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 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 . 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. 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. Results are plotted as a percentage of [3H]TdR incorporation in FIG. 2. Effect of EP on DNA, RNA, and protein cultures receiving LPS or ConA alone and represent syntheses in murine spleen cells preincubated with EP the mean ± the standard error for six assays. Incorpo- for 30 min before the addition of an optimal mitogenic ration of [3H]TdR averaged 59,812 dpm in cultures dose of LPS. Results are plotted as a percentage of receiving ConA alone and 59,474 dpm in those receiv- radiolabeled precursor incorporation compared with ing LPS alone. cultures that received LPS only. Each point represents the mean value the standard error for triplicate assays from two experiments. Incorporation in cells nate explanations include EP binding to cell receiving LPS alone averaged 99,921, 5,026, and 2,284 surface glycoproteins, with subsequent inhibi- dpm for [3H]TdR, [3H]uridine, and [3H]leucine, re- tion of B cell activation by interferene with spectively. essential membrane conformational changes, or

changes in viability or in DNA, RNA, and -'100 0 protein syntheses in 24-h cultures (Table 1). cr The suppression of lymphocyte responses to z 0 ConA may have resulted from the interference O 80 - of EP with the binding of ConA to lymphocyte U- surfaces. Supportive of such a hypothesis are our observations that the greatest suppression z 60 occurred when the cells were preincubated with 0 F EP before the addition of ConA, that the sup- t= pression persisted, and that EP alone produced R 40 F o-a CON A no significant effects on cell viability or on o-o LPS 0 DNA, RNA, or protein synthesis. Binding inhi- 0 z bition by EP is further supported by the signifi- _ cant levels of mannose found in our preparation, a 20 since a-D-mannose is known to inhibit ConA binding and mitogenicity (13). r-)I The EP-mediated suppression of mitogenic 24 48 72 96 120 was not result of responses to LPS probably the INCUBATION TIME (hr) direct inhibition of LPS binding to cell surfaces. LPS interactions with lymphocytes involve a FIG. 4. Kinetics of [3H]TdR incorporation in mu- nonspecific lipid A-membrane lipid binding, rine spleen cell cultures preincubated with 10 p.g of EP for 30 min before the addition of either LPS or ConA. can be inhibited with phospholipid (6), as which A pulse of [3H]TdR was added during the final 4 h. opposed to ConA binding to cell surface glyco- Results are plotted as a percentage of radiolabeled proteins, which is inhibitable by a-D-mannose precursor incorporation compared with cultures that (13). Thus, it would be unlikely that EP could received LPS or ConA alone and represent the mean interfere with LPS-lymphocyte binding. Alter- ± the standard error for triplicate assays. VOL. 39, 1983 NOTES 479 TABLE 1. Effect of EP on the uptake of phocyte responses, thus lending credibility to radiolabeled precursors by murine spleen cells in the hypothesis that Capnocytophaga species vitro may contribute to periodontal pathology by EP conc Incorporation (dpm) (mean ± SE) causing altered immune reactions to substances (L,a) [3H]TdR [3H]uridine [3H]leucine entering the gingival tissue. 0 5,819 ± 756 14,982 ± 1,076 1,096 ± 53 LITERATURE CITED 5 6,000 ± 685 13,538 ± 1,145 1,227 ± 77 1. Battisto, J., and F. Pappas. 1973. Regulation of immuno- 10 6,247 ± 117 13,149 ± 1,311 943 ± 60 globulin synthesis by dextran. J. Exp. Med. 138:176-193. 20 6,1% ± 473 14,431 ± 1,003 895 ± 30 2. Bolton, R. W. 1981. Modulation of murine lymphocyte 40 5,416 ± 387 14,176 ± 901 985 ± 63 mitogen responses by glycerol-teichoic acid. Immunol. Commun. 10:631-640. a Per 2 x 105 cells. Cell viability remained greater 3. Celesk, R. A., R. M. McCabe, and J. London. 1979. than 85% at all concentrations used after 48 h of Colonization of the surface of teeth by oral incubation. gram-negative bacteria. Infect. Immun. 26:15-18. 4. Chisari, F. V., R. S. Northrup, and L. C. Chen. 1974. The modulating effect of cholera enterotoxin on the immune response. J. Immunol. 113:729-739. 5. Franzl, R. W., and P. D. McMaster. 1968. The primary EP inhibition of responses to LPS by interfer- immune response in mice. I. The enhancement and sup- ence with the generation of regulatory T cells (8, pression of hemolysin production by bacterial endotoxin. 9). J. Exp. Med. 127:1087-1107. An alternate site of action for the immuno- 6. Kabir, S., and D. L. Rosenstreich. 1977. Binding of bacte- rial endotoxin to murine spleen lymphocytes. Infect. modulatory effects of EP may be the macro- Immun. 15:156-164. phage. EP may interfere with production of 7. Leadbetter, E. R., S. C. Holt, and S. S. Socransky. 1979. immunoregulatory substances (reviewed in ref- Capnocytophaga: new genus of gram-negative gliding erence 18) and, in the ConA experiments, with bacteria. I. General characteristics, taxonomic consider- ations and significance. Arch. Microbiol. 122:9-16. presentation of the mitogen to T lymphocytes 8. McGhee, J. R., H. Kiyono, S. M. Michalek, J. L. Baab, (11). The results presented here are inconclusive D. L. Rosenstreich, and S. E. Mergenhagen. 1980. Lipo- with regard to cellular adherence and the site of polysaccharide regulation of the immune response: T action of EP. Cell separation and EP adherence lymphocytes from normal mice suppress mitogenic and immunogenic responses to LPS. J. Immunol. 124:1603- experiments are in progress and should clarify 1611. the mechanism of action. 9. Norcross, M. A., and R. T. Smith. 1977. Regulation of B- It may be hypothesized that EP-mediated im- cell proliferation responses to lipopolysaccharide by a munosuppression is an experimental artifact re- subclass of thymus cells. J. Exp. Med. 145:1299-1315. 10. Page, R. C., and H. E. Schroeder. 1981. Current status of sulting from alteration of the natural biological the host response in chronic marginal periodontitis. J. properties of EP by the trichloroacetic acid Periodontol. 52:477-491. treatment. Our preliminary studies would refute 11. Rosenstreich, D. L., J. J. Farrer, and S. Dougherty. 1976. such an argument, since we initially used crude Absolute macrophage dependancy of T lymphocyte acti- vation by mitogens. J. Immunol. 116:131-139. ethanol precipitates of spent culture fluid and 12. Schwab, J. H. 1975. Suppression of the immune response found them to be immunosuppressive (unreport- by microorganisms. Bacteriol. Rev. 39:121-143. ed data). In addition, we have found that further 13. Sharon, N. 1976. Lectins as mitogens, p. 31-41. In J. J. purification of EP enhances its immunosuppres- Oppenheim and D. L. Rosenstreich (ed.), Mitogens in immunobiology. Academic Press, Inc., New York. sive properties as well as its reactivity in agar- 14. Shenker, B. J., W. P. McArthur, and C. C. Tsai. 1982. gel immunodiffusion. The validity of our obser- Immune suppression induced by Actinobacillus Actino- vations is further supported by the failure of mycetemcomitans 1. Effects on human peripheral blood extracts from uninoculated culture fluid to pro- lymphocyte responses to mitogens and antigens. J. Immu- nol. 128:148-154. duce immunosuppression. 15. Socransky, S. S., S. C. Holt, E. R. Leadbetter, A. C. R. Components of the Capnocytophaga species Tanner, E. Savitt, and B. F. Hammond. 1979. Capnocyto- found in the oral cavity may elicit a complex phaga: new genus of gram-negative gliding bacteria. III. series of immunological events, some of which Physiological characterization. Arch. Microbiol. 122:19- 33. may contribute to periodontal disease. The pres- 16. Spiro, R. G. 1976. Analysis of sugars found in glycopro- ence of LPS (17), an immunostimulatory agent teins. Methods Enzymol. 8:3-26. (5), and immunosuppressive EP in the same 17. Stevens, R. H., M. N. Sela, W. P. McArthur, A. Nowotny, species poses a considerable challenge in dis- and B. F. Hammond. 1980. Biological and chemical char- the influence this organism may have on acterization of endotoxin from Capnocytophaga sputi- secting gena. Infect. Immun. 27:246-254. the immune system. Our preliminary studies 18. Unanue, E. R. 1981. The regulatory role of macrophages indicate that EP can also suppress human lym- in antigenic stimulation. Adv. Immunol. 31:1-136.