a novel component different from endotoxin extracted from ... · acetate buffer (ph 4.4) at 100°c...

INFECTION AND IMMUNITY,0019-9567/97/$04.0010

Nov. 1997, p. 4531–4538 Vol. 65, No. 11

Copyright © 1997, American Society for Microbiology

A Novel Component Different from Endotoxin Extracted fromPrevotella intermedia ATCC 25611 Activates Lymphoid Cellsfrom C3H/HeJ Mice and Gingival Fibroblasts from Humans

KOHICHI IKI,1,2 KAZUYOSHI KAWAHARA,3 SHOICHIRO SAWAMURA,4 RIEKO ARAKAKI,1

TETSUYA SAKUTA,1 AKIKO SUGIYAMA,1 HIROSHI TAMURA,5 TAKESHI SUEDA,2

SHIGEYUKI HAMADA,4 AND HARUHIKO TAKADA1*

Department of Microbiology and Immunology1 and Department of Peridontology,2 Kagoshima University Dental School,Kagoshima 890, Department of Bacteriology, The Kitasato Institute, Minato-ku, Tokyo 108,3 Department of

Oral Microbiology, Osaka University Faculty of Dentistry, Suita, Osaka 565,4 and Research Biologicalsand Diagnostics Division, Seikagaku Cooperation, Cyuoh-ku, Tokyo 103,5 Japan

Received 3 March 1997/Returned for modification 8 May 1997/Accepted 7 August 1997

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermediaATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate andprotein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) ondeoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. Wedesignated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity onsplenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeNmice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction wereresistant to heat inactivation (100°C for 1 h) and protease treatments and were scarcely inhibited by polymyxinB. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with aphenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages fromC3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeNmice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hotphenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. Thesefindings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reportedto date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not fromthe LPS itself.

Black-pigmented bacteria (BPB) such as Porphyromonas gin-givalis and Prevotella intermedia are dominant bacteria in theperiodontal pockets of patients with periodontitis and havebeen implicated as pathogens in periodontal diseases (14, 31).Lipopolysaccharides (LPS) of BPB and related Bacteroidesspecies have been reported to possess chemical and biologicalproperties different from those of the classical endotoxin of thefamily Enterobacteriaceae (12, 20, 48). Many investigators re-ported that BPB LPS activate the lymphocytes and macro-phages of classical LPS-nonresponsive C3H/HeJ mice as wellas LPS-responsive C3H/HeN mice (1, 5, 6, 10, 11, 13, 17). Wealso found that BPB LPS activate human gingival fibroblasts toproduce interleukin-1a/b (IL-1a/b), IL-6, and IL-8 whereasLPS of the Enterobacteriaceae are scarcely active in this respect(39, 40).

We recently found that LPS extracted from the P. intermediaATCC 25611 with a phenol-chloroform-petroleum ether mix-ture (LPS-PCP) showed marked Limulus activity and activatedLPS-responsive cells, but not LPS-nonresponsive cells, as doclassical endotoxins. In contrast, LPS extracted from P. inter-media ATCC 25611 with phenol-water (LPS-PW) activatedboth LPS-responsive and LPS-nonresponsive cells. Therefore,we attempted to isolate a fraction with mitogenicity against

splenocytes of C3H/HeN and C3H/HeJ mice, but devoid ofLimulus activity, from the hot phenol-water extract of the bac-teria. We then examined the bioactivities of the fraction, whichare not exhibited by the LPS of the Enterobacteriaceae, com-pared with those of LPS-PCP and LPS-PW of the bacteria.

MATERIALS AND METHODS

Preparation of fractions of P. intermedia ATCC 25611. Lyophilized P. inter-media ATCC 25611 cells (10 g) were extracted twice with phenol-water at 67°Cfor 20 min (46). The pooled extract in the water phase was dialyzed againstdistilled water and ultracentrifuged at 140,000 3 g for 3 h to remove LPS. TheLPS as a sediment was resuspended in water and ultracentrifuged twice more,and 90 mg of LPS-PW was obtained. The resultant supernatant (S1 fraction) waslyophilized, and 730 mg of dry product was obtained. A portion of the S1 fraction(24.2 mg) was dissolved in 4 ml of 0.2% sodium deoxycholate (DOC)–100 mMTris–5 mM sodium EDTA (pH 8.5), subjected to chromatography on a 1.5- by90-cm column of Sephadex G-100 (Pharmacia, Uppsala, Sweden), and eluatedwith the same buffer at a rate of 15 ml/h. Aliquots (2 ml) of the eluate werecollected, and selected fractions were dialyzed against distilled water at 4°C todetermine their mitogenicity on splenocytes from C3H/HeN and C3H/HeJ mice,subjected to determination of Limulus activity with an Endospecy kit (SeikagakuKogyo Co., Tokyo, Japan), and subjected to DOC-polyacrylamide gel electro-phoresis (DOC-PAGE) by the method of Komuro and Galanos (18). For themitogenic assay, the dialysates were sterilized with UV irradiation and diluted1:15 with culture medium (see the description for mitogenic activity). For DOC-PAGE, 10-ml aliquots of the dialysates were applied to 12% polyacrylamide gels,electrophoresed at a constant current of 12 mA at 4°C for 2 h, and stained witha silver-staining kit (Wako Pure Chemicals, Osaka, Japan). The target fraction(F1) which showed mitogenic effects on the splenocytes from both strains of miceand was devoid of Limulus activity was treated with NP1 nuclease (Yamasa,Choshi, Japan) and rechromatographed on Sephadex G-100 to prepare the finalfraction, designated Prevotella glycoprotein (PGP). Another LPS preparation

* Corresponding author. Present address: Department of Microbi-ology and Immunology, Tohoku University School of Dentistry, 4-1Seiryo-machi, Aoba-ku, Sendai 980-77, Japan. Fax: 81-22-717-8309.

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was extracted from the same bacteria with PCP, electrodialyzed, and convertedto the triethylamine salt by the method of Galanos et al. (7, 8); it was designatedLPS-PCP.

The above procedures for the preparation of test materials are summarized inFig. 1.

Characterization of the PGP fraction. To characterize the active componentsin the PGP fraction, the PGP or F1 fraction, treated as follows, was also testedin the mitogenicity assay.

(i) Heat treatment. The F1 fraction, suspended in distilled water (2 mg/ml),was heated at 100°C for 60 min.

(ii) Protease treatment. To the PGP fraction (6 mg/ml), suspended in 100 mMTris-HCl buffer (pH 7.0), was added 2 volumes of pronase E from Streptomycesgriseus attached to carboxymethyl cellulose beads (25 mg/ml) (P-0387; Sigma, St.Louis, Mo.) and 10 ml of chloroform; the mixture was then incubated at 37°C for18 h with continuous shaking. After centrifugation at 500 3 g for 10 min, thesupernatant was dialyzed against distilled water at 4°C and filtered through amembrane filter (pore size, 0.22 mm). A similar procedure was used in theproteinase K treatment, except that the F1 fraction (6 mg/ml), suspended in 50mM Tris-HCl buffer (pH 8.0) and supplemented with 0.5% sodium dodecylsulfate, was added to an equal volume of proteinase K solution (250 mg/ml) andincubated at 37°C for 1 h. In another experiment, the F1 fraction was treatedsequentially with nuclease P1 and pronase E and then rechromatographed onSephadex G-100.

(iii) Periodate treatment. To the F1 fraction (5 mg/ml), suspended in 50 mMacetic acid–sodium acetate buffer (pH 4.0), was added 1 ml of sodium periodate(43 mg/ml), and the mixture was incubated at 4°C for 5 days with continuousshaking. The reaction mixture was dialyzed against distilled water and filteredthrough a membrane filter (pore size, 0.22 mm).

Chemical analyses and estimation of the molecular sizes of PGP and LPS-PCP. The molecular sizes of the main components of PGP and LPS-PCP wereestimated on the basis of migration profiles of the fraction on the DOC-PAGEas described above, except that an 18% polyacrylamide gel was used instead ofa 12% gel, by comparison with the S-form LPS of Salmonella abortusequi andvarious R-form LPS of Salmonella minnesota (supplied by C. Galanos, MaxPlanck Institut fur Immunbiologie, Freiburg, Germany). Chemical analyses ofLPS-PCP, LPS-PW, F1, and PGP fractions were performed by methods de-scribed previously (16). Briefly, the neutral sugars in test fractions were analyzedafter 0.1 M HCl hydrolysis (at 100°C for 48 h, in the form of alditol acetate bygas-liquid chromatography (GLC). For the estimation of the glucosamine con-tent, the test materials were treated with 4 M HCl at 100°C for 16 h and thecontent was determined by two methods: the colorimetric method (33) and GLCof glucosamine in the form of alditol acetate. For the estimation of the 3-deoxy-D-manno-octulosonic acid content, the test fractions were treated with 0.1 Macetate buffer (pH 4.4) at 100°C for 1 h and the content was determined by thethiobarbituric acid method (45). Phosphorus levels were determined by themethod of Lowry et al. (21). Fatty acids were analyzed after 4 M HCl hydrolysis(at 100°C for 5 h) in the form of methyl ester by GLC (GC-14A chromatographyapparatus; Shimadzu, Kyoto, Japan). For the identification of sugar and fattyacid derivatives, GLC-mass spectrometry (MS) on an instrument (JMS-DX 300;JEOL, Tokyo, Japan) equipped with a capillary column (CBP-1; Shimadzu) wasused. The protein, hexose, and hexosamine contents in the PGP fraction wereanalyzed with the bicinchoninic protein assay reagent (Pierce Chemical Co.,Rockford, Ill.) by the method of Sorensen and Brodbeck (32), with the anthronereagent by the method of Roe (29), and by the Elson-Morgan method as de-scribed by Strominger et al. (33), respectively.

Mitogenic activity. The mitogenic activity of test materials on splenocytes ofC3H/HeN and C3H/HeJ mice (6 to 10 weeks old; Clea Japan, Osaka, Japan), wasdetermined by measuring the incorporation of 5-125I-iodinated deoxyuridine

(125I-UdR; Daiichikagaku-yakuhin, Tokyo, Japan) by the cells. Briefly, spleno-cytes were suspended in RPMI 1640 medium (Nissui Seiyaku, Tokyo, Japan)supplemented with 200 mg of kanamycin sulfate per ml (Meiji Seika, Tokyo,Japan) (in the following experiments, the antibiotics was added) and 1% fetalcalf serum (FCS; GIBCO Laboratories, Grand Island, N.Y.) in 96-well micro-culture plates (no. 25860; Corning Glass Works, Corning, N.Y.) at 5 3 105/100ml/well. The cells were added with test materials solubilized or suspended in 100ml of the medium and were cultured for 48 h in a CO2 incubator. For the final8 h of the culture, 125I-UdR was added to the culture (0.5 mCi/well). At the endof the culture period, the cells were collected on a fiberglass filter with anauto-cell harvester (Labomash; Labo Science, Tokyo, Japan) and the 125I-UdRuptake by the cells was measured with a gamma counter (Auto Well GammaSystem ARC-300; Aloka Co., Tokyo, Japan). The stimulation index (SI) wascalculated as follows: 125I-UdR uptake in test culture/125I-UdR uptake in controlculture (medium alone).

Assays of cytokine-inducing activities. Similar procedures to those describedabove, except for the culture conditions, were used, and triplicate cultures in thepresence or absence of test materials in 96-well microculture plates were carriedout for 24 h to obtain the test culture supernatant for the cytokine assays.Thioglycolate medium-induced peritoneal exudate cells (105/100 ml/well) fromC3H/HeN and C3H/HeJ mice were preincubated in alpha-modified minimumessential medium (a-MEM; Flow Laboratories, Inc., McLean, Va.) for 2 h. Afterthe nonadherent cells were washed out, residual adherent cells, which wereshown in a separate experiment to be composed of more than 85% macrophages,were stimulated with test materials in a-MEM supplemented with 5% FCS asdescribed previously (36). Heparinized human peripheral whole blood obtainedfrom a healthy adult volunteer (25 ml of blood in 100 ml of RPMI 1640 mediumwithout FCS) was stimulated with test materials for 24 h as described previously(36). Primary cultures of human gingival fibroblasts were prepared from thehealthy human gingival tissue of a patient (an 8-year-old girl) during a fenestra-tion procedure under informed consent as described previously (39). The con-fluent cells (5 3 104/100 ml/well) in a-MEM supplemented with 1% FCS werestimulated for 24 h as described previously (39). The culture plates were thencentrifuged at 800 3 g for 10 min, and the supernatants of triplicate cultures werepooled for the cytokine assays. Tumor necrosis factor alpha (TNF-a) activity wasdetermined in terms of cytotoxicity on actinomycin D-treated L929 cells asdescribed previously (36). The TNF-a activity in a test specimen was calculatedas nanograms per milliliter on the basis of the ratio of a 50% cytotoxic dose ofthe test specimen to that of standard recombinant human TNF-a (rHuTNF-a)which was supplied by Dainippon Pharmaceutical Co. (Osaka, Japan). The IL-6activity of test specimens was determined by measuring the in vitro growth of theIL-6-dependent cell line MH60.BSF2 by the method of Matsuda et al. (23) withminor modifications for the calorimetric assay and with 3-(4,5-dimethyl-2-thia-zolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) as described previously (36).The IL-6 activity in a test specimen was determined on the basis of a comparisonwith the titration curve for standard rHuIL-6 (a gift from T. Hirano, OsakaUniversity, Osaka, Japan) and is expressed as units per milliliter. The level ofhepatocyte growth factor was measured in duplicate with an enzyme-linkedimmunosorbent assay kit (Otsuka Pharmaceutical Co., Tokushima, Japan).

Inhibition assay by use of polymyxin B. In some of the experiments, in whichthe reference classical endotoxin exhibited bioactivities, cells were stimulatedwith test materials in the presence of polymyxin B sulfate (ICN PharmaceuticalsInc., Irvine, Calif.) to examine the possibility that the bioactivities of test mate-rials are attributable to endotoxin contamination of the materials.

Northern blot analysis. The expression of IL-8 mRNA by human gingivalfibroblasts stimulated with test materials was examined by Northern blot analysisas described previously (40) with minor modifications. Briefly, the confluenthuman gingival fibroblasts (about 6 3 106 cells) in a 150-mm-diameter plate(Iwaki Glass, Tokyo, Japan) were stimulated for 8 h with test materials ina-MEM containing 1% FCS. Total cellular RNA was prepared from cells by theguanidinium isothiocyanate-cesium chloride procedure (2). Total RNA (10 mgper lane) was electrophoresed in a 1.2% agarose gel and transferred electro-phoretically to a nylon membrane. The membrane was hybridized with 32P-labeled HuIL-8 cDNA as a probe (24). The blots were exposed to an imagingplate for 1 h for analysis by an imaging analyzer or exposed to X-ray film at280°C for several days for autoradiography. Equal loading in lanes was checkedby successive hybridization with 32P-labeled glyceraldehyde-3-phosphate dehy-drogenase cDNA (41) by the method of Sambrook et al. (30). Photographs of theimaging plates were analyzed with a Bioimaging analyzer (model BAS-1000 Mac;Fujifilm Inc., Tokyo, Japan).

Statistical analyses. In most assays, the mean 6 standard error of the meanwas obtained and the significance of the difference between each test result andits respective control was examined by Student’s t test. In other experiments, inwhich calculation of the standard error of the mean was not practical, the rangeof fluctuations of the activity was within 20% of the respective mean.

RESULTS

Preparation and chemical analyses of P. intermedia frac-tions. The water phase of the hot phenol-water extract of P.intermedia ATCC 25611 was ultracentrifuged at 140,000 3 g

FIG. 1. Outline of the procedures for the preparation of test materials fromP. intermedia ATCC 25611.

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for 3 h to remove LPS. The sediment was washed by twofurther ultracentrifugation steps to prepare LPS-PW. The re-sultant supernatant, the S1 fraction, was composed of a low-molecular-weight band comparable to LPS-PCP and a high-molecular-weight band in DOC-PAGE (Fig. 2A). Comparisonwith the migration profile of the reference S. abortusequi LPSin DOC-PAGE, the molecular weight of the high-molecular-weight band was estimated to be 10,000 to 12,000 (10K to12K). To estimate the molecular weight of the low-molecular-weight band which was comparable to the main band of LPS-PCP of P. intermedia, the LPS-PCP and various chemotypes ofLPS of S. minnesota were subjected to PAGE (18% polyacryl-amide). The main band of LPS-PCP migrated between those ofRc and Rd1 LPS (Fig. 2B); the molecular weight was thereforeestimated to be about 3K. The profile of S1 was similar to thatof LPS-PW, although high- and low-molecular-weight bandswere clear in the former and latter, respectively.

The S1 fraction solubilized in 0.2% sodium DOC was sub-jected to Sephadex G-100 column chromatography, and twofractions, F1 and F2, were prepared for monitoring of themitogenicity on splenocytes from C3H/HeN and C3H/HeJmice, Limulus activity, and DOC-PAGE (Fig. 3). The F1 frac-tion, composed of the high-molecular-weight bands on DOC-PAGE, exhibited mitogenicity on splenocytes of both C3H/HeN and C3H/HeJ mice and only weak Limulus activity,whereas the F2 fraction, composed of low-molecular-weightbands on DOC-PAGE, exhibited mitogenicity on splenocytes

of C3H/HeN but not C3H/HeJ mice and showed definiteLimulus activity. We suspected that the F1 fraction containedthe target material; therefore, we treated this fraction withnuclease P1 and rechromatographed it on a Sephadex G-100column to obtain the PGP fraction. As expected, the PGPfraction was scarcely active in the Limulus test, whereas theLimulus activities of LPS-PCP and LPS-PW were strongerthan and comparable to that of the reference LPS of Esche-richia coli O111:B4, respectively (Table 1).

The chemical analytical data of the F1 and PGP fractions

FIG. 2. DOC-PAGE of P. intermedia preparations and reference LPS spec-imens. Aliquots (1 mg) of test materials were ultrasonicated in sample buffer for1 min and applied to 12% (A) or 18% (B) polyacrylamide gels. Electrophoresiswas performed with Tris-glycine buffer containing 0.2% DOC with a constantcurrent of 12 mA at 4°C for 2 h, and then the gels were subjected to silverstaining. (A) Lanes: 1, S. abortusequi LPS; 2, LPS-PCP; 3, LPS-PW; 4, S1fraction; 5, PGP fraction; 6, F2 fraction. (B) Lanes: 1, LPS-PCP; 2-7, LPS ofR-chemotypes of S. minnesota: 2, R595 (Re); 3, R3 (Rd2); 4, R7 (Rd1); 5, R5(Rc); 6, R345 (Rb); 7, R60 (Ra).

FIG. 3. Elution profiles of a hot phenol-water extract of P. intermedia ATCC25611 with monitoring mitogenic activities on splenocytes from C3H/HeN andC3H/HeJ mice, Limulus activity, and DOC-PAGE. A hot phenol-water extract(24 mg) of P. intermedia ATCC 25611 with most of its LPS removed by ultra-centrifugation (S1 fraction) was resolved in 0.2% DOC and subjected to Seph-adex G-100 column chromatography. Aliquots of eluates selected were dialyzedagainst distilled water to remove DOC, diluted by 15-fold with RPMI 1640medium, and subjected to an assay for mitogenicity in murine splenocyte cul-tures. The mitogenic activity is expressed as the SI. The levels of radioactivityincorporated in the control cultures with medium alone were 345 6 18 cpm(C3H/HeN) and 252 6 36 cpm (C3H/HeJ). Limulus activity was determined withan Endospecy kit and is expressed as nanograms per milliliter equivalent to thereference LPS of E. coli O111:B4. Aliquots (5 ml) of some eluates were subjectedto DOC-PAGE with a 12% polyacrylamide gel. For further details, see the text.The eluates marked by arrows were pooled as F1 and F2 fractions.

TABLE 1. Limulus activity of test fractions prepared fromP. intermedia ATCC 25611

Test material Limulus activitya

LPS-PCP................................................................................. 4.08LPS-PW.................................................................................. 1.13S1 fraction.............................................................................. 0.520F1 fraction.............................................................................. 0.000047F2 fraction.............................................................................. 0.400PGP fraction .......................................................................... 0.0000223S. abortusequi LPS................................................................. 3.83

a Limulus activity was determined with the Endospecy kit and is expressed asthe ratio to the activity of the reference LPS of E. coli O111:B4.

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also showed that they were not contaminated by LPS, becauseonly trace amounts of the fatty acids were detected in both(Table 2). The F1 fraction was thought to contain RNA, be-cause it showed rather high levels of phosphorus and ribose.The amounts of these components were reduced by nucleasetreatment during the preparation of PGP. In addition, the F1and PGP fractions contained rhamnose, galactose, and glucoseas neutral sugars and small amounts of glucosamine (Table 2).Glucose was also detected in LPS-PW, but rhamnose and ga-lactose were found in neither LPS-PW nor LPS-PCP. These

observations indicated that F1 and PGP fractions containedoligo- or polysaccharide moieties. The amount of protein inthe PGP fraction was estimated to be 11%, suggesting that thisfraction was a complex of carbohydrate and protein. It must bementioned here that in the analysis of fatty acids in the PGPfraction on GLC, the characteristic peak with a retention timelonger than that of normal fatty acid derivatives was detected.The molecular weight of the substance showing the peak wasestimated by chemical ionization MS to be 390, but the frag-mentation profile as estimated by electron impact MS wasdifferent from that of fatty acid derivative. This unknown peakwas not detected in LPS-PCP but was detected in LPS-PW,suggesting that LPS-PW also contains target material otherthan LPS itself. In contrast, the LPS-PCP showed typical prop-erties of BPB-LPS; it contained branched fatty acids repre-sented by 3-OH-i-C17:0, more than half of which were amidelinked to glucosamine, and neither 3-OH-C14:0 nor C14:0 wasdetected.

Bioactivities of the PGP fraction compared with the LPSspecimens. The PGP fraction exhibited strong mitogenic activ-ity on splenocytes from both C3H/HeN and C3H/HeJ mice,while LPS-PCP exhibited moderate mitogenic activity on thecells from C3H/HeN but not C3H/HeJ mice, similar to thereference S. abortusequi LPS (Fig. 4). LPS-PW exerted strongand moderate mitogenicity on the cells of C3H/HeN and C3H/HeJ mice, respectively. The PGP fraction stimulated perito-neal macrophages from both C3H/HeN and C3H/HeJ mice torelease IL-6 and TNF-a activity (Fig. 5). In contrast, LPS-PCP

FIG. 4. Mitogenic activities of P. intermedia preparations and reference S.abortusequi LPS (S-LPS) on splenocytes from C3H/HeN and C3H/HeJ mice.Splenocytes (5 3 105/200 ml/well) were cultured in RPMI 1640 medium with testmaterials for 48 h in 96-well microculture plates. During the final 8 h of theculture, the cultures were pulsed with 125I-UdR. Assays were performed intriplicate, and the activities are expressed as stimulation index (SI). The levels ofradioactivity incorporated in the control cultures with medium alone were 311 628 cpm (C3H/HeN) and 350 6 29 cpm (C3H/HeJ). Asterisks indicate that theresults were significantly different from the control by Student’s t test (p, P ,0.05; pp, P , 0.01). The results are representative of five independent experiments.

FIG. 5. Production of IL-6 (A) and TNF-a (B) activities by murine perito-neal macrophages stimulated with P. intermedia preparations and reference S.abortusequi LPS (S-LPS). Thioglycolate-induced peritoneal macrophages (105/100 ml/well) were cultured with test materials in RPMI 1640 medium for 24 h in96-well microculture plates. The IL-6 activity and TNF activity in the culturesupernatant were determined in terms of the proliferation of the IL-6-dependentcell line MH60.BSF2 and cytotoxicity on actinomycin D-treated L929 cells asdescribed in the text. The results are representative of three independent exper-iments.

TABLE 2. Chemical analytical data of LPS-PCP, LPS-PW, F1, andPGP fractions prepared from P. intermedia ATCC 25611

ComponentAmt (mmol/mg) of component in:

LPS-PCP LPS-PW F1 PGP

Sugars and aminosugars

Rhamnose 0 ,0.005 0.11 0.19Ribose 0 0.01 0.42 0.08Mannose 0.03 0.01 0.01 0.06Galactose ,0.005 0 0.04 0.03Glucose 0.03 2.36 0.32 0.16Glucosamine 0.44a (0.56b) 0.18 (0.22) 0.06 (0.08) 0.02 (0.06)KDO 0.01 ,0.005 0 ,0.005

Phosphorus 1.94 0.84 1.31 0.12

Fatty acidsc

i-C14:0 0.12 (0.17) 0.03 (0.06) 0 0i-C15:0 0.02 (0.02) 0.01 (0.01) 0 0a-C15:0 0.04 (0.04) 0.01 (0.02) ,0.005 0.01C16:0 0.01 (0.04) 0.01 (0.02) 0.01 ,0.005C17:0 0.03 (0.03) 0.01 (0.01) 0 03-OH-C16:0 0.12 (0.13) 0.06 (0.06) 0 03-OH-i-C17:0 0.25 (0.11) 0.12 (0.05) 0.01 0.013-OH-i-C18:0 0.02 (0.01) 0.01 (0.01) 0 0

a Determined by the gas-liquid chromatography method.b Determined by the colorimetric method.c The amount of fatty acid liberated by 0.1 M NaOH hydrolysis (100°C for 30

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exhibited a definite activity over a wide range of concentrationson the cells from C3H/HeN mice but at only one concentration(10 mg/ml) on those from C3H/HeJ mice. LPS-PW showed aprofile intermediate between those of PGP and LPS-PCP. Onthe peripheral human blood cells, all three P. intermedia frac-tions and the reference S. abortusequi LPS exhibited clear

activity to induce IL-6 and TNF-a production, although theactivities of the LPS-PCP were weaker than those of the otherpreparations (Fig. 6). On human gingival fibroblasts, the PGPfraction induced IL-6 activity, IL-8 mRNA expression, andhepatocyte growth factor production (Fig. 7). LPS-PCP wasscarcely active in this respect, similar to the reference S. abor-tusequi LPS, and the LPS-PW fraction exhibited a profile in-termediate between those of PGP and LPS-PCP.

Characterization of active material in the PGP fraction. Toelucidate the physicochemical properties of the active materialin the PGP fraction, the influence of various treatments ofPGP on its mitogenic activity against the splenocytes fromC3H/HeJ mice was examined. The mitogenic activity of thePGP fraction was resistant to heating at 100°C for 60 min andto pronase E and proteinase K but was almost completelyabolished by treatment with sodium periodate (Fig. 8). Thesefindings suggested that the protein(s) was not important for themitogenicity of the PGP fraction whereas the saccharide moi-ety was important in this respect. When the F1 fraction wassequentially treated with nuclease P1 and pronase E, the high-molecular-weight band in DOC-PAGE disappeared (data notshown) but the mitogenic activity remained, although the elu-tion profile of the active fraction in Sephadex G-100 columnchromatography shifted to the low-molecular-weight side (Fig.9).

Failure to inhibit the activities of the PGP fraction by poly-myxin B. To estimate the contribution of endotoxin to thebioactivities of the test materials, the bioactivities on C3H/HeN cells of the P. intermedia fractions and the reference S.

FIG. 6. Production of IL-6 (A) and TNF-a (B) activities by human periph-eral blood cells stimulated with P. intermedia preparations and reference S.abortusequi LPS (S-LPS). Heparinized human peripheral whole-blood cultures(25 ml/well) were cultured with test materials in RPMI 1640 medium for 24 h in96-well microculture plates. Other procedures were the same as those describedin the legend to Fig. 5. The results are representative of three independentexperiments.

FIG. 7. Activation of human gingival fibroblasts to produce IL-6 (A), expressIL-8 mRNA (B), and release HGF (C) by P. intermedia preparations and refer-ence S. abortusequi LPS (S-LPS). (A) Human gingival fibroblasts (53104/100ml/well) were cultured for 24 h with test materials in a-MEM supplemented with1% FCS in 96-well microculture plates. The IL-6 activity in culture supernatantswas determined in terms of the proliferation of MH60.BSF2 cells. (B) Fibro-blasts (6 3 106 cells/culture) in a 150-mm-diameter culture plate were stimulatedfor 8 h with the following test materials in a-MEM containing 1% FCS: a,medium alone; b, S. abortusequi LPS (10 mg/ml); c, LPS-PCP (10 mg/ml); d,LPS-PW (10 mg/ml); e to g, PGP fraction (e, 0.1 mg/ml; f, 1.0 mg/ml; g, 10 mg/ml).Northern blotting was performed as described in the text. The results are ex-pressed as relative mRNA accumulation with GAPDH mRNA as an internalstandard. (C) Test culture supernatants prepared as described for panel A weresubjected to an enzyme-linked immunosorbent assay to determine the HGFlevels. rHuIL-1a (10 ng/ml) and rHuTNF-a (200 ng/ml) were used as referencestimulants. Asterisks indicate that the results were significantly different from thecontrol (medium alone) by Student’s t test (p, P , 0.05; pp, P , 0.01). The resultsare representative of three (A) or two (B and C) independent experiments.



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abortusequi LPS were examined in the presence of gradedconcentrations of polymyxin B. Both the mitogenic activity onsplenocytes and the IL-6-inducing activity on macrophages ofthe PGP fraction were scarcely inhibited by polymyxin B, whilethe activities of the LPS-PCP and S. abortusequi LPS werecompletely inhibited (Fig. 10). Partial inhibitions were ob-served with LPS-PW.

DISCUSSIONThe present results provide evidence that contributes to an

explanation of the unique bioactivities of P. intermedia “LPS.”Specifically, a novel immunobiologically active material whichis composed mainly of polysaccharide and protein and an un-avoidably contaminated LPS fraction prepared from P. inter-media ATCC 25611 by hot phenol-water extraction methodsstimulated the lymphocytes and macrophages from classicalLPS-nonresponsive C3H/HeJ mice and human gingival fibro-blasts whereas the purified LPS prepared from the same bac-teria by PCP extraction was inactive in this respect. Theseunique activities were commonly observed with BPB LPS butnot with the LPS of members of the Enterobacteriaceae such asE. coli and Salmonella species. There have been various reportsof unusual LPS other than BPB LPS which activated classicalLPS-nonresponsive C3H/HeJ lymphoid cells. Vogel et al. (43)reported that a lipid A precursor, later designated lipid IVA,activated splenocytes and macrophages of C3H/HeJ mice.However, a chemically synthesized lipid IVA was completelyinactive in this respect (38). Immunostimulatory activities ofR-form LPS specimens on lymphocytes and macrophages fromC3H/HeJ mice were reported by several investigators (3, 4, 34,42). However, Manthey et al. (22) demonstrated that the un-usual activities could be attributable to the trace quantities of

endotoxin protein(s) in the LPS preparations. Concerning BPBLPS, the possible contamination of endotoxin proteins hasbeen discussed in almost every paper in this field published todate. In the present study, we first focused on the importanceof polysaccharide rather than protein to interpret the unusualactivities of BPB LPS. Our preparatory procedures for thePGP fraction are similar to those used for the preparation ofcapsular polysaccharide from Bacteroides fragilis described byPantosti et al. (27). Gibson et al. (9) reported that the capsularpolysaccharide of B. fragilis induced cytokine production inhuman monocyte and murine macrophage cultures. Therefore,the active component in the PGP fraction in this study mighthave been capsular polysaccharide. However, the weak activityof PCP-LPS, especially IL-6-inducing activity on murine mac-rophages (Fig. 5), may be attributable to endotoxin protein(s)rather than the PGP fraction because the LPS-PCP containedconsiderable amounts of protein.

A brief historical overview of the unique chemical and bio-logical properties of BPB LPS may help to further clarify theresults of this study. In the present taxonomy, the term “oralBPB” encompasses the genera Prevotella, Porphyromonas, andBacteroides; these three genera were previously classified asBacteroides. In fact, LPS of enteric Bacteroides species such asB. fragilis possess several of the properties of LPS of oral BPB,but their properties are different from those of LPS of theEnterobacteriaceae (20). The lipid A moiety in “Bacteroides”species possesses a b(1,6)-linked D-glucosamine disaccharidebackbone, like those of the members of the Enterobacteriaceae.However, they usually possess 3-hydroxylated branched andnonbranched long-chain fatty acids such as (R)-3-hydroxy-15-methylhexadecanoic (3-OH-i-C17:0), (R)-3-hydroxyhexadecanoic(3-OH-C16:0), and (R)-3-hydroxy-13-methyltetradecanoic (3-OH-i-C15:0) acids but not (R)-3-hydroxytetradecanoic acid (3-OH-14:0), which is a common constituent of the normal lipid A.Furthermore, “Bacteroides” lipid A molecules are usually com-posed of fewer fatty acids (three to five) than are those ofEnterobacteriaceae (six to seven), and they frequently lack thephosphate group at the C-4 position of the nonreducing aminosugar (C-49 position). Several studies of the structure-activityrelationships of lipid A by using various synthetic lipid A-related compounds (reviewed in references 28 and 37) dem-onstrated that monophosphoryl lipid A and fewer and longer

FIG. 8. Influences of various treatments of PGP or F1 fraction on theirmitogenic effect on splenocytes from C3H/HeJ mice. (A) The F1 fraction washeated at 100°C for 60 min. (B) The F1 fraction was treated with proteinase Kat 37°C for 1 h. (C) The PGP fraction was treated with pronase E at 37°C for18 h. (D) The F1 fraction was treated with sodium periodate at 4°C for 5 days.Detailed procedures are described in the text. The mitogenic activities of thetreated materials were compared with those of the respective nontreated mate-rial. The incorporation of 125I-UdR in the control cultures was as follows: (A),350 6 29 cpm; (B), 298 6 25 cpm; (C) 339 6 16 cpm; (D) 168 6 16 cpm.Asterisks indicate that the results were significantly different from the controlwith medium alone by Student’s t test (p, P , 0.05; pp, P , 0.01). The results arerepresentative of two independent experiments.

FIG. 9. Comparison of elution profiles of the F1 fractions before and afterenzyme treatment, with monitoring of the mitogenic activity on splenocytes fromC3H/HeJ mice. The F1 fraction (10.5 mg) was sequentially treated with nucleaseP1 and pronase E and then rechromatographed on Sephadex G-100. Aliquots ofthe eluates selected were dialyzed against distilled water, diluted 25-fold withRPMI 1640 medium, and subjected to the mitogenicity assay. The radioactivityincorporated in the control cultures with medium alone was 339 6 16 cpm. Theprofile of nontreated F1 was derived from Fig. 3.



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fatty acids resulted in low endotoxicity of the compounds. Infact, “Bacteroides” LPS have been observed to have a low levelof endotoxicity (20, 35, 48). The chemical structure of lipid Aof P. gingivalis was proposed by two Japanese groups (19, 25).The structures possess the above properties common to “Bac-teroides” lipid A, although considerable differences betweenthese two reports were noted. Ogawa (25) described the re-markably unique lipid A structure of Porphyromonas gingivalis381; the absence of phosphate at the C-49 position and ester-linked fatty acids at the C-3 and C-39 positions led to theconclusion that the lipid A was a 1-monophosphoryl lipid Acarrying a 3-OH-i-C17:0 at the C-2 and C-29 positions and aC16:0 to form an acyloxyacyl group at the C-29 position. Ku-mada et al. (19) proposed the more usual lipid A structure ofthe strain SU 63;1,49-diphosphoryl lipid A carrying five ester-and amide-linked fatty acids in total. These two groups ofinvestigators noted considerable variations concerning thelipid A structures, and therefore the two reports may not nec-essarily conflict with each other. The present results of thechemical analyses of P. intermedia LPS (LPS-PCP) indicatedstructural similarity to P. gingivalis, as expected.

The most controversial point about “Bacteroides” LPS is theunique biological activities, different from those of LPS of theEnterobacteriaceae. Such activities on C3H/HeJ cells were firstreported by Joiner et al. (15), who found that B. fragilis LPSwas mitogenic on splenocytes from C3H/HeJ mice. Interestingfindings were reported by McGhee’s group (44, 47); purifiedsplenic B cells from C3H/HeJ mice were hyporesponsive to B.fragilis LPS, suggesting that B. fragilis lipid A does not stimu-late B cells from C3H/HeJ mice whereas the polysaccharidemoiety of the LPS exerts B-cell activation via macrophages.Therefore, the above-described reports on the bioactivities ofBPB LPS on C3H/HeJ mice were an extension of LPS ofenteric Bacteroides to LPS of oral Bacteroides (BPB). Surpris-ingly, however, Ogawa et al. (25, 26) found that lipid A as wellas LPS of P. gingivalis was mitogenic on splenocytes fromC3H/HeJ mice. In the present study, we obtained evidence thatsupports another possible explanation of the unique activitiesof BPB LPS. The reason for the discrepancies between ourfindings and those of Ogawa et al. (25, 26) is not clear. The

differences in the bacterial species and genera used is perhapsa factor. It must be emphasized, however, that the LPS-PCPused in this study, which is scarcely active on C3H/HeJ cells,shared lipid A structures with the “Bacteroides” lipid A, includ-ing that of P. gingivalis. As suggested by Ogawa et al. (26) andKumada et al. (19), the chemical synthesis of “Bacteroides”lipid A may resolve the issue of unique bioactivities of BPBLPS.

ACKNOWLEDGMENTS

We thank Y. Daikuhara (Kagoshima University Dental School) forhis assistance in obtaining the HuHGF ELISA kit. We are indebted toJ. Aketagawa (Seikagaku Co.) and Y. Isshiki (Kitasato Institute) fortheir assistance with the Endospecy test and the chemical analyses,respectively. We also thank D. Mrozek (Medical English Service,Kyoto, Japan) for reviewing this paper.

This work was supported in part by Grants-in-Aid for ScientificResearch (05671507) from the Ministry of Education, Science, Sports,and Culture of Japan and by a Grant-in-Aid for scientific researchfrom Seikagaku Co., Ltd., Tokyo, Japan.

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FIG. 10. Influences of polymyxin B (PB) on the mitogenicity (A) and IL-6-inducing activity (B) of P. intermedia fractions and the reference S. abortusequi LPS. (A)Test materials (10 mg/ml) were incubated with the indicated concentrations of PB at 37°C for 60 min and subjected to a mitogenic assay with splenocytes from C3H/HeNmice. The activities are expressed as percentages, where 100% indicates the radioactivity incorporated in the respective positive control without PB minus that in thenegative control with medium alone. The radioactivity of the positive controls was 3,412 6 16 cpm (PGP fraction), 1,977 6 120 cpm (LPS-PW), 2,085 6 37 cpm(LPS-PCP), and 2,131 6 394 cpm (S. abortusequi LPS), and that in the negative control was 292 6 16 cpm. (B) The test materials (0.01 mg/ml with S. abortusequi LPSand 1 mg/ml with others) were incubated with PB as in panel A and then subjected to cultures with peritoneal macrophages from C3H/HeN mice to examine theIL-6-inducing activities of the materials. The activities are expressed as percentages, where 100% indicates the IL-6 activity in the respective positive control withoutPB minus that in the negative control with medium alone. The IL-6 activity of the positive controls was 9,461 6 204 U/ml (PGP fraction), 7,292 6 335 U/ml (LPS-PW),5.542 6 833 U/ml (LPS-PCP), and 7,546 6 718 U/ml (S. abortusequi LPS), and that in the negative control was 256 6 3 U/ml. Symbols: h, PGP; Œ, LPS-PW; ‚,LPS-PCP; F, S. abortusequi LPS. Asterisks indicate that the results were significantly different from the respective positive control by Student’s t test (p, P , 0.05; pp,P , 0.01). The results are representative of two independent experiments.



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