Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

Overnight incubation of mouse spleen cells in recombinant IL-2 generates cytotoxic cells with NK characteristics from precursors enriched with or devoid of LGL.

Interleukin-2 (IL-2) augments natural killer (NK) activity as well as generating effector cells named lymphokine activated killer cells (LAK) which are capable of lysing a wide spectrum of target cells. A large body of evidence has been accumulated to evaluate the relationship between NK and LAK cells and conflicting results have been reported. Our study was addressed to further analyse this relationship and in particular to investigate whether in a short incubation IL-2 is merely capable of augmenting the activity of pre-existing killer cells, or whether it can also promote the differentiation of precursor cells. Eighteen-hour culture of mouse spleen cells in human recombinant IL-2 induced a DNA-synthesis-independent generation of cytotoxic cells bearing an NK phenotype (aGM-1+, Thy1.2+/-, CD8-, CD4-). These were generated from precursor cells also bearing an NK phenotype, recovered either from low density Percoll fractions enriched in lytic cells with LGL morphology as well as from high density fractions devoid of LGL and cytotoxic activity.     

Dissociation of natural killer and lymphocyte-activated killer cell lytic activities in human CD3− large granular lymphocytes

CD3^? large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short-term exposure to interleukin-2, NK cells were shown to acquire lymphocyte-activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca²⁺ dependent; however, they could be dissociated by a Ca²⁺ channel blocker or a Ca²⁺ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3^? LGL, the most likely candidate being the pore-forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.     

Morphological and functional analysis of PTA granules in human NK cells

 Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3⁻, CD16⁺, CD56⁺) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNα, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation. Accepted: 24 March 1997     

Interleukin-4 differentially regulates interleukin-2-mediated and CD2-mediated induction of human lymphokine-activated killer effectors.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.     

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.     

Interleukin-12 up-regulates perforin- and Fas-mediated lymphokine-activated killer activity by intestinal intraepithelial lymphocytes

Human intraepithelial lymphocytes (IELs) comprise a unique compartment of memory T cell receptor (TCR)-alphabeta( +)CD8(+) T lymphocytes interspersed between intestinal epithelial cells. They develop potent lymphokine-activated killer (LAK) activity with interleukin (IL)-15, a cytokine that is found in excess in certain mucosal inflammatory states. IL-12, released by activated antigen-presenting cells, is known to potentiate perforin-induced cytotoxicity. This study evaluates the mechanism by which IL-12 up-regulates LAK activity. When IELs were stimulated with IL-15, the CD94(+) IEL subset expanded and carried out cytotoxic activity in redirected lysis against P815 cells as well as Fas ligand (FL)- and tumour necrosis factor (TNF)-alpha-mediated lysis of Jurkat and WEHI cells, respectively. IL-12 enhanced the perforin- and FL-, but not TNF-alpha-mediated events. In addition, the up-regulated killing of HT-29 cells by IL-12 was reduced by concanamycin (which targets perforin) and antibody neutralizing FL but not by anti-TNF-alpha antibody. Furthermore, IL-12 augmented IL-15-stimulated release of serine esterases as well as expression of perforin and FL by IELs, but not TNF-alpha. This study shows that LAK activity, carried out by the CD94(+) IELs, involves perforin, FL and TNF-alpha. IL-12 up-regulates the first two mechanisms of action, showing for the first time its effect on FL production and lytic activity.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

Regulation of Natural Killer Cell Activity by Transforming Growth Factor-β and Prostaglandin E2

The separate and combined effects of transforming growth factor-β1 (TGF-β1) and prostaglandin E₂ on human natural killer (NK) activity were studied. Peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL, 70–90% purity) were used as effector cells and K562 as targets. Overnight incubation of the effector cells with TGF-β1 resulted in a significant inhibition of NK activity. TGF-β1 did not influence the expression of CD3, CD 16, CD 18 or CD56 antigens on PBL. Combination of TGF-βl with indomethacin gave the same NK-suppressive effect as TGE-β1 alone, showing that the inhibition of NK acuvity by TGF-β1 is not due to an increase in PGE₂ levels. TGF-β did not influence cAMP level in PBL whereas PGE₂ significantly increased it. On the other hand. TGE-β1 and PGE₂ showed an additive inhibitory effect on NK activity. TGF-β1 did not reduce the binding of PBL and LGL to K562. PGE₂ suppressed the binding and TGF-β1 did not influence this suppression. TGF-β1 also suppressed IL-2-induced activation of NK activity and increase of expression of the granule proteins granzyme A and perforin. PGE₂ did not appear to affect granzyme A and perforin contents. The results indicate that TGF-β1 and PGE₂ suppress NK activity by different mechanisms.     

Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

The effects of human transferrin (Tf) on lymphokine (IL-2)-activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6-day incubation in medium with recombinant human IL-2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK-resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL-2, it augmented LAK induction dose- and time-dependently. This augmenting effect was completely abolished by pretreatment with anti-Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL-2 and their expressions of receptors for IL-2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody-bound magnetic beads. Addition of Tf to cultures of CD8+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL-2. These results indicate that Tf is important in generation of IL-2-inducible killer properties and PFP activity of human CD8+ T cells.     

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.     

Signal transduction via phosphorylated adhesion molecule, LFA-1{beta} (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

It is well known that IL-2 stimulates natural killer (NK) cellsto express lymphokine activated killer (LAK) activity and thatthis stimulation prompts the acquisition of the ability to lysepreviously insensitive target cells. The possible role of adhesionmolecules in the IL-2 activation process was probed by focussingon a lymphocyte function-associated antigen (LFA)-1-dependentmodel system. A mAb to the LFA-1ß chain abrogatedLAK activity, but only moderately suppressed NK activity, suggestinga differential role for LFA-1ß In LAK compared withNK mediated lysis. Orthophosphate labeling demonstrated thatthe LFA-1ß chain was strongly phosphorylated in LAKbut not NK cells; in contrast, the chain was phosphorylatedsimilarlyin both effector cell types. At least a portion ofthe phosphorylation of the ß chain was on tyrosineresidues, as shown by Western blotting with anti-phosphotyrosineantibody of LFA-1ß immunoprecipitates. Crosslinkingof the LFA-1ß chain with plastic-adhered antibodystimulated Ca²⁺-dependent release of cytoplasmic lytic granulesand induced phosphatidyl inositol turnover in LAK but not NKcells. We conclude that the IL-2-induced phosphorylation oftheß chain of the LFA-1 adhesion molecule in LAK cellsand associated alteration in signal transduction may be importantin the stimulation of LAK cell activity in NK cells.     

Expression of granzyme B in peripheral blood polymorphonuclear neutrophils (PMN), myeloid cell lines and in PMN derived from haemotopoietic stem cells in vitro

Granzyme B and perforin are the major protagonists of cytotoxicity mediated by natural killer (NK) cells or cytotoxic T cells. More recent we described the presence of granzyme B and perforin in polymorphonuclear neutrophils (PMN), a finding in discrepancy with the credo that granzyme B and perforin expression is restricted to cytotoxic T cells and NK cells. In extension of our previous study, we now provide evidence that granzyme B is not only present in mature PMN, but also in the myeloid cell lines HL-60 and U937, in CD34+ stem cells, and in PMN derived from CD34+ cells in vitro. In agreement with the "targeting by time" hypothesis we found the bulk of granzyme B in association with primary granules, in addition to a minor membrane expression. Granzyme B, on one hand might, enhance the cytotoxic potential of PMN, on the other, it may provide PMN with additional means to degrade extracellular matrices.     

Mechanism of lymphocyte-mediated cytolysis: functional cytolytic T cells lacking perforin and granzymes.

Involvement of the lytic protein perforin (c. 65,000 MW) and of granule proteases (granzymes) in cell lysis induced by cytolytic T lymphocytes (CTL) has been suggested, but is still controversial. For example, in vivo-primed peritoneal exudate CTL (PEL) have been found to express perforin and granzyme activity in amounts comparable to those found in non-lytic lymphocytes, although PEL are the most potent of all CTL. Exploiting several cloned CTL hybridomas developed in this laboratory and newly available molecular probes for detecting perforin, granzymes, protein and mRNA, we now directly demonstrate killer T lymphocytes which kill effectively and specifically, but are free from perforin, lytic granules and granzymes, all three of which have been postulated to be involved in lymphocyte-mediated killing. The CTL hybridomas are completely devoid of perforin and granzymes prior to, during, and after activation by antigen, mitogen or interleukin-2 (IL-2). The induction of lytic granules, perforin, and granzymes in the in vivo-primed PEL, but not in the cloned CTL hybridomas, upon cultivation in IL-2, further suggests the involvement of these constituents in antigen/lymphokine-induced CTL activation and differentiation rather than directly in their cytocidal activity. Together, these findings support a perforin- and granzyme-independent CTL lytic mechanism.     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.