Clinical and Experimental Allergy, 1992, Volume 22, pages 521-524
REVIEW
An update on PAF D. A. SPENCER Birmingham Children's Hospital, Ladywood, Birmingham, U.K.
PAF and bronchial responsiveness
Platelet activating factor (PAF) is a membrane-derived phospholipid with a wide variety of biological actions which has been proposed as a mediator of central importance to the inflammatory PAF process in asthma [1]. PAF is a potent chemotactic agent for a wide range of inflammatory cells including neutrophils and the eosinophil, for which it is one of the most potent chemotactic agents known [2]. In addition it is a potent activator of these cells. For example, it causes eosinophils to adhere to endothelial cells [3] and to synthesize LTC4 and more PAF [4]. PAF causes dose-related aggregation [5] and stimulates calcium influx [6] in platelets, whereas in the neutrophil it causes exocytosis, superoxide generation and chemiluminescence [7,8]. Other properties of PAF relevant to the pulmonary inflammatory process include stimulation of airway mucus production [9], increasing pulmonary microvascular permeability [10], and impairment of mucociliary clearance in vitro [11]. PAF induces acute bronchoconstriction in many species including guinea-pig, rhesus monkey and baboon (reviewed in [12]). In man, PAFinduced bronchoconstriction is accompanied by facial flushing and tightness of the throat. It is also associated with a dramatic, although transient, fall in circulating neutrophils [13,14] due to their pulmonary sequestration [15], and accompanied by increased recovery of these cells, but importantly not eosinophils, in bronchoalveolar lavage fluid [16]. The efl'ect of PAF which has attracted most attention and controversy is its ability to cause a prolonged increase in bronchial responsiveness. This phenomenon has been demonstrated in several animal species including guinea-pig, dog, sheep, rhesus monkey and baboon (reviewed in [12]). In 1986 Cuss et al. [14] reported that inhalation of PAF caused an increase in bronchial responsiveness to methacholine of up to 2-5fold, maximum at 3 days and persisting for up to 4 weeks in five out of six normal human subjects; several further reports from this group have effectively reiterated these findings [16,17,18]. Although others have reported similar findings in abstract form, only Kaye and Smith have, as yet, published any similar findings in a complete paper Correspondence: Dr D. A. Spencer, Birmingham Children's Hospital, Ladywood Middleway, Ladywood, Birmingham B16 8ET, U.K.
[19]. These workers reported an apparent increase of 50% in responsiveness to methacholine in six out of eight normal subjects for up to 14 days following inhaled PAF. Such changes would be at the limit of the detectable changes in responsiveness as the reproducibility of a methacholine challenge is approximately one doubling concentration of the agonist. In addition, the results in this study would seem to be invalid as the data was not apparently log transformed for the analysis. Interestingly, in the same paper these workers reported similar increases in responsiveness to methacholine following LTD4 challenge in normal subjects, the only time that this has ever been reported despite numerous previous reports to the contrary. In contrast to these positive findings, several studies have now been published in which no increase in bronchial responsiveness has occurred following PAF inhalation in normal subjects [20-23]. Possible reasons for these divergent findings have been discussed previously in this journal [22,24] and include the dose of PAF, the method for assessing changes in bronchial responsiveness and the atopic status of the subjects. Perhaps the most obvious reason would be that higher doses of PAF might have been given in the positive studies, but this would not seem to be the case, as apparently higher doses were given in three of the negative studies [20,22,23], and in the remaining study similar doses to those described by Cuss [21]. Unfortunately, every centre has used a difl"erent experimental protocol involving various PAF formulations and preparation methods, nebulization and inhalation techniques so that such attempts at comparing PAF dose delivered to the airways are probably futile. The situation is further confused by diflerent studies describing the dose of PAF given as a single dose, the total cumulative dose, the dose range and the geometric mean dose [24]. Finally, ethical considerations imposed by wide intersubject variations in the acute response to this potentially injurious phospholipid have resulted in large diflerences in the doses of PAF administered between subjects within the same study, rendering meaningless any detailed comparison between studies. The effects of PAF on bronchial responsiveness in asthmatics are less controversial and arguably of greater relevance to the pathophysiology of the disease than 521
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D. A. Spencer
studies in normal man. In neither of the two published studies [25,26] has PAF inhalation resulted in any significant or prolonged increase in bronchial responsiveness. Although these findings could be partially related to the use of smaller doses of PAF in asthmatics compared to normal subjects, they are difficult to reconcile with the suggestion that this mediator is of central importance in the pathophysiology of asthma. Chung and Barnes [26] have suggested that these results could be due either to self-tachyphylaxis, presumably due to down regulation of PAF receptors, or because 'it may be more difficult to enhance the inflammatory process that. . . may already be present in the airways of those with mild asthma'. These conclusions are hard to accept; firstly, it is difficult to conceive how any mediator could exert an important pathophysiological role contemporaneously with the tissue being tachyphylactic to the effect of that mediator. Secondly, it is very well known that clinically important stimuli such as antigen readily increase bronchial responsiveness in patients with mild asthma [27]. The most convincing proof for or against the involvement of PAF in the pathophysiology of asthma will be provided by studies using potent and specific antagonists in models of asthma as well as therapeutic studies in patients with active disease. Preliminary results of such studies are now becoming available. Dermarkarian et al. [28] found that SCH-37370, a dual platelet activating factor and histamine antagonist, had no effect on the bronchoconstriction induced by isocapnic hyperventilation of cold, dry air. The PAF antagonists WEB 2086 BS [29] and MK-287 [30] have also been investigated in allergen challenge, and have been shown not to modify early or late asthmatic responses or the subsequent changes in bronchial responsiveness in asthmatic subjects. Therapeutic trials of WEB 2086 are currently in progress. Secondary generation of other mediators by PAF The mechanisms by which PAF produces its various biological effects are incompletely understood, and it is likely that PAF acts by a variety of direct and indirect mechanisms in different situations. Morley et al. [1] originally proposed that PAF was responsible for the inflammatory features of asthma as a consequence of platelet activation. However, recognition that PAF has multiple actions on many other cell types, particularly the eosinophil, led Barnes et al. [12] to suggest that this cell may be of crucial importance in producing the biological effects attributed to PAF. Interestingly, the eosinophil has not been implicated in the PAF-induced increase in bronchial responsiveness in normal man, a phenomenon which seems to involve the neutrophil [14,16], a cell not closely associated with the pathophysiology of asthma.
High affinity binding sites for PAF which are probably PAF receptors have been described on the surface of many cells including platelets, neutrophils and in homogenates from human lung membranes [31,32], although not on human bronchial smooth muscle [33]. Binding of PAF to specific receptors may trigger complex intracellular transduction mechanisms involving activation of G-proteins and protein kinase C, an increase in turnover of inositol phosphates and a rise in intracellular Ca^""". Phospholipase A2 may then be activated causing release of arachidonate from membrane phospholipids with the subsequent production ofa cascade of mediators including leukotrienes, prostaglandins and thromboxane A2 [34]. It is likely that many ofthe effects attributed to PAF are dependent on this secondary generation of leukotrienes and prostanoids. The synthesis of PAF and leukotrienes are closely linked; the first step of PAF synthesis involves the cleaving of alkylarachidonyl-GPC, effectively liberating lyso-PAF and arachidonic acid. This arachidonic acid may under certain circumstances be channelled down the 5-lipoxygenase pathway resulting in leukotriene production [35], a hypothesis supported by the finding of simultaneous production of PAF and LTB4 in activated human neutrophils [36]. Human eosinophils and neutrophils also produce LTB4 when stimulated by PAF [37]. Some in vitro studies using 5-lipoxygenase inhibitors and leukotriene receptor antagonists suggest that in animals secondary generation of leukotrienes is responsible for the biological effects of PAF [38,39], but other studies have not confirmed these findings [40]. Unfortunately, the relevance of these studies is limited by the poor activity and selectivity of the earlier inhibitors and antagonists, and the wide interspecies variation in biological effects of lipid mediators. There have been relatively few studies on the mechanism of action of PAF in man, and again seemingly conflicting findings have been obtained by different groups. Schellenberg et al. [41] found contraction of human bronchus in vitro to be platelet-dependent; whereas Johnson et al. [42] obtained contraction in the absence of these cells. PAF-induced bronchospasm and facial flushing was reduced by pretreatment with chlorpheniramine [13], suggesting that the secondary release of histamine may be importaht. However, ketotifen, an antiallergic agent with antihistamine activity, apparently had no such effect [17]. Thromboxane A2 seems to be important in mediating PAF-induced bronchoconstriction and bronchial hyperresponsiveness in the dog [43], and inhalation of PAF by asthmatics results in a threefold increase in the urinary excretion of thromboxane metabolites [44], Unfortunately, the thromboxane receptor antagonist GR3219iB
An update on PAE
had no effect on PAF-induced bronchoconstriction in normal man [21], although it is possible that this reflects incomplete antagonism of thromboxane by this agent. There seems to be agreement on the importance of leukotrienes as secondary mediators of PAF-induced bronchoconstriction in man. In two separate studies, bronchoconstriction due to inhaled PAF was signiflcantly reduced by the selective and potent cysteinylIeukotriene receptor antagonists SK&F 104353-Z2 [23] and ICI 204,219 in normal subjects [45]. Although the PAF-induced fall in circulating neutrophils was not reduced in these studies, a significant reduction was obtained using the 5-lipoxygenase inhibitor BW A4C [46], suggesting that this phenomenon is dependent on a 5lipoxygenase product other than cysteinyl-leukotrienes, presumably LTB4. Such studies have yet to be performed in asthmatics, but indirect evidence ofthe involvement of leukotrienes in the acute response to inhaled PAF is provided by the flnding of a ninefold increase in urinary LTE4 excretion following PAF compared to that obtained after methacholine in patients with mild atopic asthma [44]. The clinical relevance of these mediator interactions remains to be determined. If PAF does act largely through the secondary generation of leukotrienes, it could be argued that leukotriene antagonists and synthesis inhibitors would be more likely to be of therapeutic value than if the metabolism of these two groups of mediators were entirely independent. However, it is not yet known if any ofthe known actions of PAF other than bronchoconstriction are dependent on secondary mediator production in man. The LTD4/LTE4 antagonist LY171883 has been shown to improve pulmonary function in patients with mild asthma [47], and clinical studies using significantly more potent cysteinyl-Ieukotriene receptor antagonists are currently in progress. If these studies do yield positive results it will raise the intriguing question of whether the direct or indirect component of leukotriene generation is the most significant in producing the pathological and physiological abnormalities in asthma. References 1 Morley J, Sanjar S, Page CP. The platelet in asthma. Lancet 1984; ii: 1142-4. 2 Wardlaw AJ, Moqbel R, Cromwell O, Kay AB. Plateletactivating factor: a potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest 1986; 78:1701-6. 3 Kimani G, Tonnesen MG, Henson PM. Stimulation of eosinophil adherence to human vaseular endothelial cells in vitro by platelet-activating factor. J Immunol 1988; 140:3161-6. 4 Lee T-C, Lenihan DJ, Malone B, Roddy LL, Wasserman SI.
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Increased biosynthesis of platelet-activating factor in activated human eosinophils. J Biol Chem 1984; 259:5526-30. 5 Chesney CMcL, Pifer DD, Byers LW, Muirhead EE. Effect of platelet-activating factor (PAF) on human platelets. Blood 1982; 59:582-5. 6 Lee T-C, Malone B, Blank ML, Snyder F. l-alkyl-2-acetyl.sw-glycero-3-phosphocholine (platelet-activating factor) stimulates calcium influx in rabbit platelets. Biochem Biophys Res Commun 1981; 102:1262-8. 7 Shaw JO, Pinekard RN, Ferrigni KS, McManus LM, Hanahan DJ. Activation of human neutrophils with 1-0hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor). J Immunol 1981; 127:12505. 8 Poitevin B, Roubin R, Benveniste J. PAF-acether generates chemiluminescence in human neutrophils in the absence of cytochalasin B. Immunopharmacology 1984; 7:135-44. 9 Goswami SK, Ohashi M, Panagiotis S, Marom Z. Platelet activating factor enhances mucous glycoprotein release from human airways in vitro. Am Rev Respir Dis 1987; 136:A159. 10 Evans TW, Chung KE, Rogers DE, Barnes PJ. Effect of platelet-activating factor on airway vascular permeability: possible mechanisms. J Appl Physiol 1987; 63:479-84. 11 Aursudkij B, Rogers DE, Evans TW, Alton EWEW, Chung KE, Barnes PJ. Reduced tracheal mucus velocity in guineapigs in vivo by platelet activating factor. Am Rev Respir Dis 1987; 136:A160. 12 Barnes PJ, Chung KE, Page CP. Inflammatory mediators and asthma. Pharmacol Rev 1988; 40:49-84. 13 Smith LJ, Rubin A-H E, Patterson R. Mechanism of platelet activating factor-induced bronchoconstriction in humans. Am Rev Respir Dis 1988; 137:1015-9. 14 Cuss EM, Dixon CMS, Barnes PJ. Effects of inhaled platelet activating factor on pulmonary function and bronchial responsiveness in man. Lancet 1986; ii: 189-92. 15 Tam EKW, Clague J, Dixon CMS et al. Neutrophil sequestration in normal human lung after inhalation of Platelet activating factor (PAE). Thorax 1990; 45:S58. 16 Wardlaw AJ, Chung KE, Moqbel R et al. Effects of inhaled PAE in humans on circulating and bronchoalveolar lavage fluid neutrophils. Am Rev Respir Dis 1990; 141:386-92. 17 Chung KE, Minette P, McCusker M, Barnes PJ. Ketotifen inhibits the cutaneous but not the airway responses to platelet-activating factor in man. J Allergy Clin Immunol 1988; 81:1192-8. 18 Chung KE, Dent G, Barnes PJ. Effects of salbutamol on bronchoconstriction, bronchial hyperresponsiveness, and leucocyte responses induced by platelet activating factor in man. Thorax 1989; 44:102-7. 19 Kaye MG, Smith LJ. Effects of inhaled leukotriene D4 and platelet-activating factor on airway reactivity in normal subjects. Am Rev Respir Dis 1990; 141:993-7. 20 Lai CKW, Jenkins JR, Polosa R, Holgate ST. Inhaled PAE fails to induce airway hyperresponsiveness in normal human subjects. J Appl Physiol 1990; 68:919-26. 21 Stenton SC, Ward C, Duddridge M et al. The actions of
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GR32191B, a thromboxane receptor antagonist, on the effects of inhaled PAE on human airways. Clin Exp Allergy 1990; 20:311-7. 22 Spencer DA, Green SE, Evans JM, Piper PJ, Costello JE. Platelet-activating factor does not cause a reproducible increase in bronchial responsiveness in normal man. Clin Exp Allergy 1990; 20:525-32. 23 Spencer DA, Evans JM, Green SE, Piper PJ, Costello JE. Participation of the cysteinyl-leukotrienes in the acute bronchoconstrictor response to inhaled platelet-activating factor in man. Thorax 1991; 46:441-5. 24 Lai CKW, Holgate ST. Does inhaled PAE cause airway hyperresponsiveness in humans? Clin Exp Allergy 1990; 20:449-52. 25 Rubin AE, Smith LJ, Patterson R. The bronchoconstrictor properties of platelet-activating factor in humans. Am Rev Respir Dis 1987; 136:1145-51. 26 Chung KE, Barnes PJ. Effects of platelet activating factor on airway calibre, airway responsiveness, and circulating cells in asthmatic subjects. Thorax 1989; 44:108-15. 27 Cockcroft DW, Ruffin RE, Dolovieh J, Hargreave EE. Allergen-induced increase in non-allergic bronchial reactivity. Clin Allergy 1977; 7:503-13. 28 Dermarkarian RM, Israel E, Rosenberg M A er al. The effect of SCH-37370, a dual platelet activating factor and histamine antagonist, on the bronchoconstriction induced in asthmatics by cold, dry air isocapnic hyperventilation (ISH). Am Rev Respir Dis 1991; 143:A812. 29 Wilkens H, Wilkens JH, Bosse S et al. Effects of an inhaled PAE-antagonist (WEB 2086 BS) on allergen-induced early and late asthmatic responses and increased bronchial responsiveness to methacholine. Am Rev Respir Dis 1991; 143:A812. 30 Bel EH, De Smet M, Rossing TH, Timmers MC, Dijkman JH, Sterk PJ. The effect ofa specific oral PAF-antagonist, MK-287, on antigen-induced early and late asthmatic reactions in man. Am Rev Respir Dis 1991; 143:A811. 31 Hwang S-B, Lam M-H, Shen TY. Specific binding sites for platelet activating factor in huirian lung tissues. Biochem Biophys Res Commun 1985; 128:972-9. 32 Dent G, Ukena D, Barnes PJ. PAE receptors. In: Barnes PJ, Page CP, Henson PM, eds. Platelet activating factor and human disease. Oxford: Blackwell Scientific Publications, 1989:58-81. 33 Goldie RG, Pedersen KE, Self GJ, Rigby PJ, Paterson JW. Autoradiographic distribution of specific binding sites for the PAE receptor antagonist ['H]-WEB 2086 in human nondiseased and asthmatic bronchi and peripheral lung. Am Rev Respir Dis 1990; 141:A725. 34 O'Elaherty JT, Wykle RL. PAE and cell activation. In: Barnes PJ, Page CP, Henson PM, eds. Platelet activating
factor and human disease. Oxford: Blackwell Scientific Publications, 1989:117-37. 35 Bratton D, Henson PM. Cellular origins of PAE. In: Barnes PJ, Page CP, Henson PM, eds. Platelet activating factor and human disease. Oxford: Blackwell Scientific Publications, 1989:23-57. 36 Sisson JH, Prescott SM, Mclntyre TM, Zimmerman GA. Production of platelet-activating factor by stimulated human polymorphonuclear leukocytes. J Immunol 1987; 138:3918-26. 37 Chilton EH, O'Elaherty JT, Walsh CE et al. Plateletactivating factor: stimulation ofthe lipoxygenase pathway in polymorphonuclear leukocytes by l-0-alkyl-2-0-acetyl-5/jglycero-3-phosphocholine. J Biol Chem 1982; 257:5402-7. 38 Cammussi G, Montrucchio G, Antro C, Bussolino E, Tetta C, Emanuelli G. Platelet-activating factor-mediated contraction of rabbit lung strips: pharmacologic modulation. Immunopharmacology 1983; 6:87-96. 39 Voelkel NE, Worthen S, Reeves JT, Henson PM, Murphy RC. Nonimmunological production of leukotrienes induced by platelet-activating factor. Science 1982; 218:286-8. 40 Eitzgerald ME, Payne AN, Garland LG, Whittle BJR. Eailure of 5-lipoxygenase inhibition with BW A4C to reduce bronchoconstriction induced by inhaled platelet-activating factor. Am Rev Respir Dis 1988; 137:A28. 41 Schellenberg RR, Walker B, Snyder E. Platelet-dependent contraction of human bronchus by platelet-activating factor. J Allergy Clin Immunol 1983; 71:145. 42 Johnson PRA, Armour CL, Black JL. PAE causes contraction and increased responsiveness to histamine in human isolated bronchus. Clin Exp Pharmacol Physiol 1988; Suppl 12:A72. 43 Chung KE, Aizawa H, Leikauf GD, Ueki IE, Evans TW, Nadel JA. Airway hyperresponsiveness induced by plateletactivating factor: role of thromboxane generation. J Pharmacol Exp Ther 1986; 236:580-4. 44 Taylor IK, Ward PS, Taylor GW, Dollery CT, Euller RW. Inhaled PAE stimulates leukotriene and thromboxane A2 production in man. J Appl Physiol, in press. 45 Kidney JC, Ridge S, Chung KE, Barnes PJ. Inhibition of PAF-induced bronchoconstriction by the oral leukotriene D4 receptor antagonist ICI 204,219 in normal subjects. Am Rev Respir Dis 1991; 143:A81I. 46 Spencer DA, Evans JE, Sampson AP, Garland LG, Piper PJ, Costello JE. Effects ofa 5-lipoxygenase inhibitor, BW A4C, on the acute response to inhaled PAE in man. Proc Natl Acad Sci (USA) 1991; 629:430-1. 47 Cloud ML, Enas GC, Kemp J et al. A specific LTD4/LTE4receptor antagonist improves pulmonary function in patients with mild chronic asthma. Am Rev Respir Dis 1989; 140:1336-9.
REVIEW
An update on PAF D. A. SPENCER Birmingham Children's Hospital, Ladywood, Birmingham, U.K.
PAF and bronchial responsiveness
Platelet activating factor (PAF) is a membrane-derived phospholipid with a wide variety of biological actions which has been proposed as a mediator of central importance to the inflammatory PAF process in asthma [1]. PAF is a potent chemotactic agent for a wide range of inflammatory cells including neutrophils and the eosinophil, for which it is one of the most potent chemotactic agents known [2]. In addition it is a potent activator of these cells. For example, it causes eosinophils to adhere to endothelial cells [3] and to synthesize LTC4 and more PAF [4]. PAF causes dose-related aggregation [5] and stimulates calcium influx [6] in platelets, whereas in the neutrophil it causes exocytosis, superoxide generation and chemiluminescence [7,8]. Other properties of PAF relevant to the pulmonary inflammatory process include stimulation of airway mucus production [9], increasing pulmonary microvascular permeability [10], and impairment of mucociliary clearance in vitro [11]. PAF induces acute bronchoconstriction in many species including guinea-pig, rhesus monkey and baboon (reviewed in [12]). In man, PAFinduced bronchoconstriction is accompanied by facial flushing and tightness of the throat. It is also associated with a dramatic, although transient, fall in circulating neutrophils [13,14] due to their pulmonary sequestration [15], and accompanied by increased recovery of these cells, but importantly not eosinophils, in bronchoalveolar lavage fluid [16]. The efl'ect of PAF which has attracted most attention and controversy is its ability to cause a prolonged increase in bronchial responsiveness. This phenomenon has been demonstrated in several animal species including guinea-pig, dog, sheep, rhesus monkey and baboon (reviewed in [12]). In 1986 Cuss et al. [14] reported that inhalation of PAF caused an increase in bronchial responsiveness to methacholine of up to 2-5fold, maximum at 3 days and persisting for up to 4 weeks in five out of six normal human subjects; several further reports from this group have effectively reiterated these findings [16,17,18]. Although others have reported similar findings in abstract form, only Kaye and Smith have, as yet, published any similar findings in a complete paper Correspondence: Dr D. A. Spencer, Birmingham Children's Hospital, Ladywood Middleway, Ladywood, Birmingham B16 8ET, U.K.
[19]. These workers reported an apparent increase of 50% in responsiveness to methacholine in six out of eight normal subjects for up to 14 days following inhaled PAF. Such changes would be at the limit of the detectable changes in responsiveness as the reproducibility of a methacholine challenge is approximately one doubling concentration of the agonist. In addition, the results in this study would seem to be invalid as the data was not apparently log transformed for the analysis. Interestingly, in the same paper these workers reported similar increases in responsiveness to methacholine following LTD4 challenge in normal subjects, the only time that this has ever been reported despite numerous previous reports to the contrary. In contrast to these positive findings, several studies have now been published in which no increase in bronchial responsiveness has occurred following PAF inhalation in normal subjects [20-23]. Possible reasons for these divergent findings have been discussed previously in this journal [22,24] and include the dose of PAF, the method for assessing changes in bronchial responsiveness and the atopic status of the subjects. Perhaps the most obvious reason would be that higher doses of PAF might have been given in the positive studies, but this would not seem to be the case, as apparently higher doses were given in three of the negative studies [20,22,23], and in the remaining study similar doses to those described by Cuss [21]. Unfortunately, every centre has used a difl"erent experimental protocol involving various PAF formulations and preparation methods, nebulization and inhalation techniques so that such attempts at comparing PAF dose delivered to the airways are probably futile. The situation is further confused by diflerent studies describing the dose of PAF given as a single dose, the total cumulative dose, the dose range and the geometric mean dose [24]. Finally, ethical considerations imposed by wide intersubject variations in the acute response to this potentially injurious phospholipid have resulted in large diflerences in the doses of PAF administered between subjects within the same study, rendering meaningless any detailed comparison between studies. The effects of PAF on bronchial responsiveness in asthmatics are less controversial and arguably of greater relevance to the pathophysiology of the disease than 521
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D. A. Spencer
studies in normal man. In neither of the two published studies [25,26] has PAF inhalation resulted in any significant or prolonged increase in bronchial responsiveness. Although these findings could be partially related to the use of smaller doses of PAF in asthmatics compared to normal subjects, they are difficult to reconcile with the suggestion that this mediator is of central importance in the pathophysiology of asthma. Chung and Barnes [26] have suggested that these results could be due either to self-tachyphylaxis, presumably due to down regulation of PAF receptors, or because 'it may be more difficult to enhance the inflammatory process that. . . may already be present in the airways of those with mild asthma'. These conclusions are hard to accept; firstly, it is difficult to conceive how any mediator could exert an important pathophysiological role contemporaneously with the tissue being tachyphylactic to the effect of that mediator. Secondly, it is very well known that clinically important stimuli such as antigen readily increase bronchial responsiveness in patients with mild asthma [27]. The most convincing proof for or against the involvement of PAF in the pathophysiology of asthma will be provided by studies using potent and specific antagonists in models of asthma as well as therapeutic studies in patients with active disease. Preliminary results of such studies are now becoming available. Dermarkarian et al. [28] found that SCH-37370, a dual platelet activating factor and histamine antagonist, had no effect on the bronchoconstriction induced by isocapnic hyperventilation of cold, dry air. The PAF antagonists WEB 2086 BS [29] and MK-287 [30] have also been investigated in allergen challenge, and have been shown not to modify early or late asthmatic responses or the subsequent changes in bronchial responsiveness in asthmatic subjects. Therapeutic trials of WEB 2086 are currently in progress. Secondary generation of other mediators by PAF The mechanisms by which PAF produces its various biological effects are incompletely understood, and it is likely that PAF acts by a variety of direct and indirect mechanisms in different situations. Morley et al. [1] originally proposed that PAF was responsible for the inflammatory features of asthma as a consequence of platelet activation. However, recognition that PAF has multiple actions on many other cell types, particularly the eosinophil, led Barnes et al. [12] to suggest that this cell may be of crucial importance in producing the biological effects attributed to PAF. Interestingly, the eosinophil has not been implicated in the PAF-induced increase in bronchial responsiveness in normal man, a phenomenon which seems to involve the neutrophil [14,16], a cell not closely associated with the pathophysiology of asthma.
High affinity binding sites for PAF which are probably PAF receptors have been described on the surface of many cells including platelets, neutrophils and in homogenates from human lung membranes [31,32], although not on human bronchial smooth muscle [33]. Binding of PAF to specific receptors may trigger complex intracellular transduction mechanisms involving activation of G-proteins and protein kinase C, an increase in turnover of inositol phosphates and a rise in intracellular Ca^""". Phospholipase A2 may then be activated causing release of arachidonate from membrane phospholipids with the subsequent production ofa cascade of mediators including leukotrienes, prostaglandins and thromboxane A2 [34]. It is likely that many ofthe effects attributed to PAF are dependent on this secondary generation of leukotrienes and prostanoids. The synthesis of PAF and leukotrienes are closely linked; the first step of PAF synthesis involves the cleaving of alkylarachidonyl-GPC, effectively liberating lyso-PAF and arachidonic acid. This arachidonic acid may under certain circumstances be channelled down the 5-lipoxygenase pathway resulting in leukotriene production [35], a hypothesis supported by the finding of simultaneous production of PAF and LTB4 in activated human neutrophils [36]. Human eosinophils and neutrophils also produce LTB4 when stimulated by PAF [37]. Some in vitro studies using 5-lipoxygenase inhibitors and leukotriene receptor antagonists suggest that in animals secondary generation of leukotrienes is responsible for the biological effects of PAF [38,39], but other studies have not confirmed these findings [40]. Unfortunately, the relevance of these studies is limited by the poor activity and selectivity of the earlier inhibitors and antagonists, and the wide interspecies variation in biological effects of lipid mediators. There have been relatively few studies on the mechanism of action of PAF in man, and again seemingly conflicting findings have been obtained by different groups. Schellenberg et al. [41] found contraction of human bronchus in vitro to be platelet-dependent; whereas Johnson et al. [42] obtained contraction in the absence of these cells. PAF-induced bronchospasm and facial flushing was reduced by pretreatment with chlorpheniramine [13], suggesting that the secondary release of histamine may be importaht. However, ketotifen, an antiallergic agent with antihistamine activity, apparently had no such effect [17]. Thromboxane A2 seems to be important in mediating PAF-induced bronchoconstriction and bronchial hyperresponsiveness in the dog [43], and inhalation of PAF by asthmatics results in a threefold increase in the urinary excretion of thromboxane metabolites [44], Unfortunately, the thromboxane receptor antagonist GR3219iB
An update on PAE
had no effect on PAF-induced bronchoconstriction in normal man [21], although it is possible that this reflects incomplete antagonism of thromboxane by this agent. There seems to be agreement on the importance of leukotrienes as secondary mediators of PAF-induced bronchoconstriction in man. In two separate studies, bronchoconstriction due to inhaled PAF was signiflcantly reduced by the selective and potent cysteinylIeukotriene receptor antagonists SK&F 104353-Z2 [23] and ICI 204,219 in normal subjects [45]. Although the PAF-induced fall in circulating neutrophils was not reduced in these studies, a significant reduction was obtained using the 5-lipoxygenase inhibitor BW A4C [46], suggesting that this phenomenon is dependent on a 5lipoxygenase product other than cysteinyl-leukotrienes, presumably LTB4. Such studies have yet to be performed in asthmatics, but indirect evidence ofthe involvement of leukotrienes in the acute response to inhaled PAF is provided by the flnding of a ninefold increase in urinary LTE4 excretion following PAF compared to that obtained after methacholine in patients with mild atopic asthma [44]. The clinical relevance of these mediator interactions remains to be determined. If PAF does act largely through the secondary generation of leukotrienes, it could be argued that leukotriene antagonists and synthesis inhibitors would be more likely to be of therapeutic value than if the metabolism of these two groups of mediators were entirely independent. However, it is not yet known if any ofthe known actions of PAF other than bronchoconstriction are dependent on secondary mediator production in man. The LTD4/LTE4 antagonist LY171883 has been shown to improve pulmonary function in patients with mild asthma [47], and clinical studies using significantly more potent cysteinyl-Ieukotriene receptor antagonists are currently in progress. If these studies do yield positive results it will raise the intriguing question of whether the direct or indirect component of leukotriene generation is the most significant in producing the pathological and physiological abnormalities in asthma. References 1 Morley J, Sanjar S, Page CP. The platelet in asthma. Lancet 1984; ii: 1142-4. 2 Wardlaw AJ, Moqbel R, Cromwell O, Kay AB. Plateletactivating factor: a potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest 1986; 78:1701-6. 3 Kimani G, Tonnesen MG, Henson PM. Stimulation of eosinophil adherence to human vaseular endothelial cells in vitro by platelet-activating factor. J Immunol 1988; 140:3161-6. 4 Lee T-C, Lenihan DJ, Malone B, Roddy LL, Wasserman SI.
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