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Expression of perforin and granzymes in vivo: potential diagnostic markers for activated cytotoxic cells
Expressionof perforinandgranzymes iI viva:potentialdiagnosticmarkersfor activatedcytotoxiccells Gillian M. Griffiths and Christoph Mueller Perforin and granzymes are considered to be instrumental in cell-mediated cytolysis by cytotoxic Tcells and natural killer cells. Here, Gillian Griffith and Christoph Mueller describe the expression of perforin and granzymes, emphasizing studies in vivo, and discuss the possibility that these proteins are useful diagnostic markers for immune responses involving cytolytic cells. Specialized granules within the cytoplasm of cytolytic lymphocytes contain a unique set of proteins that are responsible for the cytolytic activity of these cells. When isolated from the cell these granules can lyse target cells in much the same way as the intact killer cell’J. Although other mechanisms of killing may exist, it is clear that the exocytosis of these granules provides one important mechanism. In the last few years, most granuleassociated proteins have been isolated and characterized. The major components are the pore-forming protein, perforin (also known as cytolysin), and a family of highly homologous serine proteases, termed granzymes (Table 1). Serine proteases and perforins have been isolated as proteins and cDNA clones by several different groups, resulting in a highly confusing nomenclature. In this review the nomenclatures ‘granzyme’, as proposed by Masson and Tschopp3, and ‘perforin’, as proposed by Podack and Dennert4, have been adopted. The cell specificity of granzyme and perforin expression has been analysed and it has been shown that lymphokines and immunosuppressive drugs strongly influence their expression. The purpose of this review is, first, to describe the expression of perforin and granzymes in viva and, second, to examine the possibility that these proteins provide markers that can be used to identify functional cytotoxic lymphocytes-cells that may play a central role in transplant rejection and in various viral and autoimmune diseases. The structures and functions of perforin and the granzymes have been extensively reviewed elsewhere5m7. A direct role for perforin in cell-mediated cytotoxicity has been suggested by experiments using anti-sense ohgonucleotides to downregulate perforin expression and simultaneously reduce the cytotoxicity of stimulated celW. Seven serine proteases have been isolated (granzymes A-G) %iOfrom cytolytic granules of murine cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and four serine protcases have been identified in human cytoiytic lymphocytes (Refs 6,11-16 and Table 1). Proteases are implicated in cell-mediated lysis: lysis by cloned T cells and isolated granules is inhibited by pretreatment with serine protease inhibitors*7-20. Although purified granzyme A alone has no cytolytic effects on a 0 1991, El\rwrrScwm
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Table 1. Perforins and serine proteases characterized from mouse and human cytolytic lymphocytes
Mouse Perforin
Granzyme A
Granzyme B Granzyme C Granzyme D Granzyme E Granzyme F Granzyme G Human Perforin
Granzyme A
Granzyme B
Synonyms
Molecular mass (kDa)
Cytolysin, PFP,C9related protein HF, CTLA-3, mTSP-1, SE-1 CCP-1, CTLA-1, Cll, SE-2 BlO, CTLA-5
70-75 (reduced) 60-64 (nonreduced) 35 (reduced) + 60 (nonreduced)
Cytolysin, PFP,C9related protein HuHF, CSP-A, Granzyme-1, HuTSP CSP-B, CCP-2, HLP, HSE-26.1, SECT, Granzyme-2
Granzyme 3 Granzyme H
CSP-c
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Fig. 1. In situ hybridization of granzyme A and perforin expression in vivo is shown. Granzyme-A- and perforin-expressing lymphocytes are present in the infiltrate of cardiac biopsies from a patient undergoing transplant rejection ((a) and (b)) and from a murine heart five days after heterotopic transplantation in an allogeneic recipient (H2 J into H2b)s9 ((c) and (d)). Anti-sense probes for granzyme and perforin mouse and human mRNA illustrate the similar levels of granzyme A (a) and perforin (b) expression in the human, but higher levels of granzyme A (c) than perforin (d) expression in the mouse.
Variety of normal and tumor target cells 21, in combination with purified perforin it can affect the release of target cell DNA, which occurs during cell-mediated lysis 22. The exact role of granzymes is still a matter of conjecture.
Perforin and granzyme expression characterizes cytolytic lymphocytes There is a good correlation between the expression of granzyme A and perforin, and functional cytolytic ability: these proteins are expressed after T cells have been stimulated by recognition of their targets. The genes encoding both proteins are expressed 1-2 days after primary stimulation and the proteins appear on day 3-4, with the highest levels apparent on day 6-7 (Refs 23-26). In short-term functional assays in vitro, for example mixed lymphocyte cultures, the appearance of perforin and granzyme A correlates well with cytolytic ability 22,24,27,28.However, in many studies in which lymphocytes cultured for some time have been used, perforin/ granzyme expression does not always correlate with cytotoxicity~2,29-3J. One major problem in interpreting the data from experiments performed in vitro is that both granzyme and perforin mRNAs are rapidly induced by interleukin 2 (IL-2) 25,32-3s. In addition, granzyme expression has been shown to be increased by tL-4 (Refs 36,37). Since most T-cell lines require tL-2 for mainten-
Immunology 7bday
ance in culture, the significance of granzyme and perforin expression in these cells is difficult to interpret. Clearly, it is important to examine the expression of these proteins in vivo. In situ hybridization to mRNA can be used to detect single cells expressing granzymes and perforin in vivo (Fig. 1). These in vivo experiments support the idea that
expression of the proteins is linked to cytotoxicity even more strongly than do the in vitro data: in animal models of allograft rejection and viral infection, expression preceded maximal cytotoxicity by two days 38-41. Furthermore, other cytolytic cell types express these proteins in vivo. First, y8 T cells isolated directly from human peripheral blood were shown to express perforin using a monoclonal antibody raised against mouse perforin 42,43 and the cytolytic potential of these cells was confirmed by lysis of an NK-sensitive human target cell, K562. Second, granzyme A mRNA can be detected in highly cytolytic peritoneal exudate lymphocytes 44, despite earlier reports to the contrary 45. Third, freshly-isolated peripheral blood lymphocytes from patients with granular lymphocyte proliferative disorders also show a correlation between perforin gene expression and cytotoxicity 46. This correlation was less clear after just one day in culture; cytotoxicity increased whereas the perforin mRNA, although still present, decreased. This suggests either a selective expansion of one particular subset of cells or an
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increase in another mechanism of cytolysis. In either event, these findings illustrate the differences between studies performed in vitro and in vivo, as does the cellular specificity of the granzymes. In vivo, only granzymes A and B are expressed in cytolytic populations; in contrast, mRNAs for all of the mouse granzymes can be detected in in-vitro-cultured T-cell lines 2~,44. A variety of normal tissue has also been examined for granzyme expression, and the expression is consistent with the presence of cytolytic lymphocytes. For instance, granzyme A mRNA can be detected in mouse intestine, lung and spleen 26 as well as in the metrial gland 47 (which may be involved in an immune response during pregnancy47), but expression is not seen in tissues devoid of lymphoid activity such as heart, kidney or brain 26. Low levels of mRNA have been detected in a population of purified, unstimulated B cells, although no transcript could be detected after stimulation with lipopolysaccharide (LPS), leaving open the possibility that the transcripts detected initially came from T-celt contamination in the starting population 26. In situ hybridization has been used to demonstrate granzyme A expression in a small population of CD4 CD8- thymocytes 4s but whether these cells are at some early stage of thymus maturation or are mature cells with cytolytic function remains an open question. The data that have emerged so far strongly support the notion that perforin and granzyme A expression can be used as markers for activated cytolytic lymphocytes in vivo. The method of detection is important, since small populations of lymphocytes will only be detected by relatively sensitive methods. With in situ hybridization and antibodies against these proteins, it is possible to detect single cells in immune responses in vivo. Granzyme A and perforin in infectious disease One important function of cytolytic lymphocytes is the recognition and destruction of virally-infected cells. Consequently, infection of mice with lymphotropic choriomeningitis virus (LCMV) provides an excellent model for studying cytolytic immune responses in vivo. Granzyme-A- and perforin-expressing lymphocytes can be detected in LCMV-infected mice by in situ hybridization. When a hepatotropic LCMV strain is used, these cells are most frequent in the liver lymphocytic infiltrate on day 6 after infection. This timing coincides with the onset of a detectable anti-LCMV cytolytic response, preceding the maximal LCMV-specific response by two days. After intracerebral inoculation with a different LCMV strain (LCMV-Armstrong), granzyme-A- and perforin-expressing lymphocytes first appear in the central nervous system infiltrate after 6-7 days. lmmunohistochemistry and hybridization of serial sections suggest that the granzyme- and perforin-expressing lymphocytes are probably CD8 + T cells in close apposition to the virus-infected cells of the meninges and choroid plexus :3s. Thus, perforin and granzyme A are involved in the cytolytic response to LCMV-infected cells in vivo. Similar results have been obtained using a monoclonal antibody against granzyme A39 and a potyclonal antiserum against perforin 4°. Myocardial biopsies from patients with cytomegalovirus infection have also been found to contain lymphocytes expressing granzyme A Immunology Today
and perforin 49, and similar results have been obtained using an antiserum raised against an epitope of human perforin on biopsies from patients with myocarditis s°. Finally, in tuberculous leprosy patients, activated, granzyme-A-expressing cells are present in lesions. These presumptive cytolytic lymphocytes might account for the nerve cell damage frequently observed in patients with this form of the disease 51.
Granzyme A and perforin in autoimmune disease In vivo studies have demonstrated the presence of granzyme- and perforin-expressing lymphocytes in autoimmune diseases. The nonobese diabetic (NOD) mouse provides an animal model for studying the molecular and cellular mechanisms of pathogenesis in insulindependent diabetes mellitus (IDDM). At the age of 4-6 weeks, NOD mice show the first signs of mononuclear infiltration of the islets of Langerhans of the pancreas and, by 12-16 weeks after birth, the pancreatic [3 cells are destroyed, resulting in the onset of clinical diabetes. Perforin-containing CD8 + T cells can be detected in the islet infiltrate of NOD animals with spontaneous IDDM or following adoptive cell transfer into irradiated recipients 52. The presence of activated CTLs in the islet infiltrate has also been demonstrated by in situ hybridization with radiolabeled probes for the granzyme A gene 53. However, most of these granzyme-A-expressing cells were found at a distance from the ~3cell area in the islet, whereas a significant fraction of the cells in direct contact with [3 cells expressed the tumor necrosis factor (x (TNF-~) gene and were, mostly, CD4 + T cells. Adoptive transfer studies have demonstrated the requirement for both CD4 + and CD8 + T cells for the development of diabetes in NOD mice, and it is possible that cytotoxic, TNF-~-expressing CD4 + T cells and perforin-containing CD8 + T cells act synergistically to eliminate [3 cells during the autoimmune process. Perforin and granzyme A have also been detected in lymphocytes obtained from the synovial fluid of patients with rheumatoid arthritis. The lymphocytes examined were isolated directly from the synovial fluid, without being cultured, and the expression seems to be affected by immunosuppressive treatments. These results suggest not only that cytolytic lymphocytes play a role in the pathogenesis of this disease but also that granzyme A and perforin expression are regulated by immunosuppressive drugs in vivo -s4. Granzyme A and perforin during transplantation One of the first studies to demonstrate clearly the importance of granzyme-expressing lymphocytes in vivo was carried out in a mouse allograft model <. Myocardial tissue from newborn mice of one strain was transplanted under the kidney capsule of adult mice of either the same, or a mismatched, strain. In the allogeneic transplant, the myocardial tissue was rejected between eight and 12 days after transplantation. The expression of two different granzymes (A and B) was examined at different times after rejection by means of in situ hybridization and the time course of expression was found to parallel closely the rejection process. Although most granzymeexpressing lymphocytes were CD8 +, only about 10% of the CD8 + cells expressed granzyme A, indicating that the
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majority of CD8 ÷ cells in the infiltrate are not activated killers. This may explain why CD4:CD8 ratios do not provide information about the state of rejection 55. Given the close correlation between granzyme expression and the rejection process, granzyme measurement may provide a more accurate marker of the rejection process than the surface phenotype of the infiltrating lymphocytes. Several different methods have been used to detect granzyme and, more recently, perforin expression in rejecting allografts. Granzyme A can be detected not only by in situ hybridization but also by its enzymatic reactivity with the substrate N-benzyloxy-carbonyl-L-lysinethiobenzylester (BLT) ~s6. Two studies have used this activity to monitor allograft rejection. In one study, on renal allografts 57, an increase in the number of granzymeA-expressing peripheral blood lymphocytes was observed in patients undergoing rejection episodes. This number decreased two days after treatment with methylprednisolone, an immunosuppressive drug used to prevent rejection. In the other study, a rat model of renal allograft rejection 58, the enzymatic activity of cell lysates from the spleen, peripheral blood and graft infiltrate increased in comparison with the controls. However, these increases were often relatively small and it is not likely that this will provide a reliable method for monitoring rejection. The presence of granzyme-A- and perforin-expressing lymphocytes in biopsies from cardiac transplants appears to be related to the state of rejection both in a mouse model and in samples from cardiac transplant patients. In the mouse, the increase in the number of granzymeA-expressing lymphocytes peaks immediately before rejection sg. Similarly, in cardiac transplant patients, large numbers of granzyme-A- and perforin-expressing lymphocytes are detected in biopsies from rejecting tissue, whereas no expression is seen in the infiltrate'of nonrejecting tissue. Interestingly, in biopsies from patients about to undergo a rejection episode, granzyme-A- and perforin-expressing lymphocytes were seen in the infiltrate 49. However, an earlier attempt to detect cells expressing these markers in peripheral blood had proved unsuccessful. This was partly due to a relatively large fluctuation in 'normal' individuals used as controls, where viral infection led to a large number of granzyme-/ perforin-expressing lymphocytes in the periphery (R. Namikawa, unpublished) plus the fact that the number of granzyme-/perforin-expressing lymphocytes in the periphery of patients undergoing rejection did not always increase significantly. A similar lack of correlation in peripheral blood was also observed in a mouse model (C. Mueller and J. Shelby, unpublished). The problem of viral infection was noted in studies of kidney transplants s7 as well as cardiac transplants 49. Since cytolytic lymphocytes respond to virally-infected cells, the presence of granzyme-/perforin-expressing lymphocytes in virally-infected individuals is not surprising. If granzyme and perforin expression are to be used as diagnostic tools to predict imminent rejection then it will also be necessary to screen for viral infection. The effect of immunosuppressive drugs on granzyme and perforin expression is very important if these are to be used as markers for transplantation rejection. In vitro, cyclosporine treatment downregulates expression of the
Immunology Today
mRNA for both proteins 2s,6° although the effects are probably indirect. The same correlation seems to exist in vivo as, in human studies, a reduction in granzyme A and perforin expression occurred after successful immunosuppressive treatmentS0, s7. In animal models, it is possible to test the effects of immunosuppression directly. In the rat, heterotopic cardiac allograft rejection can be averted by treatment with either cyclosporine or anti-OKT4 antibody (Ref. 61) and, despite the fact that there is still a lymphocytic infiltrate in these tolerated hearts, the granzyme-A-expressing population is no longer present (Chen et al., unpublished). Similarly, in a mouse heterotopic transplant model, both granzyme A and perforin expression have been shown to be selectively depleted in cyclosporine-A-treated animals (C. Mueller et al., unpublished). Conclusions Within the past two to three years, data from a wide variety of systems, both in vitro and in vivo, have strongly indicated a role for perforin and granzyme A in cellmediated cytolysis. Although the precise mechanism of granule-mediated cytolysis and the relative contributions of other cytolytic mechanisms are still being debated, it is clear that the expression of perforin and granzyme A can be used as markers for functional, activated cytotoxic lymphocytes in vivo. This has important implications for their use as potential diagnostic markers in transplantation, and for identifying cytolytic lymphocytes that may be involved in autoimmunity and viral infections.
The authors would like to thank Jim Kaufman and Alexandra Livingstone for many helpful comments on this manuscript, Irving Weissman for introducing them to this work at Stanford, and Craig Okada, Howard Gershenfeld and John Carlson for many interesting ideas during the Stanford killer cell years. Christoph Mueller is supported by The Swiss National Science Foundation. The Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche, Switzerland. Gillian Griffiths is at the Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, CH-4005 Basel and Christoph Mueller is at the Dept of Pathology, University of Bern, Murtenstrasse 31, CH-3010 Bern, Switzerland.
References 1 Podack, E.R. and Konigsberg, P.J. (1984)J. Exp. Med. 160, 695-710 2 Henkart, P.A., Millard, P.J., Reynolds, C.W. and Henkart, M.P. (1984) J. Exp. Med. 160, 75-93 3 Masson, D. and Tschopp, J. (1987) Cell 79,679-685 4 Podack, E.R. and Dennert, G. (1983) Nature 302, 442-444 5 Berke, G. (1989) in Fundamental immunology (Paul, W., ed.), pp. 735-764, Raven Press 6 Podack, E.R. (1989) Curr. Opin. Cell Biol. 1,929-933 7 Tschopp, J. and Nabholz, M. (1990) Annu. Rev. lmmunol. 8, 279-302 8 Acha-Orbea, H., Scarpellino, L., Hertig, S., Dupuis, M. and Tschopp, J. (1990) EMBOJ. 9, 3815-3819 9 Jenne, D.E. and Tschopp, J. (1988) lmmunol. Rev. 103, 53-71 10 Garcia, S.J., Ptaetinck, G., Velotti, F. et al. (1987) EMBO ]. 6, 933-938
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11 Krahenbuhl, O., Rey, C., Jenne, D. etal. (1988)
J. lmmunol. 141, 3471-3477
12 Ferguson, W.S., Verret, C.R., Reilly, E.B. et al. (1988) J. Exp. Med. 167, 528-540
13 Hameed, A., Lowrey, D.M., Lichtenheld, M. and Podack, E.R. (1988) J. Immunol. 141, 3142-3147 14 Klein, J.L., Selvakumar, A., Trapani, J.A. and Dupont, B. (1990) Tissue Antigens 35,220-228 15 Haddad, P., Clement, M.V., Bernard, O. et al. (1990) Gene 87, 265-271 16 Poe, M., Blake, J.T., Boulton, D.A. et al. (1991)J. Biol. Chem. 266, 98-103 17 Chang, T. and Eisen, H. (1980) J. Immunol. 124, 1028-1033 18 Redelman, D. and Hudig, D. (1980)J. Immunol. 124, 870-878 19 Acha-Orbea, H., Grosscurth, P., Lang, R., Stitz, L. and Hengarmer, H. (1983) J. Immunol. 130, 2952-2959 20 Simon, M., Fruth, U., Simon, H.G. and Kramcr, M.D. (1987) Ann. Inst. Pasteur lmmunol. 138,309-314 21 Simon, M.M., Fruth, U., Simon, H.G. and Kramer, M.D. (1986) EMBO J. 5, 3267-3274 22 Hayes, M.P., Berrebi, G.A. and Henkart, P.A. (1989) J. Exp. Med. 170, 933-946 23 Gershenfeld, H.K. and Weissman, I.L. (1986) Science 232, 854-858 24 Garcia, S.J., Velotti, F., MacDonald, H.R. et al. (1988) Immunology 64, 129-134 25 Liu, C.C., Rafii, S., Granelli, P.A., Trapani, J.A. and Young, J.D. (1989)J. Exp. Med. 170, 2105-2118 26 Garcia, S.J., MacDonald, H.R., Jenne, D.E., Tschopp, J. and Nabholz, M. (1990) J. Immunol. 145, 3111-3118 27 Masson, D. and Tschopp, J. (1985) J. Biol. Chem. 260, 9069-9072 28 Velotti, F., Palmieri, G., Morrone, S. et al. (1989) Eur. J. 1mmunol. 19, 575-578 29 Brunet, J-F., Denizot, F., Suzan, M. et al. (1987) J. 1mmunol. 138, 4102-4105 30 Somersalo, K. and Saksela, E. (1989) Scand. J. Immunol. 29, 459-467 31 Zychlinsky, A., Joag, S., Liu, C.C. and Young, J.D. (1990) Cell. Immunol. 126, 377-390 32 Lichtenheld, M.G., Olsen, K.J., Lu, P. et al. (1988) Nature 335,448-451 33 Manyak, C.L., Norton, G.P., Lobe, C.G. et al. (1989) .]. 1mmunol. 142, 3707-3713 34 Eisenthal, A. and Rosenberg, S.A. (1989) J. Immunol. 142, 2307-2313 35 Smyth,M.J., Ortaldo, J.R., Shinkai, Y. et al. (1990) J. Exp. Med. 171, 1269-1281 36 Trenn, G., Takayama, H., Hu, L.J., Paul, W.E. and Sitkovsky, M.V. (1988)]. immunol. :140, 1101-1106 37 Mule, J.J., Krosnick, J.A. and Rosenberg, S.A. (1989) J. lmmunol. 142, 726-733 38 Muller, C., Kagi, D., Aebischer, T. et al. (1989) Eur. J. Immunol. 19, 1253-1259 39 Kramer, M.D., Fruth, U., Simon, M.G. and Simon, M.M. (1989) Eur. J. Immunol. 19, 151-156 40 Young, L.H., Klavinskis, L.S., Oldstone, M.B. and Young, J.D. (1989).I. Exp. Med. 169, 2159-2171 41 Mueller, C., Gershenfetd, H.G., Lobe, C.G. etal. (1988) J. Exp. Med. 167, 1124-1136 42 Kawasaki, A., Shinkai, Y., Kuwana, Y. et al. (1990) lnl. Immunol. 2, 677-684 43 Nakata, M., Smyth, M.J., Norihisa, Y. et al. (1990) J. Exp. Med. 172, 1877-1880 44 Ebnet, K., Tapia, J.C., Hurtenbach, U, Kramer, M.D. and Simon, M.M. (1991) Int. Immunol. 3, 9-19 45 Dennert, G., Anderson, C.G. and Prochazka, G. (1987)
Proc. Natl Acad. Sci. USA 84, 5004-5008 46 Oshimi, K., Shinkai, Y., Okumura, K., Osbimi, Y. and Mizoguchi, H. (1990) Blood 75,704-708 47 Zheng, L.M., Ojcius, D.M., Liu, C.C. etal. (1991) FASEB J. 5, 79-85 48 Held, W., MacDonald, H.R. and Mueller, C. (]990) Int. Immunol. 2, 57-62 49 Griffiths, G.M., Namikawa, R., Mueller, C. et al. (1991) Eur. J. lmmunol. 21,687-692 50 Young, L.H., Joag, S.V., Zheng, L.M. etal. (1990) Lancet 336, 1019-1021 51 Cooper, C.L., Mueller, C., Sinchaisri,T. et al. (1989) J. Exp. Med. 169, 1565-1581 52 Young, L.H., Peterson, L.B., Wicker, L.S., Persechini, P.M. and Young, J.D. (1989)J. Immunol. 143, 3994-3999 53 Held, W., MacDonald, H.R., Weissman, I.L., Hess, M.W. and Mueller, C. (1990) Proc. Natl Acad. Sci. USA 87, 2239-2243 54 Griffiths, G.M., Alpert, S. and Weissman, I.L. Proc. Natl Acad. Sci. USA (in press) 55 McWhinnie, D.L., Carter, N.P., Taylor, H.M. et at. (1985) Transplant. Proc. 1985, 2548-2549 56 Green, G. and Shaw, E. (1975) Anal. Biochem. 93, 223-226 57 Sunder-Plassmann, G., Wagner, L., Hruby, K., Balcke, P. and Worman, C.P. (1990) Kidney Int. 37, 1350-1356 58 Nakajima, H., Arakawa, K., Yasumura, T. and Oka, T. (1989) Transplant. Proc. 21, 1187-1188 59 Mueller, C., Shelby,J., Weissman, I.L., P&inat-Frey, T. and Eichwald, E.J. (1991) Transplantation 51,514-517 60 Hooton, J.W., Miller, C.L., Helgason, C.D. et al. (1990) J. Immunol. 144, 816-823 61 Flavin, T., Shizuru, J., Seydel,K. et al. (1990)J. Heart Transplant. 9,482-488
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Table 1. Perforins and serine proteases characterized from mouse and human cytolytic lymphocytes
Mouse Perforin
Granzyme A
Granzyme B Granzyme C Granzyme D Granzyme E Granzyme F Granzyme G Human Perforin
Granzyme A
Granzyme B
Synonyms
Molecular mass (kDa)
Cytolysin, PFP,C9related protein HF, CTLA-3, mTSP-1, SE-1 CCP-1, CTLA-1, Cll, SE-2 BlO, CTLA-5
70-75 (reduced) 60-64 (nonreduced) 35 (reduced) + 60 (nonreduced)
Cytolysin, PFP,C9related protein HuHF, CSP-A, Granzyme-1, HuTSP CSP-B, CCP-2, HLP, HSE-26.1, SECT, Granzyme-2
Granzyme 3 Granzyme H
CSP-c
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Fig. 1. In situ hybridization of granzyme A and perforin expression in vivo is shown. Granzyme-A- and perforin-expressing lymphocytes are present in the infiltrate of cardiac biopsies from a patient undergoing transplant rejection ((a) and (b)) and from a murine heart five days after heterotopic transplantation in an allogeneic recipient (H2 J into H2b)s9 ((c) and (d)). Anti-sense probes for granzyme and perforin mouse and human mRNA illustrate the similar levels of granzyme A (a) and perforin (b) expression in the human, but higher levels of granzyme A (c) than perforin (d) expression in the mouse.
Variety of normal and tumor target cells 21, in combination with purified perforin it can affect the release of target cell DNA, which occurs during cell-mediated lysis 22. The exact role of granzymes is still a matter of conjecture.
Perforin and granzyme expression characterizes cytolytic lymphocytes There is a good correlation between the expression of granzyme A and perforin, and functional cytolytic ability: these proteins are expressed after T cells have been stimulated by recognition of their targets. The genes encoding both proteins are expressed 1-2 days after primary stimulation and the proteins appear on day 3-4, with the highest levels apparent on day 6-7 (Refs 23-26). In short-term functional assays in vitro, for example mixed lymphocyte cultures, the appearance of perforin and granzyme A correlates well with cytolytic ability 22,24,27,28.However, in many studies in which lymphocytes cultured for some time have been used, perforin/ granzyme expression does not always correlate with cytotoxicity~2,29-3J. One major problem in interpreting the data from experiments performed in vitro is that both granzyme and perforin mRNAs are rapidly induced by interleukin 2 (IL-2) 25,32-3s. In addition, granzyme expression has been shown to be increased by tL-4 (Refs 36,37). Since most T-cell lines require tL-2 for mainten-
Immunology 7bday
ance in culture, the significance of granzyme and perforin expression in these cells is difficult to interpret. Clearly, it is important to examine the expression of these proteins in vivo. In situ hybridization to mRNA can be used to detect single cells expressing granzymes and perforin in vivo (Fig. 1). These in vivo experiments support the idea that
expression of the proteins is linked to cytotoxicity even more strongly than do the in vitro data: in animal models of allograft rejection and viral infection, expression preceded maximal cytotoxicity by two days 38-41. Furthermore, other cytolytic cell types express these proteins in vivo. First, y8 T cells isolated directly from human peripheral blood were shown to express perforin using a monoclonal antibody raised against mouse perforin 42,43 and the cytolytic potential of these cells was confirmed by lysis of an NK-sensitive human target cell, K562. Second, granzyme A mRNA can be detected in highly cytolytic peritoneal exudate lymphocytes 44, despite earlier reports to the contrary 45. Third, freshly-isolated peripheral blood lymphocytes from patients with granular lymphocyte proliferative disorders also show a correlation between perforin gene expression and cytotoxicity 46. This correlation was less clear after just one day in culture; cytotoxicity increased whereas the perforin mRNA, although still present, decreased. This suggests either a selective expansion of one particular subset of cells or an
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increase in another mechanism of cytolysis. In either event, these findings illustrate the differences between studies performed in vitro and in vivo, as does the cellular specificity of the granzymes. In vivo, only granzymes A and B are expressed in cytolytic populations; in contrast, mRNAs for all of the mouse granzymes can be detected in in-vitro-cultured T-cell lines 2~,44. A variety of normal tissue has also been examined for granzyme expression, and the expression is consistent with the presence of cytolytic lymphocytes. For instance, granzyme A mRNA can be detected in mouse intestine, lung and spleen 26 as well as in the metrial gland 47 (which may be involved in an immune response during pregnancy47), but expression is not seen in tissues devoid of lymphoid activity such as heart, kidney or brain 26. Low levels of mRNA have been detected in a population of purified, unstimulated B cells, although no transcript could be detected after stimulation with lipopolysaccharide (LPS), leaving open the possibility that the transcripts detected initially came from T-celt contamination in the starting population 26. In situ hybridization has been used to demonstrate granzyme A expression in a small population of CD4 CD8- thymocytes 4s but whether these cells are at some early stage of thymus maturation or are mature cells with cytolytic function remains an open question. The data that have emerged so far strongly support the notion that perforin and granzyme A expression can be used as markers for activated cytolytic lymphocytes in vivo. The method of detection is important, since small populations of lymphocytes will only be detected by relatively sensitive methods. With in situ hybridization and antibodies against these proteins, it is possible to detect single cells in immune responses in vivo. Granzyme A and perforin in infectious disease One important function of cytolytic lymphocytes is the recognition and destruction of virally-infected cells. Consequently, infection of mice with lymphotropic choriomeningitis virus (LCMV) provides an excellent model for studying cytolytic immune responses in vivo. Granzyme-A- and perforin-expressing lymphocytes can be detected in LCMV-infected mice by in situ hybridization. When a hepatotropic LCMV strain is used, these cells are most frequent in the liver lymphocytic infiltrate on day 6 after infection. This timing coincides with the onset of a detectable anti-LCMV cytolytic response, preceding the maximal LCMV-specific response by two days. After intracerebral inoculation with a different LCMV strain (LCMV-Armstrong), granzyme-A- and perforin-expressing lymphocytes first appear in the central nervous system infiltrate after 6-7 days. lmmunohistochemistry and hybridization of serial sections suggest that the granzyme- and perforin-expressing lymphocytes are probably CD8 + T cells in close apposition to the virus-infected cells of the meninges and choroid plexus :3s. Thus, perforin and granzyme A are involved in the cytolytic response to LCMV-infected cells in vivo. Similar results have been obtained using a monoclonal antibody against granzyme A39 and a potyclonal antiserum against perforin 4°. Myocardial biopsies from patients with cytomegalovirus infection have also been found to contain lymphocytes expressing granzyme A Immunology Today
and perforin 49, and similar results have been obtained using an antiserum raised against an epitope of human perforin on biopsies from patients with myocarditis s°. Finally, in tuberculous leprosy patients, activated, granzyme-A-expressing cells are present in lesions. These presumptive cytolytic lymphocytes might account for the nerve cell damage frequently observed in patients with this form of the disease 51.
Granzyme A and perforin in autoimmune disease In vivo studies have demonstrated the presence of granzyme- and perforin-expressing lymphocytes in autoimmune diseases. The nonobese diabetic (NOD) mouse provides an animal model for studying the molecular and cellular mechanisms of pathogenesis in insulindependent diabetes mellitus (IDDM). At the age of 4-6 weeks, NOD mice show the first signs of mononuclear infiltration of the islets of Langerhans of the pancreas and, by 12-16 weeks after birth, the pancreatic [3 cells are destroyed, resulting in the onset of clinical diabetes. Perforin-containing CD8 + T cells can be detected in the islet infiltrate of NOD animals with spontaneous IDDM or following adoptive cell transfer into irradiated recipients 52. The presence of activated CTLs in the islet infiltrate has also been demonstrated by in situ hybridization with radiolabeled probes for the granzyme A gene 53. However, most of these granzyme-A-expressing cells were found at a distance from the ~3cell area in the islet, whereas a significant fraction of the cells in direct contact with [3 cells expressed the tumor necrosis factor (x (TNF-~) gene and were, mostly, CD4 + T cells. Adoptive transfer studies have demonstrated the requirement for both CD4 + and CD8 + T cells for the development of diabetes in NOD mice, and it is possible that cytotoxic, TNF-~-expressing CD4 + T cells and perforin-containing CD8 + T cells act synergistically to eliminate [3 cells during the autoimmune process. Perforin and granzyme A have also been detected in lymphocytes obtained from the synovial fluid of patients with rheumatoid arthritis. The lymphocytes examined were isolated directly from the synovial fluid, without being cultured, and the expression seems to be affected by immunosuppressive treatments. These results suggest not only that cytolytic lymphocytes play a role in the pathogenesis of this disease but also that granzyme A and perforin expression are regulated by immunosuppressive drugs in vivo -s4. Granzyme A and perforin during transplantation One of the first studies to demonstrate clearly the importance of granzyme-expressing lymphocytes in vivo was carried out in a mouse allograft model <. Myocardial tissue from newborn mice of one strain was transplanted under the kidney capsule of adult mice of either the same, or a mismatched, strain. In the allogeneic transplant, the myocardial tissue was rejected between eight and 12 days after transplantation. The expression of two different granzymes (A and B) was examined at different times after rejection by means of in situ hybridization and the time course of expression was found to parallel closely the rejection process. Although most granzymeexpressing lymphocytes were CD8 +, only about 10% of the CD8 + cells expressed granzyme A, indicating that the
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majority of CD8 ÷ cells in the infiltrate are not activated killers. This may explain why CD4:CD8 ratios do not provide information about the state of rejection 55. Given the close correlation between granzyme expression and the rejection process, granzyme measurement may provide a more accurate marker of the rejection process than the surface phenotype of the infiltrating lymphocytes. Several different methods have been used to detect granzyme and, more recently, perforin expression in rejecting allografts. Granzyme A can be detected not only by in situ hybridization but also by its enzymatic reactivity with the substrate N-benzyloxy-carbonyl-L-lysinethiobenzylester (BLT) ~s6. Two studies have used this activity to monitor allograft rejection. In one study, on renal allografts 57, an increase in the number of granzymeA-expressing peripheral blood lymphocytes was observed in patients undergoing rejection episodes. This number decreased two days after treatment with methylprednisolone, an immunosuppressive drug used to prevent rejection. In the other study, a rat model of renal allograft rejection 58, the enzymatic activity of cell lysates from the spleen, peripheral blood and graft infiltrate increased in comparison with the controls. However, these increases were often relatively small and it is not likely that this will provide a reliable method for monitoring rejection. The presence of granzyme-A- and perforin-expressing lymphocytes in biopsies from cardiac transplants appears to be related to the state of rejection both in a mouse model and in samples from cardiac transplant patients. In the mouse, the increase in the number of granzymeA-expressing lymphocytes peaks immediately before rejection sg. Similarly, in cardiac transplant patients, large numbers of granzyme-A- and perforin-expressing lymphocytes are detected in biopsies from rejecting tissue, whereas no expression is seen in the infiltrate'of nonrejecting tissue. Interestingly, in biopsies from patients about to undergo a rejection episode, granzyme-A- and perforin-expressing lymphocytes were seen in the infiltrate 49. However, an earlier attempt to detect cells expressing these markers in peripheral blood had proved unsuccessful. This was partly due to a relatively large fluctuation in 'normal' individuals used as controls, where viral infection led to a large number of granzyme-/ perforin-expressing lymphocytes in the periphery (R. Namikawa, unpublished) plus the fact that the number of granzyme-/perforin-expressing lymphocytes in the periphery of patients undergoing rejection did not always increase significantly. A similar lack of correlation in peripheral blood was also observed in a mouse model (C. Mueller and J. Shelby, unpublished). The problem of viral infection was noted in studies of kidney transplants s7 as well as cardiac transplants 49. Since cytolytic lymphocytes respond to virally-infected cells, the presence of granzyme-/perforin-expressing lymphocytes in virally-infected individuals is not surprising. If granzyme and perforin expression are to be used as diagnostic tools to predict imminent rejection then it will also be necessary to screen for viral infection. The effect of immunosuppressive drugs on granzyme and perforin expression is very important if these are to be used as markers for transplantation rejection. In vitro, cyclosporine treatment downregulates expression of the
Immunology Today
mRNA for both proteins 2s,6° although the effects are probably indirect. The same correlation seems to exist in vivo as, in human studies, a reduction in granzyme A and perforin expression occurred after successful immunosuppressive treatmentS0, s7. In animal models, it is possible to test the effects of immunosuppression directly. In the rat, heterotopic cardiac allograft rejection can be averted by treatment with either cyclosporine or anti-OKT4 antibody (Ref. 61) and, despite the fact that there is still a lymphocytic infiltrate in these tolerated hearts, the granzyme-A-expressing population is no longer present (Chen et al., unpublished). Similarly, in a mouse heterotopic transplant model, both granzyme A and perforin expression have been shown to be selectively depleted in cyclosporine-A-treated animals (C. Mueller et al., unpublished). Conclusions Within the past two to three years, data from a wide variety of systems, both in vitro and in vivo, have strongly indicated a role for perforin and granzyme A in cellmediated cytolysis. Although the precise mechanism of granule-mediated cytolysis and the relative contributions of other cytolytic mechanisms are still being debated, it is clear that the expression of perforin and granzyme A can be used as markers for functional, activated cytotoxic lymphocytes in vivo. This has important implications for their use as potential diagnostic markers in transplantation, and for identifying cytolytic lymphocytes that may be involved in autoimmunity and viral infections.
The authors would like to thank Jim Kaufman and Alexandra Livingstone for many helpful comments on this manuscript, Irving Weissman for introducing them to this work at Stanford, and Craig Okada, Howard Gershenfeld and John Carlson for many interesting ideas during the Stanford killer cell years. Christoph Mueller is supported by The Swiss National Science Foundation. The Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche, Switzerland. Gillian Griffiths is at the Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, CH-4005 Basel and Christoph Mueller is at the Dept of Pathology, University of Bern, Murtenstrasse 31, CH-3010 Bern, Switzerland.
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