Actinobacillus actinomycetemcomitans Y4 and N27 - NCBI

INFECTION AND IMMUNITY, Dec. 1980, p. 862-873 0019-9567/80/12-0862/12$02.00/0

Vol. 30, No. 3

Characterization of the Lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27 PATRICIA KILEY AND S. C. HOLT* Department ofMicrobiology, University of Massachusetts, Amherst, Massachusetts 01003

The lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans strains Y4 and N27 was isolated by the phenol-water procedure. Morphologically, the molecule consisted of ribbon and branched filaments which comprised 3% of the cellular dry weight. Chemical analysis of the isolated and purified LPSs of both strains showed them to consist of carbohydrate, lipid, 2-keto-3-deoxyoctonate, heptose, hexosamine, and phosphate. The major fatty acids of the lipid A moiety were saturated C14 and f-OH C14 compounds. Rhamnose, fucose, galactose, glucose, heptose, glucosamine, and galactosamine comprised the monosaccharide portion of the LPS. Biological activity studies revealed both LPS molecules to be active in the Schwartzman reaction and in in vitro 45Ca bone resorption, as well as in macrophage activation and lethality and in platelet aggregation.

Actinobacillus actinomycetemcomitans is a gram-negative, capnophilic, fermentative, pleomorphic rod. In recent years it has been recovered from isolated cases of pneumonia and abscesses (57), and endocarditis (8) as well as osteomyelitis (37). In addition, A. actinomycetemcomitans occurs as a member of the normal flora of the upper respiratory tract and oral cavity. Moreover, a compromise or clinical manipulation of the oral cavity may serve as the origin or initiator of the above infections (8). Actinobacillus actinomycetemcomitans was originally isolated by Klinger (26a) from an actinomycotic lesion, where it was cultivated in conjunction with Actinomyces. In recent years, A. actinomycetemcomitans has been cultivated from active lesions of periodontal disease (38, 50, 55), especially from lesions occurring in children and young adults, where it may serve as a clinical indicator of juvenile periodontitis. Two strains of A. actinomycetemcomitans, Y4 and N27, have been shown effective in causing classical periodontal disease in gnotobiotic animal model systems (23, 39). The characteristic severe alveolar bone loss and migration of epithelial attachment observed in these germfree rats was accompanied by a marked increase in the number of osteoclasts in the periodontium (23). What role(s) microorganisms such as the actinobacilli play in these histological and undoubtedly biochemical alterations is unknown, especially in light of the observations that intact bacteria do not penetrate affected tissues (13). However, bacterial components (cell wall fragments, lipopolysaccharides [LPS], peptidoglycans, hydrolytic enzymes, or bacterial end products) could cross epithelial barriers and elicit pathologies

characteristic of periodontal disease (22, 24, 34, 41). Recently, Tsai et al. (56) and Taichman et al. (54) have demonstrated that strain Y4 of A. actinomycetemcomitans possesses a soluble cytotoxic factor for human polymorphonuclear leukocytes and monocytes. This "leukotoxin" in in vivo situations could compromise the host's ability to eliminate or control invading bacteria, making it susceptible to disease, such as periodontal disease. Other studies by Braunthal et al. (4) reveal that several of the actinobacilli possess large amounts of branched-chain fatty acids and presumptive lyso-containing phosphatides, compounds with known toxicities for mammalian membranes. LPSs of gram-negative bacteria have a high potential for causing the alteration or destruction of an array of host cells and tissues, such as rabbit hemorrhagic necrosis, platelet aggregation, and the elicitation of macrophages, as well as the activation of complement (3, 35, 36, 58). Moreover, the potential role of LPS in the production of periodontal disease is supported by its presence in dental plaque (9, 24), its ability to penetrate gingival crevicular epithelial tissue (47), and its recognized ability to stimulate bone resorption in in vitro assays (15-18). These latter observations have prompted us to initiate this study on the chemistry and biological activity of the LPS from A. actinomycetemcomitans Y4 and N27 to define and distinguish it from that observed in other gram-negative bacteria of known pathogenic potential. The interaction of isolated and purified LPS from strains of this recognized periodontopathogen with cells and tissues will be the first step in defining the role 862

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LPS OF ACTINOBACILLUS STRAINS

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of this microorganism in one of the periodontal disease processes. MATERIALS AND METHODS Bacterial strains. A. actinomycetemcomitans strains Y4 and N27 were isolated and characterized from individuals diagnosed as having active localized juvenile periodontitis lesions (38). The original strains were obtained from S. S. Socransky, Forsyth Dental Center, Boston, Mass. Growth conditions. A. actinomycetemcomitans was grown into late logarithmic phase as described by Holt et al. (21). Cells were harvested by centrifugation (7,500 x g, 20 min, 50C), washed twice in Ringer solution and once in distilled water, and then lyophilized to dryness. Approximately 0.4 g (dry weight) of cells per liter of medium was obtained. Isolation of LPS. LPS was extracted by the hot phenol-water procedure of Westphal (60). Briefly, lyophilized cells were suspended in distilled water to a concentration of approximately 50 mg/ml. The cell suspension was mixed with an equal volume of 90% phenol (wt/wt) at 680C for 15 min. After cooling in an ice bath, the phases were separated by centrifugation (3,000 x g, 30 min, 50C). The aqueous phase was removed, and the phenol portion was extracted twice more with equal volumes of distilled water. The combined aqueous phases were dialyzed (48 h, 40C) against several changes of distilled water and lyophilized. This fraction constituted the crude LPS extract. The crude extract was purified by ultracentrifugation (100,000 x g, 1 h, 5VC), and the pellet washed twice with distilled water, and treated with deoxyribonuclease (Sigma Chemical Co., St. Louis, Mo.; 20 ,ug/ml, final concentration, 25°C, 1 h), pH 4.5, and ribonuclease A (Sigma Chemical Co., St. Louis, Mo.; 20 ,ug/ml, final concentration, 37°C, 30 min), in 0.05 M tris(hydroxymethyl)aminomethane- hydrochloride in 0.1 mM ethylenediaminetetraacetic acid, pH 7.3. The resulting suspension was washed twice in distilled water, pelleted by ultracentrifugation (100,000 x g, 1 h, 5°C), and lyophilized to dryness. The fluffy white pellet constituted the purified LPS. Commercially prepared phenol-water-extracted Escherichia coli 055:B5 LPS was obtained from Difco Laboratories (Detroit, Mich.). Analytical methods. The analytical methods listed in Table 1 were used in this study. Gel filtration. The molecular size of the purified

LPS was determined by elution through a Sepharose 4B 1.7- by 27-cm gel column (Pharmacia Fine Chemical AB, Uppsala, Sweden). Sepharose 4B was equilibrated with 0.15 M NaCl in 0.05 M tris(hydroxymethyl)aminomethane.hydrochloride, pH 7.3. Fractions (1 ml) were collected at a flow rate of 0.2 ml/ min. The void volume of 15 ml was determined by calibration with blue dextran. Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a vertical slab gel apparatus (12-cm width, 16-cm height) was performed by a modification of the Laemmli procedure (29). Polyacrylamide (Gallard-Schlesinger Chemical Manufacturing Corp.) was incorporated into the gel solutions (5.0 ml of 3.0% polyacrylamide per 30 ml of gel solutions) for added strength and flexibility. A 4% (5.0 mi) pH 6.8 stacking gel over a 7 to 15% (22 ml) pH 8.8 running gel was electrophoresed at 30 A. LPS (1 mg) of sodium dodecyl sulfatewas suspended in 100 polyacrylamide gel buffer for analysis. Gels were stained with Coomassie blue. Lipid A hydrolysis. LPS (50 mg) was cleaved into its lipid A and polysaccharide moieties by hydrolysis in 1% (vol/vol) acetic acid (25 ml) for 1.5 h at 105°C. The resulting precipitate was collected by centrifugation (8,000 x g, 15 min, 5°C) and washed twice in distilled water, and the aqueous supernatants containing the polysaccharide moiety were combined and lyophilized. The pellet containing the crude lipid A moiety was suspended in chloroform-methanol (2:1). Repeated evaporation of the chloroform-methanol removed any residual water. The purified lipid A was brought to dryness under vacuum dessication at 48°C. The percent lipid A of the LPS was determined gravimetrically. Fatty acids. LPS and lipid A were hydrolyzed in 4 N NaOH (5 h, 100°C) in vials sealed under N2. The fatty acids were extracted and methylated by the method of Flesher and Insel (10). The methylated fatty acids were analyzed in a Varian model 3700 gas chromatograph equipped with a flame ionization detector and integrated with a Varian CDS-111 Chromatography Data System. Stainless steel columns ('I inch by 6 feet [0.32 by 183 cm]) consisting of 10% SP2330 and 10% SP-2100 on 100/120 and 100/200 mesh, respectively, Supelcoport (Supelco Inc., Bellefonte, Pa.) were employed for fatty acid separation. The carrier gas was nitrogen at a flow rate of 30 ml/min. The injector and detector temperatures were 250 and

TABLE 1. Procedures used for the analysis of the chemical composition of LPS Assay

Protein Nucleic acids

Total carbohydrate Hexosamine Total phosphate Heptose 2-Keto-3-deoxyoctonate a All reference standards were albumin; RNA, ribonucleic acid.

Method

Lowry et al. Ultraviolet absorbance (200-300 nm) Phenol-sulfuric acid Elson-Morgan

Standard'

BSA RNA

Reference

32

7 Glucose 5 Glucosamine 1 Ames Glycerophosphate 61 Sedoheptulose Cysteine-sulfuric acid 25 KDO Thiobarbituric acid obtained from Sigma Chemical Co., St. Louis, Mo. BSA, Bovine serum

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KILEY AND HOLT

220'C, respectively. The column temperature was held at an initial temperature of 1650C for 3 min and then programmed at a rate of 50C/min to 2250C and held at this latter temperature for 15 min. The fatty acids were identified by comparison of their relative retention times with known methylated standards (Applied Science Division, State College, Pa.). Percent composition was determined by the CDS-111 Chromatography Data System. Sugar analysis. Samples of purified LPS, fractionated lipid A, and polysaccharide were suspended to a concentration of 1 mg/ml in 0.1 N HCl for analysis of neutral sugars and in 4 N HCl for amino sugar determination. The samples were sealed in vials under nitrogen and hydrolyzed at 100'C for 48 and 4 h, respectively. The resulting hydrolysates were neutralized to approximately pH 5.8 by repeated freeze-drying over NaOH pellets. The neutralized samples were resuspended to approximately 8 mg/ml in distilled water and used for thin-layer chromatographic and gas-liquid chromatographic analysis of alditol acetate derivatives. Thin-layer chromatography. Approximately 25 pl of the neutralized samples (above) was spotted onto activated (110'C, 30 min) Analsil G (20 by 20 cm, 500Im thickness) precoated thin-layer chromatography plates (Analabs, Inc.). Neutral sugars were developed in a solvent system consisting of butanol-acetic acidether-water (9:6:3:1) and amino sugars developed twice in ethyl acetate-pyridine-n-butanol-butyric acid-water (8:8:4:4:1). Neutral sugars were visualized with phenol-sulfuric acid and amino sugars with ninhydrin. Gas-liquid chromatography of monosaccharide derivatives. Alditol acetate derivatives of the LPS and polysaccharide fraction were prepared by the method of Laine et al. (30) except that glacial acetic acid was used to destroy excess borohydride. All samples were analyzed in a Varian 3700 gas-liquid chromatograph (Varian Associates, Palo Alto, Calif.) on a glass column (6 feet, 2-mm inside diameter) packed with 3% SP-2340 on 100/120 Supelcoport (Supelco Inc., Bellefonte, Pa.). The injector and flame ionization temperatures were both maintained at 250'C. The column temperature was maintained at 180°C for 4 min after initial sample injection and then linear programmed from 180 to 240°C at a program rate of 5°C/ min. Unknown alditol acetates were determined by comparison of the retention time of alditol acetate standards (Supelco Inc., Bellefonte, Pa., or made in our laboratory). Quantitative analysis was again determined by integration by the Varian CDS-111 Chromatography Data System. Biological activity-lethality in mice. Lethality in mice was estimated by 50% lethal dose (LD50) procedures. Groups of five white CD1 mice of both sexes weighing between 20 and 24 g were used. LPS concentrations (between 500 and 2,000 jig/ml) prepared in 0.5 ml of sterile 0.85% NaCl (physiological saline) were injected intraperitoneally in a volume of 0.5 ml. Deaths were recorded 48 h after injection, and the LD50 was calculated by the method of Reed and Muench (46). Actinomycin D was also tested along with the LPS to ascertain whether the antibiotic predisposed the mice to an increased LPS sensitivity (44).

INFECT. IMMUN. Schwartzman reaction. New Zealand white female rabbits (2 to 3 kg) were injected intradermally on their shaved ventral surface with concentrations of LPS varying between 31.25 to 200 jig in 0.2 ml of sterile physiological saline. After 24 h they were injected intravenously with 250 jig of LPS in the right marginal ear vein. Positive responses of necrosis were observed up to 24 h after the provoking dose. Bone resorption assay. The release of 45Ca from rat fetal long bones was used as a measure of bone destructive ability (45). Briefly, pregnant rats were injected with 4'Ca on day 18 of gestation. Within 24 h, the partially calcified bones were dissected and explanted in a modified BGJ culture medium and incubated for 24 h at 37°C in 5% CO2 and air. This reduced the amount of rapidly exchangeable 45Ca. The labeled bones were then transferred to culture vessels containing test (LPS) and control substances and cultured for varying periods of time to 6 days. The percent bone resorption was determined as percent 4'Ca released compared with control compounds (i.e., a known LPS) and uninoculated culture medium. Macrophage cytotoxicity. Normal peritoneal cells were obtained by intraperitoneal lavage of CD1 white mice with 5 ml of sterile Dulbecco-modified Eagle medium supplemented with glucose and L-glutamine (1,000 mg/ml) and sodium pyruvate. Penicillin (100 U/ml) and streptomycin (100 ,g/ml) were also added. Approximately 1.4 x 106 cells per ml were then plated to 1-ml wells of Costar chambers containing glass cover slips. The macrophages were allowed to adhere for 2 h in a 15% CO2 chamber at 37°C and then were washed with fresh medium and reincubated for an additional 24 h. The cultures were washed again, and the adherent macrophage population was determined by Diff Quik differential stain. LPS (50 to 400 jig) in 0.1 ml of sterile saline was then added, and the cells were reincubated. Morphological changes were observed for 48 h with a Leitz inverted microscope. Culture viability was determined by trypan blue (GIBCO Laboratories, Grand Island, N.Y.) exclusion (medium was removed, and 0.1 ml of trypan blue was added). Diff Quik staining was also performed after aspirating media and washing in phosphate-buffered saline. Glass cover slips were then removed for differential staining. Percent cell cytotoxicity was determined by dividing the total number of viable cells in control plates by the total number of viable cells (i.e., those adhering and excluding trypan blue) after 48 h. Platelet aggregation. Rabbit platelets were isolated by the procedure of Blumenthal et al. (2). Briefly, New Zealand female white rabbits (approximately 3 kg) were bled by cardiac puncture, and the blood was immediately placed into Nacitrate to a final concentration of 0.38%. The platelet-rich plasma was separated by centrifugation (250 x g, 17 min) and placed into sterile plastic tubes. Platelet concentration was determined in a Petroff-Hauser counting chamber, and the cells were diluted with sterile 0.85% saline to a platelet concentration of approximately 2 x 108 platelets per ml. LPS platelet aggregation was determined in a Chronolog aggregometer (model 300, Chronolog Corp., Broomall, Pa.) attached to an Omniscribe linear recorder. After a steady base line was established for the approximately 0.6 ml of platelet


VOL. 30, 1980

suspension incubated in the aggregometer at 370C with constant stirring, 0.1 ml of varying concentrations (3 to 200,ug) of LPS in physiological saline was added. Platelet aggregation and shape change were determined as a change in recorder deflection and width of recorder oscillations. Electron microscopy. Platelets were isolated and separated as described above. Both control platelets and platelets aggregated in the presence of LPS were fixed in 0.1% (final concentration) glutaraldehyde for 15 min at room temperature (approximately 220C). After this initial fixation, the glutaraldehyde concentration was brought to 1%, and fixation continued at room temperature for 1 h. The platelets were washed twice with 0.2 M phosphate buffer, pH 7.2, and sedimented at low speed (480 x g, 20 min). The resulting pellets were postfixed in 1% (wt/vol) osmium tetroxide-phosphate buffer, 1 h, room temperature. After an additional wash in phosphate buffer, the fixed platelets were dehydrated through a graded ethanol-propylene oxide series and embedded in Epon 812. All samples were thin-sectioned on a Porter-Blum MT-2 automatic ultramicrotome (I. Sorvall, Inc., Norwalk, Conn.) with a diamond knife. The thin-sections were stained with lead citrate and uranyl acetate and examined and photographed in a JEOL 100S transmission electron microscope (JEOL Ltd., Tokyo, Japan). Negative-staining. Samples for negative staining were suspended in bacitracin (100 tyg/ml) in 0.1 M ammonium acetate, pH 7.0. In some instances the LPS samples were sonicated for 1 min, and the resulting solution was negatively stained with 4% (wt/vol) ammonium molybdate, pH 7.0. The negatively stained material was routinely examined at 40 kV in the JEOL 100S transmission electron microscope. Light microscopy. Light microscopy of cultured macrophages was taken on a Zeiss Opton inverted .,,!N

C'

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microscope (Zeiss, Oberkocken, West Germany) equipped with camera back and Tri-X Pan photographic film.

RESULTS Electron microscopy. When purified LPS from both A. actinomycetemcomitans strains Y4 and N27 was negatively stained with NH4MoO4, ribbons and stranded configurations were observed (Fig. 1). The N27 LPS appeared to form strands and globular structures to a much greater extent than Y4 LPS, which was consistently more stranded in configuration. Both LPS molecules were similar to that observed for other gram-negative LPS molecules (48). Staining with phosphotungstic acid, uranyl acetate, or sodium silicotungstate at varying pH's did not increase the contrast of the LPSs. NH4MoO4 at pH 7 was not as effective in revealing the molecule as it was at pH 5.8. In addition, sonication was required to visualize the Y4 molecule by negative staining, most probably disaggregating it, permitting greater stain penetration. N27 LPS was easily stained without having to resort to sonication. Chemical composition of purified LPS. The chemical compositions of purified LPS from Y4 and N27 are seen in Table 2. Both molecules contained carbohydrate, lipid, and a small amount of protein. Scanning of aqueous solutions between 200 and 300 nm of both LPSs revealed no increases in absorbency at 260 or 280 nm, indicating the relative absence of con-

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lab FIG. 1. Isolated LPS from A. actinomycetemcomitans. Strain Y4 LPS (a) occurs as thin filaments and ribbons with slight branching (arrows), whereas N27 LPS (b) has a greater tendency toward branching and membrane configuration (arrows). Strain Y4 LPS is also partially positively stained. 4% NH4MoO4, pH 5.8. Bar = 0.5 pm.

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INFECT. IMMUN.

KILEY AND HOLT

TABLE 2. Chemical composition of the LPS ofA. actinomycetemcomitans Y4 and N27 % Dry wt of strain:' Compound

Y4

N27

E. coli: 055:B5

34.8 Carbohydrate 42.3 31.1 Lipid A 32.0 0.4 0.5 KDO 2.5 7.3 7.1 Heptose 0.5 Hexosamine 12.0 11.8 15.5 Total phosphate 10.3 10.7 Protein Tr Tr ND Nucleic acid ND a Tr, Less than 1% dry weight; ND, Not detectable.

laminating nucleic acids and proteins. Further, sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal any protein bands down to a concentration of approximately 0.1%. On a dry weight basis, the purified LPS of both strains accounted for 3% of the cellular dry weight. Carbohydrate comprised the major chemical constituent for both molecules, accounting for approximately 35 and 42% of the dry weight of Y4 and N27 LPS, respectively. Total phosphate as measured by Ames technique (1) accounted for approximately 11% of the LPS dry weight in both strains. Hexosamine accounted for approximately 12% of the LPS dry weight, whereas the two classical LPS markers, heptose and 2-keto-3-deoxyoctonate (KDO), were present in lower amounts, 7 and 0.5%, respectively. Interestingly, KDO has been found in low amounts in other gram-negative bacteria isolated from the human oral cavity (53). The lipid A content of Y4 and N27 was determined after precipitation from mild hydrolysis in 1% acetic acid and purification from chloroform-methanol (2:1). In both strains it consisted of a light brown material of waxy consistency which accounted for approximately 31% of the LPS dry weight. The lipid region was examined for its amino sugar content as well as fatty acids. The fatty acids present in the lipid A moiety consisted of only two major acids, myristic acid and /3-OH myristic acid (Table 3). Minor amounts of palmitic and palmitoleic acids were also detected. Further analysis of the lipid A by thin-layer chromatography (see below) revealed the amino sugar, glucosamine. The individual sugars comprising the N27 and Y4 LPS were identified by thin-layer chromatography (Table 4). When hydrolyzed in 0.1 N HCl for 48 h and chromatographed, the resulting spots visualized with phenol-sulfuric acid revealed the presence of fucose, rhamnose, galactose, and glucose. Analysis of the whole LPS, the cleaved lipid A and extracted carbohydrate

fractions after hydrolysis for 4 h in 4 N HCl for amino sugars revealed the LPS to consist of at least two amino-positive spots which comigrated with galactosamine and glucosamine. The lipid A region yielded one ninhydrin-positive spot, identical with glucosamine, whereas the carbohydrate fraction had a ninhydrin-positive spot corresponding to galactosamine. Phosphatidylethanolamine was not detected in any of our fractions by thin-layer chromatographic analysis. The neutral sugars were quantitated by gasliquid chromatographic analysis of alditol acetate derivatives of 0.1 N HCl, 48-h-hydrolyzed samples (Table 5). Rhamnose, fucose, and glucose were equally distributed in the N27 LPS, TABLE 3. Fatty acid composition of the lipid A moiety from A. actinomycetemcomitans Y4 and N27 LPS % Composition of fraction Carbon no.

Y4 66.8

14 Saturated

fOH-14 Saturated 16 Saturated 16 Unsaturated

26.2 3.9 2.3

N27

53.9 42.3 4.8 1.3

TABLE 4. Monosaccharide composition of A. actinomycetemcomitans Y4 and N27 LPSW Relative Rf

Monosaccharide

Rhamnose Fucose Glucose Galactose Galactosamine

Glucosamine

Standard

Y4

N27

0.639 0.556 0.509 0.446 0.496 0.536

0.647 0.561 0.505 0.439 0.483 0.536

0.647 0.559 0.503

0.441 0.487 0.536

a Solvents: 1, rhamnose, fucose, galactose, glucose, butanol-ether-acetic acid-water (9:3:6:1); 2, galactosamine, glucosamine, ethyl acetate-butanol-pyridinebutyric acid-water (8:4:8:4:1).

TABLE 5. Quantitative gas-liquid chromatographic analysis of the monosaccharide composition of A. actinomycetemcomitans Y4 and N27 LPS % of total fractiona

Monosaccharide

Rhamnose Fucose Galactose Glucose Unknown 1 Unknown 2 Heptose

Y4 13.8 14.7

14.3 27.0 3.1 10.5 15.4 a Samples hydrolyzed in 0.1 N

N27

20.0 18.4 10.6 22.7 3.2 9.9 15.3

HCl, 48 h, 100°C.

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VOL. 30, 1980

with galactose representing only 10% of the neutral sugars. In Y4, glucose comprised 27% of the neutral sugar fraction, with rhamnose, fucose, and galactose each representing approximately 15% of the LPS molecule. The two unknown compounds, which represented approximately 13% of the monosaccharide fraction (Table 5i), may be hydrolytic degradation products. These unknowns did not have retention times similar to any of our known sugar alditol derivatives. Heptose accounted for 15% of the neutral sugars in both Y4 and N27. The relative molecular weight and homogeneity of the purified Y4 and N27 aggregated LPS molecules were determined by gel filtration column chromatography on Sepharose 4B. Both molecules eluted just after the void volume, indicative of an LPS of aggregated molecular weight just under 2 x 106 as determined by blue dextran elution. Biological activities of A. actinomycetemcomitans Y4 and N27 LPS. Both Y4 and N27 LPS were active in the biological assays employed in this investigation. Schwartzman reactions. The LPS preparations prepared the skin for elicitation of localized Schwartzman reaction at a minimum dose of 31.2 ,ug. Both preparations resulted in positive hemorrhagic necrosis within 24 h of intravenous injection of 250 ,ug of an LPS-provoking dose. In all LPS doses tested (31 to 200 jig), tissue necrosis was observed. Mice LD50 lethality. The lethal endotoxic activity of Actinobacillus Y4 and N27 LPS was compared with that of commercially prepared LPS from E. coli. The LDso for Y4 and N27 was 54.5 and 63.6 ,g/g of body weight, respectively. In comparison, E. coli 055:B5 LPS had an LD50 lethality of 45.5 ,ug/g of body weight. The LDso was 1.2 and 1.4 mg for Y4 and N27, respectively, as compared with 1 mg for E. coli 055:B5 LPS when calculated by the method of Reed and

Meunch (46). When actinomycin D was injected concomitantly with LPS, there was a potentiation of endotoxin lethality; the LDso for Y4 and N27 now being 104 and 73 ,ug, respectively. This corresponded to a 10-fold increase in endotoxin lethality. Bone resorption. In 45Ca fetal bone resorption assay (Table 6), the N27 LPS was very active in its ability to release 45Ca (approximately 42%) from fetal long bones similar to that observed for control Salmonella typhimurium and E. coli LPS. When compared on an absolute basis, Y4 LPS did not appear as active as the N27 LPS. However, in comparison to their respective controls, Y4 appeared to possess a similar ability to release 45Ca in this assay. Macrophage toxicity. The effect of varying concentrations of Y4 and N27 LPS on the morphology and viability of mouse peritoneal macrophages was examined (Fig. 2 and Table 7). As is apparent, both Y4 and N27 LPS had essentially identical effects on macrophage viability. When exposed to 200 jig of LPS, only 42% of the macrophage population was viable as measured by trypan blue exclusion and adherent macrophages, as compared with 83% viability when 50 jg of LPS was used. Phase-contrast examination of the effect of N27 LPS (as well as Y4, data not shown) on macrophages is seen in Fig. 2. The LPS from both strains gave essentially identical morphological results. After 24 h of incubation of control and LPS (N27)-treated macrophages, there was a significant increase in the size, spreading, and vacuolation of the cells. The numbers of internal dense granules increased significantly (being absent in control macrophages). Concentrations of 400 Atg of LPS resulted in a marked rounding, clumping, and aggregation of the cells, indicative of cell death. At 48 h, the macrophages were completely activated, possessing numerous large vacuoles and dense granules. In addition to the

TABLE 6. Stimulation of 45Ca release from fetal rat long bone cultures by LPSs from A. actinomycetemcomitans Y4 and N27 % of total 4"Ca released" at LPS dilution: LPS preparation 0.1

0.3

1.0

3.0

10.0

30.0

32.7 ± 3.4 22.5 ± 2.5 30.8 ± 4.5 25.0 + 1.5 Y4 22.6 ± 0.6 29.8 ± 3.0 29.6 ± 3.2 22.1 ± 1.2 22.8 ± 1.7 30.6 ± 2.8 N27 Control medium 1 24.5 ± 0.8 18.3 ± 0.8 Control medium 2 Control LPS 39.3 ± 4.1 S. typhimurium 29.5 ± 2.3 E. coli a Values are means ± standard error for 6 to 10 bone assays cultured for 48 h. All values are significantly different from control at P < 0.01. LPS dilutions are shown in micrograms of LPS per milliliter (final concentration).

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INFECT. IMMUN.

KILEY AND HOLT

b

4.

if

J

e

I: r

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f

4

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FIG. 2. Effect of incubation of mouse peritoneal macrophages with A. actinomycetemcomitans strain N27 LPS. b to d are representative of 50, 200, and 400 pg of LPS per 1.1 ml (24 h of incubation), whereas f to i are representative of 48 h of incubation of macrophages in the presence of 50, 100, 200, and 400 pg of LPS per 1.1 ml. Twenty-four- and forty-eight-hour controls (no LPS added) are seen in a and e, respectively. See Table 7 for macrophage viability data and text for discussion of macrophage morphology. Bar = 10 ,um.


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TABLE 7. Viability of mouse peritoneal macrophages in the presence of varying concentrations of A. actinomycetemeomitans LPS Viable rnacrophageh V'

Dose LPS" N27,

Y4

18 400 42 43 200 60 60 100 83 78 50 100 100 Control' Micrograms of LPS per 1.1 ml of culture. Expressed as total number of viable cells by trypan blue exclusion at 48 h in the presence of experimental agent divided by total number of viable cells by trypan blue exclusion as control multiplied by 100. Total number of viable macrophages by trypan blue exclusion at 48 h in absence of experimental agent.

characteristic rounding of most of the macrophages at 48 h, many of them produced long, thick extensions or pseudopodia (e.g., Fig. 2f and g). Also characteristic of macrophages at this time was the loss of attached cells (Fig. 2i), clearly related to the loss of macrophage viability. In the presence of 10% heat-inactivated fetal calf serum, similar morphological results were obtained as that observed in the absence of serum (data not shown). However, serum appeared to preserve the overall morphological integrity of the macrophage with the discoid shape of the macrophage being maintained. There were, however, numerous internal dense granules and luscent vacuoles, identical to macrophages incubated in the absence of serum. Platelet aggregation. The effect of the Actinobacillus Y4 and N27 LPS on rabbit and human platelets was determined by aggregometry and by morphological observation in the light and electron microscope. For these latter observations, platelets were examined at their maximum aggregation as revealed by the aggregometer tracing (Fig. 3). Both Y4 and N27 LPS at concentrations between 140 and 18 pg/ ml resulted in both a shape change and aggregation as revealed by a decrease in recorder oscillations and progressive decrease in optical density, respectively. Below 9 fig of LPS per ml, there was no further aggregation as measured with the aggregometer or by light microscopic observation. Human platelets failed to aggregate in the presence of both 140 pg of Y4 and N27 LPS per ml. Aggregated platelets, when observed by both phase-contrast and Nomarski interference microscopy (not shown), were aggregated into tight

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masses and clumps, the individual cells containing numerous dense bodies and protuberances of various shapes. By transmission electron microscopy (Fig. 4a, b), control platelets (incubated in the absence of LPS) were for the most part elliptical. In some instances platelets with one or two pseudopodia were observed; however, this was a rare observation. Dense granules, lysosomal granules, and

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35jig/mi

70.pg/mi

2 min

140jg/ml

FIG. 3. Rabbit platelet aggregation induced by A. actinomwcetemcomitans strains Y4 and N27 LPS. Standard concentrations (2 x 10 platelets per mll) of platelets were incubated iith carving amounts (3 to 140 yglml) of LPS. Platelet aggregation and shape change were recorded as a change in slope and compression of recorder oscillation, respectiuelv. The arrow indicates the point at which LPS was added to the platelet suspension.

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