LETTER TO THE EDITOR |
Cytoplasmic granules of cytotoxic lymphocytes contain several constituents, including perforin, granzyme A (GrA), and granzyme B (GrB). After granule exocytosis, GrA and GrB are believed to enter the target cell through perforin-derived transmembrane channels to induce DNA fragmentation and apoptosis (
Cytocentrifuge preparations of lymphokine-activated killer (LAK) cells were fixed in 4% buffered formalin for 10 min. Part of the preparations were left untreated, and either enzymatic or non-enzymatic antigen retrieval was employed on the other part. Two enzymatic antigen retrieval methods were applied: digestion with 0.1 mg/ml pepsin A in 10 mM HCl, pH 2.0, at 37C, and digestion with 0.1 mg/ml protease Type XIV in PBS, pH 7.4, at 37C. In both cases, digestion was stopped after either 30 sec, 1 min, 2 min, or 4 min. Non-enzymatic antigen retrieval consisted of boiling the preparations in 10 mM sodium citrate, pH 6.0, for 10 min. Single-color stainings were performed with monoclonal antibodies (MAbs) GrA-6 and GrB-7, raised against recombinant human GrA and GrB proteins, respectively, which recognize GrA and GrB in paraffin-embedded, formalin-fixed tissue sections (Sanbio/Monosan; Uden, The Netherlands).
No staining was observed in the majority of untreated LAK cells (Figure 1A). Enzymatic antigen retrieval did not have a beneficial effect, and extension of digestion time, especially in the pepsin A-containing antigen retrieval solution, led to loss of cell number and loss of morphology (not shown). After non-enzymatic antigen retrieval, the majority of LAK cells displayed a strong granular staining pattern, but cell loss and loss of morphology were not observed (Figure 1B).
|
The mechanisms underlying the effects of enzymatic and non-enzymatic antigen retrieval differ, and a selective benefit from one treatment over the other has also been demonstrated in immunohistochemistry for other antigens (
MAbs GrA-6 and GrB-7 were raised against recombinant granzymes produced in a prokaryotic expression system, and the fact that reactivity of these MAbs is enhanced after non-enzymatic antigen retrieval suggests that they specifically recognize granzymes with a denatured conformation. Several factors can be pointed out that might have induced conformational changes in the recombinant granzymes and may have contributed to this specificity, e.g., incorrect folding of the recombinant granzymes in the prokaryotic expression system or partial degeneration of the recombinant gran-zymes after immunization. When we extended fixation time to 7 days, we still did not observe an influence on staining results when we employed non-enzymatic antigen retrieval (not shown). This observation implies that use of MAbs GrA-6 and GrB-7 in immunocytochemistry is not hampered by masking of the respective antigens due to formalin fixation, and explains the lack of beneficial effect of enzymatic antigen retrieval.
Subsequently, non-enzymatic antigen retrieval was performed on formalin-fixed cytocentrifuge preparations of peripheral blood mononuclear cells from 10 healthy individuals. Two-color stainings were performed combining polyclonal rabbit anti- human CD3 antibodies with MAb GrA-6 or GrB-7. Two observers independently scored 300 staining cells per slide. Cells staining doubly positive were clearly distinguishable from cells staining singly positive (Figure 1C). Identical staining patterns were observed for MAbs GrA-6 and GrB-7. Table 1 shows the distribution of CD3+ granzyme-, CD3+ granzyme+, and CD3- granzyme+ cells among staining cells. Our data imply that the same cells express both GrA and GrB. This assumption is supported by the observation that expression of both granzymes, as well as expression of perforin, is upregulated after activation of T-cells in vitro (
|
GrA and GrB were expressed by 12 ± 1% (mean ± SEM) and by 9 ± 1% of CD3+ T-cells, respectively. Analogous to the distribution pattern of perforin (
Granzyme-expressing CD3- cells most likely represent NK cells. The majority of cells in this subset have a granular morphology and have been shown to express perforin (
In conclusion, we have immunocytochemically demonstrated expression of GrA and GrB in peripheral blood lymphocytes from healthy individuals using non- enzymatic antigen retrieval of formalin-fixed cells. Application in immunocytochemistry of antigen retrieval methods routinely used in immunohistochemistry may prove useful for detection of other antigens in cytocentrifuge preparations and cell smears.
Acknowledgments
Supported by the Dutch Kidney Foundation (grant C93.1278)
Received for publication November 13, 1996; accepted December 2, 1996.
Literature Cited
Berthou C, Legros-Maïda S, Soulié A, Wargnier A, Guillet J, Rabian C, Gluckman E, Sasportes M (1995) Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes. Blood 85:1540-1546[Summary]
Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, D'Amato L, Sabattini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissues sections. J Pathol 171:83-98[Medline]
Heusel JW, Wesselschmidt RL, Shresta S, Russel JH, Ley TJ (1994) Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977-987[Medline]
Kern F, Döcke WD, Reinke P, Volk HD (1994) Discordant expression of LFA-1, VLA-4, VLA-ß1, CD45RO and CD28 on T-cell subsets: evidence for multiple, subsets of "memory" T-cells. Int Arch Allergy Immunol 104:17-26[Medline]
Kummer JA, Kamp AM, Tadema TM, Vos W, Meijer CJLM, Hack CE (1995) Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood. Clin Exp Immunol 100:164-172[Medline]
Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH (1986) The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 136:4480-4486[Summary]
Lebow LT, Bonavida B (1990) Purification and characterization of cytolytic and non-cytolytic human natural killer cell subsets. Proc Natl Acad Sci USA 87:6063-6067[Summary]
Liu C-C, Rafii S, Granelli-Piperno A, Trapani JA, Young JD-E (1989) Perforin and serine esterase gene expression in stimulated human T-cells. Kinetics, mitogen requirements, and effects of cyclosporin A. J Exp Med 170:2105-2118[Summary]
Liu C-C, Walsh CM, Young JD-E (1995) Perforin: structure and function. Immunol Today 16:194-201[Medline]
Nakajima H, Park HL, Henkart PA (1995) Synergistic roles of granzymes A and B in mediating target cell death by rat basophilic leukemia mast cell tumors also expressing cytolysin/perforin. J Exp Med 181:1037-1046[Summary]
Nakata M, Kawasaki A, Azuma M, Tsuji K, Matsuda H, Shinkai Y, Yagita H, Okumura K (1992) Expression of perforin and cytolytic potential of human peripheral blood lymphocyte subpopulations. Int Immunol 4:1049-1054[Summary]
Reichert T, DeBruyère M, Deneys V, Tötterman T, Lydyard P, Yuksel F, Chapel H, Jewell D, Van Hove L, Linden J, Buchner L (1991) Lymphocyte subset reference ranges in adult caucasians. Clin Immunol Immunopathol 60:190-208[Medline]
Smyth MJ, Trapani JA (1995) Granzymes: exogenous proteinases that induce target cell apoptosis. Immunol Today 16:202-206[Medline]
Sunder-Plassmann G, Wagner L, Hruby K, Balcke P, Worman CP (1990) Upregulation of a lymphoid serine protease in kidney allograft recipients. Kidney Int 37:1350-1356[Medline]