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. 1995 Jan;63(1):134–141. doi: 10.1128/iai.63.1.134-141.1995

Monoclonal antibodies that inhibit mitogenic activity of Mycoplasma pulmonis.

Z Lapidot 1, R Siman-Tov 1, Y Naot 1
PMCID: PMC172969  PMID: 7806349

Abstract

Previous studies have suggested a correlation between mitogenic, polyclonal activation of host lymphocytes and the respiratory tract inflammatory diseases induced by Mycoplasma pulmonis. This study describes the generation of monoclonal antibodies (MAbs) to M. pulmonis membrane antigens with different capacities to inhibit stimulation of cultured rat lymphocytes by mycoplasmal membranes and with variable effects on M. pulmonis growth. We show that the inhibitory effects exerted on mitogenesis by purified MAbs are inversely related to the effects of MAbs on M. pulmonis growth. Immunoblotting of electrophoretically separated membrane proteins, with both growth- and mitogenesis-inhibiting antibodies, revealed significant changes in the reactions obtained with both types of MAb following short exposure of membranes to heat. Growth-inhibiting MAbs strongly react with heat-labile antigenic complexes with molecular weights of 65,000 to 75,000. Inhibition of mitogenesis is mainly associated with recognition of membrane complexes of 84 to 113 kDa that exhibit disperse smears and variable heat sensitivities. Following brief heating of membranes, more distinct bands of 103, 90, and 84 kDa are obtained with MAbs that inhibit mitogenesis. Experiments with other mitogenic mycoplasma species and MAb 3.3.10.2, a potent inhibitor of mitogenesis reveal that whereas the antigenic epitope recognized by this antibody is present on unheated membranes from different mycoplasmas, with heated membranes the MAb yields reactions only with M. pulmonis and M. arthritidis. Our studies suggest that M. pulmonis mitogens are unique membrane complexes of variable molecular weights, highly susceptible to heat and less sensitive to reducing agents.

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Selected References

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  1. Blazek R., Schmitt K., Krafft U., Hadding U. Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody. J Immunol Methods. 1990 Aug 7;131(2):203–212. doi: 10.1016/0022-1759(90)90191-w. [DOI] [PubMed] [Google Scholar]
  2. Cassell G. H., Davis J. K. Protective effect of vaccination against Mycoplasma pulmonis respiratory disease in rats. Infect Immun. 1978 Jul;21(1):69–75. doi: 10.1128/iai.21.1.69-75.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cole B. C., Aldridge K. E., Ward J. R. Mycoplasma-dependent activation of normal lymphocytes: mitogenic potential of mycoplasmas for mouse lymphocytes. Infect Immun. 1977 Nov;18(2):393–399. doi: 10.1128/iai.18.2.393-399.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cole B. C., Golightly-Rowland L., Ward J. R. Arthritis of mice induced by Mycoplasma pulmonis: humoral antibody and lymphocyte responses of CBA mice. Infect Immun. 1975 Nov;12(5):1083–1092. doi: 10.1128/iai.12.5.1083-1092.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davis J. K., Cassell G. H. Murine respiratory mycoplasmosis in LEW and F344 rats: strain differences in lesion severity. Vet Pathol. 1982 May;19(3):280–293. doi: 10.1177/030098588201900306. [DOI] [PubMed] [Google Scholar]
  6. Davis J. K., Parker R. F., White H., Dziedzic D., Taylor G., Davidson M. K., Cox N. R., Cassell G. H. Strain differences in susceptibility to murine respiratory mycoplasmosis in C57BL/6 and C3H/HeN mice. Infect Immun. 1985 Dec;50(3):647–654. doi: 10.1128/iai.50.3.647-654.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis J. K., Simecka J. W., Williamson J. S., Ross S. E., Juliana M. M., Thorp R. B., Cassell G. H. Nonspecific lymphocyte responses in F344 and LEW rats: susceptibility to murine respiratory mycoplasmosis and examination of cellular basis for strain differences. Infect Immun. 1985 Jul;49(1):152–158. doi: 10.1128/iai.49.1.152-158.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis J. K., Thorp R. B., Maddox P. A., Brown M. B., Cassell G. H. Murine respiratory mycoplasmosis in F344 and LEW rats: evolution of lesions and lung lymphoid cell populations. Infect Immun. 1982 May;36(2):720–729. doi: 10.1128/iai.36.2.720-729.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Denny F. W., Taylor-Robinson D., Allison A. C. The role of thymus-dependent immunity in Mycoplasma pulmonis infections of mice. J Med Microbiol. 1972 Aug;5(3):327–336. doi: 10.1099/00222615-5-3-327. [DOI] [PubMed] [Google Scholar]
  10. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  11. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871–874. doi: 10.1016/0019-2791(71)90454-x. [DOI] [PubMed] [Google Scholar]
  12. Galfrè G., Milstein C., Wright B. Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG. Nature. 1979 Jan 11;277(5692):131–133. doi: 10.1038/277131a0. [DOI] [PubMed] [Google Scholar]
  13. Gershon H., Naot Y. Induction of interleukin-2 and colony-stimulating factors in lymphoid cell cultures activated by mitogenic mycoplasmas. Isr J Med Sci. 1984 Sep;20(9):882–885. [PubMed] [Google Scholar]
  14. Ginsburg H., Naot Y., Hollander N. Lysis and necrosis: analysis of two cytotoxic phenomena mediated by lymphocytes. Isr J Med Sci. 1976 Apr-May;12(4-5):435–453. [PubMed] [Google Scholar]
  15. Ginsburg H., Nicolet J. Extensive transformation of lymphocytes by a mycoplasma organism. Nat New Biol. 1973 Dec 5;246(153):143–146. doi: 10.1038/newbio246143a0. [DOI] [PubMed] [Google Scholar]
  16. Hayflick L. Tissue cultures and mycoplasmas. Tex Rep Biol Med. 1965 Jun;23(Suppl):285+–285+. [PubMed] [Google Scholar]
  17. Horowitz S. A., Garrett B., Davis J. K., Cassell G. H. Isolation of Mycoplasma pulmonis membranes and identification of surface antigens. Infect Immun. 1987 May;55(5):1314–1320. doi: 10.1128/iai.55.5.1314-1320.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kamla V., Henrich B., Hadding U. Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies. J Immunol Methods. 1992 Feb 14;147(1):73–81. doi: 10.1016/s0022-1759(12)80031-7. [DOI] [PubMed] [Google Scholar]
  19. Katz R., Siman-Tov R., Naot Y. Comparison of mitogens from Mycoplasma pulmonis and Mycoplasma neurolyticum. Yale J Biol Med. 1983 Sep-Dec;56(5-6):613–621. [PMC free article] [PubMed] [Google Scholar]
  20. Keystone E. C., Cunningham A. J., Metcalfe A., Kennedy M., Quinn P. A. Role of antibody in the protection of mice from arthritis induced by Mycoplasma pulmonis. Clin Exp Immunol. 1982 Feb;47(2):253–259. [PMC free article] [PubMed] [Google Scholar]
  21. Lachmann P. J., Oldroyd R. G., Milstein C., Wright B. W. Three rat monoclonal antibodies to human C3. Immunology. 1980 Nov;41(3):503–515. [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lai W. C., Bennett M., Pakes S. P., Murphree S. S. Potential subunit vaccine against Mycoplasma pulmonis purified by a protective monoclonal antibody. Vaccine. 1991 Mar;9(3):177–184. doi: 10.1016/0264-410x(91)90150-5. [DOI] [PubMed] [Google Scholar]
  24. Naot Y., Davidson S., Lindenbaum E. S. Mitogenicity and pathogenicity of Mycoplasma pulmonis in rats. I. Atypical interstitial pneumonia induced by mitogenic myeoplasmal membranes. J Infect Dis. 1981 Jan;143(1):55–62. doi: 10.1093/infdis/143.1.55. [DOI] [PubMed] [Google Scholar]
  25. Naot Y., Merchav S., Ben-David E., Ginsburg H. Mitogenic activity of Mycoplasma pulmonis. I. Stimulation of rat B and T lymphocytes. Immunology. 1979 Mar;36(3):399–406. [PMC free article] [PubMed] [Google Scholar]
  26. Naot Y., Remington J. S. Use of enzyme-linked immunosorbent assays (ELISA) for detection of monoclonal antibodies: experience with antigens of Toxoplasma gondii. J Immunol Methods. 1981;43(3):333–341. doi: 10.1016/0022-1759(81)90181-2. [DOI] [PubMed] [Google Scholar]
  27. Naot Y., Siman-Tov R., Ginsburg H. Mitogenic activity of Mycoplasma pulmonis. II. Studies on the biochemical nature of the mitogenic factor. Eur J Immunol. 1979 Feb;9(2):149–154. doi: 10.1002/eji.1830090211. [DOI] [PubMed] [Google Scholar]
  28. Naot Y., Tully J. G., Ginsburg H. Lymphocyte activation by various Mycoplasma strains and species. Infect Immun. 1977 Nov;18(2):310–317. doi: 10.1128/iai.18.2.310-317.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nichols E. J., Kenny G. E. Immunochemical characterization of a heat-stable surface antigen of Mycoplasma pulmonis expressing both species-specific and strain-specific determinants. Infect Immun. 1984 May;44(2):355–363. doi: 10.1128/iai.44.2.355-363.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Sasaki T. Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen. J Bacteriol. 1991 Apr;173(7):2398–2400. doi: 10.1128/jb.173.7.2398-2400.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shai S., Bar-Yoseph N., Peer E., Naot Y. A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions. Fertil Steril. 1990 Nov;54(5):894–901. doi: 10.1016/s0015-0282(16)53952-1. [DOI] [PubMed] [Google Scholar]
  34. Simecka J. W., Davis J. K., Cassell G. H. Serum antibody does not account for differences in the severity of chronic respiratory disease caused by Mycoplasma pulmonis in LEW and F344 rats. Infect Immun. 1989 Nov;57(11):3570–3575. doi: 10.1128/iai.57.11.3570-3575.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Singer S. H., Ford M., Kirschstein R. L. Respiratory diseases in cyclophosphamide-treated mice. I. Increased virulence of Mycoplasma pulmonis. Infect Immun. 1972 Jun;5(6):953–956. doi: 10.1128/iai.5.6.953-956.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Tully J. G., Whitcomb R. F., Clark H. F., Williamson D. L. Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science. 1977 Mar 4;195(4281):892–894. doi: 10.1126/science.841314. [DOI] [PubMed] [Google Scholar]
  38. Watson H. L., Davidson M. K., Cox N. R., Davis J. K., Dybvig K., Cassell G. H. Protein variability among strains of Mycoplasma pulmonis. Infect Immun. 1987 Nov;55(11):2838–2840. doi: 10.1128/iai.55.11.2838-2840.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Watson H. L., Dybvig K., Blalock D. K., Cassell G. H. Subunit structure of the variable V-1 antigen of Mycoplasma pulmonis. Infect Immun. 1989 Jun;57(6):1684–1690. doi: 10.1128/iai.57.6.1684-1690.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Watson H. L., McDaniel L. S., Blalock D. K., Fallon M. T., Cassell G. H. Heterogeneity among strains and a high rate of variation within strains of a major surface antigen of Mycoplasma pulmonis. Infect Immun. 1988 May;56(5):1358–1363. doi: 10.1128/iai.56.5.1358-1363.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Williamson J. S., Davis J. K., Cassell G. H. Polyclonal activation of rat splenic lymphocytes after in vivo administration of Mycoplasma pulmonis and its relation to in vitro response. Infect Immun. 1986 May;52(2):594–599. doi: 10.1128/iai.52.2.594-599.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. de StGroth S. F., Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J Immunol Methods. 1980;35(1-2):1–21. doi: 10.1016/0022-1759(80)90146-5. [DOI] [PubMed] [Google Scholar]

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