J. Periodontal Res. 12: 340-350, 1977

The capacity of lipopolysaccharides from bacteroides, fusobacterium and veillonella to produce skin inflammation and the local and generalized Shwartzman reaction in rabbits KJEIX SVEEN

The Gade Institute, Department of Microbiology, Laboratory of Oral Microbiology, University of Bergen, Norway Purified lipopolysaccharides (LPS) from strains of Bacteroides, Fusobacterium and Veillonella were tested for endotoxic activity, utilizing the primary skin reaction and the Shwartzmam phenomena in rabbits. Comparisons were made to Salmonella LPS, Each LPS preparation produced the primary skin reaction in a typical dose-response fashion. Skin sites injected with 400 \ig of Fusobacterium LPS often produced necrotic lesions. In the comparisons made between the skin lesion doses (SLDjj) of the Veillonella LPS and the Salmonella LPS, the difference was either non-significant or the Salmonella LPS was only slightly more active (P < 0.02), LPS from the Fusobacterium and the Bacteroides were significantly less toxic than the Salmonella LPS (P < 0,005), All LPS preparations did prepare the skin for the local and the specific organs for the generalized Shwartzman reaction. The toxicity of the Bacteroides LPS was, however, very low in the local reactioti (P < 0,005). For all LPS preparations the sensitivity of the primary skin test (SLDsj) was superior to the local Schwartzman reaction {SPD^o) (P < 0,005). Twenty-four hours after challenge for the generalized Shwartzman reaction, extensive fibrin thrombi in the capillaries of the renal glomemli were found, as well as thrombi containing necrotic blood cells, both in the veins of the liver and in the arteries of the lungs. The high endotoxic activity of the Fusobacterium and the Yeillonella LPS may reflect a similarity between their lipid A and that of the Salmonella LPS, (Accepted for publication December 4,1976)

Introduction

rate the O-antigens of the bacteria atid a j^^^.^^ nttmber of toxic and other host reac-

Lipopolysaccharides (LPS) of the cell wall of Gratn-negative bacteria are macromolectilar strtictures contaitiing polysaccbaride, phospholipid and stnall quanitittes of protein, These tnacromolectilar cotnplexes incorpo-

tive properties, collectively described as endotoxjc. LPS are released both from autolyzing bacterial cells antl from live orgatiisms as well (Cmtchley et al. 1967, Fraok & Houver

BACTERIAL LIPOPOLYSACCHARIDES AND INFLAMMATION 1969), Most likely, such release takes place into the plaque matrix (Mergenhagen et al, 1961, Berglund et al. 1969, Selvig et al, 1971), Endotoxic activity of human gingival exudate has also been demonstrated (Simon et al, 1969), Gingival tissue is highly susceptible to endotoxin derived from plaque bacteria (Rizzo & Mergenhagen 1964, Taichman & Courant 1965), and endotoxic LPS has, therefore, been discussed as a possible pathogenetic factor in periodontal disease (Mergenhagen et al. 1961, Scherp 1962, Rizzo & Mergenhagen 1964, Gustafson et al, 1966, Rizzo 1968, Selvig et al, 1971, Schwartz et al, 1972, Simon et al, 1972). In addition, endotoxin may have etiological significance in the resorption of bone (Rizzo & Mergenhagen 1964, Hausmann et al, 1972), Few studies have dealt with the endotoxic activity of LPS isolated and purified from Gram-negative plaque bacteria. Either crude preparations have been used, or a restricted number of endotoxic activities have been examined (Mergenhagen 1960, Mergenhagen et al, 1961, Mergenhagen & Varah 1963, de Araujo et al, 1963, Hofstad & Kristoffersen 1970a, Hofstad 1970), A systematic and comparative study of the endotoxic activity of LPS purified from Bacteroides, Fusobacterium and Veillonella has therefore, been carried out. As part of this study the prodtiction of primary skin inflammation, and the Shwartzman phenomena in rabbits have been examined, A description of the electron microscopical appearance and the chemical composition of the preparations used is included.

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species intermedius strain B 10 (Hofstad 1968), Bacteroides

fragiUs subspecies fra-

gilis strains NCTC 9343 and Lille E323 (Shinjo et al, 1971), Fusobacterium nucleatum strains F l (Kristoffersen 1969) and Fev 1 (de Araujo et al, 1963), Cultural Conditions The microorganisms were grown in 500 or 1000 ml screw-cap bottles, except for NCTC 9343 which was grown in a cbemostat at pH 7,0 and at dilution rates ranging from 0.05 to 0.11 h-i (Dallarkd & Hofstad 1974), F 1 and Fev 1 were cultured in the following medium, pH 7,0 (g/1): Tryptone (Oxoid) 15,0; yeast extract (Oxoid) 3,0; NaCl 5,0; glucose 2,5; KH^POj 1.5; NajHPOj-aHaO 3.5; (NH4)2SO4 0,5; L-cysteineHCll.O; Haemin 0.016; menadione 0.0006, The same medium with 5 % human plasma was used for cultivation of B 10. For cultivation of NCTC 9343 and E 323, the coocentration of glucose was increased to 5 % and the yeast extract omitted. The Veillonella medium, pH 7.5, contained (g/1): Tryptone (Oxoid) 5.0; yeast extract 4.0; C^HgNaO^S 0.75; CHgCHOHCOaNa 20 ml from a 63 % solution.

Isolation of LPS Centrifuged, washed organisms of B10, NCTC 9343 and E 323 were suspended in distilled water (1 g/ml) and extracted with ao equal volume of 90 % phenol (Westphal et al. 1952) by constant stirring at room temperature for 15 minutes (Hofstad and Kristoffersen 1970). After centrifugation at 1000 X g for 30 minutes at 4°C, the water phase was pipetted off and dialysed for 48 hotirs against tap water at room Materials and Methods temperature, LPS was purified byultracentrifugation at 100 OOO X g for 90 minutes. Organisms The microbial strains used were: Veillonella The pellet (LPS) was suspended in 0.1 M parvula strain Ve 9 and Veillonella alcales- phosphate buffer pH 7.0 containing ribocens strain Ve 5 (Hofstad & Kristoffersen nuclease (5 X cryst,, ex-bovine pancreas, 1970b), Bacteroides melaninogenicus sub- Sigma Chemical Company, St, Louis, Mo,,

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USA) and deoxyribonucleasc (25 % activity of crystalline material deoxyribonuciease, ex-bovine pancreas, Koch-Light & Co., Ltd., Colnbrook, Bucks., England) to give a final enzyme: substrate ratio of about 1:50, and incubated in a water-bath at 37°C for 2 hours. (Hofstad & Kristoffersen 197O.a). After washing, the pellets were taken up in distilled water and lyophilized. LPS was prepared in the same way from aceton-dried cells of Ve 5 and Ve 9 (Hofstad & Kxistoffersen 1970b) and from crushed and btifferextracted cells of F 1 and Fev 1 (Kristoffersen 1969). LPS from Salmonella enteritidis strain S-795 was kindly supplied by K. C. Mihier, Rocky Moimtain Laboratory, Hamilton, Mont., USA. Chemical Analyses Neutral sugars were measured by tbe acidorcinol method (Winzler 1955) with glucose: galactose (1:1) as standard. Fatty acid esters were determined by the method of Snyder & Stephens (1959), and 2-keto-3-deoxy octonate (KDO) by the malonealdehyde-thiobarbituric acid method as modified by Weissbach & Hurwitz (1959). Protein was estimated by the Folin-Ciocalteu phenol method according to Lowry et al. (1951), using bovine serum albumin as standard. Determination of total phosphorus was performed according to Fiske & Subbarow (1925) with some of the modifications of Youngburg & Youngburg (1930). Gas Liquid Chromatography (GLC) For detection and quantitative determination of hexosamines, samples were hydrolysed in 6 N HCl for 4 hours in an oil bath at 100°C, The alditol acetates were prepared according to Niedermeier (1971), The specimens were analyzed on a glass column (0,2 X 180 cm) packed with 3 % Poly A-103 on Sapelcon AW, DMCS, Lot D 329, 100/120 mesh (Supelco Ine, Bellefonte. Pa,, USA) at a temperature of 190°C.

For detection and quantitatioti of aldoses, samples of LPS were hydrolyzed in 0,1 N HCl for 48 hours at 100°C, The aldoses were converted to alditol acetates as described by Sawardecker et al, (1965), and the samples analyzed on a glass column (0,2 X 180 cm) packed with 3 % ECNSSM on a Gas-Chrom Q, 100/120 mesh (Applied Science Laboratories, State College, Pa,, USA) at a column temperature of 170°C, Gas liquid chromatography (GLC) was run in a Perkin-Blmer 900 Gas Chromatograph equipped with flame ionazation detector. The detector temperature was 260°C and the flow of the gas (N^) was 30 ml/h. Electron Microscopy Lyophilized LPS was suspeoded in distilled water (0.5 mg/ml) by sonication (MSE/ MULLARD, 60 W, 20 kc/s) at 0°C, for up to 2 mintttes at maximum output, positively stained with 2 % luranyl acetate in distilled water, pH 4.5, and prepared for electron microscopy as described by Shands et al. (1967). The studies were carried out on a Philips 300 electron microscope at 80 kV. Bio-Assays Animals. Both primary skin inflammation and the local Shwartzman reaction were produced in New Zealand White rabbits, 3-6-months-old, weighing 2,0-3,5 kg, caged separately and maintained on a standard laboratory diet and water ad lib. The temperature and the relative humidity were kept between 20-22°C and 45-50% respectively. In order to avoid seasonal variations in rabbit susceptibility to endotoxin, the experiments were carried otit in the months of May and June (Vegh & Kovats 1967). Rabbits of both sexes were used, and an equal distribution of males and females was aimed at for each LPS preparation tested. The day before injection, the skin areas to be used were carefully clipped

BACTERIAL LIPOPOLYSACCHARIDES AND INFLAMMATION with an electric clipper. Thereafter a hair remover (Nair®, Folkestone, Kent, England) was used to facilitate tile readings. Preparation of LPS suspensions.. One mg of LPS was suspended in 1 ml of sterile isotonic saline (0.85 % NaCl) and sonicated at 0°C for 2 miautes. If necessary, the pH was adjusted to 7.2 using triethylamine. Serial two-fold dilutions were made in sterile isotonic saline with concentrations ranging from 400 to 0.195 jig in 0.2 ml. Primary skin inflammation. Suspensions of LPS (0.2 ml) were injected intradermaily in horizontal rows below the median line of the dorsum. Consecutively starting with the greatest concentration, each rabbit received 9-11 two-fold dilutions on each side. Only one LPS of the purified preparations was tested on one side; LPS-S-795 was administered on the contraiateral side for comparison. Sterile saline was used as a negative control. Each preparation was tested in six rabbits. The first reading was made 20 hours after inoculation, then every 24th hour, with a final reading at 68 hours. Measurement of the lesions were performed in horizontal and vertical directions, and those with an average diameter of 5 mm or more were considered to be positive reactions. Local Shwartzman reaction. The local Shwartzman reaction was elicited on tbe ventral surface. The two-fold dilutions of the LPS suspensions (0.2 ml) were injected 15 mm from the ventral midline, starting with the greatest dose foremost. Only one preparation was administered on one side; serial two-fold dilutions of LPS-S-795 were inoculated on the other side for comparison. Twenty-four hours after preparing the local site of the skin with 0.2 ml of the two-fold dilutions of the LPS suspensions, the homologous LPS preparation to be tested was administered in the same volume in the marginal ear vein. The inflammatory reactions were read immediately before, and

343

24 hours after provocation. Any degree ol gross hemorrhage appearing 24 hours after challenge were considered to be positive local Shwartzman reactions. Each LPS preparation was tested in six rabbits. Experiments were also performed whereby preparative doses from all LPS were inoculated in the skin of the ventral surface of the same rabbits in doses corresponding to the Shwartzman preparing dose (SPDjJ minus the standard deviation (cf. Table 3). General Shwartzman reaction. Rabbits were given two intravenous injections ol the same LPS in 0.2 ml volumes in the marginal ear vein 24 hours apart. Twentyfour hours after the last injection, the rabbits were sacrificed by bleeding thoroughly. Gross examination of liver, kidneys, suprarenal glands and lungs were made, and selected tissues were fixed in 10 % formalin in phosphate buffered saline, pH 7.2. After dehydration, the tissue was embedded in paraffin, sectioned at 7 jim and stained with hematoxylin and eosin, or phosphotungstic acid hematoxylin (PTAH) (Mallory) (Lille 1965). Statistical Methods The skin lesion dose (SLDg,,) and the Shwartzman preparing dose (SPD^,) were determined according to the method of Larson et al. (I960). The dilutions of LPS were numbered, dilution 1 equaled 400 ^ig, 2 = 200 (ig, 3 = 100 ng etc. The dose halfway between tbe dilution giving lesion and the next greatest dilution was chosen as the determining dose since the serial dilution numbers are functions of the log doses. The total number of rabbits used for each LPS preparation was the basis for determining the standard deviation of the doses, calculated as: 2 (x-x)2 n-1

(Hill 1961). The significance of the differ-

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tween the transformed dose XT and the lesion Y, Results

Fig, 1, Lyophifized LPS from B, fragUss ss, Iragiilis E 323 stasned wrth uranyl acetate. Arrows indioate rodlike parljcles One doughnut-shaped particle wilh triiaminaisd surface structure is otearly seen. Magnification X 100,000. Horizontal bar represents 1000 A.

ence between the various LPS preparations tested and LPS-S-795 was calculated according to the Wilcoxon two-sample test (two-tailed) (Documenta Geigy 1962). The dose X in the dose-response curve was transformed to XT according to the formula: XT = log (1-fX). The Pearson correlation coefficient (r) (Nie et al, 1970) was caictilated according to the relationship be-

Electron Microscopic and Chemical Characterization of the LPS Preparations The LPS preparations showed varying solubility when suspended in saline, whereas ultrasonic treatment at maximum output rendered highly dispersible suspensions. Electron microscopical examinations revealed particles mostly of a trilaminar stirface structure in all LPS preparations. Doughnut-shaped particles predominated, but also discs and rods were seen, Veillonella LPS contained mainly doughnutshaped particles, with diameters ranging from 250-300 A, The main structures observed in LPS prepared from B. melaninogenicus and Fusobacterium were discs and doughnut-shaped particles with diameters in the range of 270-1050 A. Triple-layered rod-like particles together with doughnutshaped particles ranging in length and diameters from 200—900 A, were the structures most often seen in LPS preparations from B. fragiUs strains (Fig, 1). The quantitative chemical analysis, shown

Tabie 1

Chemical data on LPS isolated from different oral .bacteria, (Mean ± s,cf. of three or four determinations) iMeutral sugar Fatty acid ester Protein Mean ± s,d, itflean ± s,d. Mean + s.d.

Source of LPS

KDO Mean ± s.d.

Ptiospiiorus Mean ± s.d.

Veilionella

VeS Ve9

23.3 ± 2.3 19.9 ± 1.3

19..B ± 0.7 29.9 ± 1.7

16.4 ± 1.3 7.6 + 0.4

9.6 ± 0.20 7.2 ± O.SO

0.67 ± 0.20 1.6B ± 0.20

Foso bacterium

Fl Fev1

18.0 ± 1.1 22.0 ± 0.7

15.1 ± 1.4 19.8 + D.7

16.0 ± 1.7 7.1 :+ 1.4

1.05 ± 0.20 2.4 ± 0.07

0.70 ± o.oo 0.35 ± O.K

B. nielaninogenious

BIO

33.1 ± 1.5

8.9 ± 1.4

12.3 + 0'.3

n.d.

0.44 ± 0.08

8. fragilis ss fragilis NCTC 9343 E323

37.6 :± 2.6 16.6 ± 2.1

23.4 ± 0.3 22.6 ± 1.0

7.3 ± 0.3 33.1 ± 0.2

n.d. n.d.

o.or ± 0.01 0.62 ± 0.00

S. enteritidis

29.7 ± 2.0Z

10.7 ± 1.67

12.B ± 0.7

7.2 ± 0.6

0.40 ± 0.00

S-795

n.d. = not detected. KDO = 2-keto-3-deoxyoctonate.

BACTERIAL LIPOPOLYSACCHARIDES AND INFLAMMATION

345

Table 2 Primary skin inflammation in rabbits produced by LPS purified from strains of Veillonella, Fusobacterium and Bacteroides Least si

The capacity of lipopolysaccharides from bacteroides, fusobacterium and veillonella to produce skin inflammation and the local and generalized Shwartzman reaction in rabbits.

J. Periodontal Res. 12: 340-350, 1977 The capacity of lipopolysaccharides from bacteroides, fusobacterium and veillonella to produce skin inflammatio...
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