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. 1991 May;138(5):1069–1075.

Immunohistochemical localization of granzyme B antigen in cytotoxic cells in human tissues.

A Hameed 1, L D Truong 1, V Price 1, O Kruhenbuhl 1, J Tschopp 1
PMCID: PMC1886029  PMID: 2024702

Abstract

Human granzyme B antigen is expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme B was generated using a prokaryotic expression vector under the control of T7 transcription and translation signals. The 25-kd recombinant protein (granzyme B) was used to develop a rabbit polyclonal antiserum. Purified anti-granzyme B antibodies were used to detect the antigen expression in cytotoxic cells in human tissues. Using the avidin-biotin-complex peroxidase technique, formalin-fixed, paraffin-embedded tissue sections from patients with acute mild or moderate allograft cardiac rejection were stained. A constant cytoplasmic staining of the lymphocytic allograft infiltrate was observed. These results provide a basis for using the anti-granzyme B antibodies for detection of cytotoxic cells in human tissues. The detection and quantitative analysis of the granzyme-associated cytotoxic cells may help to evaluate the significance of these functionally distinct cytotoxic cells in human tissues associated with increased expression of cytoplasmic granule effector molecules.

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Selected References

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

  1. Acha-Orbea H., Scarpellino L., Hertig S., Dupuis M., Tschopp J. Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides. EMBO J. 1990 Dec;9(12):3815–3819. doi: 10.1002/j.1460-2075.1990.tb07599.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson P., Nagler-Anderson C., O'Brien C., Levine H., Watkins S., Slayter H. S., Blue M. L., Schlossman S. F. A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes. J Immunol. 1990 Jan 15;144(2):574–582. [PubMed] [Google Scholar]
  3. Chadburn A., Inghirami G., Knowles D. M. Hairy cell leukemia-associated antigen LeuM5 (CD11c) is preferentially expressed by benign activated and neoplastic CD8 T cells. Am J Pathol. 1990 Jan;136(1):29–37. [PMC free article] [PubMed] [Google Scholar]
  4. Clément M. V., Haddad P., Ring G. H., Pruna A., Sasportes M. Granzyme B-gene expression: a marker of human lymphocytes "activated" in vitro or in renal allografts. Hum Immunol. 1990 Jun;28(2):159–166. doi: 10.1016/0198-8859(90)90013-f. [DOI] [PubMed] [Google Scholar]
  5. Ferguson W. S., Verret C. R., Reilly E. B., Iannini M. J., Eisen H. N. Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes. J Exp Med. 1988 Feb 1;167(2):528–540. doi: 10.1084/jem.167.2.528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fruth U., Sinigaglia F., Schlesier M., Kilgus J., Kramer M. D., Simon M. M. A novel serine proteinase (HuTSP) isolated from a cloned human CD8+ cytolytic T cell line is expressed and secreted by activated CD4+ and CD8+ lymphocytes. Eur J Immunol. 1987 Nov;17(11):1625–1633. doi: 10.1002/eji.1830171116. [DOI] [PubMed] [Google Scholar]
  7. Garcia-Sanz J. A., MacDonald H. R., Jenne D. E., Tschopp J., Nabholz M. Cell specificity of granzyme gene expression. J Immunol. 1990 Nov 1;145(9):3111–3118. [PubMed] [Google Scholar]
  8. Gerdes J., Lemke H., Baisch H., Wacker H. H., Schwab U., Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984 Oct;133(4):1710–1715. [PubMed] [Google Scholar]
  9. Gershenfeld H. K., Hershberger R. J., Shows T. B., Weissman I. L. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1184–1188. doi: 10.1073/pnas.85.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hameed A., Lowrey D. M., Lichtenheld M., Podack E. R. Characterization of three serine esterases isolated from human IL-2 activated killer cells. J Immunol. 1988 Nov 1;141(9):3142–3147. [PubMed] [Google Scholar]
  11. Hayes M. P., Berrebi G. A., Henkart P. A. Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A. J Exp Med. 1989 Sep 1;170(3):933–946. doi: 10.1084/jem.170.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henkart P. A. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985;3:31–58. doi: 10.1146/annurev.iy.03.040185.000335. [DOI] [PubMed] [Google Scholar]
  13. Jenne D. E., Tschopp J. Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation. Immunol Rev. 1988 Mar;103:53–71. doi: 10.1111/j.1600-065x.1988.tb00749.x. [DOI] [PubMed] [Google Scholar]
  14. Krähenbühl O., Rey C., Jenne D., Lanzavecchia A., Groscurth P., Carrel S., Tschopp J. Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes. J Immunol. 1988 Nov 15;141(10):3471–3477. [PubMed] [Google Scholar]
  15. 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]
  16. Lichtenheld M. G., Olsen K. J., Lu P., Lowrey D. M., Hameed A., Hengartner H., Podack E. R. Structure and function of human perforin. Nature. 1988 Sep 29;335(6189):448–451. doi: 10.1038/335448a0. [DOI] [PubMed] [Google Scholar]
  17. Mueller C., Gershenfeld H. K., Lobe C. G., Okada C. Y., Bleackley R. C., Weissman I. L. A high proportion of T lymphocytes that infiltrate H-2-incompatible heart allografts in vivo express genes encoding cytotoxic cell-specific serine proteases, but do not express the MEL-14-defined lymph node homing receptor. J Exp Med. 1988 Mar 1;167(3):1124–1136. doi: 10.1084/jem.167.3.1124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ordóez N. G., Manning J. T., Jr, Brooks T. E. Effect of trypsinization on the immunostaining of formalin-fixed, paraffin-embedded tissues. Am J Surg Pathol. 1988 Feb;12(2):121–129. doi: 10.1097/00000478-198802000-00005. [DOI] [PubMed] [Google Scholar]
  19. Pinkus G. S., Thomas P., Said J. W. Leu-M1--a marker for Reed-Sternberg cells in Hodgkin's disease. An immunoperoxidase study of paraffin-embedded tissues. Am J Pathol. 1985 May;119(2):244–252. [PMC free article] [PubMed] [Google Scholar]
  20. Redelman D., Hudig D. The mechanism of cell-mediated cytotoxicity. I. Killing by murine cytotoxic T lymphocytes requires cell surface thiols and activated proteases. J Immunol. 1980 Feb;124(2):870–878. [PubMed] [Google Scholar]
  21. Redelman D., Hudig D. The mechanism of cell-mediated cytotoxicity. III. Protease-specific inhibitors preferentially block later events in cytotoxic T lymphocyte-mediated lysis than do inhibitors of methylation or thiol-reactive agents. Cell Immunol. 1983 Oct 1;81(1):9–21. doi: 10.1016/0008-8749(83)90206-x. [DOI] [PubMed] [Google Scholar]
  22. Schmid J., Weissmann C. Induction of mRNA for a serine protease and a beta-thromboglobulin-like protein in mitogen-stimulated human leukocytes. J Immunol. 1987 Jul 1;139(1):250–256. [PubMed] [Google Scholar]
  23. Schmidt C., Burman K., Moss D. J., Ryan J., Burrows S. R., Misko I. S. Interleukin-2 receptors in infectious mononucleosis. Immunol Lett. 1989 Dec;23(2):139–142. doi: 10.1016/0165-2478(89)90126-0. [DOI] [PubMed] [Google Scholar]
  24. Schmidt R. E., MacDermott R. P., Bartley G., Bertovich M., Amato D. A., Austen K. F., Schlossman S. F., Stevens R. L., Ritz J. Specific release of proteoglycans from human natural killer cells during target lysis. Nature. 1985 Nov 21;318(6043):289–291. doi: 10.1038/318289a0. [DOI] [PubMed] [Google Scholar]
  25. Schwab U., Stein H., Gerdes J., Lemke H., Kirchner H., Schaadt M., Diehl V. Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells. Nature. 1982 Sep 2;299(5878):65–67. doi: 10.1038/299065a0. [DOI] [PubMed] [Google Scholar]
  26. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  27. Tschopp J., Jongeneel C. V. Cytotoxic T lymphocyte mediated cytolysis. Biochemistry. 1988 Apr 19;27(8):2641–2646. doi: 10.1021/bi00408a001. [DOI] [PubMed] [Google Scholar]
  28. Turbitt M. L., Mackie R. M. An assessment of the diagnostic value of the monoclonal antibodies Leu 8, OKT9, OKT10 and Ki67 in cutaneous lymphocytic infiltrates. Br J Dermatol. 1986 Aug;115(2):151–158. doi: 10.1111/j.1365-2133.1986.tb05711.x. [DOI] [PubMed] [Google Scholar]
  29. Utsunomiya N., Nakanishi M. A serine protease triggers the initial step of transmembrane signalling in cytotoxic T cells. J Biol Chem. 1986 Dec 15;261(35):16514–16517. [PubMed] [Google Scholar]
  30. Waldmann T. A. The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes. Science. 1986 May 9;232(4751):727–732. doi: 10.1126/science.3008337. [DOI] [PubMed] [Google Scholar]
  31. Wood G. S., Mueller C., Warnke R. A., Weissman I. L. In situ localization of HuHF serine protease mRNA and cytotoxic cell-associated antigens in human dermatoses. A novel method for the detection of cytotoxic cells in human tissues. Am J Pathol. 1988 Nov;133(2):218–225. [PMC free article] [PubMed] [Google Scholar]
  32. Young J. D., Liu C. C. Multiple mechanisms of lymphocyte-mediated killing. Immunol Today. 1988 May;9(5):140–144. doi: 10.1016/0167-5699(88)91201-7. [DOI] [PubMed] [Google Scholar]
  33. Young L. H., Joag S. V., Zheng L. M., Lee C. P., Lee Y. S., Young J. D. Perforin-mediated myocardial damage in acute myocarditis. Lancet. 1990 Oct 27;336(8722):1019–1021. doi: 10.1016/0140-6736(90)92486-2. [DOI] [PubMed] [Google Scholar]

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