclear neutrophils in the blood stream may adhere to the vascular wall and interact with it, even at high speed of arterial flow. Finally, polymorphonuclear neutrophils in arterial blood flow may touch the endothelium and leave it, or roll upon it. Polymorphonuclear neutrophils and the endothelium may interact to produce vasoactive substances, especially prostaglandin E, and leukotriene, mainly from the vascular wall. Vasoactive substances may keep to maintain the coronary arterial tone. However, the basal tone of the coronary arteries in vivo results from the balance between vasoconstrictor and vasodilator factors. If one or more factor(s) break down, coronary arterial tone increases and sometimes coronary vasospasm may result. We clearly showed that unstimulated polymorphonuclear neutrophils constrict the proximal coronary artery. Cyclooxygenase products, particularly the prostaglandin E, produced in the vascular wall via the interaction between the polymorphonuclear neutrophils and the endothelium, may be importantly involved as a vasoconstrictor. Polymorphonuclear neutrophils may regulate coronary arterial tone in the in vivo basal condition, as their concentration in the organ bath (lo6 to 5 x 106/ml) was within the physiological range for polymorphonuclear neutrophils in the circulation. However, further in vivo studies should be done to confirm this possibility.
References [l] Gillespie MN, Kojima S, Kunitomo M, Jay M. Coronary and myocardial effects of activated neutrophils in perfused rabbit hearts. .I Pharmacol Exp Ther 1986; 239: 836-840.
121Ohlstein EH, Nichols AJ. Rabbit polymorphonuclear neutrophils elicit endothelium-dependent contraction in vascular smooth muscles. Circ Res 1989; 65: 917-924. 131 Sobey CC, Dusting GJ, Stewart AG, Woodman OL. Rabbit polymorphonuclear leukocytes cause endothelium-dependent contraction of rabbit aorta. J Vast Med Biol 1990; 2: 107115. [41 Sessa WC, Mullane KM. Release of a neutrophil-derived vasoconstrictor agent which augments platelet-induced contractions of blood vessels in vitro. Br J Pharmacol 1990; 99: 553-559. 151 Nishida M, Kuzuya T, Hoshida S, Kim Y, Kitabatake A, Kamada T, Tada M. Polymorphonuclear leukocytes induced vasoconstriction in isolated canine coronary arteries. Circ Res 1990; 66: 253-258. 161Ma X-L, Tsao PS, Viehman GE, Lefer AM. Neutrophilmediated vasoconstriction and endothelial dysfunction in low-flow perfusion-reperfused cat coronary artery. Circ Res 1991; 69: 95-106. t71 Sobey CG, Dusting GJ, Stewart AG. Tumor necrosis factor a augments the release of an endothelium-dependent vasoconstrictor from human polymorphonuclear leukocytes. J Cardiovasc Pharmacol 1992; 20: 813-819. PI Grossman HJ, Zambetis M. Leukocyte-induced endotheliumdependent vasodilatation and post-ischaemic vasospasm in the isolated rat superior mesenteric artery. Br J Exp Path01 1989; 70: 515-523. 191Mehta JL, Lawson DL, Nichols WW, Mehta P. Modulation of vascular tone by neutrophils: dependence on endothelial integrity. Am J Physiol 1989; 257: H1315-1320. 1101McCall TB, Boughton-Smith NK, Palmer RMJ, Whittle BJR, Moncada S. Synthesis of nitric oxide from L-arginine by neutrophils. Release and interaction with superoxide anion. Biochem J 1989; 261: 293-296. I111 Rimele TJ, Sturm RJ, Adams LM, Hemy DE, Heaslip RJ, Weichman BM, Grimes D. interaction of neutrophils with vascular smooth muscle: identification of a neutrophil-derived relaxing factor. J Pharmacol Exp Ther 1988; 245: 102-111. I121 Ishibashi T, Miller SL, Burstein SA. Type p transforming growth factor is a potent inhibitor of murine megakatyocytopoiesis in vitro. Blood 1987; 69: 1737-1741. 1131 Berliner JA, Territo M, Almada L, Carter A, Shatonsky E, Fogelman AM. Monocyte chemotactic factor produced by large vessel endothelial cells in vitro. Arteriosclerosis 1986; 6: 254-259. Cl41 Fujioka M, Nagao T, Huriyama H. Actions of the novel thromboxane A, antagonists, ONO-1270 and ONO-3708, on smooth muscle cells of the guinea-pig basilar artery. Naunyn-Schmiedeberg’s Arch Pharmacol 1986; 334: 468474. v51 Toda N, Nakajima M, Okamura T, Miyazaki M. Interactions of thromboxane A, analogs and prostaglandins in isolated dog arteries. J Cardiovasc Pharmacol 1986; 8: 818-825. t161 Kondo K, Seo R, Naka M, Kitagawa T, Wakitani K, Sakata M, Kira H, Okegawa T, Kawasaki A. Effects of ONO-3708, an antagonists of the thromboxane A,/prostaglandin endoperoxide receptor, on platelet aggregation and thrombosis. Eur J Pharmacol 1989; 163: 253-261
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[ 171 Rubanyi GM, Vanhoutte PM. Nature of endothelium-derived relaxing factor: Are there two relaxing mediators? Circ Res 1987; 6l(suppl II): 11-61-11-67. [18] Beny JL, Brunet PC, Van der Bent V. Hemoglobin causes both endothelium-dependent and endothelium-independent contraction of the pig coronary arteries, independently of an inhibition of EDRF effects. Experientia 1989; 45: 132-134. [19] Piomelli D, Feinmark SJ, Cannon PJ. Leukotrien biosynthesis by canine and human coronary arteries. J Pharmacol Exp Ther 1987; 241: 763-770. [20] Chiu PJS, Vemulapalli S, Bryant RW. Augmentation of leukotriene C, and D, release due to severe stenosis in the canine coronary artery stimulated by the calcium ionophore A23187. Biochem Pharmacol 1986; 35: 3593-3599. [21] Kanao M, Watanabe Y, Kimura Y, Saegusa J, Yamamoto K, Kanno H, Hanaya N, Kubo H, Ashida S, Ishikawa F. Thromboxane A, synthetase inhibitors. 2. syntheses and activities of tetrahydronaphthalene and indan derivatives. J Med Chem 1989; 32: 1326-1334. [22] Sakuma K, Kawamoto S, Watanabe K, Ashida S. FMLPinduced coronary flow reduction in isolated hearts of hypercholesterolemic rabbits and its inhibition by DP1904. Eur J Pharmacol 1990; 183: 791. [23] Douglas SA, Hiley CR. Endothelium-dependent vascular activities of endothelium-like peptides in the isolated superior mesenteric arterial bed of the rat. Br J Pharmacol 1990; 101: 81-88. [24] Sanchez-Madrid F, Nagy JA, Robbins E, Simon P, Springer TA. A human leukocyte differentiation antigen family with distinct cu-subunits and a common P-subunit: The lymphocyte function-associated antigen(LFA-1), the C3bi complement receptor (OKMl/Mac-1), and the ~150, 95 molecule. J Exp Med 1983; 158: 1785-1803. [25] Lunney JK. Characterization of swine leukocyte differentiation antigens. Immunol Today 1993; 14: 147-148. [26] Babior BM, Kipnes RS, Cunutte JT. The production by leukocytes of superoxide, a potential bacterialicidal agent. J Clin Invest 1973; 52: 741-744. [27] Gottlieb AL, Lippton HL, Parey SE, Paustian PW, Kadowitz PJ. Blockade of vasoconstrictor responses by prostacyclin (PGI,), PGE, and PGE, in the rabbit hindquarters vascular bed. Prostaglandins Med 1980; 4: l-11.
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[28] Dusting GJ, Moncada S, Vane JR. Vascular actions of arachidonic acid and its metabolites in perfused mesenteric and femoral beds of the dog. Eur J Pharmacol 1978; 49: 65-72. [29] Kulkarni PS, Roberts R, Needleman P Paradoxical endogenous synthesis of a coronary dilating substance from arachidonate. Prostaglandins 1976; 12: 337-353. [30] Lopez JAG, Armstrong ML, Harrison DG, Piegors DJ, Heistad DD. Vascular responses to leukocyte products in atherosclerotic primates. Circ Res 1989; 65: 1078-1086. [31] Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-l and interferon-gamma: Tissue distribution, biochemistry, and function of a natural adherence molecule (IQ&I-l). J Immunol 1986; 137: 245-254. [32] Fantone JA, Ward PA. Role of oxygen derived free radicals and metabolites in leukocyte-depedent inflammatory reactions. Am J Path01 1982; 107: 397-418. [33] Lucchesi BR, Mullane KM. Leukocytes and ischemia-induced myocardial injury. Ann Rev Pharmacol Toxic01 1986; 26: 201-224. [34] Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65: 513-525. [35] KIoner RA, Giacomelli F, Alker KJ, Hale SL, Matthews R, Bellows S. Influx of neutrophils into the walls of large epicardial coronary arteries in response to ischemia/reperfusion. Circulation 1991; 84: 1758-1772. [36] Gerrity RG, Gass JA, Soby L. Control of monocyte recruitment by chemotactic factor(s) in lesion-prone areas of swine aorta. Arteriosclerosis 1985; 5: 55-66. [37] Mazzone T, Jensen M, Chait A. Human arterial wall cells secrete factors that are chemotactic for monocytes. Proc Natl Acad Sci USA 1983; 80: 5094-5097. [38] Jauchem JR, Lopez M, Sprague EA, Schwartz CJ. Mononuclear cell chemoamactant activity from cultured arterial smooth muscle cells. Exp Mol Path 1982; 37: 166-170.
Journal
of
cardiology ELSEVIER
International Journal of Cardiology 55 (1996) 15-27
Unstimulated polymorphonuclear neutrophils regulate proximal coronary arterial tone Yukihiko Abe”, Tomiyoshi Saito”, Satoshi Kurodaa, Toshiyuki Ishibashi”, Mitsumasa Keitokub, Yukio Maruyamaa’* “First Department of Internal Medicine, Fukushima Medical College, 1 Hikarigaoka, Fukushima, 960-12, Japan bFirst Department of Internal Medicine, Tohoku University, Sendai, Miyagi, Japan
Received 26 September 1995; revised 16 January 1996; accepted 5 March 1996
Abstract Our objective is to clarify, by measuring the superoxide production as a marker of active state of polymorphonuclear neutrophils, whether unstimulated polymorphonuclear neutrophils would influence coronary arterial tone. We recorded the isometric tension of the porcine coronary arterial ring in a bath of oxygenated Krebs Ringer solution. Unstimulated porcine polymorphonuclear neutrophils that contained little superoxide were added to the bath. We also analyzed the prostaglandins produced in the bath. The isometric tension of arterial rings increased dose-dependently when polymorphonuclear neutrophils were added to the bath. The vasoconstriction induced by unstimulated polymorphonuclear neutrophils was inhibited by endothelial denudation, indomethacin, anti-CD1 la/l&like antibody. Thromboxane A, synthetase inhibitor and superoxide dismutase did not affect the vasoconstriction. Prostaglandin E, predominated among the prostaglandins produced in the bath; its production was significantly inhibited by indomethacin (without vs. with indomethacin; 3898 + 1704 vs 1956 + 715 pg/ml, P < 0.05, n = 6). Pretreatment of vascular rings with indomethacin blocked the interaction of the coronary artery with polymorphonuclear neutrophils. Results suggested that unstimulated polymorphonuclear neutrophils constrict the proximal coronary artery. Such vasoconstriction may be produced by cyclooxygenase products, especially prostaglandin E, produced in the vascular wall via the interaction between the polymorphonuclear neutrophils and the endothelium. Polymorphonuclear neutrophils may regulate coronary arterial tone. Keywords:
Unstimulated polymorphonuclear
neutrophils; Endothelium; Prostaglandin E,; Coronary arterial tone; Pig
1. Introduction Interaction between the polymorphonuclear neutrophils and the blood vessels releases a variety of substances that affect vascular tone. However, results of studies of polymorphonuclear neutrophils isolated *Corresponding author. Tel.: +0245 48 2111; fax: +0245 48 1821.
from various species do not necessarily coincide [l-lo]. Studies have reported vasoaction that was endothelium-dependent or -independent, i.e. the endothelium-dependent vasoconstrictor responses in rabbit and dog polymorphonuclear neutrophils [2,3,5], the endothelium-independent vasoconstrictor responses by rat and rabbit polymorphonuclear neutrophils [4,8]. Endothelium-dependent vasodilation has been observed in human polymorphonuclear
0167-5273/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO167-5273(96)02624-l
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neutrophils [9], while endothelium-independent vasodilation has been reported in rat and rabbit polymorphonuclear neutrophils [3,10]. The effects of polymorphonuclear neutrophils on vessel tone seem to be influenced by their condition. The functional status of polymorphonuclear neutrophils is thought to influence their interaction with the vessels. According to Sessa and Mullane [4], polymorphonuclear neutrophils activated by the calcium ionophore A23187 constricts aortic rings via the release of a protein-like contractile factor. Polymorphonuclear neutrophils activated by phorbol myristate acetate induce the contraction of rabbit coronary arteries via the generation of hydroxyl radical [l]. Ohlstein and Nichols reported that the contraction of rabbit aortic rings induced by fMLPactivated polymorphonuclear neutrophils was blocked by superoxide dismutase 121.In contrast, rat polymorphonuclear neutrophils elicited by the intra peritoneal injection of oyster glycogen relaxes aortic rings via the release of a substance resembling endothelium-derived relaxing factor [ 111. Unstimulated, spontaneous polymorphonuclear neutrophils reportedly produce differing changes in vascular tone; i.e. contraction and relaxation by polymorphonuclear neutrophils of the rabbit [3], contraction by these of the dog [5] and of the rabbit [4], and vasorelaxation by the supernatants of human polymorphonuclear neutrophils [7]. Thus, polymorphonuclear neutrophils may produce a variety of vasoactive effects in the unstimulated as well as the stimulated condition. No report has described whether the polymorphonuclear neutrophils used were determined to be unstimulated, especially during the interaction between polymorphonuclear neutrophils and blood vessels. Polymorphonuclear neutrophils may be stimulated by mechanical manipulation during isolation [3] or are upon addition to the organ bath [4]. Differing stimuli may then produce differing profiles of the polymorphonuclear neutrophils and blood vessel interactions. Thus, our objective was to clarify, by measuring the superoxide production as a marker of active state of polymorphonuclear neutrophils, whether unstimulated polymorphonuclear neutrophils would influence coronary arterial tone and regulate the basal tone. We conducted in vitro experiments to exclude the role of various factors which alter vascular tension, and
investigated the mechanism(s) of the vascular response produced between unstimulated polymorphonuclear neutrophils and the vascular endothelium.
2. Materials
and methods
2.1. Preparation of polymorphonuclear
neutrophils
Polymorphonuclear neutrophils were freshly prepared as follows [ 121. Thirty milliliters of porcine blood anti-coagulated with 17% volume of acidcitrate-dextrose (ACD) solution was mixed with 20ml of 6% dextran 70 in normal saline in a 60-ml syringe. The syringe was maintained upright for 1 h. Following sedimentation, the straw-colored upper layer was carefully removed and centrifuged at 150 g for 10 min. The pellet was washed with phosphatebuffered saline. After centrifugation, the cells were overlaid on Mono-poly-resolving-medium (Flow Laboratories Inc., Costa Mesa, CA.) and centrifuged at 300g for 20 min. The cells of the fraction below the interface were washed with phosphate-buffered saline and centrifuged at 150g for 10 min. To exclude mononuclear cells, the pellet was overlaid on Lymphoprep (NYCOMED AS, Oslo, Norway) and centrifuged at 300g for 20 min. The bottom pellet was washed with phosphate-buffered saline and adjusted to 10’ cells/ml. All procedures were carried out at room temperature. In this procedure, the purity of polymorphonuclear neutrophils, as determined by May-Giemsa staining, exceeded 98%. Cellular viability was determined by the trypan blue exclusion method and exceeded 95% in the final fraction, The prepared polymorphonuclear neutrophils did not contain measurable levels of either hemoglobin (
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NaHCO, 2.1, Ca-N, 0.00973, CaCl, 0.166, and glucose 2.0. The right coronary artery was dissected free and cleaned of adherent connective tissue, then cut into ring preparations 2Smm in length. Care was taken to avoid stretching and to keep endothelial cells intact and unrubbed. The endothelial cells of some vascular rings were mechanically damaged by gently rubbing the intimal surface with a wooden stick to create de-endothelialized rings. 2.3. Tension measurement
We used 115 coronary arterial rings from 36 pigs in the experiments on tension measurement. Coronary arterial rings were suspended in water-jacketed organ chambers filled with 10 ml Krebs Ringer bicarbonate solution at 37°C and continuously gassed with 95% 02-5%CO,. Each ring was suspended on a pair of stainless steel hooks, one of which was fixed to the side wall of the chamber, while the other was connected to a force-displacement transducer (Model T7-15-240, Orientec Corporation, Tokyo, Japan). Changes in isometric tension were recorded on a polygraph system (Model 1363, NEC-SANEI, Tokyo, Japan). The arterial rings were gradually stretched until the resting force reached 3g. The solution in the organ chamber was replaced every 20 min to wash out blood cells adherent to the vascular wall. After equilibration for 180 min, the arterial rings were contracted with 35 mM KCl, followed by the addition of 0.01 PM substance P to the organ chamber for the estimation of endothelial integrity. Endothelium-unrubbed rings relaxed in response to substance P, whereas the de-endothelialized rings did not. After washing the arterial rings and observing a steady resting state, we recorded changes in tension following the addition of polymorphonuclear neutrophils. The change in tension was expressed as a percentage of the contraction induced by 35 mM KCl. No polymorphonuclear neutrophils stimulants were used during the measurement of tension.
(2) Indomethacin was used to block cyclooxygenase. Since various concentrations of indomethacin (1 PM, 10 ,LLM, or 100 PM) [2,5,13] had been used, to determine the concentration appropriate for blocking the action of cyclooxygenase in porcine coronary arterial rings, we studied the concentration-dependency of indomethacin required to inhibit the vasoconstriction induced by arachidonic acid. Arachidonic acid at concentration of 0.1, 1 and 10 ,uM, respectively, was added cumulatively to the organ chamber. Changes in isometric vascular tension were recorded before and after the addition of indomethacin, 2 or 20 ,uM. (3) To detect the vasoactive substances that altered the isometric vascular developed tension, we added the antagonist to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml. Seven antagonists were used: 1 ,uM ON03708 (a TXA,/PGH, receptor antagonist, n=6) [14-161, 20 PM indomethacin (a cyclonordioxygenase inhibitor, n=6), 20 PM hydroguaiaretic acid (a lipoxygenase inhibitor, n = 6) [17-201, 100 PM DP-1904 (a TXA, synthetase inhibitor, n=5) [21,22], 1 ,uM ketanserin (an S,-blocker, n=5), 1 ,uM pyrilamine (an H,blocker, n=5) or 2OOU/ml superoxide dismutase (n=5). The concentrations of ON03708 and nordihydroguaiaretic acid used were obtained from previous reports [14-201. We confirmed that the
lo5
2.4. Experimental
protocol
(1) A cumulative volume of polymorphonuclear neutrophils, lo’--5 x lo6 cells/ml, was added to the organ chamber, and the changes in isometric vascular tension were recorded (n = 7).
11
55 (1996) 15-27
5x105 Concentration
105 of
5X106ceIIs,mI PYNs
Fig. 1. Relation between polymorphonuclear neutrophil concentration and polymorphonuclear neutrophils-induced developed tension in porcine coronary arterial rings. Polymorphonuclear neutrophils-induced vasoconstriction (mean ? S.D.) was expressed as % of the contraction induced by 35mM KC1 (n=7).
18
Journal of Cardiology 55 (1996) 15-27
Y. Abe et al. I International
.
.. ..
. . .. . .
PMNs
b -te..f . PMNs
IM
wo
wo
PMNs
.. . . wo
ON0
PMNs
c . PMNs
. . . wo
. NDGA
. .
.
wo
PMNs I 1 Omin
Fig. 2. Typical traces of polymorphonuclear neutrophils-induced vasoconstriction suppressed by several blockers; a, IM; b, ONO; c, NDGA. PMNs, 106/ml polymorphonuclear neutrophils; wo, washout; IM, 20 PM indomethacin; ONO, 1 PM ONO-3708; NDGA, 20 PM nordihydroguaiaretic acid.
concentrations of the antagonists used in this study did not affect the vasoconstriction induced by 35 mM KCl. (4) To detect the main site of production of vasoactive substances, we pretreated the vascular rings or the polymorphonuclear neutrophils with 20pM indomethacin, then observed the interaction between the vascular rings and polymorphonuclear neutrophils as follows. Vascular rings were pretreated with indomethacin for 15 min followed by repeated washings. Polymorphonuclear neutrophils were then added to the vascular rings (n = 5), or a suspension of polymorphonuclear neutrophils pretreated with indomethacin for 15 min was washed and added to the vascular rings not exposed to indomethacin (n = 5). (5) Rings denuded of endothelium were used to study the role of the vascular endothelium in the polymorphonuclear neutrophils-induced alteration of vascular tone. Following denudation as described previously, the vascular ring was suspended. Polymorphonuclear neutrophils, lo6 cells/ml, were then added to the chamber, with the vascular tension
recorded by the procedure used without denudation (n = 6). (6) To examine the effect of NO on the poly-
morphonuclear neutrophils-induced alteration of vascular tone, we added 100 ,uM Nw-nitro-L-arginine methyl ester (NO blocker) [23] to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml (n = 5). (7) To examine the effect of stimulated polymorphonuclear neutrophils with a stimulant on the polymorphonuclear neutrophils-induced alteration of vascular tone, we added 2 ,uM A23187 to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils, lo6 cells/ml (n = 5). (8) To examine the role of receptors on the polymorphonuclear neutrophils and vascular endothelium on the polymorphonuclear neutrophils-induced alteration of vascular tone, 35 mM KC1 was first added to the organ chamber. After vascular tension had reached a plateau, a concentration of 1 or long/ml anti-CD 1la/ 18-like antibody was added to the organ chamber, followed by the addition of polymorphonuclear neutrophils, lo6 cells/ml, 15-20
Y. Abe et al. I International
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19
%
% 40
35-i
35
T
pco.01 l-r
30
25
25 20 15 10 5 0 1 control
control
IM
control
ON03708
NDGA
%
% 30
40 -
N.S.
pco.01
35 -
contol
DPI 904
control
SOD
control
denuded
Fig. 3. Bar graphs showing the effects of several antagonists or de-endothelialization on polymorphonuclear neutrophils-induced vasoconsttiction. IM, ON03708, NDGA, DP1904, SOD; 20 PM indomethacin, 1 PM ONO-3708, 20 PM nordihydroguaiaretic acid, 100 PM DP-1904 or 2OOU/ml superoxide dismutase was introduced into the chamber, respectively, and polymorphonuclear neutrophils-induced vasoconsttiction was compared with that without intervention (‘control’); denuded, contraction of ring denuded of endothelium, control; vascular ring and polymorphonuclear neutrophils were untreated.
min later (II = 6). Anti-CD 1la/ 18-&e antibody was selected because CD lla/18 is present on the surface of polymorphonuclear neutrophils, even spontaneous polymorphonuclear neutrophils [24], and only this antibody was commercially available for use in pigs. This antibody is active against porcine as well as equine leukocytes [25]. We conducted this experiment at the concentrations of
anti-CD lla/l8-like antibody that did not affect the vasoconstriction induced by 35 mM KCl. 2.5. Determination of superoxide production We used 28 coronary arterial rings from 21 pigs to evaluate the production of superoxide from polymorphonuclear neutrophils, which was measured
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Fig. 4. Bar graphs showing the effects of indomethacin pretreatment of vascular ring (n = 5) or polymorphonuclear neutrophils (n = 5) alone on polymorphonuclear neutrophils-induced vasoconstriction. IM-vascular, IM-PMNs; vascular ring or polymorphonuclear neutrophils alone was pretreated with 20 PM indomethacin respectively. Control; neither the vascular ring nor the polymorphonuclear neutrophils were treated with indomethacin.
spectrophotometrically using ferricytochrome c [26]. A porcine coronary arterial ring was mounted in a 20-ml chamber containing Krebs-Ringer bicarbonate buffer. The solution in that chamber was maintained at 37°C and continuously aerated with 95% O,-5% CO, under the conditions described earlier. Polymorphonuclear neutrophils, lo6 cells/ml, and ferricytochrome c, 50 PM, were added to the chamber, which was composed of a couple of small chambers connected to each other. Vascular tension was con-
Table 1 Superoxide production and vasoconstriction Min.
0 5 10 15 20 30 40 50 60
observed following
55 (1996) 15-27
tinuously measured in one of chambers. At the same time, 3 ml of the solution was taken from another chamber, decanted into a small tube, and the absorbance was measured at 550 nm every 5 min until 60 min after administration of polymorphonuclear neutrophils. In 14 cases, polymorphonuclear neutrophils were stimulated with 2 PM A23187. In 7 of those cases, 200U/ml superoxide dismutase was added to the chambers. Thereafter, superoxide production was measured in the same way. Data were calculated as nanomoles of superoxide released per lo6 polymorphonuclear neutrophils, based on an extinction coefficient of 15.5mM-‘cm-’ for cytochrome c reduction [26]. 2.6. Microscopic observation of polymorphonuclear neutrophils In 21 preparations from 21 pigs, we microscopically observed the polymorphonuclear neutrophils in a smear from each chamber, which was stained with May-Giemsa solution, before and after the experiment on tension measurement. The number of degranulated polymorphonuclear neutrophils per 1000 cells was counted. 2.7. Measurement of prostaglandins We analyzed the prostaglandins produced by the 67 coronary arterial rings from 14 pigs in the organ chamber using radioimmunoassay. A volume of 106/
administration of polymorphonuclear
Production of superoxide (n mol/ lo6 PMNs) VandPandCa (n=7)
VandPandCaandS (n=7)
VandP (n=7)
0+-o 14.6 t 9.2 16 5 9.2 17 _t 8.6 18.3 I 8.4 19.6 2 10.5 19.6 +- 10.7 19.6 ? 10.7 19.4 k 11.1
o-t-0 020 020 o-co 020 Ok0 020 oio oio
Ok0 0.1 t 0.3 0.6 -c 0.8 0.7 lb 1.0 0.7 2 1.5 0.7 i 1.1 0.5 t 1.2 0.5 i 1.2 0.6 k 1.2
V, coronary arterial ring; P or PMNs, polymorphonuclear
neutrophils Vasoconstriction (%) produced by PMNs without stimulant (n = 7) Ok0 0.6 IT 0.8 6.4 i 1.4 8.4 i- 1.8 10.0 ? 1.9 20.2 2 2.2 22.9 k 34 24.8 i 4.6 25.1 i 4.6
neutrophils; Ca, Ca ionophor A23187; S, superoxide dismutase.
Journal of Cardiology
Y. Abe et al. I International p
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p<0.05.n=4
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21
p
n
n
N.S.
PGE: wlml
PGhs n=5 wlm n
pco.01 n=5
N.S. n=4
n
n
I,
T
200
cant
IM
100 I L, pco.05 n=4 i---7
T
cant
IM
cant
CA
cant
PMNs
cant
IM
cant
CA
cant
p
T
cant
PMNs
p
T
CA
cant
PMNs
Fig. 5. Production of prostaglandin E, (PGE,, left panel), prostaglandin F,, (PGF,,, right upper panel), and tbromboxane B, (TXB,, right lower panel). cant, produced in the solution with lo6 polymorphonuclear neutrophils and coronary arterial ring; TM, produced in the solution with polymorphonuclear neutrophils, coronary arterial ring and 20 PM indomethacin; CA, produced in the solution with coronary arterial ring alone; PMNs, produced in the solution with polymorphonuclear neutrophils alone.
ml polymorphonuclear neutrophils was added to the organ chamber in which the porcine coronary artery ring was mounted. When the vascular tension reached a maximum, the solution was removed to a tube containing indomethacin at -20°C. The concentrations of thromboxane B, and prostaglandin E, in the solution were then measured with a lz51 assay system (Amersham, Buckinghamshire, England). ProstaglandinF,, was measured with a 3H radioimmunoassay kit (Baxter Travenol Diagnostics, Inc., Cambridge, MA). The prostaglandins released during the interaction between the arterial ring and the polymorphonuclear neutrophils were measured both without and with the addition of 20 ,uM indomethacin. We also determined the concentration of
prostaglandins in the chamber containing only the vascular ring or the polymorphonuclear neutrophils. 2.8. Agents
The following agents were purchased from Sigma Chemical, St. Louis, MO: calcium ionophore A23 187 (free acid), indomethacin, nordihydroguaiaretic acid (NDGA), pyrilamine maleate salt, ketanserin, prostaglandin Fza, prostaglandin E,, ferricytochrome c (equine heart type VI), Nw-nitro-Larginine methyl ester. Recombinant human superoxide dismutase (4000 units/mg) was a gift from Ube IL, Tokyo, Japan. ONO-3708 was kindly supplied by the OPCL, Osaka, Japan. DP-1904 was a gift from
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DPCL, Tokyo, Japan. Anti-CD 1la/ lx-like antibody was purchased from VMRD, Inc., Pullman, WA. 2.9. Analysis of data Results are expressed as mean + S.D. The vascular contraction induced by polymorphonuclear neutrophils was compared with that induced by 35mM KCl. We compared the tension developed in porcine coronary arterial rings at differing concentrations of polymorphonuclear neutrophils, or the polymorphonuclear neutrophils-induced vasoconstriction with differing concentrations of anti-CD1 la/ 18-like antibody, using one-way analysis of variance followed by Fisher’s PLSD. Other results were analyzed by the paired Student’s t-test. A P value less than 0.05 was considered statistically significant.
3. Results
3.1. Tension induced by polymorphonuclear neutrophils in porcine coronary arterial rings The isometric tension of porcine arterial rings increased dose-dependently when porcine polymorphonuclear neutrophils were added to the organ chamber at concentrations of lo5 to 5 x 106/ml (Fig. 1, n =7). The vasoconstriction induced by polymorphonuclear neutrophils achieved a maximum tension in approximately 60 min, and persisted for at least 30 min. The sustained contraction returned to basal tension following repeated washing. To examine the reproducibility of the polymorphonuclear neutrophils-induced vasoconstriction, the arterial rings were repeatedly exposed to polymorphbnuclear neutrophils. The arteries after the exposure to polymorphonuclear neutrophils produced the contraction with same magnitude as observed before the exposure (data not shown). 3.2. Concentration of indomethacin required to inhibit cyclooxygenase in porcine coronary arterial rings The contraction of porcine coronary arterial rings was dependent on the concentration of arachidonic acid; that is, it was 10.1 +- 8.8, 24.9 ? 12.4 and 52.9 + 8.7% (n=7) at doses of 0.1, 1.0 and 10 PM,
of Cardiology 55 (1996) 15-27 respectively. A concentration of 20 PM indomethacin suppressed the vasoconstriction induced by arachidonic acid (0.0 t 0.0, 1.0 ? 0.97, 1.7 +1.25%, n=7, P < 0.01 vs without indomethacin). However, 2 PM indomethacin did not significantly suppress vasoconstriction (4.0 ‘-t 1.0, 13.9 2 4.8, 36.4 + 17.0%, n=7, N.S.). The vasoconstriction induced by 35mM KC1 was not affected by exposure to 20 PM indomethacin (data not shown). From these results, we chose a concentration of 20 PM indomethacin for study. 3.3. Effect of vasoactive substances on vasoconstriction induced by polymorphonuclear neutrophils The tracing in Fig. 2 shows the effects of indomethacin (IM in Fig. 2a), ONO-3708 (ON0 in Fig. 2b) and nordihydroguaiaretic acid (NDGA in Fig. 2c) on the vasoconstriction induced by polymorphonuclear neutrophils. Fig. 3 shows the mean values of the effects of the various blockers on the polymorphonuclear neutrophils-induced vasoconstriction. It was significantly suppressed by indomethacin (IM in Fig. 3, n=6), ONO-3708 (ON03708 in Fig. 3, n=6), and nordihydroguaiaretic acid (NDGA in Fig. 3, n=6). By contrast, the polymorphonuclear neutrophils-induced vasoconstriction was unaffected by DP-1904 (DP1904 in Fig. 3, n=5), ketanserin (data not shown), pyrilamine (data not shown) and superoxide dismutase (SOD in Fig. 3, n=5). 3.4. Detection of site of production of vasoactive substances When a coronary ring alone was pretreated with indomethacin (‘IM-vascular’, in Fig. 4), the vasoconstriction induced by polymorphonuclear neutrophils was suppressed. However, pretreatment of the polymorphonuclear neutrophils suspension alone (‘IMPMNs’ in Fig. 4) did not alter the development of isometric tension. 3.5. Role of endothelium in polymorphonuclear neutrophils-induced vasoconstriction When polymorphonuclear neutrophils were added to the chamber in which a vascular ring denuded of
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endothelium was suspended (‘denuded’ in Fig. 3), the vascular ring contracted only slightly (P < 0.01, n=6). 3.6. Effect of NO in polymorphouclear induced vasoconstriction
neutrophils-
When the Nw-nitro-L-arginine methyl ester was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was not affected (control vs Nw-nitro-L-arginine methyl ester; 20.7 5 5.8 vs 21.8 2 6.5%, n = 5, NS). 3.7. Comparison between unstimulated and stimulated polymorphonuclear neutrophils-induced vasoconstriction When A23187 was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was increased (unstimulated vs A23187-stimulated; 20.5 + 8.2 vs 52.2 2 18.5%, n = 5, P < 0.01). 3.8. Role of receptors on polymorphonuclear neutrophils and vascular endothelium in vasoconstriction induced by polymorphonuclear neutrophils When the anti-CD lla/l8-like antibody was added to the organ chamber 15-20 min before the addition of lo6 cells/ml polymorphonuclear neutrophils, the vasoconstriction induced by polymorphonuclear neutrophils was suppressed dose-dependently in the range of 1 to 10 rig/ml of the antibody (control, 1 rig/ml, long/ml; 16.7 i. 4.8, 11.8 -+ 6.4, -3.2 + 5.4%*, n = 6, *P < 0.01 vs control).
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3.10. Microscopic observation of polymorphonuclear neutrophils Degranulated polymorphonuclear neutrophils comprised less than 1% of the sample as observed on microscopic examination before and after the experiments without and with polymorphonuclear neutrophils-stimulant (without and with stimulant; n = 10, n = 11). Thus, their cellular structure of polymorphonuclear neutrophils remained virtually intact during these experiments. 3.11. Measurement of prostaglandins Fig. 5 shows the production of prostaglandins. Prostaglandin E, was produced most prominently in the chamber that contained a vascular ring and polymorphonuclear neutrophils (‘cant’ in the left panel). However, the amount of prostaglandin E, produced by either the vascular ring or the polymorphonuclear neutrophils alone was significantly less than that containing a vascular ring and polymorphonuclear neutrophils. Indomethacin pretreatment (IM in the left panel) inhibited the production of prostaglandin E,. With the vascular ring and polymorphonuclear neutrophils (right upper panel), prostaglandin F,, was produced, but to a lesser extent than prostaglandin E,. Production of prostaglandin F,, was not inhibited by indomethacin for an unknown reason. Little prostaglandin F,, was produced by either the vascular ring or the polymorphonuclear neutrophils alone. With the vascular ring and polymorphonuclear neutrophils, thromboxane B, was produced (right lower panel), but to a lesser extent than prostaglandin E,. Thromboxane B, production was inhibited by indomethacin. Little thromboxane B, was produced by either the vascular ring or the polymorphonuclear neutrophils alone.
3.9. Measurement of superoxide anion
4. Discussion
In this experiment conducted without the polymorphonuclear neutrophils-stimulant, the calcium ionophore A23 187, the vascular tension increased, but only a trace of superoxide was detected. Use of A23187 led to the marked production of superoxide which was suppressed by 2OOU/ml superoxide dismutase (Table 1).
The effects of polymorphonuclear neutrophils on vessel tone seem to be influenced by their condition. Polymorphonuclear neutrophils in the present study were considered to be unactivated from (a) evidence of only a trace of superoxide production, even after tension development, and (b) microscopic observation of small number (i.e. less than 1%) of degranu-
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lated polymorphonuclear neutrophils after tension development due to administration of polymorphonuclear neutrophils to the bath containing a vessel. These unactivated polymorphonuclear neutrophils elicited contraction of the large epicardial coronary artery. The magnitude of the endothelium-dependent vasoconstriction depended upon the number of polymorphonuclear neutrophils administered. The vasoconstriction induced by polymorphonuclear neutrophils was inhibited by a cyclooxgenase inhibitor, a thromboxane A,/prostaglandin H, blocker or an anti-CD 1la/ 1&-like antibody, which showed that cyclooxygenase products, which are produced in the vascular wall via the interaction of polymorphonuclear neutrophils to the endothelium of the coronary artery, may be responsible for the vasoconstriction. From the results of the reduction in arterial tension induced by esculetin and the AA861, Nishida et al. considered that the polymorphonuclear neutrophilsinduced vasoconstriction in isolated canine coronary arterial rings was produced by the release of vasoconstrictive substances generated by the polymorphonuclear neutrophils-endothelial system, such as the leukotriene A, steal mechanism, in which leukotriene A, synthesized by polymorphonuclear neutrophils was transferred intercellularly to endothelial cells and served as the substrate of the leukotriene C, synthetase system that exists in endothelial cells [5]. As the vasoconstriction induced by polymorphonuclear neutrophils was also suppressed in our study by the lipoxygenase inhibitor, nordihydroguaiaretic acid, we considered that this mechanism plays a partial role. In Nishida’s report, indomethacin did not reduce the vasoconstriction induced by polymorphonuclear neutrophils. However, in our study, the vasoconstriction induced by polymorphonuclear neutrophils was suppressed by indomethacin and by the thromboxane A, /prostaglandin H, receptor antagonist, ONO-3708. Since Toda et al. previously reported that the coronary arterial contractions induced by prostaglandin F 2a, prostaglandin E, and prostaglandin D, were also suppressed by ONO-3708 u51, our findings indicated that cyclooxygenase products, as well as leukotriene, participate in the vasoconstriction induced by polymorphonuclear neutrophils. When only the coronary ring was pretreated with indomethacin, the polymorphonuclear neutrophils-induced vasocohstriction was suppressed. In
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contrast, pretreatment of the polymorphonuclear neutrophils suspension with indomethacin did not alter the development of isometric tension. These results strongly suggest that the constriction of the coronary artery induced by polymorphonuclear neutrophils is brought about by the cyclooxygenase products generated in the vascular wall. The reason for the discrepancy between our findings and those of Nishida regarding the reduction in polymorphonuclear neutrophils-induced vasoconstriction by indomethacin is not clear. A difference in methodology, such as species differences and differing doses of indomethacin, may be responsible. Before using indomethacin in our protocol, we evaluated the concentration of this agent that would inhibit the vasoconstriction induced by arachidonic acid. An indomethacin concentration of 20 ,uM, but not of 2 ,uM, suppressed the vasoconstriction induced by arachidonic acid. Accordingly, we used 20 PM of indomethacin. However, a lower dose of 1 PM indomethacin was used in Nishida’s study [5]. The functional status of the polymorphonuclear neutrophils used in their study was also not defined. Our differing results may be related to the differing characteristics of the polymorphonuclear neutrophils evaluated. To study the effect of NO in the unstimulated polymorphonuclear neutrophils-induced vasoconstriction, we added 100 PM Nw-nitro-L-arginine methyl ester to the organ chamber 15-20 min before adding the polymorphonuclear neutrophils. The polymorphonuclear neutrophils-induced vasoconstriction was unnaffected by Nw-nitro-L-arginine methyl ester. We considered that NO did not participate in the unstimulated polymorphonuclear neutrophils-induced vasoconstriction. By contrast, Ohlstein and Nichols reported that activated polymorphonuclear neutrophils can release superoxide anion and produce endothelium-dependent contraction. They considered that the endothelium-dependent contraction may be the result of superxide anion inactivation of endothelium-derived relaxing factor [2]. From our study, A23187 (polymorphonuclear neutrophils-stimulant) increased polymorphonuclear neutrophils-induced vasoconstriction. We speculate that the mechanism of unstimulated polymorphonuclear neutrophils-induced vasoconstriction may differ from that of stimulated polymorphonuclear neutrophils.
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Prostaglandin E, produces vasodilation in the artery of the hind leg of the rabbit [27] and the dog [28]. This substance is a potent vasoconstrictor in the coronary arteries of the cow, dog and human [29]. Lopez et al. reported that fMLP contracts the atherosclerotic artery of the monkey in vivo and that the prostaglandin E, released from the leukocytes participates in that phenomenon [30]. Prostaglandin E, and thromboxane B, were the predominant prostaglandins detected in our study; such production was inhibited by indomethacin. We also confirmed that lo-‘M prostaglandin E, contracted the porcine coronary arterial ring (46.8 t 16.8%, n = 4). However, doses of prostaglandin E, below 10M6 M had no effect. The concentrations of prostaglandin E, (left panel in Fig. 5, 3.9 + 1.7 x 1O-6 mg/ml = 1.1 + 0.5 x lo-‘M) were well below that value. We suggest that the tissue concentration of prostaglandin E, in the vascular wall produced by the interaction between polymorphonuclear neutrophils and the coronary arterial ring (not measured in our study) may be sufficient to induce vasoconstriction, and that the concentration of prostaglandin E, measured in the bath may have been diluted. As the polymorphonuclear neutrophils-induced vasoconstriction was not inhibited by DP-1904, a selective thromboxane A, synthetase inhibitor, we consider cyclooxygenase products, especially prostaglandin E,, to be major participants in the vasoconstriction induced by polymorphonuclear neutrophils. In the present experiment, anti CD lla/l&like antibody reduced the vasoconstriction induced by polymorphonuclear neutrophils. Our data showed that the receptor mechanism on the polymorphonuclear neutrophils and the vascular endothelium may participate in the polymorphonuclear neutrophils-induced vasoconstriction. It has been considered that CD 1la/ 18 emerges when the polymorphonuclear neutrophils are stimulated. However, another report holds that the intracellular adhesion molecules, i.e. CDlla, CDllb, CDllc and CDl8, are present on the surface of polymorphonuclear neutrophils without stimulation [24], and that ICAMis generally present on the vascular endothelium [31]. We suggest that the CDlla/CDl& which is present on the surface of unstimulated polymorphonuclear neutrophils, and the ligand on the vascular endothelium may participate in the vasoconstriction induced by
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unstimulated polymorphonuclear neutrophils. That is, if polymorphonuclear neutrophils adherence to the endothelium is considered to be the first step in the polymorphonuclear neutrophils/blood vessel interaction, the present data appear to be reasonable. Several methodological problems should be pointed out. First, it is difficult to assess how much the polymorphonuclear neutrophils were activated. In the present experiment, polymorphonuclear neutrophils were isolated under germ-free conditions. No polymorphonuclear neutrophils stimulants were used in the measurement of vascular contraction, resulting in the generation of very little superoxide. Further, A23 187 (polymorphonuclear neutrophils-stimulant) increased polymorphonuclear neutrophils-induced vasoconstriction, that was useful to provide additional evidence that mechanical stimuli during isolation process did not stimulate polymorphonuclear neutrophils in the present study. The reason of microscopic observation of small number (i.e. less than 1%) of degranulated polymorphonuclear neutrophils with Ca-ionophore A23 187 is unclear. We speculate that polymorphonuclear neutrophils have various stimulated stages and polymorphonuclear neutrophils used in our experiments might not reach the degranulated stage, even if A23187 was administered. We thus consider that unstimulated polymorphonuclear neutrophils can change vascular tone. There are several reports which described that polymorphonuclear neutrophils migrate to sites of tissue damage and release oxygen free radicals, arachidonic acid metabolites and lysosomal proteases, which exacerbate tissue injury during coronary artery occlusion [32,33]. Thus, we compared the difference of stimulated and unstimulated polymorphonucIear neutrophils. As a result, developed vascular tension due to stimulated polymorphonuclear neutrophils was clearly different, as mentioned above. Although we studied mainly unstimulated polymorphonuclear neutrophils-induced vasoconstriction, it is necessary to further investigate the difference between stimulated and unstimulated polymorphonuclear neutrophils-induced alteration of vascular tone in more detail. Second, concerning the interaction between the vessel and the polymorphonuclear neutrophils, it is considered that, in small veins, the rolling and sliding of polymorphonuclear neutrophils occur initially along the-endothelial surface during inflamma-
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tion which is then followed by their extravascular migration [34]. Velocity of flow in the coronary artery is rapid relative to that of venous flow; therefore, it is not known whether an interaction between polymorphonuclear neutrophils and the arterial vascular wall is normally induced in vivo. Kloner et al. showed in vivo that the neutrophils migrated into the large epicardial coronary arteries after ischaemia and reperfusion, and that this migration occurred after reperfusion and not during ischaemia [35]. Monocytes migrate into the vascular wall during formation of the atherosclerotic plaque [13,36-381, suggesting that polymorphonuclear neutrophils in the blood stream may adhere to the vascular wall and interact with it, even at high speed of arterial flow. Finally, polymorphonuclear neutrophils in arterial blood flow may touch the endothelium and leave it, or roll upon it. Polymorphonuclear neutrophils and the endothelium may interact to produce vasoactive substances, especially prostaglandin E, and leukotriene, mainly from the vascular wall. Vasoactive substances may keep to maintain the coronary arterial tone. However, the basal tone of the coronary arteries in vivo results from the balance between vasoconstrictor and vasodilator factors. If one or more factor(s) break down, coronary arterial tone increases and sometimes coronary vasospasm may result. We clearly showed that unstimulated polymorphonuclear neutrophils constrict the proximal coronary artery. Cyclooxygenase products, particularly the prostaglandin E, produced in the vascular wall via the interaction between the polymorphonuclear neutrophils and the endothelium, may be importantly involved as a vasoconstrictor. Polymorphonuclear neutrophils may regulate coronary arterial tone in the in vivo basal condition, as their concentration in the organ bath (lo6 to 5 x 106/ml) was within the physiological range for polymorphonuclear neutrophils in the circulation. However, further in vivo studies should be done to confirm this possibility.
References [l] Gillespie MN, Kojima S, Kunitomo M, Jay M. Coronary and myocardial effects of activated neutrophils in perfused rabbit hearts. .I Pharmacol Exp Ther 1986; 239: 836-840.
121Ohlstein EH, Nichols AJ. Rabbit polymorphonuclear neutrophils elicit endothelium-dependent contraction in vascular smooth muscles. Circ Res 1989; 65: 917-924. 131 Sobey CC, Dusting GJ, Stewart AG, Woodman OL. Rabbit polymorphonuclear leukocytes cause endothelium-dependent contraction of rabbit aorta. J Vast Med Biol 1990; 2: 107115. [41 Sessa WC, Mullane KM. Release of a neutrophil-derived vasoconstrictor agent which augments platelet-induced contractions of blood vessels in vitro. Br J Pharmacol 1990; 99: 553-559. 151 Nishida M, Kuzuya T, Hoshida S, Kim Y, Kitabatake A, Kamada T, Tada M. Polymorphonuclear leukocytes induced vasoconstriction in isolated canine coronary arteries. Circ Res 1990; 66: 253-258. 161Ma X-L, Tsao PS, Viehman GE, Lefer AM. Neutrophilmediated vasoconstriction and endothelial dysfunction in low-flow perfusion-reperfused cat coronary artery. Circ Res 1991; 69: 95-106. t71 Sobey CG, Dusting GJ, Stewart AG. Tumor necrosis factor a augments the release of an endothelium-dependent vasoconstrictor from human polymorphonuclear leukocytes. J Cardiovasc Pharmacol 1992; 20: 813-819. PI Grossman HJ, Zambetis M. Leukocyte-induced endotheliumdependent vasodilatation and post-ischaemic vasospasm in the isolated rat superior mesenteric artery. Br J Exp Path01 1989; 70: 515-523. 191Mehta JL, Lawson DL, Nichols WW, Mehta P. Modulation of vascular tone by neutrophils: dependence on endothelial integrity. Am J Physiol 1989; 257: H1315-1320. 1101McCall TB, Boughton-Smith NK, Palmer RMJ, Whittle BJR, Moncada S. Synthesis of nitric oxide from L-arginine by neutrophils. Release and interaction with superoxide anion. Biochem J 1989; 261: 293-296. I111 Rimele TJ, Sturm RJ, Adams LM, Hemy DE, Heaslip RJ, Weichman BM, Grimes D. interaction of neutrophils with vascular smooth muscle: identification of a neutrophil-derived relaxing factor. J Pharmacol Exp Ther 1988; 245: 102-111. I121 Ishibashi T, Miller SL, Burstein SA. Type p transforming growth factor is a potent inhibitor of murine megakatyocytopoiesis in vitro. Blood 1987; 69: 1737-1741. 1131 Berliner JA, Territo M, Almada L, Carter A, Shatonsky E, Fogelman AM. Monocyte chemotactic factor produced by large vessel endothelial cells in vitro. Arteriosclerosis 1986; 6: 254-259. Cl41 Fujioka M, Nagao T, Huriyama H. Actions of the novel thromboxane A, antagonists, ONO-1270 and ONO-3708, on smooth muscle cells of the guinea-pig basilar artery. Naunyn-Schmiedeberg’s Arch Pharmacol 1986; 334: 468474. v51 Toda N, Nakajima M, Okamura T, Miyazaki M. Interactions of thromboxane A, analogs and prostaglandins in isolated dog arteries. J Cardiovasc Pharmacol 1986; 8: 818-825. t161 Kondo K, Seo R, Naka M, Kitagawa T, Wakitani K, Sakata M, Kira H, Okegawa T, Kawasaki A. Effects of ONO-3708, an antagonists of the thromboxane A,/prostaglandin endoperoxide receptor, on platelet aggregation and thrombosis. Eur J Pharmacol 1989; 163: 253-261
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[ 171 Rubanyi GM, Vanhoutte PM. Nature of endothelium-derived relaxing factor: Are there two relaxing mediators? Circ Res 1987; 6l(suppl II): 11-61-11-67. [18] Beny JL, Brunet PC, Van der Bent V. Hemoglobin causes both endothelium-dependent and endothelium-independent contraction of the pig coronary arteries, independently of an inhibition of EDRF effects. Experientia 1989; 45: 132-134. [19] Piomelli D, Feinmark SJ, Cannon PJ. Leukotrien biosynthesis by canine and human coronary arteries. J Pharmacol Exp Ther 1987; 241: 763-770. [20] Chiu PJS, Vemulapalli S, Bryant RW. Augmentation of leukotriene C, and D, release due to severe stenosis in the canine coronary artery stimulated by the calcium ionophore A23187. Biochem Pharmacol 1986; 35: 3593-3599. [21] Kanao M, Watanabe Y, Kimura Y, Saegusa J, Yamamoto K, Kanno H, Hanaya N, Kubo H, Ashida S, Ishikawa F. Thromboxane A, synthetase inhibitors. 2. syntheses and activities of tetrahydronaphthalene and indan derivatives. J Med Chem 1989; 32: 1326-1334. [22] Sakuma K, Kawamoto S, Watanabe K, Ashida S. FMLPinduced coronary flow reduction in isolated hearts of hypercholesterolemic rabbits and its inhibition by DP1904. Eur J Pharmacol 1990; 183: 791. [23] Douglas SA, Hiley CR. Endothelium-dependent vascular activities of endothelium-like peptides in the isolated superior mesenteric arterial bed of the rat. Br J Pharmacol 1990; 101: 81-88. [24] Sanchez-Madrid F, Nagy JA, Robbins E, Simon P, Springer TA. A human leukocyte differentiation antigen family with distinct cu-subunits and a common P-subunit: The lymphocyte function-associated antigen(LFA-1), the C3bi complement receptor (OKMl/Mac-1), and the ~150, 95 molecule. J Exp Med 1983; 158: 1785-1803. [25] Lunney JK. Characterization of swine leukocyte differentiation antigens. Immunol Today 1993; 14: 147-148. [26] Babior BM, Kipnes RS, Cunutte JT. The production by leukocytes of superoxide, a potential bacterialicidal agent. J Clin Invest 1973; 52: 741-744. [27] Gottlieb AL, Lippton HL, Parey SE, Paustian PW, Kadowitz PJ. Blockade of vasoconstrictor responses by prostacyclin (PGI,), PGE, and PGE, in the rabbit hindquarters vascular bed. Prostaglandins Med 1980; 4: l-11.
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[28] Dusting GJ, Moncada S, Vane JR. Vascular actions of arachidonic acid and its metabolites in perfused mesenteric and femoral beds of the dog. Eur J Pharmacol 1978; 49: 65-72. [29] Kulkarni PS, Roberts R, Needleman P Paradoxical endogenous synthesis of a coronary dilating substance from arachidonate. Prostaglandins 1976; 12: 337-353. [30] Lopez JAG, Armstrong ML, Harrison DG, Piegors DJ, Heistad DD. Vascular responses to leukocyte products in atherosclerotic primates. Circ Res 1989; 65: 1078-1086. [31] Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-l and interferon-gamma: Tissue distribution, biochemistry, and function of a natural adherence molecule (IQ&I-l). J Immunol 1986; 137: 245-254. [32] Fantone JA, Ward PA. Role of oxygen derived free radicals and metabolites in leukocyte-depedent inflammatory reactions. Am J Path01 1982; 107: 397-418. [33] Lucchesi BR, Mullane KM. Leukocytes and ischemia-induced myocardial injury. Ann Rev Pharmacol Toxic01 1986; 26: 201-224. [34] Harlan JM. Leukocyte-endothelial interactions. Blood 1985; 65: 513-525. [35] KIoner RA, Giacomelli F, Alker KJ, Hale SL, Matthews R, Bellows S. Influx of neutrophils into the walls of large epicardial coronary arteries in response to ischemia/reperfusion. Circulation 1991; 84: 1758-1772. [36] Gerrity RG, Gass JA, Soby L. Control of monocyte recruitment by chemotactic factor(s) in lesion-prone areas of swine aorta. Arteriosclerosis 1985; 5: 55-66. [37] Mazzone T, Jensen M, Chait A. Human arterial wall cells secrete factors that are chemotactic for monocytes. Proc Natl Acad Sci USA 1983; 80: 5094-5097. [38] Jauchem JR, Lopez M, Sprague EA, Schwartz CJ. Mononuclear cell chemoamactant activity from cultured arterial smooth muscle cells. Exp Mol Path 1982; 37: 166-170.