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Generation of platelet activating factor (PAF) by a human lung epithelial cell line
European Journal of Pharmacology, 175 (1990) 253-259
253
Elsevier EJP 51140
Generation of platelet activating factor (PAF) by a human lung epithelial cell line i H a s s a n Salari 2 a n d A n n e W o n g Department of Medicine, University of British Columbia, Vancouver, B.C., Canada
Received 10 October 1989, accepted 24 October 1989
A human lung epithelial cell line (ATC-CCL-185) was cultured in nutrient Ham-F12 medium. Cells in monolayers were stimulated with either ionophore A23187 (1/~M) or phorbol myristate acetate (PMA, 0.2 btM) for various periods of time. Samples were analysed by HPLC and the presence of platelet activating factor (PAF) was detected by bioassay of the release of [3H]serotonin from rabbit platelets undergoing aggregation. The ATC-CCL 185 cells were found to synthesize PAF following activation with either PMA or ionophore. Ionophore at 1 /xM was found to be more potent than PMA at 0.2 /xM in the induction of PAF synthesis (= 80 n g / m g protein). The synthesis of PAF through ionophore stimulation reached a maximum at 5 rain, whereas PMA stimulation peaked at 15-20 rain. PMA induced approximately one third the level of PAF synthesis by the ionophore. The PAF synthesized by these CCL185 cells was found to be mainly associated with the cell membrane with less than 10% released into the medium. Release of PAF into cell supernatant was dependent on the presence of bovine serum albumin (BSA). In the absence of BSA, a large portion ( - 90%) of PAF was found to be cell associated, and only 60% when BSA concentration reached >~0.2%. These results demonstrate the ability of this lung epithelial cell line to synthesis PAF thus, suggesting that epithelial cells might participate in the process of inflammatory lung diseases, through the generation of this important mediator. Lung epithelial cell line (human); Platelet-activating factor (PAF-aceter, PAF); Phorbol myristate acetate; lonophore A23187
1. Introduction Platelet activating factor (PAF) has been characterized as a potent activator of platelets (Benveniste et al., 1972), neutrophils (Camussi et al., 1981), macrophages (Arnoux et al., 1980) and endothelial cells (Camussi et al., 1983a). In p u l m o n a r y tissue P A F has been found to induce constriction of bronchi (Vargaftig et al., 1980), increased microvascular permeability ( O ' D o n n e l l
1 This work was supported by the B.C. Lung Association. 2 H. Salari is a scholar of the B.C. Health Care Research Foundation. Correspondence to: H. Salari, Jack Bell Research Centre, 2660 Oak Street, Vancouver, B.C., Canada V6H 3Z6.
and Barnett, 1987) and a long-lasting non-specific bronchial hyperresponsiveness (Mazzoni et al., 1985; Coyle et al., 1988; C h u n g et al,, 1986; Christman et al., 1987; Cuss et al., 1986). Exposure of experimental animals to aerosolized P A F has been shown to cause recruitment of eosinophils and platelets into airways, reminiscent of the histopathological changes observed following allergen challenge of sensitized animals (Lellouch-Tubiana et al., 1985; Metzger et al., 1985). W h e n P A F was instilled into the airways of rabbits, it caused an i n f l a m m a t o r y response consisting of d e s q u a m a t i o n of epithelial cells into the lumen with accumulation of p o l y m o r p h o n u c l e a r cells, eosinophils and macrophages (Camussi et al., 1983b). In vitro studies have shown that P A F
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
254 is a potent chemotactic agent for human eosinophils (Wardlaw et al., 1986) and is chemokinetic for sheep neutrophils (Burhop et al., 1986). Therefore, the ability of PAF to act as a chemotactic agent for eosinophils raised the possibility that PAF may elicit the eosinophil recruitment into airway epithelium. This, it is important to exmaine whether epithelial cells synthesize PAF. Herein, we report results of studies that examined PAF synthesis by a human lung epithelial cell line, which biochemically and functionally resembles bronchial type II epithelial cells (Lieber et al., 1976). 2. Materials and methods 2.1. Materials
[3H]Serotonin or [3H]5-hydroxytryptamine (1020 Ci/mmol), [3H]PAF or [3H]l-O-alkyl-2-acetylsn-glycerophosphocholine (70-110 C i / m m o l ) and [3H]Iyso-PAF (80-120 C i / m m o l ) were purchased from Amersham Radiochemicals (Arlington Height, IL). Thrombin, fatty acid-free bovine albumin (fraction V), serotonin, 1-O-hexadecyl-2acetyl-sn-glycerophosphocholine PAF, lyso-PAF, phosphatidylcholine (1-stearyl 2-arachidonol) ionophore A23187 and phorbol mysistate acetate were purchased from Sigma Chemical Co. (St. Louis, MO). The PAF antagonist, CV3988, was a gift of Takeda Chemical Co. (Tokyo, Japan). Tissue culture medium (Ham-F12), Hanks balanced salt solution and fetal bovine serum were purchased from Gibco Laboratories (Grand Island, NY). HPLC-grade organic solvents were purchased from Fisher Scientific Co. (Fairlawn, N J). 2.2. Cell culture and treatment
A human bronchial epithelial cell line (ATCCCCL-185) was obtained from the American type culture collection (Rockville, MD). These cells were originally derived from human carcinoma and found to biochemically and morphologically resemble type II epithelial cells (Lieber et al., 1976). Characterization work was performed by the American type culture collection, and no further identification was done by the authors. Cells
were grown in Ham's FI2 medium, supplemented with 10% fetal bovine serum (FBS). Medium was changed 2-3 times weekly until a confluent monolayer was formed. Cells were dislodged with a rubber policeman and challanged with various concentrations of ionophore A23187 or phorbol myristate acetate (PMA) in Hanks balanced salts solution (HBSS) for 30 min. The HBSS also contained 0.25% bovine serum albumin (BSA) unless otherwise indicated. Incubations were performed with 0.5 mg cell protein in 1 ml buffer in polypropylene tubes. Cell protein was determined by the method of Bradford (Bradford, 1976). Incubations were terminated by addition of 2 ml solution of m e t h a n o l / c h l o r o f o r m (Bligh and Dyer, 1959). After separation of phases the organic phase (chloroform) was collected. Chloroform was evoporated under nitrogen and the residue was dissolved in 100/*1 of methanol/chloroform (1 : 9). The samples were then analysed by liquid chromatography as reported (Alam et al., 1982). 2.3. Characterization and measurement of PA F
The extracted lipids were analyzed by normal phase high performance liquid chromatography (NP-HPLC) as reported by Alam et al. (1982), except that the first mobile phase was omitted. By using only solvent B (isopropanol/ toluene/ acetic acid/water: 93 : 110 : 15 : 15) PAF was eluted after approximately 28 min. The HPLC column used in this study was a silica G (4.6 × 25 cm, 10 t~M size, Altech Associates, Dearfield, IL) with a flow rate of 2 ml/min. The recovery of [3H]PAF from this HPLC column was found to be 93 __+_4% (n = 5). One minute HPLC eluates were collected and the solvent was evaporated to dryness. Residues were dissolved in 100 ~tl of a solution of 0.25% BSA for determination of PAF bioactivity. In order to define PAF recovery after various manipulations, 0.01 /,Ci of [3H]lyso-PAF was added to each sample at the end of the incubation, and the radioactivity was monitored during subsequent manipulations. 2.4. Bioassays 2.4.1. Platelet aggregation
PAF bioactivity was determined by aggregation of washed rabbit platelets. Platelets were prepared
255
as reported (Pinckard et al., 1979) and approximately 108 platelets in 0.5 ml were used to assay aggregation using a Bio-Data aggregometer. PAF was quantified using a standard curve constructed with synthetic PAF (hexadecyl) and expressed as ng equivalents.
100
80
60 "0
._c 40
2.4.2. Serotonin release Washed rabbit platelets at 2 × 109/ml were incubated with 0.3 t~Ci/ml of [3H]serotonin for 3 h at 37°C. The non-incorporated [3H]serotonin was discarded by washing the platelets 3 times with Tyrode buffer (Pinckard et al., 1982). Platelets at 3 × 108/ml were challenged with PAF for 1 min and centrifuged in a microfuge at 15 000 x g for 15 s. The radioactivity in the supernatant was determined and then converted to the total [3H]serotonin incorporated into platelets. PAF generated by epithelial cells was quantified using a standard curve obtained with synthetic PAF and expressed as ng equivalents.
3. Results
The HPLC profile of [3H]PAF was shown to have a small radioactive peak with retention time of approximately 5 rain and a major radioactive peak with retention time of approximately 28 rain. Materials extracted from the 5 rain HPLC peak did not cause aggregation of rabbit platelets or release of [3H]serotonin. Thus this peak was considered to contain PAF degradation products. The major radioactive peak (retention time approximately 28 min) was found to be PAF as determined by thin layer chromatography (TLC) (Satouchi et al., 1981) using a solvent system of c h l o r o f o r m / m e t h a n o l / w a t e r (65 : 35 : 6). On the basis of the results obtained from known amounts of standard PAF, this HPLC system was used for the analysis of PAF generated by ATC-CCL 185 cells. In order to assay PAF generation, samples were analyzed by HPLC and eluates were collected at 25-30 rain. The solvent was evaporated and the residue was dissolved in 0.25% BSA. This solution was used for bioassay studies. Using washed rabbit platelets, PAF from 10-13 M caused aggregation of platelets (fig. 1). Maximal aggrega-
E 2O
-14
-13
-12
-11
-10
PAF [log M]
Fig. 1. Effect of various concentrations of PAF on aggregation of rabbit platelets (200×106/ml). Results are expressed as total percent light transmission induced by thrombin (2 units/ml). Each point is the mean of three different experiments.
tion was observed at 10 -11 M, i.e. higher concentrations of PAF did not proportionally increase the aggregation. For this reason, PAF derived from epithelial cells was diluted until the activity fell into the range of 10-11-10 13 M in order to quantitate PAF production. Similarly, serotonin release was maximum at 10 11 M PAF in which approximately 80% of total cellular platelet serotonin was released into the medium. Higher ( > 10 -11 M) concentrations of PAF did not increase serotonin release from platelets, suggesting that PAF concentration had reached the saturation level (data not shown). When platelets were incubated with CV3988 (10 .5 M) 5 min prior to the addition of cell derived PAF, no aggregation or release of serotonin was observed (n = 3), further confirming the identity of PAF. Generation of PAF from CCL-185 cells was studied by incubation of cells (0.5 mg protein) with various concentrations of ionophore A23187. Incubations were carried out for 30 rain at 3 7 ° C and terminated by the addition of c h l o r o f o r m / methanol solution (Satouchi et al., 1981). Both cell-associated and secreted PAF were assayed. As can be seen from fig. 2, ionophore stimulated the synthesis of PAF by epithelial cells from concentrations of 0.1-50/~M. lonophore at concentrations above 50 # M caused cell lysis as determined by the release of 51Cr from labeled cells. The
256
ionophore effect was maximum at about 1 /~M in which about 70-100 ng P A F / m g of cell protein was synthesized. In addition to ionophore treatment, PMA also caused the generation of PAF from CCL-185 cells (fig. 2). However, the effect of PMA was maxim u m at about 0.2 t~M which resulted in the generation of 20-40 ng P A F / m g cell protein. PAF was measured using both bioassay techniques (i.e. platelet aggregation and release of serotonin). The presence of PAF in these samples was further confirmed by its inactivation with phospholipase A z treatment (Thuren et al., 1987), and inhibition by CV3988 at 10 .5 M (data not shown). In order to determine the maximum time required for PAF generation by CCL185 cells in response to ionophore or PMA a time course experiment was performed. The kinetics of PAF synthesis in response to treatment with ionophore A23187 (1 /~M) or PMA (0.2 /~M) are shown in fig. 3. In these studies sample reactions were terminated by addition of c h l o r o f o r m / m e t h a n o l and PAF was extracted and quantified as described above. PAF production peaked at approximately 5 min of ionophore stimulation which resulted in the generation of approximately 82 ± 23 ng P A F / m g cell protein. After approximately 15
8O
"E" 60
)'
40 /
~ 2o 10
0.04
0.08
0,12
0.16
0.18
1
5
10
50
100
Agonist concentration (p.M)
Fig. 2. Effect of various concentrations of ionophore A23187 (e) or P M A (o), on the generation of PAF from cultured epithelial cells. Total PAF present in whole cell suspension (medium + cells) after cell lysis with a sonicator. Mean ___S.D., n=8.
100
o 80 {3_ (33
E c
60
c
40
Q.
20
I 5
I 10
I 15
- -
[ 20
I _ _ J 25 30
Time (rain)
Fig. 3. Time course of PAF generation from epithelial cells stimulated with 1 /xM ionophore A23187 (O) and 0.2 p.M PMA (o). Mean_+ S.E., n = 10.
rain the amount of PAF in the samples decreased, presumably due to conversion into lyso-PAF or acyl-PAF. The response to PMA was delayed in comparison to ionophore and PAF production reached a m a x i m u m between 15-20 rain, with approximately 33 ng PAF generated/rag of cell protein. After approximately 20 rain the amount of PAF in the samples decreased again presumably due to metabolism into lyso-PAF. In several cell systems (Camussi et al., 1987; Bussolino et al., 1988) it has been shown that PAF is mainly retained by cells rather than released into cell supernatants. This phenomena was investigated with CCL-185 cells. In these studies epithelial cells were centrifuged after challenge and both the supernatant and cell pellets were assayed for the presence of P A R As can be seen from fig. 4, greater than 90% of the PAF was cell-associated when no BSA was present in the medium. However, as the amount of BSA in the culture medium was increased, greater amounts of PAF were released into the supernatant. When the concentration of BSA in the medium was increased to greater than 0.2%, approximately 40% of the PAF generated was recovered in the medium with the remainder in the cell pellet (fig. 4). Increasing the concentration of BSA over 0.5% did not significantly increase PAF release from cells, suggesting that the remaining PAF was tightly
257 lOO
1--
8O J=
i
60
~ 40 <
~a . 20 .
0
0
0.001
0.01
0.1
0.2
0.3
0.5
% BSA in Medium
Fig. 4. Effect of addition of BSA on the dissociation of PAF from cell materials into the medium. M e a n + S.D., n = 10.
bound to the cell membrane or in the cell cytoplasm. To address this question experiments were carried out in which cells were incubated with 0.1 /~Ci of [3H]PAF (0.5 mg protein) for 15 min. Ceils were then washed 3 times with HBSS and sonicated for 30 s. Broken cells were centrifuged in a TL 100 bench top ultracentrifuge (Beckman Instrument Co.) for 15 min at 200000 × g. Pellets of sonicated cells that contained greater than 90% membrane, possessed approximately 70-80% of the radioactivity and the remaining 20% was associated with the supernatant (data not shown) suggesting that PAF is predominantly bound to the plasma membrane, presumably in the form of lipids or lyso-PAF.
4. Discussion
The results of the present study demonstrated that CCL-185 of human lung epithelial cell origin, like several other pulmonary cells (1-6) is capable of generating PAF. The identification of PAF was based on the ability of the HPLC-eluted materials with the same retention time as [3H]PAF, to cause the aggregation of platelets and release of serotonin, as well as by inactivation with a PAF antagonist (CV3988). CCL-185 cells synthesized PAF in response to stimulation with calcium ionophore. The concentration of ionophore needed to stimulate these ceils was similar to the concentrations shown to activate several other cell types such as neu-
trophils or macrophages. Indeed very high concentrations of ionophore (>/50 #M) were toxic to the cells as was determined by the release of 51Cr from labelled cells into medium (data not shown) and by failure exclusion of trypan blue dye. Thus the maximum concentration of ionophore used without causing cell toxicity was 1.5 /~M. The synthesis of PAF after ionophore activation of cells was rapid ( - 5 min). PMA was also found to activate epithelial cell production of PAF. PMA has been shown to activate protein kinase C (PKC), an essential enzyme signal transduction in cells (Tapley and Murray, 1985). PMA was a less potent than ionophore for the induction of PAF synthesis. Similar results have been reported for several other cell types. For example in polymorphonuclear cells, PMA has been shown to cause the generation of approximately 5-fold less PAF than ionophore. The finding that PMA can cause the generation of PAF suggests than possibly many agents that activate PKC might also cause the synthesis of PAF. For example, epithelial cells have been shown to respond to bradykinin or acetylcholine with generation of prostaglandins and chloride secretion through the activation of PKC (Leikaur et al., 1985). It would therefore be of interest to examine whether epithelial cells release PAF in response to acetylcholine or bradykinin. We found that CCL-185 cells, retain the majority of synthesized PAF in the plasma membrane; this result is similar to findings reported for endothelial cells (Camussi et al., 1987). Addition of BSA to the cell cultures was required in order for PAF to be released into the medium. Without BSA, almost all the synthesized PAF was retained in the plasma membrane. However with the addition of BSA to the medium, large amounts ( - 40%) of synthesized PAF was released into the culture medium. Although the amount of PAF released by this epithelial cell line is less than that synthesized by endothelial cells or macrophages, these concentrations of PAF should nevertheless still be sufficient to induce biological responses. For example PAF has been shown to cause eosinophil migration (chemotactic factor) in the low ng range (Wardlaw et al., 1986). Thus the amount of PAF synthesized by the epithelial cell line would be sufficient to
258
cause inflammation of the epithelium and recruitment of inflammatory cells into the epithelial layer if normal bronchial epithelial cells synthesize PAF in similar amount (Patterson and Harris, 1983; McManus et al., 1980). The migration of neutrophils and eosinophils into airways and epithelium has been shown to be mainly associated with the late phase asthmatic reaction (Dunnill, 1960; Dunnill et al., 1969), suggesting that PAF might be involved in this late stage. In this regard, Nakamura et al. (1987) have detected PAF in the plasma of asthmatics during the late phase reaction. PAF has also been shown to cause bronchial hyperresponsiveness in both human and animals (Chung et al., 1986; Rubin et al., 1987). It would be of interest therefore to examine whether PAF is detectable in pulmonary secretions. In conclusion this report demonstrates that a cell line of lung epithelial origin is capable of PAF synthesis. Therefore it is important to demonstrate the ability of normal lung epithelial cells (type 1 and II) to synthesise PAF in the hope of better understanding the role of PAF and epithelial cells in inflammatory lung disease such as asthma.
References Alam, I., J.B. Smith and M.J, Silver, 1982, Novel system for separation of phospholipids by high-performance liquid chromatography, J. Chromatogr. 234, 218. Arnoux, B., D. Duval and J. Benveniste, 1980, Release of platelet activating factor (PAF-acether) from alveolar macrophages by the calcium ionophore A23187 and phagocytosis, European J. Clin. Invest. 10, 437. Benveniste, J., P.M. Henson and C.G. Cochrane, 1972, Leukocyte-dependent histamine release from rabbit platelets: The role of IgE, basophils, and a platelet-activating factor, J. Exp. Med. 136, 1356. Bligh, E.G. and W.J. Dyer, 1959, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37, 911. Bradford, M.M., 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248. Burhop, K.E., H. Van der Zee, R. Bizios, J.E. Kaplan and A.B. Malik, 1986, Pulmonary vascular response to platelet activating factor in awake sheep and the role of cyclooxygenase metabolites, Am. Rev. Respir. Dis. 134, 548. Bussolino, F., G. Camussi and C. Baglioni, 1988, Synthesis and
release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1, J. Biol. Chem. 263, 11856. Camussi, G., M. Aglietta, R. Coda, F. Bussolino, W. Piacibello and C. Tetra, 1981, Release of platelet activating factor and histamine. II The cellular origin of human PAF: Monocytes, polymorphonuclear cells, neutrophils and basophils, Immunology 42, 191. Camussi, G., M. Aglietta, F. Malavasi, A. Tetta, W. Piacibello, F. Sanavio and F. Bussolino, 1983a, The release of platelet activating factor from human endothelial cells in culture, J. lmmunol. 31, 2397. Camussi, G., F. Bussolino, G. Salvidio and C. Baglioni, 1987, Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet activating factor, J. Exp. Med. 166, 1390. Camussi, G., I. Pawlowski, C. Tetta, C. Roffinello, M. Alberton, J. Brentjens and G. Andres, 1983b, Acute lung inflammation induced in the rabbit by local instillation of l-0-actadecyl-2-acetyl-sn-glycerol-3-phosphocholine or of native platelet-activating factor, Am. J. Pathol. 112, 75. Christman, B.W., P.L. Lefferts and J.R. Snapper, 1987, Effect of platelet activating factor on aerosol histamine responsiveness in awake sheep, Am. Rev. Respir. Dis. 13, 1267. Chung, K.F., H. Aizawa, G.D. Leikauf, I.F. Ueki, T.W. Evans and J.A. Nadel, 1986, Airway hyperresponsiveness induced by platelet activating factor: Role of thromboxane generation, J. Pharmacol. Exp. Ther. 236, 580. Coyle, A.J., S.C. Urwin, C.P. Page, C. Touvay, B. Villain and P. Braquet, 1988, The effect of the selective PAF antagonist BN52021 and PAF- and antigen-induced bronchial hyperreactivity and eosinophil accumulation, European J. Pharmacol. 148, 51. Cuss, F.M., C.M.S. Dixon and P.J. Barnes, 1986, Effects of inhaled platelet activating factor on pulmonary function and bronchial hyperresponsiveness in man, Lancet ii, 189. Dunnill, M.S., 1960, The pathology of asthma with special reference to changes in the bronchial mucosa, J. Clin. Pathol. 13, 27. Dunnill, M.S., G.R. Massarella and J.A. Anderson, 1969, A comparison of the quantitative anatomy of the bronchi in normal subjects in status asthmaticus, in chronic bronchitis and in emphysema, Thorax 24, 176. Leikaur, G.D., LF. Ueki, J.A. Nadel and J.H. Widdicombe, 1985, Bradykinin stimulates C1 secretion and prostaglandin E 2 release by canine tracheal epithelium, Am. J. Physiol. 248, F48. Lellouch-Tubiana, A., J. Lefort, E. Pirotzky, B.B. Vargaftig and A. Pfister, 1985, Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet activating factor in the guinea pig, Br. J. Exp. Pathol. 66, 345. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees and G. Todoro, 1976, A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells, Int. J. Cancer 17, 62.
259 Mazzoni, L., J. Morley, C.P. Page and S. Sanjar, 1985, Induction of airway hyperreactivity by platelet activating factor in the guinea pig, J. Physiol. 365, 107P. McManus, L.M., D.J. Hanahan, C.A. Demopoulos and R.N. Pinckard, 1980, Pathobiology of the intravenous infusion of acetylglyceryl ether phosphorylcholine (AGEPC), a synthetic platelet-activating factor (PAF) in the rabbit, J. Immunol. 124, 2919. Metzger, W.J., G.W. Hunninghake and H.B. Richerson, 1985, Late asthmatic responses: Inquiry into mechanisms and significance, Clin. Rev. Allergy 3, 155. Nakamura, T., Y. Morita, M. Kuriyama, K. Ishihara, K. lto and T. Miyamoto, 1987, Platelet activating factor in late asthmatic response, Int. Arch. Allergy Appl. Immunol. 82, 57. D'Donnell, S.R. and C.J.K. Barnett, 1987, Microvascular leakage to platelet activating factor in guinea-pig trachea and bronchi, European J. Pharmacol. 138, 385. Patterson, R. and K.E. Harris, 1983, The activity of aerosolized and intracutaneous synthetic platelet-activating factor (AGEPC) in rhesus monkeys with IgE-mediated airway responses and normal monkeys, J. Lab. Clin. Med. 102, 933. Pinckard, R.N., R.S. Farr and D.J. Hanahan, 1979, Physiochemical and functional identity of rabbit platelet activating factor (PAF) released in-vivo during lgE anaphylaxis with PAF released in-vitro from IgE sensitized basophils, J. Immunol. 123, 1947.
Pinckard, R.N., L.M. McManus and D.J. Hanahan. 1982, Chemistry and biology of acetylglycerylether phosphorylcholine (platelet activating factor), Adv. Inflam. Res. 4, 147. Rubin, A.H., L.J. Smith and R. Patterson, 1987, The bronchoconstrictor properties of platelet-activating factor in humans, Am. Rev. Respir. Dis. 136, 1145. Satouchi, K., R.N. Pinckard, L.M. McManus and D.J. Hanahan, 1981, Modification of the polar head group of acetylglycerol ether phosphorylcholine and subsequent effects upon platelets activation, J. Biol. Chem. 256, 4425. Tapley, P.M. and A.W. Murray, 1985, Evidence that treatment of platelets with phorbolester causes proteolytic activation of Ca2+-activated, phospholipid-dependent protein kinase, European J. Biochem. 15l, 419. Thuren, T., J.A. Virtanen, R, Verger and P.K.J, Kinnuene, 1987, Hydrolysis of 1-palmitoyl-2-[6-(pyren-l-yl) hexanoylsn-glycero-3-phospholipids] by phopsholipase A2: effect of polar head-group, Biochem. Biophys. Acta 917, 411. Vargaftig, B.B., J. Lefort, M. Chignard and J. Benveniste, 1980, Platelet activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives, European J. Pharmacol. 65, 185. Wardlaw, A.J., R. Modbel, P. Cromwell and A.B. Kay, 1986, Platelet activating factor: a potent chemotactic and chemokinetic factor for human eosinophils, J. Clin. Invest. 78, 1701.
253
Elsevier EJP 51140
Generation of platelet activating factor (PAF) by a human lung epithelial cell line i H a s s a n Salari 2 a n d A n n e W o n g Department of Medicine, University of British Columbia, Vancouver, B.C., Canada
Received 10 October 1989, accepted 24 October 1989
A human lung epithelial cell line (ATC-CCL-185) was cultured in nutrient Ham-F12 medium. Cells in monolayers were stimulated with either ionophore A23187 (1/~M) or phorbol myristate acetate (PMA, 0.2 btM) for various periods of time. Samples were analysed by HPLC and the presence of platelet activating factor (PAF) was detected by bioassay of the release of [3H]serotonin from rabbit platelets undergoing aggregation. The ATC-CCL 185 cells were found to synthesize PAF following activation with either PMA or ionophore. Ionophore at 1 /xM was found to be more potent than PMA at 0.2 /xM in the induction of PAF synthesis (= 80 n g / m g protein). The synthesis of PAF through ionophore stimulation reached a maximum at 5 rain, whereas PMA stimulation peaked at 15-20 rain. PMA induced approximately one third the level of PAF synthesis by the ionophore. The PAF synthesized by these CCL185 cells was found to be mainly associated with the cell membrane with less than 10% released into the medium. Release of PAF into cell supernatant was dependent on the presence of bovine serum albumin (BSA). In the absence of BSA, a large portion ( - 90%) of PAF was found to be cell associated, and only 60% when BSA concentration reached >~0.2%. These results demonstrate the ability of this lung epithelial cell line to synthesis PAF thus, suggesting that epithelial cells might participate in the process of inflammatory lung diseases, through the generation of this important mediator. Lung epithelial cell line (human); Platelet-activating factor (PAF-aceter, PAF); Phorbol myristate acetate; lonophore A23187
1. Introduction Platelet activating factor (PAF) has been characterized as a potent activator of platelets (Benveniste et al., 1972), neutrophils (Camussi et al., 1981), macrophages (Arnoux et al., 1980) and endothelial cells (Camussi et al., 1983a). In p u l m o n a r y tissue P A F has been found to induce constriction of bronchi (Vargaftig et al., 1980), increased microvascular permeability ( O ' D o n n e l l
1 This work was supported by the B.C. Lung Association. 2 H. Salari is a scholar of the B.C. Health Care Research Foundation. Correspondence to: H. Salari, Jack Bell Research Centre, 2660 Oak Street, Vancouver, B.C., Canada V6H 3Z6.
and Barnett, 1987) and a long-lasting non-specific bronchial hyperresponsiveness (Mazzoni et al., 1985; Coyle et al., 1988; C h u n g et al,, 1986; Christman et al., 1987; Cuss et al., 1986). Exposure of experimental animals to aerosolized P A F has been shown to cause recruitment of eosinophils and platelets into airways, reminiscent of the histopathological changes observed following allergen challenge of sensitized animals (Lellouch-Tubiana et al., 1985; Metzger et al., 1985). W h e n P A F was instilled into the airways of rabbits, it caused an i n f l a m m a t o r y response consisting of d e s q u a m a t i o n of epithelial cells into the lumen with accumulation of p o l y m o r p h o n u c l e a r cells, eosinophils and macrophages (Camussi et al., 1983b). In vitro studies have shown that P A F
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
254 is a potent chemotactic agent for human eosinophils (Wardlaw et al., 1986) and is chemokinetic for sheep neutrophils (Burhop et al., 1986). Therefore, the ability of PAF to act as a chemotactic agent for eosinophils raised the possibility that PAF may elicit the eosinophil recruitment into airway epithelium. This, it is important to exmaine whether epithelial cells synthesize PAF. Herein, we report results of studies that examined PAF synthesis by a human lung epithelial cell line, which biochemically and functionally resembles bronchial type II epithelial cells (Lieber et al., 1976). 2. Materials and methods 2.1. Materials
[3H]Serotonin or [3H]5-hydroxytryptamine (1020 Ci/mmol), [3H]PAF or [3H]l-O-alkyl-2-acetylsn-glycerophosphocholine (70-110 C i / m m o l ) and [3H]Iyso-PAF (80-120 C i / m m o l ) were purchased from Amersham Radiochemicals (Arlington Height, IL). Thrombin, fatty acid-free bovine albumin (fraction V), serotonin, 1-O-hexadecyl-2acetyl-sn-glycerophosphocholine PAF, lyso-PAF, phosphatidylcholine (1-stearyl 2-arachidonol) ionophore A23187 and phorbol mysistate acetate were purchased from Sigma Chemical Co. (St. Louis, MO). The PAF antagonist, CV3988, was a gift of Takeda Chemical Co. (Tokyo, Japan). Tissue culture medium (Ham-F12), Hanks balanced salt solution and fetal bovine serum were purchased from Gibco Laboratories (Grand Island, NY). HPLC-grade organic solvents were purchased from Fisher Scientific Co. (Fairlawn, N J). 2.2. Cell culture and treatment
A human bronchial epithelial cell line (ATCCCCL-185) was obtained from the American type culture collection (Rockville, MD). These cells were originally derived from human carcinoma and found to biochemically and morphologically resemble type II epithelial cells (Lieber et al., 1976). Characterization work was performed by the American type culture collection, and no further identification was done by the authors. Cells
were grown in Ham's FI2 medium, supplemented with 10% fetal bovine serum (FBS). Medium was changed 2-3 times weekly until a confluent monolayer was formed. Cells were dislodged with a rubber policeman and challanged with various concentrations of ionophore A23187 or phorbol myristate acetate (PMA) in Hanks balanced salts solution (HBSS) for 30 min. The HBSS also contained 0.25% bovine serum albumin (BSA) unless otherwise indicated. Incubations were performed with 0.5 mg cell protein in 1 ml buffer in polypropylene tubes. Cell protein was determined by the method of Bradford (Bradford, 1976). Incubations were terminated by addition of 2 ml solution of m e t h a n o l / c h l o r o f o r m (Bligh and Dyer, 1959). After separation of phases the organic phase (chloroform) was collected. Chloroform was evoporated under nitrogen and the residue was dissolved in 100/*1 of methanol/chloroform (1 : 9). The samples were then analysed by liquid chromatography as reported (Alam et al., 1982). 2.3. Characterization and measurement of PA F
The extracted lipids were analyzed by normal phase high performance liquid chromatography (NP-HPLC) as reported by Alam et al. (1982), except that the first mobile phase was omitted. By using only solvent B (isopropanol/ toluene/ acetic acid/water: 93 : 110 : 15 : 15) PAF was eluted after approximately 28 min. The HPLC column used in this study was a silica G (4.6 × 25 cm, 10 t~M size, Altech Associates, Dearfield, IL) with a flow rate of 2 ml/min. The recovery of [3H]PAF from this HPLC column was found to be 93 __+_4% (n = 5). One minute HPLC eluates were collected and the solvent was evaporated to dryness. Residues were dissolved in 100 ~tl of a solution of 0.25% BSA for determination of PAF bioactivity. In order to define PAF recovery after various manipulations, 0.01 /,Ci of [3H]lyso-PAF was added to each sample at the end of the incubation, and the radioactivity was monitored during subsequent manipulations. 2.4. Bioassays 2.4.1. Platelet aggregation
PAF bioactivity was determined by aggregation of washed rabbit platelets. Platelets were prepared
255
as reported (Pinckard et al., 1979) and approximately 108 platelets in 0.5 ml were used to assay aggregation using a Bio-Data aggregometer. PAF was quantified using a standard curve constructed with synthetic PAF (hexadecyl) and expressed as ng equivalents.
100
80
60 "0
._c 40
2.4.2. Serotonin release Washed rabbit platelets at 2 × 109/ml were incubated with 0.3 t~Ci/ml of [3H]serotonin for 3 h at 37°C. The non-incorporated [3H]serotonin was discarded by washing the platelets 3 times with Tyrode buffer (Pinckard et al., 1982). Platelets at 3 × 108/ml were challenged with PAF for 1 min and centrifuged in a microfuge at 15 000 x g for 15 s. The radioactivity in the supernatant was determined and then converted to the total [3H]serotonin incorporated into platelets. PAF generated by epithelial cells was quantified using a standard curve obtained with synthetic PAF and expressed as ng equivalents.
3. Results
The HPLC profile of [3H]PAF was shown to have a small radioactive peak with retention time of approximately 5 rain and a major radioactive peak with retention time of approximately 28 rain. Materials extracted from the 5 rain HPLC peak did not cause aggregation of rabbit platelets or release of [3H]serotonin. Thus this peak was considered to contain PAF degradation products. The major radioactive peak (retention time approximately 28 min) was found to be PAF as determined by thin layer chromatography (TLC) (Satouchi et al., 1981) using a solvent system of c h l o r o f o r m / m e t h a n o l / w a t e r (65 : 35 : 6). On the basis of the results obtained from known amounts of standard PAF, this HPLC system was used for the analysis of PAF generated by ATC-CCL 185 cells. In order to assay PAF generation, samples were analyzed by HPLC and eluates were collected at 25-30 rain. The solvent was evaporated and the residue was dissolved in 0.25% BSA. This solution was used for bioassay studies. Using washed rabbit platelets, PAF from 10-13 M caused aggregation of platelets (fig. 1). Maximal aggrega-
E 2O
-14
-13
-12
-11
-10
PAF [log M]
Fig. 1. Effect of various concentrations of PAF on aggregation of rabbit platelets (200×106/ml). Results are expressed as total percent light transmission induced by thrombin (2 units/ml). Each point is the mean of three different experiments.
tion was observed at 10 -11 M, i.e. higher concentrations of PAF did not proportionally increase the aggregation. For this reason, PAF derived from epithelial cells was diluted until the activity fell into the range of 10-11-10 13 M in order to quantitate PAF production. Similarly, serotonin release was maximum at 10 11 M PAF in which approximately 80% of total cellular platelet serotonin was released into the medium. Higher ( > 10 -11 M) concentrations of PAF did not increase serotonin release from platelets, suggesting that PAF concentration had reached the saturation level (data not shown). When platelets were incubated with CV3988 (10 .5 M) 5 min prior to the addition of cell derived PAF, no aggregation or release of serotonin was observed (n = 3), further confirming the identity of PAF. Generation of PAF from CCL-185 cells was studied by incubation of cells (0.5 mg protein) with various concentrations of ionophore A23187. Incubations were carried out for 30 rain at 3 7 ° C and terminated by the addition of c h l o r o f o r m / methanol solution (Satouchi et al., 1981). Both cell-associated and secreted PAF were assayed. As can be seen from fig. 2, ionophore stimulated the synthesis of PAF by epithelial cells from concentrations of 0.1-50/~M. lonophore at concentrations above 50 # M caused cell lysis as determined by the release of 51Cr from labeled cells. The
256
ionophore effect was maximum at about 1 /~M in which about 70-100 ng P A F / m g of cell protein was synthesized. In addition to ionophore treatment, PMA also caused the generation of PAF from CCL-185 cells (fig. 2). However, the effect of PMA was maxim u m at about 0.2 t~M which resulted in the generation of 20-40 ng P A F / m g cell protein. PAF was measured using both bioassay techniques (i.e. platelet aggregation and release of serotonin). The presence of PAF in these samples was further confirmed by its inactivation with phospholipase A z treatment (Thuren et al., 1987), and inhibition by CV3988 at 10 .5 M (data not shown). In order to determine the maximum time required for PAF generation by CCL185 cells in response to ionophore or PMA a time course experiment was performed. The kinetics of PAF synthesis in response to treatment with ionophore A23187 (1 /~M) or PMA (0.2 /~M) are shown in fig. 3. In these studies sample reactions were terminated by addition of c h l o r o f o r m / m e t h a n o l and PAF was extracted and quantified as described above. PAF production peaked at approximately 5 min of ionophore stimulation which resulted in the generation of approximately 82 ± 23 ng P A F / m g cell protein. After approximately 15
8O
"E" 60
)'
40 /
~ 2o 10
0.04
0.08
0,12
0.16
0.18
1
5
10
50
100
Agonist concentration (p.M)
Fig. 2. Effect of various concentrations of ionophore A23187 (e) or P M A (o), on the generation of PAF from cultured epithelial cells. Total PAF present in whole cell suspension (medium + cells) after cell lysis with a sonicator. Mean ___S.D., n=8.
100
o 80 {3_ (33
E c
60
c
40
Q.
20
I 5
I 10
I 15
- -
[ 20
I _ _ J 25 30
Time (rain)
Fig. 3. Time course of PAF generation from epithelial cells stimulated with 1 /xM ionophore A23187 (O) and 0.2 p.M PMA (o). Mean_+ S.E., n = 10.
rain the amount of PAF in the samples decreased, presumably due to conversion into lyso-PAF or acyl-PAF. The response to PMA was delayed in comparison to ionophore and PAF production reached a m a x i m u m between 15-20 rain, with approximately 33 ng PAF generated/rag of cell protein. After approximately 20 rain the amount of PAF in the samples decreased again presumably due to metabolism into lyso-PAF. In several cell systems (Camussi et al., 1987; Bussolino et al., 1988) it has been shown that PAF is mainly retained by cells rather than released into cell supernatants. This phenomena was investigated with CCL-185 cells. In these studies epithelial cells were centrifuged after challenge and both the supernatant and cell pellets were assayed for the presence of P A R As can be seen from fig. 4, greater than 90% of the PAF was cell-associated when no BSA was present in the medium. However, as the amount of BSA in the culture medium was increased, greater amounts of PAF were released into the supernatant. When the concentration of BSA in the medium was increased to greater than 0.2%, approximately 40% of the PAF generated was recovered in the medium with the remainder in the cell pellet (fig. 4). Increasing the concentration of BSA over 0.5% did not significantly increase PAF release from cells, suggesting that the remaining PAF was tightly
257 lOO
1--
8O J=
i
60
~ 40 <
~a . 20 .
0
0
0.001
0.01
0.1
0.2
0.3
0.5
% BSA in Medium
Fig. 4. Effect of addition of BSA on the dissociation of PAF from cell materials into the medium. M e a n + S.D., n = 10.
bound to the cell membrane or in the cell cytoplasm. To address this question experiments were carried out in which cells were incubated with 0.1 /~Ci of [3H]PAF (0.5 mg protein) for 15 min. Ceils were then washed 3 times with HBSS and sonicated for 30 s. Broken cells were centrifuged in a TL 100 bench top ultracentrifuge (Beckman Instrument Co.) for 15 min at 200000 × g. Pellets of sonicated cells that contained greater than 90% membrane, possessed approximately 70-80% of the radioactivity and the remaining 20% was associated with the supernatant (data not shown) suggesting that PAF is predominantly bound to the plasma membrane, presumably in the form of lipids or lyso-PAF.
4. Discussion
The results of the present study demonstrated that CCL-185 of human lung epithelial cell origin, like several other pulmonary cells (1-6) is capable of generating PAF. The identification of PAF was based on the ability of the HPLC-eluted materials with the same retention time as [3H]PAF, to cause the aggregation of platelets and release of serotonin, as well as by inactivation with a PAF antagonist (CV3988). CCL-185 cells synthesized PAF in response to stimulation with calcium ionophore. The concentration of ionophore needed to stimulate these ceils was similar to the concentrations shown to activate several other cell types such as neu-
trophils or macrophages. Indeed very high concentrations of ionophore (>/50 #M) were toxic to the cells as was determined by the release of 51Cr from labelled cells into medium (data not shown) and by failure exclusion of trypan blue dye. Thus the maximum concentration of ionophore used without causing cell toxicity was 1.5 /~M. The synthesis of PAF after ionophore activation of cells was rapid ( - 5 min). PMA was also found to activate epithelial cell production of PAF. PMA has been shown to activate protein kinase C (PKC), an essential enzyme signal transduction in cells (Tapley and Murray, 1985). PMA was a less potent than ionophore for the induction of PAF synthesis. Similar results have been reported for several other cell types. For example in polymorphonuclear cells, PMA has been shown to cause the generation of approximately 5-fold less PAF than ionophore. The finding that PMA can cause the generation of PAF suggests than possibly many agents that activate PKC might also cause the synthesis of PAF. For example, epithelial cells have been shown to respond to bradykinin or acetylcholine with generation of prostaglandins and chloride secretion through the activation of PKC (Leikaur et al., 1985). It would therefore be of interest to examine whether epithelial cells release PAF in response to acetylcholine or bradykinin. We found that CCL-185 cells, retain the majority of synthesized PAF in the plasma membrane; this result is similar to findings reported for endothelial cells (Camussi et al., 1987). Addition of BSA to the cell cultures was required in order for PAF to be released into the medium. Without BSA, almost all the synthesized PAF was retained in the plasma membrane. However with the addition of BSA to the medium, large amounts ( - 40%) of synthesized PAF was released into the culture medium. Although the amount of PAF released by this epithelial cell line is less than that synthesized by endothelial cells or macrophages, these concentrations of PAF should nevertheless still be sufficient to induce biological responses. For example PAF has been shown to cause eosinophil migration (chemotactic factor) in the low ng range (Wardlaw et al., 1986). Thus the amount of PAF synthesized by the epithelial cell line would be sufficient to
258
cause inflammation of the epithelium and recruitment of inflammatory cells into the epithelial layer if normal bronchial epithelial cells synthesize PAF in similar amount (Patterson and Harris, 1983; McManus et al., 1980). The migration of neutrophils and eosinophils into airways and epithelium has been shown to be mainly associated with the late phase asthmatic reaction (Dunnill, 1960; Dunnill et al., 1969), suggesting that PAF might be involved in this late stage. In this regard, Nakamura et al. (1987) have detected PAF in the plasma of asthmatics during the late phase reaction. PAF has also been shown to cause bronchial hyperresponsiveness in both human and animals (Chung et al., 1986; Rubin et al., 1987). It would be of interest therefore to examine whether PAF is detectable in pulmonary secretions. In conclusion this report demonstrates that a cell line of lung epithelial origin is capable of PAF synthesis. Therefore it is important to demonstrate the ability of normal lung epithelial cells (type 1 and II) to synthesise PAF in the hope of better understanding the role of PAF and epithelial cells in inflammatory lung disease such as asthma.
References Alam, I., J.B. Smith and M.J, Silver, 1982, Novel system for separation of phospholipids by high-performance liquid chromatography, J. Chromatogr. 234, 218. Arnoux, B., D. Duval and J. Benveniste, 1980, Release of platelet activating factor (PAF-acether) from alveolar macrophages by the calcium ionophore A23187 and phagocytosis, European J. Clin. Invest. 10, 437. Benveniste, J., P.M. Henson and C.G. Cochrane, 1972, Leukocyte-dependent histamine release from rabbit platelets: The role of IgE, basophils, and a platelet-activating factor, J. Exp. Med. 136, 1356. Bligh, E.G. and W.J. Dyer, 1959, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37, 911. Bradford, M.M., 1976, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248. Burhop, K.E., H. Van der Zee, R. Bizios, J.E. Kaplan and A.B. Malik, 1986, Pulmonary vascular response to platelet activating factor in awake sheep and the role of cyclooxygenase metabolites, Am. Rev. Respir. Dis. 134, 548. Bussolino, F., G. Camussi and C. Baglioni, 1988, Synthesis and
release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1, J. Biol. Chem. 263, 11856. Camussi, G., M. Aglietta, R. Coda, F. Bussolino, W. Piacibello and C. Tetra, 1981, Release of platelet activating factor and histamine. II The cellular origin of human PAF: Monocytes, polymorphonuclear cells, neutrophils and basophils, Immunology 42, 191. Camussi, G., M. Aglietta, F. Malavasi, A. Tetta, W. Piacibello, F. Sanavio and F. Bussolino, 1983a, The release of platelet activating factor from human endothelial cells in culture, J. lmmunol. 31, 2397. Camussi, G., F. Bussolino, G. Salvidio and C. Baglioni, 1987, Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet activating factor, J. Exp. Med. 166, 1390. Camussi, G., I. Pawlowski, C. Tetta, C. Roffinello, M. Alberton, J. Brentjens and G. Andres, 1983b, Acute lung inflammation induced in the rabbit by local instillation of l-0-actadecyl-2-acetyl-sn-glycerol-3-phosphocholine or of native platelet-activating factor, Am. J. Pathol. 112, 75. Christman, B.W., P.L. Lefferts and J.R. Snapper, 1987, Effect of platelet activating factor on aerosol histamine responsiveness in awake sheep, Am. Rev. Respir. Dis. 13, 1267. Chung, K.F., H. Aizawa, G.D. Leikauf, I.F. Ueki, T.W. Evans and J.A. Nadel, 1986, Airway hyperresponsiveness induced by platelet activating factor: Role of thromboxane generation, J. Pharmacol. Exp. Ther. 236, 580. Coyle, A.J., S.C. Urwin, C.P. Page, C. Touvay, B. Villain and P. Braquet, 1988, The effect of the selective PAF antagonist BN52021 and PAF- and antigen-induced bronchial hyperreactivity and eosinophil accumulation, European J. Pharmacol. 148, 51. Cuss, F.M., C.M.S. Dixon and P.J. Barnes, 1986, Effects of inhaled platelet activating factor on pulmonary function and bronchial hyperresponsiveness in man, Lancet ii, 189. Dunnill, M.S., 1960, The pathology of asthma with special reference to changes in the bronchial mucosa, J. Clin. Pathol. 13, 27. Dunnill, M.S., G.R. Massarella and J.A. Anderson, 1969, A comparison of the quantitative anatomy of the bronchi in normal subjects in status asthmaticus, in chronic bronchitis and in emphysema, Thorax 24, 176. Leikaur, G.D., LF. Ueki, J.A. Nadel and J.H. Widdicombe, 1985, Bradykinin stimulates C1 secretion and prostaglandin E 2 release by canine tracheal epithelium, Am. J. Physiol. 248, F48. Lellouch-Tubiana, A., J. Lefort, E. Pirotzky, B.B. Vargaftig and A. Pfister, 1985, Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection of platelet activating factor in the guinea pig, Br. J. Exp. Pathol. 66, 345. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees and G. Todoro, 1976, A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells, Int. J. Cancer 17, 62.
259 Mazzoni, L., J. Morley, C.P. Page and S. Sanjar, 1985, Induction of airway hyperreactivity by platelet activating factor in the guinea pig, J. Physiol. 365, 107P. McManus, L.M., D.J. Hanahan, C.A. Demopoulos and R.N. Pinckard, 1980, Pathobiology of the intravenous infusion of acetylglyceryl ether phosphorylcholine (AGEPC), a synthetic platelet-activating factor (PAF) in the rabbit, J. Immunol. 124, 2919. Metzger, W.J., G.W. Hunninghake and H.B. Richerson, 1985, Late asthmatic responses: Inquiry into mechanisms and significance, Clin. Rev. Allergy 3, 155. Nakamura, T., Y. Morita, M. Kuriyama, K. Ishihara, K. lto and T. Miyamoto, 1987, Platelet activating factor in late asthmatic response, Int. Arch. Allergy Appl. Immunol. 82, 57. D'Donnell, S.R. and C.J.K. Barnett, 1987, Microvascular leakage to platelet activating factor in guinea-pig trachea and bronchi, European J. Pharmacol. 138, 385. Patterson, R. and K.E. Harris, 1983, The activity of aerosolized and intracutaneous synthetic platelet-activating factor (AGEPC) in rhesus monkeys with IgE-mediated airway responses and normal monkeys, J. Lab. Clin. Med. 102, 933. Pinckard, R.N., R.S. Farr and D.J. Hanahan, 1979, Physiochemical and functional identity of rabbit platelet activating factor (PAF) released in-vivo during lgE anaphylaxis with PAF released in-vitro from IgE sensitized basophils, J. Immunol. 123, 1947.
Pinckard, R.N., L.M. McManus and D.J. Hanahan. 1982, Chemistry and biology of acetylglycerylether phosphorylcholine (platelet activating factor), Adv. Inflam. Res. 4, 147. Rubin, A.H., L.J. Smith and R. Patterson, 1987, The bronchoconstrictor properties of platelet-activating factor in humans, Am. Rev. Respir. Dis. 136, 1145. Satouchi, K., R.N. Pinckard, L.M. McManus and D.J. Hanahan, 1981, Modification of the polar head group of acetylglycerol ether phosphorylcholine and subsequent effects upon platelets activation, J. Biol. Chem. 256, 4425. Tapley, P.M. and A.W. Murray, 1985, Evidence that treatment of platelets with phorbolester causes proteolytic activation of Ca2+-activated, phospholipid-dependent protein kinase, European J. Biochem. 15l, 419. Thuren, T., J.A. Virtanen, R, Verger and P.K.J, Kinnuene, 1987, Hydrolysis of 1-palmitoyl-2-[6-(pyren-l-yl) hexanoylsn-glycero-3-phospholipids] by phopsholipase A2: effect of polar head-group, Biochem. Biophys. Acta 917, 411. Vargaftig, B.B., J. Lefort, M. Chignard and J. Benveniste, 1980, Platelet activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives, European J. Pharmacol. 65, 185. Wardlaw, A.J., R. Modbel, P. Cromwell and A.B. Kay, 1986, Platelet activating factor: a potent chemotactic and chemokinetic factor for human eosinophils, J. Clin. Invest. 78, 1701.