Bacterial Endotoxin Molecular Relationships of Struetui To

8/3/2019 Bacterial Endotoxin Molecular Relationships of Struetui To

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Bacterial endotoxin: mOle..:ar p. iatio ships of struetui toactivity and fction;

ERNST T. RIETSCHEL , TERUO Ki , F , T JL RI CH S(HADE, UW E M AM Ai, G#{220}NTERSCRMHY1HARALD LOEPNO AR?URJ ULMER, ULRICH ZHRJN(ER , ULIUCH SEYDEL ,FRAN(O. D I PAVA MAX.SCfl E . AND. HELMUT BRADEF or sc hu np in sti tu t B or ste l5 l njt it ut. fll r E xp er im e nte lle Biologic un d Medizin, D-23845 Boned5 Germany; an d*5dc, P re clin ic al R es ea rc h, C H -4 00 2 B as e!, S witz e# {2 41 }a nd

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ABSTRACT Endotoxins of Gram -negative m icrobesfu lfill as co mp onen ts of the outer m embrane a vital func-tion for bacterial viability and, if set free, induce inm ammalians potent pathophysiological effects. Chem i-cally, they are lipopolysaccharides (LPS) consisting of an0-specific chain , a core oligosaccharide, and a lipid com -ponent, termed lip id A . The latter determ ines the endo-toxic activ ities and, together with the core constituentKdo, essentia l functions for bacteria . The primary struc-ture of lip id A of various bacterial origin has been eluci-dated and lip id A of Escherich ia coli has been chem icallysynthesized . The biological analysis of synthetic lipid Apartia l structures proved that the expression of endotoxicactiv ity depends on a unique primary structure and apeculiar endotoxic conformation. The biological lip id Aeffects are m ediated by macrophage-derived bioactivepeptides such as tum or necrosis factor a (TNF). M acro-phages possess LPS receptors, and the lipid A regions in-volved in specific b inding and cell activation have beencharacterized . Synthetic lip id A partia l structures com -pete the specific binding of LPS or lip id A and antago-nistically inhib it the production of LPS-induced TNF.LPS toxicity, in general, and the ability of LPS to induceTNF are also suppressed by a recently developed mono-clonal antibody (IgG2a), which is d irected against an epi-tope located in the core oligosaccharide. At present w edeterm ine m olecular and submolecular details of thespecificity of the interaction of lip id A w ith responsivehost cells w ith the ultimate aim to provide pharm acologi-cal or immunological therapeutics that reduce or abolishthe fatal in flammatory consequences of endotoxicosis.- Rietschel, E . Th., K irikae, T ., Schade, F . U ., M amat,U ., Schm idt, G ., Loppnow , H ., Ulmer, A . J., Zihringer,U ., Seydel, U ., D i Padova, F., Schreier, M ., Brade, H .Bacterial endotoxin: m olecu lar relationships of structureto activity and function. FASEB J. 8: 217-225; 1994.Key Wo rd s. - i ip op oi ys ac c/ za ri de . lipid A tumor necrosis factorin terleukin 1 anti-LPS antibodies LPS antagonists - monocytessphingogiycoiipid

BA CT ER IA , L IKE OTHER C EL LS, A RE SU RR OU ND ED by an enve l-ope that guarantees the shape and integrity o f the m icrob ia lbody. In the cas#{231}f G ram -negative bacteria , wh ich comprisethe human pathogens H aem ophilus in ftuenzae, E scherichia coil,Salm onella enterica , K lebsielia pneum oniae, Bordeteila pelt ussis,P seu dom on as a eru gin osa , C hla myd ia p sitta cz, an d L eg io nelia p ne u-mophi la, th is enve lope represents a b ilayered and asym metri-cally organ ized membrane, the so-ca lled outer membrane(1). In the outer lea fle t o f th is membrane, which po ints to the

environment, certa in prote ins and, in particular, a c lass ofmacroamphiphi les - the lipopolysaccharides (LPS)2 - ar ethe dom inating constituents whereas the inner layer is com -posed of phospho lipids in add ition to prote ins. One bacteria lce ll conta ins approxim ate ly 3.5 106 LPS molecu les occupy-ing an area of 4 .9 itm2. As the surface of an E. coli cel lamounts to 6.7 im 2 it appears that three-quarters o f the bac-terial surface consists o f LPS , the rem ain ing area be ing filledby pro teins (1). The outer membrane reacts to changes of theenvironment and inhib its the entrance of tox ic compounds(such as antib iotics). Further, it p lays an important ro le innutrient transport, and it m ediates the physiological andpathophysio log ica l in teraction of bacteria w ith host organ-isms, in general. Therefore, the in tegrity of the membranesarch itecture and notably o f its LPS component is essentia lfor bacterial viab ility . In fact, m utants that ar e unable toform LPS are not v iable . Because of this cruc ia l ro le and itsexposed position , LPS represents an ideal ta rget for the at-tack of d isease-producing bacteria by antibod ies and otherimm unolog ica l or pharm aco log ica l agents.

When bacteria multip ly , bu t a lso when they d ie and lyse,LPS is set free from the surface. A century ago, liberatedLPS had a lready been recognized as a potent bacterial tox in,and therefore was termed endotoxin (2). Today, LPS isknown as a m ajor factor responsib le for toxic m anifesta tionsof severe G ram -negative infections and generalized in flam -mation (3). There fore , several current programs of researchin the field o f in fectious d iseases a im at the neutra liza tion ofendotoxin or its e lim ination from the circu lation. On theother hand, LPS represents a h igh ly active im munom odula-tor that is capable of inducing nonspecific res istance to vira land bacteria l in fections and ho lds great prom ise as a potentimmunolog ica l ad juvant (4). To make use of these beneficia le ffects o f LPS in clinica l medic ine , stud ies are currently per-form ed to reduce LPS toxic ity by chem ical or physica l ap-proaches w hile re tain ing its benefic ia l activ ities.

F or s uc h s tu di es t o b e s uc ce ss fu l,t he n at ur e a nd c hemic alarch itecture of bioactive domains of LPS must be known. Inrecent years great progress has been made in understand ingthe molecular organ ization and mechan isms underlying thedetrimenta l and benefic ial activ ities of endotoxins. In th is

T o w hom c orre sp ond enc e s hou ld b e ad dre sse d, at: F ors ch ung s-in stitu t B ors te l, In stitu t f# {2 52 }rxp erim en telle B io log ie un d M ediz in,P arka llee 2 2, D -2 38 45, B orste l, G erm an y.2Abbrevia tions: LP S, lipopo lysaccharide ; G Ic, n-g lucose; G al,D -g ala cto se; G lcN Ac , N -ac etyl-D -g luc osa min e; H ep , he ptos e; K do ,2-keto-3-deoxyocton ic acid; TNF, tum or necrosis factor a ; BP I,bactericida l/perm eability increasing pro tein ; m Ab, m onoclona lantibod ies; LB P, LP S bind ing pro te in .


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report, these advances w ill be sum marized and their imp lica-tions for the development of preventive or therapeutic ap-proaches aga inst endotoxem ia w ill be d iscussed.

CHEM ICAL STRUCTURE AND BIOLOGICALPROPERTIES OF LPS SEGMENTSLPS derived from different groups of G ram -negative bac-teria consist of a hydroph ilic heteropo lysaccharide and acovalently bound lipid component, te rmed lipid A (5), andthus conform to a common structural princip le. In the caseof Enterobacteriaceae, the heteropolysaccharide can be sub-div ided into the 0-specific chain and the core o ligosaccha-ride (F ig . 1).0-specific chainThe 0-specific cha in constitu tes a po lymer of o ligosaccha-rides, the so-ca lled repeating units consisting of one to e ightg lycosyl res idues. The structure of repeating units (nature ,ring form , sequence, substitu tion , and type of linkage of con-stituent monosaccharides residues) differs from strain tostrain w ith in a serotype, and thus exh ib its an enormousstructura l variability, de term ines the serological specific ity o fthe LPS and of bacteria conta ining it, and there fore func-tions as an im portant surface antigen. To emphasize th isp ro pe rt y, L PS i s of te n a ls o r ef er re d t o a s 0 -a nt ig en ( 5) .A nLPS prepara tion exh ibits considerable heterogeneity evenwhen isola ted from a single bacteria l cu lture . This hetero-gene ity resu lts from the fact tha t the bacteria synthesize vari-ous LPS molecu les that d iffe r in the length of 0-specificcha ins, i.e ., in the number of repeating units.

n-cifuccinI

r-1?: Outer InnerCore CoreKdo4-rAGa-OM Sic Ab.-OAc G ICNAc Ga l 14p (doceo.,1, o--bAiA I i- ..i1*nI4 Rho#{176}olGIc G ali cpdo!5tG cN i.tG IcNL0M J,, II I IFA1FA FA M,Ro I

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3 35i3?i3 ,4K12 -cirGlc . GIc-Hep,54op5..KdoFigure 1. Chem ica l struc ture of a Salmonella l ipopolysaccharideco ntain ing th e R a-c ore (A ) and of know n E sc /z er ic hi a c oi l c ore ty pe s(B). For litera ture , com pare ref 15.

Po lysaccharides prepared from LPS by m ild acid hydroly-s is and conta in ing the 0-specific chain as well as parts of thecore have been shown to possess intrinsic b io logical activ itysuch as immunopotentia tion (6). Desp ite various stud ies andproposa ls (7-9), the structures responsib le for the observedimmunostim ula tory activity have not been unequivoca llyidentified . Thus, it remains to be shown whether they are ,in fact, located in the polysaccharide region or whether b io-activ ity is due to the presence of m inute , nontoxic amountsof lip id A or LPS .

Severa l aspects of the genetic determ ination and b io-synthesis o f th is LPS segment are well understood (10).Thus, in S. enterica , V ibrio choierae, Shigeila, E . coil, an dP aeruginosa, the synthesis o f the 0-specific chain is deter-m ined by a cluster of genes term ed fb . Mutants w ith a defectin the rfb locus or lack ing it form an LPS w ithout an 0-specific cha in (these LPS are ca lled R-form LPS). Such mu-tants, however, are able to grow and multip ly in vitro , show-ing that the 0-chain , in princ iple , is d ispensable for bacteria lv iab ility . In tissue or body flu ids, however, pathogenicS a l m o n e l l a epers is t and surv ive only if they express an 0-specific cha in, wh ich in th is case protects bacteria fromphagocytos is and serum (complement) -mediated lysis .Because it is im portant for bacteria l surv iva l in v ivo , the0-cha in, there fore, represents a su itab le target fo r new anti-bacteria l drugs. Based on earlier stud ies of lysogenic strainsof Ace tobac te r me thano il cus lack ing the 0-cha in as the resu lt o fexpression of a prophage-encoded gene product, we foundrecently tha t a DNA fragment o f 263 bp derived from thebacteriophage Acm l, the natural host o f which is A . m etha-nolicus MB58/4 (11), la rge ly inhib its expression of the 0-cha in a lso in various E . c oi lsero types and S . e n t e r ic aerovars(12). The mechan ism of th is inh ib ition is presently not we llunderstood. The DNA fragment is too small to code for aprote in, but ra ther determ ines the synthesis of an RNAmolecu le as demonstra ted by Northern b lo t analys is andtranscrip tion in v itro using the excised DNA fragment ofAcm l as the templa te and purified RNA polymerase ofE . c oi l. ccord ing to our pre lim inary data , th is RNA appearsto act as an antisense RNA (13) inh ib iting the b iosynthesisof the 0-specific cha in . RNA/RNA hybrid iza tion experi-m ents revea led that the phage-encoded RNA molecu le isable to hybrid ize to at least two E. c o i lm RNAs the functionsof wh ich are currently being investiga ted. The broad hostspectrum of th is supposed antisense RNA, covering taxo-nom ica lly d istant strains, suggests that the gene (or genes)a ffected the code for conservative components of LPS suchas the core reg ion. The su itability of the phage DNA frag-ment to be used for the genetic construction of enterobac-terial o r o ther G ram -negative vaccines is presently understudy in our labora tory.

C ore regionThe core reg ion of enterobacteria l LPS consists o f a hetero-oligosaccharide that can formally be subdiv ided into anouter and an inner portion (Fig . 1). A single core-type [be ingoccasiona lly incomple tely expressed (14)] is present in a llSalmonel la sero types (F ig . 1 , Ra); five d iffe rent core types havebeen recognized for E . coil sero types (F ig . 1, R i to R4 andK-12), and a lso for Proteus, and three for Citrobacter (1 5). T hu s,the core is structurally m ore un ifo rm than the 0-cha in,structura l divers ity be ing found prim arily in the outer coreregion. As F ig. 1 shows, in Salmonel la an d E . co il s ero typ es theouter core contains the common hexoses D-g lucose (G ic), D-galactose (Gal), and N -acety l-D -glucosam ine (G lcNAc) inthe linkages shown. Desp ite structural differences w ithin the


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d iffe rent core types, a common sequence of hexose residuespossesses the general s tructure: aH exi- > 2aHexi- > 2aHexi-> 3crGlcl- > 3a-.

T he i nn er c or e i sc om po se d o f t he c ha ra ct er is ti ca nd L PS -specific components heptose (Hep), mainly in the L-glycero-D-manno configura tion , and 3-deoxy-D -m anno-octu losonic (or2-keto-3-deoxyocton ic) ac id (Kdo). The Hep and Kdo resi-dues are , in genera l, substitu ted by charged groups such asphosphate, pyrophosphate , 2-am inoethylphosphate , and 2-am inoethylpyrophosphate , lead ing to an agglomeration ofcharged residues in th is inner part of the core reg ion. Theh igh density o f negative ly charged residues is like ly to be ofphysio log ical s ign ificance, as it concentra tes b iva lent ca tionssuch as Ca2 and Mg2 in the close environment o f the ce llsurface where cations are requ ired for the structura l andfunctional in tegrity o f the outer membrane.

A somewhat d iffe rent LPS type is found in mucosa l patho-gens such as N els se ri a, H a em o ph il us , an d Bordeteila. In thesecases, even in the w ild -type-(S)-form the LPS lacks an 0-cha in and its s ize is s im ila r to that of the enterobacterial R -form LPS (16). However, it d iffe rs in structure from the outercore of enterobacterial LPS. On the other hand, the inner-most part o f the core reg ion appears to be well conservedamong all Gram-negat ive taxa, as Kdo form s the linkage tolipid A a lso in these 0-chain lacking LPS . Nevertheless,these bacteria have deve loped stra teg ies to escape the hostsdefense mechanism , such as phagocytos is and com plement-media ted killing , w ithout possessing the pro tecting 0-specific cha in . One of these strategies is now well understoodin the genus Nelsserla, where attachment o f N -acetyl neura-m in ic acid to the outer core reg ion protects bacteria aga instthe bacteric idal activ ity o f serum (17).

The m inim al LPS structure of grow ing and multiply ingbacteria consists o f one Kdo residue linked to lip id A (18, 19).In v iew of the essential ro le o f LPS and Kdo for bacterial v ia -bility, the mechan ism s of Kdo biosynthesis and its incorpora-tion in to LPS have been thorough ly stud ied, a lso w ith theaim to devise ways of its pharmaco log ica l inhib ition (20).The smallest LPS found in natura lly occurring pathogenicbacteria is that o f the genus C h i a m y d i a where the core reg ionconsists of only a Kdo trisaccharide of the sequenceaKdo(2- > 8)aKdo(2- > 4)aKdo (21). M ost surpris ing ly, thistrisaccharide is genera ted in the b iosynthesis through the ac-tion of a sing le enzyme that constitutes a multifunctiona ltransferase (22). This is in accordance w ith investiga tions onthe Kdo-transferase of E . c o i lK-12, wh ich is a bifunctiona lenzym e (23) cata lyz ing two distinct g lycosyla tion steps lead-ing to the formation of the a(2- >4)-linked Kdo disac-charide - a common constituent o f enterobacteria l LPS (24).Lipid AThe lip id A component has received worldw ide scientific a t-ten tion ; in 1954 it was a lready postu lated to constitu te thetox ic and immunomodulating princip le o f LPS (2). Afte r thechem ical structure of lip id A of E . co il LPS had been e luci-da ted, lip id A was chem ica lly synthesized (25) and the syn-thetic , i.e., homogenous, compound exh ibited identica l b io-log ica l activity compared w ith bacteria l lip id A and LPS(26). Thus, the concept o f lip id A constitu ting the endotoxiccenter of LPS was proved to be correct. Systems are known,however, where optimal lip id A activ ity is expressed on ly ifa t least one Kdo residue is cova lently a ttached, indica tingthat the inner core reg ion can up-regu la te lipid A b ioactivity(fo r lite ra ture , see ref 27).

Chem ica lly, lip id A represents a pecu liar phosphog lyco-lipid , the architecture of wh ich is un ique in nature (28). It

represents, together w ith the Kdo-conta in ing inner core por-tion , the structurally m ost conserved region of LPS . A ll en-dotoxica lly active lipid A stud ied so far conta in D-giuco-configura ted pyranosidic hexosam ine residues (D -G lcN or2,3-diam ino-2 ,3-d ideoxy-D -glucose, D-G1cN3N ), which arepresent as a 13(1-6)-linked homo- or heterodim er (F ig. 2).The d isaccharide carries an a-glycosidic (position 1) and anonglycosidic (position 4 ) phosphory l group, and in ester(positions 3 and 3) and am ide (positions 2 and 2 ) linkage(R )-3-hydroxy fa tty acids, o f wh ich , in general, two areacylated at the ir 3-hydroxyl group. Desp ite th is common ar-ch itecture , lip id A of d iffe rent bacteria l o rig in may vary inthe ir fine structure . Varia tions in structure resu lt from thetype of hexosam ine present, the degree of phosphory lation,the presence of phosphate substituents (not shown in F ig . 2),and most notably, the nature, cha in length , number, and lo-ca tion of acyl groups. In F ig. 2, two groups of hexaacyl lip idA are shown that differ in the acyla tion pattern. In one group(Fig. 2A)-E. c o i l ip id A be ing the classica l proto type-thehydroxy fatty acids possess a chain length of 14 carbon atoms[ 3- hy dr ox yt et ra de ca no ic a ci d, 1 4: 0( 3- OH )] . T he h yd ro xygroups of the two (R )-3-hydroxy fa tty acids of the distalG1cN-residue (G lcN II) are acyla ted by nonhydroxylatedfatty acids whereas those at the G lcN-res idue at the reducingside (G lcN I) are free. Thus, the overa ll acy lation pattern isasymmetric [4 + 2]. In contrast, in a second group of lipidA a symmetrica l fa tty acid distribution [3 + 3 ] w as id en tifie d(F ig . 2B). As w ith Nelsserla meningitldzs (29), G1cN I and G IcNII each carry three fatty acids that have shorter chain lengths(average of 12 carbon atoms) than the acyl groups of E. co il.The chain length of am ide-linked acyl groups (n) is con-stant in asymmetrically acyla ted lip id A whereas it variesamong stra ins w ith symmetrica l fatty acid distribution. Inthe la tte r case, the cha in length of secondary acyl groups (o )is constant in contrast to the E. c o i lgroup, where it variesfrom 12 to 16.

O f the many known varia tions w ith in the acyla tion pat-te rn of lip id A (for summary, compare ref 28), on ly twofurther examples are mentioned here . In Rhodobactersphaeroides and all species of the a-3 branch of thephylogenetic tree , the am ide-linked fa tty acids of the d isac-charide backbone were identified as 3-oxote tradecanoic acid(30). Some of these species contain , in add ition , unsatura ted

A

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Rhodocyclosgelolinosus 10 10 12 114)

Chro.noboct.yiumvIOlOCI t iW 10 12 12

menIngIlidus 12 14 I?

Figure 2. Chem ica l structure of the lip id A com ponent o f variousG ram -negative bacteria (from (6 7) w ith p erm is sio n). A) Lip id Ap os se ss in g a n a symm etric al fa tty a cid d is trib utio n. B) Lipid A pos-s es s in g a s ym me t ri ca l f a tt y a c id d i st ri b ut io n.


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fatty acids as lip id A constituents, e .g ., -7-te tradeceno icacid in Rh. sphaeroides (31). These lip id A are of particularb iolog ica l interest, as they lack endotoxic activ ity (31). Mostspecies be longing to the a-2-subclass, as well as L. pneu-m o p h i l a ,conta in long-chain n-2 hydroxyla ted fatty acids (32).This type of unusual hydroxy fatty acid carries the functiona lhydroxy group at the penultimate position of the hydrocar-bon chain , and not a t position 3. The most prom inent repre-senta tive , 27-hydroxyoctacosano ic acid [28:0(27-OH )], pos-sesses the doub le length of the usual 14:0(3-OH ) acid andmay stretch through the entire outer membrane. It has beensuggested that th is could resu lt in a more stable anchorageof LPS and an increased stab ility o f the b ilayered outermembrane (33).

Essentia l fea tures of the biosynthesis of lip id A have beene luc idated, and it is known that mutants defective in earlysteps of lip id A biosynthesis are not ab le to grow and multi-p ly (18). Th is shows that m ature lipid A, together w ith a tleast one Kdo residue (19), constitutes the m inimal LPSstructure requ ired for bacteria l viability.

MECHANISM OF ENDOTOXIC ACTION OF LPS

The basic princip les of LPS bioactivity are nowadays wellunderstood (9, 18, 34-38). Endotoxins do not e licit theirto xic e ffe cts - as one m ight suspect and as it is known formany pro teinous exotoxins-by killing host ce lls or by in-h ibiting ce llu lar functions. Rather, LPS requ ires the activeresponse of host cells . Accord ing to present know ledge LPS ,through its lip id A component, in teracts w ith various hostce ll types includ ing mononuclear ce lls, endothelia l andsmooth muscle ce lls , po lymorphonuclear granulocytes, andthrombocytes, among which macrophages/monocytes are ofparticu lar im portance (34, 36). Thus, LPS-induced activationof macrophages resu lts in the production of bioactive lip ids,reactive oxygen species, and in particu lar, peptide m edia torssuch as tumor necrosis factor a (TNF), in terleukin 1 (IL-i),IL-6 , IL -8, an d IL-b . These secondary, hormone-like pro-teins are endowed w ith potent bioactiv ities and are capab leof inducing many of the typical endotoxin effects by actingindependently , in sequence, synerg is tica lly or antagonisti-ca lly (35). It appears that benefic ial effects (e .g ., induction ofres is tance to in fection, adjuvant activity) are elic ited whenlow levels o f media tors are produced and that detrimenta le ffects (e .g., h igh fever, hypotension, irrevers ib le shock) areinduced when h igh leve ls o f media tors reach the circula tion .However, low mediator concentra tions may a lso becomeharm ful when the host organ ism is in a hyperreactive sta teto LPS. Hyperreactivity to endotoxin may be caused by exo-toxin s, ch ro nic infection, and by grow ing tumors, and oneimportant factor contribu ting to sensitiza tion to LPS hasbeen identified as y-in terfe ron (9 , 34).

STRUCTURE-ACTIV ITY -RELATIONSHIPS OFLIPID AL ipid A possesses a complex structure and the question aroseas to which of its constituents or structura l features is impor-tan t for endotoxic activ ity, i.e., peptide m ediator induction .Th is problem was studied by quantita tive analyses of them edia tor-inducing capacity of various lipid A prepara tions,and notab ly synthetic partia l structures d iffe ring , e .g ., in thephosphoryla tion and acyla tion pattern. Such tests were per-fo rmed using a murine macrophage ce ll line and humanperiphera l monocytes under serum -free cond itions. The

resu lts o f our stud ies in the human system are schematica llysummarized in Fig . 3 (compare refs 9 , 37, 38). Changes in thehydrophilic backbone (F ig. 3, upper pane l), i.e ., substitu tionof the a-anomeric phosphate group by the 13-anomer (in theform of the oxyethyl deriva tive) or om ission of a phosphorylgroup sign ificantly reduced b ioactiv ity (factor 10 as com -pared to lip id A), monosaccharide structures be ing the leastactive (reduction of activ ity by a factor of >107). A lte rationin the hydrophob ic region also had dramatic effects. Abstrac-tion of one acyl group (y ield ing pentaacyl lip id A ) or add i-tion of a fatty acid (yie lding heptaacyl lip id A) reduced bio-activity by a factor of 10 2 (F ig . 3 , lower pane l). The samedegree of reduction of b ioactiv ity is seen on d islocation of asingle acyl group. A tetraacyl prepara tion (precursor Ia,com pound 406) resu lting from the abstraction of two fattyacids comple te ly lacks mediator-inducing capacity (reduc-tion of b ioactiv ity by a factor of >107).

C ollective ly , these data suggest that full endotoxic activ ityi s e xp re s se d by a m olecule conta ining tw o (D-g iuco-configu-ra ted) hexosam ine residues, two phosphory l groups, and sixfa tty acids (saturated, and in part, 3-hydroxylated) includ ing3-acyloxyacyl groups w ith a defined cha in length and in adistinct location . Endotoxica lly less active or inactive arep artia l s tru ctu re s 1) lack ing on ly one such constituent, ir-respective of its chem ica l nature , 2) mole cu le s c on ta in in g

102AcyI-H 2AcyI-2HJ 1 AcyI-H

0 H - ACyIPentoaCyl Tetroacy) Hexaocyl

Figure 3 . S ch em atic re pre se nta tio n o f lip id A structure-activityrela tionsh ips.Sh own are chem ical ch anges of the E . c oi llipid Astructure and the factor by w hich the structure genera ted is less ac-tive than lip id A . A) Modifications of the hydrophilic reg ion oflip id A . B) M od ific atio ns of the h yd rop hob ic region of lip id A.


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different, although chem ica lly re la ted, constituents, and 3)ana logs w ith a diffe rent arrangement of constituents. Aun ique molecular structure and conformation (endotoxicconformation) o f lip id A , there fore , seems to allow optim alexpression of endotoxicity in v ivo . W hether the endotoxicconformation re la tes to a single endotoxin molecule or to apecu liar aggregation sta te remains to be eluc idated. Amolecular m odel o f E . c oi lip id A , w ith the attached polysac-charide portion, is shown in F ig. 4 (39). Th is model struc-tu re, ca lcu lated by energy m in im ization , fits experimentaldata such as the arrangement o f acyl groups and the dim en-s ions of the m olecule .

The concept o f a unique lip id A conform ation implies thata fitting peculiar recognition molecu le should be presente ither in the circu la tion or be expressed on endotoxin-s en sitiv e m on oc yte s and m acrophages. Thus, the in teractionbetween lip id A and the humoral or cellu la r ta rget shou ld beh igh ly spec ific , invo lving a unique m olecular conformationof lip id A on the one side and a pecu liar humora l or cellula rreceptor for lip id A on the other.

LPS AND LIP ID A BINDING TOMONOCYTES/MACROPHAGESTo study the nature of the associa tion of lip id A w ith targetcells we have estab lished a b ind ing system invo lving rad io-or F ITC -labe led Re-mutant or S-form LPS , respective ly, tothe mouse macrophage ce ll line (J774.1) or human periph-era l m onocytes. We found for both ce ll types that the associa-tion of LPS w ith phagocytes exhib ited saturab le b ind ingkinetics (40). The bind ing of labe led LPS cou ld be com -p le tely inh ib ited by nanogram amounts of non labe led R-and S-form LPS (F ig. 5). The fact that the kinetics of bind-

F igure 4. Molecu lar model of an E scherichia coil S -f orm l ip o po ly sac -charide (39). In th is model the 0-specific chain is bent by an angleof approxim ate ly 45# {176 }Celative to the stre tched core-lip id Adomain. In the bacteria-bound form the 0-chain would cover thecell surface as ha irs cove r the head or sk in .

200

F re e Ba cte ria lL ipid A

Amount of Com petitor Eng/well]Figure 5 . C omp etitio n o f s pe cific b in din g b y L PS , fr ee lip id A, andpartial structures of Re LPS to a murine macrophage ce ll line .

in g an d its inhib ition reached a pla teau conform s w ith theconcept that a specific humoral or ce llu la r LPS recogn itionmolecu le is invo lved in binding. As the human monocyteassay exh ib its features identica l to those of the m ouse macro-phage system , and as the former assay is perform ed underserum -free cond itions, it appears that the LPS bind ing sitestudied here is ce ll associa ted.

Next, we studied the structura l requ irements of lip id A forspecific b ind ing to monocytes/m acrophages by determ in ingthe capacity o f synthetic partia l s tructures to compete w ithlabe led LPS for the association w ith ce lls. We found thatstructura l changes in the hydrophilic region of lip id A dra-matica lly reduced its competitive capacity . In fact, the samestructure-activity re la tionsh ip was found as in the system ofmonokine induction (F ig . 3A). A lso , the hydrophob ic regionappeared to be invo lved in b ind ing capacity, a lthough in ad iffe rent m anner from what on e would expect from the datapresented in F ig. 3B. The tetraacyl lip id A p artia l s tru ctu reprecursor Ia (synthetic compound 406) proved to be an ex-cellent competitor o f LPS bind ing (Fig . 5) although it wasnot ab le to induce monokine production (Fig . 3B). As ex-pected, remova l o f the 1- or 4 -phosphory l group of com -pound 406 abo lished its h igh competit ive ca pac ity. B is acy llip id A partia l s tructures still exhib it inh ibito ry capacitywhereas deacylated LPS was inactive (F ig. 5).

These results suggest tha t the specificity o f lip id A b ind ingto the recogn ition molecule is la rge ly media ted by itshydroph ilic backbone (phosphory la ted-D -glucosam ine d isac-charide). The hydrophob ic region of lip id A (acyl groups),on the other hand, is of centra l s ign ificance in the activationof ce lls , which follows the b inding event. The exact ro le ofacyl groups for the induction of cytok ines presently is notunderstood. It may rela te to a pecu lia r 3-dim ensional physi-cal s tructure (41). Th is particu lar organ ization may a llow in-corpora tion in to the cellu la r membrane or even the passageof lip id A in to the cytop lasm , in ternalization possib lyrepresenting an im portant event requ ired for ce ll activation .These data a lso show that b ind ing of lip id A or LPS to ce llsis a necessary but not su ffic ient step for ce ll activa tion andc yto kin e p ro du ctio n.


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REVIEWSINH IBIT ION OF MED IATOR INDUCTIONThe experimenta l separa tion of the phenomena bind ing andactivation lead to the idea of inh ib ition of ce llula r activa tionby partial s tructures. It appeared possib le that oligoacyl par-tia l s tructures of lip id A such as precursor Ia (compound406), wh ich by themselves are inactive in cytok ine induction ,are able to inh ib it LPS- or lip id A-induced monokine pro-duction in human peripheral m onocytes, as they are potentcompetito rs o f LPS binding. Indeed, we could demonstratethat the tetraacyl compound 406 inhib ited , in a dose-dependent manner, the monokine production by humanmonocytes induced by LPS but not by S t a p h y l o c o c c u s p i d e r -midis, lipopro tein , or BCG (for lite rature , compare (38)).These resu lts ind ica te that the inhib ito ry capacity o f com -pound 406 is h igh ly specific for LPS-ce ll interaction and isnot due to suppression of monokine re lease. Rather, as wasshown by Northern b lot ana lys is , compound 406 suppressedthe LPS-induced form ation of mRNA for TNF and IL-i,and there fore monokine production was inh ibited at thetranscrip tional level (38). S im ila r results were reported forthe natural precursor Ia (so-called lip id IVa) iso la ted fromS. typhimurium (42) and a lso for nontoxic lipid A species ofRh. sphaeroides (43) and Rh. capsulatus (44). In recent studieswe could show that compound 406 a lso inhib ited LPS- butnot IL -i-induced expression of the adhesion molecu leICAM-1 and the formation of IL -6 in endothelia l cells( V . S ch# {2 46} nbe ck ,E . Brandt, H . -D . Flad, E. Th. R ie tsche l,and H . Loppnow, unpublished resu lts). These observationsare in accordance w ith the previous demonstration that par-tially deacyla ted LPS is capable of inh ibiting LPS-inducedadherence of granu locytes to vascu lar endothe lium (cited in(45)) and also w ith the concept that the observed inh ibitioni s LPS -sp ec if ic .As to the mechan ism of the inhib itory action of lip id Apartia l structures such as compound 406, we favor the viewthat a t least in the case ofJ774.i ce lls and hum an m onocytes,inh ib ition is based on competitive binding to a specific LPSbind ing pro te in . This concept is supported by the find ingthat the degree of inh ib ition is dependent on the dose of thecompetitor and that inh ib ition is overcome by higher LPSdoses. A true antagonistic m echan ism is a lso suggested bythe fact tha t if the LPS-b ind ing data obtained in the presenceor absence of an inh ib itor are p lo tted in a doub le reciprocalm anner, both regression lines cross on the Y-axis (H . Heineand A . J. U lm er, unpublished results). These considera tionsled us to conclude that antagonistica lly active compoundss uch a s pre cu rso r Ia b ind as do lip id A and LPS to the samehumora l or ce llu lar recognition site or receptor, bu t tha tlip id A partia l s tructures are not capab le to functiona lly en-gage this receptor. Due to this associa tion , however, o ligoacylprepara tions block the receptor binding site and therebydeny access of LPS , i.e., they inh ib it the activa tion of cells .The nature of the ce llu lar bind ing molecule involved remainsto be established. There is ample evidence that the CD14m olecule p lays a crucia l role in serum -dependent activa tionof monocytes/m acrophages by LPS due to the presence ofLPS bind ing prote in (LBP) (18, 46, 47). Our data presentlydo not a llow us to decide whether the specific ity o f lip id Ab ind ing re la tes to its associa tion w ith LBP (the lipid A/LBPcomplex would subsequently interact w ith ce llula r CD14 fo l-lowed by cytok ine production) or to the d irect binding to acellu lar receptor (such as CD14). As endothelia l ce lls do notd ispose of membrane-bound CD14, yet are responsive toLPS and the inhib itory e ffect o f compound 406, it appearsa lso that b ind ing/activa tion molecules d istinct from CD14

play a ro le [fo r summary compare (48)]. However , endothe-hal cells dispose of a receptor for solub le CD14 (49) that maybe present in sera used for ce ll cu ltiva tion . Recent studies ofthe human macrophage cell line THP-1 indicate that CD14is not necessarily involved in the antagonistic action ofte traacyl lip id A structures (50).

On biodegradation of E . co il lip id A by macrophages andgranu locytes, two fa tty acids (12:0 and 14:0) are removed bythe enzym e 3-acyloxyacyl-hydro lase, yie ld ing the bacteria lcounterpart o f prepara tion 406 (45). As discussed, this com -pound not on ly lacks stim ula tory (endotoxic) activ ity forhuman ce lls , bu t ra ther represents a potent endotoxin an-tagon ist. Thus, in the case of lip id A the physio log ica llyremarkab le s itua tion exists that on enzymatic degradation apartia l s tructure is genera ted that is not on ly detoxified butis even ab le to counteract endotoxin b ioactiv ity (45). Studiesof LPS antagonists are important because 1) the y p ro vid edeeper insight into the in teraction of endotoxins w ith hostce lls and the mechan ism of endotoxin bioactivity , and 2) theymay lead to new stra tegies to pharmaco logically contro l en-dotoxin effects a t a very early step. In fact, it has a lreadybeen shown that antagon istica lly active preparations arecapable of in terfe ring w ith endotoxin toxic ity in v ivo (43).

ANTI-ENDOTOXIN ANTIBODIESIt is o f present worldw ide concern that the incidence ofG ram -negative septicem ia , which represents the lead ingcause of le tha lity among hosp ita lized patien ts, is stead ily in-creasing (for lite ra ture, see re f 51). In the United Sta tesa lone, 425,000 cases of bacteria l septicem ia were reported in1987. In the group of G ram -negative bacterem ia, the le tha l-ity ra te is the order of 30% , which is particularly h igh. Th isfigure has remained unchanged over the past years desp iteimprovements in antib io tics and critica l care therapy (52). Inv iew of the lim ita tions encountered w ith the presently avail-able therapeutic m easures, new stra tegies m ust be envisagedin the fight aga inst G ram -negative sepsis, in general, andaga inst those bacterial components, such as endotoxin ,wh ich are he ld responsib le for many of the manifestations ofthe septic shock syndrome, in particular. Due to progressmade in the molecu lar characteriza tion of the bacterial andhost cell s tructures invo lved in in fection-caused in flam ma-tion , the mode of interplay of these structures, and the b io-log ica l consequences of th is in teraction, the design of newpreventive and therapeutic stra tegies is, in fact, rea lis tic to -day. Such new stra teg ies include the development o ff) an ti-bodies to toxic cytokines (e.g ., TNF, IL-i), 2) solublecytok ine receptors (e.g ., TN Fr), 3) cytok ine receptor antago-n is ts (e .g., IL-ira), 4 ) block ing antibod ies to CD i4 and LBP ,5) LPS-neutra liz ing compounds such as the bactericida l/permeability increasing pro te in (BP I), 6 ) LPS antagon istssuch as compound 406, and 7) in particular, o f antiendotoxinantibodies.

Immunological a ttempts to reduce the h igh le tha lity fromsepsis have inc luded the use of enriched imm unoglobu lins(53), human hyperimmune antisera to mutant E . c oiiJ 5 (54),and more recently , m onoclonal antibodies (mAb) to lipid Aof IgM class of human (55) or mouse orig in (56). The tar-geting of endotoxin w ith mAb has several potentia l advan-t ag es b ec au se 1) an early step in the deve lopment o f m ultipleorgan fa ilu re and death is b locked, 2) m Ab have low toxic ityand a prolonged ha lf life in the circu lation, and 3) they pos-sess b io logical activ ity such as complem ent activa tion and in-te raction w ith Fc receptors. The resu lts of recently per-


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formed clinical tria ls w ith immunoglobulins orig ina lly hadinduced hopes for the treatm ent o f septic patients (57).However, critic ism s and uncertainties about the specificityand m echan ism of protection of ava ilab le po lyc lona l antiseraor monoclonal antibod ies have been ra ised (58, 59) and,there fore, the search for new and more effective immuno-g lobulin -based agents is ongoing.In princip le, a ll reg ions of the LPS molecu le are immuno-gen ic and antigenic. Po lyc lona l and monoclona l antibod iesto the 0-specific chain are h igh ly protective in endotoxin andin fection m odels (60). Despite the ir pro tective properties, 0-specific mAb are not like ly to ga in broad app lica tion becauseof the enormous structura l variability of the 0-specific chainof diffe rent pathogenic bacteria .

Because the lip id A component is a common structura le lement o f a ll endotoxins and it represents their endotoxi-ca lly active center, th is LPS segment was considered an idea lta rget for the genera tion of antibod ies that m ight cross-reactw ith many sero log ica lly d is tinct LPS and that m ight even(cross-)protect against endotoxin effects. In fact, m onoclonallip id A antibod ies have been prepared, and their ep itopeshave been characterized (for litera ture , see ref 61). As ex-pected, lip id A monoclona l antibodies cross-react w ith alarge varie ty o f free lip id A of d istinct bacteria l orig in.However, under well-contro lled sero log ical cond itions, inour hands none of these antibodies cross-reacted w ith LPS ,i.e ., w ith lip id A carrying the saccharide portion . Thus, inLPS the lip id A epitopes are not expressed, indicating thatsaccharide-free lipid A represents a neoantigen expressingdeterm inants not present in LPS (such as the prim ary hy-droxyl group of G1cN II). A lternatively , the lip id A ep itopesare not exposed in LPS , i.e., Kdo or o ther LPS constituentssterically h inder the b ind ing of lip id A antibod ies.

The inner core reg ion of G ram -negative bacteria , suchas the Enterobacteriaceae, exhib its a largely conservativestructure that, because of modula ting effects, m ay a lso par-tic ipa te in the induction of endotoxic e ffects. Therefore , alsothe core portion represented an attractive target for theproduction of cross-reactive monoclona l antibod ies. MAb toKdo were prepared and proved to exh ibit cross-reactiv ity(62). They d id, however, not express anti-endotoxin activityin c lass ica l endotoxin test systems such as pyrogen ic ity(E . Th. R ie tsche l, B . Appe lmelk, H . B rade, and F . U .Schade, unpub lished resu lts).

Recently we were ab le to generate broadly cross-reactivemurine mAb, recogniz ing the core reg ion of E. coil an dS a l m o n e l l a R- and S-form LPS , which also exh ib ited anti-endotoxic properties (63, 64). One of these mAbs (WN1-222-5 , IgG2a) has been se lected for further eva luation invivo (64). It was found to effectively inhib it le tha lity in m ice(Table 1) and pyrogen icity in rabb its induced by LPS ofdifferent E. coil core and chemotypes and Salmonella sero-types. To reduce the inc idence of possib le negative sideeffects such as the appearance of h um an anti-m ou se antibod-ies and to increase its ha lf-life , mAb WN1-222-5 has beench im erized in to a human IgG i (SDZ 219-800), which re-ta ined the b ind ing specific ity and cross-reactiv ity of WN1-222-5 . In ELISA , it recognizes the LPS of a ll c lin ical feca lan d urinary isolatesof E . coil, S a l m o n e l l a ,an d S h i g e i i aa s w ellas the ir R mutants in the nanogram range. The smallest LPSstructure bound is that o f the RcP mutant of S . t yp h imu ri um .After DOC-PAGE and immunoblo tting of S -form LPS, SDZ219-800 reacts w ith the h igh molecu lar we ight bands andw ith the fraction contain ing no 0-side cha ins. This showsthat the pro tective ep itope is not covered by add itiona l coresugars or by the 0-specific cha in. SDZ 219-800 re ta ins neu-

TABLE 1. In hib itio n o f L PS -in du ce d le th ality b y m on oc lo na l a nti-L PSantibodies i n m i ce # {1 7 6}

Mo no clo na l a ntib od y,

S urvivin g o ve rtre ate d a nim als afterc ha lle ng e w ith e nd oto xin o fS. cborus equi E . coil 01 61 mg /mo u se (1 ng/mouse) (2 ng /mouse )

WN1-222-5 6/ 6 5/ 6S DZ 219-800 6/6 5/6HA-lA 0/6 1/6Contro l 0/6 1/6

# {176 }M Abere adm in istered iv . 2 h before iv. application of LPS andip a dm in is tra tio n o f D -g ala cto sa min e (16 m g/m ouse) in C 57bI6 m ice.

tra liz ing activ ity as it inh ib its in v itro the Limulus amoebo-cyte lysate assay and the LPS-induced secre tion of m ono-k ines (TNF, IL-6) by mouse peritoneal cells in the presenceor absence of LBP. In v ivo , SDZ 219-800 effectively neutral-izes the pyrogen ic response of rabb its to LPS, b locks TNFand IL-6 production after LPS challenge in guinea pigs, andinhib its the lethal toxic ity of LPS in D -ga lactosam ine-sensitized m ice (Table 1).

These data have important theore tica l and practica l im pli-ca tions. Thus, the cross-reactivities of mAb WNI-222-5 andSD Z 219-800 show that LPS of E . c oi l, S al mo ne ll a, an d S h i g e l i aspecies share an antigen ica lly identical determ inant locatedin the core reg ion. Th is epitope is immunogen ic and anti-gen ic, and the antibody against it is capab le of effectively in-h ib iting endotoxic activ ity . There fore , b ind ing to lip id A isnot a prerequis ite for endotoxin neutralization. The mode ofaction of these antibod ies remains to be studied. It is possib lethat they alte r the endotoxic conformation of lip id A, and inv ivo afte r b ind ing to LPS , they are likely to promote thee lim ination of endotoxin from the circu lation via Fc and C3breceptors. As both mAb WNI-222-5 and SDZ 219-800 be-long to the IgG class, it a lso fo llows that anti-endotoxic ac-tiv ity is not confined to the IgM isotype.

In summary, the potent neutra liz ing properties of themAb WN1-222-5 and SDZ 219-800 unequivoca lly show thatcross-reactive and cross-pro tective anti.-LPS antibod ies canindeed be genera ted. Due to their broad sero log ical reac-tivity and their potent pro tective characteris tics, such mu-rine , and in particu lar chim erized, m Ab represent prom is ingcand idates for immunotherapy of patients suffe ring fromlife -th re ate nin g e nd oto xe mia .

FINAL REMARKSThe sign ificance of endotoxin in in flammatory processes in-duced by Gram -negative bacteria is now w ide ly apprecia ted.Endotoxin has been recognized as a p le io tropic macro-amphiphile that in teracts w ith severa l types of host ce lls andinduces d iffe rent b ioactive endogenous lipid , peptide , andoxygen-derived media tors. In its capacity to a ffect immunecells it constitu tes perhaps the most potent and multiva lentmolecu le of bacteria l orig in. In v iew of the lim ited target cellspectrum and biolog ica l e ffects o f the pro teinous superanti-gens (65), endotoxins deserve to be ca lled hyperantigens ormegami togens.

In addition to the acquired know ledge on the mechan ism slead ing to endotoxic m anifestations, great progress has beenmade in our understand ing of the structure and activ ity ofendotoxin. L ipid A has been proved to represent the toxic


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and immunomodula tory princ iple of endotoxin , its primarystructure has been elucidated, it has been chem ica lly synthe-s ized, and the essentia l structura l requ irements for its bio-log ic activities were elucidated. As a resu lt o f these stud iesthe concept emerged that, compared w ith other phospho-g lyco lip ids, the constitu tion of lip id A is un ique and thatlip id A adopts a peculia r conformation that is responsible forthe induction of characteris tic (endotoxic) bio log ic activities.The existence of humora l and ce llula r bind ing molecu lesw ith h igh affin ity fo r lip id A verifies the exquis ite specific ityo f the interaction of th is m icrobia l tox in w ith host factors,a lthough we are still fa r from understanding a ll m oleculardeta ils o f this interaction and its in tra- and extracellu la r con-sequences.

Phototroph ic G ram -negative bacteria that exis ted a b illionyears ago have typ ica l LPS in the ir outer m embrane (27, 30).Thus, LPS constitu tes phylogenetically an extremely o ldmolecu le that ex is ted long before h igher organisms ap-peared. Th is a lso shows that bacteria produced LPS not be-cause they wanted to irrita te mammalians but simply be-cause it was necessary for the ir survival. The sign ificance ofLPS for bacteria l v iab ility has been intensively stud ied andis now well apprecia ted. However, it is not understood at themolecular leve l which structural or physica l features endowLPS with its v ita l function for bacterial growth , m ultiplica-tion, or surv iva l. Perhaps comparative stud ies w ith mole-cu les d isposing of s im ilar activities or fu lfilling sim ila r func-tions as LPS will he lp to e lucidate re levant chem ica l andphysica l fea tures. W e have recently stud ied a Gram -negativebacterium - S ph in go mo na s p au cim ob ilis - w hich u ne xpe cte dlylacks LPS in its outer membrane. However, S. paucimobiiisproduces a new type of glycolip id consisting of a lip id (cera-m ide) component carrying an acid ic sugar (g lucuron ic acid)to which a trisaccharide is a ttached. In some general fea-tures, therefore , the g lycolip id resembles LPS (66). Threemolecules of this g lycosph ingolip id occupy the same area asone LPS molecule and the g lycolip id forms, as do LPS ,asym metric m em branes in conjunction w ith phospho lipids.It is our hope that analys is o f the chem ica l, physical, and b io-log ica l properties of th is g lyco lipid w ill help to elucidatesome secre ts of the activ ity and, notab ly, the function of bac-terial LPS.

The financial support o f t he D e ut sc he F or sc hu ng sg eme in sc ha ft(B r 731/9 , Scha 402/1-4, Schm 897/1-1, Lo 385/4 -1, Se 532/2-2, andprojects B l [B rade], B2 [S chm id t/R ietsch el], and CS [U lmerl o fSFB 367 is grate fully acknow ledged. T. K irikae was a research fe l-low of the A lexander von Hum boldt-S tiftung. W e apprecia te theprepara tion of art and photograph ic work by M . Lohs andB . K# {246 }h l er ,nd the excellent secre tarial ass is tance of I. Bendt.

REFERENCES1 . N ik aid o, H ., a nd V aa ra , M . ( 19 87 )O ut er m em br an e. I n Escherichza coiian d Salm onella typhim urium . C ellular and M olecular B iology (N eidhardt, C .,Ingraharn, J. L.,Brooks Low, K., Magasanik, B.,Schaechter,M., andUm barge r, H . E ., eds) pp. 7-22, Am erican Socie ty fo r M icrob io logy,W as hin gto n, D .C .2. W estphal, 0 ., W estphal, U ., and Som mer, Th. (1978)The historyofp yr og en re se ar ch . In M ic ro bi ol og y - 19 77 (S chless inger, D ., ed ) pp.221-238, A meric an S ocie ty for M icrob io logy , W ashington, D .C .3. R ietsc hel, E . Th ., and B rade, H . (1992) B acte ria l endoto xin s. &i. Am .

2 67 , 2 6-3 34. A lv ing, C . R . (1993) L ipopolysaccharide, lip id A , and liposom es C on-ta in ing lip id A as im munolo gic adju vants . Immunobiology 1 87 , 4 30 -4 465. L#{252}deritz,. , F re ud en be rg , M . A ., Galanos, C ., Lehmann, V., Riet-schel, E. Th., and Shaw, D. (1982) Lipopolysaccharides of G ram -negati vebacte ria.In Current Topics in M em bran es an d T ra up ort (Razin, S. ,

and Ro ttem , S ., eds) Vo l. 17, pp. 79-151, Academ ic, New York6. F rank, S ., Specter,S ., N ow otny, A ., and Friedm an, H . (1977) Immuno-cyte stim ula tion in v itro by nontox ic bacteria l Iipopo lysaccharidederivatives.]. Immunol. 1 19 , 8 55 -8 607. N ow otny , A . (1987) R eview of the m olecular requirem ents of endo toxicact ions. R ev . I nfe ct. Div. 9, S503-S511

8 . L e bb ar d , S ., C a v ai ll on , I. M . , Caroff , M., Led ur, A., Brade, H.,S ar fa ti, R ., an d H ae ffn er-C av aillo n, N . (1 98 3) Molecular requirementfor in terleuk in I induction by Iipopolysaccharide-s tim ulated hum anm o no cy te s: in vo lv em e nt o f th e h ep to sy l-2 -k eto -3 -d eo xy -o ctu lo so na teregion.Eur. j Immunol. 16 , 87-91

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