Vol. 50, No. 1INFECTION AND IMMUNITY, OCt. 1985, p. 262-2700019-9567/85/100262-09$02.00/0Copyright © 1985, American Society for Microbiology

Functional Role of Interleukin 1 in Periodontal Disease: Induction ofInterleukin 1 Production by Bacteroides gingivalis

Lipopolysaccharide in Peritoneal Macrophages from C3H/HeN andC3H/HeJ Mice

SHIGEMASA HANAZAWA,l* KOHJIN NAKADA,2 YOSHIHIRO OHMORI,1 TAKEHITO MIYOSHI,' SHIGERUAMANO,' AND SHIGEO KITANO'

Departments of Oral Microbiologyl and Oral Diagnosis,2 Josai Dental University, Keyakidai, Sakado City,Saitama 350-02, Japan

Received 1 May 1985/Accepted 28 June 1985

Hot phenol-water-extracted lipopolysaccharide (LPS) from Bacteroides gingivalis 381 was purified bySephadex G-100 chromatography with Tris buffer supplemented with sodium deoxycholate and EDTA(B-LPS). In the present study, B-LPS was examined for its ability to induce interleukin 1 (IL-1) production, a

mitogenic response, and macrophage activation in LPS high-responder C3H/HeN and low-responder C3H/HeJmice. A significant increase in IL-1 production was observed in C3H/HeN and C3H/HeJ peritonealmacrophages treated with various doses (1.0 to 50 ,ug/ml) of B-LPS. IL-1 production by C3H/HeNmacrophages treated with B-LPS (10 pg/mI) was about seven times greater than that by C3H/HeJmacrophages. However, the IL-1 production induced by B-LPS (10 ,ug/mI) in C3H/HeN macrophages was fourtimes lower compared with that induced by Escherichia coli 0111 B4 LPS. Also, a significant increase in IL-Iproduction was found in human monocytes stimulated with B-LPS. That B-LPS-induced IL-1 exhibits some

molecular weight heterogeneity was indicated from Sephadex G-75 gel filtration profiles. A significant, highmitogenic response by whole spleen cells with 1 x 105 to 5 x 104 cells of either mouse strain per well treatedwith B-LPS (10 to 50 ,ug/ml) was observed. However, the response of C3H/HeJ mice was less than that of theC3H/HeN strain. Also, glucose consumption assays indicated that enhanced macrophage activation occurred inC3H/HeN but not in C3H/HeJ mice treated with B-LPS. In light of recent studies showing that IL-1 stimulatesbone resorption in a mouse calvaria system and collagenase production in fibroblasts, we suggest thatB-LPS-induced IL-1 may play a significant role in the pathogenesis of adult periodontal disease.

Since the gram-negative, anaerobic bacterium Bacteroidesgingivalis was predominantly isolated from subgingival sitesof advancing lesions in adult periodontitis patients (24, 25,27, 31) and since the serum antibody titer against thisorganism is significantly increased in sera of patients havingthis disease (12, 18; S. Hanazawa, S. Tanaka, K. Saitoh, Y.Ohmori, K. Nakano, K. Nakada, T. Masuda, and S. Kitano,submitted for publication), B. gingivalis is believed to beclosely associated with adult periodontal disease. However,the pathogenic mechanisms of B. gingivalis in the initiationand development of adult periodontal disease are not yetcompletely understood.

Bacterial lipopolysaccharides (LPS) have a high potentialfor causing the alteration and destruction of host tissues. Thestructure and immunological activities of LPS from aerobic,gram-negative bacteria, especially members of the familyEnterobacteriaceae, have been investigated in detail (17).The involvement of LPS in the pathogenesis of periodontaldisease is supported by the ability of this macromolecule topenetrate into gingival tissues and to stimulate bone resorp-tion in vitro. The structure and immunological activities ofLPS from B. gingivalis are incompletely understood. Re-cently, Nair et al. (19) have purified LPS from the outermembrane of B. gingivalis and have examined its chemicalproperties and biological activities.

Interleukin 1 (IL-1) is a monokine that exerts biologicaleffects on a variety of target cells in vivo and in vitro.

* Corresponding author.

262

Recently, Oppenheim et al. (20) and Charon et al. (3) havereported a significant increase of IL-1 in gingival fluids frominflamed sites in periodontitis patients. Most recently,Gowen et al. (4), using a mouse calvaria in vitro system,have suggested that human IL-1-like factors stimulate boneresorption. Also, a recent study has shown that IL-1 is apotent stimulator of collagenase production of fibroblasts(21). These findings have suggested the possibility that IL-1may play a significant role in the initiation and developmentof periodontal disease. It is well known that Enterobacteri-aceae-derived LPS is a potent IL-1 inducer. Therefore, itseems very important for understanding the role played byB. gingivalis in pathogenic mechanism(s) operating in adultperiodontal disease to determine whether or not LPS from B.gingivalis is also a potent inducer of IL-1 production.

In this report, we show that B. gingivalis-purified LPS is apotent inducer of IL-1 production and propose that B.gingivalis LPS-induced IL-1 plays a significant role in thepathogenesis of adult periodontal disease.

MATERIALS AND METHODS

Bacterial strain and cultivation procedures. B. gingivalis 381(originally isolated by S. S. Socransky, Forsyth DentalCenter, Boston, Mass.) was cultivated in EX-1 diffusatemedium (13) for 48 h at 37°C under anaerobic conditions. Thecells were harvested at the end of the culture period, washedwith physiological saline, and then lyophilized.

Preparation of LPS. B. gingivalis LPS was extracted bythe hot phenol-water procedure of Westphal and Jann (30)

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0 1.D 1 50 0 1.0 1050 0 0.1 1.0 10 0 0.1 1.0 10

Dose of B-LPS(Ug/m1) Dose of E-LPS(iug/ml)

FIG. 1. IL-1 activity of culture supernatants of C3H/HeN and C3H/HeJ peritoneal macrophages treated with B-LPS or E-LPS. C3H/HeNand C3H/HeJ peritoneal macrophages were cultured for 24 h in the presence of various concentrations of LPS. Each culture supernatant wasdiluted (1:4), and the IL-1 activity was determined. Thymocytes cultured only with B-LPS at 1, 10, and 50 pg/ml incorporated 2,750 + 350,4,106 + 1,023, and 2,592 + 117 cpm of [3H]thymidine, respectively; those with only E-LPS at 0.1, 1, and 10 pg/ml incorporated 5,021 + 621,3,750 ± 450, and 2,940 ± 112 cpm of [3H]thymidine, respectively.

and purified as described by Mansheim et al. (11) and Nair etal. (19). Briefly, lyophilized intact cells were treated with hotphenol-water, and the aqueous phase was collected andconcentrated. Thereafter, the concentrated materials weretreated with ethanol containing sodium acetate and subse-quently with acetone. The obtained precipitates (crude LPS)were suspended in water and washed five times by centrif-ugation at 100,000 x g for 2 h. The pelleted materials werechromatographed on a Sephadex G-100 column (90 by 2.2cm) equilibrated with 0.3% sodium deoxycholate in Trisbuffer (pH 8.0) supplemented with 1 mM EDTA, and 5-mlfractions were collected. Sodium deoxycholate in each frac-tion was removed by dialysis against distilled water contain-ing an anion-exchange resin (Dowex 1-X8). The elutionprofile was obtained by measuring the total carbohydratecontent of each fraction with anthrone reagent. The fractions(no. 40 to 55) without absorption at an optical density at 280nm in the first carbohydrate-containing major peak were

pooled, concentrated, and then lyophilized. This materialwas then used as purified LPS (B-LPS). Escherichia coli0111 B4 LPS (E-LPS) extracted by the Westphal procedurewas obtained from LBL, Inc. (Campbell, Calif.).

Mice. C3H/HeN mice (CLEA Japan Co., Tokyo, Japan)and C3H/HeJ mice (supplied by Animal Center, Josai DentalUniversity) were used at 6 to 8 weeks of age.

Preparation of peritoneal macrophages. Mice were injected

intraperitoneally with 3 ml of thioglycolate medium (NisuiSeiyaku Co., Tokyo, Japan). At day 4 after injection, theperitoneal macrophages were prepared as described previ-ously (5). The peritoneal exudate cells were harvested bywashing the peritoneal cavity with RPMI 1640 mediumcontaining heparin (50 U/ml). The harvested cells werewashed and suspended in RPMI 1640 medium supplementedwith penicillin (100 U/ml) and streptomycin (100 ,ug/ml).One-milliliter volumes of this cell suspension (106 cells perml) were placed in Falcon culture dishes (Becton DickinsonLabware, Oxnard, Calif.) and incubated for 2 h at 37°C in 5%CO2. After the incubation, the nonadherent cells were re-moved by vigorous washing with RPMI 1640. The adherentcells were judged to be 90 to 95% macrophages from theirphagocytic activity with latex particles.

Induction of IL-1 production by peritoneal macrophages.Peritoneal adherent macrophages were prepared as de-scribed above and were incubated in RPMI 1640 with B-LPSor E-LPS at 37°C in 5% CO2. At various times of incubation,the culture medium was harvested and centrifuged, and thesupernatant was passed through a filter membrane (0.22 ,um[pore size], Millipore Corp., Bedford, Mass.). In someexperiments, the supernatant was concentrated with poly-ethylene glycol and then chromatographed on a SephadexG-75 column (98 by 2.8 cm) equilibrated with 20 mMphosphate-buffered saline (pH 7.2). Columns were calibrated

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FIG. 2. Time course of IL-1 activity in culture supernatants of C3H/HeN (0, *) and C3H/HeJ (0, O) peritoneal macrophages untreated(C1, *) or treated with B-LPS (0, 0). Macrophages were cultured for various times in the presence of B-LPS (10 ,ug/ml). The culturesupernatants were harvested at the end of each culture period and diluted (1:4), and then the IL-1 activity was examined.

with the following markers: aldolase (molecular weight[MW], 158,000), bovine serum albumin (MW, 68,000), henegg albumin (MW, 45,000), chymotrypsinogen A (MW,25,000), and cytochrome c (MW, 12,500).

Induction of IL-1 production by human monocytes. Periph-eral blood from clinically healthy donors was diluted in 2volumes of RPMI 1640, layered on Ficoll-Hypaque(Pharmacia Fine Chemicals, Uppsala, Sweden), and centri-fuged for 30 min at 600 x g. The cell-containing interphasewas harvested, and the cells were then washed twice inRPMI 1640 and suspended at a concentration of 106 cells perml in RPMI 1640 supplemented with 10% fetal bovine serum(Flow Laboratories, Inc., McLean, Va.). The cells wereincubated in Falcon plastic dishes for 2 h in 5% C02, andthereafter the nonadherent cells were removed by rinsingwith RPMI 1640. The adherent cells were incubated for 24 hand then harvested by pipetting. The harvested cells (3 x 104cells) were placed in each well of a Falcon microculture plateand were incubated in the presence of various concentra-tions of B-LPS or E-LPS. After 24 h, the culture mediumwas harvested and centrifuged. IL-1 activity in the culturesupernatant was then measured as described below.Assay for IL-1 activity. IL-1 activity was quantitated by

measuring the incorporation of [3H]thymidine into C3H/HeJmouse thymocytes. The cells were cultured for 72 h at 37°Cin RPMI 1640 containing 5% fetal bovine serum (FlowLaboratories, Inc.), 2-mercaptoethanol, and phytohemag-

glutinin-p (final concentration, 2,000-fold; Difco Laborato-ries, Detroit, Mich.) and in test samples. Cell proliferationwas measured by pulsing with 0.5 ,uCi of [3H]thymidine(New England Nuclear Corp., Boston, Mass.) for the last 24h of the culture period. The results were expressed as themean incorporation with standard deviation obtained intriplicate cultures.

Glucose consumption by peritoneal macrophages. Macro-phage activation was evaluated by a modification of theglucose consumption assay described by Ryan et al. (22).Peritoneal macrophages (3 x 104 cells) were cultured asmonolayers in Eagle minimal essential medium in each wellof a Falcon microculture plate. After 48 h of culture with thetest samples, 20 p.l of culture medium was taken from eachwell for measurement of glucose content, which was donewith a glucose B test Wako kit (Wako Junyaku Co., Osaka,Japan). This kit consists of glucose oxidase, peroxidase,phenol, and p-aminoantipyrine. Each assay was carried outin duplicate cultures, and the results were expressed aspercentages of the glucose consumption found in controlcultures.Mitogenic activity assay. Single spleen cells from

C3H/HeN and C3H/HeJ mice were prepared by passing thespleen cells through a stainless steel screen. The cells werecultured in RPMI 1640 (Nisui Seiyaku Co.) supplementedwith 10% fetal bovine serum (Flow Laboratories, Inc.) and2-mercaptoethanol in Nunc U-bottomed microculture plates

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E-LPS

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2 3 4 5 6 7 2 3 4 5 6 7

Log2 dilution of test s#mpleFIG. 3. Amounts of IL-1 activity present in culture supernatants of C3H/HeN (@) and C3H/HeJ (0) peritoneal macrophages treated with

B-LPS or E-LPS. C3H/HeN and C3H/HeJ peritoneal macrophages were cultured for 24 h in the presence of B-LPS (10 ,ug/ml) or E-LPS (10,ug/ml). Each culture supernatant was serially diluted, and then IL-1 activity was determined. IL-1 units were expressed as percentagesrelative to the values obtained with E-LPS in C3H/HeN mice. The number of IL-1 units in each case is as follows: C3H/HeN E-LPS, 100 U;C3H/HeN B-LPS, 25 U; C3H/HeJ E-LPS 12.5 U; C3H/HeJ B-LPS, 3.5 U.

and incubated with various doses of LPS for 72 h at 37°C in5% CO2. The cultures were pulsed with 0.5 ,uCi of[3H]thymidine (New England Nuclear Corp.) for 24 h beforethe end of the culture period. The cells were then harvestedonto glass filters with a multiple-sample cell harvester (LaboScience Co., Tokyo, Japan), and the incorporation of[3H]thymidine into the cultured cells was measured by liquidscintillation counter.

SDS-polyacrylamide gel electrophoresis. The Laemmli so-dium dodecyl sulfate (SDS)-polyacrylamide gel electropho-resis system incorporating 4 M urea in a 14% separating gelwas used in a slab gel apparatus. Samples were boiled for 5min in 0.1 M Tris hydrochloride buffer (pH 6.8) containing2% SDS, 2% 2-mercaptoethanol, and 10% glycerol. Samples(20 RI) were applied to sample wells and subjected toelectrophoresis until the bromophenol blue marker migrated5.5 cm. LPS was visualized by the silver stain described byTsai and Frasch (28).

Analytical assays. Protein was determined by the methodof Bradford (1), 2-keto-3-deoxyoctonate was determined bythe thiobarbituric acid method (10), and heptose was deter-mined by the cysteine-sulfuric acid method (34).

RESULTSChemical characterization of B-LPS. Hot phenol-water

extracts of B. gingivalis were chromatographed on a Seph-

adex G-100 column equilibrated with Tris hydrochloridebuffer containing 0.3% sodium deoxycholate and 0.1 mMEDTA. The eluted fractions (no. 40 to 55) were pooled, andthe material was lyophilized. Purified B-LPS was then ana-lyzed chemically and examined for some of its biologicalactivities.The protein concentration, determined by the method of

Bradford, was 2%. No 2-keto-deoxyoctonate or heptose wasdetected in B-LPS. The SDS-polyacrylamide gel electropho-resis patterns of B-LPS and E-LPS detected by silverstaining show that B-LPS possesses a simpler pattern thandoes E-LPS. Also, no protein bands were detected in theB-LPS and E-LPS preparations as judged by silver stainingof SDS-polyacrylamide gels (data not shown).

IL-1 production by B-LPS-treated peritoneal macrophagesfrom C3H/HeN and C3H/HeJ mice. Peritoneal macrophagesfrom C3H/HeN and C3H/HeJ mice were cultured in thepresence of various doses of B-LPS or E-LPS. The cell-freeculture supernatants were obtained after 24 h and assayedfor IL-1 activity (Fig. 1). The IL-1 production showeddose-response curves with regard to both types of macro-phages. Although the IL-1 activity in C3H/HeJ macrophageswas generally lower than that in C3H/HeN macrophages, asignificantly high IL-1 activity was observed in C3H/HeJmacrophages. The inducing activity of B-LPS (50 p.g/ml) forIL-1 production in C3H/HeN macrophages was significantly

30 h B-LPS

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monocytes treated with B-LPS (0) or E-LPS (0). Humanmonocytes were prepared as described in Materials and Methods.Human monocytes were cultured for 24 h in the presence of variousconcentrations of B-LPS or E-LPS. Each culture supernatant wasdiluted (1:4), and then IL-1 activity was determined.

lower than that of E-LPS (1.0 ,ug/ml). These results demon-strate that B-LPS stimulates IL-1 production in peritonealmacrophages from C3H/HeN and C3H/HeJ mice.

Kinetics of IL-1 production by peritoneal macrophagesstimulated with B-LPS. The IL-1 activity present in culturesupernatants of peritoneal macrophages from C3H/HeN andC3H/HeJ mice treated with B-LPS reached a maximum at 24h and decreased thereafter (Fig. 2). Also, a similar timecourse was observed for IL-1 activity in peritoneal macro-phages treated with E-LPS (data not shown). The reasonsfor the decrease in IL-1 activity are unclear.Amount of IL-1 activity present in culture supernatants of

C3H/HeN and C3H/HeJ peritoneal macrophages stimulatedwith B-LPS. The IL-1 activity was determined in variousdilutions of the supernatants (24-h culture) from C3H/HeNor C3H/HeJ peritoneal macrophages stimulated with 10-jig/ml concentrations of B-LPS (Fig. 3). This figure showsthat supernatant samples of C3H/HeN macrophages stimu-lated with B-LPS exhibited significantly higher IL-1 activitythan did those of C3H/HeJ macrophages in all samplestested. However, the IL-1 activity induced by B-LPS inC3H/HeN macrophages was significantly lower than thatinduced by E-LPS. We estimated IL-1 units from these databy using the method of Mizel (16). C3H/HeN macrophages

stimulated with B-LPS had about seven times more IL-1units than did similarly treated C3H/HeJ macrophages.However, the number of IL-1 units produced with B-LPS inC3H/HeN mice was about four times lower than that elicitedby E-LPS.

IL-1 production by human monocytes treated with B-LPS.Human adherent monocytes were stimulated for 24 h withB-LPS or E-LPS at various concentrations (0.1, 1.0, and 10,ug/ml), and then the culture supernatants were measured forIL-1 activity (Fig. 4). A significant production of IL-1 wasobserved in culture supernatants of human monocytestreated with 0.1-p.g/ml concentrations of B-LPS. However,the activity was lower than that of human monocytes treatedwith E-LPS.ApparentMW of IL-1 induced by B-LPS. To determine the

MW of IL-1 from peritoneal macrophages stimulated withB-LPS, C3H/HeN macrophage culture supernatants gener-ated under serum-free conditions were pooled and concen-trated 50-fold. The concentrated supernatant was then sub-jected to gel filtration chromatography on Sephadex G-75.The major peak of B-LPS-induced IL-1 activity was detectedin the fraction corresponding to an MW of ca. 15,000 (Fig. 5).Also, there was detected a minor peak of activity in the33,000-dalton region. This gel filtration pattern indicates thatB-LPS-induced IL-1 exhibits some heterogeneity in size.

Effect of B-LPS on macrophage activation and on mitogenicresponses of spleen cells from C3H/HeN and C3H/HeJ mice.Previous studies have shown that purified LPS from B.gingivalis 381 outer membranes does not induce splenic cellproliferation in either C3H/HeN or C3H/HeJ mice (19). Wereexamined in detail the ability of B-LPS to induce mitogenicresponses in spleen cells from both strains of mice. Signifi-cantly high mitogenic responses of spleen cells from eitherstrain treated with B-LPS (10 and 50 ,ug/ml) were detected atcell concentrations ranging from 1 x 105 to 5 x 104 spleencells per well. However, the C3H/HeJ mice were lessresponsive than the C3H/HeN mice (Fig. 6). The mitogenicresponses induced by B-LPS in C3H/HeN mice were usuallylower than those observed for E-LPS.We also examined the effect of B-LPS on peritoneal

macrophage activation of C3H/HeN and C3H/HeJ mice invitro. The glucose consumption assay described by Ryanwas employed for evaluation of macrophage activation (Fig.7). When C3H/HeN peritoneal macrophages were culturedwith B-LPS for 48 h, glucose consumption was enhancedsignificantly. The degree of enhancement was lower thanthat with E-LPS. However, neither LPS had any significanteffect on glucose consumption in C3H/HeJ macrophages.

DISCUSSION

B. gingivalis is the predominant gram-negative, anaerobicorganism isolated from gingival pockets of adult periodonti-tis patients. The finding that the number of B. gingivalisorganisms is increased significantly among the subgingivalmicroflora in cases of alveolar bone resorption has suggestedthe possibility that B. gingivalis may play a functional role inalveolar bone resorption in adult periodontitis patients.Also, previous investigations have shown that LPS fromgram-negative bacteria stimulates bone resorption in mousecalvaria (6, 7). These findings led us to postulate that LPSreleased by B. gingivalis present predominantly in sub-gingival sites of adult periodontitis patients might be impor-tant with regard to alveolar bone resorption as observed inchronic periodontal disease. Nair et al. (19) and lino andHopes (8) indicated in their mouse calvaria system that LPSfrom B. gingivalis exhibited bone resorption activity. Sev-eral mechanisms for LPS-induced bone resorption have beenpostulated (15, 23). However, the mechanisms are not un-derstood in detail.Macrophages and osteoclasts are prominent in neonatal

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Fraction No.

FIG. 5. Sephadex G-75 chromatography of a concentrated culture supernatant containing IL-1 derived from C3H/HeN peritonealmacrophages treated with B-LPS. C3H/HeN peritoneal macrophages were cultured for 24 h in the presence of B-LPS (10 j±g/ml). The culturesupernatant (50 ml) was concentrated 50-fold and applied onto a Sephadex G-75 column. Five-milliliter fractions were collected. A sampleof every fraction was examined for its effect on the uptake of [3H]thymidine by mouse thymocytes.

mouse calvaria. Many investigations have shown that mac-rophages release many soluble factors, including prosta-glandins, IL-1, and cytotoxic factors. It is well known thatLPS strongly induces the secretion of these macrophage-derived factors. Therefore, macrophage lineages and theirsoluble factors may play an important role in LPS-inducedbone resorption. A recent study has indicated that IL-1 fromE. coli LPS-treated human monocytes stimulates bone re-sorption in neonatal mouse calvaria (4). These findingssuggest that IL-1 may be an important factor involved inalveolar bone resorption as observed in periodontal dis-eases. Therefore, it is very important for an understanding ofthe mechanism(s) by which B-LPS stimulates bone resorp-tion to examine whether B-LPS is a potent inducer of IL-1production and to determine whether B-LPS-induced IL-1stimulates bone resorption.

In the present study, we have shown that B-LPS inducesIL-1 production in LPS high-responder C3H/HeN and low-responder C3H/HeJ mouse peritoneal macrophages. How-ever, the IL-1 production in C3H/HeJ mice was always

significantly lower than that which occurred in the C3H/HeNstrain. Also, our present results indicate that the IL-1-inducing activity of B-LPS in C3H/HeN mice was lower thanthat of E-LPS (Fig. 1 to 3).Many investigators have demonstrated different effects of

LPS on immunological activities in C3H/HeN and C3H/HeJmice (29). We also examined the effects of B-LPS onmitogenic responses of spleen cells and macrophage activa-tion in these mouse strains. B-LPS induced mitogenic re-sponses at cell concentrations ranging from 1 x 105 to 5 x104 spleen cells per well for either C3H/HeN or C3H/HeJmice, but the responses in C3H/HeJ mice were significantlylower than those that occurred in C3HI/HeN mice. Also, wefound that B-LPS-induced mitogenic responses in C3H/HeNspleen cells were remarkably low in comparison to theresponses induced by E-LPS (Fig. 6). These results wereconsistent with earlier observations (32, 33) that LPS fromBacteroides fragilis induces mitogenic responses in mousespleen cell cultures derived from both C3H/HeN andC3H/HeJ mice, though the responses were significantly

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Dose of LPS (jg/ml)FIG. 6. Effect of B-LPS on mitogenic responses by spleen cells from C3H/HeN (rzi) and C3H/HeJ () mice. Spleen cells (5 x 104 to

1 x 106 cells per well) were incubated for 72 h in the presence of various concentrations of B-LPS. [3H]thymidine (0.5 ,uCi/well) was addedto each culture 24 h before termination of the culture.

lower in C3H/HeJ than in the C3H/HeN mice. HIowever,Nair et al. (19) have reported that LPS prepared from B.gingivalis outer membranes did not induce nmitogenic re-sponses by spleen cells from C3H/HeN and C3H/HeJ mice.

50

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LPS(-) B-IFS E-LPS LPS(-) B-LPS E-U'S

FIG. 7. Glucose consumption of C3H/HeN and C3H/HeJ perito-neal macrophages treated with I3-LPS or E-LPS. C3H/HeN andC3H/HeJ peritoneal macrophages were cultured for 48 h in thepresence of B-LPS (10 ,ug/ml) or E-LPS (10 ,ug/ml). Glicoseconsumption was assayed as described in Materials and Methods.

Although the LPS used in both groups was prepared by thesame phenol extraction procedure, we used lyophilizedwhole cells of B. gingivalis as the starting material. Also,Nair et al. did not test for mitogenic effects of their LPS athigh doses, nor did they vary the number of spleen cells.However, the reason(s) for the discrepancy in our findings isstill unclear. Further, our results for B-LPS-induced macro-phage activation have shown that the C3H/HeJ macrophageis more resistant to activation than the C3H/HeN macro-phage. However, macrophage activation by B-LPS inC3H/HeN was significantly lower than by E-LPS (Fig. 7).Although the reason for the significant differences be-

tween B-LPS and E--LPS for some immunobiological activ-ities is now unclear, such differences may result fromdifferences in chemical properties between the two. In fact,some investigators (10, 11, 26) have demonstrated thatchemical properties of LPS from Bacteroides species differfrom those of enterobacterial species. For example, heptoseand 2-keto-oxyoctonate, as found in LPS of E. coli andSalmonella species, are not detectable in B-LPS. In entero-bacterial LPS, lipid A is considered to be the region respon-sible for at least some of the biological activities of themacromolecule. Therefore, our results concerning certainimmunobiological activities of B-LPS suggest that the LPSeither may be lipid A defective or may contain a lipid moietywhich is different frorn that of the lipid A of enterobacterialLPS. In fact, Williamson et al. (32) have shown that the lipidA structure of Bacteroides LPS does differ from that ofenterobacterial LPS.Recent studies (3, 20) have shown that a significantly

greater amount of IL-1 can be detected in the gingival fluidfrom inflamed sites than from noninflamed sites in periodon-titis patients. Most recently, we have observed that human

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INTERLEUKIN 1 AND PERIODONTAL DISEASE 269

gingival fibroblasts in culture produce large amounts of IL-1(unpublished data). We propose from these findings that thesignificant increase of IL-1 in gingival sites of periodontitispatients may be derived from their gingival fibroblasts andinflammatory macrophages. Therefore, it is very importantto understand the physiological roles of IL-1 present ingingival tissues and fluids. Some possible mechanisms havebeen postulated in regard to the significant role of IL-1-likefactor in immunological and inflammatory reactions. Thisfactor may presumably serve to enhance immunological andinflammatory responses in gingival tissues. Also, IL-1 mayattract polymorphonuclear leukocytes to migrate into thegingival tissues, and then enzymes released from theseleukocytes may be involved in the inflammation of gingivaltissues. Also, it has been demonstrated that human IL-1stimulates bone resorption in a calvaria system (4), andPostlethwaite et al. (21) have shown that IL-1 is a potentstimulator of fibroblast collagenase production in vitro. Incontrast, a recent study has also indicated that IL-1 fromLPS-treated human monocytes increases collagen type IVproduction in mammary epithelial cells (14). Further, it hasalso been reported that IL-1 inhibits the production of thebone-specific protein osteocalcin in human osteoblast-likecells (2). These findings suggest that IL-1 may be a novelregulatory factor in the bone remodeling system. B-LPSinduced significant IL-1 production by mouse macrophagesand human monocytes (Fig. 1 and 4). Therefore, we proposefrom these results that B-LPS may be involved in theregulation of human gingival fibroblast metabolism and instimulation of alveolar bone resorption in human periodontaldisease.

In conclusion, we have demonstrated that B-LPS is apotent inducer of IL-1 in peritoneal macrophages fromC3H/HeN and C3H/HeJ mice and suggest that B-LPS-induced IL-1 may play a significant role in the pathogenesisof chronic adult periodontitis.

ACKNOWLEDGMENTSWe acknowledge the excellent technical assistance of Sadako Itoh

in these experiments.This work was supported in part by a grant from the Ministry of

Education, Science, and Culture of Japan.

LITERATURE CITED1. Bradford, M. N. 1976. A rapid and sensitive method for the

quantitation of microgram quantities of principles of protein dyebinding. Anal. Biochem. 72:248-254.

2. Bresford, J. L., J. A. Gullangher, M. Gowen, M. Couch, J.Poser, D. D. Wood, and R. G. G. Russell. 1984. The effects ofmonocyte-conditioned medium and interleukin 1 on the synthe-sis of collagenous and noncollagenous proteins by mouse boneand human bone cells in vitro. Biochim. Biophys. Acta801:58-65.

3. Charon, J. A., T. A. Luger, S. E. Mergenhagen, and J. J.Oppenheim. 1982. Increased thymocyte-activating factor in hu-man gingival fluid during gingival inflammation. Infect. Immun.38:1190-1195.

4. Gowen, M., D. D. Wood, E. J. Ihrie, M. K. B. McGuire, andG. G. Russell. 1983. An interleukin 1-like factor stimulates boneresorption in vitro. Nature (London) 306:378-380.

5. Hanazawa, S., H. Kato, K. Yamaura, and Y. Yamaguchi. 1978.Kinetics and mechanisms of macrophage activation by Coryne-bacterium anaerobium. Microbiol. Immunol. 22:155-166.

6. Hausmann, E., L. G. Raisz, and W. A. Miller. 1970. Endotoxinstimulation of bone resorption in tissue culture. Science168:862-864.

7. Hausmann, E., N. Weinfeld, and W. A. Miller. 1972. Effects oflipopolysaccharides on bone resorption in tissue culture. Calcif.

Tissue Res. 9:277-282.8. lino, Y., and R. M. Hopes. 1984. The bone resorbing activities in

tissue culture of lipopolysaccharides from the bacteriaActinobacillus actinomycetemcomitans, Bacteroides gingivalisand Capnocytophaga ochracea isolated from human mouths.Arch. Oral Biol. 29:59-63.

9. Karkhanis, Y. D., J. Y. Zeltner, J. J. Jackson, and D. J. Carlo.1978. A new and improved microassay to determine 2-keto-3deoxyoctonate in lipopolysaccharide of gram negative bacteria.Anal. Biochem. 85:595-601.

10. Kasper, D. G. L. 1976. Chemical and biological characterizationof the lipopolysaccharide of Bacteroides fragilis subspeciesfragilis. J. Infect. Dis. 134:59-66.

11. Mansheim, B. J., A. B. Onderdonk, and D. L. Kasper. 1978.Immunochemical and biological studies of the lipopolysaccha-ride of Bacteroides melaninogenicus subspecies asac-charolyticus. J. Immunol. 120:72-78.

12. Mansheim, B. J., M. L. Stenstrom, S. B. Low, and W. B. Clark.1980. Measurement of serum and salivary antibodies to the oralpathogen Bacteroides asaccharolyticus in human subjects.Arch. Oral Biol. 25:553-557.

13. Mashimo, P. A., and S. A. Ellison. 1972. Diffusate media forcultivation of oral anaerobic bacteria. J. Dent. Health 22:38-45.

14. Matsushima, K., M. Bano, W. R. Kidwell, and J. J. Oppenheim.1985. Interleukin 1 increases collagen type IV production bymurine manmmary epthelial cells. J. Immunol. 134:903-909.

15. Meryon, S. D., and D. P. Perris. 1981. Lipopolysaccharideinduced bone resorption is mediation by prostaglandin. Life Sci.28:1061-1065.

16. Mizel, S. B. 1980. Studies on the purification and structure-function relationships of murine lymphocyte activating factor(interleukin 1). Mol. Immunol. 17:571-577.

17. Morrison, D. C., and J. L. Ryan. 1979. Endotoxins and hostimmune responses. Adv. Immunol. 28:293-450.

18. Mouton, C., P. G. Hammond, J. Slots, and R. J. Genco. 1981.Serum antibodies to oral Bacteroides asaccharolyticus (Bacte-roides gingivalis): relationship to age and periodontal disease.Infect. Immun. 31:182-192.

19. Nair, B. C., W. R. Mayberry, R. Dziak, P. B. Chen, M. J.Levine, and E. Hausmann. 1983. Biological effects of a purifiedlipopolysaccharide from Bacteroides gingivalis. J. PeriodontalRes. 18:40-49.

20. Oppenheim, J. J., J. A. Charon, and T. A. Luger. 1982.Evidence for in vivo inflammatory role of interleukin 1 (IL-1).Tranplant. Proc. 14:553-555.

21. Postlethwaite, A. E., L. B. Lachman, C. L. Mainaldi, and A. H.Kang. 1983. Interleukin 1 stimulation of collagenase productionby cultured fibroblasts. J. Exp. Med. 157:801-806.

22. Ryan, J. L., L. M. Glode, and J. R. Rosenstreich. 1979. Lack ofresponsiveness of C3H/HeJ macrophages to lipopolysaccha-ride: the cellular basis of LPS-stimulated metabolism. J. Immu-nol. 122:932-935.

23. Sandberg, A. L., L. G. Raisz, J. M. Goodson, H. A. Simmons,and S. E. Mergenhagen. 1977. Initiation of bone resorption bythe classical and alternative C pathway and its mediation byprostaglandins. J. Immunol. 119:1378-1381.

24. Slots, J. 1977. The predominant cultivable microflora of ad-vanced periodontitis. Scand. J. Dent. Res. 85:114-121.

25. Slots, J. 1979. Subgingival microflora and periodontal disease. J.Clin. Periodontol. 6:351-382.

26. Sveen, K. 1977. The capacity of lipopolysaccharides from Bac-teroides, Fusobacterium, and Veillonella to produce skin in-flammation and the local and generalized Schwarzman reactionin rabbits. J. Periodontal Res. 12:340-350.

27. Tanner, A. C. R., C. Haffer, G. T. Bratthal, R. A. Visconti, andS. S. Socransky. 1979. A study of the bacteria associated withadvancing periodontitis in man. J. Clin. Periodontal. 6:278-307.

28. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal.Biochem. 119:115-119.

29. Vogel, S. N., and S. E. Mergenhagen. 1982. Cellular basis ofendotoxin susceptibility, p. 160-168. In R. J. Genco and S. E.Mergenhagen (ed.), Host-parasite interactions in periodontal

VOL. 50, 1985

on July 28, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

270 HANAZAWA ET AL. INFECT. IMMUN.

diseases. American Society for Microbiology, Washington,D.C.

30. Westphal, O., and K. Jahn. 1%5. Bacterial lipopolysaccharideextraction with phenol water and further applications of theprocedures. Methods Carbohydr. chem. 5:83-91.

31. White, D., and D. Mayrand. 1981. Association of oral Bacte-roides with gingivitis and adult periodontitis. J. Periodontal Res.16:259-265.

32. Williamson, S. I., M. J. Wannemuehier, E. Jirillo, D. G.Pritchard, S. M. Michalek, and J. R. McGhee. 1984. LPSregulation of the immune response: separate mechanisms for

murine B cell activation by lipid A (direct) and polysaccharide(macrophage-dependent) derived from Bacteroides LPS. J. Im-munol. 133:2294-2300.

33. Wonnemuehler, H. J., S. M. Michalek, E. Jirillo, S. I. Wil-liamson, M. Hirasawa, and J. R. McGhee. 1984. LPS regulationof the immune response: Bacteroides endotoxin induces mito-genic, polyclonal, and antibody responses in classical LPSresponsive but not C3H/HeJ mice. J. Immunol. 133:299-305.

34. Wright, B. G., and P. A. Robers. 1972. Procedure for determin-ing heptose and hexose in lipopolysaccharide. A modification ofthe cysteine-sulfuric acid method. Anal. Biochem. 492:307-319.

on July 28, 2020 by guesthttp://iai.asm

.org/D

ownloaded from