INFECTION AND IMMUNITY, Jan. 1983, p. 476-479 Vol. 39, No. 1 0019-9567/83/010476-04$02.00/0 Copyright C 1983, American Society for Microbiology Suppression of Murine Lymphocyte Mitogen Responses by Exopolysaccharide from Capnocytophaga ochracea RONALD W. BOLTON* AND JOHN K. DYER Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska 68583-0740 Received 26 July 1982/Accepted 5 October 1982 An extracellular polysaccharide was purified from culture supernatants of Capnocytophaga ochracea 25, a gram-negative bacillus associated with human periodontal disease. The extracellular polysaccharide suppressed in vitro mito- genic responses of murine splenic lymphocytes to concanavalin A and lipopoly- saccharide. This suppression was dose dependent, persisted up to 120 h, and was not caused by direct toxicity of the extracellular polysaccharide. Modulation of immune responses by bacterial C. ochracea 25, a gift from S. S. Socransky, components has received considerable attention Forsythe Dental Center, Boston, was cultivated in recent years (1, 2, 4, 5). Since a number of at 37°C in Trypticase soy broth supplemented these substances are produced by members of with 1% yeast extract and 0.1% NaHCO3. After the normal bacterial flora, they may have con- 48 h the cultures were centrifuged at 10,000 x g siderable impact on host-parasite interactions. for 15 min at 4°C. EP was isolated from liquid Indeed, there are several diseases of microbial culture supernatants by cold 95% ethanol pre- etiology, both clinical and experimental, which cipitation. The precipitate was dissolved in wa- have been associated with immune suppression ter and treated with cold 10% trichloroacetic or enhancement (reviewed in reference 12). acid for 2 h, dialyzed against water, and repre- A consequence of the immunomodulatory ac- cipitated with ethanol. The ethanol precipitate tion of bacterial products may be human peri- was dissolved in 0.05 M Tris buffer (pH 7.2) and odontal disease. Bacteria are essential agents in further purified by Sephadex G-100 gel filtration. the etiology of this disease; however, the prog- Fractions were analyzed for carbohydrate by the ress of the lesion has been attributed to altered anthrone method (16) and for protein by the host responses to bacterial products which may BioRad protein assay. Purified EP was hydro- penetrate gingival tissue (10). Recently, much lyzed in 1 N HCI for 4 h at 100°C. The hydroly- attention has been given to identifying the bacte- sates were dried in vacuo over P205 and NaOH ria associated with periodontal disease and char- pellets. Monosaccharides in the reconstituted acterizing host responses to these organisms hydrolysates were determined by thin-layer (reviewed in reference 10); however, little atten- chromatography with silica gel 60 (EM Labora- tion has been given to the immunomodulatory tories, Elnsford, N.Y.) and n-butanol-95% etha- potential of these bacteria. Recently, sonicated nol-water (52:32:16, three ascends). Purified EP cells of Actinobacillus actinomycetemcomitans, was examined for the presence of endotoxin by a gram-negative bacterium implicated in certain the Limulus amoebocyte lysate test. Pyrogen- forms of periodontal disease, were shown to free water was used for the preparation of all suppress human lymphocyte activation in vitro reagents used in this investigation. The antigenic (14). The authors suggested that such immuno- purity of the Sephadex G-100 column fraction suppressive agents may act in vivo by enhancing was evaluated by agar-gel immunodiffusion the pathogenicity of their parent microorganism against rabbit anti-C. ochracea 25 serum pre- or that of some other opportunistic pathogen. pared by subcutaneous injection of whole cells In this investigation an extracellular polysac- mixed with incomplete Freund adjuvant. charide (EP) was purified from culture superna- Spleen cells from C57BL/6 mice (aged 5 tants of Capnocytophaga ochracea 25, a gram- months) were cultured at a concentration of 2 x negative bacterial species found in the human 106 cells per ml of RPMI 1640 medium supple- oral cavity and associated with certain forms of mented with gentamycin sulfate (100 ug/fml), L- periodontal disease (3, 7, 15). The modulatory glutamine (2 mM), and 10% fetal calf serum, as effect of EP on murine splenic lymphocyte re- described previously (2). Optimal concentra- sponses to the mitogens concanavalin A (ConA) tions of ConA (0.25 ,ug per culture) (Pharmacia and lipopolysaccharide (LPS) was evaluated in Fine Chemicals, Piscataway, N.J.) or LPS (7 jig vitro. per culture) (Escherichia coli 055:B5; Difco Lab- 476 VOL. 39, 1983 NOTES 477 oratories, Detroit, Mich.) were added in 0.1-ml 3H-TdR volumes to the appropriate tubes, and the cul- loor 0-c---Do~ tures were incubated for 72 h or as indicated. 3H- URIDINE I During the last 4 h of incubation, each culture 0 3H-LEUCINE received a 1-,uCi pulse of either methyl-[3H]thy- 80- midine (TdR) (specific activity, 2 Ci/mmol), z 0 [3H]uridine (specific activity, 5 Ci/mmol), or 0 [3H]leucine (specific activity, 10 Ci/mmol) (all LL 0 6OF from New England Nuclear Corp., Boston, 0R Mass.) for evaluation of DNA, RNA, and pro- z tein syntheses, respectively. The radioactivity 0 of trichloroacetic acid-precipitable material was IZ- 401 evaluated by liquid scintillation, and counting m 0 efficiency was determined by channels ratio. a- Modulation experiments were performed by diluting EP in culture medium (see the figure 0 20 F legends) and adding 0.1 ml of each dilution to z triplicate spleen cell cultures at various times before or after the addition of LPS or ConA; however, the elapsed time from addition of 2 5 10 20 4050 mitogen to culture termination remained con- EXOPOLYSACCHARIDE (y.g /CULTURE) stant except in the kinetics study, when cultures FIG. 1. Effect of EP on DNA, RNA, and protein were terminated at 24-h intervals. A control syntheses in murine spleen cells preincubated with EP preparation extracted from the bacterial culture for 30 min before the addition of an optimal mitogenic medium was evaluated similarly. Results were dose of ConA. Results are plotted as a percentage of compared with controls which contained only radiolabeled precursor incorporation compared with LPS or ConA. The effect of EP alone on murine cultures that received ConA only. Each point repre- spleen cell activity was evaluated by incubating sents the mean value ± the standard error for triplicate various concentrations of EP with the cells in assays from two experiments. Incorporation in cells the absence of LPS or ConA for 48 h, with a receiving ConA alone averaged 98,713, 26,720, and pulse of radiolabeled precursor during the final 4 5,882 disintegrations per min (dpm) for [3H]TdR, h. Representative cultures were then evaluated [3H]uridine, and [3H]leucine, respectively. for uptake of labeled precursor, and others were examined for viability by trypan blue dye exclu- sion. C. ochracea produced 5.2 g of EP per 100 g of (Fig. 2). The time at which EP was added to the cells (dry weight). EP eluted from Sephadex G- cell cultures in relation to that of mitogen deter- 100 as a single peak immediately after the void mined the degree of suppression (Fig. 3). The volume. This fraction produced a single band most significant suppression of both the LPS against rabbit anti-strain 25 serum in agar-gel and ConA responses was observed when the immunodiffusion. Preliminary analyses revealed cells were preincubated with EP for 30 min. EP this material to be a polysaccharide, with man- added to the cultures after LPS had little effect nose as a major constituent (80%). Protein was on stimulation; however, suppression of ConA present at a concentration of 27 ,ug/mg of EP. responses remained significant, although dimin- Our preparation was free of endotoxin at the ished, when EP was added afterward. The con- concentrations used in this investigation, since 1 trol preparation extracted from bacterial culture mg/ml produced a negative Limulus test. A medium produced no significant effects on lym- complete chemical analysis of EP will be forth- phocyte responses when used at the same con- coming. The extraction procedure for EP was centrations as EP. used on uninoculated culture medium and pro- EP did not mediate the observed suppression duced a precipitate, which was used as a control of cell activation by delaying the response. Cul- in the lymphocyte stimulation experiments. tures preincubated with 10 ,ug of EP before LPS EP produced a dose-dependent suppression of or ConA was added and harvested at 24-h inter- murine spleen cell responses to the T lympho- vals revealed a continual increase in suppression cyte mitogen ConA (Fig. 1). DNA, RNA, and up to 120 h (Fig. 4). Suppression was not caused protein syntheses were suppressed to similar by toxic effects produced by EP, as cell viability degrees, with 80% suppression occurring at 40 did not significantly differ from controls during ,g of EP per culture. EP also produced signifi- the incubation times and at the EP concentra- cant but less marked suppression of radiolabeled tions employed in this investigation. EP in the precursor uptake in LPS-stimulated cultures absence of LPS or ConA produced no significant 478 NOTES INFECT. IMMUN. lo-o 3H-TdR 02100 loor D-O I. KLPS 3H-URIDINE z 3H-LEUCINE 0 0 i L) 80/ 80F 0 z 0 0 a20C0NA U- I- 0 cr 601 0 0-40- z 0 0 0 z 40F cr 20- 0~ 0 z 20F -24 -I 0 i 24 TIME (hr) FIG. 3. Effect of pre- and post-mitogen exposure of l1 I I I " cells to EP (20 ,ug per culture). Cultures were incubat- 1 2 5 10 20 40 50 ed for 72 h after the addition of LPS or ConA regard- EXOPOLYSACCHARI DE (Q.g/CULTURE) less of the time at which EP was added.