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. 1997 Nov;65(11):4531–4538. doi: 10.1128/iai.65.11.4531-4538.1997

A novel component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans.

K Iki 1, K Kawahara 1, S Sawamura 1, R Arakaki 1, T Sakuta 1, A Sugiyama 1, H Tamura 1, T Sueda 1, S Hamada 1, H Takada 1
PMCID: PMC175651  PMID: 9353030

Abstract

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermedia ATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate and protein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) on deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. We designated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity on splenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction were resistant to heat inactivation (100 degrees C for 1 h) and protease treatments and were scarcely inhibited by polymyxin B. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with a phenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages from C3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeN mice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hot phenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. These findings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reported to date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not from the LPS itself.

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Selected References

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  1. Bramanti T. E., Wong G. G., Weintraub S. T., Holt S. C. Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277. Oral Microbiol Immunol. 1989 Dec;4(4):183–192. doi: 10.1111/j.1399-302x.1989.tb00250.x. [DOI] [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Flebbe L. M., Chapes S. K., Morrison D. C. Activation of C3H/HeJ macrophage tumoricidal activity and cytokine release by R-chemotype lipopolysaccharide preparations. Differential effects of IFN-gamma. J Immunol. 1990 Sep 1;145(5):1505–1511. [PubMed] [Google Scholar]
  4. Flebbe L., Vukajlovich S. W., Morrison D. C. Immunostimulation of C3H/HeJ lymphoid cells by R-chemotype lipopolysaccharide preparations. J Immunol. 1989 Jan 15;142(2):642–652. [PubMed] [Google Scholar]
  5. Fujiwara T., Nishihara T., Koga T., Hamada S. Serological properties and immunobiological activities of lipopolysaccharides from black-pigmented and related oral Bacteroides species. J Gen Microbiol. 1988 Nov;134(11):2867–2876. doi: 10.1099/00221287-134-11-2867. [DOI] [PubMed] [Google Scholar]
  6. Fujiwara T., Ogawa T., Sobue S., Hamada S. Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains. J Gen Microbiol. 1990 Feb;136(2):319–326. doi: 10.1099/00221287-136-2-319. [DOI] [PubMed] [Google Scholar]
  7. Galanos C., Lüderitz O. Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur J Biochem. 1975 Jun;54(2):603–610. doi: 10.1111/j.1432-1033.1975.tb04172.x. [DOI] [PubMed] [Google Scholar]
  8. Galanos C., Lüderitz O., Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem. 1969 Jun;9(2):245–249. doi: 10.1111/j.1432-1033.1969.tb00601.x. [DOI] [PubMed] [Google Scholar]
  9. Gibson F. C., 3rd, Tzianabos A. O., Onderdonk A. B. The capsular polysaccharide complex of Bacteroides fragilis induces cytokine production from human and murine phagocytic cells. Infect Immun. 1996 Mar;64(3):1065–1069. doi: 10.1128/iai.64.3.1065-1069.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamada S., Koga T., Nishihara T., Fujiwara T., Okahashi N. Characterization and immunobiologic activities of lipopolysaccharides from periodontal bacteria. Adv Dent Res. 1988 Nov;2(2):284–291. doi: 10.1177/08959374880020021301. [DOI] [PubMed] [Google Scholar]
  11. Hamada S., Okahashi N., Fujiwara T., Nishihara T., Koga T. Selection induction of prostaglandin E production in C3H/HeJ mouse macrophages by lipopolysaccharides from Bacteroides gingivalis. Oral Microbiol Immunol. 1988 Dec;3(4):196–198. doi: 10.1111/j.1399-302x.1988.tb00010.x. [DOI] [PubMed] [Google Scholar]
  12. Hamada S., Takada H., Ogawa T., Fujiwara T., Mihara J. Lipopolysaccharides of oral anaerobes associated with chronic inflammation: chemical and immunomodulating properties. Int Rev Immunol. 1990;6(4):247–261. doi: 10.3109/08830189009056635. [DOI] [PubMed] [Google Scholar]
  13. Hanazawa S., Nakada K., Ohmori Y., Miyoshi T., Amano S., Kitano S. Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice. Infect Immun. 1985 Oct;50(1):262–270. doi: 10.1128/iai.50.1.262-270.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holt S. C., Bramanti T. E. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit Rev Oral Biol Med. 1991;2(2):177–281. doi: 10.1177/10454411910020020301. [DOI] [PubMed] [Google Scholar]
  15. Joiner K. A., McAdam K. P., Kasper D. L. Lipopolysaccharides from Bacteroides fragilis are mitogenic for spleen cells from endotoxin responder and nonresponder mice. Infect Immun. 1982 Jun;36(3):1139–1145. doi: 10.1128/iai.36.3.1139-1145.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kawahara K., Brade H., Rietschel E. T., Zähringer U. Studies on the chemical structure of the core-lipid A region of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305. Detection of a new 2-octulosonic acid interlinking the core oligosaccharide and lipid A component. Eur J Biochem. 1987 Mar 16;163(3):489–495. doi: 10.1111/j.1432-1033.1987.tb10895.x. [DOI] [PubMed] [Google Scholar]
  17. Koga T., Nishihara T., Fujiwara T., Nisizawa T., Okahashi N., Noguchi T., Hamada S. Biochemical and immunobiological properties of lipopolysaccharide (LPS) from Bacteroides gingivalis and comparison with LPS from Escherichia coli. Infect Immun. 1985 Mar;47(3):638–647. doi: 10.1128/iai.47.3.638-647.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Komuro T., Galanos C. Analysis of Salmonella lipopolysaccharides by sodium deoxycholate-polyacrylamide gel electrophoresis. J Chromatogr. 1988 Oct 26;450(3):381–387. doi: 10.1016/s0021-9673(01)83593-7. [DOI] [PubMed] [Google Scholar]
  19. Kumada H., Haishima Y., Umemoto T., Tanamoto K. Structural study on the free lipid A isolated from lipopolysaccharide of Porphyromonas gingivalis. J Bacteriol. 1995 Apr;177(8):2098–2106. doi: 10.1128/jb.177.8.2098-2106.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROBERTS N. R., LEINER K. Y., WU M. L., FARR A. L. The quantitative histochemistry of brain. I. Chemical methods. J Biol Chem. 1954 Mar;207(1):1–17. [PubMed] [Google Scholar]
  21. Lindberg A. A., Weintraub A., Zähringer U., Rietschel E. T. Structure-activity relationships in lipopolysaccharides of Bacteroides fragilis. Rev Infect Dis. 1990 Jan-Feb;12 (Suppl 2):S133–S141. doi: 10.1093/clinids/12.supplement_2.s133. [DOI] [PubMed] [Google Scholar]
  22. Manthey C. L., Perera P. Y., Henricson B. E., Hamilton T. A., Qureshi N., Vogel S. N. Endotoxin-induced early gene expression in C3H/HeJ (Lpsd) macrophages. J Immunol. 1994 Sep 15;153(6):2653–2663. [PubMed] [Google Scholar]
  23. Matsuda T., Hirano T., Kishimoto T. Establishment of an interleukin 6 (IL 6)/B cell stimulatory factor 2-dependent cell line and preparation of anti-IL 6 monoclonal antibodies. Eur J Immunol. 1988 Jun;18(6):951–956. doi: 10.1002/eji.1830180618. [DOI] [PubMed] [Google Scholar]
  24. Matsushima K., Morishita K., Yoshimura T., Lavu S., Kobayashi Y., Lew W., Appella E., Kung H. F., Leonard E. J., Oppenheim J. J. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J Exp Med. 1988 Jun 1;167(6):1883–1893. doi: 10.1084/jem.167.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ogawa T. Chemical structure of lipid A from Porphyromonas (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett. 1993 Oct 11;332(1-2):197–201. doi: 10.1016/0014-5793(93)80512-s. [DOI] [PubMed] [Google Scholar]
  26. Ogawa T., Nakazawa M., Masui K. Immunopharmacological activities of the nontoxic monophosphoryl lipid A of Porphyromonas gingivalis. Vaccine. 1996 Jan;14(1):70–76. doi: 10.1016/0264-410x(95)00128-n. [DOI] [PubMed] [Google Scholar]
  27. Pantosti A., Tzianabos A. O., Onderdonk A. B., Kasper D. L. Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis. Infect Immun. 1991 Jun;59(6):2075–2082. doi: 10.1128/iai.59.6.2075-2082.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. ROE J. H. The determination of sugar in blood and spinal fluid with anthrone reagent. J Biol Chem. 1955 Jan;212(1):335–343. [PubMed] [Google Scholar]
  29. Rietschel E. T., Kirikae T., Schade F. U., Mamat U., Schmidt G., Loppnow H., Ulmer A. J., Zähringer U., Seydel U., Di Padova F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994 Feb;8(2):217–225. doi: 10.1096/fasebj.8.2.8119492. [DOI] [PubMed] [Google Scholar]
  30. STROMINGER J. L., PARK J. T., THOMPSON R. E. Composition of the cell wall of Staphylococcus aureus: its relation to the mechanism of action of penicillin. J Biol Chem. 1959 Dec;234:3263–3268. [PubMed] [Google Scholar]
  31. Sultzer B. M., Castagna R. Inhibition of activated nonresponder C3H/HeJ lymphocytes by lipopolysaccharide endotoxin. Infect Immun. 1988 Dec;56(12):3040–3045. doi: 10.1128/iai.56.12.3040-3045.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takada H., Hirai H., Fujiwara T., Koga T., Ogawa T., Hamada S. Bacteroides lipopolysaccharides (LPS) induce anaphylactoid and lethal reactions in LPS-responsive and -nonresponsive mice primed with muramyl dipeptide. J Infect Dis. 1990 Aug;162(2):428–434. doi: 10.1093/infdis/162.2.428. [DOI] [PubMed] [Google Scholar]
  33. Takada H., Kawabata Y., Tamura M., Matsushita K., Igarashi H., Ohkuni H., Todome Y., Uchiyama T., Kotani S. Cytokine induction by extracellular products of oral viridans group streptococci. Infect Immun. 1993 Dec;61(12):5252–5260. doi: 10.1128/iai.61.12.5252-5260.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takada H., Kotani S. Structural requirements of lipid A for endotoxicity and other biological activities. Crit Rev Microbiol. 1989;16(6):477–523. doi: 10.3109/10408418909104475. [DOI] [PubMed] [Google Scholar]
  35. Takada H., Kotani S., Tsujimoto M., Ogawa T., Takahashi I., Harada K., Katsukawa C., Tanaka S., Shiba T., Kusumoto S. Immunopharmacological activities of a synthetic counterpart of a biosynthetic lipid A precursor molecule and of its analogs. Infect Immun. 1985 Apr;48(1):219–227. doi: 10.1128/iai.48.1.219-227.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takada H., Mihara J., Morisaki I., Hamada S. Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides. Infect Immun. 1991 Jan;59(1):295–301. doi: 10.1128/iai.59.1.295-301.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tamura M., Tokuda M., Nagaoka S., Takada H. Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival fibroblast cultures. Infect Immun. 1992 Nov;60(11):4932–4937. doi: 10.1128/iai.60.11.4932-4937.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tokunaga K., Nakamura Y., Sakata K., Fujimori K., Ohkubo M., Sawada K., Sakiyama S. Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res. 1987 Nov 1;47(21):5616–5619. [PubMed] [Google Scholar]
  39. Tomai M. A., Johnson A. G., Ribi E. Glycolipid induced proliferation of lipopolysaccharide hyporesponsive C3H/HeJ splenocytes. J Leukoc Biol. 1988 Jan;43(1):11–17. doi: 10.1002/jlb.43.1.11. [DOI] [PubMed] [Google Scholar]
  40. Vogel S. N., Madonna G. S., Wahl L. M., Rick P. D. In vitro stimulation of C3H/HeJ spleen cells and macrophages by a lipid A precursor molecule derived from Salmonella typhimurium. J Immunol. 1984 Jan;132(1):347–353. [PubMed] [Google Scholar]
  41. WEISSBACH A., HURWITZ J. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J Biol Chem. 1959 Apr;234(4):705–709. [PubMed] [Google Scholar]
  42. Wannemuehler M. J., Michalek S. M., Jirillo E., Williamson S. I., Hirasawa M., McGhee J. R. LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice. J Immunol. 1984 Jul;133(1):299–305. [PubMed] [Google Scholar]
  43. Williamson S. I., Wannemuehler M. J., Jirillo E., Pritchard D. G., Michalek S. M., McGhee J. R. LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS. J Immunol. 1984 Nov;133(5):2294–2300. [PubMed] [Google Scholar]

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