NK cell-induced cytotoxicity is dependent on a Ca2+ increase in the target

In previous work we showed that programmed cell death (PCD) in thymocytes is mediated by a sustained increase in cytosolic Ca2+ concentration, resulting in the activation of an endogenous endonuclease, DNA fragmentation, and cell death. In this study we investigated the roles of Ca2+ and DNA fragmentation in target cell killing by natural killer (NK) cells. The effector cells induced a rapid, sustained increase in cytosolic Ca2+ concentration in Jurkat target cells. Buffering the target cell cytosolic Ca2+ with the Ca2(+)-selective dye, quin-2, prevented target cell killing. Extensive DNA fragmentation was associated with killing in every target tested, and this response was also blocked by quin-2. The endonuclease inhibitor, aurintricarboxylic acid, inhibited both DNA fragmentation and killing without influencing the Ca2+ increase in target cells. Thus, it is concluded that NK cell killing depends on a Ca2+ increase and appears to involve endogenous endonuclease activation in target cells.     

Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.     

Modulation of CD3- large granular lymphocyte functions by agonist and antagonists of protein kinase C: effects on NK and lymphokine-activated killer activity and production of IFN-gamma

The biochemical mechanisms involved in the activation and killing of tumor targets by large granular lymphocytes (LGL) have not yet been clearly defined. This laboratory has investigated these processes by analyzing the effects of protein kinase C (PKC) inhibitors (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride and retinol) on LGL cytotoxicity and IFN-gamma production. We now report that PKC inhibitors block the LGL functions of 1) NK activity, 2) IFN-gamma production, and 3) LAK activity induced by IL-2. Complete inhibition of cytotoxic activity occurs rapidly because only 2.5 h treatment of the LGL with the inhibitors was required. However, the inhibition of NK activity by the PKC inhibitors could be reversed by IL-2 or the synthetic diacylglycerol, L-gamma-1-oleyl-2-acetol-sn-3-glycerol (OAG), but not by IFN-alpha. The reversal of inhibition observed with OAG indicates that, in these studies, (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride is inhibiting PKC activity and not the activity of other cellular kinases. Furthermore, inhibition of LGL functional activity with PGE2 could not be reversed with OAG, supporting the contention that PG inhibition of NK activity is mediated by a pathway that does not directly involve PKC. These results indicate, in addition to IL-2-mediated events, that basal NK activity is under PKC regulatory control.     

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Characterization of a monoclonal antibody enhancing porcine natural killer cell activity (PNK-E)

A monoclonal antibody, termed PNK-E, that functionally enhances porcine natural killer (NK) cell activity but not antibody-dependent cellular cytotoxicity (ADCC) is investigated in this report. When PNK-E and K562 target cells were simultaneously added to effector cells, killing of target cells could be detected as early as 30 min, and a dramatic enhancement of killing activity was observed in short term 51Cr-release assays. When a panel of five NK-sensitive targets were tested, PNK-E enhanced the killing of K562, MOLT-4, and U937 cells, but not the killing of CEM and YAC-1. F(ab)'2 fragments of PNK-E did not enhance NK activity, indicating a requirement for the Fc portion of PNK-E to elicit enhancement of NK. Immunofluorescence analysis shows that PNK-E antigen is expressed on approximately 15% of peripheral blood lymphocytes with a relatively dull fluorescence staining pattern. PNK-E-positive sorted cells were enriched for large granular lymphocytes (LGL) and contained all detectable NK activity as compared to the PNK-E-negative sorted cells. When analyzed by polyacrylamide gel electrophoresis, PNK-E antibody immunoprecipitated a protein from 125I-labeled peripheral blood lymphocyte (PBL) cell lysates that resolved as a single band of approximately 205 kDa under nonreducing conditions and as two bands of approximately 50 kDa and 47 kDa under reducing conditions. The present data demonstrate a functional association between PNK-E antigen and NK cell activation.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium

NKR-P1 is a 60-kDa homodimer expressed on all rat NK cells. Previous studies by others suggest that NKR-P1 may play a role in NK cell activation because antibody to NKR-P1 stimulates the release of granules from NK cells, and anti-NKR-P1 causes redirected lysis by activated NK cells against targets that express FcR. To examine the mechanism of transmembrane signaling by NKR-P1, we studied the rat NK cell line, RNK-16. We here demonstrate that F(ab')2 antibody to NKR-P1 stimulates phosphoinositide turnover and a rise in intracellular calcium within RNK-16 cells. The response is augmented by cross-linking the F(ab')2 antibody. The phosphoinositide/calcium pathway is also stimulated by NKR-P1 in activated rat NK cells, although no response is detectable in polymorphonuclear cells, which also express NKR-P1. We also demonstrate that RNK-16 cells kill the anti-NKR-P1 (3.2.3) hybridoma and that exposure to the hybridoma target cells stimulates phosphoinositide turnover in RNK-16 cells. Both killing and phosphoinositide turnover are inhibited by F(ab')2 anti-NKR-P1, implicating NKR-P1 in both responses. In contrast, neither cytotoxicity nor phosphoinositide turnover is appreciably blocked by F(ab')2 anti-NKR-P1 in response to YAC-1 targets. Thus, with either target, killing is linked to phosphoinositide turnover, but killing of YAC-1 involves pathways that differ from those that direct killing of the anti-NKR-P1 hybridoma. Our studies support the hypothesis that NKR-P1 may serve as an activating cell-surface receptor on NK cells, and they clarify the mechanisms by which it activates NK cells.     

Direct induction of lymphocyte killing by calcium ionophore and phorbol ester treatment

To analyze transduction mechanisms in human lymphocyte killing, intracellular Ca2+ levels were increased by ionophore A23187 treatment and protein kinase C activated by phorbol ester 12-O-tetradecanoylphorbol-acetate (TPA). Drugs were tested either alone or in combinations on effector cells active in natural, antibody-dependent, and lectin-dependent killing. TPA suppressed killing in all systems at 100 ng/ml whereas A23187 was only suppressive for NK killing at concentrations higher than 0.1 microM. TPA combined with A23187, above 10 ng/ml and 0.5 microM, respectively, induced killing of all tested target cell lines with a slower kinetic than NK killing of K562 cells. Drug-induced killing did not increase optimal lectin and antibody-dependent killing and was demonstrated most easily on NK-resistant target cell lines. Fractionation of effector lymphocytes into NK cell-depleted, T3-positive and NK cell-enriched, T3-negative cells demonstrated that similar levels of TPA/A23187-dependent killing could be induced in both fractions. It is concluded that TPA/A23187 induce normal lymphocytes to nonselective killing of different target cells in similarity to the triggering effect these drugs have in many other cell systems. Whether the induced killing is representative of NK killing is discussed in relation to the presence of other potential effector cells and effector molecules in peripheral blood lymphocytes.     

Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.     

IFN increases class I MHC antigen expression on adenovirus-infected human cells without inducing resistance to natural killer cell killing.

It has been previously shown that unstimulated NK cells cannot preferentially lyse adenovirus serotypes 2 and 5-infected human cells. In this study, the ability of IFN to promote the selective NK cell-mediated lysis of adenovirus-infected human cells was determined. The relationship between target cell susceptibility to NK cell-mediated killing and class I Ag expression was also analyzed through the use of adenovirus serotype 2 and 5 mutants that do not make the adenovirus early region 3 19-kDa class I binding protein. IFN induced the selective lysis of adenovirus serotype 2 and 5-infected human cells by activating NK cells (IFN-alpha) and protecting uninfected, but not adenovirus-infected cells, from NK cell-mediated lysis (IFN-gamma). IFN-gamma increased the expression of class I Ag on the surface of cells infected with the adenovirus early region 3 deletion mutants, dl327 or dl801, to a level equal to or greater than that expressed on uninfected cells. Despite the increased expression of class I Ag, IFN-gamma could not protect these adenovirus-infected cells from NK cell-mediated lysis. Thus, dl327 or dl801 infection prevented IFN-gamma's induction of cytolytic resistance to NK cell-mediated killing but left IFN-gamma's induction of class I Ag intact. Surface class I Ag levels were substantially higher on IFN-gamma-treated, dl327-, and dl801-infected cells in comparison to cells infected with wild type adenovirus serotype 5. Again, higher target cell levels of class I Ag did not correlate with increased resistance to NK cell-mediated lysis because there was equivalent NK cell-mediated killing of IFN-gamma-treated adenovirus serotype 5-, dl327-, or dl801-infected cells. Thus, IFN-gamma only protects uninfected cells from NK cell-mediated killing, irrespective of target class I Ag levels, and thereby concentrates NK lytic activity on just adenovirus-infected cells. These data demonstrate that IFN-gamma's ability to protect target cells from NK cell-mediated cytolysis is unrelated to IFN-gamma's induction of surface class I MHC Ag.     

Interferon-gamma upregulates the susceptibility of human neuroblastoma cells to interleukin-2-activated natural killer cells

The influence of interferon-gamma on the susceptibility of neuroblastoma cells in cell-mediated killing was investigated. Neuroblastoma cells were only weakly susceptible targets for peripheral mononuclear cells. However, enrichment of natural killer (NK) cells or activation of NK cells with interleukin-2 resulted in a considerable increase of neuroblastoma cell lysis. Pretreatment of neuroblastoma targets with interferon-gamma additionally increased the susceptibility to enriched NK cells as well as to interleukin-2-activated NK cells. The conjugate formation between enriched NK cells and the neuroblastoma targets was not affected by the pretreatment of the targets with interferon-gamma. Concomitantly, treatment of the neuroblastoma targets with interferon-gamma resulted in a strong induction of otherwise poorly expressed major histocompatibility complex (MHC) class I antigen expression. These results suggest that the increased expression of MHC class I antigens on target cells is not always correlated with decreased sensitivity for NK cells but can also be followed by an increased susceptibility for NK cells.     

Perforin-dependent cryptococcal microbicidal activity in NK cells requires PI3K-dependent ERK1/2 signaling

Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the alpha-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.     

Studies on the lethal hit stage of natural killer cell-mediated cytotoxicity. I. Both phorbol ester and ionophore are required for release of natural killer cytotoxic factors (NKCF), suggesting a role for protein kinase C activity

Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC.     

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target structures which trigger killing activity of NK cells are determined by the phenotype of the target cell and are dependent on its MHC class I expression disregarding the haplotype of the cell.     

Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (Fc gamma RIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with Fc gamma RIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.     

Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis.

The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.     

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.     

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.