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Platelet-Activating Factor or a Platelet-Activating Factor Antagonist Decreases Tumor Necrosis Factor-a in the Plasma of Mice Treated with Endotoxin Kim M. Ferguson-Chanowitz, Andrew S. Katocs, Jr., Walter C. Pickett, Jeffrey B. Kaplan, Philip M. Sass, Arnold L. Oronsky, and S. S. Kerwar*
From the Oncology and Immunology Research Section, Medical Research Division, American Cyanamid Company, Lederle Laboratories, Pearl River, New York
Recent studies by Beutler et al. [1] and Tracey et al. [2] have shown that administration ofan antibody directed against tumor necrosis factor (TNFa) protects mice and primates from mortality induced by endotoxin. Additional studies have shown that the administration of TNFa to mice and dogs can induce shock-like lesions [3, 4]. TNFa has also been detected in the plasma of human volunteers administered small amounts of endotoxin [5, 6]. These observations suggest that TNFa plays a key role in the pathogenesis of endotoxin shock. In addition to TNFa, platelet-activating factor (PAF) has been implicated as a key mediator of this disease. Studies have shown that pretreatment of mice or rats with PAF antagonists can prolong the survival of animals treated with endotoxin [7-9]. Recently TNFa was shown to stimulate the synthesis of PAF in human vascular endothelial cells and rat peritoneal macrophages suggesting that TNFa can increase the levels ofPAF in the systemic circulation [10, 11]. In a rat model of bowel necrosis, Sun and Hsueh [12] demonstrated that the intestinal necrosis associated with lipopolysaccharide (LPS) administration can be reproduced by TNFa and that PAF antagonists can prevent the clinical necrosis induced by either TNFa or LPS. In view of the pivotal role of TNFa and PAF in the pathogenesis of murine endotoxin shock, we examined the interrelationship of these key mediators in a murine model. We also investigated the effects of a PAF antagonist (alprazolam) on plasma TNFa of LPS-treated mice.
Received 23 January 1990; revised 3 April 1990. Reprints or correspondence: Dr. S. S. Kerwar, Oncology and Immunology Research Section, Building 60B, Room 109, Lederle Laboratories, Pearl River, NY 10965. * Also a guest investigator at the Rockefeller University, New York City. The Journal of Infectious Diseases 1990;162:1081-1086 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6205-0013$01.00
Materials and Methods BALB/c mice were used throughout these studies. Groups of25-50 mice were injected intraperitoneally with Escherichia coli LPS (0111:B4; Sigma Chemical, St. Louis) at a dose of 50 mg/kg; 1 h later blood was collected in heparinized tubes and chilled to 4°C. Plasma was isolated in the cold and assayed for TNFa using an ELISA assay. Briefly, each well of Immulon II plates (Dynatech, Alexandria, VA) was incubated with 0.2 ml of PBS containing 10 JLg/ml monoclonal murine anti-TNFa. After 16 h at 4°C, the wells were washed extensively with PBS containing 0.05 % Tween 20 and 0.02 % sodium azide (buffer A). To block sites in the well that were still available for binding, the wells were incubated for 2 h at room temperature with 0.2 ml of PBS containing 10% horse serum. The wells were then washed extensively with buffer A. Plasma (0.2 ml diluted in PBS containing 0.05 % Tween 20, 0.02 % sodium azide, and 1% bovine serum albumin [buffer B] was added and the plates were incubated for 6 h at 4°C. After washing with buffer A, 0.2 ml of polyclonal rabbit anti-mouse TNFa (500 units diluted in buffer B; Genzyme, Boston) was added. After incubation at 4°C for 18 h, the wells were washed with buffer A. Alkaline phosphatase-conjugated anti-rabbit IgG (Sigma), 0.2 ml diluted 1:1000 in buffer B, was added to each well. The plates were incubated for 3 h at room temperature, the wells were washed extensively with buffer A, and 0.2 ml ofp-nitrophenyl phosphate (1 mglml) was added. The plates were incubated at room temperature for 15 min, and the amount ofp-nitrophenol generated was measured by ELIDA reader (405 nm; Physica Biomed, New York). Pure recombinant murine TNFa (Genzyme) was used as a standard in each assay. Blank wells contained either normal mouse plasma diluted in buffer B or buffer B alone. Plasma TNFa was also assayed using WEHI 164 cells (gift of Dr. V. Tomazic, University of Maryland, Baltimore). Briefly 8 x 105 cells/ml were suspended in supplemented RPMI-1640 medium containing 10% fetal calf serum and 2 JLg/ml actinomycin; 0.1 ml of the cell suspension was added to microtiter wells. RPMI-1640 medium (0.1 ml), containing various amounts of recombinant murine TNFa (standard) or diluted mouse plasma samples was added to each well. The culture was incubated at 37°C for 24 h in a tissue culture incubator. The medium was removed by aspiration, and 0.1
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When L-platelet-activating factor (PAF) or alprazolam (a PAF antagonist) was administered to lipopolysaccharide (LPS)-treated mice, the level of plasma tumor necrosis factor (TNFa) determined by either ELISA or a cytotoxic assay using WEHI cells was significantly lowered. The inactive stereoisomer, D-PAF, was not effective in lowering plasma TNFa levels in LPS-treated mice. The decrease in plasma TNFa induced by L-PAF or alprazolam was partly reversed by indomethacin. Despite a decrease in plasma TNFa, L-PAF or alprazolam caused an increase in the amount of TNFa mRNA present in the kidneys and the livers of LPS-treated mice, suggesting that a posttranscriptional event leading to the synthesis or release of TNFa was inhibited by these agents.
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Cyanamid (Pearl River, NY). All other reagents were obtained from commercial sources.
Results The effect of various amounts of intraperitoneal LPS on mouse plasma TNFa levels was first examined. Mice treated with high doses (25-50 mg/kg) of LPS developed shocklike symptoms (hypotension, tachycardia, hypothennia, diarrhea) beginning rv2-4 h after LPS administration. Plasma TNFa peaked at an LPS dose of 25-50 mg/kg (figure 1A). When 50 mg/kg was administered, plasma TNFa peaked 60-90 min after LPS treatment (figure lB). Thus the increase in plasma TNFa levels preceded the c1inicallesions associated with LPS treatment. These observations are consistent with others [1-6] that indicate that TNF a is one of the mediators responsible for the lesions associated with endotoxin shock. In vitro studies conducted with alveolar macrophages have shown that PAF, when added to LPS-containing cultures, stimulates the synthesis of TNFa [19]. The effect of L-PAF on plasma levels of TNFa in LPS-treated mice was examined. Contrary to the in vitro observations, the plasma of mice injected with L-PAF contained significantly lower amounts of TNFa (figure 2A). This effect was restricted to the active isomer: Administration of D-PAF (inactive isomer) at doses as high as 80 I-'g/kg had no effect on plasma TNFa (figure 2B). Mice treated with low doses of L-PAF (1-10 I-tg/kg) appeared normal and at these doses, TNFa levels in plasma were significantly lower (figure 2A). At the higher dose (20 I-'g/kg) , their fur was rumed and the mice were prostrate. However, these mice recovered 20 min after PAF administration and appeared completely nonnal. High doses of L-PAF (50-100 I-'g/kg) were lethal to mice; death occurred 2-4 h after administration. Plasma of mice administered L-PAF in the absence of LPS did not contain TNFa (data not shown). Since L-PAF is known to induce the synthesisofprostaglandins, the effect of indomethacin on L-PAF-dependent suppression of plasma TNFa of LPS-treated mice was examined. Indomethacin pretreatment partly reversed the suppressive effects of L-PAF on plasma TNFa (figure 3); however, levels were still significantly lower than those observed with mice treated with LPS alone. These observations suggest that the decrease in TNFa levels due to L-PAF in LPS-treated mice results from two mechanisms, one mediated by prostaglandins and sensitive to indomethacin and the other prostaglandinindependent. In control studies, administration of arachidonic acid, a substrate for prostaglandin synthesis, to LPS-treated mice also caused a decrease in plasma TNFa (figure 3). However, this decrease was completely abolished in mice pretreated with indomethacin, suggesting that it was due to arachidonic acid's conversion in vivo to prostaglandins. Mice treated with arachidonic acid (100 I-'g/kg) appeared completely nonnal. In our laboratories, the LDso for intravenous arachidonic
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ml of crystal violet (0.1 % in ethanol and water, 1:4) was added to each well. After 20 min at room temperature, the crystal violet solution was removed by aspiration and the wells were washed three times with cold water. The wells were dried and 0.1 ml of 95 % methanol was added. The absorbance of each well was measured at 600 nm using an ELIDA reader. The amount of bioactive TNFa in the plasma sample was determined using recombinant murine TNFa as a standard. To determine whether PAF or a PAF antagonist would suppress the levels of plasma TNFa in LPS-treated mice, PAF or alprazolam (a PAF antagonist) was administered intraperitoneally; 1 h later mice were injected intraperitoneally with LPS and then bled 1 h after LPS treatment. The level of plasma TNFa was determined. In some studies, indomethacin (7 mg/kg) was administered intraperitoneally; 30 min later mice were treated with various doses of alprazolam or L-PAF and 1 h after that with LPS. Plasma was collected 1 h after LPS treatment and assayed for TNFa The effects of arachidonic acid on plasma TNFa levels of mice treated with LPS were also examined. In these studies arachidonic acid was administered intravenously at a dose of 100 /Lg/kg. The mice were immediately treated with LPS, and the amount of TNFa in the plasma was determined 1 h later. The effect of indomethacin was ascertained by pretreating these mice with indomethacin (7 mg/kg intraperitoneally) 30 min before the administration of arachidonic acid. Clearance of labeled bovine serum albumin in mice treated with LPS and those treated with LPS and PAF was ascertained. Treated mice were injected intravenously with 12sI-Iabeled bovine serum albumin (1 /LCi; New England Nuclear, Boston) 1 h after LPS and L-PAF treatment. They were bled at 2,4,8, and 16 min after treatment with labeled albumin, and the plasma was assayed for radioactivity. To determine TNFa mRNA levels in kidneys and liver; mice treated with LPS as above were sacrificed 15 min after LPS treatment. The kidneys and the liver were removed and snap-frozen in liquid nitrogen. This time point and tissues were chosen because Ulich et al. [13] found that the level of TNFa mRNA in kidneys and liver of endotoxin-treated rats was high. In addition, TNFa mRNA is relatively unstable and is degraded at 30 min after LPS administration [13]. Total RNA was isolated by homogenizing the tissue in 4 M guanidine isothiocyanate followed by phenol extraction and precipitation with ethanol. Polyadenylate-rich RNA was isolated by chromatography on oligodeoxythymidine cellulose using procedures described by Jacobsen [14]. Polyadenylate-rich RNA (20 /Lg) was fractionated on 1% agarose formaldehyde gels [15] and transferred to Optibind-supported nitrocellulose membranes (Schleicher & Schuell, Keene, NH) according to the method of Southern [16]. Nitrocellulose membranes were hybridized at 65°C for 18 h with a random primer-labeled murine TNFa cDNA [17, 18] in 50% formamide, 5 x SSC (lX SSC is 0.15 MNaCI, 0.015 M sodium citrate' 2 H20), Ix PE (50 mM Tris-CI, pH 7.5, 0.1 % sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrolidone, 0.2% ficoll, and 5 mM EDTA) , and 150 /Lg/ml denatured salmon sperm DNA. After hybridization, filters were washed twice at 65°C in 2x SSC and 0.1% SDS and twice at 65°C in 0.1 x SSC containing 0.1 % SDS. The washed filter was exposed to autoradiography. Alprazolam and other PAF antagonists were provided by the Chemical Research Department, Medical Research Division, American
110 1990;162 (November)
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Platelet-Activating Factor & Plasma TNF
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abrogated by pretreatment with indomethacin, indicating that the decrease was dependent in part on prostaglandin synthesis (figure 4). The concentration of TNFa in plasma was ascertained using an ELISA. To determine whether levels determined by ELISA would correlate with a bioassay, a cytotoxic assay using WEHI cells was established. Plasma samples of LPS-treated mice and those treated with L-PAF or alprazolam were assayed for bioreactive TNFa using WEHI cells. An excellent correlation in plasma TNFa levels was found between the ELISA and the cytotoxic assay (not shown). L-PAF is known to increase vascular permeability, in part by its effect on endothelial cells. To determine whether the
acid in mice is rv50 mglkg. Thus the effects observed with arachidonic acid occurred at doses several orders of magnitude lower than its LDso. Alprazolam, a benzodiazepine tranquilizer, is a potent PAF antagonist. Benzodiazepine receptor antagonists do not block PAF antagonist activity of alprazolam, suggesting that this activity is unrelated to its ability to bind to benzodiazepine receptors [20, 21]. Alprazolam was used as an example of a PAF antagonist, and its effect on plasma TNFa of LPS-treated mice was investigated. Administration at doses of 5-20 mglkg caused a dose-dependent reduction in plasma TNFa of LPStreated mice (figure 4). At 1 mg/kg, the drug had no effect. This decrease in plasma TNFa levels by alprazolam was partly
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Figure 3. Effect of pretreatment with indomethacin (7 mglkg) on plasma tumor necrosis factor (TNF) levels of lipopolysaccharidetreated mice administered L-platelet-activating factor (L-PAF; 20 JLglkg) or arachidonic acid (100 JLg/kg). Lane 1, control; lane 2, arachidonic acid; lane 3, arachidonic acid and indomethacin; lane 4, L-PAF; lane 5, L-PAF and indomethacin; lane 6, indomethacin. For each determination, n = 25. * = significantly different from control (P < .05, Student's t test).
L-PAF-induced decreases in plasma TNFa in LPS-treated mice were due to extravasation of plasma proteins from the systemic circulation, the clearance of radioiodinated bovine serum albumin was determined in mice treated with LPS (50 mg/kg) with and without L-PAF (20 Ilg/kg). The half-life of bovine serum albumin in LPS-treated mice was ""21 min. In mice treated with LPS and PAF, it was ""23 min. These results indicate that the L-PAF-dependent decrease in plasma TNFa in LPS-treated mice is probably not due to the extravasation of this lymphokine into tissues. To determine whether the decreases in plasma TNFa were due to an effect of PAF or alprazolam on TNFa mRNA levels, polyadenylate-rich RNA was isolated from kidneys and liver, subjected to gel electrophoresis, transferred to nitrocellulose membranes, and hybridized with a random primer-labeled murine TNFa cDNA pr:obe (figure 5). Densitometric analysis of the autoradiograph indicated that in kidneys of mice treated with PAF or alprazolam, the levels of TNFa mRNA were ""12 times higher than in kidneys of LPS-treated mice. Results of control hybridization of the filters with actin cDNA probe indicated that equal amounts of polyadenylate RNA were used in these hybridization experiments. Similar to the observations in kidneys, TNFa mRNA levels were higher in livers of mice treated with L-PAF and LPS or alprazolam and LPS compared with levels observed in livers of mice treated with LPS alone (not shown).
Discussion This is the first demonstration to our knowledge that L-PAF in vivo can decrease plasma TNFa levels in mice treated with
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Figure 4. Effect of alprazolam and indomethacin on plasma tumor necrosis factor (TNF) levels of lipopolysaccharide-treated mice. Lane 1, control; lanes 2-5, alprazolam at 20, 10, 5, and 1 mg/kg, respectively; lanes 6-8, indomethacin (7 mg/kg) and alprazolam at 20, 10, and 5 mglkg, respectively. For each determination, n = 35. * = significantly different from control (P < .05, Student's t test). t = significantly different from corresponding group receiving alprazolam alone.
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Figure 5. Northern hybridization analysis with tumor necrosis factor (TNF) or actin cDNA probe of polyadenylate-rich RNA (20 JLg) extracted from kidneys pooled from five lipopolysaccharide (LPS)treated mice administered L-platelet-activating factor (L-PAF; 20 JLg/kg) or alprazolam (20 mg/kg). Lane A, control (no LPS); lane B, LPS and L-PAF; lane C, LPS; lane D, LPS and alprazolam. Arbitrary densitometric units after scanning TNF panel: lane A, 564; lane B, 16,940; lane C, 1330; lane D, 16,583. Data were similar for actin panel.
LPS. Two mechanisms appear to be involved; one mediated by prostaglandins and sensitive to indomethacin and the other prostaglandin-independent. L-PAF is known to induce the synthesis of prostaglandin Ez [22], and exogenous prostaglandin Ez can decrease the amount of TNFa synthesized by LPS-stimulated macrophages [23, 24]. This effect appears to be due to decreases in the levels of TNFa mRNA synthesized in the presence of prostaglandin E z. Arachidonic acid also decreases the amount of plasma TNFa, and this decrease ap-
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described [29]. It is possible that PAF or alprazolam destabilizes this AU-rich sequence or inhibits the AU-specific ribonuclease such that TNFa mRNA degradation is inhibited, leading to its accumulation in tissues. Alternatively, PAF or alprazolam may cause accumulation of TNFa mRNA by stimulating transcription pathways (reviewed in [30]). A sequela to the perturbation of these transcription or posttranscriptional events by either L-PAF or alprazolam would be the net accumulation of TNFa mRNA in tissues. The mechanism(s) by which PAF or alprazolam causes a decrease in plasma TNFa of mice treated with LPS is also not known. We suggest three potential mechanisms to account for this decrease. PAF or alprazolam may induce a translational blockade such that TNFa mRNA is not translated. Studies by Beutler et al. [31] have shown that LPS-stimulated (1 JLg/ml) macrophages isolated from endotoxin-resistant C3H/HeJ mice contain a small but detectable pool of TNFa mRNA that is not translated. It has been suggested that a posttranscriptional blockade prevents the synthesis of TNFa by LPS-stimulated macrophages of C3H/HeJ mice. If PAF or alprazolam were to induce a posttranscriptional blockade of TNFa mRNA translation, plasma TNFa levels would be decreased. A second possible mechanism is related to the clearance of TNFa. PAF or a PAF antagonist may stimulate the clearance of TNFa from the circulation. Beutler et al. [32] have shown that the half-time of TNFa in mice is rv7 min, and a major portion ofthe injected TNFa was detected in the liver and skin of treated mice. If L-PAF or the PAF antagonist were to accelerate the clearance of TNFa in vivo, the levels of circulating TNF a would be expected to decrease, as observed in the present study. Finally, a third mechanism relates to the processing of TNFa. Recent studies conducted by Kreigler et al. [33] indicate that activated monocytes synthesize a precursor form of TNFa with a molecular mass of26 kDa. Pulse chase studies have shown that this form of TNFa (transmembrane TNFa) is cleaved and the biologically active 17-kDa protein is released from the membrane. If PAF or alprazolam were to inhibit the processing of this 26-kDa protein or the release of the 17kDa protein, the levels of TNFa in plasma would be expected to decrease. Although the precise mechanism(s) by which PAF or alprazolam decrease plasma TNFa levels remains to be established, we describe for the first time that these agents could decrease the levels of circulating TNFa in mice treated with LPS. The observation that a PAF antagonist could decrease these levels suggests that these agents may have a dual mechanism of action: PAF antagonism and suppression of TNFa. This may account for the attenuation of the clinical lesions associated with septic shock when LPS-treated mice are treated with a PAF antagonist. Clinical trials with PAF antagonists will determine whether they are useful in the clinical management of human septic shock. If they attenuate the
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pears to be entirely dependent on its conversion in vivo to prostaglandins. A second noteworthy observation is that the PAF antagonist alprazolam could decrease the amount of TNF a in plasma of LPS-treated mice. This decrease, like that induced by PAF, was partly reversed by indomethacin. While alprazolam was used as an example of a PAF antagonist, other novel PAF antagonists synthesized by the Chemical Research Department, American Cyanamid, or reported elsewhere [9] were examined for their effect on TNFcx. These PAF antagonists also were able to decrease the amount of plasma TNFa in endotoxin-treated mice (data not shown). While the structures of these PAF antagonists were different (and, unlike alprazolam, they do not bind to the tissue benzodiazepine receptor), they share the common property of binding to the PAF receptor. Thus we speculate that agents (such as PAF or PAF antagonists) that bind to the PAF receptor of tissues decrease the amount of TNFa synthesized and released into the plasma of endotoxin-treated mice, perhaps by a membranedependent event. The observation that a PAF antagonist can decrease plasma TNFa levels in endotoxin-treated mice has clinical significance. TNFa levels in the plasma of endotoxin-treated volunteers and those suffering from endotoxin shock are high, and TNFa appears to be responsible, at least in part, for the clinical lesions associated with shock. If PAF antagonists were to decrease plasma TNFa and, in addition, block the deleterious effects of L-PAF, they have potential application, in the clinical management of endotoxin shock. The mechanism by which PAF or alprazolam decreases plasm~ TNFa of LPS-treated mice is not entirely established. The amounts of TNFa mRNA present in kidneys and the livers of LPS-treated mice, were increased. This result was unexpected in that decrease in TNFa by PAF or alprazolam was partly reversed by indomethacin (figures 3 and 4), suggesting the involvement of prostaglandins, and prostaglandins are known to decrease TNFa mRNA levels [23, 24]. Since the decrease in TNFa levels in the plasma of LPS-treated mice by PAF and alprazolam was due to two mechanisms, one dependent on prostaglandin synthesis and the other prostaglandinindependent, the increase in TNFa mRNA observed (figure 5) must reflect the net effect of these disparate mechanisms on TNFa mRNA synthesis. Most important, however, despite an apparent increase in TNFa mRNA levels, the amount of TNFa in plasma of LPS-treated mice treated with these agents was decreased. The mechanism(s) by which L-PAF or alprazolam induces an apparent increase in TNFa mRNA in tissues of LPS-treated mice is not known. mRNAs of many lymphokines and protooncogenes contain AU-rich sequences at the 3' untranslated region, and it has been suggested that these sequences induce instability to the message [18, 25-28]. An AU-specific ribonuclease may be responsible for the degradation of these mRNAs, and an assay specific for this ribonuclease has been
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clinical lesions of shock, it would be of interest to determine whether these PAF antagonists decrease TNFa in the plasma of septic shock patients. Acknowledgment We thank A. Cerami and G. Davatelis (Rockefeller University) for providing murine TNFa eDNA-containing plasmid; V. Tomazic (University of Maryland) for the WEHI cells used in these studies; and S. K. Hu of this section for conducting experiments related to the effects of L-PAF and alprazolam on the TNFa receptor of murine peritoneal macrophages and macrophage cell lines. We also thank Marie Black for manuscript preparation.
1. Beutler B, Milsark IW, Cerami A. Passive immunization against cachectin/tumor necrosis factor (TNF) protects mice from lethal effect of endotoxin. Science 1985;229:869-871 2. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature 1987;330:662-664 3. Tracey KJ, Beutler B, Lowry SF, Milsark IW, Cerami A. Shock and tissue injury induced by recombinant human cachectin. Science 1986;234:470-474 4. Tracey KJ, Lowry SF, Fahey T1. Cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog. Surg Gynecol Obstet 1987;164:415-422 5. Michie HR, Manogue K, Spriggs DR, Revaug A, O'Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988;318:1481-1486 6. Michie HR, Spriggs DR, Manogue KR, Sherman ML, Revhaug A, O'Dwyer ST, Arthur A, Dinarello CA, Cerami A, Wolff SM, Kufe OW, Wl1more DW. Thmor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery 1988;104:280-286 7. Terashita Z, Imura Y, Nishikawa K, Sumida S. Is platelet activating factor a mediator of endotoxin shock: Eur J PharmacoI1985;109:257-261 8. Doebber TW, Wu MS, Robbins JC, Choy BM, Chang MN, Shen TY. Platelet activating factor involvement in endotoxin induced hypotension in rats: studies with PAF receptor antagonist, kadsurenone. Biochern Biophys Res Commun 1985;29:799-808 9. Braquet P, Chabrier PE, Mencia-Huerta JM. The promise ofPAF-Acether antagonists. Adv Inflam Res 1988;12:135-149 10. Bussolino F, Camussi G, Baglioni C. Synthesis and release of platelet activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1 alpha. J BioI Chern 1988; 263:11856-11861 11. Camussi G, Bussolino F, Salvidio G, Baglioni C. Thmor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils and vascular endothelial cells to synthesize and release platelet activating factor. J Exp Med 1987;166:1390-1404 12. Sun XM, Hsueh W. Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet activating factor. J Clin Invest 1988; 81:1328-1331
13. Ulich TR, Guo K, del Castillo 1. Endotoxin induced cytokine gene expression in vivo. Am J Pathol 1989;134:11-14 14. Jacobsen A. Purification and fractionation of poly A RNA. Methods Enzymol 1987;152:254-261 15. Goldberg D. Isolation and partial characterization of Drosophila alcohol dehydrogenase gene. Proc Natl Acad Sci USA 1980;77:5794-5799 16. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol BioI 1975;98:503-518 17. Feinberg AP, Vogelstein B. cDNA probes for hybridization. Anal Biochern 1983;132:6-14 18. Caput D, Beutler B, Hartog K, Thayer KR, Shimer-Brown S, Cerami A. Identification of a common nucleotide sequence in the 3' untranslated region of mRNA molecules specifying inflammatory mediators. Proc Natl Acad Sci USA 1986;83:1670-1674 19. Dubois C, Bissonnette E, Rolapleszczynski M. PAF enhances TNF production by alveolar macrophages: prevention by PAP receptor antagonists and lipooxygenase inhibitors. J ImmunoI1989;143:964-970 20. Weber KH, Heuer H. Hetrazepines as antagonists of platelet activating factor. Med Res Rev 1989;9:181-218 21. Griffin K, Hong T, Levy JV. The benzodiazepine receptor ligand Ro 5-4864 and Ro 15-1788 do not block the inhibition of PAF induced platelet aggregation seen with hetrazepine WEB 2086. Biochem Biophys Res Commun 1989;160:263-267 22. Levine L. Platelet activating factor stimulates arachidonic acid metabolism in rat liver cells (C-9 cell line) by a receptor mediated mechanism. Mol Pharmacol 1988;43:793-799 23. Kunkel SL, Wiggins RC, Chensue SW, Larrick 1. Regulation of macrophage tumor necrosis factor production by prostaglandin E2. Biochern Biophys Res Commun 1986;137:404-409 24. Kunkel SL, Spengler M, May MA, Spengler R, Larrick J, Remick D. Prostaglandin E2 regulates macrophage derived tumor necrosis factor gene expression. J BioI Chern 1988;263:5380-5384 25. Shaw G, Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 1986;46:659-667 26. Malter JS. Identification of an AUUUA specific messenger RNA binding protein. Science 1989;246:664-666 27. Brawerman G. mRNA decay: finding the right targets. Cell 1989;57:9-10 28. Kruys V, Marinx 0, Shaw G, Deschamps J, Huez G. Translational blockade imposed by cytokine derived UA rich sequences. Science 1989;245:852-855 29. Beutler B, Thompson P, Keyes J, Hagerty K, Crawford O. Assay of a ribonuclease that preferentially hydrolyzes messenger-RNAs containing cytokine-derived UA-rich instability sequences. Biochem Biophys Res Commun 1988;152:973-980 30. Muegge K, Durum SK. From the cell code to the gene code: cytokines and transcription factors. New Biologist 1989;1:239-247 31. Beutler B, Krochin N, Milsark IW, Leudke C, Cerami A. Control of cachectin (tumor necrosis factor) synthesis: mechanism of endotoxin resistance. Science 1986;232:977-980 32. Beutler BA, Milsark IW, Cerami A. Cachectin/tumor necrosis factor: production, distribution and metabolic fate in vivo. J Immunol 1985;135:3972-3977 33. Kriegler M, Perez C, Defay K, Albert I, Lu SD. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology ofTNF. Cell 1988;53:45-53
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References
1ID 1990;162 (November)
Platelet-Activating Factor or a Platelet-Activating Factor Antagonist Decreases Tumor Necrosis Factor-a in the Plasma of Mice Treated with Endotoxin Kim M. Ferguson-Chanowitz, Andrew S. Katocs, Jr., Walter C. Pickett, Jeffrey B. Kaplan, Philip M. Sass, Arnold L. Oronsky, and S. S. Kerwar*
From the Oncology and Immunology Research Section, Medical Research Division, American Cyanamid Company, Lederle Laboratories, Pearl River, New York
Recent studies by Beutler et al. [1] and Tracey et al. [2] have shown that administration ofan antibody directed against tumor necrosis factor (TNFa) protects mice and primates from mortality induced by endotoxin. Additional studies have shown that the administration of TNFa to mice and dogs can induce shock-like lesions [3, 4]. TNFa has also been detected in the plasma of human volunteers administered small amounts of endotoxin [5, 6]. These observations suggest that TNFa plays a key role in the pathogenesis of endotoxin shock. In addition to TNFa, platelet-activating factor (PAF) has been implicated as a key mediator of this disease. Studies have shown that pretreatment of mice or rats with PAF antagonists can prolong the survival of animals treated with endotoxin [7-9]. Recently TNFa was shown to stimulate the synthesis of PAF in human vascular endothelial cells and rat peritoneal macrophages suggesting that TNFa can increase the levels ofPAF in the systemic circulation [10, 11]. In a rat model of bowel necrosis, Sun and Hsueh [12] demonstrated that the intestinal necrosis associated with lipopolysaccharide (LPS) administration can be reproduced by TNFa and that PAF antagonists can prevent the clinical necrosis induced by either TNFa or LPS. In view of the pivotal role of TNFa and PAF in the pathogenesis of murine endotoxin shock, we examined the interrelationship of these key mediators in a murine model. We also investigated the effects of a PAF antagonist (alprazolam) on plasma TNFa of LPS-treated mice.
Received 23 January 1990; revised 3 April 1990. Reprints or correspondence: Dr. S. S. Kerwar, Oncology and Immunology Research Section, Building 60B, Room 109, Lederle Laboratories, Pearl River, NY 10965. * Also a guest investigator at the Rockefeller University, New York City. The Journal of Infectious Diseases 1990;162:1081-1086 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6205-0013$01.00
Materials and Methods BALB/c mice were used throughout these studies. Groups of25-50 mice were injected intraperitoneally with Escherichia coli LPS (0111:B4; Sigma Chemical, St. Louis) at a dose of 50 mg/kg; 1 h later blood was collected in heparinized tubes and chilled to 4°C. Plasma was isolated in the cold and assayed for TNFa using an ELISA assay. Briefly, each well of Immulon II plates (Dynatech, Alexandria, VA) was incubated with 0.2 ml of PBS containing 10 JLg/ml monoclonal murine anti-TNFa. After 16 h at 4°C, the wells were washed extensively with PBS containing 0.05 % Tween 20 and 0.02 % sodium azide (buffer A). To block sites in the well that were still available for binding, the wells were incubated for 2 h at room temperature with 0.2 ml of PBS containing 10% horse serum. The wells were then washed extensively with buffer A. Plasma (0.2 ml diluted in PBS containing 0.05 % Tween 20, 0.02 % sodium azide, and 1% bovine serum albumin [buffer B] was added and the plates were incubated for 6 h at 4°C. After washing with buffer A, 0.2 ml of polyclonal rabbit anti-mouse TNFa (500 units diluted in buffer B; Genzyme, Boston) was added. After incubation at 4°C for 18 h, the wells were washed with buffer A. Alkaline phosphatase-conjugated anti-rabbit IgG (Sigma), 0.2 ml diluted 1:1000 in buffer B, was added to each well. The plates were incubated for 3 h at room temperature, the wells were washed extensively with buffer A, and 0.2 ml ofp-nitrophenyl phosphate (1 mglml) was added. The plates were incubated at room temperature for 15 min, and the amount ofp-nitrophenol generated was measured by ELIDA reader (405 nm; Physica Biomed, New York). Pure recombinant murine TNFa (Genzyme) was used as a standard in each assay. Blank wells contained either normal mouse plasma diluted in buffer B or buffer B alone. Plasma TNFa was also assayed using WEHI 164 cells (gift of Dr. V. Tomazic, University of Maryland, Baltimore). Briefly 8 x 105 cells/ml were suspended in supplemented RPMI-1640 medium containing 10% fetal calf serum and 2 JLg/ml actinomycin; 0.1 ml of the cell suspension was added to microtiter wells. RPMI-1640 medium (0.1 ml), containing various amounts of recombinant murine TNFa (standard) or diluted mouse plasma samples was added to each well. The culture was incubated at 37°C for 24 h in a tissue culture incubator. The medium was removed by aspiration, and 0.1
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When L-platelet-activating factor (PAF) or alprazolam (a PAF antagonist) was administered to lipopolysaccharide (LPS)-treated mice, the level of plasma tumor necrosis factor (TNFa) determined by either ELISA or a cytotoxic assay using WEHI cells was significantly lowered. The inactive stereoisomer, D-PAF, was not effective in lowering plasma TNFa levels in LPS-treated mice. The decrease in plasma TNFa induced by L-PAF or alprazolam was partly reversed by indomethacin. Despite a decrease in plasma TNFa, L-PAF or alprazolam caused an increase in the amount of TNFa mRNA present in the kidneys and the livers of LPS-treated mice, suggesting that a posttranscriptional event leading to the synthesis or release of TNFa was inhibited by these agents.
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Cyanamid (Pearl River, NY). All other reagents were obtained from commercial sources.
Results The effect of various amounts of intraperitoneal LPS on mouse plasma TNFa levels was first examined. Mice treated with high doses (25-50 mg/kg) of LPS developed shocklike symptoms (hypotension, tachycardia, hypothennia, diarrhea) beginning rv2-4 h after LPS administration. Plasma TNFa peaked at an LPS dose of 25-50 mg/kg (figure 1A). When 50 mg/kg was administered, plasma TNFa peaked 60-90 min after LPS treatment (figure lB). Thus the increase in plasma TNFa levels preceded the c1inicallesions associated with LPS treatment. These observations are consistent with others [1-6] that indicate that TNF a is one of the mediators responsible for the lesions associated with endotoxin shock. In vitro studies conducted with alveolar macrophages have shown that PAF, when added to LPS-containing cultures, stimulates the synthesis of TNFa [19]. The effect of L-PAF on plasma levels of TNFa in LPS-treated mice was examined. Contrary to the in vitro observations, the plasma of mice injected with L-PAF contained significantly lower amounts of TNFa (figure 2A). This effect was restricted to the active isomer: Administration of D-PAF (inactive isomer) at doses as high as 80 I-'g/kg had no effect on plasma TNFa (figure 2B). Mice treated with low doses of L-PAF (1-10 I-tg/kg) appeared normal and at these doses, TNFa levels in plasma were significantly lower (figure 2A). At the higher dose (20 I-'g/kg) , their fur was rumed and the mice were prostrate. However, these mice recovered 20 min after PAF administration and appeared completely nonnal. High doses of L-PAF (50-100 I-'g/kg) were lethal to mice; death occurred 2-4 h after administration. Plasma of mice administered L-PAF in the absence of LPS did not contain TNFa (data not shown). Since L-PAF is known to induce the synthesisofprostaglandins, the effect of indomethacin on L-PAF-dependent suppression of plasma TNFa of LPS-treated mice was examined. Indomethacin pretreatment partly reversed the suppressive effects of L-PAF on plasma TNFa (figure 3); however, levels were still significantly lower than those observed with mice treated with LPS alone. These observations suggest that the decrease in TNFa levels due to L-PAF in LPS-treated mice results from two mechanisms, one mediated by prostaglandins and sensitive to indomethacin and the other prostaglandinindependent. In control studies, administration of arachidonic acid, a substrate for prostaglandin synthesis, to LPS-treated mice also caused a decrease in plasma TNFa (figure 3). However, this decrease was completely abolished in mice pretreated with indomethacin, suggesting that it was due to arachidonic acid's conversion in vivo to prostaglandins. Mice treated with arachidonic acid (100 I-'g/kg) appeared completely nonnal. In our laboratories, the LDso for intravenous arachidonic
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ml of crystal violet (0.1 % in ethanol and water, 1:4) was added to each well. After 20 min at room temperature, the crystal violet solution was removed by aspiration and the wells were washed three times with cold water. The wells were dried and 0.1 ml of 95 % methanol was added. The absorbance of each well was measured at 600 nm using an ELIDA reader. The amount of bioactive TNFa in the plasma sample was determined using recombinant murine TNFa as a standard. To determine whether PAF or a PAF antagonist would suppress the levels of plasma TNFa in LPS-treated mice, PAF or alprazolam (a PAF antagonist) was administered intraperitoneally; 1 h later mice were injected intraperitoneally with LPS and then bled 1 h after LPS treatment. The level of plasma TNFa was determined. In some studies, indomethacin (7 mg/kg) was administered intraperitoneally; 30 min later mice were treated with various doses of alprazolam or L-PAF and 1 h after that with LPS. Plasma was collected 1 h after LPS treatment and assayed for TNFa The effects of arachidonic acid on plasma TNFa levels of mice treated with LPS were also examined. In these studies arachidonic acid was administered intravenously at a dose of 100 /Lg/kg. The mice were immediately treated with LPS, and the amount of TNFa in the plasma was determined 1 h later. The effect of indomethacin was ascertained by pretreating these mice with indomethacin (7 mg/kg intraperitoneally) 30 min before the administration of arachidonic acid. Clearance of labeled bovine serum albumin in mice treated with LPS and those treated with LPS and PAF was ascertained. Treated mice were injected intravenously with 12sI-Iabeled bovine serum albumin (1 /LCi; New England Nuclear, Boston) 1 h after LPS and L-PAF treatment. They were bled at 2,4,8, and 16 min after treatment with labeled albumin, and the plasma was assayed for radioactivity. To determine TNFa mRNA levels in kidneys and liver; mice treated with LPS as above were sacrificed 15 min after LPS treatment. The kidneys and the liver were removed and snap-frozen in liquid nitrogen. This time point and tissues were chosen because Ulich et al. [13] found that the level of TNFa mRNA in kidneys and liver of endotoxin-treated rats was high. In addition, TNFa mRNA is relatively unstable and is degraded at 30 min after LPS administration [13]. Total RNA was isolated by homogenizing the tissue in 4 M guanidine isothiocyanate followed by phenol extraction and precipitation with ethanol. Polyadenylate-rich RNA was isolated by chromatography on oligodeoxythymidine cellulose using procedures described by Jacobsen [14]. Polyadenylate-rich RNA (20 /Lg) was fractionated on 1% agarose formaldehyde gels [15] and transferred to Optibind-supported nitrocellulose membranes (Schleicher & Schuell, Keene, NH) according to the method of Southern [16]. Nitrocellulose membranes were hybridized at 65°C for 18 h with a random primer-labeled murine TNFa cDNA [17, 18] in 50% formamide, 5 x SSC (lX SSC is 0.15 MNaCI, 0.015 M sodium citrate' 2 H20), Ix PE (50 mM Tris-CI, pH 7.5, 0.1 % sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrolidone, 0.2% ficoll, and 5 mM EDTA) , and 150 /Lg/ml denatured salmon sperm DNA. After hybridization, filters were washed twice at 65°C in 2x SSC and 0.1% SDS and twice at 65°C in 0.1 x SSC containing 0.1 % SDS. The washed filter was exposed to autoradiography. Alprazolam and other PAF antagonists were provided by the Chemical Research Department, Medical Research Division, American
110 1990;162 (November)
JIO 1990;162 (November)
Platelet-Activating Factor & Plasma TNF
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abrogated by pretreatment with indomethacin, indicating that the decrease was dependent in part on prostaglandin synthesis (figure 4). The concentration of TNFa in plasma was ascertained using an ELISA. To determine whether levels determined by ELISA would correlate with a bioassay, a cytotoxic assay using WEHI cells was established. Plasma samples of LPS-treated mice and those treated with L-PAF or alprazolam were assayed for bioreactive TNFa using WEHI cells. An excellent correlation in plasma TNFa levels was found between the ELISA and the cytotoxic assay (not shown). L-PAF is known to increase vascular permeability, in part by its effect on endothelial cells. To determine whether the
acid in mice is rv50 mglkg. Thus the effects observed with arachidonic acid occurred at doses several orders of magnitude lower than its LDso. Alprazolam, a benzodiazepine tranquilizer, is a potent PAF antagonist. Benzodiazepine receptor antagonists do not block PAF antagonist activity of alprazolam, suggesting that this activity is unrelated to its ability to bind to benzodiazepine receptors [20, 21]. Alprazolam was used as an example of a PAF antagonist, and its effect on plasma TNFa of LPS-treated mice was investigated. Administration at doses of 5-20 mglkg caused a dose-dependent reduction in plasma TNFa of LPStreated mice (figure 4). At 1 mg/kg, the drug had no effect. This decrease in plasma TNFa levels by alprazolam was partly
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Figure 3. Effect of pretreatment with indomethacin (7 mglkg) on plasma tumor necrosis factor (TNF) levels of lipopolysaccharidetreated mice administered L-platelet-activating factor (L-PAF; 20 JLglkg) or arachidonic acid (100 JLg/kg). Lane 1, control; lane 2, arachidonic acid; lane 3, arachidonic acid and indomethacin; lane 4, L-PAF; lane 5, L-PAF and indomethacin; lane 6, indomethacin. For each determination, n = 25. * = significantly different from control (P < .05, Student's t test).
L-PAF-induced decreases in plasma TNFa in LPS-treated mice were due to extravasation of plasma proteins from the systemic circulation, the clearance of radioiodinated bovine serum albumin was determined in mice treated with LPS (50 mg/kg) with and without L-PAF (20 Ilg/kg). The half-life of bovine serum albumin in LPS-treated mice was ""21 min. In mice treated with LPS and PAF, it was ""23 min. These results indicate that the L-PAF-dependent decrease in plasma TNFa in LPS-treated mice is probably not due to the extravasation of this lymphokine into tissues. To determine whether the decreases in plasma TNFa were due to an effect of PAF or alprazolam on TNFa mRNA levels, polyadenylate-rich RNA was isolated from kidneys and liver, subjected to gel electrophoresis, transferred to nitrocellulose membranes, and hybridized with a random primer-labeled murine TNFa cDNA pr:obe (figure 5). Densitometric analysis of the autoradiograph indicated that in kidneys of mice treated with PAF or alprazolam, the levels of TNFa mRNA were ""12 times higher than in kidneys of LPS-treated mice. Results of control hybridization of the filters with actin cDNA probe indicated that equal amounts of polyadenylate RNA were used in these hybridization experiments. Similar to the observations in kidneys, TNFa mRNA levels were higher in livers of mice treated with L-PAF and LPS or alprazolam and LPS compared with levels observed in livers of mice treated with LPS alone (not shown).
Discussion This is the first demonstration to our knowledge that L-PAF in vivo can decrease plasma TNFa levels in mice treated with
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Figure 4. Effect of alprazolam and indomethacin on plasma tumor necrosis factor (TNF) levels of lipopolysaccharide-treated mice. Lane 1, control; lanes 2-5, alprazolam at 20, 10, 5, and 1 mg/kg, respectively; lanes 6-8, indomethacin (7 mg/kg) and alprazolam at 20, 10, and 5 mglkg, respectively. For each determination, n = 35. * = significantly different from control (P < .05, Student's t test). t = significantly different from corresponding group receiving alprazolam alone.
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Figure 5. Northern hybridization analysis with tumor necrosis factor (TNF) or actin cDNA probe of polyadenylate-rich RNA (20 JLg) extracted from kidneys pooled from five lipopolysaccharide (LPS)treated mice administered L-platelet-activating factor (L-PAF; 20 JLg/kg) or alprazolam (20 mg/kg). Lane A, control (no LPS); lane B, LPS and L-PAF; lane C, LPS; lane D, LPS and alprazolam. Arbitrary densitometric units after scanning TNF panel: lane A, 564; lane B, 16,940; lane C, 1330; lane D, 16,583. Data were similar for actin panel.
LPS. Two mechanisms appear to be involved; one mediated by prostaglandins and sensitive to indomethacin and the other prostaglandin-independent. L-PAF is known to induce the synthesis of prostaglandin Ez [22], and exogenous prostaglandin Ez can decrease the amount of TNFa synthesized by LPS-stimulated macrophages [23, 24]. This effect appears to be due to decreases in the levels of TNFa mRNA synthesized in the presence of prostaglandin E z. Arachidonic acid also decreases the amount of plasma TNFa, and this decrease ap-
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JID 1990;162 (November)
Platelet-Activating Factor & Plasma TNF
described [29]. It is possible that PAF or alprazolam destabilizes this AU-rich sequence or inhibits the AU-specific ribonuclease such that TNFa mRNA degradation is inhibited, leading to its accumulation in tissues. Alternatively, PAF or alprazolam may cause accumulation of TNFa mRNA by stimulating transcription pathways (reviewed in [30]). A sequela to the perturbation of these transcription or posttranscriptional events by either L-PAF or alprazolam would be the net accumulation of TNFa mRNA in tissues. The mechanism(s) by which PAF or alprazolam causes a decrease in plasma TNFa of mice treated with LPS is also not known. We suggest three potential mechanisms to account for this decrease. PAF or alprazolam may induce a translational blockade such that TNFa mRNA is not translated. Studies by Beutler et al. [31] have shown that LPS-stimulated (1 JLg/ml) macrophages isolated from endotoxin-resistant C3H/HeJ mice contain a small but detectable pool of TNFa mRNA that is not translated. It has been suggested that a posttranscriptional blockade prevents the synthesis of TNFa by LPS-stimulated macrophages of C3H/HeJ mice. If PAF or alprazolam were to induce a posttranscriptional blockade of TNFa mRNA translation, plasma TNFa levels would be decreased. A second possible mechanism is related to the clearance of TNFa. PAF or a PAF antagonist may stimulate the clearance of TNFa from the circulation. Beutler et al. [32] have shown that the half-time of TNFa in mice is rv7 min, and a major portion ofthe injected TNFa was detected in the liver and skin of treated mice. If L-PAF or the PAF antagonist were to accelerate the clearance of TNFa in vivo, the levels of circulating TNF a would be expected to decrease, as observed in the present study. Finally, a third mechanism relates to the processing of TNFa. Recent studies conducted by Kreigler et al. [33] indicate that activated monocytes synthesize a precursor form of TNFa with a molecular mass of26 kDa. Pulse chase studies have shown that this form of TNFa (transmembrane TNFa) is cleaved and the biologically active 17-kDa protein is released from the membrane. If PAF or alprazolam were to inhibit the processing of this 26-kDa protein or the release of the 17kDa protein, the levels of TNFa in plasma would be expected to decrease. Although the precise mechanism(s) by which PAF or alprazolam decrease plasma TNFa levels remains to be established, we describe for the first time that these agents could decrease the levels of circulating TNFa in mice treated with LPS. The observation that a PAF antagonist could decrease these levels suggests that these agents may have a dual mechanism of action: PAF antagonism and suppression of TNFa. This may account for the attenuation of the clinical lesions associated with septic shock when LPS-treated mice are treated with a PAF antagonist. Clinical trials with PAF antagonists will determine whether they are useful in the clinical management of human septic shock. If they attenuate the
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pears to be entirely dependent on its conversion in vivo to prostaglandins. A second noteworthy observation is that the PAF antagonist alprazolam could decrease the amount of TNF a in plasma of LPS-treated mice. This decrease, like that induced by PAF, was partly reversed by indomethacin. While alprazolam was used as an example of a PAF antagonist, other novel PAF antagonists synthesized by the Chemical Research Department, American Cyanamid, or reported elsewhere [9] were examined for their effect on TNFcx. These PAF antagonists also were able to decrease the amount of plasma TNFa in endotoxin-treated mice (data not shown). While the structures of these PAF antagonists were different (and, unlike alprazolam, they do not bind to the tissue benzodiazepine receptor), they share the common property of binding to the PAF receptor. Thus we speculate that agents (such as PAF or PAF antagonists) that bind to the PAF receptor of tissues decrease the amount of TNFa synthesized and released into the plasma of endotoxin-treated mice, perhaps by a membranedependent event. The observation that a PAF antagonist can decrease plasma TNFa levels in endotoxin-treated mice has clinical significance. TNFa levels in the plasma of endotoxin-treated volunteers and those suffering from endotoxin shock are high, and TNFa appears to be responsible, at least in part, for the clinical lesions associated with shock. If PAF antagonists were to decrease plasma TNFa and, in addition, block the deleterious effects of L-PAF, they have potential application, in the clinical management of endotoxin shock. The mechanism by which PAF or alprazolam decreases plasm~ TNFa of LPS-treated mice is not entirely established. The amounts of TNFa mRNA present in kidneys and the livers of LPS-treated mice, were increased. This result was unexpected in that decrease in TNFa by PAF or alprazolam was partly reversed by indomethacin (figures 3 and 4), suggesting the involvement of prostaglandins, and prostaglandins are known to decrease TNFa mRNA levels [23, 24]. Since the decrease in TNFa levels in the plasma of LPS-treated mice by PAF and alprazolam was due to two mechanisms, one dependent on prostaglandin synthesis and the other prostaglandinindependent, the increase in TNFa mRNA observed (figure 5) must reflect the net effect of these disparate mechanisms on TNFa mRNA synthesis. Most important, however, despite an apparent increase in TNFa mRNA levels, the amount of TNFa in plasma of LPS-treated mice treated with these agents was decreased. The mechanism(s) by which L-PAF or alprazolam induces an apparent increase in TNFa mRNA in tissues of LPS-treated mice is not known. mRNAs of many lymphokines and protooncogenes contain AU-rich sequences at the 3' untranslated region, and it has been suggested that these sequences induce instability to the message [18, 25-28]. An AU-specific ribonuclease may be responsible for the degradation of these mRNAs, and an assay specific for this ribonuclease has been
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clinical lesions of shock, it would be of interest to determine whether these PAF antagonists decrease TNFa in the plasma of septic shock patients. Acknowledgment We thank A. Cerami and G. Davatelis (Rockefeller University) for providing murine TNFa eDNA-containing plasmid; V. Tomazic (University of Maryland) for the WEHI cells used in these studies; and S. K. Hu of this section for conducting experiments related to the effects of L-PAF and alprazolam on the TNFa receptor of murine peritoneal macrophages and macrophage cell lines. We also thank Marie Black for manuscript preparation.
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References
1ID 1990;162 (November)
