Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities

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1 Eur. J. Biochem. 148, 1-5 (1985) 0 FEBS 1985 Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities Chris GALANOS, Otto LUDERITZ, Ernst Th. RIETSCHEL, Otto WESTPHAL, Helmut BRADE, Lore BRADE, Marina FREUDENBERG, U. SCHADE, Masahiro IMOTO, Hiroyuki YOSHIMURA, Shoichi KUSUMOTO and Tetsuo SHI BA Max-Planck-Institut fur Immunbiologie, Freiburg; Forschungsinstitut Borstel, Institut fur Experimentelle Biologie und Medizin, Borstel; and Osaka University, Faculty of Science, Toyonaka, Osaka (Received November 6/December 19, 1984) - EJB The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests. Lipopolysaccharides (endotoxins) of gram-negative bacteria which consist of a heteropolysaccharide and a lipid component (termed lipid A) elicit multiple acute pathophysiological effects such as fever, lethality, Shwartzman reactivity, macrophage and B-lymphocyte activation, and other activities [I]. In 1954 it was proposed that for the induction of these effects the polysaccharide portion is dispensable and that the lipid A component represents the active center responsible for the endotoxic properties of lipopolysaccharides [2]. Evidence for this was then obtained in numerous investigations [2-41 and this concept is now generally accepted. The chemical structure of the lipid A component of several enterobacterial lipopolysaccharides has been analysed during recent years in great detail (for reviews see [5, 61) and it was recognized that lipid A of Escherichiu coli possesses a comparatively simple structure. Free E. coli lipid A consists of a 8(1-6)-linked D-glucosamine disaccharide which is substituted by two phosphoryl groups, one being bound to position 4 of the nonreducing glucosamine residue (GlcN 11) and one being a-linked [7] to the glycosidic hydroxyl group of the reducing glucosaminyl group (GlcN I) (Fig. 1). (In the lipopolysaccharide the glycosidically linked phosphoryl group carries a further phosphoryl residue in nonstoichiometric amounts [7,8].) This hydrophilic backbone is acylated by (R)- 3-hydroxy or (R)-3-acyloxyacyl residues. Specifically, GlcN I1 carries at position 2 amide-linked (R)-3-dodecanoyloxytetradecanoic and at position 3 ester-linked (R)-3-tetrddecanoyloxytetradecanoic acid [ GlcN I carries at both positions 2 and 3 non-acylated (R)-3-hydroxytetradecanoic acid [9, 1 I]. In free lipid A the hydroxyl groups at positions 4 (GlcN I) and 6 (GlcN 11) are free [12], the latter representing the attachment site of the polysaccharide portion in E. coli [I31 (U. Zahringer and S. Kusumoto, unpublished observations) and other lipopolysaccharides (for literature see [6]). A compound structurally corresponding to free E. coli lipid A has recently been synthesized [14]. Here, we report on results obtained with the synthetic lipid A preparation in comparison with bacterial free E. coli lipid A in various in vitro and in vivo endotoxin test systems. Abbreviation. docla, 3-deoxy-~-manno-octulosonic acid. MATERIALS AND METHODS Synthetic lipid A The chemical synthesis of E. coli lipid A has recently been described [14]. Structurally it corresponds to the anticipated formula of bacterial free E. coli lipid A (Fig. 1). The synthetic preparation was dissolved in pyrogen-free water as described previously for synthetic lipid A part structures [15]. Lipopolysaccharide and free lipid A of E. coli Free lipid A was prepared by subjecting the lipopolysaccharide of an E. coli Re mutant (strain F515) [lo, Ill to treatment with acetate buffer (0.1 M, ph 4.4; 100 C; [16]). It consisted of D-glucosamine ( nmol/mg), phosphate Fig. 1. Chernicalstruclure of synthetic andnatural free Escherichia coli lipid A. In E. coli lipopolysaccharide the glycosidic phosphate group of the lipid A backbone is nonstoichiometrically substituted by a phosphoryl residue [8, 131. Numbers in circles indicate the number of carbon atoms in acyl chains. The primary hydroxyl group of the nonreducing glucosaminyl residue (position 6 ) represents the attachment site of docla and, thus, of the polysaccharide portion in lipopolysaccharide

2 2 (1030 nmol/mg), and fatty acids [12:0, 14:0, (3-OH) 14:O; 2651 nmol/mg] and was essentially free of docla (10 nmol/ md. General test systems All chemicals and sera were as described previously [I 51. Biological tests were carried out under the conditions also described in Tumouv necrotizing activity The tumouricidal action of synthetic and natural lipid A was investigated in BALB/c mice employing a syngeneic methylcholanthrene-induced fibrosarcoma (Meth A tumour) obtained originally from Dr L. Old, Sloan Kettering Institute, New York. The tumour, perpetuated as peritoneal ascites, was exudated, the cells washed three times with Pi/NaC1, and suspended in Pi/NaCI (2 x lo6 cells/ml). From this suspension 50 pl (lo5 cells) were injected intradermally in 8-week-old female BALB/c mice. On day 8 after inocculation, solid tumours of 6-mm average diameter had developed. For the tumour necrosis test, groups of six tumour-bearing animals received on day 8 intraperitoneally different amounts (10 pg to 50 pg) of the lipid A sample under test in distilled water (0.2 ml). Six mice receiving distilled water served as control. Limulus amoebocyte lysate gelation Limulus amoebocyte lysate was a gift from Pyroquant, Associates of Cape Cod Inc. After reconstitution according to manufacturer's instructions, 100 p1 of Limulus amoebocyte lysate were added to dilutions (0.01 pg to 0.1 pg) of synthetic or natural lipid A in 100 p1 distilled water in pyrogen-free test tubes. As controls 100 p1 Limulus amoebocyte lysate and 100 p1 distilled water were used. After mixing, the samples were incubated at 37 C (1 h). Gelation was assessed by tilting the tubes and observing for gel formation. Pyrogen-free distilled water and glass-ware were used throughout the experiment. RESULTS Lethal toxicity test The lethal properties of the synthetic lipid A preparation and of the natural free lipid A in galactosamine-sensitized C57B1/6 mice are depicted in Table 1. Both preparations exhibit comparable activity, inducing 100% lethality with 0.1 pg and about 50% lethality with 0.01 pg. Also the kinetics of lethality were very similar for the two preparations, all deaths occurring within 10 h of injection. Pyrogenic properties The pyrogenic activity of the synthetic lipid A was compared to that of natural lipid A in chinchilla rabbits. Table 2 shows that the two preparations induced almost identical fever responses, exhibiting a biphasic rise in rectal temperature at 1 h and 3 h of between 1 "C and 2 C with the two higher concentrations (0.1 and 0.01 pg/kg). With the lowest concentration (0.001 pg/kg) only the first peak (at 1 h) of fever response was obtained with an average rise in temperature of 0.7"C for the synthetic and natural lipid A. Table 1. Lethal toxicity of synthetic and natural lipid A in galactosamine-sensitized mice The animals received D-galactosamine (2.6 mmol/kg) intraperitoneally in 0.4 ml P,/NaCI and immediately afterwards the various amounts of the two lipid A preparations intravenously in 0.2 ml Pi/ NaCI. Lethality is shown as number dead per number tested Preparation Lethality with 0.1 PLg 0.01 pg pg Synthetic lipid A lo/lo 13/20 OjlO Natural lipid A lo/lo 10/20 O/lO Table 2. Pyrogenicity of synthetic and natural lipid A Chinchilla rabbits ( kg) received intravenously 0.1, 0.01, and pg/kg of either lipid A preparation. Fever responses are presented as the rise in temperature at 1 h and 3 h after injection Preparation Dose Fever response at Ih Pg/kg "C Synthetic lipid A o Natural lipid A 0.1 I Local Shwartzman reaction The property of the synthetic and natural lipid A in preparing the skin for or in eliciting the Shwartzman reaction was investigated in New Zealand white rabbits both in homologous and cross-reacting systems. To enable better comparison, identical amounts of the two preparations were applied intradermally, alongside in the same animal. The results of Table 3 are representative of three different experiments. Both preparations were active to a comparable extend, inducing strong skin lesions in concentrations between 50 pg and 12.5 pg, administered intradermally. In fact, in the lower concentrations, 6.2 pg and 3.1 pg, the synthetic preparation was reproducibly more active than natural lipid A, both in the homologous and cross-reacting systems. Results very similar to the above were also obtained in a cross-shwartzman system in which synthetic lipid A and intact lipopolysaccharide from Salmonella abortus equi were used, respectively, as preparative and eliciting reagent and vice versa (results not shown). Limulus amoebocyte lysate gelating activity Both preparations induced complete gelation of Limulus amoebocyte lysate in amounts up to 10 pg. Thus, also in this test system the activity of synthetic lipid A was identical to that of natural lipid A. Tumouricidal activity Following treatment of tumour-bearing mice with synthetic or natural lipid A, depending on the amount injected, 3h

3 3 a hemorrhagic necrosis appeared in the tumour mass 24 h later. In some cases this initial necrosis caused only a retardation in tumour growth (compared to controls) and after several days of a stationary phase, the tumours began to grow again. In other animals the tumours became completely necrotic after 3-6 days and the animals were completely healed 3-4 weeks later. For this reason, in Table 4 the results are expressed both as a hemorrhagic necrosis regardless of final outcome, and as healing. The results of Table 4 show that synthetic lipid A is indistinguishable from natural free lipid A in its turnour Table 3. Induction of local Shwartzrnan reaction by synthetic and natural lipid A New Zealand white rabbits were prepared by an intrddermal injection of different amounts of the two preparations in a volume of 100 pl. After 24 h the animals were challenged by an intravenous injection (50 pg) of either preparation. Number of crosses indicates the degree of skin necrosis 4 h after the intravenous injection Pretreatment (day 0, intrddermal injection) Skin reaction after intravenous challenge (day 1, 50 pg) with preparation dose synthetic natural lipid A lipid A pg Synthetic lipid A Natural lipid A necrotizing properties. In amounts of 50 pg, both preparations lead to tumour necrosis in 80% and to complete healing in 70% of the animals. Endotoxin cross-tolerance Endotoxin tolerance was induced by pretreatment of groups of NMRI mice on day 0 with synthetic or natural lipid A. As Table 5 shows, significant tolerance to a 150 pg challenge with S. abortus equi lipopolysaccharide had developed 4 days after pretreatment in all groups. This is indicated by the significant reduction of lipopolysaccharide lethality after pretreatment with synthetic (92% survival) and natural lipid A (100%) versus the control group (8%). Mitogen icity Table 6 shows the mitogenic activity of the synthetic and natural lipid A preparations for spleen cells of C3H/TiF mice (expressed as mitogenic index). Controls without mitogen had a background value of 932 cpm. Natural lipid A elicited a dose-dependent mitogenic response which was maximal with 2 pg, resulting in a fifteenfold increase of thymidine incorporation (14635 cpm) as compared to controls. With the synthetic compound a very similar dose-response curve was obtained (14030 and cpm for 2 pg and 10 pg, respectively). As expected neither the synthetic nor the natural lipid A preparation stimulated spleen cells from the lipopolysaccharide-nonresponder mouse strain C3H/HeJ (data not shown). Table 6. Mitogenicity of synthetic and natural lipid A Spleen cells (8 x lo5) of C3H/TiF lipopolysaccharide-responder mice were incubated (37"C, 48 h) with the preparations followed by a pulse with [3H]thymidine (16 h) Table 4. Anti-turnour activity of synthetic and natural lipid A Balb/c mice (6 in each group) received lo5 Meth A cells intradermally. Eight days later the animals received the preparation (50 pg) intraperitoneally. The results represent the accumulative values of two experiments Preparation Necrosis Necrosis and healing Synthetic lipid A (50 pg) 9/ Natural lipid A (50 pg) /12 Dose <I Mitogenic index synthetic lipid A natural lipid A <1 Table 5. Induction of cross-tolerance to S. abortus equi lipopolysaccharide by synthetic and natural lipid A Groups of 12 mice were treated on day 0 with 30 pg of each synthetic or natural free lipid A and challenged on day 4 with S. abortus equi lipopolysaccharide (1 50 pg). Survival was recorded for 72 h Pretreatment (day 0) Challenge (day 4) Number surviving per animals tested preparation dose lipopolysaccharide dose pg Synthetic lipid A 30 S. abortus equi /12 Natural lipid A 30 S. abortus equi /12 Pi/NaC1 (control) 0.2 ml S. abortus equi 150 1/12

4 4 Table 7. Induction of prostaglandin synthesis in mouse peritoneal macrophages by synthetic and natural lipid A Cells (1 x lo6) were incubated with 1, 10 and 50 pg of each lipid A sample at 37 C for 24 h. Prostaglandins E2 and Fzcl were determined in supernatants by radioimmunoassay Preparation Dose Amount released of prostaglandin Synthetic lipid A Natural lipid A Pi/NaC1 (control) Induction of prostaglandin synthesis Mouse peritoneal macrophages were incubated with different doses (1 pg, 10 pg, 50 pg) of the natural and synthetic lipid A preparation and the release of prostaglandins Ez and Fza was determined (Table 7). It was found that both preparations were of similar activity in inducing a significant dosedependent increase in formation of prostaglandins Ez and Fza over control levels. Antigenic properties The property of synthetic lipid A to cross-react with natural lipid A were assessed by the passive hemolysis and passive hemolysis inhibition tests as described earlier [15]. In the passive hemolysis test, sheep erythrocytes coated with synthetic lipid A were lyzed by anti-salmonella lipid A antiserum to the same extent (hemolytic titre 1028) as those coated with free lipid A from E. coli. In the inhibition test the lysis of erythrocytes coated with either natural or synthetic lipid A was inhibited to the same extent ( bg) by both natural or synthetic lipid A, showing the presence of complete serological cross-reactivity between the two lipid A preparations. DISCUSSION A synthetic lipid A having the complete structure of E. coli lipid A (Fig. 1) was found to be fully active as endotoxin, and indistinguishable from natural lipid A in all biological parameters tested. Lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction, i.e. the three classical parameters of endotoxic activity, were all expressed by the synthetic preparation to the same degree as by natural free lipid A. The synthetic preparation was also active in a number of other parameters that are known to be expressed by endotoxin. Thus, synthetic lipid A was mitogenic for mouse B-lymphocytes and induced prostaglandin synthesis in mouse peritoneal macrophages. Similarly, the synthetic preparation induced cross-tolerance in mice to the lethal effects of S. abortus equi lipopolysaccharide, and tumour necrosis in Meth A tumour-bearing BALBc mice. Finally, synthetic lipid A induced gelation of Limulus amoebocyte lysate and showed complete serological cross-reaction with natural free lipid A in the passive hemolysis and passive hemolysis inhibition tests. In all above parameters the activity of the synthetic preparation was identical to that of natural free lipid A. Previous studies on synthetic lipid A analogues [19, 201 or on structures representing lipid A in different degrees of completion [15, 17, 181 had revealed that some preparations exhibited biological activity, like interaction with the complement system, induction of prostaglandin synthesis and mitogenicity. Also, most preparations were antigenically active. This would be expected because it had already been established that for the expression of lipid A antigenicity the presence of ester-linked fatty acids and phosphate are not necessary [21]. None of the incomplete forms of lipid A, however, expressed all activities of natural lipid A to the full extent. Thus, significant lethal toxicity comparable to that of natural lipid A was expressed only by the synthetic lipid A precursor. This preparation, however, was of low pyrogenic activity, and the property to induce the local Shwartzman reaction was completely absent. Synthetic and natural lipid A precursor contain a B(1-6)-linked D-glucosamine disaccharide, bisphosphorylated at positions 1 and 4 and carrying 4mol of (R)-3-hydroxytetradecanoyl residues at positions 2, 3, 2 and 3. Therefore, the lipid A precursor differs from E. coli free lipid A by the absence of two non-hydroxylated fatty acids, dodecanoic and tetradecanoic acid. The low pyrogenicity and absence of the property to induce the Shwartzman reaction from the synthetic and natural lipid A precursor, and the full expression of these properties by the synthetic and natural lipid A make it evident that the presence of the additional two fatty acids endows full pyrogenic activity and confers the property to induce the local Shwartzman reaction. Therefore, full expression of endotoxic activity is exhibited by a structure in which all constituents of lipid A, glucosamine, fatty acids and phosphate occupy specific positions in the molecule. The synthetic preparation tested here fullfils the requirements and represents the first synthetic lipid A exhibiting classical endotoxic activity to a full extent. The data provide proof that lipid A represents the endotoxic principle of lipopolysaccharides and they support the structure anticipated for E. coli lipid A (Fig. 1). The expert technical assistance of M.-L. Gundelach, H. Stubig, U. Berger and B. Henselmann are gratefully acknowledged. We thank Dr T. J. Novitsky for the generous gift of Limulus amoebocyte lysate. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Br 73113) and by the Fond der Chemischen Industrie (OLu, EThR). REFERENCES 1. Luderitz, O., Freudenberg, M. A., Galanos, C., Lehmann, V., Rietschel, E. Th. & Shaw, D. H. (1982) Curr. Top. Membr. 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