Neutrophil aggregating properties of PAF-acether and leukotriene B4

Int. J. lmmunopharmac., Vol. 5, No. I, pp. 17-21,1983. Printed in Great Britain.

0192-0561/83/010017-05 $03.00/0 © 1983 International Society for lmrnunopharmacology.

NEUTROPHIL AGGREGATING PROPERTIES OF PAF-ACETHER AND LEUKOTRIENE B4 A. W. FORD-HUTCHINSON* Department of Chemical Pathology, King's College Hospital Medical School, Denmark Hill, London, SE5 8RX, U.K. (Received 18 November 1981 and in final form 12 January 1982)

Abstract - - Both leukotriene B4 (LTB4) and platelet activating factor (PAF-acether) induce the aggregation of rat polymorphonuclear leucocytes. PAF-acether, but not LTB4, causes the aggregation of washed rabbit platelets. The results obtained from cross-desensitisation studies and experiments using inhibitors of arachidonic acid metabolism support the concept that LTB4 and PAF-acether have separate receptor sites on the neutrophil cell surface and show that PAF-acether does not induce neutrophil aggregation by releasing LTB,. A number of substances have been reported to induce the aggregation of polymorphonuclear leucocytes (PMNs). These include chemotactic factors which induce a rapid reversible response, such as the complement derived peptide C5a, the synthetic peptide formyl-methionyl-leucyl-phenylalanine (F-metleu-phe) and leukotriene B4 (5S,12R-dihydroxy-6,14cis-8, lO-trans-eicosatetraenoic acid (LTB4)) (O'Flaherty, Kreutzer & Ward, 1977; Craddock, Hammerschmidt, White, Dalmasso & Jacob, 1977; Ford-Hutchinson, Bray, Doig, Shipley & Smith, 1980). Other substances such as the calcium ionophore A23187, concanavalin A and phorbol myristate acetate induce irreversible aggregation responses (O'Flaherty, Showell, Becker & Ward, 1978; O'Flaherty, Cousart, Lineberger, Bond, Bass, Dechatalet, Leake & McCall, 1980). A number of platelet aggregating agents including ADP, adrenaline and thrombin have been reported to have no effect on neutrophil aggregation (O'Flaherty, Showell, Becker & Ward, 1979). Arachidonic acid, a potent platelet aggregating agent, also causes the aggregation of PMNs (O'Flaherty et al., 1979) following its metabolism by lipoxygenase pathways to LTB4 (Ford-Hutchinson, Bray & Smith, 1979; FordHutchinson et al., 1980). Recently another platelet aggregating agent, 1-0-alkyl-2-0-acetyl-sn-glyceryl-3phosphorylcholine (platelet activating factor: PAFacether) has also been shown to aggregate PMNs (Camussi, Tetta, Bussolino, Cappio, Coda, Masera & Segolini, 1981; O'Flaherty, Wykle, Miller, Lewis, Waite, Bass, McCall & DeChatelet, 1981). PAF-

acether, like LTB4, can also be released by PMNs following exposure to a number of agents including C5a (Lynch, Lotner, Betz & Henson, 1979) and it is possible that either agent may induce the aggregation of PMNs by releasing the other. EXPERIMENTAL PROCEDURES

Neutrophil cell suspensions (>85o70 PMNs) were prepared from peritoneal exudates obtained 24 h after the injection of 12O7o(wt/vol) sodium caseinate into 250-400g male Wistar rats (Cunningham, Smith, Ford-Hutchinson & Walker, 1979). The cells were washed and resuspended at a concentration of 1 × 10 r cells/ml in Eagle's minimum essential medium buffered to pH 7.4 with 30 mM N'-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid. Neutrophil aggregation was assessed by nephelometry in a Payton aggregometer as previously described (Cunningham, Shipley & Smith, 1980). The results are expressed as the increase in light transmission measured in millimeter on the recorder. Leukotriene B, was generated from rat peritoneal PMNs, elicited with sodium caseinate, by incubation with the calcium ionophore A23187. The leukotriene was purified by ether extraction, silicic acid chromatography and reverse phase high pressure liquid chromatography as previously described (FordHutchinson et al., 1980). The concentration of leukotriene B,was determined spectrophotometricalm~or~oAtl 39,500 (Borgeat & Samuelsson, ly using a ~_ 281nrn 1979).

*Present address and address for reprint requests: Laboratoires Merck Frosst, P.O. Box 1005, Pointe Claire, Dorval, Quebec, H9R 4P8, Canada. 17

A. W. FORD-HUTCHINSON

18

!80

Washed rabbit platelets were isolated from citrated rabbit blood as previously described (Bills, Smith & Silver, 1976). The platelets were suspended at a concentration of 2 × 108 cells/ml in Tris-Tyrode's solution, pH 7.4, containing 0.2507o bovine serum albumin. Platelet aggregation was assessed by nephelometry in a Payton aggregometer and the results are expressed as the increase in light transmission measured in millimeter on the recorder.

Materials Drugs used were: nordihydroguaiaretic acid (Sigma); 5,8,11,14-eicosatetraynoic acid (Roche); indomethacin (Merck, Sharpe and Dohme); 3-amino- 1-(3-trifluoromethyl phenyl)-2-Pyrazolone hydrochloride (BW755C) (Wellcome Research Laboratories). Stock solutions of drugs were made up in dimethylsulphoxide (DMSO). The final concentration of D M S O in cell suspensions was always <0.1070 (vol/vol). Platelet aggregating agents used were: PAF-acether and lyso-PAF-acether (Dr. J. Benveniste); thrombin (Sigma, Grade II; 1000 U / m l in 0.05M phosphate buffer, p H 7.0, obtained from bovine plasma); adrenaline (Sigma); carrageenan (Viscarin marine colloids); collagen [acid soluble extract obtained from calf achilles tendon prepared as described by Cazenave, P a c k h a m & Mustard (1973)]. PAF-acether was stored at - 20°C in chloroform:methanol (80:20) and dilutions were made in buffered solutions containing 0.25°7o (wt/vol) bovine serum albumin.

RESULTS Both LTB, and PAF-acether caused a partially reversible aggregation of rat peritoneal P M N s at concentrations as low as 100 and 300 p g / m l respectively (Fig. 1). The shape of the aggregation curves was similar to that previously observed following the addition of F-met-leu-phe or zymosan activated serum to rat neutrophils (Cunningham et al., 1980). The EDs0 values for LTB, and PAF-acether were

~, j,~~~

0.1

10 100 Concentration ng/rnl

'

1000

Fig. 1. The effects of PAF-acether ( • ), LTB4(A) and lysoPAF-acether (B) on the aggregation of rat peritoneal polymorphonuclear leucocytes. Results are shown with standard errors (n = 5-37). 230 p g / m l (9 × 10-'°M) and 2 n g / m l (3.6 × 10-'M) respectively. In contrast the non-acetylated analogue of PAF-acether (lyso-PAF-acether) caused a small neutrophil aggregation response only at 1 g g / m l and was inactive at a concentration of 300 p g / m l . A number of other platelet aggregating agents were tested for their ability to induce rat neutrophil aggregation. Thrombin (1, 10 and 100 U / m l ) , adrenaline (10 -6, 10.8 and 10-4) carrageenan (50 and 500/~g/ml) and collagen (0.6, 1.2 and 2.4/~g/ml) all failed to induce P M N aggregation. The ability of LTB, and PAF-acether to induce platelet aggregation was tested using washed rabbit platelets. PAF-acether caused significant aggregation at concentrations as low as 300 p g / m l (EDs0, 1.2 × 10-~M). In contrast LTB,, over the concentration range 100 p g / m l to 100 ng/ml, caused no platelet aggregation and did not affect the response to P A F acether. LTB, also failed to aggregate human platelet rich plasma over the same concentration range. In order to investigate the possibility that LTB, and PAF-acether have different receptor sites, desensitisation tests were carried out. The response to either 1 n g / m l LTB, or 10 n g / m l PAF-acether (maximal responses) was measured 4 min after an aggregation response to the same or a different aggregating agent (Table 1). There was a 73 07o reduction

Table 1. Rat PMN aggregation: desensitisation studies Second aggregating agent LTB, (1 ng/ml) PAF-acether (10 ng/ml) First aggregating agent LTB~ (1 ng/ml) PAF-acether (10 ng/ml)

/~o !

Control response Second aggregation response 41.2 _+ 0.9 (30) 11.0 _+ 0.3 (7) 73°/o ~* 57.3 _+ 1.8 (7) 11%~ 64.5 _+ 1.2 (25) 26.6 _+ 0.4 (7) 350/0 ~* 14.6 _+ 1.0 (7) 77°/o ~ *

Results are expressed as the height of the aggregation response as measured in mm on the chart recorder +_ S.E.M. The number of observations is shown in brackets. * P<0.0001.

Neutrophil Aggregating Properties of PAF-Acether and Leukotriene B, in the response to LTB, added 4 min after LTB4 and a 77°7o reduction in the second response to PAFacether. In contrast, the aggregation responses to LTB4 after the addition of PAF-acether and PAFacether after LTB4 were reduced by only 35 and 11 °70 respectively. Administration of 1 ng/ml LTB, together with 10 ng/ml PAF-acether significantly enhanced the response to PAF-acether (P<0.0001; PAF-acether alone, 64.5 _+ 1.2 (n = 25): PAFacether + LTB4 85.7 _ 1.7 (n = 5). In order to determine whether PAF-acether causes neutrophil aggregation by releasing LTB4, neutrophil aggregation responses to both agents were determined in the presence of a number of drugs known to affect lipoxygenase pathways of arachidonic acid metabolism. The results of these experiments are shown in Table 2.

DISCUSSION The present work shows that PAF-acether, a potent platelet aggregating agent, also causes the aggregation of rat PMNs, confirming previous work using human and rabbit PMNs (Camussi et aL, 1981; O'Flaherty et al., 1981). LTB, in common with other potent chemotactic factors, such as the complement derived peptide C5a and the synthetic peptide F-metleu-phe, differs from PAF-acether in that it causes no platelet aggregation. Not all platelet aggregating agents, however, cause neutrophil aggregation. Thus in the present work thrombin, adrenaline, carrageenan and collagen all failed to induce PMN aggregation. Arachidonic acid, a potent platelet aggregating agent, induces neutrophil aggregation by a different mechanism, through the formation of LTB4 (Ford-Hutchinson et al., 1980; Bray, FordHutchinson & Smith, 1981a). The only other platelet

19

aggregating agent known to induce PMN aggregation is ADP (Camussi et al., 1981) and the PMN aggregating activity of a number of purine nucleotides will be the subject of a separate manuscript. PAF-acether has been shown to be released from PMNs following addition of a number of stimuli including the chemotactic peptide C5a (Lynch et al., 1979). Other workers have suggested that PAFacether is the final common mediator of neutrophil aggregation following stimulation of these cells with immune complexes, CSa or cationic proteins (Camussi et al., 1981). The possibility must be considered that all chemotactic factors, including LTB,, induce neutrophil aggregation by releasing PAFacether. A number of pieces of evidence argue against this. Intravenous administration of C5a, F-met-leu-phe (O'Flaherty, Showell & Ward, 1977) or LTB4 (Bray, Ford-Hutchinson & Smith, 1981b) causes a profound neutropenia with no change in platelet numbers. In contrast intravenous administration of PAF-acether induces both thrombocytopenia and neutropenia. In the present work the desensitisation studies and the additive effects of the two agents strongly suggest that LTB4 and PAF-acether cause neutrophil aggregation through independent mechanisms by interacting with separate receptor sites. Preincubation of rat neutrophils with nordihydroguaiaretic acid 0 0 -5 and 10-eM), indomethacin (10-'M) and BW755C (10-4M) had either no effect on or caused a small significant inhibition of the aggregation response to PAF-acether. These drug concentrations completely inhibit the release of LTB, from rat neutrophils stimulated with the calcium ionophore A23187 (Bray et al., 1980; FordHutchinson et al., 1980) and will inhibit the aggregation of rat neutrophils by arachidonic acid (FordHutchinson et al., 1979). PAF-acether cannot

Table 2. Effects of drugs on the aggregation of rat PMNs induced by PAF-acether and LTB4 Aggregating agent

10 ng/ml

PAF-acether

10 ng ml

LTB4

64.5 _+ 1.2 64.5 _+ 1.4 57.1 _+ 1.0 56.6 -+ 1.8

(25) (5) (5) 110704* (5) 12%$*

41.2 _+0.9 36.5 -+ 1.6 33.3 + 0.7 5.5 ± 0.4

(30) (5) 11%4** (5) 110704** (5) 87%4**

10-SM 5,8,11,14 eicosatetraynoic acid

29.6 ± 1.9

(5) 65%~**

7.2 ± 0.3

(5) 82%4**

10-4M BW755C

41.7 ± 3.1

(5) 35%4*

22.2 ± 2.1

(5) 46% **

Drug Control 10-eM nordihydroguaiaretic acid 10-SM nordihydroguaiaretic acid 10-'M indomethacin

Results are expressed as the height of the aggregation response as measured in mm on the chart recorder _+ S.E.M. The number of observations is shown in brackets. * P<0.01 ** P<0.0001 when compared to control.

20

A. W. FORD-HUTCHINSON

therefore cause n e u t r o p h i l aggregation by releasing LTB,. T h e effects o f these drugs o n the aggregation induced by L T B , differed from those seen with P A F acether, arguing against the possibility that b o t h m e d i a t o r s act by a final c o m m o n pathway. Ind o m e t h a c i n (10-'M) a n d B W 7 5 5 C (10-'M) b o t h inhibit the action of L T B , a n d at these high concentrations m a y inhibit the binding of LTB, to its receptor site or the cell surface. N o r d i h y d r o g u a i a r e t i c acid (10 -8 a n d 10-SM) h a d little effect o n the aggregation responses a l t h o u g h it is a p o t e n t inhibitor of LTB, synthesis. 5,8,11,14-eicosatetraynoic acid inhibited the action o f b o t h aggregating agents a n d m a y inhibit the binding o f these agents to the n e u t r o p h i l cell membrane. These results show clearly t h a t LTB, a n d P A F acether have different biological actions a n d that P A F - a c e t h e r does n o t induce the aggregation o f P M N s by releasing LTB,. They also suggest t h a t L T B , a n d P A F - a c e t h e r have separate receptor sites o n the P M N cell surface a n d t h a t b o t h agents should

be considered as i m p o r t a n t mediators o f leucocyte function. -200 J

/ 0.1

loo

A.

•

1

10

Con~l"~on

100

ng/rnl

Fig. 2. The effects of PAF-acether ( • ) and LTB4 (A) on the aggregation of washed rabbit platelets. Results are shown with standard errors (n = 4 - 6 ) .

Acknowledgements - - The author would like to thank Dr. J. Benveniste for the supply of synthetic PAF-acether and lyso-PAF-acether and Professor M. J. H. Smith for his help and advice.

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Neutrophil Aggregating Properties of PAF-Acether and Leukotriene B,

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