Cyclooxygenase and lipoxygenase metabolite synthesis by polymorphonuclear neutrophils: in vitro effect of dipyrone

Rostaghdins 0 Lawman

Leukotrienes and Essential Group UK Ltd 1990

0952.3278/90/0041-w89/$10.00

Fatty Acids (1990) 41.89-93

Cyclooxygenase and Lipoxygenase Metabolite Synthesis by Poiymorphonuclear Neutrophils: in vitro Effect of Dipyrone R. Abbate, A. M. Gori, S. Pinto, M. Attanasio, M. Boddi and G. G. Neri Serneri Clinica Medica I requests to RA)

University of Florence,

R. Paniccia, M. Coppo. S. Castellani,

Viale Morgagni 85, 50134 Florence,

B. Giusti,

Italy (Reprint

ABSTRACT.

Functional activity of polymorphonuclear neutrophils (PMN) is associated with the metabolism of Arachidonic Acid (AA) released from membrane phospholipids. In this study the in vitro effect of dipyrone, a non steroidal anti-inflammatory drug, on the production of AA metabolites through cyclooxygenase (CO) and lipoxygenase (LO) pathways by stimulated PMN has been investigated. PMN isolated by counterIIow centrifuge elutriator were > 98% pure and vIable. MetaboIite production was evaluated by RIA of Thromboxane A2 (TxAz), ProstagIandin Ez (PGEz), Leukotriene Bz (LTB4) and Leukotriene C4 (LTCJ after PMN stimulation with calcium ionophore A 23187 (20 PM). The levels of beta-thromboglobulin (RIA) lower than 5 ng/ml allowed us to rule out activation of residual contaminant platelets. In these experimental conditions, in the absence of dipyrone the products (ng/106 cells) of AA metabolism were LTb (3.51+0.22), LTCl (0.81 + O&8), TxB2 (0.144 + 0.025) and PG& (0.150 + 0.017). Incubation with dipyrone induced changes of PGE2 and TX& production in a dose dependent fashion (r=0.83 and r=0.87, p
INTRODUCTION

Among prostaglandin metabolites prostaglandin Ez (PGE?) appears of particular importance due to its vasodilation activity and its potentiating effects on vascular permeability (6) and algesia induced by bradykinin and other mediators of the inflammatory process (7, 8). Moreover PGE-, also stimulates collagenase activity and bone destruction

Human neutrophilic polymorphonuclear (PMN) leukocytes provide an effective host defense against bacterial and fungal infections, but they are also important in the pathogenesis of tissue damage in certain noninfectious diseases (1). Several functional activities, such as chemotaxis and opsonization are associated with the release of arachidonic acid (AA) from the phospholipid fraction of the phagocytic vacuole membranes (2). The release of AA is a result of membrane perturbations, which can be brought about by inflamimmunological matory or stimuli, calcium ionophores or mechanical agitation (3). PMN have the capacity to transform AA into its biologically active derivatives prostaglandins and leukotriene B4 through both cyclooxygenase (CO) and lipoxygenase (LO) pathway (4, 5). Date Date

received accepted

(9). Leukotriene B4 (LTB d), produced by oxidation of arachidonic acid through the lipoxygenase pathway, is a potent chemotactic and chemokinetic agent for neutrophils, eosinophils and monocytes and it also stimulates lysosomal enzyme release and superoxide-ion production in leukocytes (10). The anti-inflammatory action of nonsteroidal antiinflammatory drugs (NSAID) is associated with CO inhibition, whereas a possible activity on LO remains to be clarified. Dipyrone, a pirazolone derivative, has substantial anti-inflammatory activity (1 l), but its mechanism of action is still under investigation. The aim of this study was to investigate whether dipyrone in vitro affects the formation

JO January IYYO 17 June IYYO XY

90

Prostaglandins Leukotrienes and Essential Fatty Acids

of AA metabolites produced through both cyclooxygenase and lipoxygenase pathways human stimulated neutrophils.

the by

viability was assessed by Trypan blue exclusion and was over 95%. Incubation conditions

MATERIALS

AND METHODS

Polymorphonuclear leukocyte isolation Blood was collected by venipuncture from healthy volunteers who had abstained from any drugs for at least two weeks before the sampling. Polymorphonuclear leukocytes (PMN) were isolated from titrated (ACD : acid-citrate-dextrose NIH, formula A) venous blood by countertlow centrifuging using a Beckman JEGelutriator (Palo Alto, California) rotor held in a J2iB centrifuge (12). The how rates of cell suspensions passing through the rotor were controlled by a Cole-Palmer 7545 Masterflex pump. Cells with different physical properties (size, shape, density) equilibrate at different radii and can be removed from the rotor by altering the flow rate at a constant rotor speed. Separation of neutrophils from cell populations of different diameters was obtained by elutriation. balancing centrifugal force against a moving stream of isotonic phosphate buffered sodium chloride solution containing 0.15 (w/v) per cent bovine serum albumin (BSA) Fraction V (Sigma, St Louis, MO, USA). The elutriation medium was pressure-filter sterilized through a 0.2 urn filter assembly (Millipore S.A.‘, Molsheim, France). Blood was injected into rotor by a syringe taking care to eliminate air bubbles. During the loading the rate of the fluid flow was maintained at 14 ml/min, whereas the rotor speed was set and maintained at 2500 rpm throughout the entire operation. After blood loading the flow rate was increased to 16 ml/min and 200 ml of suspension was collected and then discarded. Due to a great individual variability in flow rate necessary to obtain neutrophil separation, in a subsequent step of the purification process, the increase of the flow was adapted under observation of the separation chamber. In fact flow rate was increased until the boundary of turbid zone was displaced to the top of the wide side of the chamber (range 18-23 ml/min). The suspension (150 ml) so elutriated was discarded. The pump and centrifuge were simultaneously stopped and cells remaining in the chamber (granulocyte suspension virtually free of red cells and lymphocytes) were aspirated and transferred in a polypropylene tube, resuspended in 10 ml of buffer and submitted to a second elutriation identical to the first one. After the elutriation procedure the purity of PMNs was checked by counting 200 cells on a Wright stained film and it was > 98%. Neutrophil

PMNs (1 x 10h) in Dulbecco’s phosphate-buffered saline (components in g/l: CaCl* 0.1, KC1 0.2, KH2P04 0.2, MgC12x6H20 0.1, NaCl 8.0, Na*HPO, x 12 Hz0 3.43) were incubated at 37°C for 15 minutes with different dipyrone concentrations (5, 10, 20, 40, 100, 200 pg/ml, final concentrationf.c.) and dissolving solution. Then neutrophils were stimulated by adding 20 PM (f.c.) calcium ionophore A23187. Incubation was terminated after ten minutes by centrifuging the cellular suspension at 6000 g x 15 minutes at 4°C. The cell-free supernatant was removed and stored at -80°C until the assays were performed. In order to rule out activation of residual conplatelets, taminant Beta-thromboglobulin (Beta-TG) assay in supernatant has been performed (n=5 experiments). Beta-TG was assayed by ELISA using a commercial kit (Boehringer Mannheim, FRG). TxBz assay in supernatant Neutrophil TxB2 production in the supernatant was measured by radioimmunoassay (RIA) according to Granstrom et al. (13), using a commercial kit (American Biomedical Technologies, FRG). Cross reactivity of antibody was 0.06% for PGD2 and 0.05% for PGFialpha, PGFzalpha, PGE ,, PGE2 and 6-keto-PGF, alpha. The minimum detectable concentration was 10 pg/mL.

Prostaglandin E2 assay Neutrophil PGEz produced by neutrophil cells in supernatant was measured by RIA according to the method described by Patron0 et al. (14). The antiserum kindly provided by Prof. Peskar (Lehrstuhle fur Pharmakologie und Toxicologic, Ruhr-Universitaet Bochum, FRG), showed the following cross reactivities: PGE, 1.2%; PGA2 1.5%; PGA, 0.008%; PGFzalpha 1%; PGFialpha 0.14%; PGB2 0.03%; TxB, 0.015%; 6-keto-PGFialpha 0.014%. The detection limit for PGEz was 2 pg/ml. The variation coefficients of intra-assay and inter-assay were 9% and 10%. Standard PGE;! was purchased from Upjohn Co (Kalamazoo, USA). A 100% inhibition was arbitrary attributed when metabolite concentration was below the detection limit. Leukotriene B4 assay LTB4 production

by neutrophil

was measured

by

CO

and LO Metaholite

Synthesis by PMN

Neutrophils

91

RIA, according to Salmon et al. (15), using a commercial kit (Amersham, UK). Cross reactivity of antibody was 0.004% for LTB4 and 0.06% for LTE.1. The minimum detectable concentration was 100 pg/ml. Leukotriene C4 assay LTC, production by neutrophil was measured by RIA, according to Lindgren et al. (16), using a commercial kit (Amersham, UK). Cross reactivity of antibody was 0.03% for LTC4 and LTD4. The minimum detectable concentration was 125 pg!ml. Fig. 1 Inhibition of TxB, production by stimulated neutrophils induced dy different doses of dipyrone.

Statistical analysis Statistical analysis of the results was carried out by the Student’s t-test for paired data and polynomial fitting. The statistical evaluation of the data was performed by the one-way analysis of variance. Results are reported as mean + S.E.M.

10

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11 .

,080 ‘0

PMN suspensions were obtained free from platelets as demonstrated by beta-TG concentration, which was found to be lower than 5 ng/ml in all experiments. After calcium ionophore A23187 stimulation highly purified human PMN produced detectable amounts of CO and LO metabolites (Table 1). Whereas TxA2 (measured as TxB2) and PGE? were formed in equivalent amounts, the formation of LTB4 was clearly preeminent in comparison to that of LTC+ Table

1

Arachidonic

by neutrophils (n = 10)

Mean S.E.M.

. 1

&xi

RESULTS

acid ‘metabolites

stimulated

with A23187

TXB:

PGEz

0.144 0.025

0.150 0.017

(q/l@ calcium

LTB, 3.51 0.22

cells) produced ionophore

LTC, 0.81 0.08

Incubation with dipyrone induced changes of TxB2 production by stimulated neutrophils in a dose-dependent fashion (Fig. 1): a slight (33%), but significant (p
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rzO.87

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236

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CONCENTRATIONS ~g/d

160

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Fig. 2 Inhibition of PGE, production by stimulated neutrophils (calcium ionophore A23187 20 FM) induced different doses of dipyrone).

by

Prostaglandin E2 production by stimulated neutrophils was found to be inhibited dose-dependently by dipyrone (Fig. 2): incubation of neutrophils in the presence of the lowest concentration tested of dipyrone (5 pg) reduced PGE:! synthesis by 40% (p
92

Prostaglandins Leukotrienes and Essential Fatty Acids Table 2 LTB, production (n 10” cells) by stimulated neutrophils with A23187 calcium ionophore in the presence of dipyrone (p buffer X

3.51 SEM 0.22

Dip 5

Dip 10

Dip 20

Dip 40

Dip 100

Dip 200

3.83 0.34

3.69 0.31

3.57 0.32

3.49 0.26

3.64 0.32

3.66 0.31

F = 1.34

Table 3 LTC4 production (n lo6 cells) by stimulated neutrophils with A23187 calcium ionophore in the presence of dipyrone (p buffer X 0.81 SEM 0.08

Dip 5

Dip 10

Dip 20

Dip 40

0.91 0.13

0.88 0.10

0.93 0.15

0.86 0.11

Dip 100 0.94 0.12

Dip 200 0.90 0.1

F = 0.91

DISCUSSION In this study stimulated neutrophils have been found to convert endogenous arachidonic acid into both CO (PGE2 and TxA2) and LO (LTB4 and LTC,) metabolites and the predominant was LTB4. In our experimental conditions the dipyrone inhibitory activity on CO metabolite production by neutrophils was dose-dependent and the IC 50 was similar to that found for washed platelets by us (17). Moreover this IC.50 was equivalent to plasma dipyione concentrations found after oral administration of therapeutic dosages. In this study the amounts of AA metabolites produced by stimulated neutrophils were lower than those reported in other studies (10, 18, 19). This finding seems to be attributable to the different experimental procedure. Our cellular suspensions were completely free from platelets, due to an extensive washing of white blood cell suspensions by means of counterflow centrifuge elutriation. The complete removal of platelets is confirmed by the undetectable levels of beta-thromboglobulin, a specific sign of platelet activation. The absence of platelets in cellular suspensions eliminates TxB2 and PGE2 contamination, deriving from platelets, so allowing measurement of the real amounts of TxB2 and PGE2 produced by neutrophils. In previous papers higher amounts of LTs were reported (10, 18, 19). This difference could be attributed to the absence of platelets, as platelets provide LO metabolites (12-HETE) which are transformed by neutrophils into LTs (20). On the other hand, neither the duration of incubation time, which was the same (10 minutes) as that used by the other authors (10, 15, 18) and appropriate to obtain the maximal. LT leukocytes synthesis (19), so that it cannot be responsible for the lower amounts of LTs detected in our cellular

suspensions. The present findings indicate a significant inhibitory effect of dipyrone on the formation of arachidonic acid metabolites by human stimulated leukocytes through CO pathway. Dipyrone inhibitory activity on PGE* production could account for the well known antipyretic action of the drug. In fact PGE,! is one of the most potent pyretic agents and some evidence has been accumulated suggesting that PGEz is an endogenous mediator of fever (21) which acts on the anterior hypothalamus. However it must be considered that PGE2 is 80-90s inactivated already during the first passage through the lung (22). It is not clearly demonstrated that the concentrations of the drug or its active metabolites, adequate to inhibit PGE*, reach the central nervous system. The inhibition of PGE2 synthesis by PMN observed in this study could contribute, at least in part, to clarify the antiinflammatory activity of dipyrone, as PGE:, is a prostaglandin directly involved in the inflammatory process (4,7). PGE2 has been found in inflamed tissues in sufficient concentrations to contribute to the erythema by its vasodilating activity and its enhancement of vascular permeability and plasma exudation induced by histamine or bradykinin (4, 7). In our study leukotriene production was not depressed by dipyrone, even at concentrations much higher than those reached in therapeutic use, in keeping with the results obtained by Weithmann and Alpermann (11) in a different investigation system. However, it is likely that the effect of dipyrone observed in vitro on neutrophil AA metabolism is lower than the effect which takes place in vivo. In fact in the in vitro experimental procedure cell stimulation occurs in a medium free of albumin, which is able to decrease the conversion of AA into CO and LO metabolites and, in addition, plasma proteins enhance the stability of dipyrone and its

CO

_

metabolites. The in vivo capacity of dipyrone to exert a therapeutic antiinflammatory action, may, therefore, be ascribed to the relevant inhibition of production of arachidonic acid neutrophil metabowhich contribute to PGE,, lites, essentially leukocyte* activation associated with inflammatory feactions.

Acknowledgement This investigation was supported Fondazione Hoechst Milano.

by a grant from

References 1. Malech 2.

3.

4.

5.

6.

7.

H L, Gallin J I. Neutrophils in human disease. New England Journal of Medicine 317: 687-694, 1987. Henricks P A J, Van Der Tol M E, Van Kats-Renaud J H, Nijkamp F P, Verhoef J. Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function. Biochimica et Biophysics Acta 801: 206-214. 1984. Weissmann G, Smolen J, Korchak H. Prostaglandins and inflammation: receptor/cyclase coupling as an explanation of why PGEs and PGI, inhibit functions of inflammatory cells. ~I637 in: Advances in prostaglandin and thromboxane research. (P W Ramwell, R Paoletti eds) Raven Press, New York. 1980. Higgs G A, Moncada S, Salmon J A, Seager K. The source of thromboxane and prostaglandins in experimental inflammation. British Journal of Pharmacology, 79: 863-868. 1983. Simmons P M. Salmon J A, Moncada S. The release of leukotriene B, during experimental inflammation. Biochemical Pharmacology 32: 1353-1359, 1983. Trang L E. Prostaglandin and inflammation. Seminars in Arthritis and Rheumatology 9: 153-190,198O. Lewis G P. lmmunoregulatory activity of metabolites of arachidonic acid and their role in inflammation. British Medical Bulletin 3Y: 243-248.

1983. 8. Higgs G A, Moncada S. Vane J R. Eicosanoids in inflammation. Annual Clinical Research 16: 287-299.1984. Y. Krane S M. Mechanisms of tissue destruction in

and LO

rheumatoid

Metabolite arthritis.

Synthesis by PMN p 593-604

allied conditions (D J McCarty Philadelphia, 1985.

In: Arthritis

Neutrophils and

ed) Lea & Febiger.

IO. Moilanen E. Alanko J. Seppala E, Vapaatalo H. Effects of antirheumatic drugs on leukotriene B, and prostanoid synthesis in human polymorphonucldar leukocytes in vitro. Agents and Actions. 24 (314): 387-394. 1988. II. Weithmann i<’ U. Alpermann H G. Biochemical and pharmacological effects of dipyrone and its metabolites in model system related to arachidonic acid cascade. Arzneimittel Forschung Drug Research 35 (6): 947-952. 1985. 12. Persidsky M D, Olson L S. Granulocyte separation by modified centrifugal elutriation system. Proceeding of the Society for Experimental Biology and Medicine 157: 599-604, 1978. 13. Granstrom E, Kindhal H, Samuelsson B. A radioimmunoassay for thromboxane B? Analytical Letters. 9: 61 l-627, 1976. 14. Patron0 C, Grossi-Belloni D, Ciabattoni G, Serra G B. Latorre E, Bombardieri S, Mancuso S. Gorga C. Radioimmunoassay measurement of F prostaglandin in the human body fluid. Hormon. Meta Research 5: IYO-lY5. 1974. 15. Salmon J A. Simmons P M, Palmer R M J. A radioimmunoassay for leukotriene B,. Prostaglandins 24: 225-235. 1982. S. Goetze J. A 16. Lindgren J A, Hammarstrom sensitive and specific radioimmunoassay for leukotriene C,. FEBS Letters 152: 83-88, 1983. 17. Abbate R. Pinto S. Gori A M, Paniccia R, Coppo M. Neri Serneri G G. Activity of dipyrone on intraplatelet arachidonic acid metabolism: an in vitro study. Pharmacological Research Communications 21: 43-50. lY89. 18. Lewis R A, Austen K F. The biologically active leukotrienes. Biosynthesis. metabolism, receptors, functions and pharmacology. Journal Clinical Investigation 73: 889-897. 1984. IO. Salmon J A. Simmons P M. Palmer R M J. Synthesis and metabolism of leukotriene B4 in human neutrophils measured by specific radioimmunoassay. FEBS Letters 146: 18-22, 1082. 20 Marcus A J, Broekman M J. Safier L B, Ullman H L. Islam N. Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro. Biochemical Biophysical Research Communications l(r): 130-137. 1982. 21. Feldberg W. Fever. prostaglandins and antipyretics. p 197-204 in: Prostaglandin synthetase inhibitors. (H J Robinson J R Vane eds) Raven Press. New York 1974. 22. Vane J R. The release and fate of vasoactive hormones in the circulation. British Journal of Pharmacology 35: 209-242. 1969.

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