Clin. exp. Immunol. (1991) 86, 304-3 10

Actinomycin D upregulates lipopolysaccharide induction of macrophage procoagulant expression and tumour necrosis factor-alpha production H. R. WHEELER*, E. J. ROCKETTt, I. CLARK & C. L. GECZY§ *Department of Clinical Oncology, Royal North Shore Hospital, Sydney, IDepartment of Zoology and WCell Biology Division, Australian National University, Canberra and §Heart Research Institute, Sydney, Australia

(Acceptedfor publication 9 May 1991)

SUMMARY

The antitumour antibiotic actinomycin D (Act D) and the aminosugar D-galactosamine both enhance the sensitivity of animals to bacterial lipopolysaccharide (LPS). Lipopolysaccharide stimulates macrophage membrane-bound procoagulant activity (MPCA) and tumour necrosis factor-alpha (TNF-a) production in vitro. We investigated the effects of LPS combined with either Act D or D-galactosamine on procoagulant and TNF-cx production in vitro. Actinomycin D directly induced procoagulant on the malignant monocytoid cell line WEHI 265, and synergized with LPS to enhance MPCA on both WEHI 265 cells and thioglycollate-induced peritoneal exudate macrophages. In the presence of Act D, exudate macrophages expressed procoagulant in response to concentrations of LPS 100000-fold lower than normally required. Pulsing experiments demonstrated that LPS primed these cells within 4 h to respond to Act D, whereas 4 h priming with Act D inhibited subsequent procoagulant induction by LPS. Although its effects on TNF-a production were less intense, low levels of Act D more than doubled TNF-oc produced by LPS-stimulated exudate macrophages. Procoagulant expression and TNF-c production were not always co-ordinately expressed; interferon-gamma (IFN-y) synergized with LPS to enhance both responses but when IFNy was combined with Act D only procoagulant was upregulated. D-galactosamine failed to affect these macrophage responses. Results indicate different in vivo mechanisms of enhancement of LPS toxicity by these two agents. Keywords macrophage procoagulant tumour necrosis factor actinomycin D bacterial lipopolysaccharide

Lipopolysaccharide also stimulates expression of murine membrane-bound procoagulants (MPCA), with tissue factor (TF) and factor VIla-like properties (review by Shands, 1984). Interferon (IFN-y) primes murine macrophages to express high levels of cell surface MPCA in response to low levels of LPS (Moon & Geczy, 1988). Macrophage procoagulants are potent activators of the extrinsic coagulation cascade, and are responsible for fibrin deposition in many cell-mediated immune responses (review by Ryan & Geczy, 1987). Peritoneal macrophages (Robinson, Rapaport & Brown, 1978) and peripheral blood mononuclear cells (Osterud, Olsen & Tindall, 1985) from animals injected with LPS express high levels of procoagulant activity. Furthermore, anti-TF antibodies prevent disseminated intravascular coagulation (DIC) in animals treated with LPS (Warr, Rao & Rapaport, 1990). This is consistent with the major role which macrophage procoagulants are thought to play in DIC and haemorrhagic necrosis associated with endotoxic shock. This study examined the effects of Act D and D-galactosamine on LPS-induced MPCA and TNF-ci production in vitro. Actinomycin D enhanced both procoagulant expression and

INTRODUCTION The sensitivity of mice to the lethal effects of lipopolysaccharide (LPS) is enhanced when either actinomycin D (Act D) or Dgalactosamine is administered together with LPS (Pieroni et al., 1970; Galanos, Freudenberg & Reutter, 1979). The effects of Dgalactosamine are confined to the liver, where depletion of hepatic UTP leads to inhibition of RNA and protein synthesis (Decker & Keppler, 1974). Actinomycin D also inhibits RNA synthesis, but its effects are not confined to hepatocytes (Galbraith & Mellet, 1975). Both these agents have been used to study mechanisms of LPS toxicity (Agarwal & Berry, 1968; Galanos et al., 1979). Products of activated macrophages may mediate the lethal effects of LPS (Freudenberg & Galanos, 1988) and tumour necrosis factor-alpha (TNF-ax) has been implicated as a major contributory factor (Beutler, Milsark & Cerami, 1985). In addition to TNF-Lx, other products related to, or induced by, LPS may be involved in the induction of toxicity by LPS (Rothstein & Schreiber, 1988; Kiener et al., 1988). Correspondence: H. R. Wheeler, Department of Clinical Oncology, Royal North Shore Hospital, Sydney, Australia.

304

Upregulation of macrophage procoagulant and TNF-c by actinomycin D TNF-a secretion by macrophages, implicating these two pathways in LPS lethality. The failure of D-galactosamine to affect these macrophage responses confirms reports that this agent acts via mechanisms which are different to Act D. MATERIALS AND METHODS

Cell preparation

BALB/c or C57B1/6J mice were obtained from the Animal Resources Centre (Willeton, Western Australia) and used when 6-8 weeks of age. Thioglycollate broth (TG, 2 ml; BBL, Microbiology Systems, Baltimore, MD) was injected i.p. 3 days before harvest. The peritoneal cavities were lavaged with 10 ml Dulbecco's modified Eagles medium (DME; GIBCO, Grand Island, NY) containing 388% sodium citrate, and peritoneal exudate cells (PEC) washed twice in DME. Cell suspensions contained approximately 80-850X viable macrophages, as assessed by nigrosine blue exclusion. WEHI 265, a murine monocytoid cell line (derived from an Abelson-induced tumour in a BALB/c mouse), was obtained from the Walter and Eliza Hall Institute, Melbourne, Australia). It was maintained in RPMI 1640 (GIBCO), supplemented with I0"X, heated (56-C, 30 min) fetal calf serum (Flow Laboratories, Irvine, Scotland) in 75 cm2 tissue culture flasks (Nunc, Roskilde, Denmark). Cells were diluted 1:20 v/v (1 -2 x 104/ml) with fresh medium twice a week and were always used 1-2 days after subculturing when in logarithmic growth phase. The WEHI 265 cells became more sensitive to LPS after 10- 15 passages and were discarded. Further details regarding induction of procoagulant activity on these cells are described by Farram et al. (1983). All batches of cells were routinely cultured to test for mycoplasma contamination. The cells (. 90'S viable) were washed twice in serum-free DME before MPCA assays.

Induction of MPCA Conditions (e.g. cell concentrations) for optimal induction of MPCA were those established by Geczy et al. (1983) for TGPEC and by Farram et al. (1983) for WEHI 265 cells. Briefly, TG-PEC (1 5 x 106) or WEHI 265 cells (4 x 105) were cultured together with varying concentrations of LPS (LPS W, Escherichia coli 055:B5; DIFCO Laboratories, Detroit, MI), Act D (Calbiochem Behring Corp., La Jolla, CA), D-galactosamine (Sigma Chemical Co., St Louis, MO), recombinant murine IFN-y or recombinant murine TNF-ac (both E. coli-derived); 100 U IFN-y (bioassay: mouse L cells/EMCV) was equivalent to 10 ng IFN-y and 500 U TNF-at (bioassay: murine L-M cells) was equivalent to 10 ng TNF-ax; gifts from Dr G. Adolf, Boehringer Ingelheim, Vienna, Austria), alone or in combination. Cultures were conducted in a total volume of 0-5 ml serum-free RPMI 1640, in polypropylene tubes (Nunc, Roskilde, Denmark) at 37 C in an atmosphere of 5% CO2 in air. Cells were routinely tested after 16-18 h culture. Timecourses of MPCA induction were conducted by testing cells for fixed time intervals. For pulsing experiments, cells were cultured with LPS or Act D alone and, after 4 h, washed twice in warm DME. They were then resuspended in 0-5 ml RPMI 1640, and the second stimulus added. Membrane-bound procoagulant activity was assessed after a further 14 h culture. At the end of culture, cells were pelleted at 400 g for 10 min, supernatants collected for TNF-a assays and stored at -70 C

305

until tested. Cell pellets were washed twice in DME, resuspended in 0 5 ml RPMI 1640 and tested for MPCA. Only samples containing > 70%, viable cells were assayed. Measurement of MPCA The ability of washed cells to enhance the recalcification time of pooled, citrated, platelet-poor rat plasma was determined using a one-stage recalcification assay, described in detail previously (Moon & Geczy, 1988; Wheeler & Geczy, 1990). The results were expressed as milliunits (mU) of TF by comparing the recalcification times (RT) with a standard curve. The RT of plasma alone was designated as 0 mU and that of 1 jg mouse brain extract as 10 mU. The stimulation index (SI) was calculated as

SI=

mU TF of stimulated cells

mU TF of non-stimulated cells

A stimulation index of 1 7 was considered positive on the basis of observations made in 10 experiments performed by using increasing dilutions of LPS. Measurement of TNF-a production Murine TNF-o was assayed by a method modified from Sheenan, Ruddle & Schreiber (1989). Briefly, 96-well flatbottomed plates (Immulon 11; Dynatech Laboratory, Alexandria, VA) were coated by overnight incubation at 4 C with 0-2 pg per well of TN319: 12, a TNF-specific monoclonal antibody, which had been made up in carbonate buffer (pH 9 6). The plates were then washed six times with phosphate-buffered saline containing 0 050 Tween 20 and again between each subsequent treatment. They were then incubated overnight at 4 C with 100 I1 of a 1 in 5 dilution of the test samples per well in RPMI 1640 supplemented with 10% fetal calf serum, 10 mM penicillin and streptomycin, and 10 mm Hepes (RIO). A standard curve of recombinant murine TNF-ac (Genzyme, Boston, MA) starting at 25 ng/ml and followed by eight doubling dilutions was used. Rabbit anti-murine TNF-oc (100 ml; Genzyme) in R 10 1/750 v/v was added to each well for 2 h at 25 C. The same volume of goat anti-rabbit alkaline phosphatase (Silenus, Victoria, Australia) diluted to 1/ 1000 in RI 0 was added for 2 h at 25 C. Colour was developed using 100 pl per well phosphatase substrate tablets (Sigma, St Louis, MO) dissolved in substrate buffer to a concentration of I mg/ml. The plates were read with a Titertek MC ELISA reader (Flow Laboratories, McLean, VA) at a wavelength of 450 nm with a reference of 620 nm, and the TNF-x in the test samples calculated from the standard curve.

RESULTS Induction of MPCA bY LPS and Act D on TG-PEC and WEHI 265 cells In agreement with earlier studies (Moon & Geczy, 1988), LPS (I .0 piglml) increased basal levels of surface procoagulant activity on TG-PEC approximately six-fold. In contrast, only up to 2 5-fold enhancement was evident on WEHI 265 cells (Fig. 1). Less than 1 0 pig/ml LPS induced only weak procoagulant activity on both cell types. Actinomycin D alone had a negligible effect on TG-PEC, whereas it directly induced MPCA on WEHI 265 cells (an approximately seven-fold increase with 0 25 pg/ml, Fig. 1). Strong synergy was evident on both cell types when LPS

H. R. Wheeler et al.

306 30

40

-

(a)

(b)

30-

20

-

(n 20-

I

I0

I0

0

0

1

0-25 0.5 0 75 1-0 0*05 0 0 0.05 0.I 0*25 Q.5 0.75 1V0 Actinomycin D (pg/ml) Actinomycin D (pLg/mI) Fig. 1. Induction of membrane-bound procoagulant activity (expressed as stimulation index (SI)) by increasing concentrations of actinomycin D, assayed after 18 h culture, either alone (-) or in combination with lipopolysaccharide (1 0 pg/ml (0) on (a) TG-PEC or (b) WEHI 265 cells. Results represent the mean + standard error of the mean of five experiments. 0

14

( a)

12 I0

T

8 U)

H

U)

6

2 0

0*001

0-01

0.1

1-0

10

0

0-001 0.01 0.1 LPS (ng/ml)

I*0

10

LPS (ng/ml) Fig. 2. Induction of membrane-bound procoagulant activity (expressed as stimulation index (SI)) by increasing concentrations of lipopolysaccharide (LPS) assayed after 18 h culture, either alone (-) or in combination with actinomycin D (0 25 Pg/ml) (El) on (a) TGPEC or (b) WEHI 265 cells. Results represent the mean + standard error of the mean of three experiments.

and Act D were combined in culture. The optimal effect on both cell types was with Act D at 0 25 Mg/ml and weaker synergy was observed with increasing doses (Fig. 1). When the optimal concentration of Act D was added to levels of LPS that failed to induce detectable MPCA, the response was amplified such that TG-PEC expressed procoagulant activity with as little as 0 01 ng/ml LPS (SI= 5; Fig. 2a). Cells stimulated with LPS alone required 1-0 pg/ml LPS to achieve similar levels of activity. Thus, the sensitivity of TG-PEC to LPS was increased approximately 100000-fold in the presence of Act D. WEHI 265 cells required higher doses of LPS (10 ng/ml) than TG-PEC to achieve levels of MPCA significantly higher (P=0-02) than those induced by Act D alone (Fig. 2b). Lipopolysaccharide induced low levels of MPCA on TGPEC after 2 h, and the enhancing effect of Act D was evident after 5 h (Fig. 3a). Membrane-bound procoagulant activity induction was optimal after 8 h, and slowly declined. A similar time-course of response was evident with WEHI 265 cells (not shown). Lipopolysaccharide primed both TG-PEC and WEHI 265 cells after 4 h to respond synergistically to Act D added to the culture as the second signal (P < 0-03; Table 1). Membrane-

bound procoagulant activity of LPS-primed TG-PEC reached similar levels to those observed with both stimuli present for the entire culture period. In contrast, WEHI 265 cells primed with LPS and cultured with Act D expressed only 50% of the procoagulant activity obtained when cells were cultured for 18 h with both stimuli. Induction of MPCA by IFN-y, TNF-oc and Act D on TG-PEC IFN-y did not directly induce MPCA on TG-PEC, but it markedly enhanced MPCA induction by Act D (Table 2). As little as 01 U IFN-y/ml approximately doubled the MPCA induced by Act D, and adding 10 U/ml IFN-y induced procoagulant levels similar to those obtained when LPS (1 0 Pg/ ml) was combined with Act D (approx. 15-fold increase above basal levels). Neither TNF-a (up to 500 U/ml) nor TNF-ac combined with Act D activated procoagulant expression (Table 2).

Effect of D-galactosamine and TNF-o on the procoagulant response induced by LPS on TG-PEC D-galactosamine (25-100 mg/ml) did not alter basal procoagu-

307

Upregulation of macrophage procoagulant and TNF-ax by actinomycin D 30

( a)

20 E H U)

L.5 -

z

10

0

5

10

15

20

25

30

0

5

15

10

20

25

Time ( h )

Time ( h )

Fig. 3. Time-course of induction of (a) membrane-bound procoagulant activity (expressed as stimulation index (SI)) by lipopolysaccharide (LPS) (1 0 pg/ml) (0); actinomycin D (Act D) (0-25 pg/ml) (*) or LPS (1 0 pg/ml) plus Act D (0 25 pg/ml) (es) on TG-PEC cells. Results are expressed as SI and are representative of three experiments. (b) Tumour necrosis factor-alpha (TNF-a) production (pg/ml per 106 TG-PEC) by LPS (1 0 pg/ml) (0); Act D (0-1 pg/ml) (O) or LPS (1 0 pg/ml) plus Act D (0 1 pg/ml) (A). Results are expressed as the mean + s.d. of three experiments. Table 1. Membrane-bound procoagulant activity response of TG-PEC and WEHI 265 cells to lipopolysaccharide (LPS) or actinomycin D (Act D) priming

Table 2. Interferon-gamma (IFN-y) but not tumour necrosis factor-alpha (TNF-a) synergize with actinomycin D (Act D) to induce membrane-bound procoagulant activity

SI + s.e.m.t Stimulant

TG-PEC

WEHI 265

18 h incubation LPS* Act Dt LPS +Act D

5-3+0 9 2 0+0 4 14 3 + 5-0

16+0 3 4 7+0 3 19 5+4 4

4 h LPS pulse LPS alone Add Act D

2-0+0-8 17 6+6 9

10-2+2 8

4 h Act D pulse Act D alone Add LPS

1-2+004 29+04

1-4+005

3-7+ 1-4 59+1 7

* Lipopolysaccharide used at 1 0 Mg/ml and Act D at 0-5 pg/ml. t Results expressed as stimulation index (SI)±standard error of mean (s.e.m.) of duplicate assays of four experiments of procoagulant induction. t Analysis of variance between Act D pulse and LPS pulse, P-= 0025.

lant levels (1 6 + 1-1 mU TF/ 105 TG-PEC) and, when combined with LPS, MPCA expression (6-0 + 3 mU TF/105 TG/PEC) was similar to that induced by LPS alone (6-2 + 0-8 mU TF/1 05 TGPEC) in three separate experiments. TNF-x (up to 500 U/ml) failed to enhance the LPS response. Production of TNF-aftom TG-PEC by LPS, IFN-y, Act D andDgalactosamine Lipopolysaccharide (1 0 yg/ml) activated TG-PEC to produce

mu

TF/105 cellst

Stimulant

TG-PEC

TG-PEC+Act D*

RPMI 1640

2 2+1 0

2 1+1 0

01 10 10 100

17+08 18+1 1 25+19 1-7+0-8

32+11 62+26 291+110 31 8+13 7

TNF-a 10 100 500

27+02 28+1 1 22+1 1

22+08 22+1 1 24+05

IFN-yt

* 01 pg/ml Act D was used. t Results expressed as mean+ s.d. of three

experiments. $ Increasing doses of IFN-y or TNF-a in U/ ml. TF, Tissue factor.

TNF-a. The response was evident after 2 h, and remained elevated for a further 22 h (Fig. 3b). Actinomycin D (0-01-0-5 ,g/ml) alone did not induce TNF-a, but low concentrations (0-1-0-25 pg/ml) more than doubled the LPS (1-0 pg/ml) response (P = 0-03; Fig. 3b). Optimal TNF-a levels were evident after 4-12 h culture. At very low doses of Act D (0-05 pg/ml) the synergy was no longer observed. In contrast, D-galactosamine alone did not affect TNF-cx production or significantly alter its induction by LPS (Table 3).

H. R. Wheeler et al.

308 Table 3. Interferon-gamma (IFN-y) and actinomycin D (Act D), but not D-galactosamine (Dgal), synergise with lipopolysaccharide (LPS) to produce tumour necrosis factor-alpha (TNF-a) TNF-a (ng/ml/106 TG-PEC)

Stimulant

Alone

LPS*

Act Dt

04

194

09

01 10 100

05 04 0-2

21 7 18 5 256

ND ND ND

Act DI 0.1

09

43-4

ND

03

37-1

04

RPMI 1640 D-gal:

IFN-y$ 100

* Lipopolysaccharide used at 1 0 pg/ml. t Actinomycin D used at 0 1 jpg/ml. I Increasing doses of D-galactosamine and Act D (pg/ml) or IFN-y (U/ml). Results are representative of three experi-

ments. ND, Not done.

Up to 100 U/ml IFN-y alone failed to induce TNF-a. In marked contrast to the enhanced MPCA expression Act D caused when incubated in combination with IFN-y, this mixture did not stimulate TNF-a production (Table 3). IFN-y and LPS synergized to approximately double TNF-a production. DISCUSSION Macrophages and their products mediate many of the pathophysiological reactions that lead to shock, and ultimately death, in LPS-treated animals (Freudenberg, Keppler & Galanos, 1986). Actinomycin D and D-galactosamine both directly enhance LPS toxicity in animal models (Agarwal & Berry, 1968; Galanos et al., 1979), through different mechanisms. We showed that Act D directly upregulated MPCA and TNF-a production by LPS-treated macrophages, whereas D-galactosamine had no effect. Lipopolysaccharide-induced monocyte TF activates the extrinsic coagulation cascade, triggering DIC in Gram-negative endotoxemia (Warr et al., 1990), and TNF-a induces hypovolemic shock, watery diarrhoea, and a capillary leak syndrome (Remick et al., 1987). We report that Act D elevated the production of these two mediators. Actinomycin D enhanced the expression of macrophage procoagulant in the presence of LPS at LPS concentrations several thousand-fold lower than those effective alone (Fig. 2a). In addition, WEHI 265 cells, normally poorly responsive to LPS (Farram et al., 1983; Geczy et al., 1983), responded directly to optimal concentrations of Act D (0-25 pg/ml; Fig. lb) and these MPCA responses were significantly enhanced by LPS. Although neutralizing antibodies to murine TF are currently unavailable, studies indicate it to be the major procoagulant on murine macrophages (Moon & Geczy, 1988). Optimal procoagulant induction on both cell types occurred after 6-12 h (Fig. 3a). The differences in responses of exudate

(inflammatory) macrophages and the monocytoid tumour cell line may reflect changes in monocytoid cells to a neoplastic phenotype (which may be exaggerated by long-term tissue culture) or differences in the maturation and differentiation states of these two cell types as previously suggested (Edgington et al., 1981; Farram et al., 1983; Lyberg et al., 1983; Geczy & Jones, 1988; Wijermans et al., 1989). Our earlier studies showed that, as for many functions acquired by activated macrophages (review by Hamilton & Adams, 1987), IFN-y-primed exudate macrophages express elevated procoagulant responses to levels of LPS that alone are inactive (Moon & Geczy, 1988). This pathway of procoagulant induction may contribute to the intravascular thrombosis characteristic of the Schwartzman reaction (review by Billiau, 1988; Rapaport & Hjort, 1967; Niemetz, 1972). In contrast to the priming sequence necessary to observe the IFN/LPS synergy, LPS-primed TG-PEC and WEHI 265 cells expressed elevated MPCA in response to Act D whereas Act D-primed cells were unresponsive to LPS (Table 1). Furthermore, IFN-y synergized with Act D to enhance MPCA (Table 2), indicating its capacity to amplify the procoagulant response. Because of its established role in endotoxin shock, we compared some of the parameters of TNF-oc production with induction of procoagulant expression on TG-PEC. Figure 3b shows that although Act D had little direct effect, it increased the TNF-a antigen levels induced with LPS approximately twofold. Production of TNF-ac followed a similar time course to that observed for procoagulant expression (Fig. 3). These experiments compared total TNF-a antigen levels produced under various conditions. The possibility that Act D may regulate the production of TNF-a-binding protein (Seckinger et al., 1990) warrants further investigation. TNF-a and procoagulant were apparently not co-ordinately expressed. Whereas IFN-y increased both TNF-ot antigen levels (from 19-4 to 37-1 ng/ml, Table 3) and MPCA levels by LPSstimulated TG-PEC, it enhanced only MPCA in the presence of Act D (Tables 2 and 3). D-galactosamine also enhances the lethal effects of LPS (Galanos et al., 1979), and these may be mediated by a product of LPS-stimulated macrophages (Freudenberg et al., 1986). In sharp contrast to Act D, D-galactosamine had no potentiating effect on MPCA (not shown) or TNF-a production (Table 3). Dgalactosamine causes depletion of hepatic UTP (Hoffman, Wale & Decker, 1976), and the toxic effects of LPS in D-galactosamine-treated animals can be prevented by uridine (Galanos et al., 1979). In contrast, Act D inhibits DNA-dependent RNA synthesis by binding to guanine residues (de Fraine, Diekman & Gaynor, 1986) and, although taken up by the liver, is excreted unchanged via the biliary system (Galbraith & Mellett, 1975). Dgalactosamine may act either by increasing sensitivity to LPSinduced macrophage products or by impairing hepatic clearance of these products. A number of drugs which intercalate with DNA enhance procoagulant expression (Wheeler & Geczy, 1990). Inflammatory monokine synthesis is apparently highly regulated with multiple signals required to induce certain functions. Low doses of cycloheximide, an inhibitor of protein synthesis, superinduce MPCA in response to LPS or IFN-y (Moon & Geczy, 1988). Gregory, Morrissey & Edgington (1989) reported that adding LPS and cycloheximide to human monocytes induced 5-10 times more TF m-RNA then did LPS alone. Furthermore,

Upregulation of macrophage procoagulant and TNF-L* by actinomycin D cycloheximide and I FN-y increase transcription of the genes for TNF-cx and IL-I by murine TG-PECs (Collart et al., 1986) suggesting control of gene transcription by short-lived repressor proteins. Accordingly, Act D may act by altering the synthesis of regulatory proteins controlling TF and TNF-a transcription. Monocyte TF contributes to the intravascular coagulation occurring in sepsis (review by Lyberg, 1984) and recent studies with neutralizing antibodies have confirmed the role of TF in the coagulopathies associated with septic shock (Warr et al., 1990). We suggest that elevated macrophage procoagulant activity contributes to the potentiating effects of Act D on LPS toxicity in vivo. High levels of TNF-ot, proportional to the degree of intravascular coagulation seen in patients with meningococcal sepsis (Waage, Halstensen & Espevik, 1987), have been reported. Although TNF-ac did not directly influence macrophage procoagulant (Table 2), the prothrombotic response may be amplified by virtue of its capacity to upregulate TF (Nawroth & Stern, 1986) and plasminogen activator inhibitor Type I (van Hinsberg et al., 1988) and to downregulate plasminogen activator and thrombomodulin expression (Naworth & Stern, 1986; Moore, Esmon & Esmon, 1989) by endothelial cells. We propose that the enhanced sensitivity of mice to LPS following Act D treatment is modulated, at least in part, by the increased production of TNF-c and TF which can activate a prothrombotic response. ACKNOWLEDGMENTS This work was supported by grants from the Leo & Jenny Leukaemia & Cancer Foundation, the Cancer Council NSW and The National Health and Medical Research Council of Australia. We thank Dr G. Adolf (Boehringer Ingelheim, Vienna) for gifts of recombinant IFN-y and TNF-a.

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