A Neutrophil-Derived Cytochrome P450 - NCBI

American Journal of Pathology, Vol. 128, No. 3, September 1987 Copyright © American Association of Pathologists

A Neutrophil-Derived Cytochrome P450-

Dependent Metabolite of Arachidonic Acid Modulates Neutrophil Behavior From the Department of Pharmacology, New York Medical College, Valhalla, New York

ROSEMARY KRAEMER, MS, MARTIN M. BEDNAR, MD, PhD, MARY ANN HATALA, MS, and KEVIN M. MULLANE, PhD

acid to two Recently, the metabolism of 1arachidonic and Peak 2) by a cy(Peak products tochrome P-450 dependent mixed function oxidase has been described in canine polymorphonuclear leukocytes (PMNs). This study assessed the biologic activity of one ofthese metabolites, Peak 2, on PMN function. Peak 2 was formed biologically following addition of exogenous arachidonic acid to canine PMNs pretreated with BW755c to inhibit lipoxygenase and cyclooxygenase enzymes, and purified by high performance liquid chromatography following separation by column Peak 2 (20-200 ng/ml) inhibited calchromatography. cium ionophore A23187-induced PMN aggregation and the second phase of LTB4-induced aggregation. Additionally, Peak 2 inhibited A23187-induced PMN

adhesion to columns of Sephadex G-25. BW755c (94 #M), which increased the formation of Peaks 1 and 2 by almost 300%, also inhibited A23187-induced PMN adhesion. In contrast, Peak 2 did not inhibit the release of superoxide anions or immunoreactive LTB4 after stimulation of the PMNs with A23187. Thus, Peak 2 may modulate some activities of canine PMNs. Because the biologic activity of Peak 2 is opposite to that of LTB4, which promotes PMN aggregation and adhesion, and because LTB4 may be metabolized by a cytochrome P-450-dependent mixed function oxidase to less active metabolites, this enzyme system may play a central role in the control of PMN function. (Am J Pathol 1987, 128:446-454)

UPON activation with a chemotactic stimulus, poly-

perior to the conventional aspirinlike cyclooxygenase inhibitors because they possess the additional propertv of suppressing PMN infiltration into an inflammatory lesion.9-" This activity is important because the aspirinlike drugs, while providing symptomatic relief in chronic inflammation, reducing swelling, for example, do little to ameliorate the underlying progression of the disease.12 In contrast, drugs that prevent the cellular infiltration may actually retard the

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morphonuclear leukocytes (PMNs) undergo margination, directional movement (chemotaxis), adhesion to the vascular endothelium, and migration from the vascular compartment. In addition, activated PMNs undergo a "respiratory burst" with the generation of superoxide anions, together with the release of a variety oflysosomal enzymes and the liberation ofarachidonic acid (AA) from membrane phospholipids.l'2 PMNS contain a variety oflipoxygenase enzymes capable of metabolizing free arachidonate. The presence of 5-, 12-, and 15-lipoxygenases have been described,3-5 although attention has been focused primarily on the 5-lipoxygenase enzyme, because it gives rise to leukotriene B4 (LTB4; 5,12-diHETE), a potent chemotactic agent which promotes neutrophil

Address

reprint requests to

Dr. K.

Mullane, Director,

Cardiovascular Research, Ciba-Geigy Pharmaceuticals, 556 Morris Ave., Summit, NJ 07901. Supported by U.S. Public Health Service Grants HL31591 and HL34300 from the National Heart, Lung and Blood Institute. Kevin Mullane is the recipient of a Pharmaceutical Manufacturers Association Faculty Development Award and Rosemary Kraemer of a Pharmaceutical Manufacturer's Association Pre-doctoral Fellowship. Accepted for publication April 24, 1987.

adhesion and degranulation.68 Dual inhibitors of the lipoxygenase and cyclooxygenase enzymes such as BW755c are considered antiinflammatory drugs su-

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PMN MODULATION BY P450-DERIVED EICOSANOIDS

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development of the disease.' Moreover, studies with lipoxygenase inhibitors suggest that lipoxygenase metabolites of arachidonic acid play a central role in PMN activation.4,14 Recently, an additional pathway ofAA metabolism has been described in canine PMNs, mediated by a cytochrome P-450-dependent mixed function oxidase (MFO).'5 This enzyme system gives rise to two products, designated Peak 1 (P1) and Peak 2 (P2), which can be separated from the lipoxygenase metabolites by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) procedures,'5 although structural analysis has not yet been completed. In addition, these products do not possess the ultraviolet (UV) absorption spectra typical of li-

poxygenase products.16 Studies on the time course of formation of these two products indicate that P2 is a metabolite of P .16 Inhibition of the lipoxygenase enzymes enhances the formation of PI and P2, while

their synthesis is attenuated by SKF-525A, an inhibitor of cytochrome P-450-dependent MFO.'5 Because these products accounted for about 50% of the recovered radiolabeled material after incubation of unstimulated canine PMNs with '4C-AA,15 it was important to determine their effects on PMN function. This study examined the effects of P2, purified by HPLC, on PMN aggregation and adhesion, the release of superoxide anions, and the formation of LTB4.

Materials and Methods Formation of Peak 2 Generation of Peak 2for Assessment of

Biologic Activity Canine peritoneal PMNs were harvested by the method of Bednar et al.'5 Briefly, 400 ml of 0.1% glycogen (Sigma, St. Louis, Mo) in sterile saline containing heparin (2.5 U/ml) was injected through an angiocatheter (14 g, 2 /4 inches, Critikon; Tampa, Fla) into the peritoneal cavity. After 3 hours, 600 ml of sterile saline was administered, and the PMN-rich fluid was harvested. Following hypotonic lysis of contaminating erythrocytes, the PMNs were washed in normal saline and resuspended in Hanks' balanced salt solution (composition [g/l]: HEPES, 7.15; NaCl, 6.67; KCI, 0.33; Na2HPO4, 0.08; KH2PO4, 0.08; CaCL2, 0.17; MgSO4, 0.17; glucose, 0.92, NaHCO2, 1.17; pH adjusted to 7.4 with 1 N NaOH) at a concentration of 5 X 107 PMNs/ml. Histologic analysis by Wright-Giemsa stain revealed the suspension comprised 95% PMNs with 97% cell viability, as assessed by trypan blue exclusion. One-milliliter aliquots ofthe neutrophil suspension were preincubated for 10 minutes with the dual cy-

447

clooxygenase and lipoxygenase inhibitor BW755c (47 #M in distilled H20; Wellcome Research Laboratories, Beckenham, UK). All incubations were carried out at 37 C. Subsequently, a mixture of radiolabeled l-'4C-arachidonic acid (0.05 uCi; specific activity 60.1 mCi/mmol, Amersham, Arlington Heights, Ill) as a detector and nonradiolabeled arachidonic acid (20 ,uM, Nu Check) prepared in 0.01 N NaOH, was added to the cell suspensions. The reaction was terminated after 20 minutes by centrifugation at 10,000g on a Brinkman microcentrifuge. The supernatants were combined and acidified to pH 3.5-4.0 with 1 M citric acid. (Separate incubation of 1 -ml volumes were found to produce more of P1 and P2 than one large incubation.) Peaks 1 and 2 and other AA metabolites were extracted three times with three volumes of ethyl acetate. After evaporation of the ethyl acetate on a rotary evaporator, the products were reconstituted in 200 1u ethyl acetate for storage in -70C until separa-

tion and purification. Effect ofB W755c on Formation ofPeak 1 and Peak 2 One-milliliter aliquots of harvested peritoneal and peripheral PMNs, the latter separated by Ficoll-Hy-

paque procedures,17 were suspended at 107 PMNs/ml

in Hanks' buffer and preincubated with or without BW755c (47 uM and 94 ,M) for 10 minutes at 37 C. Radiolabeled AA (0.3 #Ci, 6 uM) was then added, the PMNs incubated for an additional 10 minutes, and the reaction terminated by centrifugation at 10,000g. Metabolites were extracted and resuspended in 150#1u of ethyl acetate, for separation by TLC. The samples were applied to Brinkman Silica Gel TLC plates (Brinkman, Westbury, NY) and the plates subsequently developed in the A9 solvent system (organic phase of ethyl acetate/ isooctane/acetic acid/water; 55/25/10/50 vol/vol/vol/vol) and the metabolites visualized by autoradiography. Previously determined zones for cytochrome P-450 monooxygenase, lipoxygenase, and cyclooxygenase products were cut and radioactivity determined by standard liquid scintilation techniques. Product formation was expressed as percentage oftotal radioactivity recovered for control and experimental values.

Separation and Purification of Peak 1 and Peak 2 Separation ofArachidonic Acid metabolites by Silicic Acid Chromatography Separation of metabolites was achieved in two steps. Initially, PI and P2 were separated from residual AA and prostaglandins with a 5-ml column of silicic acid gel (Silicar CC-4 Special; Mallinckrodt;

448

KRAEMER ET AL

Pans, Kentucky) packed in hexane:ethyl acetate (95:5). P1 and P2 were eluted from the column with hexane: ethyl acetate (2: 1), while AA was eluted with hexane: ethyl acetate, 95:5, and prostaglandins eluted with 100% ethyl acetate. The eluant containing P I and P2 was evaporated, and the products reconstituted in acetonitrile for separation by HPLC.

Purification of Peak I and Peak 2 by HPLC Separation ofP1 and P2 was achieved using reverse phase HPLC, performed on a C-18/u Bondapack column (4.6 mm X 20 mm, Waters Associates, Morristown, NJ). A linear gradient of 1.25% from acetonitrile: water ( 1: 1) plus 0.1% acetic acid to acetonitrile plus acetic acid (0.1%) at a flow rate of 1 ml/min was used for maximum separation of products. Radioactivity was monitored by a radioactive flow detector (Radiometric Instrument and Chemical Co., Inc., Tampa, Fla). The fractions containing P2 eluting from the HPLC column were pooled and extracted three times with three volumes of ethyl acetate. After evaporation of ethyl acetate under nitrogen, P2 was reconstituted in 200 ul ethyl acetate and stored at

-70 C.

Quantification of Peak 2 The amount of P2 formed was quantified based on the specific activity of the '4C-AA substrate, and assuming a molecular weight of 300 for P2. The molecular weight is likely to be higher than that of AA (304), but a rounded figure of 300 was used for convenience and provides an indication of relative potency. Peak 2 and Leukocyte Function

Neutrophil Aggregation Harvested peritoneal neutrophils were treated with 0.3 ug/ml of prostacyclin (PGI2; Wellcome Research Laboratories), prior to the washing procedure, and finally suspended in Hanks' buffer without PGI2 at a concentration of 1.5 X 107 PMNs/ml for aggregation studies. Preliminary experiments revealed that suspensions of elicited peritoneal PMNs (and consequently "activated") frequently exhibit unstable basal levels of light transmission and are prone to spontaneous clumping. Pretreatment with PGI2 stabilized the PMNs, giving rise to more consistent aggregation responses. Moreover, due to its chemical instability, the 1-hour interval between PMN harvesting and commencement of aggregation was sufficient to degrade any PGI2 that had been added to the initial incubate. Aggregation was determined with a Payton

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September 1987

light aggregometer (Payton Assoc., Buffalo, NY), as previously described.'4 A 500-1l aliquot, representing 0% light transmission, was equilibrated for 2-3 minutes at 37 C and stirred at 600 rpm with a siliconized stir bar. A 20% dilution of the neutrophil suspension was used to represent 100% transmission with the range of0-100% set to span 35 arbitrary units. Aggregation was induced with 5-10 ,1 of calcium ionophore A23187 (A23187: dissolved in ethanol and diluted 1: 300 in Hanks' buffer: final concentration 3.8 X 10-8 to 3.8 X 10-7 M; Sigma) or LTB4 (gift of Dr. P.Y-K. Wong, Department of Pharmacology, NYMC) (3 X 10 -8 to 3 X 10 -7M), in the presence or absence of cytochalasin B (10 ,M; Sigma) and increases in light transmission were recorded. Aggregation induced by these agonists was compared in the presence or absence of P2 (10-100 ng), preincubated

with the PMNs for 2 minutes before the addition of the stimulus.

Neutrophil Adhesion Isolation of Peripheral PMNs From

Canine Whole Blood Peripheral PMNs were isolated from 50 ml canine whole blood according to the procedure of Boyum.'7 Briefly, canine whole blood was collected in 3.15% sodium citrate ( 1:9 volume). Erythrocytes were sedimented using 6% dextran (MW 500,000, in normal saline, pH 7.4; Sigma). After hypotonic lysis of residual erythrocytes, leukocytes were washed with 0.9% saline and layered onto a Ficoll-Hypaque density gradient (Histopaque 1077; Sigma) and centrifuged at 200g for 20 minutes. Histologic analysis revealed the final cell pellet to be composed of 80-85% PMNs, with 95% viability as assessed by trypan blue exclusion. The PMN pellet was resuspended in Hanks' buffer supplemented with Tris-HCl (0.01 M, pH 7.4; Sigma) to 6 X 106 PMNS/ml. One-milliliter aliquots ot this PMN suspension were used in the adhesion assays. PMN Adhesion

Assays

Adhesion of PMNs to preswollen columns of Sephadex G-25 (Pharmacia, Piscataway, NJ) as described by Valone et all8 was used as an index of neutrophil adhesion. One-milliliter aliquots of the PMN suspension were preincubated with either P2 (100-200 ng in Hanks' buffer), BW755c (47 or 94 ,uM), or Hanks' buffer alone for 10 minutes and subsequently stimulated with A23187 (1.9-3.8 X 10-7 M) for an additional 5 minutes. Basal adhesion (without stimulation by A23187) and its modification

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by P2 and BW755c was also assessed. The cell suspensions were then applied to the Sephadex columns, and

the columns were washed with 2 ml of the Hanks'Tris buffer. The eluants were centrifuged at 400g at 4 C for 5 minutes, 0.04 mg sodium dodecyl sulfate (National Diagnostics, Somerville, NJ) in 2 ml of water was added to the pellet, and UV absorbance at 280 nm was determined. Percent adhesion was calculated from the following: % adhesion = 100 X 1Absorbance of solution of PMN eluting from the column absorbance of PMN suspension prior to application to the column To determine whether A23187 had any effect on the protein concentration of PMNs, cells (6 X 106) were incubated in the presence or absence of A23187 (3.8 X 10 -7M) for 5 minutes. After centrifugation, the cell pellets were treated with sodium dodecyl sulfate (SDS) resulting in protein concentrations of 0.92 ± 0.1 and 0.78 ± 0.1 mg/ml for control and A23187treated cells, respectively (P = 0.46, n = 6). A standard curve was constructed with BSA (Sigma).

Peak 2 and Superoxide Anion Generation The effects of P2 on A23187-induced generation of 0O was determined by the superoxide dismutase (SOD) inhibitable reduction offerricytochrome C according to the method of Babior et al.'9 Peripheral PMNs were resuspended to 2 X 107 cells/ml. Aliquots of 400 ul were preincubated with ferricytochrome C (1.4 ug/ml, Sigma) in the presence or absence ofcytachalasin B (20 ,M) for 15 minutes at 37 C and then stimulated with A23187 (10-6 M). Some aliquots were preincubated with 100 ng P2. The reaction was terminated by the addition of SOD (62.5 ug/ml; Sigma). A duplicate set of tubes contained SOD throughout. Superoxide generation was then determined spectrophotometrically, by the difference in absorbance between the duplicate samples. Results are expressed as nanomoles 02 per 106 cells per minute.

A23187-Induced Release of LTB4 by Activated PMNs Peripheral PMNs were isolated and resuspended to a concentration of 5 X 106 PMNs/ml. Aliquots of 400 Iul were preincubated with or without P2 (100 ng) for 15 minutes. Subsequently, PMNs were stimulated by A23187 (1.8 X 10-7 M) for 0, 5, and 10 minutes, and the reaction was terminated by centrifugation at

449

10,000g. The supernatants were assayed for LTB4 by radioimmunoassay (LTB4 [3H] Assay Reagents System, Amersham). Cross-reactivity of the LTB4 antiserum with other known AA metabolites products is less than 3.3%.

Statistics Statistical analysis was performed with a Student t test for independent samples.

Results Formation of Peak 2 Generation of Peak 2 HPLC separation of P1 and P2 on C-18 , Bondapak following the incubation of BW755c-treated canine PMNs with AA (20 uM with 0.05 ,Ci '4C AA added as a tracer) for 20 minutes at 37 C is shown in Figure 1. Peak 2, the major peak of radioactivity, showed a retention time of approximately 16 minutes. The amount of P2 synthesized in 5 different experiments ranged from 0.9 to 5.3 ug/109 PMNs (mean 3.08 ug P2/109 cells). This material was subsequently used for assessments of biologic activity.

Effect of B W755c on Formation of Peaks 1 and 2 The effect ofBW755c on the conversion ofradiolabeled AA to P1 and P2 is depicted in Figure 2. When the PMNs were pretreated with 47 gM BW755c, the formation of products comigrating with lipoxygenase standards was decreased from 21% ± 2% to 11% ± 2% radioactivity recovered (mean + SEM, P < 0.004; n = 6). Thus, 47 uM BW755c represents the approximate IC50 for the PMN lipoxygenase enzymes. At this concentration, BW755c increased the formation of Peaks I and 2 from 33% ± 6% to 60 ± 9% (P= 0.27, n = 6). There was a similar enhancement of formation of both PI and P2. Increasing the concentration of BW755c to 94 uM resulted in approximately 80% inhibition of lipoxygenase activity from 18% ±2% to 6% 1% (P< 0.008; n = 4), with a concomitant increase of approximately 300% in P1 and P2 formation from 22 ± 15% to 62 ± 8% radioactivity recovered (P < 0.05, n = 3) (Figure 2). Assessment of Biologic Activity

Neutrophil Aggregation The addition of calcium ionophore A23187 (3.8 X

10-8 to 3.8 X 10-7 M) to 500 #1 of a suspension of canine PMNs (1.5 X 107 PMNs/ml) resulted in a

dose-dependent increase in light transmission, of 33-60 units. The

high

dose of A23187 was subse-

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KRAEMER ET AL

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PEAK 2

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September 1987

Figure 1-Purification of P2 by reverse phase HPLC. P2 was formed from BW755C-treated canine PMNs incubated for 20 minutes with 20 ,M AA plus 0.05 #Ci '4C-AA as a tracer, separated and purified as described in Materials and Methods. This representative tracing shows P2 with a retention time of -16 minutes. The shaded area represents those fractions collected from the HPLC column as P2 for use in the biologic studies. Based on

PEAK

specific activity, approximately 2.4 ug P2 was formed in this experiment.

32

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quently used for aggregation studies (Figure 3). The addition of 10-100 ng of P2 to the cell suspension did not promote neutrophil aggregation. Rather, P2 inhibited the aggregation usually observed upon the subsequent addition of A23187 to PMNs (Figure 3, inset). Preincubation of the PMN suspension with 10-100 ng of P2 caused a dose-dependent inhibition of A23187-stimulated PMN aggregation ranging from 33% ± 3% to 68% ± 3% (Figure 3). The ability of P2 to inhibit neutrophil aggregation was also observed when LTB4 was used as the stimu-

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lus. LTB4 induced a two phase aggregatory response as previously observed by Ringertz et al,20 each phase separated by a lag period of approximately 45 seconds, in contrast to the single wave of aggregation provoked by A23187. Pretreatment with 100-200 ng of P2 had no effect on the initial aggregatory response, but inhibited the second phase of aggregation (Table 1). PMN Adhesion Basal adherence ofunstimulated PMNs to columns of Sephadex G25 was 26% ± 3%. Preincubation with 100 ng or 200 ng P2 had no effect on this basal adherence. Stimulation of PMNs with A23187 (1.9 X 10-7 to 3.8 X 10-7 M) caused a dose-dependent increase in adhesion of PMNs to columns of Sephadex G-25 (Figure 4), with 3.8 X 10-7 M A23187 increasing PMN adhesion to 66% ± 4% (P < 0.001, n = 12). This increase in adhesion was not secondary to the formation of aggregates, as determined by changes in light transmission in the Payton aggregometer. Preincubation of the PMN suspension with 200 ng or 100 ng P2 prior to stimulation with A23187 (3.8 X 10-7 M) caused a dose-dependent inhibition of the adhesion response. However, only the high dose of P2 caused a significant

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Figure 2-Increased formation of P1 and P2 in the presence of the dual cyclooxygenase/lipoxygenase inhibitor, BW755c. The broken lines represent individual experiments, and the solid lines represent the mean.

decreasing the adhesion response Stimulation of the YMN suspension with 1.9 X 10-7 M A23187 increased adhesion to 40% ±4% (P < 0.02, n = 9). Preincubation with 200 ng of P2

inhibition of 59%,

to 41% ± 4% (P< 0.005, n = 7).

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decreased this response to a level slightly above basal adherence, while 100 ng P2 had no effect on adhesion induced by the low dose ofA23187 (P = 0.64, n = 3). Because the pretreatment of canine PMNs with BW755c increased the formation of P2, the effects of this drug on A23187-induced adhesion was assessed. Preincubation of the PMNs with 94 ,M BW755c inhibited the response to A23187 (3.8 X 10-7 M) decreasing the adhesion to 35% ± 2% (P < 0.005, n = 3). BW755c, at 47 #M, which did not significantly increase P2 formation, failed to reduce adhesion induced by A23187 (P = 0.68, n = 4).

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A23187 +BW755C +BW755C 3.8 x 167M 47,uM 94,uM to columns of SeFigure 4A-Dose-dependent increase in PMN adhesion phadex G-25 following stimulation with 1.9 X 10 -7 (n = 9) and 3.8 X 10-7 M (n = 12) of A23187. B-Inhibition of A23187 (3.8 X 10-7 M) induced PMN adhesion after preincubation of the PMNs with 100 ng (n 6) and 200 ng (n = 7) P2. C-Inhibition of A23187 (3.8 X 10-7 M) induced PMN adhesion after preincubation of PMNs with 47 /M (n 4) and 94 pM (n = 3) BW755c. *P < 0.02; **P < 0.005; ***P < 0.001; N.S., not significant (Student t test). =

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Table 1-Inhibition of the Second Phase of LTB4-lnduced

n Cytochalasin B + Cytochalasin B

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Neutrophil Aggregation by Peak 2 LTB4-induced aggregation (arbitrary units) LTB4 + vehicle LTB4 + P2 (100 ng) 1st phase 2nd phase 1st pnase 2nd phase 15 ±2.6 25 ±1.9

18.1 ±5.0 27 ±2.9

13 ±2.0 21 ±3.3

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Aggregation induced by 3 X 10-7 M LTB4 in the absence of cytochalasin B and 3 X 10-8 M in its presence. *

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LTB4 + P2 (200 ng) 2nd phase

1st phase 15+ 2.5

1.25 ±4.0t

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KRAEMER ET AL

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September 1987

ide anions and release and metabolism of AA. Aggregation shares some similarities with chemotaxis,21 because addition of a chemotactic factor such as LTB4, Superoxide anion generation (nmol O°/106 cells/min) or the calcium ionophore A23187, to a suspension of PMNs causes the cells to adhere together. This ren Control +A23187 A23187 + P2 (150 ng) was attenuated in a dose-dependent manner sponse 0.9 ±0.2t Cytochalasin B 3 0.05 ± 0.01 0.3 ± 0.1* by pretreatment of the PMNs with P2. Differences in 0.35 ± 0.15t + Cytochalasin B 4 0.02 ± 0.01 0.42 ± 0.1* the nature of this inhibitory response were noted. A23187 concentration 10 M. Whereas A23187 induced a monophasic aggregatory *P < 0.005 versus control. response, that evoked by LTB4 was biphasic, as ret P > 0.05 (NS) versus A23187 alone. ported previously,20 and only the second-wave of LTB4-induced aggregation was inhibited by P2. A23187-Stimulated Release of Superoxide Anions P2 also reduced the increased adhesiveness of The rate of superoxide anion (0O) formation by A23187-treated PMNs to columns of Sephadex G-25. canine neutrophils was increased upon incubation A23187-induced PMN adhesion is not merely a rewith A23187 (Table 2). Pretreatment with P2 did not flection of aggregation and the trapping of cell aggreattenuate A23187-induced 0O generation. In the abgates on the column, because the concentration of PMNs used in this assay (6 X 106/ml) was too low to sence of cytochalasin B, there was a tendency for P2 to increase O° release, but this was not significant (P= permit aggregation to occur,22 and no visible aggre0.07). P2 was without any effect in the presence of gates were formed after the addition of A23187 to the cell suspension. cytochalasin B. The adhesion of PMNs to the vascular endotheA23187-Stimulated Generation of lium is a key event in the migration of PMNs from the Immunoreactive LTB4 venules into an inflammatory lesion. PMNs adhere Stimulation of PMNs with 2 X 10-7 M A23187 preferentially to endothelial cells, giving rise to the significantly increased the formation ofimmunoreac- proposal that PMN adhesion represents an interactive (i) LTB4 over basal levels. For example, at 5 min- tion between the leukocyte and the endothelial cell.23 utes iLTB4 levels increased from 0.47 ± 0.1 to 4.1 + Consequently, our study of adhesion to Sephadex 0.7 ug/106 cells (P < 0.05, n = 8). Preincubation with G-25 may not accurately reflect the adhesion re100 ng P2 had no effect on A23187-induced forma- sponse in vivo, and any effect of P2 on the endothelial cell component of the response cannot be detertion of iLTB4 at any time point (Table 3). Table 2-A23187-Induced Inhibited by Peak 2

Superoxide Anion Generation Is Not

-

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Discussion This study demonstrates that a PMN-derived metabolite of arachidonic acid formed via a cytochrome P450-dependent mechanism'5 can modulate PMN behavior. This activity was only observed against responses-aggregation and adhesion-which may be considered indicative of PMN margination, movement, and attachment, and not against the phase of PMN activation related to the generation of superoxTable 3-A23187-Induced Release of iLTB4 Is Not Inhibited Peak 2

responses.67 Moreover, monohydroxyeicosatetraen-

by

Immunoreactive LTB4 Release

(ng/106 cells)

Control

A23187

A23187 + P2 (100 ng)

0.47 ± 0.1

4.1 ± 0.7*

4.0 ± 1.0 t

A23187 concentration of 1.9 X 10-7 M, incubated with PMNs for 5 minutes. *

P < 0.05 versus control.

t P = 0.5 versus A23187 alone.

mined. However, Tonneson and co-workers24 found that treatment ofendothelial cells with the chemotactic factors, f-Met-Leu-Phe and C5a des Arg, had no effect on PMN adhesion, whereas incubation with the PMNs augmented the response. These authors24 concluded that the chemotactic factors act primarily on the PMN, although differences may be observed with other stimuli. The basal PMN adhesion to Sephadex columns was 26% + 3%, which compares favorably with the 35% ± 1% reported using cultures of umbilical vein endothelial cells.24 LTB4 can induce PMN aggregation and adhesion

oic acid (mono-HETE) metabolites of AA inhibit LTB4 synthesis stimulated by A23187.25 The cytochrome P-450-dependent MFO system can also produce mono-HETEs.26'27'28 Consequently, the possibility that P2 attenuated aggregation and adhesion responses through inhibition of A23187-induced LTB4 formation was examined. In addition, the generation of superoxide anions, another biochemical mechanism stimulated by A23187 and other chemotactic factors, which could influence PMN behav-

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ior, 114 was examined. P2 had no effect on either iLTB4 or superoxide anion formation at concentrations which significantly inhibited PMN aggregation. Consequently, the ability ofP2 to inhibit cell aggregation and adhesion cannot be correlated with a biochemical change, and suggests that this metabolite of AA selectively impairs the initial responses of the PMN to a chemotactic factor, but not the secondary responses relating to mediator release. Inhibition of lipoxygenase and cyclooxygenase activity in the canine PMNs with BW755c increased the formation of Peak 1 and Peak 2 in a dose-dependent manner. BW755c inhibits PMN aggregation elicited by f-Met-Leu-Phe or A2318714 and reduces A23187induced PMN adhesion. These activities are attributed to inhibition of lipoxygenase product formation, such as LTB4, but may also be due, in part, to the increased synthesis of Peaks 1 and 2. SKF-525A inhibits the formation of Peaks 1 and 2.15 If SKF-525A attenuated the inhibitory effects of BW755c on cell adhesion and aggregation, this may infer that the increased production of Peaks 1 and 2 does participate in these responses. However, the addition of SKF-525A to a suspension of PMNs produced an intense aggregation (results not shown) which may be a nonspecific effect of the drug. Therefore, it is not yet possible to determine the contribution of these cytochrome P450-dependent metabolites to the antiinflammatory effects of BW755c without more selective and specific inhibitors of this pathway. Another limitation of the present studies is that we investigated the activities of P2, which is a metabolite of P1. By analogy with other eicosanoids, the conversion of 12-HPETE to 12-HETE, for example, where the more stable metabolite generally shows less biologic activity,29 P2 may likewise be less active than P 1. Because P1 is rapidly converted to P2 in the PMN with a t,/, of 4 minutes,'6 it would not be possible to ascertain which effects were due to P1 or its conversion to P2. Consequently, it was decided to focus initially on the biologic properties of P2. Further studies are required to determine whether these properties are shared, or even enhanced, by P1. In addition, the purification of P2 by HPLC might not represent a homogeneous metabolite. The retention time of Pl is 2 minutes greater than that of P215 and some overlap may occur in the fractions collected. Contamination with P1 was minimized by using a 20-minute incubation time when most of the P1 was metabolized to P2. However, without knowledge of the structure of these compounds, their homogeneity cannot be determined. The structures of these compounds are currently being analyzed. Moreover, studies to date have utilized exogenous substrate, and the endogenous

453

Arachidonic Acid Peak 1

Lipoxygenase

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Cytochrome

P450

dependent MFO

LTB4

Peak 2

inhibits aggregation - suppresses adhesion w -oxidation

20-OH, 20-COOH

- induces aggregation - promotes adhesion

Figure 5-Proposed modulation of PMN behavior by cytochrome P-450-dependent (MFO) mediated metabolism of eicosanoids. Cytochrome P-450dependent enzymes inactivate LTB4 via w-hydroxylation, while converting arachidonic acid to Peaks 1 and 2, with biologic properties opposite those of

LTB4.

formation of these compounds cannot be determined because of the lack of any characteristic UV absorbance. The quantities of P2 calculated do not take into consideration any endogenous production which may reduce the apparent potency of this metabolite. These results suggest that the formation of P2 may serve to suppress PMN activation, and, if there is a basal formation of this compound, could contribute to maintaining these cells in a "quiescent" state. The inhibitory properties of P2 were observed as antiaggregatory and antiadhesive activities. In contrast, the PMN-derived 5-lipoxygenase metabolite, LTB4, induces PMN aggregation and adhesion6'7-actions opposite to those of the monoxygenase product. Moreover, LTB4 is metabolized to its 20-OH and 20-COOH metabolites via a cytochrome P450-dependent enzyme system.30 Thus, it appears that the same enzyme system may remove an agent which activates the PMNs (LTB4), while also producing an inhibitory factor (P2; see Figure 5). Additional studies are required to determine whether these two actions can be attributed to the same cytochrome P-450 isozyme. However, this enzyme system may play a key role in modulating PMN activity.

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Acknowledgments We wish to thank Pam Blank for typing the manuscript and Mike Blank for the artwork.