Endotoxin releases platelet-activating factor from human monocytes in vitro

Immunopharmacology, 20 (1990) 105-113

105

Elsevier I M P H A R 00512

Endotoxin releases platelet-activating factor from human monocytes in vitro H.A. Leaver, J.M. Qu, G. Smith, A. Howie, W.B. Ross and P.L. Yap Blood Transfusion Centre, Department of Pharmacology, Universityof Edinburgh, Edinburgh EH8 9JZ, Scotland (Received 17 January 1990; accepted 21 June 1990)

Abstract: The role ofplatelet-activating factor (PAF) in human endotoxaemia was investigated by incubating human monocytes from 27 subjects with S. minnesota endotoxin in vitro. Monocytes synthesized 1.01 + 0.4 x 10- ~o M to 5.3 + 1.51 x 10 - 9 M PAF after 60 min of incubation with endotoxin at concentrations of 1 × 10 - s M and 4.5 × 10 - 6 M respectively. The endotoxin-stimulated release of PAF was not significantly increased in monocytes cultured on a human endothelial cell monolayer. This rapid release of PAF in response to endotoxin is consistent with a role for monoeyte-derived PAF as a toxic mediator of the acute systemic changes observed in patients with endotoxin-related septic shock. Key words:

Platelet-activating factor; Monocyte; Endotoxin; Septic shock

Introduction

The incidence of septic shock due to infection by gram-negative bacteria is high (Wolff and Bennet, 1974) and treatment with antibiotics may make the situation worse by causing lysis of bacterial cell walls and the release of endotoxin (Shenep and Mogan, 1984). Endotoxin in turn may cause the release of various toxic mediators which result in the clinical effects of endotoxaemia such as hypotension, fever, leucopenia and disseminated intravascular coagulation (Morrison and Ulevitch, 1978; Ulevitch et al., 1989). The primary Correspondence: Dr. H.A. Leaver, Department of Pharmacology, University of Edinburgh, Edinburgh EH8 9JZ, Scotland, U.K. Abbreviations: PAF, platelet-activating factor; acyltransferase, acyl-CoA : l-alkyl-2-1yso-sn-Gro-3-P acyltransferase (EC 2.3.1.63); PMA, phorbol myristoyl acetate; LPS, endotoxin lipopolysaccharide.

mediator (or mediators) ofendotoxaemia remains to be fully identified although the potent activities of platelet-activating factor (PAF) on the vascular, reticuloendothelial, pulmonary, hepatic and cardiovascular systems indicate that PAF mimics many of the in vivo effects of endotoxin (Hannahan, 1986; Chang et al., 1987; Doebber etal., 1985; Vercoletti et al., 1989; Diez etai., 1988; Salari and Walker, 1989; Violi et al., 1987). The administration of picomolar concentrations of PAF is associated with hypotension, changes in vascular permeability, thrombocytopenia and leukopenia in animal models (Hannahan, 1986; Chang et al., 1987; Doebber et al., 1985). An increase in the concentration of circulating PAF has been reported during endotoxaemia in humans and animals. Elevated concentrations of PAF (10 - 8-10 - 9 M) have been detected in the plasma of patients with sepsis (Diez et al., 1988; Innarrea et al., 1985) or cirrhosis (Caramelo et al., 1987)

0162-3109/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

106 and in the plasma of rats experimentally exposed to purified endotoxins from S. enteritidis (Chang et al., 1987) or E. coli 0.111 :B4 (Doebber et al., 1985). The instability of PAF in the circulation, together with problems in the development of specific and sensitive assays for the measurement of PAF, has limited knowledge about the effect of endotoxin on PAF release. A combination of platelet aggregometry and lipid separation and characterization techniques have indicated that several cell types are capable of synthesizing PAF (Hannahan, 1986). Using stimuli such as the calcium ionophore and opsonized particles, these studies have demonstrated the capacity of a variety of human cell types (Billah et al., 1986; Camussi et al., 1988; Oda et al., 1985; Camussi etal., 1983; Bussolino etal., 1988; SanchezCrespo et al., 1987; Leyravaud et al., 1986) to generate P A F in the nanomolar concentration range detected during endotoxaemia (Chang et al., 1987; Doebber etal., 1985; Diez etal., 1988; Innarrea et al., 1985). Furthermore, these reported concentrations of PAF produced lie within the concentration range of PAF which has been reported to activate a range of human cell types (10-6_10-1o M) (Violi et al., 1987; Maziere et al., 1988; Valone and Johnston, 1987; Voelkel et al., 1986; Mossman et al., 1986; Ng and Wong, 1988; Fisher etal., 1988; RolaPieszeynski et al., 1987), and above the picomolar PAF concentrations which enhance the neutrophil oxidative response and endothelial cell adhesion (Ingeham et al., 1987). However, the main cellular sources of PAF in human endotoxaemia have not yet been identified. The monocyte has been postulated to act as a primary responding cell during endotoxaemia (Morrison and Ulevitch, 1978; Ulevitch etal., 1989), and the capacity of the mononuclear phagocyte to produce high levels of PAF may be a functional characteristic of mononuclear cells, as there is evidence that, as cells differentiate towards monocytic functions, their ability to produce PAF increases (Billah et al., 1986; Camussi et al., 1988). Mononuclear phagocytes have been

shown to produce substantial quantities of PAF (10 9-10 6 M per 106 cells) in response to ionophore A23187 (Camussi et al., 1983, 1988) and in response to opsonized particles (Camussi et al., 1983, 1988; Sanchez-Crespo et al., 1987; Doebber et al., 1986). Very little information is available about the endotoxin sensitivity of human monocyte PAF production, although two animal models (alveolar macrophages of guineapigs exposed to endotoxin aerosol; Rylander and Beijer, 1986; and a serial rat monocyte and heart perfusion system; Salari and Walker, 1989) indicate that rodent monocytes respond to endotoxin by releasing PAF. There is increasing evidence for an endotoxin receptor on human monocytes (Morrison and Ulevitch, 1978; Ulevitch et al., 1989). We detected the specific binding of picomolar concentrations of various endotoxins to human monocytes at concentrations which activated monocyte superoxide generation (Williams etal., 1989, 1990). The stimulation with S. minnesota endotoxin was associated with the intracellular formation of inositol trisphosphate (Qu et al., 1989) and the elevation of calcium (Leaver et al., 1989) in human monocytes within one minute after endotoxin stimulation. The initial response to endotoxin is important in the clinical pathology of endotoxaemia, as the cardiovascular effects are seen within 30 min after endotoxin exposure (Hannahan, 1986; Chang et al., 1987; Doebber et al., 1985; Salari and Walker, 1989; Toth and Mikulascheck, 1986). We therefore investigated the effect of endotoxin on the acute release of PAF from human monocytes using a human platelet aggregation assay system (Violi et al., 1987; Valone and Johnston, 1987). We also incubated the monocytes with serum and co-cultured the monocytes with endothelial cells to determine whether there were factors in serum or factors produced by endothelial cells which might synergize with endotoxin in stimulating the acute release of PAF.

107 Materials

and Methods

Preparation of mononuclear cell preparations Venous blood from 27 healthy volunteers was treated with 0.2~o EDTA. Mononuclear phagocytes were purified by centrifugation (600 x g, 40min, 20°C) over a discontinuous Ficoll Hypaque gradient (Lymphocyte Separation Medium, Flow, Irvine, Scotland). The cells were suspended in Hanks buffered salt solution, containing 10- 5 M indomethacin, 2 mM CaC12 and 0.1 ~o BSA at a concentration of 10 6 cells/ml. The proportion of monocytes, estimated using nonspecific esterase staining, was between 16 and 35 ~/o; they were 93-100 ~o viable by Trypan Blue exclusion. Analysis of PAF release from mononuclear cell preparations The human peripheral blood mononuclear cell preparations (1 ml, 10 6 cells) were incubated with endotoxin lipopolysaccharide prepared from Salmonella minnesota (wild type) by the Westphal method (1.8-180 #g/ml, Sigma) or calcium ionophore A23187, 1/~g/ml (Novabiochem, Nottingham, U.K.) at 37°C for 5-90 min. The synthesis of PAF was stopped by addition of 0.2~o EDTA. PAF was immediately extracted using chloroform : methanol. The supernatant was vortex-mixed with methanol (6:4, v : v ) and centrifuged (2000 x g, 20 min) to precipitate protein. Chloroform was added to the supernatant, and the proportion was adjusted to CHC13 :methanol: H20, 1.0:0.95:0.8. The mixture was shaken thoroughly and the chloroform layer was removed for platelet aggregatory analysis after resuspension in 0.2 ml of Hanks buffered salt solution containing 0.25~o BSA. The mean extraction efficiency of PAF, calculated by the addition of the PAF standard to the incubation buffer, chloroform extraction, and assay by human platelet aggregometry, was 22.6 + 10.1~o (n = 12). In experiments involving incubation of monocytes with endotoxin, extraction controls were carried out at the highest concentration of endotoxin (121 nM) which was extracted from incubation buffer. In

ten experiments, no stimulation of human platelet aggregation by the endotoxin extracts was detected. The extraction of ionophore A23187 1/~g/ml was similarly monitored in the 17 experiments in which ionophore was incubated with monocytes. Considerable platelet-aggregating activity was detected in extracts of ionophore A23187 (5.0 + 1.27 units of aggregation, n = 17), compared with a mean aggregation of 18.3 + 4.00 units in monocytes incubated with ionophore (n = 17). Therefore, in each experiment, the aggregation due to ionophore alone was subtracted from the aggregation detected in the supernatant of monocytes incubated with ionophore A23187.

Platelet aggregation assay for the detection of PAF release Venous blood was collected from healthy volunteers using citrate dextrose anticoagulant and centrifuged at 220 x g for 25 min at 4°C. The plasma layer was centrifuged at 1000 x g for 20 min. The pellet was resuspended in calciumfree Hanks buffer supplemented with citrate (5.7mM) dextrose (12.9mM). Platelets were washed in this buffer twice by centrifugation (600 × g, 15 min, 20 ° C). Platelets were resuspended in Hanks buffered salt solution containing 1 x 10 - 5 M indomethacin, 313/~g. ml - 1 creatinine phosphate (Sigma), 153/~g. m l - 1 creatine phosphokinase and 0.25~o BSA. The platelet suspension (300/~1, 3 x 10v cells) was stirred in the cuvette of a two-channel clinical platelet recorder (Malin electronics) and platelet aggregation at 37°C was detected using a Rikadenki Mitsui U K two-channel chart recorder. The test solution (100 #1) was added and the change in optical density over 2-3 min was monitored. Aggregation against standard PAF (10 pg-500 ng, Calbiochem), ADP (50nmol-50mmols) and thrombin (0.01-1 U) was monitored in each experiment. Individual variation was observed in the sensitivity of human platelets to PAF, and platelets which had a high PAF sensitivity were used for aggregometry. A typical PAF dose-response curve was described by the equation y = 4.98(log PAF) + 0.087,

108 where y = units of aggregation, and the concentration of PAF is expressed in ng. ml ~; correlation coefficient 0.84 (7 df), and regression was significant (p < 0.01). The effect of endotoxin on the platelet aggregation assay was investigated in two types of control experiment. The effect of endotoxin (1.21 x 10 5 M), extracted from the incubation buffer, on human platelet aggregation was analysed in 10 platelet preparations, and no platelet aggregation was detected (see extraction procedures for the analysis of PAF release by monocytes above). The effect of 100 ~1 of 121 nM S. minnesota endotoxin, added directly to platelets, was also analysed in 10 platelet preparations. Radioimmunoassay of PAF synthesis The analysis of LPS-stimulated monocyte PAF synthesis by platelet aggregometry was compared with PAF determination by radioimmunoassay, using mononuclear cell preparations from two individuals. The lipid extract of monocytes incubated with S. minnesota endotoxin 1 x 10 8-4.5 x 1 0 - 6 M as described above were analysed by PAF radioimmunoassay using a polyclonal sheep Anti-PAF IgG with high specificity and affinity for PAF and [ 125I]PAF radiotracer, both from N E N Dupont, NEK-062. The sensitivity of the assay was 0.02-0.05 ng and the intra- and inter-assay coefficients of variation were 6.63 + 0.44~o and 6.64 + 0.79~o respectively. Culture of monocytes on an endothelial cell layer This was carried out using a previously described technique (Howie et al., 1988). Human endothelial cells, prepared from human umbilical vein using collagenase (Jaffe et al., 1973), were plated i n t o 2 5 - c m 2 culture flasks coated with 250/lg of human fibronectin (Protein Fractionation Centre, Edinburgh). Endothelial cells were cultured to confluence (for 4-5 d) in Medium 199 containing 20~o human serum, 2 mM L-glutamine, 50 IU/ml penicillin, 50/lg/ml streptomycin, and 150 #g/ml endothelial cell growth supplement from bovine

pituitary (Sigma, Poole, UK). Culture media and supplements were obtained from Flow Laboratories, Irvine, Scotland, and human serum was prepared from healthy volunteers within 2 h of each experiment. The medium was changed at 2-day intervals. When the endothelial cells had reached confluence, medium was removed and cells were washed three times with 2 ml of phosphate-buffered saline. In five experiments, an equal number of non-specific esterase-positive cells (approximately 0.5 x 10 6 cells) were added to the endothelial cell monolayer, and incubated with endothelial cells in serum-free medium supplemented with 1 To bovine serum albumin 96-98~o, Fraction V, 20#g/ml transferrin and 10 #g/ml insulin and 0 nM, 30 nM or 300 nM S. minnesota endotoxin (all from Sigma). The supernatant was removed after 20 h of culture and centrifuged at 1000 rpm for 5 min. The cells were loaded with dichlorofluorescin, and the superoxide-generating activity of these preparations was analysed (Howie et al., 1988). Statistical analysis of data The Wilcoxon matched-pairs test was used to compare the platelet aggregatory activity of lipid extracted from the supernatant of co-cultures compared with the sum of the aggregation detected in extracts of the supernatants of monocytes and of endothelial cells cultured alone. Results are expressed as the means + SEM of n determinations.

Results

Characteristics of the human platelet aggregatory response to PAF and the release of human monocyte PAF activity In the human platelet assay, the aggregatory response was proportional to the loglo of PAF concentration, between PAF concentrations of 1 nM and 250 nM, and the lowest level of detection of the assay was 0.1 nM (Fig. 1). The lipid extract of the supernatant of human monocytes incubated with calcium ionophore stimulated platelet aggre-

109

30t t Units of Aggregation

•

5

50

PAF-acether concentration

500 nmol/I

Fig. 1. The sensitivity of human platelets to standard PAF solutions. The effects of 100#1 of PAF standard 0.5-500 nmol/l (i.e. 0.5-500 pmol PAF) (O) and PAF standard 0.5-500 nM ( 0 ) in incubation buffer, extracted into chloroform. The PAF or PAF-containing lipid extract was dried under nitrogen and resuspended in 100 #1 buffer, and 0.5-500 pmol of PAF were added to 500/~1 of stirred platelet suspension in a Malin electronics platelet aggregation recorder. 100 #1 of standard solutions of PAF 18.4 nM and thrombin 0.56 U/ml caused an aggregation of 5 units. Results are the means of triplicate determinations + SEM.

gation in a time- and dose-dependent manner characteristic of phagocyte PAF release (Billah etal., 1986; Camussi etal., 1983, 1988; Oda etal., 1985; Bussolino etal., 1988; SanchezCrespo et al., 1987; Leyravaud et al., 1989; Sturk et al., 1986) (data not shown). The incubation of monocytes with calcium ionophore A23187 1/~g/ml resulted in the release of a lipophilic factor which significantly stimulated aggregation (p<0.01, n = 17) with maximal release 3.02 + 0.426 x 10-7 M PAF per 106 leucocytes (n = 15) occurring after 30 min of incubation. In these experiments, the stimulation of platelet aggregation due to extracted ionophore was subtracted from the aggregatory activity detected in the lipid extract of monocytes incubated with ionophore (see Methods). No PAF activity was detected in the supernatants of monocytes incubated in the absence of ionophore (21 determinations in monocytes from 17 indi-

viduals), using the platelet aggregation assay. The PAF radioimmunoassay indicated that 2.45 × 1 0 - l l M PAF was released by unstimulated monocytes (see below). The effect of S. minnesota endotoxin on the release of PAF activity from human monocytes The basal release of PAF by human monocytes was analysed by determining the platelet aggregatory activity of the chloroform-methanol extract of the supernatant of monocytes incubated for 5-90 min. The concentration of the lipophilic platelet-aggregating factor released by human monocytes under these conditions was below the level of detection of our assay ( < 0.1 nM of PAF per 106 monocytes, n = 16) (see Fig. 2). The incubation of human monocytes with S. minnesota endotoxin (1 × 10-8-1 x 10 -5 M) significantly stimulated the release of plateletaggregating activity in the supernatant of monocytes (p < 0.01) (Fig. 2). The platelet aggregation 0

r--

1.21nM

12.1nM

~

~

~

~

121 nM

S. minnesota endotoxin

f

S

StandardPAF-acether

0.105 nM 1.05r i M /

1

1/

Fig. 2. The effects of S. minnesota endotoxin on mononuclear cell release of PAF detected by platelet aggregation. Monocytes from three individuals were incubated for 60 min with 0-121 nM S. minnesota endotoxin. Polar lipid was extracted from the monocyte supernatants using chloroform: methanol extraction (Camussi et al., 1983). The extract was dried under nitrogen, resuspended in 100 #1 buffer and added to 500 #1 of stirred platelet suspension in a Malin electronics platelet aggregation recorder. Standard PAF 0.1-10 nM and thrombin 0.5-5 U/ml in 100 #1 buffer were added to each platelet preparation. Thrombin 0.5 U/ml caused an aggregation of 5.6-6.4 units of aggregation in these platelet preparations. S. minnesota endotoxin 0-121 nM had no effect on platelet aggregation.

110 was not due to endotoxin co-extracted with PAF, as lipid extracts of 121 nM of S. minnesota endotoxin in incubation buffer did not elicit platelet activation in 10 platelet preparations, and S. minnesota endotoxin added directly to human platelets (see Methods) did not significantly stimulate platelet aggregation. The kinetics of human platelet aggregation elicited by endotoxin-stimulated monocytes resembled those of standard PAF, and the aggregatory response was calibrated using the aggregation response curve of standard PAF. The maximum release of plateletactivating factor by human monocytes incubated with S. minnesota endotoxin was 5.3 + 1.51 × 10 9 M (n = 16), which was observed at an endotoxin concentration of 4.5 x 10- 6 M. The minimum release of PAF detectedwas 1.01 + 0.4 x 10 ~°M (n -- 14),which was released at an endotoxin concentration of 1 x 1 0 - 8 M . The dose-dependent endotoxin stimulation of P A F synthesis in mononuclear cell preparations was confirmed using P A F radioimmunoassay. Monocyte samples from two individuals were incubated with 0-4.5 x 10 6M S. minnesota endotoxin (n = 44 samples), under the conditions used for the aggregation assay, and PAF was extracted and analysed by radioimmunoassay. The basal release of PAF was 2.45 x 10 ' l M after 60min incubation, and incubation with endotoxin (1 x 10- 8-4.5 x 10 - 6 M) significantly (p < 0.05) stimulated P A F production by monocytes. In these two individuals, the mean PAF release increased 4-fold to 9.69 x 1 0 - " M after incubation in the presence of the lower concentration (10- 8 M) of endotoxin, and a 20-fold increase to 5.3 x 10-lO M P A F was detected after 60 min incubation with 4.5 x 10-6 M endotoxin. The effect of human serum and of cell culture on a human endothelial cell monolayer on the release of PAF activity by human monocytes

The effect of human serum and monocyte-endothelial cell contact was investigated in experi-

ments in which monocyte preparations from five donors were cultured on a human endothelial cell monolayer for 20 h in human serum. The release of platelet-aggregatory activity by the co-cultures was compared with that of monocytes or endothelial cells alone. Endothelial cell preparations cultured alone released platelet-aggregatory activity at concentrations which lay at the lower level of detection of the assay (0.83 + 0.31 aggregation units in 6 endothelial cell incubations), and no significant effect of S. minnesota endotoxin (p > 0.05) was observed on the acute 30 min release of PAF by endothelial cell cultures. The co-culture of monocytes with human endothelial cells did not significantly increase the synthesis of P A F in cultures incubated with endotoxin (3.3 x 10-7 M), compared with the PAF synthesis detected in cultures of monocytes and endothelial cells alone (p > 0.05).

Discussion

The primary mediator(s) of the acute effects of endotoxin have not been conclusively identified. However, it is likely that such mediator(s) would be rapidly released and have potent vasoactive properties. There are many similarities in the effects of endotoxin and PAF. The experimental administration of endotoxin or PAF can cause acute circulatory failure, leading to organ failure and death (Morrison and Ulevitch, 1978; Hannahan, 1986; Chang et al., 1987; Doebber et al., 1985; Salari and Walker, 1989; Toth and Mikulascheck, 1986). Recent pharmacological studies show that selective PAF antagonists inhibit these effects (Hannahan, 1986; Chang etal., 1987; Doebber etal., 1986; SanchezCrespo et al., 1987; Voelkel et al., 1986). Although these observations strongly suggest that P A F is involved in the pathogenesis of endotoxic shock, only a few reports have documented the release of P A F from endotoxin-challenged cells (Salari and Walker, 1989; Rylander and Beijer, 1986; Hwang, 1988). In this communication, we report that human

111 mononuclear cells respond to endotoxin by releasing PAF during short-term (60 min) incubations in a dose-dependent response. Endotoxin-stimulated synthesis of PAF was detected using two different techniques, platelet aggregometry and PAF radioimmunoassay. The concentration of PAF which was released by endotoxin-stimulated human monocytes (up to 5.3 nM per 106 monocytes) was comparable with the concentrations of PAF which have been detected in the plasma of endotoxaemic subjects (Chang et al., 1987; Doebber et al., 1985; Diez et al., 1988; Innarrea et al., 1985). There is much evidence that monocytes are the cells responsible for PAF release in mononuclear cell preparations (Camussi et al., 1983; Hannahan, 1986; SanchezCrespo et al., 1987), and this was confirmed in two preliminary experiments using adherencepurified monocytes (data not shown). Mononuclear cell preparations were used to maximize monocyte yield in our experiments. Although only a proportion of PAF synthesized by phagocytes is released (Palmantier et al., 1989; Camussi et al., 1989), the effects of cell-tocell contact on the transfer of cell-associated PAF has not been reported. The activities of released PAF are especially relevant to the pathogenesis of endotoxaemia, in which a range of effects on the cardiovascular system are distant from the site of endotoxin challenge. A paracrine action of PAF released from monocytes is supported by the elegant experiments of Salari and Walker (1989), in which the superfusion medium from endotoxinchallenged monocytes produced greater cardiac effects than the addition of endotoxin-stimulated monocytes into the heart. The calcium-sensitive release of PAF activity which we observed during ionophore stimulation of human monocytes resembled PAF synthesis reported in plastic-adherent monocytes and in other phagocytic cells stimulated by the calcium ionophore A23187 (Billah et al., 1986; Camussi etal., 1988; Oda etal., 1985; Camussi etal., 1983; Bussolino etal., 1988; Sanchez-Crespo et al., 1987; Leyravaud et al., 1989; Sturk et al., 1986). The control of PAF release by activation

of acetyltransferase sensitive to a calcium-dependent protein kinase and stimulated phosphoinositide metabolism has been demonstrated in neutrophils and macrophages (Hannahan, 1986; Sanchez-Crespo et al., 1987; Palmantier etal., 1989). Our experiments indicate that S. minnesota endotoxin may act via such a pathway. The kinetics of endotoxin stimulation of PAF release which we report in this communication are compatible with the changes in intracellular calcium (Leaver et al., 1989) and polyphosphoinositide metabolism (Qu et al., 1989) which we detected during stimulation of human monocytes with S. minnesota endotoxin. A substantial proportion of the mononuclear phagocyte population responding to endotoxin will be the resident tissue macrophages adhering to endothelial cells, and these endothelial cells may either influence mononuclear cell responses (Vercoletti et al., 1989; Howie et al., 1988), or contribute to the pool of released PAF directly (Hannahan, 1986; Bussolino et al., 1988). We observed that the endotoxin-stimulated oxidative activity of human monocytes cultured for 20 h on a human endothelial cells monolayer was significantly (400-500~o) enhanced by co-culture (Howie et al., 1988). However, under the same experimental conditions, the endotoxin-stimulated short-term (60 min) release of PAF by co-cultured human monocytes was not significantly different from PAF release of monocytes incubated with endotoxin alone. In conclusion, S. minnesota endotoxin stimulated the synthesis of PAF from human monocytes. This PAF was capable of directly stimulating the aggregation of human platelets. These experiments indicate that the mononuclear phagocyte acts as a primary responding cell producing PAF in response to endotoxin and suggest that the monocyte may contribute substantially to the PAF released during human endotoxaemia. These experiments also suggest sites of action at which therapeutic PAF antagonists may influence human endotoxaemia, and, together with other studies (Ulevitch et al., 1989; Salari and Walker, 1989; Billah et al., 1986; Camussi et al., 1988;

112 R y l a n d e r a n d Beijer, 1986; W i l l i a m s et al., 1989, 1990), o u r r e s u l t s i n d i c a t e t h a t m o n o n u c l e a r res p o n s e s to e n d o t o x i n in v i t r o m a y give i m p o r t a n t i n f o r m a t i o n a b o u t t h e b i o l o g i c a l activities o f P A F a n t a g o n i s t s in e n d o t o x a e m i a .

Acknowledgements W e a r e v e r y g r a t e f u l to t h e S c o t t i s h H o m e a n d Health Department (Grant K/MRS/50/C874) a n d t h e M e l v i l l e T r u s t for s u p p o r t i n g this s t u d y , to B o b J o n e s o f t h e D e p a r t m e n t o f P h a r m a c o l o g y , U n i v e r s i t y o f E d i n b u r g h , for v a l i d a t i o n a n a l y s i s , a n d S a n d r a E a g l e s o n for t y p i n g t h e m a n u s c r i p t .

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