- Email: [email protected]
Platelet activating factor and airway smooth muscle
Pharmac. Ther. Vol.40, pp. 373 to 382,1989 Printed in Great Britain.All rightsreserved
0163-7258/89$0.00+ 0.50 Copyright© 1988PergamonPresspie
Spec&list Subject Editor: I. RODGER
PLATELET
ACTIVATING SMOOTH
FACTOR MUSCLE
AND
AIRWAY
J. MORLEY, D. SMITH and I. CHAPMAN Preclinical Research, Sandoz AG, Basel 4002, Switzerland
1. INTRODUCTION Release of blood histamine during allergic reactions in the rabbit poses a paradox, since blood histamine in this species is stored almost exclusively within platelets (Humphrey and Jaques, 1954), yet allergic hypersensitivity is dependent upon interaction of allergen with IgE upon the surface of basophils in this, as in other species (Siraganian and Osier, 1969). Secretion by basophils of materials which have the capacity to activate platelets provides an explanation for the release of histamine into rabbit blood during an IgE-dependent allergic reaction. Platelet activating factor (PAF) was the designation given to this material (Benveniste et al., 1972). To resolve this issue, unequivocally, has necessitated the isolation and eventual synthesis of PAF whose nomenclature has persisted, despite the emergence of alternative names as a consequence of chemical characterisation (acetyl glyceryl ether phosphorylcholine, AGEPC, Paf-acether, anti-hypertension polar renomedullary lipid, APRL) (Snyder, 1985). This novel allergic mediator can affect airway smooth muscle in a number of ways. 2. PAF AND PLATELET ACTIVATION Platelet activation, in response to PAF, may be measured as aggregation & vitro. Alternatively, activation may be inferred from the release of platelet constituents in vitro or in vivo (Vargaftig et al., 1981; McManus, 1987). The potency of PAF as a stimulant of platelets is striking, since concentrations between 1-10 nM suffice to induce activation (Chignard et al., 1979). Activation of platelets by PAF causes secretion of preformed materials with biological activity and induces generation of lipid mediators. Hence, it can be anticipated that release or generation of PAF in vivo will evoke a wide range of responses that are due to the biological activity of products of platelet activation. For some time, there was a tendency to ascribe in vivo actions of PAF to platelet activation. It was difficult to exclude platelet activation by use of drugs, but the availability of platelet antisera, and the use of in vitro tests revealed that the low concentrations of PAF that activate platelets can also activate other cell types including neutrophils (O'Flaherty et al., 1987), eosinophils (Wardlow et al., 1986) and endothelial cells (Bourgain et al., 1985). Both platelet-dependent and platelet-independent events can contribute to the biological actions of PAF in vivo, as can be conveniently illustrated by consideration of the effects of PAF upon skin. Thus, picogram quantities of PAF will induce increased vascular permeability on intracutaneous injection into guinea-pig skin, irrespective of whether platelets are present within the circulatory system or not (Morley et al., 1983). As a stimulus to increased vascular permeability in guinea-pig skin, PAF is considerably more potent than bradykinin, which hitherto had been acknowledged as the most potent endogenous stimulus yet identified. At higher concentrations, intracutaneous injection of PAF induces a sustained accumulation of inflammatory cells, which may be determined by the prior accumulation of platelets at the injection site, a phenomenon which can be demonstrated using Ullndium labelled platelets (Morley et al., 1983). The accumulation 373
374
J. MORLEYet al.
of inflammatory cells at sites of intracutaneous injection can be presumed to depend upon platelet activation, since this phenomenon is not evident in the rat, a species whose platelets do not respond to PAF even though skin vessels exhibit a sensitivity to PAF that is comparable to that observed in the guinea-pig (Pirotzky et al., 1984). 3. BRONCHOSPASM The dichotomy of actions of PAF that is evident in skin extends to the lung. In a number of mammalian species, intravenous injection of PAF has proved to be a potent stimulus to airway obstruction (Vargaftig et al., 1980). For example, PAF appears to be approximately 1000 times as potent as acetylcholine when injected intravenously in a ventilated guinea-pig (Fig. 1). The bronchospasm that follows intravenous injection of PAF is platelet-dependent, since depletion of platelets by prior infusion of a lytic anti-platelet antiserum abolishes this response (Vargaftig et al., 1980). Intravenous injection of other platelet stimuli (e.g. adenosine diphosphate) is known to induce bronchospasm, hence the capacity of a potent stimulus of platelet aggregation (e.g. PAF) to induce bronchospasm is not exceptional. It has been generally accepted that such bronchospasm is of reflex origin since there is evidence that embolised materials induce reflex bronchoconstriction (Mills et al., 1969). However, three observations indicate that such reflexes need not be invoked in order to account for induction of bronchospasm by PAF. Firstly, section of the vagus nerves does not diminish the magnitude of PAF-induced bronchospasm (Vargaftig et al., 1980). Secondly, the expression of bronchospasm largely (>90%) precedes detectable accumulation of platelets (Page et al., 1984). Thirdly, PAF has not been shown to induce contraction of airway smooth muscle, except when platelets are added to the bathing fluid of tracheal smooth muscle in an isolated preparation and hence are external to the vascular system (Schellenberg et al., 1983). The mechanism whereby PAF induces bronchospasm has not been satisfactorily defined. Bronchospasm that results from exposure to PAF might be presumed to depend at least in part upon generation of cyclooxygenase products by platelets or neutrophils, since thromboxane A2 is a spasmogen for airway smooth muscle, and since the magnitude of the acute constrictor response to PAF is reduced by drugs such as indomethacin or OKY-046 (Chung et al., 1986a). It has been reported that bronchospasm is inhibited by a mixture of histamine(H1) and serotonin antagonists when aspirin or salicylate are also present (Vargaftig et aL, 1982). Such an observation does not, however, indicate a I
i
500
250
--4 el"
0
_
PAF 132ng/k~ i.v.)
0
E
&
1111 I II!lTrI'.
-
:iii l
: ;~'
'i::
'
ii+ ,
, +
,
i
. i+iill;M.rlIl!lMlllllli'['i .... ,',ili ili[[[ ++I{ IilJ H[ ~1!i!i1!~:,1i!!!I', ,~i!!itl!l}llllililHl[llfllllltlilt! • :.,,+:!i[t+,?:llt!+l t tt.,l;HtlilMtllllll ! + ++. ++! ++ ,:+lit++i; H+t+ : 't + h ;, +; ; ',; . ; .+ ,',; ,';;!'i.[". t, ',:!i!+ii"; I,,.,1 ....... HIIIIllJil+ ' ' . . . . . ' .......
FIG. 1. Comparison between the effects of platelet activating factor (PAF) and Acetylcholine (Ach) upon guinea-pig airways. Changes in airway resistance (RL) and compliance (Cdyn) in response to intravenous injection of Ach (32/,g/kg) and PAF (32ng/kg) in the ventilated, anaesthetised guinea-pig.
PAF and airway smooth muscle
375
mechanism that can account for contraction any more than does the information that ketotifen and theophylline are potent inhibitors of PAF-induced bronchospasm (Morley et al., 1985a). Currently, there is no consensus as to the mechanism that determines airway smooth muscle contraction following exposure of platelets to PAF. It is presumed that following interaction between PAF and specific binding sites on platelets there is secretion of spasmogens. However this leaves unresolved the question as to whether activation of platelets by PAF differs from activation by other stimuli, since infusion of ADP in quantities sufficient to cause comparable accumulation in vivo does not induce airway hyperreactivity (unpublished observations). The secretory pattern that results from exposure of platelets to PAF is associated with a rise in intracellular calcium, a process common to other activation stimuli. Since different platelet stimuli elevate internal calcium by distinct processes (Sage and Rink, 1986a,b), it is possible that the calcium influx that follows exposure to PAF includes a component unique to this stimulus. It is also possible that metabolism of PAF by platelets yields spasmogens, thereby differentiating the effects of PAF from other platelet stimuli. There is, however, no experimental evidence to support this speculation other than an awareness that these rapid metabolic effects do take place (Snyder, 1987). The ability of platelet depletion to abrogate fully the bronchospasm due to intravenous injection of PAF precludes any significant contribution of direct effects of PAF upon airway smooth muscle, if modest doses are employed. When larger quantities of PAF are administered by inhalation, there is a bronchospasm which is not diminished by depletion of platelets (Lefort et al., 1984). The mechanism underlying this response remains uncertain, but it is possible that higher concentrations of PAF may have a direct effect upon smooth muscle, as has been proposed on the basis of studies using parenchymal strips (Stimler and O'Flaherty, 1983).
4. AIRWAY HYPER-REACTIVITY It has been demonstrated in the guinea-pig that repeated intraperitoneal injection of PAF induces hyperplasia of smooth muscle cells within the abdominal aorta (Handley et al., 1983). This observation is reinforced by the more recent evidence that infusion of PAF from an implanted reservoir causes generalised enlargement of vascular smooth muscle, especially within the pulmonary vasculature (Ohar et al., 1987). It might be presumed that such effects of PAF are secondary to platelet activation, since platelets are known to contain and release platelet derived growth factor, which would be a plausible stimulus of smooth muscle hyperplasia (Ross et al., 1985). However, such a conclusion remains tentative in the absence of studies employing platelet depletion or selective antagonists. The capacity of PAF to cause morphological changes in smooth muscle prompted consideration of this material as a mediator of airway hyperreactivity (Morley et al., 1984). Acute intravenous injection of PAF into spontaneously breathing guinea-pigs gave no unequivocal evidence of airway hyperreactivity, even though the propensity of such animals to succumb to repeated injection of PAF might possibly have been interpreted as evidence of hyperreactivity. The response of the guinea-pig to intravenous injection of PAF is less deleterious in ventilated animals and repeated injection of PAF could be shown to give responses that were relatively constant in amplitude (Fig. 2). The consistent effect of PAF on airways contrasts with its effect upon platelet activation, which exhibits tachyphylaxis (Henson, 1976). This anomaly was resolved when it was perceived that repeated exposure to PAF must have caused an increased sensitivity of smooth muscle to spasmogens, so that any tachyphylaxis of platelet activation in response to regular injection of PAF was being offset by increased responsiveness of the airways. This interpretation has yet to be verified experimentally, but in accord with such a conclusion, infusion of PAF over a one hour period consistently induces hyperreactivity of airway smooth muscle to unrelated spasmogens such as histamine or bombesin (Mazzoni et al., 1985). J.P.T. 40/3--D
376
J. MORLEYet al. PAF-Acether (10 ng/kg l.v,)induced in the Guinea-pigs
bronchoconstrlctlon
RL
Cdyn I0
RL
d.~,:
30
30
' "5S-~:~L ~
4O
-' •
~
50
"
Time (rain)
FIG. 2. PAF (10 ng/kg i.v.) induced bronchoconstriction in the guinea-pig. Changes in airway
resistance (RLcm H20/lsec-t) and compliance (ml/cm H20) induced by intravenous injection of PAF (10 ng/kg) in the ventilated, anaesthetisedguinea-pig at 10 min intervals. The airway hyperreactivity that results from exposure to PAF is non-selective, since enhanced responses are evident whether the test spasmogen is histamine, substance P, prostaglandin F2a~pha or bombesin in addition to PAF itself. The change in airway sensitivity that results from exposure to PAF persists for days and hence is long-lasting by comparison with the transient hyperreactivity that follows exposure of the airways to peptidoleukotrienes (Fennessy et al., 1986). In the guinea-pig, hyperreactivity can be detected 24 hr after inhalation of PAF and in the rabbit it is evident 7 days after a single intravenous infusion of PAF (Mazzoni et al., 1985). In man, hyperreactivity to methacholine following inhalation of PAF persists for 14 days and can require up to 28 days to return to pre-treatment levels (Cuss et al., 1986). In these species, hyperreactivity is regularly observed following exposure to PAF. However, although the duration of the reaction to such a labile material is impressive, it must be acknowledged that the altered sensitivity to test spasmogens is of modest intensity. In this respect, the response to PAF resembles the reaction to inhalation of small doses of allergen by atopic asthmatic patients, for this clinical procedure often induces a change in sensitivity that is modest, yet of considerable duration (Cockcroft et aL, 1977). The mechanism whereby PAF can modify the response of airway smooth muscle to spasmogens remains obscure. A modest shift in the dose-response curve might be interpreted as a manifestation of a reduction in cross sectional area of the airways as a consequence of oedema or increased mucous secretion (Moreno et al., 1986). However, in animals which lack circulating platelets, exposure to PAF causes no change in airway sensitivity to spasmogens (Morley et al., 1985a), even though increased vascular permeability due to PAF is largely unaffected by such treatment (Morley et al., 1983). Equally, the infusion of histamine, in doses that can be expected to induce oedema, does not cause hyperreactivity. Similarly, increased mucous secretion seems an insufficient explanation, since substance P or carbachol can produce substantial secretion without inducing a
PAF and airway smooth muscle
377
persistent change in reactivity. Furthermore, the inability of spasmogens such as histamine or substance P to induce a change of airway reactivity indicates that the phenomenon of hyperreactivity is not an incidental consequence of sustained bronchospasm. As a corollary, it may be noted that infusion of the bronchodilator isoprenaline effects hyperreactivity of comparable magnitude to that produced by PAF (Morley and Sanjar, 1987). It follows that during the development of hyperreactivity in response to PAF in the guinea-pig, neither oedema, bronchospasm nor mucous secretion can be considered to be primary determinants of airway hyperreactivity. Altered vagal tone might be favoured, since hyperreactivity does not follow exposure to PAF in the rabbit when the vagus nerves have been sectioned. However, vagal section does not influence the expression of airway hyperreactivity in guinea-pigs that have been exposed to an infusion of PAF (Mazzoni et al., 1985). It might be anticipated that hyperreactivity in response to PAF would be secondary to activation of a haematogenous element, since platelets, neutrophils and subsequently eosinophils are entrapped within the lung following exposure to PAF (Camussi et al., 1983; McManus, 1987); indeed, the elimination of platelets by lytic antisera fully suppresses acute airway hyperreactivity (Morley et al., 1985a). Removal of neutrophils on the other hand caused no diminution of the acute response, notwithstanding the substantial neutrophil activation produced by these doses of PAF (Morley et al., 1985a). The role of eosinophils remains a matter for conjecture, but eosinophil-mediated damage to airways is well documented (Frigas and Gleich, 1986) and might intensify bronchospasm either by reflex mechanisms (Barnes, 1986) or by loss of an endogenous dilator substance (Flavahan et al., 1985). Mechanisms whereby platelets may modify the function of airway smooth muscle have not been established. It is known that platelets can rapidly convert PAF to other lipids (Snyder 1987) whose properties have yet to be evaluated in airway tissues. The endogenous platelet spasmogen, which is presumed to cause contraction of airway smooth muscle in response to intravenous infusion of PAF, seems an unlikely candidate to account for airway hyperreactivity, since drugs such as the cyclooxygenase inhibitor indomethacin and the lipoxygenase inhibitor QA 208-199 can diminish acute bronchospasm without affecting development of hyperreactivity. Furthermore, drugs such as sodium cromoglycate (DSCG) or steroids can inhibit the development of hyperreactivity even though they do not influence the acute bronchospasm that results from exposure to PAF (Morley et al., 1985b). Sustained changes in airway reactivity may stem from the migration of platelets into the airway tissues, for in these circumstances secretion of growth factors, such as platelet derived growth factor (PDGF), could serve to change the volume and hence contractile properties of airway smooth muscle. It is significant, therefore, that it has been shown that platelets migrate into lung parenchyma following an infusion of PAF (Lellouch-Tubiana et al., 1985). On the other hand, it could be considered that the small sustained change in airway sensitivity, due to a single exposure to PAF, may be secondary to epithelial damage and may persist until epithelial integrity has been reestablished (Fig. 3). 5. DRUGS AND PULMONARY RESPONSES TO PAF Four aspects of the pulmonary response to PAF (increased vascular permeability; cell and platelet accumulation; acute bronchospasm and increased airway reactivity) may contribute to airway obstruction and have been the subject of pharmacological analysis. 5. l. INCREASEDVASCULARPERMEABILITY Increased vascular permeability is a prominent component of the pulmonary response to PAF and changes in vascular permeability can be inferred from measurement of the extravasation of plasma protein into pulmonary tissues using isotope labelling or vital dye.
378
J. MORLEYet al. lEE-ALLERGEN INTERACTION
T-CELL PRODUCTS
PHAGOCYTOSIS
V¢6~ ?¢e~q
l IMPAIRED MUCOCILARY CLEARANCE
1
NKA BOMB "BRADY
HYPERREACTIVESMOOTH ~ 1 ~ MUSCLE
J
1
COUGH BRONCHOSPASM FIG. 3. Schematic representation of the biochemical events which determine cough and airway hyperreactivity in developing asthma. Eosinophils (activated by IgE or T-cell products), macrophages (activated by IRE, T-cell products or phagocytic stimuli) or neutrophils (activated by T-cell products or phagocytic stimuli) are likely sources of PAF in asthma. Two distinct effects are envisaged as contributing to protracted changes of airways: eosinophil-mediated change of airway epithelium due to major basic protein (MBP), eosinophil cationic protein (ECP) release, which contributes to hyperreactivity and also determines impaired mucociUiary clearance and cough; platelet dependent induction of smooth muscle hyperplasia following release of platelet derived growth factor (PDGF), thereby effecting increased reactivity. The capacity of hyperreactive smooth muscle in asthma to respond to diverse chemical stimuli (acetylcholine, histamine, serotonin, adenosine, prostaglandins D 2, F 2 al ha and E2, thromboxane A2, neurokinin AI, bombesin, bradykinin, leukotriene C4 and D4) ~as been depicted schematically in order to emphasise the futility of selective antagonism.
This element of the response to PAF is inhibited by competitive PAF antagonists such as gingkolide B, but skin responses may not be suppressed fully, e.g. approximately 50% being the maximum inhibition that can be achieved when gingkolide B (1 mR/site) was injected together with PAF intracutaneously in the guinea-pig. It has been reported that the inhibitory effects of gingkolide B (1 mg/kg) are more pronounced in the trachea during the response to intravenous PAF (Barnes et al., 1985). This may indicate a contribution of endogenous PAF formation by endothelial cells during responses at this site. 5.2. CELL AND PLATELET ACCUMULATION
Acute exposure of airways to PAF results in platelet and neutrophil accumulation and an increased incidence of eosinophils within the airway lumen (Arnoux et al., 1985;
PAF and airway smooth muscle
379
McManus, 1987). Measurement of accumulation of eosinophils is laborious, so the actions of drugs on this response parameter have not been extensively pursued. On the other hand, platelets can be labelled with l'qndium so that intrathoracic accumulation can be monitored continuously by use of external detectors (Morley and Page, 1984). The accumulation of platelets, even in response to large doses of PAF, is inhibited fully by gingkolide B and other selective PAF antagonists (Deeming et al., 1985). In these circumstances, other drug types (the cyclooxygenase inhibitor indomethacin, the histamine(HI) antagonist mepyramine and the beta-adrenoreceptor agonist isoprenaline) do not influence the accumulation of platelets (Deeming et al., 1986). It has been reported that the platelet aggregation that results from an infusion of PAF is inhibited by prophylactic anti-asthma drugs (Deeming et al., 1986), but use of more sensitive recording techniques has not confirmed these preliminary observations. On present evidence, only selective PAF antagonists appear able to inhibit accumulation of platelets in response to PAF. 5.3. ACUTEBRONCHOSPASM The effect of drugs upon the acute bronchospasm that results from the intravenous injection of PAF has been subjected to greater attention than other aspects of the pulmonary response to PAF. The airway response may be recorded simply as air overflow during ventilation at a fixed stroke volume. Alternatively, airway resistance and compliance can be determined, either in ventilated animals or in spontaneously breathing animals. The latter method is technically exacting, but reveals that certain anti-asthmatic drugs can inhibit acute bronchospasm in the guinea-pig at concentrations within the therapeutic range (Morley et al., 1985a). Histamine(HI) antagonists and mast cell stabilising agents are not effective inhibitors of PAF-induced bronchospasm, but other drugs have not been investigated using this preparation. A number of drug categories can effect substantial inhibition of the acute bronchospasm that follows intravenous injection of PAF, as can be shown using ventilated anaesthetised animals. These include PAF antagonists, cyclooxygenase and thromboxane synthetase inhibitors, fl-adrenoceptor agonists, theophyUine, ketotifen, FPL 55712 and certain benzodiazepines (Morley et al., 1985b; Alabaster and Keir, 1987; Casals-Stenzel, 1987). In contrast, histamine(H1) or serotonin antagonists, mast cell stabilizing drugs, lipoxygenase inhibitors, DSCG and glucocorticosteroids have proved ineffective (Morley and Aoki, 1986). It is apparent that diverse drugs can inhibit acute bronchospasm and certain effects might be anticipated since the airway response is secondary to platelet activation. Clearly, such a response parameter to PAF is inappropriate for selection of anti-asthmatic drugs, being subject to false positives and false negatives. 5.4. AIRWAY HYPERREACTIVITY The first indications that anti-asthmatic drugs might inhibit development of airway hyperreactivity in response to PAF were provided in the rabbit. In this species PAF induced an airway obstruction of late onset that was inhibited by intratracheal instillation of cromoglycate (Page et al., 1985), as was the clinical response to allergen (Larsen et al., 1984). It might be inferred that such effects of cromoglycate are predictive for inhibition of airway hyperreactivity in response to PAF, but direct evidence of an inhibition of PAF-induced hyperreactivity by cromoglycate has not been reported for this species. Upon establishing a routine preparation for PAF-induced hyperreactivity in the guinea-pig, it has been possible to establish that prophylactic anti-asthma drugs (cromoglycate, theophylline, glucocorticosteroids and ketotifen) (Mazzoni et al., 1985) impair development of hyperreactivity with a potency that is comparable with, or superior to, the existing selective PAF antagonists (Deeming et al., 1986). Other drug categories: histamine(HI) antagonists (mepyramine, oxatomide, clemastine, astemizole, azelastine), SRS-A antagonists (FPL55712, AA-673), mast cell stabilizing drugs (oxatomide, tranilast, azelastine,
380
J. MORLEYet al.
zaprinast), cyclooxygenase (indomethacin) or lipoxygenase (QA 208-198, AA-861) inhibitors and a beta-adrenoreceptor agonist (isoprenaline), did not produce significant inhibition (Morley and Aoki, 1986). On present evidence, inhibition of the effect of PAF upon airway smooth muscle can be considered a necessary feature of prophylactic drugs which are effective in asthma therapy. It should be noted that certain drugs which have been given other designations can nonetheless inhibit induction of hyperreactivity by PAF. Thus, the thromboxane synthetase inhibitor FCE-22178 inhibits PAF-induced hyperreactivity in the guinea-pig (Giorgetti, personal communication), and the thromboxane antagonist OKY-046 has been observed to inhibit induction of hyperreactivity in the dog (Chung et al., 1986b). Further, a series of benzodiazepines have been shown to inhibit PAF-induced bronchospasm (Casals-Stenzel, 1987). 6. C O N C L U S I O N The ability of PAF to modify the function of airway smooth muscle depends substantially upon an effect mediated via platelets. Such a mechanism could account not only for acute bronchospasm but also for sustained changes in smooth muscle which occur either as a direct effect or by interaction with eosinophils. PAF is also a potent activation stimulus for inflammatory cells, particularly neutrophils and eosinophils and hence may modify airway smooth muscle function indirectly as a consequence of an inflammatory reaction. Because of the extensive pharmacological effects of PAF, it is difficult 1o discern to what extent bronchospasm and hyperreactivity reflect other events, such as mucosal oedema or alteration in the formation and elimination of mucous. This is especially true since airway smooth muscle in vitro is neither contracted by PAF nor made hyperreactive by exposure to PAF in doses that correspond to in vivo studies. The capacity of this mediator to mimic symptoms of asthma contrasts sharply with the limited actions of other putative mediators of asthma. The capacity of anti-asthma drugs to inhibit responses to PAF indicates a possible central role for PAF in the recruitment and activation of platelets and eosinophils in asthma. At present, PAF remains the only mediator whose properties could account for the sustained changes of airway smooth muscle that are characteristic of asthma and that are evident to a lesser extent in bronchitis, byssyinosis and cystic fibrosis. Consequently, it may be of value to evaluate drugs which seek to pre-empt or reverse changes of lung function in these diseases by use of PAF inhalation in animals or man.
REFERENCES ALABASTER,V. and Krm, R. (1987) Comparative effect of drugs on bronchoconstriction induced by PAF, histamine and acetylcholine. Br. ]. Pharmac. 90: 145P. ARNOUX,B., DENJEAN,A., PACE,C., MORLEY,J. and BENVENIS~,J. (1985) Pulmonary effects of Platelet Activating Factor in a primate are inhibited by ketotifen. Am. Rev. Resp. Dis. 131: A2.
BARNES,P., Cnu~qG,K., ROGERS,D. and EVANS,T. (1985) Increased vascular permeability induced by platelet activating factor: effect of specificantagonism and platelet depletion. Br. J. Pharmac. 89: 764P. BARN~S,P. (1986) Asthma as an axon reflex. Lancet i: 242-244, BENVENIS~,J., HENSON,P. and COCHRANE,C. (1972) Leucocytedependenthistamine releasefrom rabbit platelets: The role of IgE, basophil and a platelet activating factor. J. Exp. Med. 136: 1356-1377. BOURGA1N,R., MAES,L., BRAQUET,P., ANDRIES,R., TOUQUI,L. and BRAQUET,M. (1985)The effectof 1-0-alkyl-snglycero-3-phosphorocholine(paf-acether) on the arterial wall. Prostaglandins 30: 185-197. CAMUSSI,G., PAWLOWSKI,I., TETTA, C., ROFFIRELLO,C., ALBERTON,i . , BRENTJENS,J. and ANDRES,G. (1983) Acute lung inflammation induced in the rabbit by local instillation of 1-o-octadecyl-2-acetyl-syn-glyceryl-3phosphorylcholine or of a native platelet activating factor. Am. J. Path. 115: 78-88. CASALS-STENZEL,J. (1987) The inhibitory activity of brotizolam and related compounds on Platelet Activating Factor induced effects in vitro amd in vivo. In: New Horizons in Platelet Activating Research, Vol. 29, pp. 277-285, WlNSLOW,C. and LEE, M. (eds). CHIGNARD,M. LE COUEDIC,J., TENCE,M., VARGAFTIG,B. and BENVENISTE,J. (1979) The role of platelet activating factor in platelet aggregation. Nature 279: 799-800. CHUNG, K., AIZAWA,H., BECKER,A., FRICK, O., GOLD, W. and NADEL,J. (1986a) Inhibition of antigen-induced airway hyperresponsiveness by a thromboxane synthetase inhibitor (OKY-046) in allergic dogs. Am. Rev. Resp. Dis. 134: 258-261. CHUNG, K., AIZAWA,H., LEIKAUF,G., UEKI, I., EVANS,T. and NADEL,J. (1986b) Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation, d. Pharmac. Exp. Ther. 236: 580-584.
PAF and airway smooth muscle
381
COCKCROFT,D., RUFFIN,R., DOLOVICH,J. and HARGREAVE,F. (1977) Allergen induced increase in non-allergic bronchial reactivity. Clin. Allergy 7: 503-513. Cuss, F., DIXON,C. and BARNES,P. (1986) Effects of inhaled platelet activating factor on pulmonary function and bronchial responsiveness in man. Lancet ii: 189-192. DEEMING, K., MAZZONI,L., MORLEY,J., PAGE,C., SANJAR,S. and SMITH,n. (1985) PAF antagonism in asthma. Prostaglandins 30: 715. DEEMING,K., MAZZONI,L., MORLEY,J., PAGE, C. and SANJAR,S. (1986) Prophylatic anti-asthma drugs impair platelet accumulation within the lung. Br. J. Pharmac. 87: 73P. FENNESSY, M., STEWART, A. and THOMPSON, D. (1986) Aerosolised and intravenously administered leukotrienes: effects on the bronchoconstrictor potency of histamine in the guinea-pig. Br. J. Pharmac. 87: 741-749. FLAVAHAN,N. A., AARHUS,L. L., RIMELE,T. J. and VANHOUTTE,P. i . (1985) Respiratory epithelium inhibits bronchial smooth muscle tone. J. Appl. Physiol. 58: 834-838. FRIGAS,E. and GLEICH,G. J. (1986) The eosinophil and the pathophysiology of asthma. J. Allergy Clin. Immun. 77: 527-537. GIORGETTI,C., Personal communication. HANDLEY,n., LEE,M. and SAUNDERS,R. (1983) Extravasation and aortic ultrastructural changes accompanying acute and sub-acute intraperitoneal administration of PAF. Platelet Activating Factor. I N S E R M Symposium No. 23, pp. 245-250, BENVENISTE,J. and ARNOUX,B. (eds) Elsevier Science Publishers, B.V. Amsterdam. HENSON, P. (1976) Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitised basophils. 1. Characteristics of the secretory response. J. Exp. Ned. 143: 937-952. HUMPHREY,J. and JACQUES,R. (1954) The histamine and serotonin content of the platelets and polymorphonuclear leucocytes of various species. J. Physiol. (Lond.) 124: 300-304. LARSEN,G., SHAMPAIN,M., MARSH,W. and BEHRENS,B. (1984) An animal model of the late asthmatic response to antigen challenge. In: Asthma IIl, pp. 245-259, KAY,A. B., LICHTENSTEIN,L. M. and AusTEN K. F. (eds) Academic Press, New York and London. LEFORT, J., ROTILIO,n. and VARGAFTIG,B. (1984) The platelet-independent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br. J. Pharmac. 82: 565-575. LELLOUCH-TUBIANA,A., LEFORT,J., PIROTZKY,E., VARGAFTIG,B. and PFISTER,A. (1985) Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet activating factor (Paf-acether) to guinea-pigs. Br. J. Exp. Path. 66: 345-356. MAZZONI,L., MORLEY,J., PAGE,C. and SANJAR,S. (1985) Induction of hyperreactivity by platelet activating factor in the guinea-pig. J. Physiol, 365: 107P. MCMANUS, L. (1987) Acute lung injury induced by intravascular platelet activating factors. In: New Horizons in Platelet Activating Research, Vol. 25, pp. 233-245, W1NSLOW,C. and LEE, M. (eds). MILLS,J., SELLICK,H. and WIDDICOMBE,J. (1969) Activity of lung irritant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstrictions. J. Physiol. 203: 337-357. MORENO,R., HOGG, J. and PARE,P. (1986) Mechanics of airway narrowing. Am. Rev. Resp. Dis. 133:1171-I 180. MORLEY, J., PAGE, C. and PAUL, W. (1983) Inflammatory actions of platelet activating factor (Paf-acether) in guinea-pig skin. Br. J. Pharmac. 80: 503-509. MORLEY,J., SANJAR,S. and PAGE,C. (1984) The platelet in asthma. Lancet ii: 1142-1144. MORLEY, J. and PAGE,C. (1984) In vivo aggregometry. Trends. Pharm. Sci. 5: 258-261. MORLEY, J., PAGE, C., MAZZONI, L. and SANJAR,S. (1985a) Anti-allergic drugs in asthma. Triangle 24: 59 70. MORLEY, J., PAGE, C. and SANJAR,S. (1985b) PAF and airway hyperreactivity. Lancet ii: 451. MORLEY, J. and AOKI, S. (1986) A central role platelet activating factor in asthma. In: Immunopharmacology Reviews of the 4th Symposium: PAF in allergy and inflammation, pp. 17-30, MIYAMOTO,T. (ed.) Deutsche Medizinische Wochenschrift. MORLEY,J. and SANJAR,S. (1987) Isoprenaline induces increased airway reactivity in the guinea-pig. J. Physiol. 390: 180P. O'FLAHERTY,J. and WYKLE,R. (1987) Metabolism of Platelet-Activating Factor. In: PAF and Asthma, MORLEY, J. and HOLMES,G. (eds) (in press). OHAR,J., PYLE,J., HYERS,T. and WEBSTER,R. (1987) AGEPC-induced pulmonary vascular injury: A rabbit model of Pulmonary Hypertension. Am. Rev. Resp. Dis. (in press). PAGE, C., PAUL, W. and MORLEYJ. (1984) Platelets and bronchospasm. Int. Archs. Allergy Appl. lmmun. 74: 347-350. PAGE,C., GUERREIRO,n., SANJAR,S. and MORLEY,J. (1985) Platelet activating factor (Paf-acether) may account for late-onset reactions to allergen inhalation. Ag. Act. 16: 30-33. PIROTZKY, E., PAGE, C., ROUBIN,R., PFIFSTER,A., PAUL, W., BONNET,J. and BENVENISTE,J. (1984) Paf-acetherinduced plasma exudation in rat skin is independant of platelets and neutrophils. Microcirc. End. Lymph. 1: 107-122. Ross, R., RAINES,E. and BOWEN-POPE,D. (1985) Macrophages, Platelets, Endothelial Injury, Growth Factors, and Cell Proliferation. Adv. Inflamm. Res. 10: 99-104. SAGE, S. O. and RINK, T. J. (1986a) Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2-1oaded human platelets. Biochem. Biophys. Res. Commun. 136:1124-1129. SAGE,S. O. and RINK, T. J. (1986b) Effects of ionic substitution on [Ca 2+ ] rises evoked by thrombin and PAF in human platelets. Fur. J. Pharmac. 128: 99-107. SCHELLENBERG,R., WALKER,B. and SNYDER,F. (1983) Platelet dependent contraction of human bronchi by platelet activating factor. J. Allergy Clin. Immun. 71: A145. SIRAGANIAN,R. and OSLER,A. (1969) Histamine release from sensitized rabbit leukocytes and associated platelet involvement. J. Allergy 43: 167-175.
382
J. MORLEYet al.
SNYDER,F. (1985) Chemical and biochemical aspects of platelet activating factor: A novel class of acetylated ether-linked choline-phospholipids. Med. Res. Rev. 5: 107-140. SNYDER,F. (1987) Biochemistry of Platelet Activating Factor. In: P A F and Asthma, MORLEY,J. and HOLMES,G. (eds) (in press). STIMLER,N. and O'FLgHERTY,J. (1983) Spasmogenic properties of platelet activating factor. Am. J. Path. 113: 75-84. VARGAFTIG,B., LEFORT,J., CHINGARD,M. and BENVENISTE,J. (1980) Platelet-activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives. Fur. J. Pharmac. 65: 185-192. VARGAFTIG,B., CHIGNARD,M. and BENVENISTE,J. (1981) Present concepts on the mechanisms of platelet aggregation. Biochem. Pharmac. 30:263 271. VARGAFTIG,B., LEFORT,J., WOLF, F., CH1GNARD,M. and MEDEIROS,M. (1982) Non steroidal anti-inflammatory drugs if combined with anti histamine and anti serotonin agents interfere with the bronchial and platelet effects of platelet activating factor (Paf acether) in the guinea pig. Eur. J. Pharmac. 82: 121-130. WARDLOW,A., MOQBEL,R., CROMWELL,O. and KAY, A. (1986) Platelet activating factor, a potent chemotactic and chemokinetic factor for human eosinophils. J. Clin. Invest. 78:1701 1706.
0163-7258/89$0.00+ 0.50 Copyright© 1988PergamonPresspie
Spec&list Subject Editor: I. RODGER
PLATELET
ACTIVATING SMOOTH
FACTOR MUSCLE
AND
AIRWAY
J. MORLEY, D. SMITH and I. CHAPMAN Preclinical Research, Sandoz AG, Basel 4002, Switzerland
1. INTRODUCTION Release of blood histamine during allergic reactions in the rabbit poses a paradox, since blood histamine in this species is stored almost exclusively within platelets (Humphrey and Jaques, 1954), yet allergic hypersensitivity is dependent upon interaction of allergen with IgE upon the surface of basophils in this, as in other species (Siraganian and Osier, 1969). Secretion by basophils of materials which have the capacity to activate platelets provides an explanation for the release of histamine into rabbit blood during an IgE-dependent allergic reaction. Platelet activating factor (PAF) was the designation given to this material (Benveniste et al., 1972). To resolve this issue, unequivocally, has necessitated the isolation and eventual synthesis of PAF whose nomenclature has persisted, despite the emergence of alternative names as a consequence of chemical characterisation (acetyl glyceryl ether phosphorylcholine, AGEPC, Paf-acether, anti-hypertension polar renomedullary lipid, APRL) (Snyder, 1985). This novel allergic mediator can affect airway smooth muscle in a number of ways. 2. PAF AND PLATELET ACTIVATION Platelet activation, in response to PAF, may be measured as aggregation & vitro. Alternatively, activation may be inferred from the release of platelet constituents in vitro or in vivo (Vargaftig et al., 1981; McManus, 1987). The potency of PAF as a stimulant of platelets is striking, since concentrations between 1-10 nM suffice to induce activation (Chignard et al., 1979). Activation of platelets by PAF causes secretion of preformed materials with biological activity and induces generation of lipid mediators. Hence, it can be anticipated that release or generation of PAF in vivo will evoke a wide range of responses that are due to the biological activity of products of platelet activation. For some time, there was a tendency to ascribe in vivo actions of PAF to platelet activation. It was difficult to exclude platelet activation by use of drugs, but the availability of platelet antisera, and the use of in vitro tests revealed that the low concentrations of PAF that activate platelets can also activate other cell types including neutrophils (O'Flaherty et al., 1987), eosinophils (Wardlow et al., 1986) and endothelial cells (Bourgain et al., 1985). Both platelet-dependent and platelet-independent events can contribute to the biological actions of PAF in vivo, as can be conveniently illustrated by consideration of the effects of PAF upon skin. Thus, picogram quantities of PAF will induce increased vascular permeability on intracutaneous injection into guinea-pig skin, irrespective of whether platelets are present within the circulatory system or not (Morley et al., 1983). As a stimulus to increased vascular permeability in guinea-pig skin, PAF is considerably more potent than bradykinin, which hitherto had been acknowledged as the most potent endogenous stimulus yet identified. At higher concentrations, intracutaneous injection of PAF induces a sustained accumulation of inflammatory cells, which may be determined by the prior accumulation of platelets at the injection site, a phenomenon which can be demonstrated using Ullndium labelled platelets (Morley et al., 1983). The accumulation 373
374
J. MORLEYet al.
of inflammatory cells at sites of intracutaneous injection can be presumed to depend upon platelet activation, since this phenomenon is not evident in the rat, a species whose platelets do not respond to PAF even though skin vessels exhibit a sensitivity to PAF that is comparable to that observed in the guinea-pig (Pirotzky et al., 1984). 3. BRONCHOSPASM The dichotomy of actions of PAF that is evident in skin extends to the lung. In a number of mammalian species, intravenous injection of PAF has proved to be a potent stimulus to airway obstruction (Vargaftig et al., 1980). For example, PAF appears to be approximately 1000 times as potent as acetylcholine when injected intravenously in a ventilated guinea-pig (Fig. 1). The bronchospasm that follows intravenous injection of PAF is platelet-dependent, since depletion of platelets by prior infusion of a lytic anti-platelet antiserum abolishes this response (Vargaftig et al., 1980). Intravenous injection of other platelet stimuli (e.g. adenosine diphosphate) is known to induce bronchospasm, hence the capacity of a potent stimulus of platelet aggregation (e.g. PAF) to induce bronchospasm is not exceptional. It has been generally accepted that such bronchospasm is of reflex origin since there is evidence that embolised materials induce reflex bronchoconstriction (Mills et al., 1969). However, three observations indicate that such reflexes need not be invoked in order to account for induction of bronchospasm by PAF. Firstly, section of the vagus nerves does not diminish the magnitude of PAF-induced bronchospasm (Vargaftig et al., 1980). Secondly, the expression of bronchospasm largely (>90%) precedes detectable accumulation of platelets (Page et al., 1984). Thirdly, PAF has not been shown to induce contraction of airway smooth muscle, except when platelets are added to the bathing fluid of tracheal smooth muscle in an isolated preparation and hence are external to the vascular system (Schellenberg et al., 1983). The mechanism whereby PAF induces bronchospasm has not been satisfactorily defined. Bronchospasm that results from exposure to PAF might be presumed to depend at least in part upon generation of cyclooxygenase products by platelets or neutrophils, since thromboxane A2 is a spasmogen for airway smooth muscle, and since the magnitude of the acute constrictor response to PAF is reduced by drugs such as indomethacin or OKY-046 (Chung et al., 1986a). It has been reported that bronchospasm is inhibited by a mixture of histamine(H1) and serotonin antagonists when aspirin or salicylate are also present (Vargaftig et aL, 1982). Such an observation does not, however, indicate a I
i
500
250
--4 el"
0
_
PAF 132ng/k~ i.v.)
0
E
&
1111 I II!lTrI'.
-
:iii l
: ;~'
'i::
'
ii+ ,
, +
,
i
. i+iill;M.rlIl!lMlllllli'['i .... ,',ili ili[[[ ++I{ IilJ H[ ~1!i!i1!~:,1i!!!I', ,~i!!itl!l}llllililHl[llfllllltlilt! • :.,,+:!i[t+,?:llt!+l t tt.,l;HtlilMtllllll ! + ++. ++! ++ ,:+lit++i; H+t+ : 't + h ;, +; ; ',; . ; .+ ,',; ,';;!'i.[". t, ',:!i!+ii"; I,,.,1 ....... HIIIIllJil+ ' ' . . . . . ' .......
FIG. 1. Comparison between the effects of platelet activating factor (PAF) and Acetylcholine (Ach) upon guinea-pig airways. Changes in airway resistance (RL) and compliance (Cdyn) in response to intravenous injection of Ach (32/,g/kg) and PAF (32ng/kg) in the ventilated, anaesthetised guinea-pig.
PAF and airway smooth muscle
375
mechanism that can account for contraction any more than does the information that ketotifen and theophylline are potent inhibitors of PAF-induced bronchospasm (Morley et al., 1985a). Currently, there is no consensus as to the mechanism that determines airway smooth muscle contraction following exposure of platelets to PAF. It is presumed that following interaction between PAF and specific binding sites on platelets there is secretion of spasmogens. However this leaves unresolved the question as to whether activation of platelets by PAF differs from activation by other stimuli, since infusion of ADP in quantities sufficient to cause comparable accumulation in vivo does not induce airway hyperreactivity (unpublished observations). The secretory pattern that results from exposure of platelets to PAF is associated with a rise in intracellular calcium, a process common to other activation stimuli. Since different platelet stimuli elevate internal calcium by distinct processes (Sage and Rink, 1986a,b), it is possible that the calcium influx that follows exposure to PAF includes a component unique to this stimulus. It is also possible that metabolism of PAF by platelets yields spasmogens, thereby differentiating the effects of PAF from other platelet stimuli. There is, however, no experimental evidence to support this speculation other than an awareness that these rapid metabolic effects do take place (Snyder, 1987). The ability of platelet depletion to abrogate fully the bronchospasm due to intravenous injection of PAF precludes any significant contribution of direct effects of PAF upon airway smooth muscle, if modest doses are employed. When larger quantities of PAF are administered by inhalation, there is a bronchospasm which is not diminished by depletion of platelets (Lefort et al., 1984). The mechanism underlying this response remains uncertain, but it is possible that higher concentrations of PAF may have a direct effect upon smooth muscle, as has been proposed on the basis of studies using parenchymal strips (Stimler and O'Flaherty, 1983).
4. AIRWAY HYPER-REACTIVITY It has been demonstrated in the guinea-pig that repeated intraperitoneal injection of PAF induces hyperplasia of smooth muscle cells within the abdominal aorta (Handley et al., 1983). This observation is reinforced by the more recent evidence that infusion of PAF from an implanted reservoir causes generalised enlargement of vascular smooth muscle, especially within the pulmonary vasculature (Ohar et al., 1987). It might be presumed that such effects of PAF are secondary to platelet activation, since platelets are known to contain and release platelet derived growth factor, which would be a plausible stimulus of smooth muscle hyperplasia (Ross et al., 1985). However, such a conclusion remains tentative in the absence of studies employing platelet depletion or selective antagonists. The capacity of PAF to cause morphological changes in smooth muscle prompted consideration of this material as a mediator of airway hyperreactivity (Morley et al., 1984). Acute intravenous injection of PAF into spontaneously breathing guinea-pigs gave no unequivocal evidence of airway hyperreactivity, even though the propensity of such animals to succumb to repeated injection of PAF might possibly have been interpreted as evidence of hyperreactivity. The response of the guinea-pig to intravenous injection of PAF is less deleterious in ventilated animals and repeated injection of PAF could be shown to give responses that were relatively constant in amplitude (Fig. 2). The consistent effect of PAF on airways contrasts with its effect upon platelet activation, which exhibits tachyphylaxis (Henson, 1976). This anomaly was resolved when it was perceived that repeated exposure to PAF must have caused an increased sensitivity of smooth muscle to spasmogens, so that any tachyphylaxis of platelet activation in response to regular injection of PAF was being offset by increased responsiveness of the airways. This interpretation has yet to be verified experimentally, but in accord with such a conclusion, infusion of PAF over a one hour period consistently induces hyperreactivity of airway smooth muscle to unrelated spasmogens such as histamine or bombesin (Mazzoni et al., 1985). J.P.T. 40/3--D
376
J. MORLEYet al. PAF-Acether (10 ng/kg l.v,)induced in the Guinea-pigs
bronchoconstrlctlon
RL
Cdyn I0
RL
d.~,:
30
30
' "5S-~:~L ~
4O
-' •
~
50
"
Time (rain)
FIG. 2. PAF (10 ng/kg i.v.) induced bronchoconstriction in the guinea-pig. Changes in airway
resistance (RLcm H20/lsec-t) and compliance (ml/cm H20) induced by intravenous injection of PAF (10 ng/kg) in the ventilated, anaesthetisedguinea-pig at 10 min intervals. The airway hyperreactivity that results from exposure to PAF is non-selective, since enhanced responses are evident whether the test spasmogen is histamine, substance P, prostaglandin F2a~pha or bombesin in addition to PAF itself. The change in airway sensitivity that results from exposure to PAF persists for days and hence is long-lasting by comparison with the transient hyperreactivity that follows exposure of the airways to peptidoleukotrienes (Fennessy et al., 1986). In the guinea-pig, hyperreactivity can be detected 24 hr after inhalation of PAF and in the rabbit it is evident 7 days after a single intravenous infusion of PAF (Mazzoni et al., 1985). In man, hyperreactivity to methacholine following inhalation of PAF persists for 14 days and can require up to 28 days to return to pre-treatment levels (Cuss et al., 1986). In these species, hyperreactivity is regularly observed following exposure to PAF. However, although the duration of the reaction to such a labile material is impressive, it must be acknowledged that the altered sensitivity to test spasmogens is of modest intensity. In this respect, the response to PAF resembles the reaction to inhalation of small doses of allergen by atopic asthmatic patients, for this clinical procedure often induces a change in sensitivity that is modest, yet of considerable duration (Cockcroft et aL, 1977). The mechanism whereby PAF can modify the response of airway smooth muscle to spasmogens remains obscure. A modest shift in the dose-response curve might be interpreted as a manifestation of a reduction in cross sectional area of the airways as a consequence of oedema or increased mucous secretion (Moreno et al., 1986). However, in animals which lack circulating platelets, exposure to PAF causes no change in airway sensitivity to spasmogens (Morley et al., 1985a), even though increased vascular permeability due to PAF is largely unaffected by such treatment (Morley et al., 1983). Equally, the infusion of histamine, in doses that can be expected to induce oedema, does not cause hyperreactivity. Similarly, increased mucous secretion seems an insufficient explanation, since substance P or carbachol can produce substantial secretion without inducing a
PAF and airway smooth muscle
377
persistent change in reactivity. Furthermore, the inability of spasmogens such as histamine or substance P to induce a change of airway reactivity indicates that the phenomenon of hyperreactivity is not an incidental consequence of sustained bronchospasm. As a corollary, it may be noted that infusion of the bronchodilator isoprenaline effects hyperreactivity of comparable magnitude to that produced by PAF (Morley and Sanjar, 1987). It follows that during the development of hyperreactivity in response to PAF in the guinea-pig, neither oedema, bronchospasm nor mucous secretion can be considered to be primary determinants of airway hyperreactivity. Altered vagal tone might be favoured, since hyperreactivity does not follow exposure to PAF in the rabbit when the vagus nerves have been sectioned. However, vagal section does not influence the expression of airway hyperreactivity in guinea-pigs that have been exposed to an infusion of PAF (Mazzoni et al., 1985). It might be anticipated that hyperreactivity in response to PAF would be secondary to activation of a haematogenous element, since platelets, neutrophils and subsequently eosinophils are entrapped within the lung following exposure to PAF (Camussi et al., 1983; McManus, 1987); indeed, the elimination of platelets by lytic antisera fully suppresses acute airway hyperreactivity (Morley et al., 1985a). Removal of neutrophils on the other hand caused no diminution of the acute response, notwithstanding the substantial neutrophil activation produced by these doses of PAF (Morley et al., 1985a). The role of eosinophils remains a matter for conjecture, but eosinophil-mediated damage to airways is well documented (Frigas and Gleich, 1986) and might intensify bronchospasm either by reflex mechanisms (Barnes, 1986) or by loss of an endogenous dilator substance (Flavahan et al., 1985). Mechanisms whereby platelets may modify the function of airway smooth muscle have not been established. It is known that platelets can rapidly convert PAF to other lipids (Snyder 1987) whose properties have yet to be evaluated in airway tissues. The endogenous platelet spasmogen, which is presumed to cause contraction of airway smooth muscle in response to intravenous infusion of PAF, seems an unlikely candidate to account for airway hyperreactivity, since drugs such as the cyclooxygenase inhibitor indomethacin and the lipoxygenase inhibitor QA 208-199 can diminish acute bronchospasm without affecting development of hyperreactivity. Furthermore, drugs such as sodium cromoglycate (DSCG) or steroids can inhibit the development of hyperreactivity even though they do not influence the acute bronchospasm that results from exposure to PAF (Morley et al., 1985b). Sustained changes in airway reactivity may stem from the migration of platelets into the airway tissues, for in these circumstances secretion of growth factors, such as platelet derived growth factor (PDGF), could serve to change the volume and hence contractile properties of airway smooth muscle. It is significant, therefore, that it has been shown that platelets migrate into lung parenchyma following an infusion of PAF (Lellouch-Tubiana et al., 1985). On the other hand, it could be considered that the small sustained change in airway sensitivity, due to a single exposure to PAF, may be secondary to epithelial damage and may persist until epithelial integrity has been reestablished (Fig. 3). 5. DRUGS AND PULMONARY RESPONSES TO PAF Four aspects of the pulmonary response to PAF (increased vascular permeability; cell and platelet accumulation; acute bronchospasm and increased airway reactivity) may contribute to airway obstruction and have been the subject of pharmacological analysis. 5. l. INCREASEDVASCULARPERMEABILITY Increased vascular permeability is a prominent component of the pulmonary response to PAF and changes in vascular permeability can be inferred from measurement of the extravasation of plasma protein into pulmonary tissues using isotope labelling or vital dye.
378
J. MORLEYet al. lEE-ALLERGEN INTERACTION
T-CELL PRODUCTS
PHAGOCYTOSIS
V¢6~ ?¢e~q
l IMPAIRED MUCOCILARY CLEARANCE
1
NKA BOMB "BRADY
HYPERREACTIVESMOOTH ~ 1 ~ MUSCLE
J
1
COUGH BRONCHOSPASM FIG. 3. Schematic representation of the biochemical events which determine cough and airway hyperreactivity in developing asthma. Eosinophils (activated by IgE or T-cell products), macrophages (activated by IRE, T-cell products or phagocytic stimuli) or neutrophils (activated by T-cell products or phagocytic stimuli) are likely sources of PAF in asthma. Two distinct effects are envisaged as contributing to protracted changes of airways: eosinophil-mediated change of airway epithelium due to major basic protein (MBP), eosinophil cationic protein (ECP) release, which contributes to hyperreactivity and also determines impaired mucociUiary clearance and cough; platelet dependent induction of smooth muscle hyperplasia following release of platelet derived growth factor (PDGF), thereby effecting increased reactivity. The capacity of hyperreactive smooth muscle in asthma to respond to diverse chemical stimuli (acetylcholine, histamine, serotonin, adenosine, prostaglandins D 2, F 2 al ha and E2, thromboxane A2, neurokinin AI, bombesin, bradykinin, leukotriene C4 and D4) ~as been depicted schematically in order to emphasise the futility of selective antagonism.
This element of the response to PAF is inhibited by competitive PAF antagonists such as gingkolide B, but skin responses may not be suppressed fully, e.g. approximately 50% being the maximum inhibition that can be achieved when gingkolide B (1 mR/site) was injected together with PAF intracutaneously in the guinea-pig. It has been reported that the inhibitory effects of gingkolide B (1 mg/kg) are more pronounced in the trachea during the response to intravenous PAF (Barnes et al., 1985). This may indicate a contribution of endogenous PAF formation by endothelial cells during responses at this site. 5.2. CELL AND PLATELET ACCUMULATION
Acute exposure of airways to PAF results in platelet and neutrophil accumulation and an increased incidence of eosinophils within the airway lumen (Arnoux et al., 1985;
PAF and airway smooth muscle
379
McManus, 1987). Measurement of accumulation of eosinophils is laborious, so the actions of drugs on this response parameter have not been extensively pursued. On the other hand, platelets can be labelled with l'qndium so that intrathoracic accumulation can be monitored continuously by use of external detectors (Morley and Page, 1984). The accumulation of platelets, even in response to large doses of PAF, is inhibited fully by gingkolide B and other selective PAF antagonists (Deeming et al., 1985). In these circumstances, other drug types (the cyclooxygenase inhibitor indomethacin, the histamine(HI) antagonist mepyramine and the beta-adrenoreceptor agonist isoprenaline) do not influence the accumulation of platelets (Deeming et al., 1986). It has been reported that the platelet aggregation that results from an infusion of PAF is inhibited by prophylactic anti-asthma drugs (Deeming et al., 1986), but use of more sensitive recording techniques has not confirmed these preliminary observations. On present evidence, only selective PAF antagonists appear able to inhibit accumulation of platelets in response to PAF. 5.3. ACUTEBRONCHOSPASM The effect of drugs upon the acute bronchospasm that results from the intravenous injection of PAF has been subjected to greater attention than other aspects of the pulmonary response to PAF. The airway response may be recorded simply as air overflow during ventilation at a fixed stroke volume. Alternatively, airway resistance and compliance can be determined, either in ventilated animals or in spontaneously breathing animals. The latter method is technically exacting, but reveals that certain anti-asthmatic drugs can inhibit acute bronchospasm in the guinea-pig at concentrations within the therapeutic range (Morley et al., 1985a). Histamine(HI) antagonists and mast cell stabilising agents are not effective inhibitors of PAF-induced bronchospasm, but other drugs have not been investigated using this preparation. A number of drug categories can effect substantial inhibition of the acute bronchospasm that follows intravenous injection of PAF, as can be shown using ventilated anaesthetised animals. These include PAF antagonists, cyclooxygenase and thromboxane synthetase inhibitors, fl-adrenoceptor agonists, theophyUine, ketotifen, FPL 55712 and certain benzodiazepines (Morley et al., 1985b; Alabaster and Keir, 1987; Casals-Stenzel, 1987). In contrast, histamine(H1) or serotonin antagonists, mast cell stabilizing drugs, lipoxygenase inhibitors, DSCG and glucocorticosteroids have proved ineffective (Morley and Aoki, 1986). It is apparent that diverse drugs can inhibit acute bronchospasm and certain effects might be anticipated since the airway response is secondary to platelet activation. Clearly, such a response parameter to PAF is inappropriate for selection of anti-asthmatic drugs, being subject to false positives and false negatives. 5.4. AIRWAY HYPERREACTIVITY The first indications that anti-asthmatic drugs might inhibit development of airway hyperreactivity in response to PAF were provided in the rabbit. In this species PAF induced an airway obstruction of late onset that was inhibited by intratracheal instillation of cromoglycate (Page et al., 1985), as was the clinical response to allergen (Larsen et al., 1984). It might be inferred that such effects of cromoglycate are predictive for inhibition of airway hyperreactivity in response to PAF, but direct evidence of an inhibition of PAF-induced hyperreactivity by cromoglycate has not been reported for this species. Upon establishing a routine preparation for PAF-induced hyperreactivity in the guinea-pig, it has been possible to establish that prophylactic anti-asthma drugs (cromoglycate, theophylline, glucocorticosteroids and ketotifen) (Mazzoni et al., 1985) impair development of hyperreactivity with a potency that is comparable with, or superior to, the existing selective PAF antagonists (Deeming et al., 1986). Other drug categories: histamine(HI) antagonists (mepyramine, oxatomide, clemastine, astemizole, azelastine), SRS-A antagonists (FPL55712, AA-673), mast cell stabilizing drugs (oxatomide, tranilast, azelastine,
380
J. MORLEYet al.
zaprinast), cyclooxygenase (indomethacin) or lipoxygenase (QA 208-198, AA-861) inhibitors and a beta-adrenoreceptor agonist (isoprenaline), did not produce significant inhibition (Morley and Aoki, 1986). On present evidence, inhibition of the effect of PAF upon airway smooth muscle can be considered a necessary feature of prophylactic drugs which are effective in asthma therapy. It should be noted that certain drugs which have been given other designations can nonetheless inhibit induction of hyperreactivity by PAF. Thus, the thromboxane synthetase inhibitor FCE-22178 inhibits PAF-induced hyperreactivity in the guinea-pig (Giorgetti, personal communication), and the thromboxane antagonist OKY-046 has been observed to inhibit induction of hyperreactivity in the dog (Chung et al., 1986b). Further, a series of benzodiazepines have been shown to inhibit PAF-induced bronchospasm (Casals-Stenzel, 1987). 6. C O N C L U S I O N The ability of PAF to modify the function of airway smooth muscle depends substantially upon an effect mediated via platelets. Such a mechanism could account not only for acute bronchospasm but also for sustained changes in smooth muscle which occur either as a direct effect or by interaction with eosinophils. PAF is also a potent activation stimulus for inflammatory cells, particularly neutrophils and eosinophils and hence may modify airway smooth muscle function indirectly as a consequence of an inflammatory reaction. Because of the extensive pharmacological effects of PAF, it is difficult 1o discern to what extent bronchospasm and hyperreactivity reflect other events, such as mucosal oedema or alteration in the formation and elimination of mucous. This is especially true since airway smooth muscle in vitro is neither contracted by PAF nor made hyperreactive by exposure to PAF in doses that correspond to in vivo studies. The capacity of this mediator to mimic symptoms of asthma contrasts sharply with the limited actions of other putative mediators of asthma. The capacity of anti-asthma drugs to inhibit responses to PAF indicates a possible central role for PAF in the recruitment and activation of platelets and eosinophils in asthma. At present, PAF remains the only mediator whose properties could account for the sustained changes of airway smooth muscle that are characteristic of asthma and that are evident to a lesser extent in bronchitis, byssyinosis and cystic fibrosis. Consequently, it may be of value to evaluate drugs which seek to pre-empt or reverse changes of lung function in these diseases by use of PAF inhalation in animals or man.
REFERENCES ALABASTER,V. and Krm, R. (1987) Comparative effect of drugs on bronchoconstriction induced by PAF, histamine and acetylcholine. Br. ]. Pharmac. 90: 145P. ARNOUX,B., DENJEAN,A., PACE,C., MORLEY,J. and BENVENIS~,J. (1985) Pulmonary effects of Platelet Activating Factor in a primate are inhibited by ketotifen. Am. Rev. Resp. Dis. 131: A2.
BARNES,P., Cnu~qG,K., ROGERS,D. and EVANS,T. (1985) Increased vascular permeability induced by platelet activating factor: effect of specificantagonism and platelet depletion. Br. J. Pharmac. 89: 764P. BARN~S,P. (1986) Asthma as an axon reflex. Lancet i: 242-244, BENVENIS~,J., HENSON,P. and COCHRANE,C. (1972) Leucocytedependenthistamine releasefrom rabbit platelets: The role of IgE, basophil and a platelet activating factor. J. Exp. Med. 136: 1356-1377. BOURGA1N,R., MAES,L., BRAQUET,P., ANDRIES,R., TOUQUI,L. and BRAQUET,M. (1985)The effectof 1-0-alkyl-snglycero-3-phosphorocholine(paf-acether) on the arterial wall. Prostaglandins 30: 185-197. CAMUSSI,G., PAWLOWSKI,I., TETTA, C., ROFFIRELLO,C., ALBERTON,i . , BRENTJENS,J. and ANDRES,G. (1983) Acute lung inflammation induced in the rabbit by local instillation of 1-o-octadecyl-2-acetyl-syn-glyceryl-3phosphorylcholine or of a native platelet activating factor. Am. J. Path. 115: 78-88. CASALS-STENZEL,J. (1987) The inhibitory activity of brotizolam and related compounds on Platelet Activating Factor induced effects in vitro amd in vivo. In: New Horizons in Platelet Activating Research, Vol. 29, pp. 277-285, WlNSLOW,C. and LEE, M. (eds). CHIGNARD,M. LE COUEDIC,J., TENCE,M., VARGAFTIG,B. and BENVENISTE,J. (1979) The role of platelet activating factor in platelet aggregation. Nature 279: 799-800. CHUNG, K., AIZAWA,H., BECKER,A., FRICK, O., GOLD, W. and NADEL,J. (1986a) Inhibition of antigen-induced airway hyperresponsiveness by a thromboxane synthetase inhibitor (OKY-046) in allergic dogs. Am. Rev. Resp. Dis. 134: 258-261. CHUNG, K., AIZAWA,H., LEIKAUF,G., UEKI, I., EVANS,T. and NADEL,J. (1986b) Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation, d. Pharmac. Exp. Ther. 236: 580-584.
PAF and airway smooth muscle
381
COCKCROFT,D., RUFFIN,R., DOLOVICH,J. and HARGREAVE,F. (1977) Allergen induced increase in non-allergic bronchial reactivity. Clin. Allergy 7: 503-513. Cuss, F., DIXON,C. and BARNES,P. (1986) Effects of inhaled platelet activating factor on pulmonary function and bronchial responsiveness in man. Lancet ii: 189-192. DEEMING, K., MAZZONI,L., MORLEY,J., PAGE,C., SANJAR,S. and SMITH,n. (1985) PAF antagonism in asthma. Prostaglandins 30: 715. DEEMING,K., MAZZONI,L., MORLEY,J., PAGE, C. and SANJAR,S. (1986) Prophylatic anti-asthma drugs impair platelet accumulation within the lung. Br. J. Pharmac. 87: 73P. FENNESSY, M., STEWART, A. and THOMPSON, D. (1986) Aerosolised and intravenously administered leukotrienes: effects on the bronchoconstrictor potency of histamine in the guinea-pig. Br. J. Pharmac. 87: 741-749. FLAVAHAN,N. A., AARHUS,L. L., RIMELE,T. J. and VANHOUTTE,P. i . (1985) Respiratory epithelium inhibits bronchial smooth muscle tone. J. Appl. Physiol. 58: 834-838. FRIGAS,E. and GLEICH,G. J. (1986) The eosinophil and the pathophysiology of asthma. J. Allergy Clin. Immun. 77: 527-537. GIORGETTI,C., Personal communication. HANDLEY,n., LEE,M. and SAUNDERS,R. (1983) Extravasation and aortic ultrastructural changes accompanying acute and sub-acute intraperitoneal administration of PAF. Platelet Activating Factor. I N S E R M Symposium No. 23, pp. 245-250, BENVENISTE,J. and ARNOUX,B. (eds) Elsevier Science Publishers, B.V. Amsterdam. HENSON, P. (1976) Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitised basophils. 1. Characteristics of the secretory response. J. Exp. Ned. 143: 937-952. HUMPHREY,J. and JACQUES,R. (1954) The histamine and serotonin content of the platelets and polymorphonuclear leucocytes of various species. J. Physiol. (Lond.) 124: 300-304. LARSEN,G., SHAMPAIN,M., MARSH,W. and BEHRENS,B. (1984) An animal model of the late asthmatic response to antigen challenge. In: Asthma IIl, pp. 245-259, KAY,A. B., LICHTENSTEIN,L. M. and AusTEN K. F. (eds) Academic Press, New York and London. LEFORT, J., ROTILIO,n. and VARGAFTIG,B. (1984) The platelet-independent release of thromboxane A2 by Paf-acether from guinea-pig lungs involves mechanisms distinct from those for leukotriene. Br. J. Pharmac. 82: 565-575. LELLOUCH-TUBIANA,A., LEFORT,J., PIROTZKY,E., VARGAFTIG,B. and PFISTER,A. (1985) Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet activating factor (Paf-acether) to guinea-pigs. Br. J. Exp. Path. 66: 345-356. MAZZONI,L., MORLEY,J., PAGE,C. and SANJAR,S. (1985) Induction of hyperreactivity by platelet activating factor in the guinea-pig. J. Physiol, 365: 107P. MCMANUS, L. (1987) Acute lung injury induced by intravascular platelet activating factors. In: New Horizons in Platelet Activating Research, Vol. 25, pp. 233-245, W1NSLOW,C. and LEE, M. (eds). MILLS,J., SELLICK,H. and WIDDICOMBE,J. (1969) Activity of lung irritant receptors in pulmonary microembolism, anaphylaxis and drug-induced bronchoconstrictions. J. Physiol. 203: 337-357. MORENO,R., HOGG, J. and PARE,P. (1986) Mechanics of airway narrowing. Am. Rev. Resp. Dis. 133:1171-I 180. MORLEY, J., PAGE, C. and PAUL, W. (1983) Inflammatory actions of platelet activating factor (Paf-acether) in guinea-pig skin. Br. J. Pharmac. 80: 503-509. MORLEY,J., SANJAR,S. and PAGE,C. (1984) The platelet in asthma. Lancet ii: 1142-1144. MORLEY, J. and PAGE,C. (1984) In vivo aggregometry. Trends. Pharm. Sci. 5: 258-261. MORLEY, J., PAGE, C., MAZZONI, L. and SANJAR,S. (1985a) Anti-allergic drugs in asthma. Triangle 24: 59 70. MORLEY, J., PAGE, C. and SANJAR,S. (1985b) PAF and airway hyperreactivity. Lancet ii: 451. MORLEY, J. and AOKI, S. (1986) A central role platelet activating factor in asthma. In: Immunopharmacology Reviews of the 4th Symposium: PAF in allergy and inflammation, pp. 17-30, MIYAMOTO,T. (ed.) Deutsche Medizinische Wochenschrift. MORLEY,J. and SANJAR,S. (1987) Isoprenaline induces increased airway reactivity in the guinea-pig. J. Physiol. 390: 180P. O'FLAHERTY,J. and WYKLE,R. (1987) Metabolism of Platelet-Activating Factor. In: PAF and Asthma, MORLEY, J. and HOLMES,G. (eds) (in press). OHAR,J., PYLE,J., HYERS,T. and WEBSTER,R. (1987) AGEPC-induced pulmonary vascular injury: A rabbit model of Pulmonary Hypertension. Am. Rev. Resp. Dis. (in press). PAGE, C., PAUL, W. and MORLEYJ. (1984) Platelets and bronchospasm. Int. Archs. Allergy Appl. lmmun. 74: 347-350. PAGE,C., GUERREIRO,n., SANJAR,S. and MORLEY,J. (1985) Platelet activating factor (Paf-acether) may account for late-onset reactions to allergen inhalation. Ag. Act. 16: 30-33. PIROTZKY, E., PAGE, C., ROUBIN,R., PFIFSTER,A., PAUL, W., BONNET,J. and BENVENISTE,J. (1984) Paf-acetherinduced plasma exudation in rat skin is independant of platelets and neutrophils. Microcirc. End. Lymph. 1: 107-122. Ross, R., RAINES,E. and BOWEN-POPE,D. (1985) Macrophages, Platelets, Endothelial Injury, Growth Factors, and Cell Proliferation. Adv. Inflamm. Res. 10: 99-104. SAGE, S. O. and RINK, T. J. (1986a) Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2-1oaded human platelets. Biochem. Biophys. Res. Commun. 136:1124-1129. SAGE,S. O. and RINK, T. J. (1986b) Effects of ionic substitution on [Ca 2+ ] rises evoked by thrombin and PAF in human platelets. Fur. J. Pharmac. 128: 99-107. SCHELLENBERG,R., WALKER,B. and SNYDER,F. (1983) Platelet dependent contraction of human bronchi by platelet activating factor. J. Allergy Clin. Immun. 71: A145. SIRAGANIAN,R. and OSLER,A. (1969) Histamine release from sensitized rabbit leukocytes and associated platelet involvement. J. Allergy 43: 167-175.
382
J. MORLEYet al.
SNYDER,F. (1985) Chemical and biochemical aspects of platelet activating factor: A novel class of acetylated ether-linked choline-phospholipids. Med. Res. Rev. 5: 107-140. SNYDER,F. (1987) Biochemistry of Platelet Activating Factor. In: P A F and Asthma, MORLEY,J. and HOLMES,G. (eds) (in press). STIMLER,N. and O'FLgHERTY,J. (1983) Spasmogenic properties of platelet activating factor. Am. J. Path. 113: 75-84. VARGAFTIG,B., LEFORT,J., CHINGARD,M. and BENVENISTE,J. (1980) Platelet-activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives. Fur. J. Pharmac. 65: 185-192. VARGAFTIG,B., CHIGNARD,M. and BENVENISTE,J. (1981) Present concepts on the mechanisms of platelet aggregation. Biochem. Pharmac. 30:263 271. VARGAFTIG,B., LEFORT,J., WOLF, F., CH1GNARD,M. and MEDEIROS,M. (1982) Non steroidal anti-inflammatory drugs if combined with anti histamine and anti serotonin agents interfere with the bronchial and platelet effects of platelet activating factor (Paf acether) in the guinea pig. Eur. J. Pharmac. 82: 121-130. WARDLOW,A., MOQBEL,R., CROMWELL,O. and KAY, A. (1986) Platelet activating factor, a potent chemotactic and chemokinetic factor for human eosinophils. J. Clin. Invest. 78:1701 1706.