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At least 19 recordsLinked to original sources
PubMed1997

Residual cytotoxicity and granzyme K expression in granzyme A-deficient cytotoxic lymphocytes.

Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and 125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A-/- mice. The minimal cytotoxic defect in granzyme A-/- CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K.

Amino Acid Sequence↗
PubMed2003

Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling.

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Amino Acid Sequence↗
PubMed2004

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.

Animals↗
PubMed1989

Isolation and complete structure of the lymphocyte serine protease granzyme G, a novel member of the granzyme multigene family in murine cytolytic T lymphocytes. Evolutionary origin of lymphocyte proteases.

A cDNA clone that is closely related to the granule-associated serine proteases of cytolytic T lymphocytes (CTL), called granzymes A-F, was isolated from a CTL expression library. The encoded serine protease, granzyme G, shows 70%-89% nucleotide identities to the granzymes C-F and, like those, consists of 228 amino acids preceded by the short propeptide Glu-Glu and a 18 residue long signal peptide. Granzyme G was identified by amino-terminal sequence analysis as a correctly processed and sorted protein stored in lysosome-like granules. The phylogenetic history of the granzyme multigene family was reconstructed by two tree-making methods and by Southern blot analyses of human, rat, and mouse DNA. Our results indicate differences in the evolutionary pathway between these species. The murine granzymes C-G descended from a progenitor present at the time of mammalian radiation. Granzyme C branched off first after the primate-rodent split and was involved in a recombination event with granzyme B before the rat-mouse divergence. Granzymes D and E have diverged after the mouse-rat speciation. However, no experimental evidence for the existence of a granzyme C-D-E-F-G equivalent was found in humans, and loss of the ancestral gene in the primate lineage is discussed. In view of the species differences in the number of granzyme gene copies during recent evolution, we propose that the murine granzymes B-G play several distinct roles in CTL-mediated effector functions as a response to quite recent changes of the biochemical environment.

Amino Acid Sequence↗
PubMed1999

The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.

Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.

Amino Acid Sequence↗
PubMed2004

Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B.

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

Amino Acid Sequence↗
PubMed1998

Down-regulation of human granzyme B expression by glucocorticoids. Dexamethasone inhibits binding to the Ikaros and AP-1 regulatory elements of the granzyme B promoter.

The serine protease granzyme B is an essential component of the granule exocytosis pathway, a major apoptotic mechanism used by cytotoxic T lymphocytes and natural killer cells to induce target cell apoptosis. Granzyme B gene transcription is induced in activated lymphocytes upon antigenic stimulation, and several regulatory regions including CBF, AP-1, and Ikaros binding sites have been shown to be essential in the control of granzyme B promoter activation. Dexamethasone, a glucocorticoid that is widely used as an immunomodulatory and anti-inflammatory agent, inhibits granzyme B mRNA transcript in phytohemagglutinin-activated peripheral blood mononuclear cells. Transfection of a reporter construct containing the -148 to +60 region of the human granzyme B promoter demonstrated that this region was the target for dexamethasone repression. Mutation of Ikaros or AP-1 binding sites in the context of the granzyme B promoter demonstrated that both sites participate in dexamethasone-mediated inhibition of the granzyme B promoter activity. Electromobility shift assay revealed that dexamethasone abolished the binding of nuclear transcription factors to the Ikaros binding site and reduced AP-1 binding activity. These results indicate that dexamethasone is able to abrogate the transcriptional activity of the human granzyme B gene promoter by inhibiting the binding of nuclear factors at the AP-1 and Ikaros sites.

CCAAT-Enhancer-Binding Proteins↗
PubMed1997

Inhibition of CPP32-like proteases prevents granzyme B- and Fas-, but not granzyme A-based cytotoxicity exerted by CTL clones.

The perforin-facilitated entry of granzymes in target cells is a major mechanism used by CTL to induce cell death. It has been reported that granzyme B can cleave and activate the apoptotic cysteine protease p32 (CPP32)/Yama and its homologues in vitro. However, the mechanism for granzyme-based cytolysis exerted by intact CTL remains unclear. In the present work, we have used anti-CD3 mAb-redirected lysis of Fas-negative L1210 cells by CTL clones as a model to study perforin/granzyme-based cytotoxicity separately from the contribution of the Fas/Fas ligand system. N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), a specific inhibitor of CPP32-like proteases, completely prevented the former type of lysis in 3-h assays, but not in long-term (16-h) assays. A combination of Ac-DEVD-CHO and the granzyme A inhibitor IGA (7-(phenyl-ureido)-4-chloro-3-(2-isothioureidoethoxy)-isocoumarin) inhibited long-term cytolysis. 3,4-Dichloroisocoumarin, a serine-protease inhibitor that efficiently inhibits granzyme B and poorly inhibits granzyme A, had similar effects as Ac-DEVD-CHO on anti-CD3 mAb-redirected lysis of L1210 cells. On the other hand, Fas-based cytolysis exerted by the same CTL clones on Fas-transfected L1210 cells (L1210Fas) was inhibited completely by Ac-DEVD-CHO, irrespective of the incubation time. These results suggest that granzyme B- and Fas-based cytotoxicity exerted by CTL clones converge at the level of CPP32-like protease activation, while granzyme A acts via a different, still undefined, pathway. We also demonstrate that perforin/granzyme-based cytolysis occurs without increase in the cellular ceramide content, ruling out the contribution of the sphingomyelinase pathway to this mechanism of cell death.

Animals↗
PubMed1999

Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.

Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions.

Alpha-Globulins↗
PubMed1992

Differential expression of granzyme A and granzyme B proteases and their secretion by fresh rat natural killer cells (NK) and lymphokine-activated killer cells with NK phenotype (LAK-NK).

Granzymes A-G are a family of serine proteases localized in the cytoplasmic granules of cytotoxic T lymphocytes (Masson, D. et al. Cell 1987. 49: 679) and granzyme A is secreted by T lymphocytes in response to antigenic stimulation. Granzyme A is also expressed by natural killer (NK) and lymphokine-activated killer (LAK) cells. Here we show that fresh rat NK cells constitutively express granzyme B and that granzyme A and granzyme B are differentially regulated in unstimulated NK cells vs. LAK cells with NK phenotype (LAK-NK cells). We also show that both granzymes A and B are secreted in a calcium-dependent manner, by NK and LAK-NK cells in response to stimuli which trigger NK cell functions.

Animals↗
PubMed1998

Efficient nuclear targeting of granzyme B and the nuclear consequences of apoptosis induced by granzyme B and perforin are caspase-dependent, but cell death is caspase-independent.

The secretory lysosomes of cytolytic lymphocytes house the principal apoptotic molecules for eliminating virus-infected cells: a membranolytic agent, perforin, and the serine protease, granzyme B. Perforin allows granzyme B access to cytosolic and nuclear substrates that, when cleaved, result in the characteristic apoptotic phenotype. Key among these substrates is a family of cytoplasmic caspases that mediate cell suicide. We have examined the caspase dependence of several nuclear and cytoplasmic parameters of apoptosis induced by purified perforin and granzyme B. Cell membrane leakage in response to perforin and granzyme B was independent of caspase activation; however, nuclear events such as DNA fragmentation and nuclear condensation and disintegration were abolished by the broad-acting caspase inhibitor, z-VAD-fmk. Despite being spared from nuclear damage, z-VAD-fmk-treated cells exposed to both cytotoxins uniformly died when they were re-cultured, while cells exposed to perforin or granzyme alone survived and proliferated as readily as untreated cells. Pretreatment of cells with z-VAD-fmk also resulted in reduced granzyme B nuclear uptake following addition of perforin; however, its uptake into the cytoplasm in the absence of perforin was unaffected. We conclude that cell death in response to perforin and granzyme B does not require caspase activation and still proceeds efficiently through non-nuclear pathways when nuclear substrate cleavage is inhibited.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.

The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.

Amino Acid Sequence↗
PubMed1998

Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.

Adenoviridae↗
PubMed1996

Extracellular activities of human granzymes. I. Granzyme A induces IL6 and IL8 production in fibroblast and epithelial cell lines.

Situated in secretory granules of cytotoxic cells, granzymes are essential for induction of target cell apoptosis with perforin. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independently of their role in the lytic event. Thrombin has been shown to function as an activation molecule by cleaving its receptor. We hypothesize that granzymes may act similarly. In this report, we show that purified human granzyme A can induce human lung fibroblasts to produce IL6 and IL8. Cytokine induction is abrogated by treating the serine protease with the suicide serine protease inhibitor 3,4-dichloroisocoumarin. Other fibroblast lines as well as epithelial cells produced cytokines in response to granzyme A. Our data suggest that granzyme A can function as an activation molecule with potentially important immunoregulatory functions.

Cell Line↗
PubMed1992

Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides.

Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.

Amino Acid Sequence↗
PubMed2005

Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells.

The mechanism of target cell lysis in cytotoxic lymphocyte-mediated death is not well understood, and the role of granzymes in this process is unclear. Chemical functional probes were thus prepared for the major granzymes A and B to deconvolute their role in natural killer cell-mediated lysis of target cells. These biotinylated and substrate specificity-based diphenyl phosphonates allowed facile evaluation of selectivity through activity-based profiling in cell lysates and intact cells. Both inhibitors were found to be extremely selective in vitro and in cells. Use of these inhibitors in cell-based assays revealed granzyme A to be a minor effector and granzyme B to be a major effector of target cell lysis by natural killer cells. These studies indicate that the proapoptotic granzyme B functions also as a pronecrotic effector of target cell death.

Apoptosis↗
PubMed1999

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.

Alcohol Drinking↗