Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Oct;63(10):3858–3863. doi: 10.1128/iai.63.10.3858-3863.1995

Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes.

D A Kostyal 1, G H Butler 1, D H Beezhold 1
PMCID: PMC173543  PMID: 7558292

Abstract

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.

Full Text

The Full Text of this article is available as a PDF (234.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abou-Zeid C., Filley E., Steele J., Rook G. A. A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles. J Immunol Methods. 1987 Apr 2;98(1):5–10. doi: 10.1016/0022-1759(87)90429-7. [DOI] [PubMed] [Google Scholar]
  2. Aida Y., Pabst M. J. Removal of endotoxin from protein solutions by phase separation using Triton X-114. J Immunol Methods. 1990 Sep 14;132(2):191–195. doi: 10.1016/0022-1759(90)90029-u. [DOI] [PubMed] [Google Scholar]
  3. Almeida R. A., Wannemuehler M. J., Rosenbusch R. F. Interaction of Mycoplasma dispar with bovine alveolar macrophages. Infect Immun. 1992 Jul;60(7):2914–2919. doi: 10.1128/iai.60.7.2914-2919.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arai S., Furukawa M., Munakata T., Kuwano K., Inoue H., Miyazaki T. Enhancement of cytotoxicity of active macrophages by mycoplasma: role of mycoplasma-associated induction of tumor necrosis factor-alpha (TNF-alpha) in macrophages. Microbiol Immunol. 1990;34(3):231–243. doi: 10.1111/j.1348-0421.1990.tb01006.x. [DOI] [PubMed] [Google Scholar]
  5. Beezhold D. H., Leftwich J. A., Hall R. E. P48 induces tumor necrosis factor and IL-1 secretion by human monocytes. J Immunol. 1989 Nov 15;143(10):3217–3221. [PubMed] [Google Scholar]
  6. Beutler B., Cerami A. The biology of cachectin/TNF--a primary mediator of the host response. Annu Rev Immunol. 1989;7:625–655. doi: 10.1146/annurev.iy.07.040189.003205. [DOI] [PubMed] [Google Scholar]
  7. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  8. Brenner T., Yamin A., Abramsky O., Gallily R. Stimulation of tumor necrosis factor-alpha production by mycoplasmas and inhibition by dexamethasone in cultured astrocytes. Brain Res. 1993 Apr 16;608(2):273–279. doi: 10.1016/0006-8993(93)91468-8. [DOI] [PubMed] [Google Scholar]
  9. Butler G. H., Kotani H., Kong L., Frick M., Evancho S., Stanbridge E. J., McGarrity G. J. Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes. Infect Immun. 1991 Mar;59(3):1037–1042. doi: 10.1128/iai.59.3.1037-1042.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dudler R., Schmidhauser C., Parish R. W., Wettenhall R. E., Schmidt T. A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells. EMBO J. 1988 Dec 1;7(12):3963–3970. doi: 10.1002/j.1460-2075.1988.tb03283.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feng S. H., Lo S. C. Induced mouse spleen B-cell proliferation and secretion of immunoglobulin by lipid-associated membrane proteins of Mycoplasma fermentans incognitus and Mycoplasma penetrans. Infect Immun. 1994 Sep;62(9):3916–3921. doi: 10.1128/iai.62.9.3916-3921.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallily R., Salman M., Tarshis M., Rottem S. Mycoplasma fermentans (incognitus strain) induces TNF alpha and IL-1 production by human monocytes and murine macrophages. Immunol Lett. 1992 Sep;34(1):27–30. doi: 10.1016/0165-2478(92)90023-h. [DOI] [PubMed] [Google Scholar]
  13. Gallily R., Sher T., Ben-Av P., Loewenstein J. Tumor necrosis factor as a mediator of Mycoplasma orale-induced tumor cell lysis by macrophages. Cell Immunol. 1989 Jun;121(1):146–153. doi: 10.1016/0008-8749(89)90012-9. [DOI] [PubMed] [Google Scholar]
  14. Herbelin A., Ruuth E., Delorme D., Michel-Herbelin C., Praz F. Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes. Infect Immun. 1994 Oct;62(10):4690–4694. doi: 10.1128/iai.62.10.4690-4694.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoffmann P., Wiesmüller K. H., Metzger J., Jung G., Bessler W. G. Induction of tumor cytotoxicity in murine bone marrow-derived macrophages by two synthetic lipopeptide analogues. Biol Chem Hoppe Seyler. 1989 Jun;370(6):575–582. doi: 10.1515/bchm3.1989.370.1.575. [DOI] [PubMed] [Google Scholar]
  16. Kostyal D. A., Beezhold D. H., Hall R. E. Differentiation-inducing cytokine P48 exists in a membrane-associated form. J Immunol. 1991 Aug 1;147(3):893–898. [PubMed] [Google Scholar]
  17. Kostyal D. A., Butler G. H., Beezhold D. H. A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes. Infect Immun. 1994 Sep;62(9):3793–3800. doi: 10.1128/iai.62.9.3793-3800.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lin Y., Collins J. L., Case P. G., Patek P. Q. Effect of mycoplasmas on natural cytotoxic activity and release of tumor necrosis factor alpha by spleen cells. Infect Immun. 1988 Dec;56(12):3072–3075. doi: 10.1128/iai.56.12.3072-3075.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mühlradt P. F., Frisch M. Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6. Infect Immun. 1994 Sep;62(9):3801–3807. doi: 10.1128/iai.62.9.3801-3807.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mühlradt P. F., Schade U. MDHM, a macrophage-stimulatory product of Mycoplasma fermentans, leads to in vitro interleukin-1 (IL-1), IL-6, tumor necrosis factor, and prostaglandin production and is pyrogenic in rabbits. Infect Immun. 1991 Nov;59(11):3969–3974. doi: 10.1128/iai.59.11.3969-3974.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nyström S., Johansson K. E., Wieslander A. Selective acylation of membrane proteins in Acholeplasma laidlawii. Eur J Biochem. 1986 Apr 1;156(1):85–94. doi: 10.1111/j.1432-1033.1986.tb09552.x. [DOI] [PubMed] [Google Scholar]
  23. Nyström S., Wieslander A. Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii. Biochim Biophys Acta. 1992 Jun 11;1107(1):39–43. doi: 10.1016/0005-2736(92)90326-h. [DOI] [PubMed] [Google Scholar]
  24. Proust J. J., Buchholz M. A., Nordin A. A. A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination. J Immunol. 1985 Jan;134(1):390–396. [PubMed] [Google Scholar]
  25. Ross S. E., Simecka J. W., Gambill G. P., Davis J. K., Cassell G. H. Mycoplasma pulmonis possesses a novel chemoattractant for B lymphocytes. Infect Immun. 1992 Feb;60(2):669–674. doi: 10.1128/iai.60.2.669-674.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ruuth E., Praz F. Interactions between mycoplasmas and the immune system. Immunol Rev. 1989 Dec;112:133–160. doi: 10.1111/j.1600-065x.1989.tb00556.x. [DOI] [PubMed] [Google Scholar]
  27. Sher T., Rottem S., Gallily R. Mycoplasma capricolum membranes induce tumor necrosis factor alpha by a mechanism different from that of lipopolysaccharide. Cancer Immunol Immunother. 1990;31(2):86–92. doi: 10.1007/BF01742371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sher T., Yamin A., Matzliach M., Rottem S., Gallily R. Partial biochemical characterization of spiroplasma membrane component inducing tumor necrosis factor alpha. Anticancer Drugs. 1990 Oct;1(1):83–87. doi: 10.1097/00001813-199010000-00014. [DOI] [PubMed] [Google Scholar]
  29. Sher T., Yamin A., Rottem S., Gallily R. In vitro induction of tumor necrosis factor alpha, tumor cytolysis, and blast transformation by Spiroplasma membranes. J Natl Cancer Inst. 1990 Jul 4;82(13):1142–1145. doi: 10.1093/jnci/82.13.1142. [DOI] [PubMed] [Google Scholar]
  30. Stuart P. M., Cassell G. H., Woodward J. G. Induction of class II MHC antigen expression in macrophages by Mycoplasma species. J Immunol. 1989 May 15;142(10):3392–3399. [PubMed] [Google Scholar]
  31. Stuart P. M., Egan R. M., Woodward J. G. Characterization of MHC induction by Mycoplasma fermentans (incognitus strain). Cell Immunol. 1993 Nov;152(1):261–270. doi: 10.1006/cimm.1993.1286. [DOI] [PubMed] [Google Scholar]
  32. Takema M., Oka S., Uno K., Nakamura S., Arita H., Tawara K., Inaba K., Muramatsu S. Macrophage-activating factor extracted from mycoplasmas. Cancer Immunol Immunother. 1991;33(1):39–44. doi: 10.1007/BF01742526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Teh H. S., Ho M., Williams L. D. Suppression of cytotoxic responses by a supernatant factor derived from Mycoplasma hyorhinis-infected mammalian cell lines. Infect Immun. 1988 Jan;56(1):197–203. doi: 10.1128/iai.56.1.197-203.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wallis R. S., Amir-Tahmasseb M., Ellner J. J. Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte western blot. Proc Natl Acad Sci U S A. 1990 May;87(9):3348–3352. doi: 10.1073/pnas.87.9.3348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang G., Coffman F. D., Wheelock E. F. Characterization and purification of a macrophage-triggering factor produced in Mycoplasma arginini-infected L5178Y cell cultures. J Immunol. 1994 Sep 15;153(6):2579–2591. [PubMed] [Google Scholar]
  37. Zielinski G. C., Ross R. F. Morphologic features and hydrophobicity of the cell surface of Mycoplasma hyopneumoniae. Am J Vet Res. 1992 Jul;53(7):1119–1124. [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES