Effect of RU 38486 on TNF production and toxicity

Gluracarticoid steroids provide considerable protection against the systemic toxicity or tumor necrosis factor‐α (TNF‐α, cachexin). In animal experiments RU 38486 (mircpristone), a steroid antagonist, increased the synthesis of TNF and sensitized the animals to the cytotoxic action of TNF. As compared to the control and methylprednisolone‐treated groups, mifepristone significantly increased the level of TNF in the serum, liver and spleen or lipopolysaccharide (LPS)‐treated animals. In tissue cultures F.U. 38486 induced the TNF synthesis of mycloid cells and increase the TNF production or genetically modified HeLa cells, which synthesize TNF constitutively. Normal and tumor cell cultures exhibited increased sensitivity toward TNF in the presence of mifepristone.


1, INTRODUCTION
Tumor necrosis factor (TNF) shows a very high spccif'lti:y in its cytotoxicity towards ccrtnin tumor cells [l]. At the same time, TNF is a lymphokinc with powerful immunostimulating effects and causes severe systemic conscqucnccs ut elcvatcd lcvcls [2]. Furthermore, TNF [3.4] and other cytokincs [5] hnvc been also implicated in the pothogcncsis of septic shock, which is one of the most common causes of death in intensive care units today.
Despite advances in technology and in antibiotic therapy, the mortality rate for septic shock remains in excess of 50% [6,7]. Glucocorticoid hormones arc krlown to protect cxgcrimcntal animals very cffcctivcly against the lethal effect of bacterial cndotoxins [a], but the bcncficial influcncc of these hormones in clinical and cxpcrimcntol fomls of septic shock remains a question of d&ate [9-l I]. Antagonists of steroid hormones provide ;t new framework of action not only because they arc of potential clinical USC, but more so bccausc they pcrmh molecular dissection of hormone-dependent proc&scs.
Tier antiglucocorticoid action of mifcpristonc, or RU 38486, has been reviewed elsewhere [12,13] Tumor necrosis factor-a is the primary mediator of the pathogcncsis of cndotoxin and scptic shock [17.18]. Glucocorticoids inhibit LDS.induced TNF production [19] and TNF can stimulate pituitary adrcnocorticotropin secretion, resulting in the rclcasc of corticostcrone [20,:!1]. All thcsc findings suggest an important role of the ncurocndocrinc axis in the secretion of steroid hormones and in the modulation of cytokinc production. On this basis, WC set out to study the influence of RU 38486 on TNF production and toxicity.

MATERIALS AND METHODS
Mnlc NMRI mice (LAW Animal HOW. CiBd6llb, Hungary). 30-35 g in weight. wcrc housed with free xccss IO pcllct food und wiltcr ut all times.

REFULTS
RU 384%d greatly increased the toxicity of hTNF both in tissue cultures and in animals. The highly scnsitivc mouse L919 tumor cells wcrc killed by a 2-3 times lower TNF concentration, if RU 38486 was nlso prcscnt. 3T3 normal mouss fibroblast. which is highly rcsistant to TNF, bccamc sensitive in the prcscncc of the drug (Fig. 1). Intravenous injection of 2 ,ug/lO g body weight of hT.NF. concurrently with 1 pg& body weight of LPS killed 4 of 20 animals (80% survival). but when it wus supplcmcntcd with RU 38486 trcatmcnt (1 mg) ull of the 20 animnb died ( Table I). lnjcction of mcthylprcdnisolonc (2 mg) concurrently with hTNF + LPS + RU rcsultcd in significant protection (70% survival). It Wi\s earlier shown that bacterial cndotoxin potcntiatcs the toxicity of TNF 124). so that the vimultnncous prcscncc of othcrwisc innocuous amounts of LPS nnd TNF triggers lethal shock. In our cxpsrimsnts, TFN + RU gave 70% survival. but if combinsd with LPS trci\tmcnt, the survival dropped to 0% The cffcct of RU 38486 was not lirnitcd to the increased TNF sensitivity of cultured cells and animals. The &err of the drug ott TNF production wns ulso dcmonstratsd in vitro. In M9 cslls, which product hTNF (as B rssult of genetic tmnsformation with the hTNF gcnc). the TNF synthesis mtc was more thun doubled as XI rcvult of RU 38486 treatment (Fig. 2). In mouss P388 myc!;id tumor cells the regulation of the TNF-a gcnc wns unbred. Them cells did not prods TNF without induction by LPS, TNF or phorbol cstcrs. The prcscncc of RU 38486 ;~!onc induced the synthesis of rclntivcly high amounts ofTNF in thcscculturcs. The cffcct of RU 38486 on the rutc of LPS-in&cd TNF production in the swum, liver and spleen is shown in Fig, 3A,B,C, At the time of pc& rcsponsc (one hour after LPS injection) RU 38486 had significtmtly incrwcd the conccntmtion of TNF in the swum. liver und spleen OS compurcd to the control or mcthylprcdnisolonc-trcptcd groups. Mcthylprednisolonc sign& cuntly decreased LPS-induced TNF production and completely abolished the effect of RU 38486. Neither RU 38486 nor mcrhylprcdnisolonc indused TNF production (data not shown).

DISCUSSION
TNF.a (cuchcctin) is il mucrophugedcrivcd peptids hormone rclcoscd in response to different stimuli. in= eluding bacteria! LPS. It has !xcn implicated as u prin. ciptl! mediator in septic and cndotoain shock. Scvcrrrl lines of cvidcncc huvc so f;lr indicatsd that the pituitary-;rdrcnal axis !~trs un important part in regulating the TNF activity. Adrcnalcctomy sensitizes mice to the !c= tha! cffcct of TNF. snd that sensitization is abolished ;lftcr addition of dcxamcthnsonc [251. Furthcrrnorc, in udrcnalcctomizcd or hypophyscctomkd mice the LPSinduced serum TNF conccntmtion rennin5 tit a high lcvcl as compnrcd to normal animals (211. In in vitro models. glucocorticoids inhibit the synthesis of TNF and another important septic shock mediator. IL-I. at t!lc !cvc!s of both transcription and tmnslution [ !9.26,27]. Morcovcr. in cxpcrimcntal animals dcxamcth;lsonc protects against Icth;llity induced by TNF [28]. Our results prove that RU 38486 significzmtly increases the cndot.oxin-induced TNF lcvcls in the strum, liver and spleen and the drug wn induct TNF syrlthcsis in mycloid cells. even in the clbscncc of cndotoxin. Thcsc observations explain tlx shock-sensitizing cffcct of this compound, and suggest the invoivcmcnt of endogcnous glucosorticoids in the regulation of TNF expression. RU 38486 acts on the receptor lcvcl, und thus the increased TNF production seems EO bc mediated via the glucocorticoid receptors. This is corroborated by the observation that mcthylprcdnisolonc complctciy abalishcd the cffcct of RU 38486 on the production of TNF, Our experiments revealed that RU 38436 enhances the toxic effects of hTNF in both cell cultures and cxpcrimcntal nnimals. It increases the expression of the TNF gene in cells producing TNF and simultaneously de= creases the tolcrancc of cells to TNF by interfering with the protcctivc cffsct of cndogcnous glusasorticoids. A recent rcccptor [29] dcmonstrntcd that TNF c~uscs abortion in pregnant mice and destroys embryonic cells in combination with IFN-r. It is conccivablc that. in addition to its action as an antiprogcstcronc, the in= creased susceptibility of the fcttli tissues to cndogcnous TNF may also play o role in the abortogsnic effect of mifcpristonc.
Our results concerning the effect of the new glucocorticoid antagonist on systemic TNF production and toxicity confirmed the important role cndogsnous glucocorticoids play in the control of the immunopathologicol proscsscs caused by high lcvcls of certain lymphokincs.