The Paradoxical Effect of Tumor Necrosis Factor Alpha (TNF-a) in Endotoxin-Induced Uveitis

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1 The Prdoxicl Effect of Tumor Necrosis Fctor Alph (TNF-) in Endotoxin-Induced Uveitis Louis Ksner, Chi-Cho Chn, Scott M. Whitcup, nd I gl Gery Purpose. To investigte the role of TNF- in endotoxin-induced uveitis (EIU) in mice. Methods. To neutrlize TNF- ctivity, mice were pretreted with either repeted injections of this cytokine or single injection of ntibody ginst it. The mice were then injected intrperitonelly with 5 /ug endotoxin, to induce lethl septic shock, or into the footpd with 2 ^g to induce EIU. Results. Although both pretretments conferred protection ginst the systemic toxic effects of LPS, TNF-resistnt mice nd mice treted with nti-tnf- ntibody demonstrted n excerbtion of EIU when compred to control nimls. Conclusions. Unlike its pprent prticiption in the systemic effect of endotoxin, TNF- is not directly involved in the pthogenesis of EIU nd my even protect ginst the inflmmtory processes of this disese. Invest Ophthlmol Vis Sci 1993;34: umor necrosis fctor lph (TNF-), 17-kD peptide cytokine, plys role in vriety of inflmmtory processes. Much work hs been reported recently in ttempts to define the function of TNF- in the process of bcteril sepsis, in which it seems to be prominently involved. Infusion of TNF- in vivo will cuse systemic symptoms mimicking septic shock. 1 Experimentl models of grm-negtive septic shock, including infusion of live bcteri 2 or bcteril lipopolyscchride (LPS), 3 ' 4 hve been shown to produce shrp elevtions in serum TNF- levels, nd ntibodies directed ginst TNF- re protective ginst the mortlity induced by mny, but not ll, of these models. 2 ' 5 Animls cn be mde resistnt to TNF- by dministrtion of multiple low doses of the cytokine over 5- to 6-dy period. 6 * 7 These nimls re then likewise protected ginst septic shock, lthough this TNF-resistnt stte does not lst beyond 16 dys fter the lst TNF- dose. Endotoxin-induced uveitis (EIU) is n cute, trnsient, inflmmtory response loclized to the eye tht peks 24 hours fter the dministrtion of single sublethl dose of LPS to n experimentl niml. Al- From the Uibnmlory of Immunology, Ntionl Eye Institute, Ntionl Institutes of Helth, liethesd, Mrylnd. Submitted for publiction October 12, 1992; ccepted Februry 17, Proprietry interest ctegory: N. Reprint requests: Igl Gery, Ph.D., Ntionl Eye Institute, Ntionl Institutes of Helth, Building 1, Room IN28, liethesd, MD though first produced in the Lewis rt, EIU cn, in vrying intensities, be induced in other rt strins, 8 s well s in mice. 9 While in the Lewis rt, EIU is chrcterized histologiclly by cute inflmmtion loclized to the nterior chmber; in the mouse, the nterior inflmmtion is usully less severe, nd prominent collection of neutrophils develops in the vitreous round the retinl vessels t the optic nerve hed. EIU is model for cute nterior uveitis nd is used in studies exmining both the pthophysiology nd potentil tretment of this disese. 1 "" 13 Despite numerous studies, however, the pthogenic mechnism of EIU remins uncler. Becuse TNF- hs been clerly implicted s meditor in LPS-relted processes, its role in EIU hs come under question. Recently, we were ble to reduce significntly the inflmmtory response in EIU through pretretment of rts with chlorpromzine, 14 phenothizine with number of immunomodultory effects, including the inhibition of TNF- relese from mcrophges. 15 A rise in both queous nd serum TNF- levels hs been mesured during EIU induction; 16 however, in nother study, ttempts to block the development of EIU with nti-tnf- ntibodies proved unsuccessful (R. Hoekzem, personl communiction). Although TNF- is cpble of inducing n inflmmtory rection when injected directly into the vitreous of rbbit eyes, the chrcter of the inflmmtion is primrily monocytic nd lymphocytic, in coninvestigtive Ophthlmology & Visul Science, September 1993, Vol. 34, No. 1 Copyright Assocition for Reserch in Vision nd Ophthlmology 2911

2 2912 Investigtive Ophthlmology & Visul Science, September 1993, Vol. 34, No. 1 trst with the neutrophilic response chrcteristic of both EIU 8-9 nd intrvitrel LPS. 17 To investigte further the role of TNF- in EIU, we exmined the effect of both nti-tnf- ntibodies nd TNF resistnce, chieved by repeted injections of subpthogenic doses of this cytokine, 7 on the development of EIU in the mouse. The mouse ws chosen over the rt becuse its smller size enbled us to minimize the required dosge of ntibody nd cytokine. We were ble to confirm the bioctivity of the TNF- nd nti-tnf- ntibody tretments by demonstrting their protective ctivity ginst LPS-induced mortlity. Our results indicte tht, prdoxiclly, both nti- TNF- ntibody nd TNF resistnce significntly worsen the inflmmtion in EIU. This finding suggests tht TNF- ppers to ply n ntiinflmmtory role in this disese model. MATERIALS AND METHODS Animls Femle C3H/HeN mice, 6 to 8 weeks of ge (18 to 22 g), were housed in 12-hour light/drk cycle environment nd llowed free ccess to utoclved NH-31 rodent chow (Zeigler Bros., Grdners, PA) nd wter. All niml experiments dhered to the NIH Guiding Principles in the Cre nd Use of Animls nd to the ARVO Sttement for the Use of Animls in Ophthlmic nd Vision Reserch. Induction of TNF Resistnce Recombinnt murine TNF- ws the generous gift of Genentech, Inc. (Sn Frncisco, CA). It hd specific ctivity of 1.2 X 1 7 U/mg nd n endotoxin concentrtion of.511 EU/mg s ssessed by the stndrd limulus ssy. Treted nimls were injected intrperitonelly twice dily for 6 dys with TNF- t dosge of 135 Mg (1-62 X 1 6 U)/kg, diluted in.2 ml of sterile pyrogen-free sline (Meditech, Herndon, VA). Control nimls received injections of sline lone on the sme schedule. Dily records were kept of the totl weight of the nimls in ech group, nd the estblishment of TNF resistnce ws confirmed by the typicl weight loss 7 nd the cquired resistnce of treted mice to mortlity induced by injection of lrge dose of LPS, s described below. Injections with LPS to induce mortlity or EIU were given 24 hours fter the lst TNF- injection. Anti-TNF- Antibody Anti-TNF- ntibody ws the generous gift of Dr. R. Gzzinelli (NIAID, NIH, Bethesd, MD). The ntibody is rt monoclonl IgG } ntibody, XT1122, which ws originlly developed by J. Abrms (DNAX, Plo Alto, CA). The ntibody preprtion ws purified from scites, did not contin ny detectble endotoxin, nd ws found to neutrlize TNF- ctivity. The ntibody ws dministered intrperitonelly, 25 ng per mouse, 6 hours before the chllenge with LPS. Control mice were similrly injected with 25 /xg of rt IgG (Sigm, St Louis, MO). LPS-induced Mortlity In two seprte experiments, groups of mice pretreted with TNF- or with ntibody to TNF- nd their corresponding controls were given single intrperitonel injection of 5 ng Slmonell typhimurium LPS (Difco, Detroit, MI). The mice were exmined dily; mortlity ws ssessed for 3 dys fter LPS injection. Induction of EIU EIU ws induced by injecting 2 ng LPS dissolved in.1 ml of sterile pyrogen-free sline into one hind footpd. EIU induction ws tested in two seprte experiments with TNF-resistnt mice nd in two other experiments in mice receiving ntibodies to TNF-. All nimls were scrificed by CO 2 inhltion 24 hours fter endotoxin injection, nd the right eye ws immeditely enucleted for histopthologic exmintion. Eyes were plced in 4% glutrldehyde for 3 to 45 minutes nd then trnsferred to 4% neutrl buffered formldehyde solution for overnight fixtion. The eyes were embedded in methcrylte, nd 3-^m thick sections were prepred through the verticl plne of the pupil nd the center of the optic nerve nd stined with hemtoxylin nd eosin. Oculr inflmmtion ws grded by two msked independent investigtors on scle of (no inflmmtion) to 4 (severe inflmmtion). The grding scle is bsed on the number of inflmmtory cells in the nterior chmber nd posterior vitreous, nd the presence of protein in the nterior chmber, s detiled in Tble 1. Grdes given by the two investigtors differed in pproximtely 1% of the eyes, but only by.5. In these cses, slides were re-reviewed by both pthologists, nd consensus score ws reched. Sttisticl Anlysis The effect of pretretment with TNF- nd nti-tnf ntibody on mortlity ws compred to seprte control groups using chi-squre nlysis in 2 X 2 contingency tble. However, the clculted P vlues must be interpreted in the context of the smll smple size used in these two experiments. The scores of oculr inflmmtion in mice pretreted with TNF- nd nti- TNF ntibody were compred to seprte control groups using the Mnn-Whitney rnk sum test for two independent smples. Becuse these dt re not normlly distributed, mens were clculted only for descriptive purposes, nd sttisticl tests compring the

3 Prdoxicl Effect of TNF- in EIU 2913 TABLE l. The Scoring System for EIU in Mice Anterior Chmber Score Cells in Chmber >1 Score Protein in chmber: dd 1. Cells in Vitreous >5 Vitreous Score Totl score: Anterior chmberscore + vitreous score 2 Score Sections from the right eye of tested mice were used for the scoring. clculted mens were not performed. The null hypothesis ws rejected in ll sttisticl nlyses when the clculted P vlue ws less thn.5. Pretretment with TNF- protected ginst the mortlity induced by intrperitonel LPS injection (Fig. 2A). Only one of the five pretreted mice died within 36 hours, wheres ll five untreted controls succumbed to the LPS effect. Despite the smll smple size, these differences pproched sttisticl significnce (P =.53). An even more potent protective effect ginst LPS ws demonstrted in the nimls treted with ntibodies to TNF- (Fig. 2B). In this experiment, ll five control mice were ded within 24 hours, wheres ll mice pretreted with the ntibodies survived the LPS injection. This difference ws sttisticlly significnt (P =.11). Pretretments with TNF- or nti-tnf- ntibody excerbted EIU Injection of LPS in ll untreted control nimls induced the chrcteristic findings of EIU, including cute inflmmtory infiltrtion loclized to the poste- 1 - RESULTS Pretretments with TNF- or nti-tnf- ntibody protected ginst systemic effects of LPS Animls receiving TNF- tretment hd n verge weight loss of 4% to 6% during the first 24 hours nd then grdully recovered towrd bseline during the subsequent 5 dys of tretment (Fig. 1). This pttern of weight chnge is similr to tht reported previously in Lewis rts undergoing TNF tretment. 7.4 cen c> Q. 75 > c/) A i 5 I Siittitiiimtitft TNF (n=5) controls (n=5) Hours 6 72 ( E ght bseline c u o Q nti-tnf (n=5) lnge u Men Tretment dy TNF (n=8) Control (n=8) FIGURE 1. Averge dily weight of TNF- group nd control group compred to bseline. There is chrcteristic initil drop in weight on the first cly of TNF- tretment, followed by stedy recovery towrd bseline. 3 CO B controls (n=5) i « Hours FIGURE 2. Mortlity curves fter chllenge with n LD IOO dose of LPS. (A) TNF resistnce conferred protection ginst mortlity in four of five mice (P =.53). (B) Pretretment with TNF-cv ntiserum conferred protection in five of five mice (P =.11).

4 2914 Investigtive Ophthlmology & Visul Science, September 1993, Vol. 34, No. 1 rior vitreous surrounding the optic nerve hed, milder cellulr infiltrtion, nd occsionl protein in the nterior chmber (Fig. 3). The LPS-induced oculr chnges were more severe in mice pretreted with TNF- or with the ntibody ginst this cytokine. The incresed inflmmtion ws noted by more intense cell infiltrtion of the nterior chmber nd by vitreous nd proteinceous exudte in the nterior chmber (Fig. 4). Anlysis of the inflmmtory scores of individul mice of the different groups (Fig. 5) showed tht the scores were significntly higher in both the TNFresistnt mice (P <.5) nd the ntibody-treted mice (P <.2) thn in their controls. DISCUSSION In light of the pprent involvement of TNF- in LPSssocited processes tht hs been demonstrted by number of studies in recent yers, '~7 it ws somewht FIGURE 4. EIU in TNF-resistnt mouse. (A) Protein (sterisk) filling the nrrow nterior chmber, Mny neutrophils (rrows) in the chmber ngle nd surrounding the prs plict of the ciliry body (scored t 2.). (B) More numerous neutrophils (rrows) in the posterior vitreous (scored t 3.). Hemtoxylin nd eosin, X4. FIGURE 3. EIU in n untreted control mouse. Microphotogrphs of the eye, 24 hours fter footpd injection with LPS 2 /xg. (A) Scttered neutrophils (rrow) in the nterior chmber (scored t.5). (B) Some neutrophils (rrows) in the posterior vitreous ner the optic nerve hed (scored t.5). Hemtoxylin nd eosin, X4. surprising tht both cquired resistnce to this cytokine, nd blocking ntibodies directed ginst it ctully excerbted the EIU disese process. A similr effect of nti-tnf- ntibodies hs recently been observed by De Vos et l.16 The induction of EIU hs been shown to be ccompnied by elevtions in TNF- levels.16 Incresed levels of the cytokine were observed in the serum t 2 nd 2 hours nd in the queous humor t 4 nd 22 hours fter injection of LPS. The ssocition of TNF- levels with EIU or other inflmmtory conditions does not necessrily imply cusend-effect reltionship. The dt on the effect of ntitnf- ntibodies in inflmmtory processes hve been mixed, depending on the type of ntibody nd the model system used. Although some studies report tht blocking the effect of TNF- protects nimls in experimentl models of sepsis,25 others find no such effect.1819 Most recently, for exmple, Eskndri et l18 filed to demonstrte protection ginst mortlity

5 Prdoxicl Effect of TNF- in EIU 2915 > to w O Q) o CO O o CO E E A B n 3-2 (n = 13) B Control (n = 15 D Q Q Q Q Control (n=13) «TNF (n = 15) Anti-TNF FIGURE 5. Scores grding oculr inflmmtion from treted groups were significntly elevted compred to controls. Points indicte the oculr inflmmtory grde of ech niml. Men vlues re shown for descriptive purposes only. (A) Scores of oculr inflmmtion from TNF-resistnt (TNF) group versus controls. The men oculr inflmmtory grdes of TNF-resistnt mice nd control mice re 3.54 nd 2.31, respectively (P <.5). (B) Scores of oculr inflmmtion from the nti-tnf- ntibody (nti-tnf) treted group versus controls. The men oculr inflmmtory grdes of mice treted with nti-tnf-: nd control mice re 2.38 nd 1.52, respectively (P <.2). by polyclonl rbbit ntibody ginst murine TNF- in CD1 mice fter either LPS injection or cecl ligtion nd puncture, despite significnt reduction in serum TNF- levels in the ntiserum-treted nimls. Bgby nd ssocites 19 reported dichotomy between septic shock models involving prenterl dministrtion of LPS or bcteri where nti-tnf- ws protective, nd eqully lethl models of peritonitis where the ntibody hd no effect on mortlity. 19 Other studies hve suggested tht TNF- ctully confers beneficil effects, with protection ginst morbidity nd mortlity in some circumstnces. 2 " 23 The pprent inconsistencies mong these vrious studies underscore the complex nture of the inflmmtory process nd the interction of the mny cytokines involved in its induction nd modultion. It is likely tht there cn be comprtmentliztion of n inflmmtory process tht cretes distinct locl milieu of cytokines nd inflmmtory meditors with different dynmics from concurrent systemic inflmmtory process. In line with this notion, Otsuk et l 24 hve reported tht TNF- inhibits neutrophil migrtion to locl inflmmtory sites. Thus, the bility of TNF- ntibody to block completely the lethl effects of n LDi dose of LPS while incresing inflmmtion in the EIU model could be explined through such comprtmentliztion. The significnce of our finding tht TNF resistnce excerbtes the inflmmtion in EIU while conferring protection ginst LPS mortlity is more difficult to interpret. TNF resistnce is probbly complex phenomenon involving n entire network of cytokines, nd its precise mechnism is not known. It hs been determined, however, tht TNF resistnce cnnot be explined on the bsis of n incresed rte of elimintion of the cytokine from the circultion or the endogenous production of nti-tnf- ntibodies. 7 There is some evidence to suggest tht the induction of heptic superoxide dismutse my be involved, which would enhnce protection ginst toxic superoxide rdicls An nlogous resistnt stte to LPS cn be induced through multiple injections, finding tht hs been known for yers, 27 nd both endotoxin-resistnt nd TNF-resistnt nimls re mutully resistnt to the lethl effects of LPS nd TNF-. 7 Despite this pprent similrity between the two phenomen, their mechnisms re different: peritonel mcrophges from mice rendered tolernt by prior injections with LPS will not secrete TNF-, mong vriety of other cytokines, in response to subsequent LPS chllenge in vitro. 28 On the other hnd, inducing TNF resistnce in mice ctully ugments circulting TNF- levels in vivo nd TNF- production in vitro fter LPS chllenge. 29 Thus, becuse the resistnce to TNF- opertes by principles different from those of resistnce to LPS, it is not surprising tht, unlike the excerbtion of EIU in mice resistnt to TNF-, recorded here, the resistnce to LPS ws reported to diminish the oculr inflmmtory response in EIU. 3 Initil exposure to TNF- stimultes mutul dherence fctors on neutrophils nd vsculr endothelium, 3132 which my contribute to the mssive influx of neutrophils into the orgns of nimls chllenged with single high dose of TNF- or LPS. Prt of the mechnism of TNF resistnce my be downregultion of

6 2916 Investigtive Ophthlmology 8c Visul Science, September 1993, Vol. 34, No. 1 these dhesion fctors, resulting in protection from the lethl inflmmtion nd necrosis. On the other hnd, it my be tht in the cse of the oculr tissues, the initil high levels of dhesion fctors re mintined in the fce of repeted TNF- tretments, leding to the observed increse in cute inflmmtion on subsequent LPS chllenge. Alterntively, TNF- my normlly ply counterregultory role in EIU through the induction of ntiinflmmtory mechnisms, such s the one described by Otsuk et l. 24 The constntly elevted levels of the cytokine during the induction of TNF resistnce my serve to desensitize these effects of TNF, perhps through receptor downregultion, thus llowing the excerbted oculr inflmmtion in pretreted eyes. A high level of circulting neutrophils could lso fcilitte n increse in oculr tissue inflmmtion during EIU induction. However, lthough mssive leukocytosis is induced 1 to 2 hours fter initil TNF- injection in rts, this response disppers with the cquisition of TNF resistnce, 33 mking it n unlikely contributing fctor in our experiments. Although the mechnisms behind the seemingly prdoxicl role of TNF- in EIU remin uncler, our findings on TNF resistnce, coupled with the dt on nti-tnf- ntibody, do suggest tht TNF- does not ply proinflmmtory role in this experimentl disese. The inflmmtory mechnisms opertive in EIU pper to be different from those involved in systemic sepsis, lthough they re both initited by LPS. Key Words endotoxin-induced uveitis, tumor necrosis fctor lph, inflmmtion, cytokine, septic shock Acknowledgments The uthors thnk Genentech, Inc., for generous supply of TNF-cv; Dr. Robert Schreiber, Wshington University, nd Dr. Ricrdo Gzzinelli, LPD, NIAID, NIH, for providing the nti-tnf- ntibodies; nd Dr. Ruth Net, AFRRI, for very helpful discussion. References 1. Trcey KJ, Beutler BA, Lowry SF, et l. Shock nd tissue injury induced by recombinnt humn cchectin. Science. 1986; 234: Trcey KJ, Fong Y, Hesse DJ, et l. Anti cchectin/ TNF monoclonl ntibodies prevent septic shock during lethl bcteremi. Nture. 1987;33: Beutler BA, Milsrk IW, Cermi A. Cchectin/tumor necrosis fctor: Production, distribution, nd metbolic fte in vivo./ Immunol. 1985; 135: Michie HR, Mnogue K.R, Spriggs DR, et l. Detection of circulting tumor necrosis fctor fter endotoxin dministrtion. N Engl J Med. 1988;318: Beutler BA, Milsrk IW, Cermi A. Pssive immuniztion ginst cchectin/tumor necrosis fctor protects mice from the lethl effects of endotoxin. Science. 1985;228: Cermi A, Beutler BA. The role of cchectin/tnf in endotoxic shock nd cchexi. Immunol Tody. 1988;9: Frker DL, Stovroff MC, Merino MJ, Norton JA. Tolernce to tumor necrosis fctor in rts nd the reltionship to endotoxin tolernce nd toxicity. J Exp Med. 1988; 168: Rosenbum JT, McDevitt HO, Guss RB, Egbert PR. Endotoxin-induced uveitis in rts s model for humn disese. Nture. 198;286: Tnouchi Y, Kogiso M, Miki S, et l. Endotoxin-induced uveitis in LPS-susceptible C3H/HeN mice. ARVO Abstrcts. Invest Ophthlmol Vis Sci. 1991; 32: Kim MK, Chn C-C, Nussenbltt RB, Plestine AG. Phrmcologicl effects of expression of clss II histocomptbility ntigen in experimentl endotoxin-induced uveitis. Clin Immunol hnmunopthol. 1987; 45: Herbort CP, Okumur A, Mochizuki M. Endotoxininduced uveitis in the rt: A study of the role of inflmmtion meditors. Grefe's Arch Clin Exp Ophthlmol. 1988;226: Herbort CP, Okumur A, Mochizuki M. Immunophrmcologicl nlysis of endotoxin-induced uveitis in the rt. Exp Eye Res. 1989;48: Chn C-C, Ni M, Miele L, et l. Effect of ntiflmmins on endotoxin-induced uveitis in rts. Arch Ophthlmol. 1991;19: Ksner L, Chn C-C, Cordell-Miele E, Gery I. The effect of chlorpromzine (CPZ) on endotoxin induced uveitis. Curr Eye Res. 1992; 11: Gdin M, Bertini R, Mengozzi M, et l. Protective effect of chlorpromzine on endotoxin toxicity nd TNF production in glucocorticoid-sensitive nd glucocorticoid-resistnt models of endotoxic shock./ Exp Med. 1991;l73: De Vos AF, Hoekzem R, Kijlstr A. Cytokines nd uveitis, review. Curr Eye Res. 1992, 11: Fleisher LN, Ferrell JB, McGhn MC. Oculr inflmmtory effects of intrvitrelly injected tumor necrosis fctor-lph nd endotoxin. Inflmmtion. 199; 14: Eskndri MK, Bolgos G, Miller C, et l. Anti-tumor necrosis fctor ntibody therpy fils to prevent lethlity fter cecl ligtion nd puncture or endotoxemi./ Immunol. 1992; 148: Bgby GJ, Plessl KJ, Wilson LA, Thompson JJ, Nelson S. Divergent efficcy of ntibody to tumor necrosis fctor-lph in intrvsculr nd peritonel models of sepsis./ Infect Dis. 1991; 163: Beutler B, Cermi A. Cchectin: More thn tumor necrosis fctor. N Engl J Med. 1987;316: Echtencher B, Flk W, Mnnel DN, Krmmer PH. Requirement of endogenous tumor necrosis fctor/ cchectin for recovery from experimentl peritonitis. / Inununol. 199; 145: Long NC, Kunkel SL, Vnder, AJ, Kluger MJ. Anti-

7 Prdoxicl Effect of TNF- in EIU 2917 serum ginst tumor necrosis fctor enhnces lipopolyscchride fever in rts. AmJ Physiol. 199; 258: Guice KS, Oldhm KT, Remick DG, Kunkel SL, Wrd PA. Anti-tumor necrosis fctor ntibody ugments edem formtion in cerulein-induced pncretitis. J Surg Res. 1991;51: Otsuk Y, Ngno K, Ngno K, et l. Inhibition of neutrophil migrtion by tumor necrosis fctor: Ex vivo nd in vivo studies in comprison with in vitro effect. J Immunol. 199; 145: Alexnder HR, Shepprd BC, Jensen JC, et l. Tretment with recombinnt tumor necrosis-lph protects rts ginst the lethlity, hypotension, nd hypothermi of grm-negtive sepsis. J Clin Invest. 1991; 88: Alexnder HR, Doherty GM, Block MI, et l. Single dose tumor necrosis fctor protection ginst endotoxin-induced shock nd tissue injury in rts. Infect Immunol. 1991; 59: Abernthy RS. Homologous nd heterologous resistnce in mice given bcteril endotoxins. J Immunol. 1957;78: Hslberger A, Syers T, Reiter H, Chung J, Sciitze E. Reduced relese of TNF nd PCA from mcrophges of tolernt mice. Circ Shock. 1988;26: Frker DL, Norton JA. TNF nd endotoxin: Cross-tolernce by different mechnisms. Surg Forum. 1988;39: Hoekzem R, Murry PI, vn Hren MA, Helie M, Kijlstr A. Anlysis of interleukin-6 in endotoxin-induced uveitis. Invest Ophthlmol Vis Sci. 1991; 32: Gmble JR, HrlnJM, KlebnoffSJ, VdsMA. Stimultion of the dherence of neutrophils to umbilicl vein endothelium by humn recombinnt tumor necrosis fctor. Proc Ntl Acd Sci USA. 1985; 82: Pohlmn TH, Sthness KA, Betty PG, Ochs HD, Hrln JM. An endothelil cell surfce fctor(s) induced in vitro by lipopolyscchride, interleukin-1 nd tumor necrosis fctor-lph increses neutrophil dherence by CDwl8-dependent mechnism. J Immunol. 1986; 136: Ptton JS, Peters PM, McCbe J, et l. Development of prtil tolernce to the gstrointestinl effects of high doses of recombinnt tumor necrosis fctor-lph in rodents./ Clin Invest. 1987;8: