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Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis
FEMS Immunology and Medical Microbiology 28 (2000) 273^281
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Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis Tomohiko Ogawa a; *, Yasuyuki Asai a , Hiroyo Yamamoto a , Yasuhiro Taiji a , Takayoshi Jinno a , Tohru Kodama b , Shinjiro Niwata b , Hidetoshi Shimauchi c , Kuniyasu Ochiai d a
c
Department of Oral Microbiology, Asahi University, School of Dentistry, 1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan b Institute for Fundamental Research, Suntory Limited, 120-1 Takahama, Shimamoto-cho, Mishima-gun, Osaka 618-0012, Japan Department of Periodontology and Endodontology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan d Department of Microbiology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan Received 21 February 2000; received in revised form 29 March 2000 ; accepted 30 March 2000
Abstract A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Synthetic lipid A; Endotoxicity; C3H/HeJ mouse; Macrophage; Gingival ¢broblast; Porphyromonas gingivalis
1. Introduction Porphyromonas gingivalis is one of the suspected periodontopathic bacteria and has been frequently isolated from the periodontal pockets of patients with chronic periodontal diseases [1]. This organism is a Gram-negative, anaerobic oral black-pigmented rod which has lipopolysaccharides (LPS) located on the cell surface. It has been reported that the chemical and biological properties of P. gingivalis LPS and its active center, lipid A, are identical to those of LPS from Bacteroides fragilis, but di¡erent from those of classical enterobacterial LPS and their lipid As [2]. LPS is a major component of the outer membrane of Gram-negative bacteria, and is well-known to exhibit various biological responses for host cells [1]. Lipid A, the lipophilic portions of LPS whose structures correspond to
those proposed for Escherichia coli-, Salmonella-, and other enterobacterial type lipid As, were chemically determined and synthesized [3^5]. These chemically well-de¢ned synthetic lipid A compounds have been investigated extensively for the structure^function relationships of lipid As. It was previously demonstrated that puri¢ed P. gingivalis lipid A exhibited a quite di¡erent phosphorylation and acylation pattern as compared with enterobacterial lipid As [6]. The present study was designed to investigate the endotoxicity and immunobiological activities of a synthetic lipid A of P. gingivalis, and compare them with those of its bacterial lipid A product and synthetic E. colitype lipid A. 2. Materials and methods 2.1. Animals
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BALB/c, C57BL/6, C3H/HeN, and C3H/HeJ mice
0928-8244 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 0 0 ) 0 0 1 6 7 - X
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(males, 8 weeks old) were obtained from Japan SLC, Hamamatsu, Japan. Domestic Japanese white rabbits were purchased from Nihon Rabbit, Osaka, Japan. 2.2. Bacteria and LPS preparation P. gingivalis strain 381 was grown anaerobically in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin and menadione at 37³C for 26 h. Bacterial cells were collected by centrifugation, washed three times with pyrogen-free water, and lyophilized. LPS was extracted from lyophilized cells by the hot phenol/water method [7], and the puri¢cation of the crude extract was performed by repeated ultracentrifugation (100 000Ug, 3 h) followed by treatment with nuclease P1 (Yamasa Shoyu, Choshi, Japan) and ¢nally lyophilized [6]. 2.3. Preparation and puri¢cation of lipid A Puri¢ed LPS was hydrolyzed in 0.6% acetic acid at 105³C for 2.5 h, then cooled and neutralized. The hydrolysate was made up to 100 ml with H2 O and added to 200 ml of CHCl3 /MeOH/H2 O/triethylamine (30/12/2/0.1). The lower phase was evaporated under reduced pressure. Subsequently, the lipid A fractions were separated by silica gel column chromatography (Silica Gel 60, 230^400 mesh: Nacalai Tesque, Kyoto, Japan) with CHCl3 /MeOH/H2 O/ triethylamine (30/12/1.5/0.1). The eluants were monitored by thin-layer chromatography (TLC) on a silica gel 60 plate. The plates were developed with CHCl3 /MeOH/ H2 O/triethylamine (30/12/2/0.1) and visualized by sulfuric acid, Dittmer^Lester reagent [8], and triphenyltetrazolium chloride (TTC) reagent [9]. The slow moving compound (Rf = 0.3), observed as a single spot, was recognized as the major component [6]. The chemical structure of lipid A from LPS of P. gingivalis strain 381 was characterized using conventional chemical procedures, gas-liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry [6]. 2.4. Synthesis of P. gingivalis strain 381 lipid A P. gingivalis derived lipid A is shown in Fig. 1A [6]. The £uoro-sugar (Fig. 1B) was condensed with 4,6-dihydroxy sugar (Fig. 1C), with bis (cyclopentadienyl) hafnium dichloride (Aldrich, Milwaukee, WI, USA) and silver perchlorate hydrate (AgClO4 , Aldrich) used to generate the disaccharide (Fig. 1D) [10]. The £uoro-sugar (Fig. 1B) was prepared from N-acetyl-D-glucosamine (Aldrich) in six steps ; methyl glycoside formation with cation exchange resin (Amberlite IR-120B from Organo, Tokyo) in methanol, benzylation of the hydroxy groups, demethylation and deacetylation with 6N-HCl/dioxane under re£ux, protection of the 2-amino group with 2,2,2-trichloroethyl chloroformate (Troc-Cl, Aldrich), acetylation of the 1-hydroxy group by usual methods, and then £uorination of
Fig. 1. Total synthesis of P. gingivalis 381 derived lipid A. Synthetic procedures for the chemically de¢ned structure of the lipid A backbone of LPS from P. gingivalis 381 are described in Section 2.
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the 1-acetoxy group with HF/pyridine (Aldrich) [11]. The 4,6-dihydroxy sugar (Fig. 1C) was prepared from D-glucosamine hydrochloride (Aldrich) in ¢ve steps; protection of the 2-amino group with 9-£uorenylmethyl chloroformate (Fmoc-Cl, Aldrich), allyl glycoside formation with HCl/dioxane/allyl alcohol, 4,6-benzylidenation with benzaldehyde dimethyl acetal (Aldrich) and p-toluenesulfonic acid in N,N-dimethylformamide (DMF), benzylation of the 3-hydroxy group with benzyl bromide (Aldrich) and Ag2 O (Aldrich) in CH2 Cl2 [12], and then deprotection of the 4,6-benzylidene group with HCl/dioxane. The two fatty acid moieties, 3-(R)-benzyloxy-15-methylhexadecanoic acid (Fig. 1E) and 3-(R)-palmitoyloxy-15methylhexadecanoic acid (Fig. 1F), were introduced into the disaccharide derivative (Fig. 1D) by respective deprotection and acylation of the protected amino groups (Fmoc-amino and Troc-amino groups) to obtain the fatty acyl disaccharide (Fig. 1G). 3-(R)-Benzyloxy-15-methylhexadecanoic acid (Fig. 1E) was obtained from 3-(R)-hydroxy-15-methylhexadecanoic acid (Wako Pure Chemical Industries, Osaka, Japan) in three steps; esteri¢cation with phenacyl bromide (Aldrich) in triethylamine (Et3 N)/ethyl acetate (AcOEt), benzylation with benzyl bromide and Ag2 O in benzene, and then de-esteri¢cation with Zn in acetic acid (AcOH). 3-(R)-Palmitoyloxy-15-methylhexadecanoic acid (Fig. 1F) was also prepared from 3-(R)-hydroxy-15-methylhexadecanoic acid in three steps; phenacyl ester formation, esteri¢cation of the 3-hydroxy group with palmitoyl chloride (Aldrich) and N,N-dimethylaniline (Aldrich) in benzene, and then deprotection of the phenacyl group. The fatty acyl disaccharide (Fig. 1G) was converted to a 1-phosphate derivative (Fig. 1H) using n-butyl lithium-dibenzyl phosphorochloridate [13], and then all of the protecting groups were reductively removed to generate the synthetic lipid A of P. gingivalis 381 (Fig. 1A) by treatment with palladium catalyst. This ¢nal product was proven by pmr analysis to contain a L-con¢guration of the glycosidic linkage and an K-con¢guration of the phosphorylated position. 2.5. Reference synthetic product The E. coli-type synthetic lipid A (compound 506) used in this study was synthesized as described by Imoto et al. [14]. The bacterial and synthetic products were dissolved at a concentration of 2 mg ml31 in 0.1% triethylamine aqueous solution. The stock solution was kept at 4³C and appropriately diluted with pyrogen-free phosphatebu¡ered saline (PBS; Biken, Osaka) or cell culture medium before use in the assay. 2.6. Preparation of peritoneal macrophages Peritoneal exudate cells were obtained from C3H/HeN and C3H/HeJ mice by washing with 5 ml of PBS, followed
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by aspiration of the wash from the peritoneal cavity [15]. The cells were washed once with and resuspended in RPMI 1640 medium (Nikken Biomedical Laboratories, Kyoto) supplemented to a ¢nal concentration of 100 U ml31 penicillin, 100 Wg ml31 streptomycin, 300 Wg ml31 L-glutamine, and 10% (v/v) fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), and then added to 24-well culture plates (FALCON 3047; Becton Dickinson, Lincoln Park, NJ, USA) at a concentration of 106 cells per well, where they were incubated for 60 min at 37³C in 5% (v/v) CO2 . Non-adherent cells were removed, and the adherent cells were washed three times with PBS and resuspended in RPMI 1640 medium supplemented with 10% FBS. The number of viable cells was determined using the trypan blue exclusion technique. 2.7. Preparation of murine gingival ¢broblasts (MGF) Gingival tissues (ca. 2^5 mg) were obtained from two or three C3H/HeN and C3H/HeJ mice. The explants were cultured in K minimal essential medium (K-MEM ; Nikken Biomedical Laboratories) supplemented to a ¢nal concentration of 50 Wg ml31 gentamicin, 50 ng ml31 amphotericin B (Sigma, St. Louis, MO, USA), and 10% (v/v) fetal calf serum (FCS; Sigma) in plastic culture dishes [16]. The MGF that showed a slim and spindle-shape morphology under a phase contrast microscope [17] were designated MGFN if from C3H/HeN mice and MGFJ if from C3H/HeJ mice. The cells were grown and maintained in K-MEM containing 10% FCS at 37³C in a 5% (v/v) CO2 atmosphere, and were used at the 5th and 13th passages in the assay. 2.8. Lethal toxicity in galactosamine-loaded mice Groups of ¢ve C57BL/6 mice were injected intraperitoneally with 16 mg of D-galactosamine hydrochloride (Wako Pure Chemicals) in 0.5 ml of PBS, followed immediately by an intravenous injection of the indicated doses of test specimens in 0.2 ml of PBS by the method of Galanos et al. [18]. The death of the mice due to intoxication was observed over a 24-h period. The 50% lethal dose (LD50 ) for each group was calculated by the method of Ka«rber [19]. 2.9. Pyrogenicity Three domestic Japanese white rabbits (male; 2^2.5 kg) per group were injected intravenously with the indicated doses of the test specimens in 10 ml of pyrogen-free PBS per kg as described previously [20]. The rectal temperature of each rabbit was measured and recorded continuously with an automatic device (EP670, 12; Iio Electric Co., Tokyo, Japan). The measurements in the present study were simpli¢ed to determine the rectal temperature of each rabbit every 30 min between 1 and 1.5 h (peak I)
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and between 2.5 and 4 h (peak II) after injection of the test specimens for the ¢rst- and second-phase temperature, respectively. An increase in rectal temperature of more than 0.6³C was taken as a positive febrile response. 2.10. Limulus test A Limulus test was performed with Pre Gel, an amebocyte lysate prepared from Tachypleus tridentatus (Seikagaku Kogyo, Tokyo, Japan) according to the manufacturer's directions. A solution of E. coli O111:B4 LPS-W (Difco Laboratories, Detroit, MI, USA) incubated in the reagent kit was used as a reference standard. The dose which caused a reaction de¢nitely stronger than the increased viscosity was taken as the minimum e¡ective dose. 2.11. Hemagglutinating assay A hemagglutination assay was performed with a 96-well round-bottom microtiter plate (Cell Wells 25850 ; Corning Glass Works, Corning, NY, USA) as described previously [21]. Test specimens (25 Wl) diluted serially twofold in PBS were added to an equal volume of a 2% rabbit erythrocyte suspension in PBS. After gentle shaking, the microtiter plate was incubated at 37³C for 2 h, and the hemagglutination titer was shown as the concentration of test specimens giving a positive reaction by visual inspection. 2.12. Mitogenicity Murine mononuclear cells (MNC) were isolated by Histopaque (Sigma) separation from spleen cells of BALB/c, C3H/HeN, and C3H/HeJ mice [22]. Splenic MNC (each 2.5U105 cells) were cultured with the indicated doses of test specimens in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS in a 96-well £at-bottom microtiter plate (Falcon 3040 ; Becton Dickinson) for 48 h at 37³C in 5% (v/v) CO2 . Thirty-seven kiloBequerel of methyl3 H-thymidine ([3 H]thymidine; 185 MBq nmol31 ; Amersham International, Amersham, UK) was added during the ¢nal 16 h of culture to estimate DNA synthesis, and all cultures were harvested onto glass paper ¢lter strips. Thymidine uptake was measured in a liquid scintillation counter (model 1215 ; LKB-Wallec, Turku, Finland). The e¡ect of polymyxin B on mitogenic responses in BALB/c, C3H/HeN, and C3H/HeJ mice was examined as follows. Splenic MNC (each 2.5U105 cells) were cultured at 37³C for 48 h in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS with the indicated doses of polymyxin B sulfate (5U105 U per vial; Wako Pure Chemicals) and 10 Wg of a test specimen, except for 5 Wg of concanavalin A (Con A; Sigma) [23]. Cultures were pulsed for the ¢nal 16 h of incubation with 37 kBq of [3 H]thymidine, and then thymidine incorporation was determined as described above.
2.13. Cytokine assay Peritoneal macrophages were suspended at a cell density of 2U106 cells ml31 of RPMI 1640 medium supplemented with 10% FBS. These cells (2U105 cells per well) were incubated with or without the indicated doses of the test specimens for 24 h at 37³C in humidi¢ed air containing 5% (v/v) CO2 in a 96-well £at-bottom microtiter plate. MGF were also suspended at 2U105 cells ml31 of K-MEM, and the indicated doses of the test specimens were added to the cell cultures (2U104 cells per well) and incubated at 37³C for 18 h. After incubation, the supernatants were collected and stored at 380³C until the assay for cytokine production. The production of interleukin-6 (IL-6) and tumor necrosis factor K (TNF-K) was measured in the culture supernatants by means of a commercial ELISA kit system (Endogen, Cambridge, MA, USA). The assay was performed according to the manufacturer's instructions, and the data were determined using a standard curve prepared for each assay. 2.14. Statistics Data were analyzed by a one-way analysis of variance (ANOVA), using the Bonferroni or Dunn method, and the results are presented as the mean þ S.E.M. When an individual study is demonstrated, it is representative of at least three independent experiments. 3. Results 3.1. Endotoxic activities Compound PG-381 lacked lethal toxicity at a dose of up to 20 Wg per mouse (data not shown), and the LD50 for the natural lipid A of P. gingivalis was 12.6 Wg per mouse [24]. The toxicity of compound PG-381 was weaker than that of its natural lipid A. Compound 506 was markedly toxic in galactosamine-loaded mice, and its LD50 was 0.0079 Wg per mouse [24]. The toxicity of compound PG-381 and its natural lipid A were considerably weaker than that of compound 506. Rabbits injected with compound PG-381 at a dose of 10 Wg kg31 showed no induction of peak I and peak II responses, while natural lipid A of P. gingivalis at a dose of Table 1 Activation of the clotting-enzyme cascade of T. tridentatus amebocyte lysate (Limulus test) by P. gingivalis natural and synthetic lipid A, and E. coli-type synthetic lipid A (compound 506) Test specimen
Minimum e¡ective dose (ng per test)
Compound PG-381 P. gingivalis natural lipid A Compound 506
10 10 0.01
Data shown were obtained by the Pre Gel test.
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Fig. 2. Mitogenic e¡ect of P. gingivalis synthetic lipid A on splenic MNC from BALB/c, C3H/HeN, and C3H/HeJ mice. Cells were cultured for 48 h with or without the indicated dose of each test specimen as follows : compound PG-381 (b), P. gingivalis natural lipid A (a), and compound 506 (F). The results are expressed as stimulation index, a value that was determined using the formula : stimulation index = counts per minute (cpm) for test culture/cpm in culture with medium alone. The counts (mean þ S.E.M.) in control cultures were 1151 þ 36 cpm in BALB/c MNC, 1997 þ 45 cpm in C3H/ HeN MNC, and 2535 þ 105 cpm in C3H/HeJ MNC. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean þ S.E.M. Signi¢cant di¡erences between the groups with or without test specimen administered (*P 6 0.01).
10 Wg kg31 exhibited very low responses at peak II and none at peak I (data not shown, [24]). However, compound 506 at a dose of 0.1 Wg kg31 induced positive peak I and peak II responses. The results obtained by a conventional Limulus test showed that compound PG-381 exhibited a weak activity as compared to that of the natural product, P. gingivalis 381 lipid A (Table 1). However, their activities were 1/1000 of that of compound 506. 3.2. Immunobiological activities Compound PG-381 strongly agglutinated rabbit erythrocytes at a minimum concentration of 0.004 Wg ml31 , and
the natural lipid A of P. gingivalis induced hemagglutination at a minimum concentration of 0.015 Wg ml31 . Compound 506 also caused hemagglutination at a concentration of 0.06 Wg ml31 . These results indicated that compound PG-381 as well as the natural lipid A of P. gingivalis exhibited stronger hemagglutinating activities than compound 506. The natural lipid A of P. gingivalis 381 exhibited almost comparable mitogenic activities as compound 506, and compound PG-381 elicited weak but signi¢cant mitogenic responses in splenic MNC from both BALB/c and LPSresponder C3H/HeN mice (Fig. 2). Compound PG-381 and its natural lipid A showed signi¢cant mitogenic responses in splenic MNC from LPS-hyporesponder C3H/
Fig. 3. E¡ect of polymyxin B on the mitogenic responses of splenic MNC from BALB/c, C3H/HeN, and C3H/HeJ mice to P. gingivalis synthetic lipid A. Cells were cultured for 48 h with 10 Wg per well of compound PG-381 (b), P. gingivalis natural lipid A (a), compound 506 (F), 5 Wg per well of Con A (R), or RPMI 1640 medium alone (O), containing the indicated dose of polymyxin B. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean þ S.E.M. of results.
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Fig. 4. Cytokine production by peritoneal macrophages from C3H/HeN and C3H/HeJ mice in response to stimulation by P. gingivalis synthetic lipid A. Cells were cultured at 37³C for 24 h in RPMI 1640 medium containing 10% FBS, with or without the indicated dose of each test specimen. After incubation, the supernatants were collected and cytokine production was determined by ELISA. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean+S.E.M. of results. Signi¢cant di¡erences between the groups with and without the test specimens (*P 6 0.01).
HeJ mice, whereas compound 506 was not found to have mitogenic activities. The antibiotic polymyxin B has been shown to bind to the lipid A region of LPS [25]. We further investigated the inhibitory e¡ect of polymyxin B on the mitogenic activities of natural and synthetic lipid As on the splenic MNC from the strains of mice used in the present study. The addition of polymyxin B to BALB/c and C3H/HeN MNC cultures markedly inhibited the thymidine incorporation by compound PG-381 and its natural product, as well as compound 506, but not by Con A (Fig. 3). In splenic MNC from C3H/HeJ mice, polymyxin B also completely inhibited the thymidine incorporation of MNC stimulated with compound PG-381 as well as the natural lipid A, but not with Con A. Cytokine production by peritoneal macrophages and gingival ¢broblasts from C3H/HeN and C3H/HeJ mice, after stimulation with the natural and synthetic lipid As,
was examined. Compound PG-381 de¢nitely induced IL-6 and TNF-K production in peritoneal macrophages from C3H/HeN mice, however, the activities were weaker than those of its natural product (Fig. 4). Natural lipid A of P. gingivalis also exhibited these cytokine-producing activities on C3H/HeJ peritoneal macrophages, whereas the stimulation of the macrophages with compound PG-381 and compound 506 resulted in no cytokine-producing activity. IL-6 production in MGF was examined after stimulation with the natural and synthetic lipid As. P. gingivalis natural and synthetic lipid As, as well as compound 506, de¢nitely exhibited cytokine-producing activities in MGFN derived from C3H/HeN mice. The natural lipid A of P. gingivalis also elicited IL-6 production in MGFJ derived from C3H/HeJ mice. However, stimulation with compound PG-381 as well as compound 506 resulted in no IL-6 production in the MGFJ from C3H/HeJ mice, as shown in Fig. 5.
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Fig. 5. IL-6 production in gingival ¢broblasts from C3H/HeN and C3H/HeJ mice in response to stimulation by P. gingivalis synthetic lipid A. Cells were cultured at 37³C for 18 h in RPMI 1640 medium containing 10% FBS with or without the indicated dose of each test specimen. After incubation, the supernatants were collected and cytokine production was determined by ELISA. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean+S.E.M. Signi¢cant di¡erences between the groups with and without the test specimens (*P 6 0.01).
4. Discussion The present study, using a chemical synthesized lipid A, showed that the lipid A portion of LPS from P. gingivalis exhibits no or very low endotoxic activities, as demonstrated by tests of lethal toxicity in galactosamine-loaded mice and pyrogenicity in rabbits as well as activity in Limulus test. These activities of P. gingivalis synthetic lipid A, like its natural lipid A, were considerably weaker than those of E. coli-type synthetic lipid A (compound 506). The P. gingivalis synthetic lipid A backbone consists of a L-(1-6)-linked glucosamine disaccharide which is phosphorylated at the 1 position of the reducing sugar, however, this lipid A structure lacks an ester-linked phosphate group which is bound to the hydroxy group at the 4P-position of the nonreducing sugar (Fig. 1). It was also found that the hydroxyl groups at the 3-, 3P-, 4-, 4P-, and 6P-positions of the P. gingivalis lipid A backbone are free. Furthermore, it was shown that the P. gingivalis lipid A molecule possesses 3-hydroxy-15-methylhexadecanoic acid and 3-hexadecanoyloxy-15-methyl-hexadecanoic acid, which are amide linked at the 2- and 2P-positions, respectively (Fig. 1). P. gingivalis lipid A has a distinctly di¡erent structure from enterobacterial lipid As. Namely, it has been reported that there is no 4-O-phosphoryl group in the lipid A backbone of B. fragilis [26^29] or Bacteroides intermedius [30]. This chemical structure is suggested to be the cause of very low toxic activities, as indicated in the present study. It has also been demonstrated that among the bisphosphoryl disaccharide compounds so far described, removal of a 1- or 4P-phosphate group from 1,4P-bisphospho-disaccharide synthesized compounds clearly reduced endotoxicity, as compared with the parent molecule [5].
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We previously demonstrated that P. gingivalis LPS and its lipid A de¢nitely caused agglutination of rabbit erythrocytes [24]. In contrast, others have reported that P. gingivalis LPS had no hemagglutinating activity [31,32]. Kirikae et al. also suggested that the non-hemagglutinating activity of P. gingivalis LPS may be attributed to differences in the chemical structure of the lipid A moiety. In this study, synthetic lipid A as well as natural lipid A of P. gingivalis possessed stronger hemagglutinating activities than compound 506. Thus, the P. gingivalis lipid A molecule was found to possess the capability to induce agglutination of rabbit erythrocytes. P. gingivalis synthetic lipid A, similar to its natural product, was found to stimulate splenic MNC of both LPS-responder C3H/HeN and LPS-hyporesponder C3H/ HeJ mice, at a level weaker than that induced by natural lipid A, whereas compound 506 stimulated splenic MNC of C3H/HeN, but not C3H/HeJ mice (Fig. 2 and [24]). We and other research groups have previously demonstrated that LPS and its natural lipid A from P. gingivalis were mitogenic for splenic MNC and B-cells from both of these murine strains [23,24,33,34]. The total protein content in P. gingivalis lipid A specimen was scarcely detectable by amino acid analysis, as described previously [35]. Whether a trace of protein has an in£uence on the mitogenic activity of C3H/HeJ mice remains to be shown. The present study appears to clarify this possibility point with synthetic lipid A, and the stimulatory e¡ect on the splenocytes of C3H/HeJ mice is suggested to be attributable to a unique structure of the lipid A portion of P. gingivalis LPS. Furthermore, the mitogenicity of P. gingivalis synthetic lipid A has shown to be inhibited by the addition of polymyxin B to all BALB/c, C3H/HeN and C3H/HeJ splenic MNC cultures so far examined. The lipid As of P. gingivalis and E. coli are the bioactive centers in their respective LPS. The present ¢ndings demonstrate that we have been able to accomplish the synthesis of a lipid A preparation with endotoxic and immunobiological properties nearly identical to those of natural lipid A of P. gingivalis 381. However, there are marked di¡erences between compound PG-381 and its natural lipid A in regard to immunobiological activities, such as their cytokine-producing activities in peritoneal macrophages and gingival ¢broblasts from C3H/HeJ mice. We found that compound PG-381 exhibited de¢nite cytokine-producing activities in both macrophages and ¢broblasts from C3H/HeN mice, as did natural lipid A of P. gingivalis and compound 506. However, both compound PG-381 and compound 506 had no response in the same cells from C3H/HeJ mice. In contrast, the natural lipid A caused an activation of C3H/HeJ peritoneal macrophages and gingival ¢broblasts. We previously reported that the natural lipid A of P. gingivalis 381 induced IL-1L production in splenic C3H/HeJ macrophages [22]. Tanamoto et al. also demonstrated that the natural lipid A derived from P. gingivalis
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activated C3H/HeJ peritoneal macrophages [33]. Recently, induction of lethal toxicity in galactosamine-loaded C3H/ HeJ mice by P. gingivalis LPS was reported [36]. Further, Kirikae et al. showed that P. gingivalis 381 LPS activated C3H/HeJ peritoneal macrophages, followed by an induction of TNF production [34]. These ¢ndings suggest that P. gingivalis LPS and its lipid A possess unique chemical structures. Manthey and Vogel [37] previously noted that LPS-associated proteins were necessary for activation in C3H/ HeJ mice. On the other hand, Kirikae et al. recently showed that repuri¢cation removed the contaminating proteins from P. gingivalis 381 LPS preparations, as opposed to di¡erent from those derived from Enterobacteriaceae, and still retained the potency to induce TNF production in C3H/HeJ peritoneal macrophages [34]. These ¢ndings together support the notion that the lipid A portion of P. gingivalis LPS may play an important role as a bioactive center. However, a synthetic counterpart of P. gingivalis 381-type lipid A, in contrast to its natural lipid A, lacked the ability to activate C3H/HeJ peritoneal macrophages in the present study. An amino acid analysis of P. gingivalis natural lipid A was previously done with an amino acid analyzing system, and the lipid A specimen was found to contain 0.32% (w/w) protein as the calculated value [35]. This suggests that only trace molecule(s) such as lipoprotein, lipopeptide, or other unknown components may be contained in the natural LPS/lipid A of P. gingivalis, and that the contaminant(s) may be associated with activation of C3H/HeJ peritoneal macrophages. In conclusion, we have demonstrated that a chemically synthesized lipid A of P. gingivalis, like natural lipid A, possesses very low endotoxicity, in contrast to E. coli type synthetic lipid A, and has potent immunostimulatory capabilities such as hemagglutinating activity, mitogenic responses with splenic MNC from both C3H/HeJ and C3H/ HeN mice, as well as cytokine-producing activities with peritoneal macrophages and gingival ¢broblasts in C3H/ HeN, but not C3H/HeJ mice. Further experiments are in progress to investigate the receptor(s) on a variety of immunocytes for P. gingivalis lipid A using synthetic components. Acknowledgements This work was supported in part by Grants-in-Aid for Scienti¢c Research from the Ministry of Education, Science and Culture of Japan (No. 11671829) and the Frontier Science from the Ministry of Education, Science, Sports and Culture of Japan (Nihon University School of Dentistry at Matsudo). We thank M. Benton for critical reading of the manuscript.
References [1] Slots, J. and Listgarten, M.A. (1988) Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J. Clin. Periodontol. 15, 85^93. [2] Hamada, S., Takada, H., Ogawa, T., Fujiwara, T. and Mihara, J. (1990) Lipopolysaccharides of oral anaerobes associated with chronic in£ammation: chemical and immunomodulating properties. Int. Rev. Immunol. 6, 247^261. [3] Imoto, M., Yoshimura, H., Sakaguchi, N., Kusumoto, S. and Shiba, T. (1985) Chemical structure of Escherichia coli lipid A. Tetrahedron Lett. 26, 1545^1548. [4] Kotani, S., Takada, H., Takahashi, I., Tsujimoto, M., Ogawa, T., Ikeda, T., Harada, K., Okamura, H., Tamura, T., Tanaka, S., Shiba, T., Kusumoto, S., Imoto, M., Yoshimura, H. and Kasai, N. (1986) Low endotoxic activities of synthetic Salmonella-type lipid A with an additional acyloxyacyl group on the 2-amino group of L(1-6)glucosamine disaccharide 1, 4P-bisphosphate. Infect. Immun. 52, 872^884. [5] Takada, H. and Kotani, S. (1992) Structure-function relationships of lipid A. In: Bacterial Endotoxic Lipopolysaccharides (Morrison, D.C. and Ryan, J.L., Eds.), vol. 1, pp. 107^134, CRC, New York. [6] Ogawa, T. (1993) Chemical structure of lipid A from Porphyromonas (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett. 332, 197^201. [7] Westphal, O. and Jann, K. (1965) Extraction with phenol and further applications of the procedure. Methods Carbohydr. Chem. 5, 83^91. [8] Dittmer, J.C. and Lester, R.L. (1964) A simple, speci¢c spray for the detection of phospholipids on thin-layer chromatograms. J. Lipid Res. 5, 126^127. [9] Fischer, F.G. and Do«rfel, H. (1954) Die quantitative bestimmung reduzierender zucker auf papierchromatogrammen. Hoppe-Seyler's Z. Physiol. Chem. 297, 164^178. [10] Suzuki, K., Maeta, H. and Matsumoto, T. (1989) An improved procedure for metallocene-promoted glycosidation. Enhanced reactivity by employing 1:2-ratio of Cp2HfCl2 -AgClO4 . Tetrahedron Lett. 30, 4853^4856. [11] Hashimoto, S., Hayashi, M. and Noyori, R. (1984) Glycosylation using glucopyranosyl £uorides and silicon-based catalysts. Solvent dependency of the stereoselection. Tetrahedron Lett. 25, 1379^1382. [12] Inage, M., Chaki, H., Imoto, M., Shimamoto, T., Kusumoto, S. and Shiba, T. (1983) Synthetic approach to lipid A: preparation of phosphorylated disaccharides containing (R)-3-hydroxyacyl and (R)3-acyloxyacyl groups. Tetrahedron Lett. 24, 2011^2014. [13] Scho¡stall, A.M. (1975) Cyclopropylmethyl dihydrogen phosphate. Preparation and use in the phosphorylation of nucleosides. J. Org. Chem. 40, 3444^3445. [14] Imoto, M., Yoshimura, N., Kusumoto, S. and Shiba, T. (1984) Total synthesis of lipid A, active principle of bacterial endotoxin. Proc. Jpn. Acad. Ser. B 60, 285^288. [15] Asai, Y., Uchida, H., Yamamoto, H., Ohyama, Y., Jinno, T., Taiji, Y., Ochiai, K. and Ogawa, T. (2000) Prevention of endotoxin-induced lethality in mice by calmodulin kinase activator.. FEMS Immunol. Med. Microbiol. 27, 201^210. [16] Ogawa, T., Ogo, H. and Kinoshita, A. (1997) Antagonistic e¡ect of synthetic peptides corresponding to the binding regions within ¢mbrial subunit protein from Porphyromonas gingivalis to human gingival ¢broblast. Vaccine 15, 230^236. [17] Tamura, M., Tokuda, M., Nagaoka, S. and Takada, H. (1992) Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival ¢broblast culture. Infect. Immun. 60, 4932^4937. [18] Galanos, C., Freudenberg, M.A. and Reutter, W. (1979) Galactosamine-induced sensitization to the lethal e¡ects of endotoxin. Proc. Natl. Acad. Sci. USA 76, 5939^5943.
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T. Ogawa et al. / FEMS Immunology and Medical Microbiology 28 (2000) 273^281 [19] Ka«rber, G. (1931) Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch. Exp. Pathol. Pharmakol. 162, 480^483. [20] Takada, H., Kotani, S., Tsujimoto, M., Ogawa, T., Takahashi, I., Harada, K., Katsukawa, C., Tanaka, S., Shiba, T., Kusumoto, S., Imoto, M., Yoshimura, H., Yamamoto, M. and Shimamoto, T. (1985) Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs. Infect. Immun. 48, 219^227. [21] Ogawa, T., Kusumoto, Y., Uchida, H., Nagashima, S., Ogo, H. and Hamada, S. (1991) Immunobiological activities of synthetic peptide segments of ¢mbrial protein from Porphyromonas gingivalis. Biochem. Biophys. Res. Commun. 180, 1335^1341. [22] Ogawa, T., Shimauchi, H., Uchida, H. and Mori, Y. (1996) Stimulation of splenocytes in C3H/HeJ mice with Porphyromonas gingivalis lipid A in comparison with enterobacterial lipid A. Immunobiol. 196, 399^414. [23] Shimauchi, H., Ogawa, T., Uchida, H., Yoshida, J., Ogoh, H., Nozaki, T. and Okada, H. (1996) Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopolysaccharide of Porphyromonas gingivalis. Experientia 52, 909^917. [24] Ogawa, T. (1994) Immunobiological properties of chemically de¢ned lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis. Eur. J. Biochem. 219, 737^742. [25] Morrison, D.C. and Jacob, D.M. (1976) Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 13, 813^818. [26] Weintraub, A., Za«hringer, U., Wollenweber, H.-W., Seydel, U. and Rietschel, E.T. (1989) Structual characterization of the lipid A component of Bacteroides fragilis strain NCTC9343 lipopolysaccharide. Eur. J. Biochem. 183, 425^431. [27] Hofstad, T., Skaug, N. and Sveen, K. (1993) Stimulation of B lymphocytes by lipopolysaccharides from anaerobic bacteria. Clin. Infect. Dis. 16, 200^202. [28] Joiner, K.A., McAdam, K.P. and Kasper, D.L. (1982) Lipopolysac-
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charides from Bacteroides fragilis are mitogenic for spleen cells from endotoxin responder and nonresponder mice. Infect. Immun. 36, 1139^1145. Ganglo¡, S.C., Hijiya, N., Haziot, A. and Goyert, S.M. (1999) Lipopolysaccharide structure in£uences the macrophage response via CD14-independent and CD14-dependent pathways. Clin. Infect. Dis. 28, 491^496. Johne, B., Olsen, I. and Bryn, K. (1988) Fatty acids and sugars in lipopolysaccharides from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loesheii. Oral Microbiol. Immunol. 3, 22^27. Okuda, K. and Kato, T. (1987) Hemagglutinating activity of lipopolysaccharides from subgingival plaque bacteria. Infect. Immun. 55, 3192^3196. Kirikae, T., Inada, K., Hirata, M., Toshida, M., Galanos, C. and Lu«deritz, O. (1986) Hemagglutination induced by lipopolysaccharides and lipid A. Microbiol. Immunol. 30, 269^274. Tanamoto, K., Azumi, S., Haishima, Y., Kumada, H. and Umemoto, T. (1997) The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide speci¢cally mediates the activation of C3H/HeJ mice. J. Immunol. 158, 4430^4436. Kirikae, T., Nitta, T., Kirikae, F., Suda, Y., Kusumoto, S., Qureshi, N. and Nakano, M. (1999) Lipopolysaccharides (LPS) of oral blackpigmented bacteria induce tumor necrosis factor production by LPSrefractory C3H/HeJ macrophages in a way di¡erent from that of Salmonella LPS. Infect. Immun. 67, 1736^1742. Ogawa, T., Nakazawa, M. and Masui, K. (1996) Immunopharmacological activities of the nontoxic monophosphoryl lipid A of Porphyromonas gingivalis. Vaccine 14, 70^76. Tanamoto, K. (1999) Induction of lethal shock and tolerance by Porphyromonas gingivalis lipopolysaccharide in D-galactosamine-sensitized C3H/HeJ mice. Infect. Immun. 67, 3399^3402. Manthey, C.L. and Vogel, S.N. (1994) Elimination of trace endotoxin protein from rough chemotype LPS. J. Endotoxin Res. 1, 84^91.
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Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis Tomohiko Ogawa a; *, Yasuyuki Asai a , Hiroyo Yamamoto a , Yasuhiro Taiji a , Takayoshi Jinno a , Tohru Kodama b , Shinjiro Niwata b , Hidetoshi Shimauchi c , Kuniyasu Ochiai d a
c
Department of Oral Microbiology, Asahi University, School of Dentistry, 1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan b Institute for Fundamental Research, Suntory Limited, 120-1 Takahama, Shimamoto-cho, Mishima-gun, Osaka 618-0012, Japan Department of Periodontology and Endodontology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan d Department of Microbiology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan Received 21 February 2000; received in revised form 29 March 2000 ; accepted 30 March 2000
Abstract A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Synthetic lipid A; Endotoxicity; C3H/HeJ mouse; Macrophage; Gingival ¢broblast; Porphyromonas gingivalis
1. Introduction Porphyromonas gingivalis is one of the suspected periodontopathic bacteria and has been frequently isolated from the periodontal pockets of patients with chronic periodontal diseases [1]. This organism is a Gram-negative, anaerobic oral black-pigmented rod which has lipopolysaccharides (LPS) located on the cell surface. It has been reported that the chemical and biological properties of P. gingivalis LPS and its active center, lipid A, are identical to those of LPS from Bacteroides fragilis, but di¡erent from those of classical enterobacterial LPS and their lipid As [2]. LPS is a major component of the outer membrane of Gram-negative bacteria, and is well-known to exhibit various biological responses for host cells [1]. Lipid A, the lipophilic portions of LPS whose structures correspond to
those proposed for Escherichia coli-, Salmonella-, and other enterobacterial type lipid As, were chemically determined and synthesized [3^5]. These chemically well-de¢ned synthetic lipid A compounds have been investigated extensively for the structure^function relationships of lipid As. It was previously demonstrated that puri¢ed P. gingivalis lipid A exhibited a quite di¡erent phosphorylation and acylation pattern as compared with enterobacterial lipid As [6]. The present study was designed to investigate the endotoxicity and immunobiological activities of a synthetic lipid A of P. gingivalis, and compare them with those of its bacterial lipid A product and synthetic E. colitype lipid A. 2. Materials and methods 2.1. Animals
* Corresponding author. Tel. : +81 (58) 329-1421; Fax: +81 (58) 329-1421; E-mail: [email protected]
BALB/c, C57BL/6, C3H/HeN, and C3H/HeJ mice
0928-8244 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 0 0 ) 0 0 1 6 7 - X
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(males, 8 weeks old) were obtained from Japan SLC, Hamamatsu, Japan. Domestic Japanese white rabbits were purchased from Nihon Rabbit, Osaka, Japan. 2.2. Bacteria and LPS preparation P. gingivalis strain 381 was grown anaerobically in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin and menadione at 37³C for 26 h. Bacterial cells were collected by centrifugation, washed three times with pyrogen-free water, and lyophilized. LPS was extracted from lyophilized cells by the hot phenol/water method [7], and the puri¢cation of the crude extract was performed by repeated ultracentrifugation (100 000Ug, 3 h) followed by treatment with nuclease P1 (Yamasa Shoyu, Choshi, Japan) and ¢nally lyophilized [6]. 2.3. Preparation and puri¢cation of lipid A Puri¢ed LPS was hydrolyzed in 0.6% acetic acid at 105³C for 2.5 h, then cooled and neutralized. The hydrolysate was made up to 100 ml with H2 O and added to 200 ml of CHCl3 /MeOH/H2 O/triethylamine (30/12/2/0.1). The lower phase was evaporated under reduced pressure. Subsequently, the lipid A fractions were separated by silica gel column chromatography (Silica Gel 60, 230^400 mesh: Nacalai Tesque, Kyoto, Japan) with CHCl3 /MeOH/H2 O/ triethylamine (30/12/1.5/0.1). The eluants were monitored by thin-layer chromatography (TLC) on a silica gel 60 plate. The plates were developed with CHCl3 /MeOH/ H2 O/triethylamine (30/12/2/0.1) and visualized by sulfuric acid, Dittmer^Lester reagent [8], and triphenyltetrazolium chloride (TTC) reagent [9]. The slow moving compound (Rf = 0.3), observed as a single spot, was recognized as the major component [6]. The chemical structure of lipid A from LPS of P. gingivalis strain 381 was characterized using conventional chemical procedures, gas-liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry [6]. 2.4. Synthesis of P. gingivalis strain 381 lipid A P. gingivalis derived lipid A is shown in Fig. 1A [6]. The £uoro-sugar (Fig. 1B) was condensed with 4,6-dihydroxy sugar (Fig. 1C), with bis (cyclopentadienyl) hafnium dichloride (Aldrich, Milwaukee, WI, USA) and silver perchlorate hydrate (AgClO4 , Aldrich) used to generate the disaccharide (Fig. 1D) [10]. The £uoro-sugar (Fig. 1B) was prepared from N-acetyl-D-glucosamine (Aldrich) in six steps ; methyl glycoside formation with cation exchange resin (Amberlite IR-120B from Organo, Tokyo) in methanol, benzylation of the hydroxy groups, demethylation and deacetylation with 6N-HCl/dioxane under re£ux, protection of the 2-amino group with 2,2,2-trichloroethyl chloroformate (Troc-Cl, Aldrich), acetylation of the 1-hydroxy group by usual methods, and then £uorination of
Fig. 1. Total synthesis of P. gingivalis 381 derived lipid A. Synthetic procedures for the chemically de¢ned structure of the lipid A backbone of LPS from P. gingivalis 381 are described in Section 2.
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the 1-acetoxy group with HF/pyridine (Aldrich) [11]. The 4,6-dihydroxy sugar (Fig. 1C) was prepared from D-glucosamine hydrochloride (Aldrich) in ¢ve steps; protection of the 2-amino group with 9-£uorenylmethyl chloroformate (Fmoc-Cl, Aldrich), allyl glycoside formation with HCl/dioxane/allyl alcohol, 4,6-benzylidenation with benzaldehyde dimethyl acetal (Aldrich) and p-toluenesulfonic acid in N,N-dimethylformamide (DMF), benzylation of the 3-hydroxy group with benzyl bromide (Aldrich) and Ag2 O (Aldrich) in CH2 Cl2 [12], and then deprotection of the 4,6-benzylidene group with HCl/dioxane. The two fatty acid moieties, 3-(R)-benzyloxy-15-methylhexadecanoic acid (Fig. 1E) and 3-(R)-palmitoyloxy-15methylhexadecanoic acid (Fig. 1F), were introduced into the disaccharide derivative (Fig. 1D) by respective deprotection and acylation of the protected amino groups (Fmoc-amino and Troc-amino groups) to obtain the fatty acyl disaccharide (Fig. 1G). 3-(R)-Benzyloxy-15-methylhexadecanoic acid (Fig. 1E) was obtained from 3-(R)-hydroxy-15-methylhexadecanoic acid (Wako Pure Chemical Industries, Osaka, Japan) in three steps; esteri¢cation with phenacyl bromide (Aldrich) in triethylamine (Et3 N)/ethyl acetate (AcOEt), benzylation with benzyl bromide and Ag2 O in benzene, and then de-esteri¢cation with Zn in acetic acid (AcOH). 3-(R)-Palmitoyloxy-15-methylhexadecanoic acid (Fig. 1F) was also prepared from 3-(R)-hydroxy-15-methylhexadecanoic acid in three steps; phenacyl ester formation, esteri¢cation of the 3-hydroxy group with palmitoyl chloride (Aldrich) and N,N-dimethylaniline (Aldrich) in benzene, and then deprotection of the phenacyl group. The fatty acyl disaccharide (Fig. 1G) was converted to a 1-phosphate derivative (Fig. 1H) using n-butyl lithium-dibenzyl phosphorochloridate [13], and then all of the protecting groups were reductively removed to generate the synthetic lipid A of P. gingivalis 381 (Fig. 1A) by treatment with palladium catalyst. This ¢nal product was proven by pmr analysis to contain a L-con¢guration of the glycosidic linkage and an K-con¢guration of the phosphorylated position. 2.5. Reference synthetic product The E. coli-type synthetic lipid A (compound 506) used in this study was synthesized as described by Imoto et al. [14]. The bacterial and synthetic products were dissolved at a concentration of 2 mg ml31 in 0.1% triethylamine aqueous solution. The stock solution was kept at 4³C and appropriately diluted with pyrogen-free phosphatebu¡ered saline (PBS; Biken, Osaka) or cell culture medium before use in the assay. 2.6. Preparation of peritoneal macrophages Peritoneal exudate cells were obtained from C3H/HeN and C3H/HeJ mice by washing with 5 ml of PBS, followed
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by aspiration of the wash from the peritoneal cavity [15]. The cells were washed once with and resuspended in RPMI 1640 medium (Nikken Biomedical Laboratories, Kyoto) supplemented to a ¢nal concentration of 100 U ml31 penicillin, 100 Wg ml31 streptomycin, 300 Wg ml31 L-glutamine, and 10% (v/v) fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), and then added to 24-well culture plates (FALCON 3047; Becton Dickinson, Lincoln Park, NJ, USA) at a concentration of 106 cells per well, where they were incubated for 60 min at 37³C in 5% (v/v) CO2 . Non-adherent cells were removed, and the adherent cells were washed three times with PBS and resuspended in RPMI 1640 medium supplemented with 10% FBS. The number of viable cells was determined using the trypan blue exclusion technique. 2.7. Preparation of murine gingival ¢broblasts (MGF) Gingival tissues (ca. 2^5 mg) were obtained from two or three C3H/HeN and C3H/HeJ mice. The explants were cultured in K minimal essential medium (K-MEM ; Nikken Biomedical Laboratories) supplemented to a ¢nal concentration of 50 Wg ml31 gentamicin, 50 ng ml31 amphotericin B (Sigma, St. Louis, MO, USA), and 10% (v/v) fetal calf serum (FCS; Sigma) in plastic culture dishes [16]. The MGF that showed a slim and spindle-shape morphology under a phase contrast microscope [17] were designated MGFN if from C3H/HeN mice and MGFJ if from C3H/HeJ mice. The cells were grown and maintained in K-MEM containing 10% FCS at 37³C in a 5% (v/v) CO2 atmosphere, and were used at the 5th and 13th passages in the assay. 2.8. Lethal toxicity in galactosamine-loaded mice Groups of ¢ve C57BL/6 mice were injected intraperitoneally with 16 mg of D-galactosamine hydrochloride (Wako Pure Chemicals) in 0.5 ml of PBS, followed immediately by an intravenous injection of the indicated doses of test specimens in 0.2 ml of PBS by the method of Galanos et al. [18]. The death of the mice due to intoxication was observed over a 24-h period. The 50% lethal dose (LD50 ) for each group was calculated by the method of Ka«rber [19]. 2.9. Pyrogenicity Three domestic Japanese white rabbits (male; 2^2.5 kg) per group were injected intravenously with the indicated doses of the test specimens in 10 ml of pyrogen-free PBS per kg as described previously [20]. The rectal temperature of each rabbit was measured and recorded continuously with an automatic device (EP670, 12; Iio Electric Co., Tokyo, Japan). The measurements in the present study were simpli¢ed to determine the rectal temperature of each rabbit every 30 min between 1 and 1.5 h (peak I)
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and between 2.5 and 4 h (peak II) after injection of the test specimens for the ¢rst- and second-phase temperature, respectively. An increase in rectal temperature of more than 0.6³C was taken as a positive febrile response. 2.10. Limulus test A Limulus test was performed with Pre Gel, an amebocyte lysate prepared from Tachypleus tridentatus (Seikagaku Kogyo, Tokyo, Japan) according to the manufacturer's directions. A solution of E. coli O111:B4 LPS-W (Difco Laboratories, Detroit, MI, USA) incubated in the reagent kit was used as a reference standard. The dose which caused a reaction de¢nitely stronger than the increased viscosity was taken as the minimum e¡ective dose. 2.11. Hemagglutinating assay A hemagglutination assay was performed with a 96-well round-bottom microtiter plate (Cell Wells 25850 ; Corning Glass Works, Corning, NY, USA) as described previously [21]. Test specimens (25 Wl) diluted serially twofold in PBS were added to an equal volume of a 2% rabbit erythrocyte suspension in PBS. After gentle shaking, the microtiter plate was incubated at 37³C for 2 h, and the hemagglutination titer was shown as the concentration of test specimens giving a positive reaction by visual inspection. 2.12. Mitogenicity Murine mononuclear cells (MNC) were isolated by Histopaque (Sigma) separation from spleen cells of BALB/c, C3H/HeN, and C3H/HeJ mice [22]. Splenic MNC (each 2.5U105 cells) were cultured with the indicated doses of test specimens in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS in a 96-well £at-bottom microtiter plate (Falcon 3040 ; Becton Dickinson) for 48 h at 37³C in 5% (v/v) CO2 . Thirty-seven kiloBequerel of methyl3 H-thymidine ([3 H]thymidine; 185 MBq nmol31 ; Amersham International, Amersham, UK) was added during the ¢nal 16 h of culture to estimate DNA synthesis, and all cultures were harvested onto glass paper ¢lter strips. Thymidine uptake was measured in a liquid scintillation counter (model 1215 ; LKB-Wallec, Turku, Finland). The e¡ect of polymyxin B on mitogenic responses in BALB/c, C3H/HeN, and C3H/HeJ mice was examined as follows. Splenic MNC (each 2.5U105 cells) were cultured at 37³C for 48 h in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS with the indicated doses of polymyxin B sulfate (5U105 U per vial; Wako Pure Chemicals) and 10 Wg of a test specimen, except for 5 Wg of concanavalin A (Con A; Sigma) [23]. Cultures were pulsed for the ¢nal 16 h of incubation with 37 kBq of [3 H]thymidine, and then thymidine incorporation was determined as described above.
2.13. Cytokine assay Peritoneal macrophages were suspended at a cell density of 2U106 cells ml31 of RPMI 1640 medium supplemented with 10% FBS. These cells (2U105 cells per well) were incubated with or without the indicated doses of the test specimens for 24 h at 37³C in humidi¢ed air containing 5% (v/v) CO2 in a 96-well £at-bottom microtiter plate. MGF were also suspended at 2U105 cells ml31 of K-MEM, and the indicated doses of the test specimens were added to the cell cultures (2U104 cells per well) and incubated at 37³C for 18 h. After incubation, the supernatants were collected and stored at 380³C until the assay for cytokine production. The production of interleukin-6 (IL-6) and tumor necrosis factor K (TNF-K) was measured in the culture supernatants by means of a commercial ELISA kit system (Endogen, Cambridge, MA, USA). The assay was performed according to the manufacturer's instructions, and the data were determined using a standard curve prepared for each assay. 2.14. Statistics Data were analyzed by a one-way analysis of variance (ANOVA), using the Bonferroni or Dunn method, and the results are presented as the mean þ S.E.M. When an individual study is demonstrated, it is representative of at least three independent experiments. 3. Results 3.1. Endotoxic activities Compound PG-381 lacked lethal toxicity at a dose of up to 20 Wg per mouse (data not shown), and the LD50 for the natural lipid A of P. gingivalis was 12.6 Wg per mouse [24]. The toxicity of compound PG-381 was weaker than that of its natural lipid A. Compound 506 was markedly toxic in galactosamine-loaded mice, and its LD50 was 0.0079 Wg per mouse [24]. The toxicity of compound PG-381 and its natural lipid A were considerably weaker than that of compound 506. Rabbits injected with compound PG-381 at a dose of 10 Wg kg31 showed no induction of peak I and peak II responses, while natural lipid A of P. gingivalis at a dose of Table 1 Activation of the clotting-enzyme cascade of T. tridentatus amebocyte lysate (Limulus test) by P. gingivalis natural and synthetic lipid A, and E. coli-type synthetic lipid A (compound 506) Test specimen
Minimum e¡ective dose (ng per test)
Compound PG-381 P. gingivalis natural lipid A Compound 506
10 10 0.01
Data shown were obtained by the Pre Gel test.
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Fig. 2. Mitogenic e¡ect of P. gingivalis synthetic lipid A on splenic MNC from BALB/c, C3H/HeN, and C3H/HeJ mice. Cells were cultured for 48 h with or without the indicated dose of each test specimen as follows : compound PG-381 (b), P. gingivalis natural lipid A (a), and compound 506 (F). The results are expressed as stimulation index, a value that was determined using the formula : stimulation index = counts per minute (cpm) for test culture/cpm in culture with medium alone. The counts (mean þ S.E.M.) in control cultures were 1151 þ 36 cpm in BALB/c MNC, 1997 þ 45 cpm in C3H/ HeN MNC, and 2535 þ 105 cpm in C3H/HeJ MNC. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean þ S.E.M. Signi¢cant di¡erences between the groups with or without test specimen administered (*P 6 0.01).
10 Wg kg31 exhibited very low responses at peak II and none at peak I (data not shown, [24]). However, compound 506 at a dose of 0.1 Wg kg31 induced positive peak I and peak II responses. The results obtained by a conventional Limulus test showed that compound PG-381 exhibited a weak activity as compared to that of the natural product, P. gingivalis 381 lipid A (Table 1). However, their activities were 1/1000 of that of compound 506. 3.2. Immunobiological activities Compound PG-381 strongly agglutinated rabbit erythrocytes at a minimum concentration of 0.004 Wg ml31 , and
the natural lipid A of P. gingivalis induced hemagglutination at a minimum concentration of 0.015 Wg ml31 . Compound 506 also caused hemagglutination at a concentration of 0.06 Wg ml31 . These results indicated that compound PG-381 as well as the natural lipid A of P. gingivalis exhibited stronger hemagglutinating activities than compound 506. The natural lipid A of P. gingivalis 381 exhibited almost comparable mitogenic activities as compound 506, and compound PG-381 elicited weak but signi¢cant mitogenic responses in splenic MNC from both BALB/c and LPSresponder C3H/HeN mice (Fig. 2). Compound PG-381 and its natural lipid A showed signi¢cant mitogenic responses in splenic MNC from LPS-hyporesponder C3H/
Fig. 3. E¡ect of polymyxin B on the mitogenic responses of splenic MNC from BALB/c, C3H/HeN, and C3H/HeJ mice to P. gingivalis synthetic lipid A. Cells were cultured for 48 h with 10 Wg per well of compound PG-381 (b), P. gingivalis natural lipid A (a), compound 506 (F), 5 Wg per well of Con A (R), or RPMI 1640 medium alone (O), containing the indicated dose of polymyxin B. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean þ S.E.M. of results.
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Fig. 4. Cytokine production by peritoneal macrophages from C3H/HeN and C3H/HeJ mice in response to stimulation by P. gingivalis synthetic lipid A. Cells were cultured at 37³C for 24 h in RPMI 1640 medium containing 10% FBS, with or without the indicated dose of each test specimen. After incubation, the supernatants were collected and cytokine production was determined by ELISA. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean+S.E.M. of results. Signi¢cant di¡erences between the groups with and without the test specimens (*P 6 0.01).
HeJ mice, whereas compound 506 was not found to have mitogenic activities. The antibiotic polymyxin B has been shown to bind to the lipid A region of LPS [25]. We further investigated the inhibitory e¡ect of polymyxin B on the mitogenic activities of natural and synthetic lipid As on the splenic MNC from the strains of mice used in the present study. The addition of polymyxin B to BALB/c and C3H/HeN MNC cultures markedly inhibited the thymidine incorporation by compound PG-381 and its natural product, as well as compound 506, but not by Con A (Fig. 3). In splenic MNC from C3H/HeJ mice, polymyxin B also completely inhibited the thymidine incorporation of MNC stimulated with compound PG-381 as well as the natural lipid A, but not with Con A. Cytokine production by peritoneal macrophages and gingival ¢broblasts from C3H/HeN and C3H/HeJ mice, after stimulation with the natural and synthetic lipid As,
was examined. Compound PG-381 de¢nitely induced IL-6 and TNF-K production in peritoneal macrophages from C3H/HeN mice, however, the activities were weaker than those of its natural product (Fig. 4). Natural lipid A of P. gingivalis also exhibited these cytokine-producing activities on C3H/HeJ peritoneal macrophages, whereas the stimulation of the macrophages with compound PG-381 and compound 506 resulted in no cytokine-producing activity. IL-6 production in MGF was examined after stimulation with the natural and synthetic lipid As. P. gingivalis natural and synthetic lipid As, as well as compound 506, de¢nitely exhibited cytokine-producing activities in MGFN derived from C3H/HeN mice. The natural lipid A of P. gingivalis also elicited IL-6 production in MGFJ derived from C3H/HeJ mice. However, stimulation with compound PG-381 as well as compound 506 resulted in no IL-6 production in the MGFJ from C3H/HeJ mice, as shown in Fig. 5.
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Fig. 5. IL-6 production in gingival ¢broblasts from C3H/HeN and C3H/HeJ mice in response to stimulation by P. gingivalis synthetic lipid A. Cells were cultured at 37³C for 18 h in RPMI 1640 medium containing 10% FBS with or without the indicated dose of each test specimen. After incubation, the supernatants were collected and cytokine production was determined by ELISA. Experiments were done at least three times, and representative results are presented. Each assay was done in triplicate, and the data are expressed as the mean+S.E.M. Signi¢cant di¡erences between the groups with and without the test specimens (*P 6 0.01).
4. Discussion The present study, using a chemical synthesized lipid A, showed that the lipid A portion of LPS from P. gingivalis exhibits no or very low endotoxic activities, as demonstrated by tests of lethal toxicity in galactosamine-loaded mice and pyrogenicity in rabbits as well as activity in Limulus test. These activities of P. gingivalis synthetic lipid A, like its natural lipid A, were considerably weaker than those of E. coli-type synthetic lipid A (compound 506). The P. gingivalis synthetic lipid A backbone consists of a L-(1-6)-linked glucosamine disaccharide which is phosphorylated at the 1 position of the reducing sugar, however, this lipid A structure lacks an ester-linked phosphate group which is bound to the hydroxy group at the 4P-position of the nonreducing sugar (Fig. 1). It was also found that the hydroxyl groups at the 3-, 3P-, 4-, 4P-, and 6P-positions of the P. gingivalis lipid A backbone are free. Furthermore, it was shown that the P. gingivalis lipid A molecule possesses 3-hydroxy-15-methylhexadecanoic acid and 3-hexadecanoyloxy-15-methyl-hexadecanoic acid, which are amide linked at the 2- and 2P-positions, respectively (Fig. 1). P. gingivalis lipid A has a distinctly di¡erent structure from enterobacterial lipid As. Namely, it has been reported that there is no 4-O-phosphoryl group in the lipid A backbone of B. fragilis [26^29] or Bacteroides intermedius [30]. This chemical structure is suggested to be the cause of very low toxic activities, as indicated in the present study. It has also been demonstrated that among the bisphosphoryl disaccharide compounds so far described, removal of a 1- or 4P-phosphate group from 1,4P-bisphospho-disaccharide synthesized compounds clearly reduced endotoxicity, as compared with the parent molecule [5].
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We previously demonstrated that P. gingivalis LPS and its lipid A de¢nitely caused agglutination of rabbit erythrocytes [24]. In contrast, others have reported that P. gingivalis LPS had no hemagglutinating activity [31,32]. Kirikae et al. also suggested that the non-hemagglutinating activity of P. gingivalis LPS may be attributed to differences in the chemical structure of the lipid A moiety. In this study, synthetic lipid A as well as natural lipid A of P. gingivalis possessed stronger hemagglutinating activities than compound 506. Thus, the P. gingivalis lipid A molecule was found to possess the capability to induce agglutination of rabbit erythrocytes. P. gingivalis synthetic lipid A, similar to its natural product, was found to stimulate splenic MNC of both LPS-responder C3H/HeN and LPS-hyporesponder C3H/ HeJ mice, at a level weaker than that induced by natural lipid A, whereas compound 506 stimulated splenic MNC of C3H/HeN, but not C3H/HeJ mice (Fig. 2 and [24]). We and other research groups have previously demonstrated that LPS and its natural lipid A from P. gingivalis were mitogenic for splenic MNC and B-cells from both of these murine strains [23,24,33,34]. The total protein content in P. gingivalis lipid A specimen was scarcely detectable by amino acid analysis, as described previously [35]. Whether a trace of protein has an in£uence on the mitogenic activity of C3H/HeJ mice remains to be shown. The present study appears to clarify this possibility point with synthetic lipid A, and the stimulatory e¡ect on the splenocytes of C3H/HeJ mice is suggested to be attributable to a unique structure of the lipid A portion of P. gingivalis LPS. Furthermore, the mitogenicity of P. gingivalis synthetic lipid A has shown to be inhibited by the addition of polymyxin B to all BALB/c, C3H/HeN and C3H/HeJ splenic MNC cultures so far examined. The lipid As of P. gingivalis and E. coli are the bioactive centers in their respective LPS. The present ¢ndings demonstrate that we have been able to accomplish the synthesis of a lipid A preparation with endotoxic and immunobiological properties nearly identical to those of natural lipid A of P. gingivalis 381. However, there are marked di¡erences between compound PG-381 and its natural lipid A in regard to immunobiological activities, such as their cytokine-producing activities in peritoneal macrophages and gingival ¢broblasts from C3H/HeJ mice. We found that compound PG-381 exhibited de¢nite cytokine-producing activities in both macrophages and ¢broblasts from C3H/HeN mice, as did natural lipid A of P. gingivalis and compound 506. However, both compound PG-381 and compound 506 had no response in the same cells from C3H/HeJ mice. In contrast, the natural lipid A caused an activation of C3H/HeJ peritoneal macrophages and gingival ¢broblasts. We previously reported that the natural lipid A of P. gingivalis 381 induced IL-1L production in splenic C3H/HeJ macrophages [22]. Tanamoto et al. also demonstrated that the natural lipid A derived from P. gingivalis
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activated C3H/HeJ peritoneal macrophages [33]. Recently, induction of lethal toxicity in galactosamine-loaded C3H/ HeJ mice by P. gingivalis LPS was reported [36]. Further, Kirikae et al. showed that P. gingivalis 381 LPS activated C3H/HeJ peritoneal macrophages, followed by an induction of TNF production [34]. These ¢ndings suggest that P. gingivalis LPS and its lipid A possess unique chemical structures. Manthey and Vogel [37] previously noted that LPS-associated proteins were necessary for activation in C3H/ HeJ mice. On the other hand, Kirikae et al. recently showed that repuri¢cation removed the contaminating proteins from P. gingivalis 381 LPS preparations, as opposed to di¡erent from those derived from Enterobacteriaceae, and still retained the potency to induce TNF production in C3H/HeJ peritoneal macrophages [34]. These ¢ndings together support the notion that the lipid A portion of P. gingivalis LPS may play an important role as a bioactive center. However, a synthetic counterpart of P. gingivalis 381-type lipid A, in contrast to its natural lipid A, lacked the ability to activate C3H/HeJ peritoneal macrophages in the present study. An amino acid analysis of P. gingivalis natural lipid A was previously done with an amino acid analyzing system, and the lipid A specimen was found to contain 0.32% (w/w) protein as the calculated value [35]. This suggests that only trace molecule(s) such as lipoprotein, lipopeptide, or other unknown components may be contained in the natural LPS/lipid A of P. gingivalis, and that the contaminant(s) may be associated with activation of C3H/HeJ peritoneal macrophages. In conclusion, we have demonstrated that a chemically synthesized lipid A of P. gingivalis, like natural lipid A, possesses very low endotoxicity, in contrast to E. coli type synthetic lipid A, and has potent immunostimulatory capabilities such as hemagglutinating activity, mitogenic responses with splenic MNC from both C3H/HeJ and C3H/ HeN mice, as well as cytokine-producing activities with peritoneal macrophages and gingival ¢broblasts in C3H/ HeN, but not C3H/HeJ mice. Further experiments are in progress to investigate the receptor(s) on a variety of immunocytes for P. gingivalis lipid A using synthetic components. Acknowledgements This work was supported in part by Grants-in-Aid for Scienti¢c Research from the Ministry of Education, Science and Culture of Japan (No. 11671829) and the Frontier Science from the Ministry of Education, Science, Sports and Culture of Japan (Nihon University School of Dentistry at Matsudo). We thank M. Benton for critical reading of the manuscript.
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