effects of tumor necrosis factor and IL-1. Sensitization and ...

0022-1767/88/1409-2994$02.00/0 THEJOURNALOF IMMUNOLWY Copyright0 1988 by The AmericanAssoclatlon of Immunologists

VOI. 140.2994-2999. No. 9. May 1. 1988 Prlnted in U.S.A.

SENSITIZATION AND DESENSITIZATION TOLETHALEFFECTSOF NECROSISFACTOR AND IL-1' DAVIDWALLACH,'

HELMUT HOLTMANN, HARTMUT ENGELMANN,

AND

TUMOR

YARON NOPHAR

From the Departmentof Virology,The Welzrnann Institute of Science, Rehouot 76100,Israel

BALB/c mice were sensitized to lethal effects of to toxic effects of LPS have shown that LPS toxicity can human rTNF-a and of human r I L l a by simultaneous be increased by various sensitizing agents (4-7). Contreatment with sublethal doses of actinomycin D versely, exposure to a sublethal dose ofLPS results in (Act D) or D-galactosamine (GalN). In contrast, treat-desensitization tothe toxic effects of this agent (reviewed ment with sublethal doses of TNF or I L L themselves in Reference 8). resulted in desensitization of the mice to the lethal In vitro studies on the cytocidal activity of TNF revealed effect of these cytokines: mice injected with TNF or a quite similar pattern.Thus, killing by TNF is markedly IL-1 in the absence of Act D or GalN responded to a potentiated by sensitizing agents, particularly thosethat second injection of TNF or ILL, this time together inhibit thesynthesis RNA of and proteins(9-12); with ActD or GalN, by a significantly delayed death,whereas killing byTNF is decreased, in a process of or even survived. Desensitization developed rapidly (0.5-1.0 h) and abated24 to 48 h postinjection. Each desensitization, when cells are preexposed to TNF (1217).GalN,3which is known to sensitize mice to the deleof the two cytokines induced hyporesponsiveness terious effectsof LPS (7), wasreported in a recent in vivo to its own lethal effectas well as to thatof the other. the of mice to a lethal Injection of TNF or IL-1 at sublethal doses resulted study to also increase vulnerability 8). This sensitizing activity is apparently effect of TNF (1 also in hyporesponsiveness to the lethal effect of mediated by depletion of hepatic UTP and, as a conseLPS on mice primed with bacillus Calmette-Guiirin, an effect which most likelyis mediated by TNF and quence, arrest of RNA synthesis in liver cells (7. 18, 19). RNA synthesis inhibitor Act D, IL-1 produced in those mice in response to theLPS. In the present study, the TNF and IL-1 in combination had an additive effect also known to potentiate the lethal effect of LPS (5).is shown to similarly sensitize mice to TNF. Furthermore, both in lethality and in desensitization of the mice. mice vulnerable These findings suggest that some of the deleteri- both Act D and GalN were found to make ous effectsof TNF and IC1 are modulated by antag-to killing by IL- 1-whose effects oncell function resemonistic mechanisms: mechanisms which be can sup- ble, in many respects, those of TNF. In contrast, protecpressed by sensitizing agents, specifically by agents tion against the lethaleffect is observed on exposure of inhibitingthe synthesis of RNA orprotein:but the mice to either cytokine in the absenceof Act D, or of which, in the absence of such agents, are found to GalN; the protective effect is demonstrated by increased be augmented in response to TNF and I L L , thus survival on subsequent exposure of the mice to those resulting in desensitization. cytokines in the presence of Act D or GalN. These findInitial findings on TNF have drawn attention to a n apparently selective antitumor functionof this cytokine: a necrotic effect on certain transplantable tumors of mice, and cytocidal activity against tumorcells in culture (1).However, more recent studies haverevealed that TNF can also mediate destructive effects on normal tissues. This activity of TNF has been clearly demonstrated in studies showing that the shock and tissue injury that occur in response to bacterial LPS are largely mediated by TNF, whose production is induced by LPS (2, 3).Very little is known about the mechanisms involved in those effects of TNF or on the factors that modulate them. However, some studieson the regulation of the response Received for publication November 12, 1987. Accepted for publication February 2. 1988. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertlsernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported in part by grants from the Minlstry of Sclence, The National Councllfor Research and Development,Israel. and H. H.and H. E. are reclpientsof fellowships from InterYeda. Ltd., Rehovot. from the Minerva Foundatton (FRG). 'To whom correspondence and reprint requests should be addressed.

ings indicate the existence of biologic mechanisms that can antagonize the lethal effects of TNF and IL-1 and may thus help to attenuate the response to those potentially deleterious cytokines. MATERIALSANDMETHODS

Materials. Human rTNF (6 x lo7 U/mg protein) produced by Genentech (South San Francisco, CA) (20). was kindly provided by Dr. G . Adolfof the Boehringer Institute, Vienna, Austria. Human rIL-la. consisting of the 154 carboxy-terminal amino acids of the 271 amino acid human IL-la precursor (21) (3 X lo7 LAF-U/mg protein) produced by Hoffmann La Roche (Nutley. N J ) was a generous gift fromDrs. A. Stern andP. T. Lomedico. By the Limulus amebocyte lysate test (22) the preparation of rIL-1 contained about 1.5.lo-' U of endotoxins/Fg IL-1: in the rTNF preparation, no endotoxin could be detected. LPS from Escherlchta colt serotype 0127:B8. prepared with the phenolicextractionprocedure (23), was obtained from Sigma Chemical Co. (St. Louis, MO).GalN hydrochloride was obtained fromP. Fanstiehl (Waukegon, IL). BCG from Pasteur Vacclns, Marnes-La Coquette, France, Act D from GIBCO (Grand Island, NY), and proteinaseK from Merck (Darmstadt, FRG). Deterrnlnatlon of the lethal and desensttlzlng eflects of TNF. IL-1,and LPS. Unless otherwise specified. BALB/c mlce. 9 to 13wk old, obtalned from HarlanOlac Ltd., Shaw's FarmBicester. UK. were used. TNF, IL-1, BCG, LPS. Act D. and GalN were solubilized in PBS and injected into the animalsin aliquots of 0.5 ml each. Except for Abbreviations used In this paper: GalN. o-galactosamine: Act D. actinomycin D; BCG, bacillus Calmette-Guerin.

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OF TNF AND IL-1

the experiment described in Figure 6.all injections were given i.p. to the lethaleffect of TNF (Fig. 1B). To determine lethality, TNF, IL-1,or LPS were administered alone, Injection of GalN which by itself is not lethal to mice, or 10 min after fnjection of Act D or GalN. In most desensitization results in effective sensitization to the lethal effect of experiments. mice were injected with TNF or IL-1 12h before a challenge following the sameprotocol as in the lethality tests. In all TNF, similar tothat mediated by ActD (18)(see also Figs. experiments, injections to determine lethality were given between 1A and 3). As shown in Figure 3, mice injected with GalN 9.00and 10.00a.m. After treatment. the mice were cmtinuously observed for a period of 72 h to determine the timeof death. Mice were also rendered vulnerableto the lethal effect of IL- 1. Desensitization to the lethal effects of TNF and of ILthat survived 72 h appeared completely normal at thattime: moreover, micethat were observedfor a further week showed no signsof 1. To elucidate the natureof the sensitizing effectsof Act deterioration in their condition. All experiments were performed in D and GalN, we injected TNF or IL-1 in the absence of duplicate andgave qualitativelythe same results. Each experimental point represents data from two animals: their individual survival these agents and checked the response of these mice to time as well as the average survival time for the two mice are a subsequent injection of TNF or IL-1 together with Act presented. D or GalN. As shown in Figures 2 and 3, mice injected

with TNF + Act D 12 h after injection of TNF survived the lethal effect of TNF + Act D for a longer time than Sensitization to the lethal eflects of TNF and IL-1by mice which had not been preexposed to TNF. That proAct D and by GalN. Vulnerability of mice to the lethal tective effect was prominent in mice which were preeffects of TNF and of IL-1 increases substantially after treated with a high dose of TNF (5 &/mouse) but could injection of Act D. A s shown in Figure 1, BALB/c mice clearly be discerned even in mice which were pretreated died within 6 h after i.p. injection of as little as 1 Fg TNF. with as little as 0.02 p g TNF (Fig. 2). Similarly, viability when injected also with 20 pg Act D. Injection of lower of the mice after injection with IL-1 + Act D was prodoses of TNF (0.2or 0.04 &/mouse) also resulted in death longedby prior injection of IL-1 (Fig. 3). Pretreatment of the Act D-treated mice, although more slowly. In con- with IL-1 also increased the ability of mice to survive a trast, themice survived injection of TNF alone, even at a subsequent injection with TNF + Act D, whereas mice dose of 25 pgfmouse. Similarly, human rIL- 1CY was lethal pretreated with TNF showed a n increased ability to surat as little a s 0.2 @/mouse, to mice also treated withAct vive the lethal effect ofIL-I + Act D (Figs. 2 and 3). D, but was not lethal evenat 5 @mouse when injected Injecting mice with TNF and IL- 1 together protected them alone (Fig. 1A). Injection of TNF and IL-1 jointly to mice I I I I , treated with Act D resulted in death more rapidly than >72 when such mice were injected with either TNF or IL-1 alone (rectangular plot, Fig. 1A). Death occurred in Act 6o D-treated mice injected with high doses of TNF rather abruptly; initial signs of deterioration wereobserved only about 0.5 h before death. In contrast, the condition of mice killed by injection of low doses of TNF, and mice whose survival was extended by pretreatment with TNF or IL-1 (see below), showed a prolonged period of deterioration extending over a period of many hours. Act D itself was not lethal to themice, within thetime range of these experiments, at 20 @/mouse, but caused -OL 002 01 0 5 2 5 125 death of some of the mice at doses of 30 wg/mouse and was invariably lethal a t a dose of 40 &/mouse. Mice TNF/IL-I In pretreotment (@/muse) injected with only 10 fig Act D/mouse remained resistant Ftgure 2. Desensitizationin response toTNF and IL-1.Micewere RESULTS

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injected with the indicated amounts ofTNF (0)or IL-1 (0)or with both TNF and IL-1. each at 0.5 &/mouse (0).Twelve hours later they were injected again with TNF (5 pg) and Act D (20 pg). Their survival times thereafter were recorded.

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Ftgure 1. Sensitizing effects of Act D and GalN. A, Dose-response curve for the lethal effect of TNF: survival time after injection of TNF. at the indicated amounts. together with Act D (20pg; O),with GalN (18 mg; r)or alone [A). Also shown is the effect of IL-1 when injected at 0.2 p g ! mouse together with Act D (20 pg: 0):with Act D and TNF (20pg and 0.2 g ,respectively; 0)or at 5 alone (V).B. Dose-response curve for the Sensitizing effectof of Act D: survival time after injection ofAct D. at the indicated amounts. either alone (0) or together with TNF (5pg: 0).

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Ftgure3. Homologous and heterologous desensitization in the response to T N F and to IL-I. Mice were injected with TNF (5fig: U )with IL1 (0.2pg; m) or with PBS (0). Twelve hours later they were again injected either with T N F (2 pg) or with IL-1 (0.4 pg). this time together witheither Act D (20 pg) or GalN ( 1 8 mg). Their survival times thereafter were recorded.

from the lethal effect of TNF + Act D more effectively then preinjection either with TNF or IL-1 alone (rectangular plot in Fig. 2). Mice injected with TNF or IL-1were also protected from the lethal effect of subsequent injection of TNF, or IL-1, in the presence of GalN (Fig. 3). even more effectively than by the cytokines in the presence of Act D. Death due to sensitization with Act D was delayed only by prior treatment with IL-1 or TNF at theconcentrations shown in Figure 3. while death due to sensitization with GalN was actually prevented. GalN-sensitized challenges were therefore chosen to examine the kinetics of the protective effect (Fig. 4). Partial protection from the lethal effect of TNF + GalN, reflected in prolongation of survival time, could be observed as soon as 30 min after injection of TNF or IL-1alone (10pg and 0.4 &/mouse, respectively). One hour afterTNF or IL-1injection, the mice were fully protected, and they remained so for 12 h. Twenty-four hours after pretreatment withTNF, one of the two mice died when injected with TNF + GalN. suggesting some decrease in the protective effect. Forty-eight hours after pretreatment, theprotective effect hadfully disappeared. Lethal and protective effect of TNF and of IL-1 are not mediated by contamfnating LPS. The effectsof TNF and of IL-1 described in the present studyare reminiscent of previously described effects of LPS: Act D and GalN potentiate the lethal effect of LPS (5, 7)(see also Fig. 5). whereas in mice injected with a sublethal dose of LPS hyporesponsiveness to LPS toxicity can be observed (8). We have therefore checked whether the effects of the bacterially derived recombinant preparationsof TNF and IL-1used in the studywere indeed mediated by the cytokines andnot by some contaminating LPS that might be present. By using the Limulus amebocyte lysate test(22) no LPS could be detected in the rTNF preparation. Only trace amountswere found in the IL-1 preparation, much lower than the minimal amounts which exert a lethal effect in the pressure of Act D or induce a decrease in vulnerability of mice to the lethal effect of TNF (about 0.1p g LPSfmouse by i.p. injection, full data not shown). To further exclude the involvement of LPS we subjected the preparations of TNF and of IL-1 to proteolytic digestion, followed byincubation at 100°C.A s shown inFigure 5, both the lethal and the desensitizing effects of TNF and of IL-1 were abolished by that treatment while the lethaleffect of identically treated LPS remained unchanged.

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Figure 5. Exclusion of a role for contaminating LPS in the lethal and desensitizingeffects of the TNF and IL-1 preparations.Mice were injected with TNF (5p g ] . with IL-1 (0.2p g ] . or with PES and 12 h later with LPS alone (10pgfirst bar at the left) or with Act D (20 fig) together with LPS (1 pg), TNF (3 pg) or IL-1 (0.2pg]. Alternatively. where indicated by an asterisk and a stippled bar. the mice were injected with preparations of LPS,TNF or 1L-1 which were subjected to proteolysis by incubation for 1 hat 37°Cwith proteinase K (ata ratio byweight of 5:1 ) and then incubated further for 10 min at 100°C.

TNF and IL-1 -mediated desensitizationto the lethal effect of LPS in BCG primed mfce.To explore the physiologic role of the desensitizing effects of TNF and IL-1 we posed the question whether that desensitization can be effective also in situations in which lethal effects are mediated by endogenously produced TNF. Such a situation is found in mice injected with LPS after theirpriming with BCG. They then produce high quantities of TNF in response tothe LPS injection (1)and arealso extensively sensitized to the lethal effect of LPS (4) which, to large extent, appears tobe mediated by the induced TNF (2.3). As shown in Figure 6, protection from death due to the desensitization effects of TNF or IL-1 could be observed also in this system. Death of the mice was significantly delayed by prior injection of a sublethal dose of TNF and was prevented by injection of IL-1, 12 h before the injection of LPS. DISCUSSION

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Figure4. Kinetics of desensitization in the response toTNF.Mice were injected with TNF ( 1 0 pg: 0) with IL-1 (0.4pg: 8) or with PBS 0.At the indicated times. they were injected with GalN ( 1 8 mg) and 10 min later again with TNF (3p g ) . Their survival tlmes thereafter were recorded.

The findings presented in this study provide information on several aspects of the mechanismswhereby deleterious effects of TNF and of IL-1 are regulated: l) a close relationship between the mechanisms which are involved in mediation of some of these effects by TNF and by IL-1.2)existence of mechanisms which can counteract the deleterious effect of these cytokines, and 3) a major role played by sensitizing agents in determining the intensity of these effects. The close relationship between the mechanisms involved in the lethal effects of TNF and IL-1 is indicated by the fact that the same agents-Act D and GalNsensitize mice to the effect of both cytokines. Further evidence for this relationship lies in thefinding that each of these cytokines can mediate heterologous desensiti-

REGULATION OF LETHAL EFFECTS OF AND TNF

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of the cytocidal effect invitro and the lethal effectvivo in may reflect that cell death in some vital organs may actually beinvolved in themediation of the lethal effects of TNF and IL- It1.may, however, be Only a reflection of similarity between or identity of the regulation mechanisms: those regulating the cytocidal effects of TNF and IL-1, and the mechanisms whereby some other,nonlytic, responses to these cytokines, whichare involved in their lethal effect,are regulated. The marked potentiation of the cytocidal as well a s of the lethaleffects of TNF and IL-1by agents which inhibit RNA/protein synthesis suggests that both effects are antagonized by certain mechanisms which are dependent on protein synthesis.Less prone to mechanistic interpretation, at present, is the desensitizing effect of pretreatment with TNF or IL-1.There is evidence that protection from the cytocidal effect of TNF by pretreatment withIL1 involves desensitization on more than one mechanistic level. IL-1 induces effective and rapid decrease in TNF Pretreatment TNF 11-1 receptors, even though it binds to different, and apparChallenge LPS ently independent, receptors. That decrease is observed within afew minutes of IL-1application and is independSemitizing w n r : BCG (2 w b . Wom -1 LPS) ent of protein synthesis. It is readily reversible, so that Ftgure 6. Desensitization to the lethal effects of LPS in response to within a few hours of removal of IL-1 from pretreated TNF and IL-1. Ten-week-old C57BL/6 mice (Harlan Olac Ltd.) were incells, their TNF receptors are found to be fully restored. jected i.v. with 0.38 mg BCG in 0 . 5 ml PBS. Two weeks later. they were Yet, even after restoration of the receptors, the cells injected 1.p. with TNF ( 1 0 p g ] or with IL-1 (0.4 pg) in 0.5 ml PBS or with PBS alone. Twelve hours later the mice were injected 1.v.with 100 pg LPS whichhave been exposed to IL-1 stillmaintaintheir in 0.5 ml PBS and their survival times thereafter were recorded. resistance to cytolysis by TNF (15,16).Similarly, the zation to the effect of the other. Vulnerability of mice to increase in resistanceof cells to cytolysis by TNF consethe lethal effect of TNF + Act D/GalN is decreased by quent to their pretreatment with TNF itself is found to prior exposure of the mice to IL-1; vulnerability to the be maintained even after free receptors for TNF have effect of IL-1can be decreasedby pretreatment withTNF. been fully restored (14).This resistance seems, therefore, Yet, the additivity of the desensitizing effectsof TNF and to involve, in addition to modulation of receptor level, IL-1 at saturating levels of the latter (Fig. 2). and the some other more slowly reversible changes of a n as yet additivity in their lethal effects (Fig. 1A)suggest that the unclear nature. A similar situation is probably present of TNF or ILmechanisms involved in theresponse toTNF and toIL-1 in vivo: desensitization to the lethal effects may not be identical.Such similarity in the natureof the 1 can be detected as early as 30’ following injection of response. and additivity or synergismin its extent,have these cytokines tomice and is maintained for at least 24 been observed with regard to some other effects of these h. It thus may reflect both rapidly induced and, perhaps, cytokines as well (e.g., cf. Refs. 17, 24-26).Further transient changes,like down-regulation of receptors, and studies will be necessary toreveal the exact mechanisms more slowly reversible changes, maintained long after of these lethal cytokine effects.Detailed examination of clearance of the injected cytokines. The fact that resistthe organs and tissues affected to pinpoint the damage ance to cytolysis and to the lethal effect do not develop caused by TNFand IL-1, as well as study of the extent to in the presence of inhibitors of RNA/protein synthesis which these effectsare altered by sensitizing agents, and may reflect involvement of some inducible proteins in in response to desensitization, may provide relevant in- mediation of that resistance. By this hypothesis, the desensitizing effectsof TNF and IL-1and thesensitizing formation. There is a n intriguing similarity betweenthe way vul- effects of inhibitors of RNA/protein synthesis are acnerability of mice to lethal effects and vulnerability of tually inverse reflectionsof the same phenomenon: preeffects of TNF and cells to cytocidal effects of TNF and of IL-1is found tobe vention of the cytocidal and the lethal modulated. The invitro cytocidal effects of TNF and IL-1 IL-1 by some “protective” or “repair” proteins, produced these cytokines. However it is also possible are extensively potentiated by inhibitors of synthesis of in response to RNA or of protein (9-17, 27). and can be found to syn- that sensitization and desensitization tothe lethal effects ergize in the presence of such inhibitors (17).Similarly, of TNF and IL-1are mediated by mechanisms which are the in vivo lethal effects of TNF and IL-1 are augmented unrelated to each other, and perhaps even function in by agents which inhibit RNA synthesis: Act D which differing cells. Desensitization may reflect a n induced inhibits the synthesis ofRNA in cells, by intercalating refractory state in cells which respond to TNF and IL-1 into their DNA, and GalN which inhibits RNA synthesis, by production of some other mediators of the inflammaprimarily in the liver, by depleting UTP (19).Further- tory response-like leukotrienes, platelet-activating factor more, just like the lethal effect of TNF in mice, its cyto- or PG; mediators whose own effects may be the actual lytic effect in the presence of inhibitors of RNA/protein cause of death. Sensitization, in such case, may reflect synthesis is found to be markedly decreased following inhibition, by protein synthesis blockers, of mechanisms exposure of the cells either toTNF or IL-1in the absence functioning in the cells which respond to those mediators of such inhibitors (12-17).This similarity in regulation or which take part in their inactivation. ” ” “ “

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REGULATION OF LETHAL EFFECTS OF

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The decrease in responsiveness to the lethal effectsof into account that, in vivo, such a situation is probably TNF and IL- 1, after prior exposure to eitherof them, quite exceptional. It may occur only when mechanisms which likely contributes to the tolerance to the toxicity of endo- are directed to "wall-off" the inflammatory region have toxin observed after injection of a sublethal dose of LPS failed. Normally, formation of inflammatory mediators (reviewed in Ref. 8). Indeed, a s shown in this study, like TNF or IL-1 is likely to be restricted, with regard to injection of TNF or IL- 1 at sublethal doses results also inthe pathogenic agents which induce their formation, both decreased responsivenessto the lethal effect of LPS (Fig. in time and space: exposure of a tissue to such agents 6). Endotoxin tolerance can be divided temporally into precedes the induction of the inflammatory mediators 48 h, and imposes that induction in a focal manner-within, two distinct phases:a n early phase, waning within which was suggested to reflect refractoriness of cells, and peripheral to, the afflicted tissue. One would then and a late increasing phase, starting after 48 h and expect the sensitizing effects of the pathogenic agent to persisting for several weeks, which could be related to result in selective mediation of the damaging effects of the production of anti-0-antibodies (28). The kineticsof TNF and IL-1 against the afflicted tissue: and the desendesensitization to the lethal effect of TNF and of its sitization toTNF and to IL- 1,mediated in the surrounding reversal (Fig. 4) place this desensitization in the early pathogen-free tissues, to result in protection of those tissues from the deleterious effects. phase of refractoriness. Evidence has been presented that thisearly refractoriness alsoinvolves a decrease in Both the effects of sensitizing agentsand theexistence responsiveness of mononuclear phagocytes to the effect of mechanisms for desensitization may have a bearing of LPS; a decrease resulting from accumulation of im- on the way TNF can be applied in therapy. Since agents mature, unresponsivemacrophages and perhaps also like Act D, which inhibit RNA and protein synthesis, can from desensitization of the maturemacrophages (29,30). sensitize tumor cells to the cytocidal effect of TNF, they activity of this cytoThis unresponsiveness is reflected in decreased induci- are likely to augment the antitumor bility of effector cytokines of mononuclear phagocytes, kine also invivo. Yet, because these agents are found to including those cytokines which were initially termed increase, just as well, the vulnerability of the whole or"endogenous pyrogen" and arenow known to be equiva- ganism to the harmful effects of TNF, their application lent toIL-1 and TNF (8.30.31).Early endotoxin tolerance in tumor therapy together with TNF may also have adthus involves desensitization on thelevel of the formation verse consequences. Conversely, in view of the desensiof TNF and IL-1 and. as suggested by the present study, tization to the lethal effects of TNF upon pretreatment also desensitization on the level of the effector function with TNF itself, or with IL-1, it is expected that the of these cytokines. The relative contribution of these deleterious effects of TNF willbe less effectively induced differing mechanisms endotoxin to tolerance may depend when thecytokine is given in escalatingdoses. They may on the specific effect of endotoxin which is being exam- also be decreased by injecting IL- 1, before the application ined. It should alsobe noted that not all responses toTNF of TNF. Yet such regimens may turn out to be of lower and IL- 1 are necessarily subject to desensitization.Some therapeutic efficacy, because the decrease in responsiveresponses may actually be potentiated by a prior exposure ness to TNF effects as a result of desensitization may to thesecytokines. Indeed, that seems to be the case with also be reflected in decreased effectivity of its antitumor regard to the local Shwartzman reaction, in which re- activities. Detailed study of the in vivo response to TNF sponse to injection of IL-1 intradermally results not in a is therefore necessary to determine by which protocols decrease, but rather inpotentiation, of the inflammatory for TNF application may sensitizing agents, on the one response to a subsequent i.v. injection of either IL-1 or hand, and desensitization to TNF effects, on the other, be most effectively directed to effect selective destruction LPS (32). Besides a role for some cellular regulatory mechanisms of tumor cells with minimal harm to the host. in determiningthe extent of response to the lethaleffects (18) also of TNF and IL- 1, the present and another study Note added inproof. Recently, M. A. Freudenberg and indicate a major role for extraneoussensitizing agents in C . Galanos have also reported that TNF induces in mice regulation of these effects. It is not known what physio- a state of hyporesponsiveness to the lethal effect which logical agents would exert sensitization to the lethal ef- TNF itself can mediate in thepresence of GalN (34). fects of TNF and IL-1 similar to that mediated by Act D and GalN. Studies on the cytocidal activity of TNF revealed that cells can be sensitized to that activity by certain viruses, similar to the way in which they are Acknowledgments. We thank Dr. G. Adolf of the Boehsensitized with artificial inhibitors of protein synthesis ringer Institute, Vienna, for the gift of rTNF, Drs. A. (33).Perhaps viruses mediate also in vivo a sensitizing Stern andP. T. Lomedico of the Roche Research Center, effect to those kinds of response to TNF and IL-1 that Nutley, N J , for the gift of rIL-la, andDr. Louis Deiss for take part in the lethal effect of these cytokines. 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