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Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man
PROSTAGLANDINS
EFFECT OF INHALED PLATELET-ACTIVATING FACTOR ON CIRCULATING NEUTROPHILS AND PLATELETS IN VIVO AND EX VIVO IN MAN
I. Kioumis,
J.W. Lammers,
Department of Thoracic Brompton Hospital, LONDON
G. Dent, K.F. Chung
Medicine, SW3, U.K.
and P.J. Barnes
Cardiothoracic
Institute,
ABSTRACT successive inhalations of We studied the effect of five platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these PAF (24 "g) caused a mean cells ex viva in normal subjects. of the expiratory flow rate at 70% vital capacity maximal fall from a partial manoeuvre (ep30) of 46.4 + 6.2% (pM PAF. inhalation, but this was did changes in Methacholine inhalation not cause any We conclude that responsiveness of neutrophils to PAF ex viva. platelet desensitisation cannot be used as an index of ex viva reduced responsiveness of endogenous PAF release, but neutrophils ex viva is not a sensitive indicator.
INTRODUCTION There is increasing interest in the possible role of platelet-activating factor (PAF) in the pathophysiology of as its asthma (1). PAF has several relevant properties, such (2.3). which capacity to cause chemotaxis of human eosinophils are prominent cells in the asthmatic airway, and to induce microvascular leakage and oedema of the airways (4). another characteristic feature of the airways in acute asthma. Recent inhaled PAF induces studies in man have shown that loo-fold bronchoconstriction, with a potency approximately In addition, inhaled greater than that of methacholine (5). PAF leads to a sustained increase in airway responsiveness to methacholine (5,6), an effect that can persist for as long as 4 weeks (5). underlying bronchoconstriction The mechanism the and in man airway hyperresponsiveness induced by PAF remain unclear. PAF has negligible contractile effects on human bronchi in vitro, although it is active in the presence of platelets (7). In viva, the bronchoconstriction and airway
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in the guinea-pig hyperresponeiveness induced by PAF are inhibited by depletion of circulating platelets with a platelet antibody (8). Neutrophils have also been implicated in the hyperresponsiveness induction of bronchial after exposure to ozone (9) and to antigen in sensitised animals (10,ll). In aerosol causes bronchoconstriction and to PAF dogs, exposure associated bronchial hyperresponsiveness, with an increased in bronchoalveolar lavage fluid (12). recovery of neutrophils Inhalation of PAF in man results in a transient but impressive fall in circulating neutrophils but no changes in circulating platelets (13): however, when a larger amount of PAF "as administered intratracheally a thrombocytopenia "as also observed (14). In the present study, we have further examined the effect in normal subjects by of PAF on neutrophils and platelets studying their activation by PAF ex viva following inhalation of PAF. We reasoned that exposure to PAF in viva may reduce their ex viva responses to PAF due to tachyphylaxis (15). METHODS Subjects Twenty-three normal non-asthmatic subjects (age range 21-35; 5 female) agreed to participate in the study which "as All approved by the Brompton Hospital Ethics Committee. informed consent. Two subjects were current subjects gave skin-prick smokers and were atopic as indicated by positive tests with several common allergens. At the start of the study had been free of upper respiratory tract period, all subjects None of the were infections for at least 4 weeks. subjects Caffeine-containing beverages were taking any medication. withheld for 2 hours prior to challenge on each day. Study design Sixteen subjects inhaled PAF occasion the and on each assessed as the flow rate obtained at 70% airway response was of vital capacity from a partial expiratory maneouvre (irp30). blood samples were and after In addition, obtained before cell counts inhalation of PAF for measurement of circulating studies of platelet or neutrophil activation in vitro. and for neutrophil Platelet studies were performed in 9 subjects and Controls for the platelet studies were studies in 7 subjects. who performed in 5 subjects (including one "ho inhaled PAF) inhaled lyso-PAF (the inactive precursor and metabolite of PAF) methacholine in order the which was mixed with to mimic bronchoconstrictor effect of PAF. For the neutrophil studies, 4 subjects (including one who inhaled PAF) inhaled methacholine alone.
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PAF inhalation
challenge
PAF and lyso-PAF (Bachem Feinchemikalen AG, Switzerland) 100% ethanol at -6O'C at a concentration of 10 were stored in mg/ml. On the study day, a 2.5 mg/ml solution was prepared in concentration of 0.9% saline containing a final 0.03% PAF or lyso-PAF aerosol heat-treated human serum albumin. Was delivered from a nebuliser attached to a dosimeter (MEFAR, of 22 Brescia, Italy) driven by compressed air at a pressure nebulisation, which lasted for 1.0 sets, the psi. On each sublect inhaled for 3 seconds from functional residual capacity to total lung capacity and held his breath for 7 seconds before breathing out normally. The output of the nebuliser was 6 ;-11 per breath. Because of the development of rapid tachyphylaxis of the bronchoconstrictor response to PAF, we administered equal doses of PAF (2 breaths: 24 fig) every 15 minutes on five occasions (total dose: 10 breaths; For lyso-PAF, 120 yg). which has no bronchoconstrictor effect (5), we mixed the first concentration of methacholine dose with an appropriate (determined from the subject's bronchial responsiveness to to induce an approximately 40% fall in methacholine) in order op30: for the second to fifth inhalations only lyso-PAF was administered. In the other control study, only methacholine was administered on a single occasion to induce a fall in +p30 similar to that induced by PAF. or lyso-PAF The response to PAF was measured from volume-standardised partial expiratory flow-volume curves (16). Partial curves were used because of their more sensitive measure of bronchoconstriction, thus reducing the necessity for administering large doses of PAF or methacholine. Flow-volume curves were measured using a spirometer (Vitalograph, UK) and were analysed using a Hewlett-Packard microcomputer (Collingwood Measurements, Leicester, UK). flow-volume The curves were stored and could be displayed after each manoeuvre. Subjects initially performed a full vital capacity, and measurements of flow were made at 70% of vital capacity, measured from total lung capacity. Flow volume manoeuvres were performed by expiration from lust above tidal inspiration to residual volume followed by inhalation to total lung capacity before breathing out normally. All subjects were fully trained in these breathing manoeuvres before entering the study. Measurements of fp30 were made at one, 3, 5, 10 and 15 minutes after each inhalation of PAF or lyso-PAF: for the control methacholine challenge only, we measured 4~30 at one, 3, 5, 10, 15, 30, 45 ,60 and 75 minutes. The responses of sp30 were expressed as the percentage change from the baseline cp30. Measurement
of circulating
cells
In order to sample venous blood, an intravenous cannula (19G, Venflon, Viggo AB, Sweden) was inserted 30 minutes prior to PAF inhalation. For the measurement of circulating cells, samples (2 ml) were taken in the baseline state, at 5 and 15 minutes after the first 2 inhalations of PAF and at 15 min after the fifth occasion, and collected into tubes containing disodium edetate. Total white cell and platelet counts were
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measured on a Coulter Counter 880 (Coulter Electronics, Hialeah, Florida, USA). Blood smears were made from each sample and with stained May-Grumwald-Giemsa stain. Differential cell counts were performed on 100 cells from each smear by an independent observer unaware of the experimental The numbers of protocol. neutrophils, eosinophils and lymphocytes per cubic mm of blood were estimated by multiplying the percentage of each cell type by the total white cell count. Platelet
responsiveness
in vitro
Blood samples (15 ml) were obtained in the baseline state at 15 mins after the first, second and last inhalations of PAF, and collected in 3.2% trisodium citrate in a volume ratio of 1 to 9 parts of blood. The blood was transferred into plastic tubes in 1.5 ml aliquots and spun on a bench Eppendorf centrifuge (Model 1914; maximal centrifugal force: 8.200 g) for timed-periods of 2 seconds. With this method, a more rapid preparation of platelet-rich plasma (PRP) was possible so that platelet function could be more quickly assessed than conventional methods plasma (PPP) was Platelet-poor (17). obtained by centrifugation of a 1.5 ml anticoagulated blood aliquot for 5 mins on the Eppendorf centrifuge. As an index of platelet response, we determined platelet aggregation by measuring light transmission through aliquots of PRP using a dual channel aggregometer Payton with the PPP sample used to define 100% transmission. Aggregation of determined to 1, 10 and 100 nM PAF and lO>M ADP platelets was at 37°C. PRP (450~1) was placed in siliconised glass cuvettes containing nickel stirring bars and light transmission PAF or ADP (50 yl) was added and the maximum recorded. increase in light transmission recorded. The results have been expressed as the deflection in arbritary units induced by the increase in light transmission for each dose of PAF or ADP. Neutrophil
activation
in vitro
Blood (20 ml) was taken before and at 15 minutes after the first and last inhalations of PAF: for the control studies, taken before and at 15 and 60 minutes after a single blood was inhalation of methacholine. Blood was added to 4 ml of dextran 110 (6% in 0.9% NaCl; Fisons PLC, Loughborough, UK) and 200 U heparin contained in sterile polysterene tubes. Sedimentation of erythrocytes was allowed for 1 hour and the leucoctye-rich plasma was then layered on to 10 ml of Ficoll-Paque (Pharmacia 40 mins at AB, Upsalla, Sweden) and centrifuged at 425 g for room temperature. Residual erythrocytes in the cell pellet were lysed with 1 ml of ice-cold distilled water for 1 min. and the osmolarity was restored by addition of 10 ml of Medium 199 Irvine, without phenol red and (Flow Laboratories, UK) HEPES (Nl-2 L-glutamine, buffered with 20 mM and hydroxyethyl-piperazine N-l-2-ethanesulphonic acid). The 190 g for 20 mins at 4" C. suspension was centrifuged at were repeated and the final pellet Washing and centrifugation was resuspended in Medium 199 to a cell density of 3 x 10' cells per ml. Viability of the neutrophils was always greater
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than 98% as assessed by Trypan Blue exclusion. As an index of neutrophil activation, measured we light emission which represents ensuing chemiluminescence or cellular oxidative respiratory burst by using a luminometer (Model 1251, LKB Wallac, Turku, Finland). To each cuvette was added 500~~1 of lOOF luminol (Sigma, Poole, UK) with 100 p luciferin (Sigma) and 100 ul of PAF. The concentrations of PAF 0.01, 0.1, 1 and 10 )IM. The reaction was started studied were by adding 300 ~1 of neutrophil suspension at 37' C and the recorded in millivolts (mV) over a generated luminescence was period of 5 mins. Peak values were used for comparison. Data analysis All results have been reported as means + SEM. In order of PAF on 3~30 or on circulating cell to assess the effect counts, paired t-tests were used to compare the data with that of baseline To determine changes in the responses of values. platelet aggregation or of neutrophil chemiluminescence at each concentration of PAF in vitro, the values at baseline and at various time-points measured after inhalation of PAF were compared using a two-factor analysis of variance and the Newman-Keuls multiple range test (18).
RESULTS PAF-induced bronchoconstriction Inhalation of the first 2 breaths of PAF resulted in a fall in 4~30 of 46.4 + 6.2% at 5 min (p< 0.001) mean maximal from a baseline value of 133.6 + 9.4 l/min, followed by partial recovery to 33.7 + 4.6% at 1S min. All subjects noticed transient facial -flushing, associated with varying degrees of cough and chest tightness. Further inhalations of PAF did not result in larger falls in cp30 (Fig 1). and caused lesser degrees of facial flushing or chest tightness. By the fifth inhalation, 0~30 had only fallen by 24.2 _+ 5.2% at 5 min. which was less than the corresponding fall after the first inhalation (p < 0.01). TIME
1----0
0
1 15 0
tminl
1 15 0
I
I
15 0
15 0
tp30 20 &_,+
(% fall) 40 L
601 Fig. Effect of repeated inhalations of platelet-activating factor (PAF) on the partial expiratory flow at 70% vital capacity (cp30) in 16 normal subjects. PAF was administered at similar doses (24 pg) every 15 min on 5 occasions, as indicated by the arrows. Mean values -+ SEM are shown.
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PAF-induced leucopenia Total white cell count fell from 6.35 _+ 0.41 to 3.96 -+ 0.48 x 103/mm3 (p< 0.001) within 5 min of PAF inhalation, with subsequent recovery at 15 min (Fig 2). Further inhalations of PAF did not cause but there was a signif‘cant leucopenia, ; 7.87 + 0.61 x increase in total cell with 10 /nun3 count The leucopenia (p
9
t
Leucocyte or
T
Neutrophil count
( 103/mm3)
260r Platelet count
240.
k_I
li
1103/mm3) 220.l Of
t-
t
244 III
I_
0-
15
0 TIME
15
15
(mini
Fig 2. Effect of inhalations of platelet-activating factor (PAF) on circulating total white cell (o), neutrophil ( .) and platelet ( A ) counts in 16 subjects. Successive inhalations of PAF are indicated by the arrows. Blood counts were obtained after the first two inhalations. Circulating platelets remain unchanged after PAF total but white count fell min bY 5 (p
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Neutrophil function ex viva The chemiluminescence response to PAF was maximal at 1pM. unchanged These responses were with some reduction at 10 PM. at 15 and 60 min after methacholine-induced bronchoconstriction At 15 min. after the first PAF inhalation, there was (Fig 3). at all a small non-significant increase in chemiluminescence However, at 15 min after the fifth concentrations of PAF. the first approximately 75 min after inhalation of PAF (i.e. chemiluminescence in there was a reduction inhalation), only the 10 JIM but response to exogenous PAF at 0.1, 1 and significantly reduced (p
40
I
30 CL (mV)
‘Cl 10.
0. L
0.01
0.1
1
10 PAF
0.01
0.1
1
4
10
(uM)
Fig 3. Chemiluminescence response (CL) from neutrophils activated by platelet-activating factor (PAF) in vitro. Neutrophils were isolated from venous blood samples before (0) and at 15 min after the first (0) and the fifth (A) inhalations of PAF aerosol as shown on the left-hand panel. Similar data are shown on the right-hand panel for control subjects who inhaled methacholine instead of PAF to induce similar degrees of bronchoconstriction. PAF exposure resulted in a significant decrease in CL response of neutrophils, isolated after the fifth inhalation, to 1 PM PAF ex viva when compared to the response of neutrophils obtained after the first inhalation Mean values -+ SEM are shown. (P
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Platelet function ex viva Baseline aggregation to PAF was 25 + 3.2, 35.5 + 3.8 and 35.6 + 3.8 units at 1, 10 and 100 nM respectively;ADP at 10 JIM resulted in 56.7 + 3.5 units of aggregation (Fig 4). There were small reductions in platelet aggregability at 15 min after the first and second inhalation of PAF, but these did not achieve statistical significance. No significant changes in platelet aggregation were observed after inhalation of lyso-PAF mixed with methacholine (Fig 4).
70.
60
50 Platelet aggregation (arbitrary
40
30
units)
10
0’
-.1
10 PAF
100 +Mt
1;
1
ADP(pM)
10 PAF
100 (JIM)
1; ADP(pM
1
Fig 4. Aggregation of platelets in vitro in response to exogenous platelet-activating factor and adenosine (PAF) Platelets prepared as platelet-rich phosphate (ADP). were plasma (see text) from venous blood sampled before (0) and at 5 minutes after the first (0). the second (A) and the fifth (0) inhalations of PAF aerosols in 9 subjects, as shown on the left-hand panel. Similar data are shown on the right-hand panel for 5 control sublects who inhaled lyso-PAF mixed with methacholine in order to induce similar degrees of bronchoconstriction as PAF. There were no significant differences in the response of the platelets to PAF or ADP in vitro following the inhalations of PAF or lyso-PAF. Mean values -+ SEM are shown.
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DISCUSSION We have shown that PAF inhalation causes no significant changes in neutrophil activation to PAF ex viva, as measured by inhalation of a single dose of PAF; chemiluminescence after however, after the fifth PAF inhalation, there was a signficant reduction of neutrophil activation induced by l>M of PAF. In terms of platelet aggregation, there was no significant effect when PAF was added to platelets in vitro. Neutrophil counts fell dramatically within 5 min of the first inhalation, but subsequently tachyphylactic to further this response was inhalations of PAF; a significant rebound neutrophilia was significant subsequently observed. No changes in platelet counts were observed. Previous investigators have assumed that, if PAF were released endogenously, then circulating platelets may become tachyphylactic to PAF ex viva because PAF activates platelets which are subsequently desensitised to its effects (19). Using this assumption, endogenous release of PAF into the circulation after antigen challenge in allergic asthmatic subjects has been previously reported (20.21). However, in our study we did not detect any reduction in ex viva platelet responsiveness to exogenous PAF after repeated PAF inhalations in viva despite the marked tachyphylactic response to PAF in the airways. in our subjects resulted in extrapulmonary Inhalation of PAF symptoms of flushing, suggesting that some of the inhaled PAF reaching systemic circulation despite its rapid must be the metabolic breakdown in plasma (22) and in the airway epithelium It may be that only a minor proportion of circulating (23). platelets is exposed in viva after inhalation, thus to PAF making it difficult to detect any small tachyphylactic response ex viva. The amount of PAF that can be administered in our study is limited by its systemic effects. Thus, the dose of PAF reaching the airways of our subjects was approximately 100 to l,OOO-fold less than that administered intratracheally in brain-dead ventilated patients by Gateau et al (14). In this study, profound thrombocytopenia and neutropenia with systemic hypotension were observed. The rapid, transient fall in circulating neutrophils after the first inhalation of PAF is probably the result of temporary sequestration of neutrophils within the pulmonary vasculature. 15 min Interestingly, neutrophils at after the first PAF inhalation were slightly more responsive to exogenous PAF, but this was not statistically significant. In a previous study, we have shown that the density of circulating neutrophils at this time-point is significantly decreased (24) suggesting that the neutrophils have been in a state of "activation". may However, the most important observation is the reduction in the chemiluminescence response to PAF ex viva of neutrophils obtained after the fifth inhalation of PAF when compared to the response after the first inhalation, particularly at the 1 uM dose. This diminished response could represent a desensitisation phenomenon. It is also possible that this could result from the presence of a new population of immature neutrophils from the bone-marrow, in response to the profound neutropenia induced by inhaled PAF. However, whether the
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numbers PAF is decreased in these affinity or receptor for immature neutrohils is not known. The relationship of these ex viva responses of circulating platelets or neutrophils to the bronchoconstrictor response can only be speculative. Human bronchial smooth muscle preparations do not contract in response to PAF in vitro, of platelets except in the presence (71, suggesting that PAF-induced bronchoconstriction in viva may be mediated by the However, the lack of tachyphylaxis activation of platelets. of the platelets ex viva observed in our study does not support this mechanism in man. Although PAF-induced bronchoconstriction induced by intravenous PAF may be mediated by circulating platelets in the guinea-pig (8). they are not involved when PAF is administered as an aerosol (25). The reduced response of the circulating neutrophils ex viva after PAF inhalation together with tachyphylactic response to the inhaled PAF in our subjects gives support for a possible role the neutrophil in mediating PAF-induced for bronchoconstriction: in addition, we have also observed an increased number of neutrophils in bronchoalveolar lavage fluid after PAF inhalation in man (24). Alveolar macrophages may involved as their activation ex viva is reduced by also be repeated inhalations of PAF in the guinea-pig (15). In summary, we have shown no evidence for ex viva platelet inhalations of PAF in desensitisation to PAF after repeated neutrophil activation to PAF was significantly viva. Ex vivo reduced at only one dose of PAF. These results suggest that studies using ex viva PAF desensitisation of platelets as an index of in viva PAF release must be treated with caution. of neutrophil activation ex viva may be more Although the "se suitable, the "se of currently available specific PAF receptor antagonists will be a more useful tool to examine the role of PAF in various pathophysiological states in man (26).
ACKNOWLEDGEMENTS for her utmost care in the We thank Madeleine Wray preparation of this manuscript. This work was supported by the Asthma Research Council and Medical Research Council (UK).
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References PAF and Chung, K.F. closely 1. Barnes, P.J. Sci. 8: Trends. Pharmacol. mimics pathology of asthma. 285 (1987). 2. Wardlaw, A.J., Moqbel, R., Cromwell, 0. and Kay, chemotactic A.B. A potent Platelet-activating factor. factor for human eosinophils. J. Clin. and chemokinetic 78: 1701 (1986). Invest. 9.9. Accumulation of 3. Henocq, E., Vargaftig, PAF-acether and eosinophils in response to intracutaneous allergens in man. Lancet 1: 1378 (1986). and Rogers, D.F. 4. Evans, T.W.. Chung, K.F., Effect of platelet-activating factor on Barnes, P.J. J. airway vascular permeability : possible mechanisms. 63: Physiol. 479 (1987). APP~. Barnes, P.J. Dixon, C.M.S. and 5. Cuss, F.M., Effects of inhaled platelet activating factor on pulmonary Lancet 2: function and bronchial responsiveness in man. 189 (1986). 6. Rubin, A.-H., Smith, L.J. and Patterson, R. The platelet-activating bronchoconstrictor properties of Rev. Respir. 136: 1145 factor in humans. Am. Dis. (1987). Airway responses to 7. Schellenberg, R.R. Respir. Rev. Dis. platelet-activating factor. Am. 136: 528 (1987). Sanjar, S. 8. Mazzoni, L., Morley, J., Page, C.P., hyper-reactivity by platelet Induction of airway Physiol. 365: activating factor in the guinea-pig. J. 107P (1985). 9. O'Byrne, P.M., Walters, E.H., Gold, B.D., Aizawa. Holtzman, H.A., Fabbri, L.M., Alpert, S.E., Nadel, J.A., Neutrophil inhibits airway M.J. depletion hyperresponsiveness induced by ozone exposure in dogs. 130: Respir. 214 (1984). Am. Rev. Dis. 10. Chung, K.F., Becker, A.B., Lazarus, S.C., Frick, Gold, W.M. Antigen-induced airway O.L., Nadel, J.A., hyperresponsiveness and pulmonary inflammation in allergic 558: dogs. J. APP~. Physiol. 1347 (1985). 11. Murphy, K.R., Wilson, M.C., Irvin, C.G., Glezen, and Larsen, L.S., Marsh, W.R., Haslett, C., Henson, P.M. G.L. requirement for polymorphonuclear leukocytes in The asthmatic response and heightened airways the late model. Am. Respir Dis. reactivity in an animal Rev. 134: 62 (1986). Ueki, 12. Chung, K.F., Aizawa, H.A., Leikauf, G.D., Evans, T.W. Nadel. Airway T.F., and J.A. factor hyperresponsiveness induced by platelet activating : a role of thromboxane generation. J. Pharmacol. Exp. 236: Therap. 580 (1986). M. and 13. Chung, K.F., Minette, P.. McCusker, Barnes, P.J. Ketotifen inhibits the cutaneous but not the airway responses to platelet-activating factor in man. J. Immunol. In press (1988). Allergy Clin. P. 14. Gateau, 0.. Arnoux, B., Deriaz, H., Viars, effects of intratracheal and Benveniste, J. Acute
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administration of paf-acether (platelet-activating factor) Rev. Respir. in humans. Am. Dis. 3: 129 (1984). I., Lagente, V., Lefort, J., 15. Maridonneau-Parini, Russo-Marie, F. Randon, J., and Vargaftig, B.B. bronchoconstriction and to Desnsitization of PAF-induced activation of alveolar macrophges by repeated inhalation of PAF in the guinea-pig. Biochem. Biophys. Res. 131: 42 (1985). comun. Gribbin, H.R., 16. Barnes, P.J., Osmanliev, D., Pride, N.B. Partial flow-volume measure curves to bronchodilator dose-response curves in normal man. J. 1193 (1981). Physiol. 50: APP~. F. 17. Whittle, B.J.R., Moncada, S.. Whiting, and Vane, J.R. Carbacyclin - a potent stable prostacyclin inhibition of platelet analogue for the aggregation. Prostaglandins 19: 605 (1980). J.H. Biostatistical 18. Zar, analysis. Prentice-Hall Inc., Englewood Cliffs, N.J. (1974). C.M., Pifer, K.M. 19. Chesney, D.D., Huch, Desensitisation of human platelets by platelet-activating Biochem.Biophys.Res.Commun 127: factor. 24 (1985). Hanson, J.M., Bilani H., 20. Thompson, P.J., Turner-Warwick, M. and Morley, J. Platelets, platelet activating factor and asthma. Am. Rev. Respir. Dis. 3 (1984). 129: Wirkungen def 21. Beer, H-J. platelet-activating factor (PAF) aus die thrombozyten def menschen. MD University of Zurich (1984). thesis. C., 22. Latrigue-Mattei, Godeneche, Chabard, D.. Berger J.A. Pharmacokinetic J.L. and study of Preliminary results PAF-acether: after the intravenous administration of a 3-H- labelled product to the rabbit. Ag Act 1982: 12: 703 (1982). 23. Haroldsen, P.E., Voelkel, N.F., Henson, J.E., Henson, P.M. and Murphy, R.C. Metabolism of platelet-activating factor in isolated perfused rat lung. 1860 (1987). J. Clin. Invest. 79: A.J., Chung, 24. Wardlaw. K.F., Moqbel, R., Macdonald, A.J., McCusker, M., Barnes, P.J., Collins, J.V. Cellular changes in and Kay, A.B. blood and bronchoalveolar lavage (BALI and bronchial responsiveness PAF in man. Am. after inhaled Rev. Respir. Dis. In press (1988). 25. Lefort, J., Rotilio, D., Vargaftig, B.B. The platelet-independent release of thromboxane A2 by PAF-acether for guinea-pig lungs involves mechanisms leukotriene C4 and bradykinin. distinct from those for 82: 525 (1984). Br. J. Pharmacol. 26. Chung, K-F., Barnes, P.J. PAF antagonists: their potential therapeutic role in asthma. Drugs 35: 93 (1988).
Editor:
354
B. Whittle
Received:
6-7-88
Accepted:7-19-88
SEPTEMBER 1988 VOL. 36 NO. 3
EFFECT OF INHALED PLATELET-ACTIVATING FACTOR ON CIRCULATING NEUTROPHILS AND PLATELETS IN VIVO AND EX VIVO IN MAN
I. Kioumis,
J.W. Lammers,
Department of Thoracic Brompton Hospital, LONDON
G. Dent, K.F. Chung
Medicine, SW3, U.K.
and P.J. Barnes
Cardiothoracic
Institute,
ABSTRACT successive inhalations of We studied the effect of five platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these PAF (24 "g) caused a mean cells ex viva in normal subjects. of the expiratory flow rate at 70% vital capacity maximal fall from a partial manoeuvre (ep30) of 46.4 + 6.2% (p
INTRODUCTION There is increasing interest in the possible role of platelet-activating factor (PAF) in the pathophysiology of as its asthma (1). PAF has several relevant properties, such (2.3). which capacity to cause chemotaxis of human eosinophils are prominent cells in the asthmatic airway, and to induce microvascular leakage and oedema of the airways (4). another characteristic feature of the airways in acute asthma. Recent inhaled PAF induces studies in man have shown that loo-fold bronchoconstriction, with a potency approximately In addition, inhaled greater than that of methacholine (5). PAF leads to a sustained increase in airway responsiveness to methacholine (5,6), an effect that can persist for as long as 4 weeks (5). underlying bronchoconstriction The mechanism the and in man airway hyperresponsiveness induced by PAF remain unclear. PAF has negligible contractile effects on human bronchi in vitro, although it is active in the presence of platelets (7). In viva, the bronchoconstriction and airway
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in the guinea-pig hyperresponeiveness induced by PAF are inhibited by depletion of circulating platelets with a platelet antibody (8). Neutrophils have also been implicated in the hyperresponsiveness induction of bronchial after exposure to ozone (9) and to antigen in sensitised animals (10,ll). In aerosol causes bronchoconstriction and to PAF dogs, exposure associated bronchial hyperresponsiveness, with an increased in bronchoalveolar lavage fluid (12). recovery of neutrophils Inhalation of PAF in man results in a transient but impressive fall in circulating neutrophils but no changes in circulating platelets (13): however, when a larger amount of PAF "as administered intratracheally a thrombocytopenia "as also observed (14). In the present study, we have further examined the effect in normal subjects by of PAF on neutrophils and platelets studying their activation by PAF ex viva following inhalation of PAF. We reasoned that exposure to PAF in viva may reduce their ex viva responses to PAF due to tachyphylaxis (15). METHODS Subjects Twenty-three normal non-asthmatic subjects (age range 21-35; 5 female) agreed to participate in the study which "as All approved by the Brompton Hospital Ethics Committee. informed consent. Two subjects were current subjects gave skin-prick smokers and were atopic as indicated by positive tests with several common allergens. At the start of the study had been free of upper respiratory tract period, all subjects None of the were infections for at least 4 weeks. subjects Caffeine-containing beverages were taking any medication. withheld for 2 hours prior to challenge on each day. Study design Sixteen subjects inhaled PAF occasion the and on each assessed as the flow rate obtained at 70% airway response was of vital capacity from a partial expiratory maneouvre (irp30). blood samples were and after In addition, obtained before cell counts inhalation of PAF for measurement of circulating studies of platelet or neutrophil activation in vitro. and for neutrophil Platelet studies were performed in 9 subjects and Controls for the platelet studies were studies in 7 subjects. who performed in 5 subjects (including one "ho inhaled PAF) inhaled lyso-PAF (the inactive precursor and metabolite of PAF) methacholine in order the which was mixed with to mimic bronchoconstrictor effect of PAF. For the neutrophil studies, 4 subjects (including one who inhaled PAF) inhaled methacholine alone.
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PAF inhalation
challenge
PAF and lyso-PAF (Bachem Feinchemikalen AG, Switzerland) 100% ethanol at -6O'C at a concentration of 10 were stored in mg/ml. On the study day, a 2.5 mg/ml solution was prepared in concentration of 0.9% saline containing a final 0.03% PAF or lyso-PAF aerosol heat-treated human serum albumin. Was delivered from a nebuliser attached to a dosimeter (MEFAR, of 22 Brescia, Italy) driven by compressed air at a pressure nebulisation, which lasted for 1.0 sets, the psi. On each sublect inhaled for 3 seconds from functional residual capacity to total lung capacity and held his breath for 7 seconds before breathing out normally. The output of the nebuliser was 6 ;-11 per breath. Because of the development of rapid tachyphylaxis of the bronchoconstrictor response to PAF, we administered equal doses of PAF (2 breaths: 24 fig) every 15 minutes on five occasions (total dose: 10 breaths; For lyso-PAF, 120 yg). which has no bronchoconstrictor effect (5), we mixed the first concentration of methacholine dose with an appropriate (determined from the subject's bronchial responsiveness to to induce an approximately 40% fall in methacholine) in order op30: for the second to fifth inhalations only lyso-PAF was administered. In the other control study, only methacholine was administered on a single occasion to induce a fall in +p30 similar to that induced by PAF. or lyso-PAF The response to PAF was measured from volume-standardised partial expiratory flow-volume curves (16). Partial curves were used because of their more sensitive measure of bronchoconstriction, thus reducing the necessity for administering large doses of PAF or methacholine. Flow-volume curves were measured using a spirometer (Vitalograph, UK) and were analysed using a Hewlett-Packard microcomputer (Collingwood Measurements, Leicester, UK). flow-volume The curves were stored and could be displayed after each manoeuvre. Subjects initially performed a full vital capacity, and measurements of flow were made at 70% of vital capacity, measured from total lung capacity. Flow volume manoeuvres were performed by expiration from lust above tidal inspiration to residual volume followed by inhalation to total lung capacity before breathing out normally. All subjects were fully trained in these breathing manoeuvres before entering the study. Measurements of fp30 were made at one, 3, 5, 10 and 15 minutes after each inhalation of PAF or lyso-PAF: for the control methacholine challenge only, we measured 4~30 at one, 3, 5, 10, 15, 30, 45 ,60 and 75 minutes. The responses of sp30 were expressed as the percentage change from the baseline cp30. Measurement
of circulating
cells
In order to sample venous blood, an intravenous cannula (19G, Venflon, Viggo AB, Sweden) was inserted 30 minutes prior to PAF inhalation. For the measurement of circulating cells, samples (2 ml) were taken in the baseline state, at 5 and 15 minutes after the first 2 inhalations of PAF and at 15 min after the fifth occasion, and collected into tubes containing disodium edetate. Total white cell and platelet counts were
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measured on a Coulter Counter 880 (Coulter Electronics, Hialeah, Florida, USA). Blood smears were made from each sample and with stained May-Grumwald-Giemsa stain. Differential cell counts were performed on 100 cells from each smear by an independent observer unaware of the experimental The numbers of protocol. neutrophils, eosinophils and lymphocytes per cubic mm of blood were estimated by multiplying the percentage of each cell type by the total white cell count. Platelet
responsiveness
in vitro
Blood samples (15 ml) were obtained in the baseline state at 15 mins after the first, second and last inhalations of PAF, and collected in 3.2% trisodium citrate in a volume ratio of 1 to 9 parts of blood. The blood was transferred into plastic tubes in 1.5 ml aliquots and spun on a bench Eppendorf centrifuge (Model 1914; maximal centrifugal force: 8.200 g) for timed-periods of 2 seconds. With this method, a more rapid preparation of platelet-rich plasma (PRP) was possible so that platelet function could be more quickly assessed than conventional methods plasma (PPP) was Platelet-poor (17). obtained by centrifugation of a 1.5 ml anticoagulated blood aliquot for 5 mins on the Eppendorf centrifuge. As an index of platelet response, we determined platelet aggregation by measuring light transmission through aliquots of PRP using a dual channel aggregometer Payton with the PPP sample used to define 100% transmission. Aggregation of determined to 1, 10 and 100 nM PAF and lO>M ADP platelets was at 37°C. PRP (450~1) was placed in siliconised glass cuvettes containing nickel stirring bars and light transmission PAF or ADP (50 yl) was added and the maximum recorded. increase in light transmission recorded. The results have been expressed as the deflection in arbritary units induced by the increase in light transmission for each dose of PAF or ADP. Neutrophil
activation
in vitro
Blood (20 ml) was taken before and at 15 minutes after the first and last inhalations of PAF: for the control studies, taken before and at 15 and 60 minutes after a single blood was inhalation of methacholine. Blood was added to 4 ml of dextran 110 (6% in 0.9% NaCl; Fisons PLC, Loughborough, UK) and 200 U heparin contained in sterile polysterene tubes. Sedimentation of erythrocytes was allowed for 1 hour and the leucoctye-rich plasma was then layered on to 10 ml of Ficoll-Paque (Pharmacia 40 mins at AB, Upsalla, Sweden) and centrifuged at 425 g for room temperature. Residual erythrocytes in the cell pellet were lysed with 1 ml of ice-cold distilled water for 1 min. and the osmolarity was restored by addition of 10 ml of Medium 199 Irvine, without phenol red and (Flow Laboratories, UK) HEPES (Nl-2 L-glutamine, buffered with 20 mM and hydroxyethyl-piperazine N-l-2-ethanesulphonic acid). The 190 g for 20 mins at 4" C. suspension was centrifuged at were repeated and the final pellet Washing and centrifugation was resuspended in Medium 199 to a cell density of 3 x 10' cells per ml. Viability of the neutrophils was always greater
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than 98% as assessed by Trypan Blue exclusion. As an index of neutrophil activation, measured we light emission which represents ensuing chemiluminescence or cellular oxidative respiratory burst by using a luminometer (Model 1251, LKB Wallac, Turku, Finland). To each cuvette was added 500~~1 of lOOF luminol (Sigma, Poole, UK) with 100 p luciferin (Sigma) and 100 ul of PAF. The concentrations of PAF 0.01, 0.1, 1 and 10 )IM. The reaction was started studied were by adding 300 ~1 of neutrophil suspension at 37' C and the recorded in millivolts (mV) over a generated luminescence was period of 5 mins. Peak values were used for comparison. Data analysis All results have been reported as means + SEM. In order of PAF on 3~30 or on circulating cell to assess the effect counts, paired t-tests were used to compare the data with that of baseline To determine changes in the responses of values. platelet aggregation or of neutrophil chemiluminescence at each concentration of PAF in vitro, the values at baseline and at various time-points measured after inhalation of PAF were compared using a two-factor analysis of variance and the Newman-Keuls multiple range test (18).
RESULTS PAF-induced bronchoconstriction Inhalation of the first 2 breaths of PAF resulted in a fall in 4~30 of 46.4 + 6.2% at 5 min (p< 0.001) mean maximal from a baseline value of 133.6 + 9.4 l/min, followed by partial recovery to 33.7 + 4.6% at 1S min. All subjects noticed transient facial -flushing, associated with varying degrees of cough and chest tightness. Further inhalations of PAF did not result in larger falls in cp30 (Fig 1). and caused lesser degrees of facial flushing or chest tightness. By the fifth inhalation, 0~30 had only fallen by 24.2 _+ 5.2% at 5 min. which was less than the corresponding fall after the first inhalation (p < 0.01). TIME
1----0
0
1 15 0
tminl
1 15 0
I
I
15 0
15 0
tp30 20 &_,+
(% fall) 40 L
601 Fig. Effect of repeated inhalations of platelet-activating factor (PAF) on the partial expiratory flow at 70% vital capacity (cp30) in 16 normal subjects. PAF was administered at similar doses (24 pg) every 15 min on 5 occasions, as indicated by the arrows. Mean values -+ SEM are shown.
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PAF-induced leucopenia Total white cell count fell from 6.35 _+ 0.41 to 3.96 -+ 0.48 x 103/mm3 (p< 0.001) within 5 min of PAF inhalation, with subsequent recovery at 15 min (Fig 2). Further inhalations of PAF did not cause but there was a signif‘cant leucopenia, ; 7.87 + 0.61 x increase in total cell with 10 /nun3 count The leucopenia (p
9
t
Leucocyte or
T
Neutrophil count
( 103/mm3)
260r Platelet count
240.
k_I
li
1103/mm3) 220.l Of
t-
t
244 III
I_
0-
15
0 TIME
15
15
(mini
Fig 2. Effect of inhalations of platelet-activating factor (PAF) on circulating total white cell (o), neutrophil ( .) and platelet ( A ) counts in 16 subjects. Successive inhalations of PAF are indicated by the arrows. Blood counts were obtained after the first two inhalations. Circulating platelets remain unchanged after PAF total but white count fell min bY 5 (p
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Neutrophil function ex viva The chemiluminescence response to PAF was maximal at 1pM. unchanged These responses were with some reduction at 10 PM. at 15 and 60 min after methacholine-induced bronchoconstriction At 15 min. after the first PAF inhalation, there was (Fig 3). at all a small non-significant increase in chemiluminescence However, at 15 min after the fifth concentrations of PAF. the first approximately 75 min after inhalation of PAF (i.e. chemiluminescence in there was a reduction inhalation), only the 10 JIM but response to exogenous PAF at 0.1, 1 and significantly reduced (p
40
I
30 CL (mV)
‘Cl 10.
0. L
0.01
0.1
1
10 PAF
0.01
0.1
1
4
10
(uM)
Fig 3. Chemiluminescence response (CL) from neutrophils activated by platelet-activating factor (PAF) in vitro. Neutrophils were isolated from venous blood samples before (0) and at 15 min after the first (0) and the fifth (A) inhalations of PAF aerosol as shown on the left-hand panel. Similar data are shown on the right-hand panel for control subjects who inhaled methacholine instead of PAF to induce similar degrees of bronchoconstriction. PAF exposure resulted in a significant decrease in CL response of neutrophils, isolated after the fifth inhalation, to 1 PM PAF ex viva when compared to the response of neutrophils obtained after the first inhalation Mean values -+ SEM are shown. (P
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Platelet function ex viva Baseline aggregation to PAF was 25 + 3.2, 35.5 + 3.8 and 35.6 + 3.8 units at 1, 10 and 100 nM respectively;ADP at 10 JIM resulted in 56.7 + 3.5 units of aggregation (Fig 4). There were small reductions in platelet aggregability at 15 min after the first and second inhalation of PAF, but these did not achieve statistical significance. No significant changes in platelet aggregation were observed after inhalation of lyso-PAF mixed with methacholine (Fig 4).
70.
60
50 Platelet aggregation (arbitrary
40
30
units)
10
0’
-.1
10 PAF
100 +Mt
1;
1
ADP(pM)
10 PAF
100 (JIM)
1; ADP(pM
1
Fig 4. Aggregation of platelets in vitro in response to exogenous platelet-activating factor and adenosine (PAF) Platelets prepared as platelet-rich phosphate (ADP). were plasma (see text) from venous blood sampled before (0) and at 5 minutes after the first (0). the second (A) and the fifth (0) inhalations of PAF aerosols in 9 subjects, as shown on the left-hand panel. Similar data are shown on the right-hand panel for 5 control sublects who inhaled lyso-PAF mixed with methacholine in order to induce similar degrees of bronchoconstriction as PAF. There were no significant differences in the response of the platelets to PAF or ADP in vitro following the inhalations of PAF or lyso-PAF. Mean values -+ SEM are shown.
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DISCUSSION We have shown that PAF inhalation causes no significant changes in neutrophil activation to PAF ex viva, as measured by inhalation of a single dose of PAF; chemiluminescence after however, after the fifth PAF inhalation, there was a signficant reduction of neutrophil activation induced by l>M of PAF. In terms of platelet aggregation, there was no significant effect when PAF was added to platelets in vitro. Neutrophil counts fell dramatically within 5 min of the first inhalation, but subsequently tachyphylactic to further this response was inhalations of PAF; a significant rebound neutrophilia was significant subsequently observed. No changes in platelet counts were observed. Previous investigators have assumed that, if PAF were released endogenously, then circulating platelets may become tachyphylactic to PAF ex viva because PAF activates platelets which are subsequently desensitised to its effects (19). Using this assumption, endogenous release of PAF into the circulation after antigen challenge in allergic asthmatic subjects has been previously reported (20.21). However, in our study we did not detect any reduction in ex viva platelet responsiveness to exogenous PAF after repeated PAF inhalations in viva despite the marked tachyphylactic response to PAF in the airways. in our subjects resulted in extrapulmonary Inhalation of PAF symptoms of flushing, suggesting that some of the inhaled PAF reaching systemic circulation despite its rapid must be the metabolic breakdown in plasma (22) and in the airway epithelium It may be that only a minor proportion of circulating (23). platelets is exposed in viva after inhalation, thus to PAF making it difficult to detect any small tachyphylactic response ex viva. The amount of PAF that can be administered in our study is limited by its systemic effects. Thus, the dose of PAF reaching the airways of our subjects was approximately 100 to l,OOO-fold less than that administered intratracheally in brain-dead ventilated patients by Gateau et al (14). In this study, profound thrombocytopenia and neutropenia with systemic hypotension were observed. The rapid, transient fall in circulating neutrophils after the first inhalation of PAF is probably the result of temporary sequestration of neutrophils within the pulmonary vasculature. 15 min Interestingly, neutrophils at after the first PAF inhalation were slightly more responsive to exogenous PAF, but this was not statistically significant. In a previous study, we have shown that the density of circulating neutrophils at this time-point is significantly decreased (24) suggesting that the neutrophils have been in a state of "activation". may However, the most important observation is the reduction in the chemiluminescence response to PAF ex viva of neutrophils obtained after the fifth inhalation of PAF when compared to the response after the first inhalation, particularly at the 1 uM dose. This diminished response could represent a desensitisation phenomenon. It is also possible that this could result from the presence of a new population of immature neutrophils from the bone-marrow, in response to the profound neutropenia induced by inhaled PAF. However, whether the
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numbers PAF is decreased in these affinity or receptor for immature neutrohils is not known. The relationship of these ex viva responses of circulating platelets or neutrophils to the bronchoconstrictor response can only be speculative. Human bronchial smooth muscle preparations do not contract in response to PAF in vitro, of platelets except in the presence (71, suggesting that PAF-induced bronchoconstriction in viva may be mediated by the However, the lack of tachyphylaxis activation of platelets. of the platelets ex viva observed in our study does not support this mechanism in man. Although PAF-induced bronchoconstriction induced by intravenous PAF may be mediated by circulating platelets in the guinea-pig (8). they are not involved when PAF is administered as an aerosol (25). The reduced response of the circulating neutrophils ex viva after PAF inhalation together with tachyphylactic response to the inhaled PAF in our subjects gives support for a possible role the neutrophil in mediating PAF-induced for bronchoconstriction: in addition, we have also observed an increased number of neutrophils in bronchoalveolar lavage fluid after PAF inhalation in man (24). Alveolar macrophages may involved as their activation ex viva is reduced by also be repeated inhalations of PAF in the guinea-pig (15). In summary, we have shown no evidence for ex viva platelet inhalations of PAF in desensitisation to PAF after repeated neutrophil activation to PAF was significantly viva. Ex vivo reduced at only one dose of PAF. These results suggest that studies using ex viva PAF desensitisation of platelets as an index of in viva PAF release must be treated with caution. of neutrophil activation ex viva may be more Although the "se suitable, the "se of currently available specific PAF receptor antagonists will be a more useful tool to examine the role of PAF in various pathophysiological states in man (26).
ACKNOWLEDGEMENTS for her utmost care in the We thank Madeleine Wray preparation of this manuscript. This work was supported by the Asthma Research Council and Medical Research Council (UK).
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References PAF and Chung, K.F. closely 1. Barnes, P.J. Sci. 8: Trends. Pharmacol. mimics pathology of asthma. 285 (1987). 2. Wardlaw, A.J., Moqbel, R., Cromwell, 0. and Kay, chemotactic A.B. A potent Platelet-activating factor. factor for human eosinophils. J. Clin. and chemokinetic 78: 1701 (1986). Invest. 9.9. Accumulation of 3. Henocq, E., Vargaftig, PAF-acether and eosinophils in response to intracutaneous allergens in man. Lancet 1: 1378 (1986). and Rogers, D.F. 4. Evans, T.W.. Chung, K.F., Effect of platelet-activating factor on Barnes, P.J. J. airway vascular permeability : possible mechanisms. 63: Physiol. 479 (1987). APP~. Barnes, P.J. Dixon, C.M.S. and 5. Cuss, F.M., Effects of inhaled platelet activating factor on pulmonary Lancet 2: function and bronchial responsiveness in man. 189 (1986). 6. Rubin, A.-H., Smith, L.J. and Patterson, R. The platelet-activating bronchoconstrictor properties of Rev. Respir. 136: 1145 factor in humans. Am. Dis. (1987). Airway responses to 7. Schellenberg, R.R. Respir. Rev. Dis. platelet-activating factor. Am. 136: 528 (1987). Sanjar, S. 8. Mazzoni, L., Morley, J., Page, C.P., hyper-reactivity by platelet Induction of airway Physiol. 365: activating factor in the guinea-pig. J. 107P (1985). 9. O'Byrne, P.M., Walters, E.H., Gold, B.D., Aizawa. Holtzman, H.A., Fabbri, L.M., Alpert, S.E., Nadel, J.A., Neutrophil inhibits airway M.J. depletion hyperresponsiveness induced by ozone exposure in dogs. 130: Respir. 214 (1984). Am. Rev. Dis. 10. Chung, K.F., Becker, A.B., Lazarus, S.C., Frick, Gold, W.M. Antigen-induced airway O.L., Nadel, J.A., hyperresponsiveness and pulmonary inflammation in allergic 558: dogs. J. APP~. Physiol. 1347 (1985). 11. Murphy, K.R., Wilson, M.C., Irvin, C.G., Glezen, and Larsen, L.S., Marsh, W.R., Haslett, C., Henson, P.M. G.L. requirement for polymorphonuclear leukocytes in The asthmatic response and heightened airways the late model. Am. Respir Dis. reactivity in an animal Rev. 134: 62 (1986). Ueki, 12. Chung, K.F., Aizawa, H.A., Leikauf, G.D., Evans, T.W. Nadel. Airway T.F., and J.A. factor hyperresponsiveness induced by platelet activating : a role of thromboxane generation. J. Pharmacol. Exp. 236: Therap. 580 (1986). M. and 13. Chung, K.F., Minette, P.. McCusker, Barnes, P.J. Ketotifen inhibits the cutaneous but not the airway responses to platelet-activating factor in man. J. Immunol. In press (1988). Allergy Clin. P. 14. Gateau, 0.. Arnoux, B., Deriaz, H., Viars, effects of intratracheal and Benveniste, J. Acute
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administration of paf-acether (platelet-activating factor) Rev. Respir. in humans. Am. Dis. 3: 129 (1984). I., Lagente, V., Lefort, J., 15. Maridonneau-Parini, Russo-Marie, F. Randon, J., and Vargaftig, B.B. bronchoconstriction and to Desnsitization of PAF-induced activation of alveolar macrophges by repeated inhalation of PAF in the guinea-pig. Biochem. Biophys. Res. 131: 42 (1985). comun. Gribbin, H.R., 16. Barnes, P.J., Osmanliev, D., Pride, N.B. Partial flow-volume measure curves to bronchodilator dose-response curves in normal man. J. 1193 (1981). Physiol. 50: APP~. F. 17. Whittle, B.J.R., Moncada, S.. Whiting, and Vane, J.R. Carbacyclin - a potent stable prostacyclin inhibition of platelet analogue for the aggregation. Prostaglandins 19: 605 (1980). J.H. Biostatistical 18. Zar, analysis. Prentice-Hall Inc., Englewood Cliffs, N.J. (1974). C.M., Pifer, K.M. 19. Chesney, D.D., Huch, Desensitisation of human platelets by platelet-activating Biochem.Biophys.Res.Commun 127: factor. 24 (1985). Hanson, J.M., Bilani H., 20. Thompson, P.J., Turner-Warwick, M. and Morley, J. Platelets, platelet activating factor and asthma. Am. Rev. Respir. Dis. 3 (1984). 129: Wirkungen def 21. Beer, H-J. platelet-activating factor (PAF) aus die thrombozyten def menschen. MD University of Zurich (1984). thesis. C., 22. Latrigue-Mattei, Godeneche, Chabard, D.. Berger J.A. Pharmacokinetic J.L. and study of Preliminary results PAF-acether: after the intravenous administration of a 3-H- labelled product to the rabbit. Ag Act 1982: 12: 703 (1982). 23. Haroldsen, P.E., Voelkel, N.F., Henson, J.E., Henson, P.M. and Murphy, R.C. Metabolism of platelet-activating factor in isolated perfused rat lung. 1860 (1987). J. Clin. Invest. 79: A.J., Chung, 24. Wardlaw. K.F., Moqbel, R., Macdonald, A.J., McCusker, M., Barnes, P.J., Collins, J.V. Cellular changes in and Kay, A.B. blood and bronchoalveolar lavage (BALI and bronchial responsiveness PAF in man. Am. after inhaled Rev. Respir. Dis. In press (1988). 25. Lefort, J., Rotilio, D., Vargaftig, B.B. The platelet-independent release of thromboxane A2 by PAF-acether for guinea-pig lungs involves mechanisms leukotriene C4 and bradykinin. distinct from those for 82: 525 (1984). Br. J. Pharmacol. 26. Chung, K-F., Barnes, P.J. PAF antagonists: their potential therapeutic role in asthma. Drugs 35: 93 (1988).
Editor:
354
B. Whittle
Received:
6-7-88
Accepted:7-19-88
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