Discordant Regulation of Granzyme H and Granzyme B Expression in Human Lymphocytes

We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3-CD56+ natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4+ and particularly CD8+ T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells. We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3-CD56+ natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4+ and particularly CD8+ T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells. Cytotoxic T lymphocytes and natural killer (NK) 1The abbreviations used are: NK, natural killer; NKT, NK T; gzm, granzyme; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin.1The abbreviations used are: NK, natural killer; NKT, NK T; gzm, granzyme; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin. cells are cytotoxic lymphocytes that are responsible for inducing rapid apoptosis of virus-infected or transformed cells (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar, 2Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (859) Google Scholar). Cytotoxic lymphocytes utilize two pathways for killing target cells, both of which require direct cell contact. The first pathway involves exocytosis of potent toxins from secretory granules stored in the effector cell cytoplasm, whereas the second is triggered by clustering of death receptors on the target cell membrane following interaction with their respective ligands (tumor necrosis factor superfamily members) expressed on the killer cell (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar, 2Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (859) Google Scholar). Cytotoxic granules contain two major types of toxin that co-operatively induce target cell apoptosis: granzymes (gzms), a family of serine proteases, and a pore-forming protein, perforin. The precise mechanism of gzm/perforin synergy has not been clarified; however, it is likely that gzms enter the target cell by endocytosis (3Motyka B. Korbutt G. Pinkoski M.J. Heibein J.A. Caputo A. Hobman M. Barry M. Shostak I. Sawchuk T. Holmes C.F. Gauldie J. Bleackley R.C. Cell. 2000; 103: 491-500Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 4Trapani J.A. Sutton V.R. Thia K.Y. Li Y.Q. Froelich C.J. Jans D.A. Sandrin M.S. Browne K.A. J. Cell Biol. 2003; 160: 223-233Crossref PubMed Scopus (92) Google Scholar), whereas perforin enables pro-apoptotic gzms access to their substrates in the target cell cytosol by destabilizing endosomes (5Froelich C.J. Orth K. Turbov J. Seth P. Gottlieb R. Babior B. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W. J. Biol. Chem. 1996; 271: 29073-29079Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 6Browne K.A. Blink E. Sutton V.R. Froelich C.J. Jans D.A. Trapani J.A. Mol. Cell Biol. 1999; 19: 8604-8615Crossref PubMed Scopus (176) Google Scholar). The more traditionally held notion of perforin permitting access to the cytosol through plasma membrane pores may also apply in some circumstances (6Browne K.A. Blink E. Sutton V.R. Froelich C.J. Jans D.A. Trapani J.A. Mol. Cell Biol. 1999; 19: 8604-8615Crossref PubMed Scopus (176) Google Scholar). Gzms are closely related to one another structurally, and their genes are clustered in several loci on distinct chromosomes (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar). In both humans and rodents, each locus encodes proteases with a single broad type of substrate cleavage. Gzms A and K are trypsin-like proteases (“tryptases”) that cleave proteins at basic residues, and their genes are linked on human chromosome 5 (8Baker E. Sayers T.J. Sutherland G.R. Smyth M.J. Immunogenetics. 1994; 40: 235-237Crossref PubMed Scopus (37) Google Scholar). Gzm M preferentially cleaves at residues with long, uncharged side chains (Met, Leu), and its gene is closely linked to the neutrophil elastase gene on human chromosome 19 (9Smyth M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar). The genes for gzms B and H are located on chromosome 14, tightly linked with the gene encoding cathepsin G, another chymotrypsin-like protease (“chymase”) that, unlike the gzms, is expressed in cells of the myeloid lineage but not in lymphocytes (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar, 10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). These three genes all map to within 60 kb of one another. Gzm B cleaves its substrates adjacent to acidic residues, particularly Asp (11Odake S. Kam C.M. Narasimhan L. Poe M. Blake J.T. Krahenbuhl O. Tschopp J. Powers J.C. Biochemistry. 1991; 30: 2217-2227Crossref PubMed Scopus (225) Google Scholar), and plays a central role in eliciting death of the target cell by perturbing mitochondria and activating caspases (12Sutton V.R. Davis J.E. Cancilla M. Johnstone R.W. Ruefli A.A. Sedelies K. Browne K.A. Trapani J.A. J. Exp. Med. 2000; 192: 1403-1414Crossref PubMed Scopus (298) Google Scholar, 13Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Its absence from the granules of lymphocytes from gene knockout mice slows the rate of target cell DNA damage (14Heusel J.W. Wesselschmidt R.L. Shresta S. Russell J.H. Ley T.J. Cell. 1994; 76: 977-987Abstract Full Text PDF PubMed Scopus (743) Google Scholar). In the mouse, the gene for gzm B is tightly linked to at least four other granzyme genes predicted to code for functional chymases (gzms C-F); however, only one such chymase, gzm H, performs this function in humans (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Gzm C is now known to possess an unusual type of pro-apoptotic activity characterized by mitochondrial swelling and disruption that does not require prior caspase activity (16Johnson H. Scorrano L. Korsmeyer S.J. Ley T.J. Blood. 2003; 101: 3093-3101Crossref PubMed Scopus (91) Google Scholar). However, no such evidence has yet come forward for human gzm H. We recently showed that recombinant gzm H expressed in cells preferentially cleaves substrates with or at the (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). this substrate with gzms gzm H has no direct in the and to a human Gzm H has a high with of the gzm B particularly the of the P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google a distinct has been from the high of that the gzmH gene by of of the of the gzmB gene and and with the of an another serine protease gene P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). The function of gzm H is and its has been by a of to it from gzm B at the protein or mRNA levels. The expression of gzm H has been in one that gzm H mRNA expression in human T cells P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). that gzm gzm H is expressed at high levels in T cells. However, in another a of the gzmH was used to preferentially direct the expression of T to the The was not in cytotoxic T lymphocytes that expressed high levels of gzm B but was expressed in NK cells and in NK and cell that in Ley T.J. Blood. 1999; PubMed Google Scholar). the genes encoding cathepsin and gzms B and H are tightly linked, cathepsin and gzm B expression cathepsin is expressed by cells of the myeloid particularly and G. Ley T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is quite that gzms B and H may expressed and regulated The of gzm H to for this a novel gzm mAb, now that gzm H is expressed in but its constitutive expression is quite distinct from that of gzm is its expression in to a of that induce gzm B used in and several by our J.A. Browne K.A. M. Smyth M.J. 1993; PubMed Scopus Google Scholar), M.J. O'Connor M.D. P. Thia K.Y. Trapani J.A. PubMed Scopus Google Scholar), M. Trapani J.A. B. PubMed Scopus Google Scholar), and M.S. Sutton V.R. Trapani J.A. Mol. PubMed Scopus Google Scholar). In was from Cell lines, and human cell used in this all in with and in a at These YT and and B and T and and cell of of human T cell lymphoma of two from also T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google Scholar). gzm human gzm B and gzm H from the of cells, (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The gzm proteins by and against The gzms for their activity by the of substrates gzm a from Kam and of or gzm H, from and to mice the with recombinant gzm H with to four the mice with the with following a a was and its cells with cells, using for of H using a in which gzm H gzm B a was the of a by and the and in of a was raised in by with recombinant human gzm B with and in of with at of Leukocyte of cell in and to at for to the cells. from the and in for to the cells. The was to and the cells and by from peripheral blood mononuclear cells with and at least T cells and CD3-CD56+ NK cells from by cell and of cells, in and by for that with by and for and and by cell and was to T and NK Cell and of T and NK cells in with recombinant human or for to prior to and for gzm on and the proteins protein was by with in at for with to in at and and was detected with to of and for the of gzm B and H by of each respective using The gzm B was using the and and was in The gzm H was similarly using the and 5 and was in was from and with by or with The used to mRNA in was from various of human cells and cell and by on and to by The membrane was to at and at for was in and at with for the at and high for and to for at In some DNA was and in The at for and and of was using H for each gene using with a at and a of using for at by of for and for 60 in the The levels of gzm H and B mRNA to that of protein and gzm and gzm H, and for gzms B and H was by the of of gzm B and H cell YT human NK cells several in PBS, and by Davis J.E. Browne K.A. Trapani J.A. J. Immunol. 2003; Google Scholar). The cell was on a and with the of the the of by of each analyzed by and the and expression of gzm H, it was first to an that it from other particularly its gzm with which it A. Trapani J.A. B. PubMed Scopus Google Scholar). We expressed and gzm H by cells (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), this protein was used an for in for in a to an that to gzm H. but one of an on gzm detected an to both granzymes not However, the by gzm H and failed to with gzm B in the which used in on gzm B (12Sutton V.R. Davis J.E. Cancilla M. Johnstone R.W. Ruefli A.A. Sedelies K. Browne K.A. Trapani J.A. J. Exp. Med. 2000; 192: 1403-1414Crossref PubMed Scopus (298) Google Scholar, 13Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, J.A. Browne K.A. M. Smyth M.J. 1993; PubMed Scopus Google Scholar, J. Sutton L. Trapani J.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, V.R. Trapani J.A. J. Immunol. Google Scholar), was for gzm B and failed to gzm H. detected of gzm H but not at all with gzm B in In the with gzm B in but not with gzm H not with both a raised against gzm B showed with gzm H, the of the two granzymes a to an with gzms B and H using other recombinant granzymes also that both and not with recombinant human gzm a more of gzms B and H data not (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar). that gzm H, used the in on cell from a of and cells The human NK cell YT and not both a 33-kDa protein that with recombinant The predicted on was consistent with a gzm H of from the and of Gzm H was not expressed by the T cell and the B lymphoma cell and the myeloid cell and or cells from two from with T cell no was in the human cell or the and and YT cells both contain of granules and also perforin and gzms B and M Davis J.E. Browne K.A. Trapani J.A. J. Immunol. 2003; Google Scholar), the of gzm H by cells was consistent with a role for gzm H in cytotoxic cell the of gzm H, on YT cells, using and was gzms B and M to granules in the of YT cells M.J. O'Connor M.D. P. Thia K.Y. Trapani J.A. PubMed Scopus Google Scholar, J. Sutton L. Trapani J.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). in YT cells was also with the gzmH however, the of the from cell to some YT cells a and the showed no The of was not due to of and cells a of not of the granzyme to cells, which used a in this gzmH to cytotoxic a of YT cells was on a The analyzed by with for gzms B and H, and the which is to the cytosol of cytotoxic lymphocytes than granules J. Sutton L. Trapani J.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Gzm H was with the proteins perforin and gzmB in at the of the data not of which is abundant in the cytosol of YT cells by the least but is not a of the granules J. Sutton L. Trapani J.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). all of the human and granzymes to are expressed by NK cells and in an by CD8+ some T cells, and an effector J.A. Int. Rev. PubMed Google Scholar). The two granzymes known to a role in target cell gzm which is by a gene that is tightly linked to that of gzm H, and gzm on a 5 (8Baker E. Sayers T.J. Sutherland G.R. Smyth M.J. Immunogenetics. 1994; 40: 235-237Crossref PubMed Scopus (37) Google are both regulated in this to this is gzm which is expressed by NK cells but not by human or T cells M.J. O'Connor M.D. P. Thia K.Y. Trapani J.A. PubMed Scopus Google Scholar, T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google Scholar). We to which in human peripheral blood both in their or following to activating unfractionated from to prior to or to agents that T cell and in from showed constitutive gzm H whereas the expression of gzm B by the cells was or gzmB is known to expressed at levels in NK cells T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google and is from levels in CD4+ and CD8+ T cells P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar, S. A. Trapani J.A. J. Exp. Med. PubMed Scopus Google Scholar, M.J. H. M. K. J. Exp. Med. PubMed Scopus Google Scholar, J.A. B. S. A. PubMed Scopus Google Scholar). with this of of to the T cell and a of gzmB The to within and from T cells and CD3-CD56+ NK cells The T cells expressed no gzm B and levels of gzm H, whereas the NK cells a for gzm H and a for gzm B. NK cells only of PBMC, it is likely that gzm B in of unfractionated in of the T cells to a and of gzm B levels the of gzm H expression not constitutive levels but In NK cells to the levels of gzm H whereas gzm B levels which cell types responsible for gzm H more gzm H was expressed at high levels in CD3-CD56+ NK cells and at far lower levels in T cells with the NK cells also expressed of gzm B and far of gzm M T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google Scholar). CD4+ and CD8+ T cells also for granzyme expression in with and consistent with both T cell expressed of gzm whereas gzm M was Gzm H was expressed in CD4+ cells, but to P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar), the of expression was not in with CD4+ cells. of gzm the CD8+ T cells expressed no gzmH NK cells expressed of gzm H, also in cells also expressed gzm H. the of cells in is only unfractionated from a in with and for in the of cells with to not cells to and analyzed by protein to expression of gzm H, whereas of gzm B expressed gzmH was that gzm H is expressed by NK cells and by CD4+ T cells but not by CD8+ T cells in human peripheral A of that induce gzm B expression no on gzm H protein levels. The absence of with myeloid cells that this does not with cathepsin G, a closely related serine protease expressed at high levels in myeloid cells but not at all in lymphocytes G. Ley T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). not in with a that that gzm H is T cell P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). was the of a gzm H and on mRNA expression with the may from of the gzm H with gzm B for gzm B and gzm H on located in the of both the respective gzmB and gzm H which and the for gzms B and H of with gzm which has a far lower of with both gzm H and gzm B. the of for of all to gzm B or H. The gzmH and gzmB genes are tightly linked to that encoding cathepsin G, and all three proteases and that gzmH protein is not expressed in myeloid cells that gene expression of gzm H and cathepsin not been is also known that myeloid cells of cathepsin in their granules but mRNA only the of gene transcription cell D. Ley T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). NK cells gzm H protein in the absence of its used gzm and gzm to of CD3-CD56+ peripheral blood NK cells. We to mRNA encoding both gzms B and H gzm H protein was more abundant than gzm B in NK cells gzm B and gzm H abundant in NK cells with a gzmH gene are in NK cells, unlike the with in myeloid cells R. I. S. Int. J. 2003; Google encoding gzms B and H are both expressed in cells. of from CD3-CD56+ peripheral blood NK cells and unfractionated of a is The is expressed the expression of to The data are of four with from two We a mAb, which gzm H, and used it to the expression of gzms B and H in a of cell and human cells. The two gzms and are predicted to both are chymotrypsin-like serine was to first that and the B used in this with their respective target was in several the by was that recombinant gzm H in that it failed to gzm B in a to a gzm mAb, for with gzm B was in the of the other to the of for gzm H was in using proteins recombinant proteins was no that was of an gzm a of the human NK cell YT of for gzms B and H. gzm B was and all cells, gzm H was only in a of cells, and the of from cell to We are to the that gzmH expression in YT cells. has raised a of The major finding is that the expression of gzms B and H is quite discordant in human both in T and NK cells and the cells with a of agents that induce and gzm H is expressed at high levels in peripheral blood NK cells, whereas gzm B is expressed at lower levels by the cells. that induce gzm B expression also on gzm H levels in T or NK cells to various and J.W. Ley T.J. J. Biol. Chem. 1991; Full Text PDF PubMed Google also used to a likely in gzm B and H in human with the levels of gzm H mRNA in NK cells but in T cells. NK cell also been to mRNA for both gzms B and H in that are to used that between the two gzms R. I. S. Int. J. 2003; Google Scholar). to the gzms expressed by NK cells are gzms H and gzms A and M T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google but not gzm B. the expression of all gzms is in both of the NK cell and that been to T.J. T. Smyth M.J. J. Immunol. PubMed Scopus Google Scholar, J.A. B. S. A. PubMed Scopus Google Scholar). The in NK cells the of NK cells target cell apoptosis levels of gzm which is to the pro-apoptotic activity of of the In the absence of gzm gzms and H all to a pro-apoptotic or CD3-CD56+ NK cells expressed of the of cells in peripheral blood that it was not to cells also gzm H. cells for the cells in and the that may gzmH expression in The of expression of gzm H protein was quite distinct from that of gzm B in peripheral blood T lymphocytes that in various in in of the in this to of gzmB protein, both in CD4+ and CD8+ However, gzm H protein was expressed at levels in CD4+ T cells but was not detected in CD8+ T cells and was not by and of T cell PHA, and used or in various our data indicate that gzms B and H, the of tightly linked are regulated in distinct the data in this also indicate that of the gzmH gene is to mRNA is not myeloid cell the of all three closely linked genes is with the monoclonal used in this raised against gzm B or gzm H not with both gzms, of the raised against recombinant that at least one between the two closely related gzms is by mice and other the between human that it to to some our was to of the gzm that to the of the and the gzms of that used to such of gzm to an between residues and the of gzm B Browne K.A. Smyth M.J. Trapani J.A. Mol. Immunol. PubMed Scopus Google Scholar). These for for gzmB used in the J.A. Browne K.A. M. Smyth M.J. 1993; PubMed Scopus Google and M.J. O'Connor M.D. P. Thia K.Y. Trapani J.A. PubMed Scopus Google Scholar). However, from the of gzm H that to the but not gzmH not The of gzmH in cells has to this with the also that used in not gzm B or H. that the of both genes are far more for and related such in other the of for may a the to gzm B are for both in and L. Froelich C.J. Exp. Immunol. 1999; PubMed Scopus Google Scholar, J. A. M. C.J. R. S. A. PubMed Scopus Google Scholar, M.V. M. 2002; PubMed Scopus Google Scholar), with or DNA the of which with other gzm family and cathepsin has not been