Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VI. Characterization of human, rat, and murine natural killer cytotoxic factors

Recent evidence has implicated natural killer cytotoxic factors (NKCF) as the lytic mediators of NK cell-mediated cytotoxicity reactions. The objective of this study was to examine and compare some of the biochemical and functional characteristics of human, rat, and murine NKCF. Supernatants containing NKCF were generated by stimulating effector cells with Con A or U937 (for human PBL) or YAC-1 (for rodent spleen cells) and tested for cytotoxic activity in a 20-hour (rodent) or 24-hour (human) 51Cr release assay. NKCF activity was inactivated by heating to 63 degrees C, 8 M urea, pH 2, and reduction and alkylation. These factors were highly sensitive to trypsin, moderately sensitive to papain and resistant to neuraminidase. Adsorption of human NKCF to U937 cells is inhibited by mannose-6-phosphate and adsorption of rodent NKCF to YAC-1 cells is inhibited by alpha-methyl-D-mannoside and fructose-6-phosphate. Oxidation of NKCF with sodium periodate abolished lytic activity. Pretreatment of NKCF with Con A but not pretreatment of target cells inhibited lytic activity. NKCF activity eluted in a single broad band of apparent MW of 15,000-40,000 after fractionation by HPLC gel permeating chromatography. Pooled fractions containing NKCF activity were subjected to some of the same tests performed on whole supernatants. Test result with semipurified NKCF confirmed that these factors are inactivated by trypsin or sodium periodate and that mannose-6-phosphate inhibits their binding to target cells. There were no major differences observed in NKCF produced by the three different species whether stimulated by Con A or NK-sensitive tumor cells. The evidence indicates that NKCF are glycoproteins in which disulfide bonding is essential for lytic activity. Furthermore, it appears that carbohydrate residues expressed on NKCF molecules are involved in the binding of these factors to the target cell membrane.     

Depressed natural cytotoxicity but normal natural killer cytotoxic factor (NKCF) production by mononuclear cells derived from patients with hepatocellular carcinoma

Summary This study investigated the relation between the production of natural killer cytotoxic factors (NKCF) and the phenomenon of natural killing (NK) activity against target K562 cells. Two different models of defective NK cell activity were employed. In the first instance, cytotoxic activity of mononuclear cells (MN) derived from patients with hepatocellular carcinoma was compared to the ability of these cells to produce NKCF. Although direct cytotoxicity was considerably impaired in these patients, the ability of their MN to produce NKCF when stimulated with K562 cells was found to be normal. In the second model, MN treated with the lysosomotropic drug monensin showed considerably reduced direct cytotoxic activity, although they were capable of producing normal amounts of NKCF when activated by K562 cells. These results therefore indicate that there is no correlation between NK activity and corresponding NKCF release, and suggest that NKCF production and activity is independent of direct NK cytotoxic activity.     

The cytotoxic activity of human natural killer cells requires an intact secretory apparatus

We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. I. Defective trigger on NK cells for NKCF production by target cells, and partial restoration by IL 2

Peripheral blood from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) exhibits poor NK activity in the 51Cr-release assay. The present studies were undertaken to investigate the mechanism underlying the observed defective NK cytotoxic activity. On the basis of our studies on the mechanism of natural killer cell-mediated cytotoxicity (NKCMC), a defective NK cell can result from lack or decreased frequency of effector cells, inability to recognize and bind the target cell, failure to be activated for the release of NK cytotoxic factors (NKCF), and/or failure to synthesize or secrete NKCF. Each of these various possibilities was examined. Single cell analysis revealed that the frequency of NK cells was comparable to controls, and although the NK cells bind to the NK-sensitive target, the bound target is not lysed. These results suggested that the defect in NK cells was not due to depletion of NK cells or to a defect in recognition structures, but that it was located at the postrecognition event. We previously demonstrated that after binding to target, the NK cell is stimulated to release NKCF in the supernatants and NKCF lyse specifically NK-sensitive targets. Accordingly, we investigated the activation of NK cells from AIDS and ARC patients for release of NKCF. After coculture with the stimulator cell, the patients' NK cells failed to release active NKCF in the supernatant. However, the cells released NKCF after stimulation with the lectin Con A or a mixture of TPA and ionophore, albeit to a lesser extent than controls. These results suggested that AIDS and ARC NK cells are defective in the trigger involved in release of NKCF. Further studies were done to investigate whether the immunomodulator IL 2 can restore the functional activity of the defective NK cells. Treatment with IL 2 resulted in augmented NK cytolytic activity, but did not reach control levels of activated cells from normal controls. Furthermore, the patients' IL 2-treated cells recover partially the ability to be stimulated by NK cells and to release NKCF. These results suggest that the trigger for NKCF production and the cytolytic function of the patients' NK cells are regulated by IL 2. By delineating the stage at which the AIDS and ARC NK cells are defective, it is now possible to monitor their recovery and to investigate the effect of various biologic response modifiers in restoring NK activity.     

Mechanism of NK activation by OK-432 (Streptococcus pyogenes). I. Spontaneous release of NKCF and augmentation of NKCF production following stimulation with NK target cells

The biological response modifier OK-432 (Picibanil) (manufactured in Japan) is produced by lyophilization of cultures of the low virulent Su strain of group A Streptococcus pyogenes of human origin. This preparation has been shown to have multiple effects on the immune system and has been used as an anti-cancer therapeutic agent in man. It has been shown that OK-432 augments the cytotoxic activity of human natural killer (NK) cells. We have proposed that natural killer cytotoxic factors (NKCF) derived from NK cells play a role in the mechanism of NK cell-mediated cytotoxicity (CMC). The present study investigates the underlying mechanism of the OK-432-mediated enhancement of NK activity by determining whether OK-432 has an effect on the induction and activity of NKCF produced by NK cells. Treatment of peripheral blood lymphocytes (PBL) with OK-432 for 20 hr and wash resulted in significant augmentation of NK CMC and this enhancement was dependent on the concentration of OK-432 used. Coculture of the OK-432-treated PBL with U937 resulted in a several-fold enhanced production of NKCF in the supernatant. The NKCF produced were similar to those produced by untreated effector cells in that they had the same NK target specificity for lysis. The time kinetics of stimulation of PBL with OK-432 for optimal production of NKCF was found to be 8-12 hr. It was also observed that culture of OK-432-treated PBL in the absence of stimulator cells spontaneously release significant amounts of NKCF into the supernatant. The supernatant containing NKCF was tested for interleukin 2 (IL-2) activity using an IL-2-dependent HT-2 line. It was found that there was no direct correlation between the levels of NKCF and IL-2 activity. The results of this study demonstrate that OK-432 stimulates NK cells to produce NKCF in the presence or absence of stimulator cells. The optimum concentration of OK-432-induced augmentation of NK CMC paralleled that seen for optimum NKCF production, suggesting that one mode of action of OK432 is to enhance NKCF production in a manner reminiscent of IFN and IL-2. The results also point out that OK-432 acts by a mechanism independent of the action of IL-2.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.     

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.     

The "lazy" NK cells of Chediak-Higashi syndrome

Natural killer (NK) function, measured in a short-term (4-hr) 51Cr-release assay, is profoundly depressed in circulating PBL of donors with Chediak-Higashi syndrome (CHS). In this study, we demonstrate that CHS NK cells can express relatively normal lytic function after prolonged exposure in vitro to high levels of activating as well as cytotoxic stimuli. After activation with the human cloned interferon (B1) for 24 hr, CHS NK cells have lytic activity comparable to unactivated normals in a 4-hr 51Cr-release assay. In addition, after 5 days of activation with mitomycin C-treated B cell lines, CHS NK cells have levels of activity similar to those of activated normals but are defective in generating cytotoxic cells capable of lysing the stimulator B cell. Even though CHS NK cells are defective in a 4-hr 51Cr-release assay, after 16 hr they enhance their killing capability 200 to 400-fold. In fact, after 16 hr of interaction with K562 target cells, CHS NK cells are capable of releasing NK soluble cytotoxic factors. These results are consistent with the hypothesis that CHS NK cells have all the necessary cellular structures and molecules required for them to function as lytic effector cells, but their lack of cytotoxic function is due to a relative refractoriness in initiating the post-binding lytic mechanism.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Effect of altered membrane structure on NK cell-mediated cytotoxicity. III. Decreased susceptibility to natural killer cytotoxic factor (NKCF) and suppression of NKCF release by membrane rigidification

We have shown recently that alteration of the membrane fluidity of either effector or target cells results in significant and selective inhibition of NK cell-mediated cytotoxicity (NK CMC). However, the localization of the defective stage in the NK lytic pathway is not known. In the present study, we show that rigidification of the NK-sensitive U937 target cell membrane by lipid modulation reduces its sensitivity to lysis by NK cytotoxic factor (NKCF). This resistance was not due to loss of NKCF binding sites on the target cell because target cells with rigid membranes absorbed more NKCF than control cells. The enhanced ability to absorb NKCF by membrane modification was supported by data showing that NK-resistant Raji cells lacking NKCF-binding sites absorb NKCF after lipid modification. Furthermore, consistent with the lipophilic nature of NKCF, synthetic lipid vesicles absorb NKCF. In contrast to membrane rigidification, membrane fluidization of the target cell did not change the target cell properties. Rigidification of the NK effector cell membrane abrogates it ability to secrete active NKCF when stimulated by target cells or by mitogens. Membrane fluidization of the NK effector cells did not inhibit their ability to release NKCF. The results of these studies demonstrate that inhibition of NK CMC by rigidification of the target cell membrane results in cells that are inhibited in processing bound NKCF to lysis. Inhibition of NK CMC by rigidification of the NK effector cell results in defective trigger for activation of the NKCF release mechanism.     

Analysis of rat natural killer cytotoxic factor (NKCF) produced by rat NK cell lines and the production of a murine monoclonal antibody that neutralizes NKCF

Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.     

Human natural killer cytotoxic factor. Studies on its production, specificity, and mechanism of interaction with target cells

Human peripheral blood lymphocytes cultured in vitro for 2 days in serum-free conditions produced a natural killer (NK) cytotoxic factor (NKCF) which selectively killed NK-susceptible targets. Optimal release of NKCF was achieved under serum-free conditions, while the presence of fetal calf serum inhibited both the production and activity of the factor. Mechanistic studies with NKCF demonstrated that the factor could be adsorbed by the target cells within 6 h, with no further exposure to NKCF required for maximal levels of lysis of the treated targets after additional 30-48 h of incubation, as assessed by a 111I release microcytotoxicity assay. NKCF adsorption to target cells and its cytotoxic activity were inhibited by some phosphorylated sugars (mannose-6PO4 and glucose-6PO4), but not by fructose-6PO4 or nonphosphorylated sugars (mannose, glucose, galactose). These results suggest a role of sugar-6PO4 at the level of interaction of NKCF with NK target cells. This was further supported by the finding that inhibition of target cell glycosylation by tunicamycin also inhibited absorption of NKCF to the target cells and direct killing by NKCF. Therefore, it appears that NKCF is a large granular lymphocyte produced factor which produces lysis as a result of the interaction with glycosylated structures on target cell membranes. Purification studies were performed to begin biochemical characterization of human NKCF. The results indicated that NKCF has an apparent molecular weight between 20,000 and 40,000 dalton. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action. The ability to elicit NKCF under serum-free conditions should facilitate its testing, purification, and biochemical characterization.     

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.     

Hyperthyroxinemic mice have reduced natural killer cell activity. Evidence for a defective trigger mechanism

Natural killer (NK) activity of peripheral blood lymphocytes from hyperthyroxinemic patients (Graves' disease or thyroxine (T4)-treated) is severely depressed. In order to study the relationship of thyroid hormone to NK activity, a model for hyperthyroxinemia was induced in mice by addition of T4 to the drinking water. Control mice were hypothyroid (fed propylthiouracil) or normal. Serum T4 levels were elevated (within 2 wk) in mice fed thyroid hormone. Six weeks after initiation of the diets, in vitro NK activity was undetectable in the peripheral blood, spleen, or lung mononuclear cell populations harvested from hyperthyroxinemic mice. Control mice had NK activity within the normal range. Spleen cells from mice fed thyroid hormone and control mice were tested for their ability to release lytic factors (natural killer cytotoxic factors). Lymphoid cells were incubated for 20 hr with unlabeled Yac-1 cells. Supernatants were tested for their capacity to lyse 51Cr-labeled Yac-1 cells in a 20-hr chromium release assay. Unlike controls, supernatants from hyperthyroxinemic spleen cells incubated with Yac-1 targets were unable to lyse 51Cr-Yac-1 cells. The NK cells from the mice fed T4 synthesized lytic factors because nonspecific stimuli, such as 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore A23187, induced release of lytic factors capable of lysing Yac-1 targets into the media. These data support the hypothesis that excess thyroid hormone interferes with the triggering mechanism used by NK targets to cause release of lytic molecules from NK cells.     

Lipoprotein lipase protects bovine endothelial cells from human NK cytotoxic activity

Human lipoprotein lipase (LPL), in a dose dependent fashion, significantly inhibited spontaneous human natural killer (NK) cells, but not lymphokine-activated killer (LAK) cytotoxic activity against bovine pulmonary endothelial cells. The effect was dependent on endothelial heparan sulfate (HS) sites, since heparitinase reverted it. When HS is added before LPL, NK and LAK cytotoxicity are markedly reduced. Endothelial and NK cell priming, with LPL and HS+LPL, significantly induced CD40 and CD154 expression, respectively. Furthermore, CD40 expression was inversely proportional to lytic units (R2 = 0.9, P < 0.001). Treating endothelial cells simultaneously with indomethacin, CD154 fusion protein, and Wortmanin prevented the CD40 effect increasing xenograft rejection. LPL and HS+LPL protect bovine endothelial cells from NK cytotoxicity by inducing CD40, CD154 expression, and secretion of soluble factors. The high, non-modulated expression of adhesion receptors and the low number of HS sites account for the minor effect of CD40 in LAK cytotoxic responses against bovine endothelial cells.     

Analysis of rat natural killer cytotoxic factor (NKCF): mechanism of action and relationship to other cytotoxic/cytostatic factors

Natural killer cytotoxic factor (NKCF) has been proposed as one of the factors that mediates lysis induced by natural killer (NK) cells. Recently, an excellent source of NKCF has been found to be the rat large granular lymphocyte (LGL) tumor (RNK) cell line. In this study, the kinetics of lysis of the NK-sensitive, tumor target YAC-1 by the RNK-NKCF was analyzed and found to parallel that seen with NK cell-mediated killing. RNK-NKCF was also capable of killing the NK-resistant target cell, MBL-2, over a longer time period. This study utilized monoclonal antibodies (mAbs) prepared against granule protein, previously termed "anti-NKCF mAbs." These mAbs established the nature of RNK-NKCF as compared to other known cytotoxic factors in combination with studies that show that RNK-NKCF causes both 51Cr release and nuclear degradation. Antibody inhibition experiments have verified that RNK-NKCF is unique from tumor necrosis factor (TNF), leukoregulin, or complement. Anti-NKCF mAbs were capable, however, of neutralizing the RNK cell granule activity against YAC-1 tumor target cells. Based on these results, the ability of anti-NKCF mAbs to neutralize the cytolytic function of pore-forming protein (PFP), a component of these granules, was analyzed. In these experiments, the antibodies were found to inhibit the hemolytic activity of granules. Interestingly, the antibodies were effective in inhibiting the activity of unbound granule proteins as well as those bound to sheep red blood cell (SRBC) targets. Further studies to examine the target lysis requirements demonstrated that in contrast to PFP, the RNK-NKCF was able to lyse the tumor target in the absence of calcium. In addition, treatment of targets with RNA and protein synthesis inhibitors indicated that the mechanism of lysis of NKCF is quite unique from other defined cytotoxic moieties.     

Evidence of a dynamic role of the target cell membrane during the early stages of the natural killer cell lethal hit

The role of membrane movement during the stages of human NK cytolysis has been examined by using the bifunctional protein cross-linking reagent, glutaraldehyde. The binding stage was inhibited when K562 target cells or NK cells were pretreated with glutaraldehyde. When added post-binding, after initiation of calcium pulse, glutaraldehyde did not dissociate conjugates, but inhibited NK cytolysis. In contrast to the early stages of NK cytolysis, glutaraldehyde enhanced lysis during the terminal stage, killer cell independent lysis ( KCIL ). Lysis of the preprogrammed target cells, however, was enhanced only when glutaraldehyde was added immediately after dispersion of the conjugates, before target cell lysis. The mechanism of enhancement of lysis during the terminal stages of cytolysis was further explored in assays for NK cell-derived cytolytic factor (NKCF). L929 target cells prebound with NKCF were lysed more readily in the presence of glutaraldehyde, but as in KCIL , maximum enhancement of lysis occurred when glutaraldehyde was added immediately after NKCF was bound to the target cell. These results suggest that the target cell membrane may play a dynamic role during the terminal stages of the NK lethal hit.     

Human natural killer cytotoxic factor (NKCF): role of IFN-alpha

The relationship between production of NKCF and IFN-alpha by human lymphocytes was studied. NKCF activity was generated in response to K562-inducer cells. The presence of NKCF in supernatants was always accompanied by antiviral activity, but in several experiments IFN was detected without concomitant NKCF. In no instance was NKCF activity detected in the absence of IFN. Cell lines which were good inducers of IFN-alpha were found to be good inducers of NKCF. NKCF activity of supernatants was completely adsorbed after incubation with MOLT-4 cells, whereas there was only minimal depletion of IFN-alpha activity. Most of the antiviral activity and all of the NKCF activity of preformed supernatants was neutralized by anti-IFN-alpha serum, whereas anti-IFN-gamma serum and pH2 inactivation had minimal effect on either activity. Addition of IFN-alpha to neutralized supernatants restored NKCF activity. These experiments support the hypothesis that IFN-alpha is involved in the modulation of NKCF-lytic activity. Both antiviral and NKCF activities were abrogated when anti-IFN-alpha serum was added to cultures of lymphocytes plus inducer cells (induction phase). The addition of purified IFN-alpha to such cultures was effective in allowing resumption of NKCF activity; however, addition of IFN-gamma to these cultures did not overcome this block. The addition of purified IFN-alpha directly to supernatants generated in the presence of anti-IFN-alpha serum could not restore their NKCF activity, thereby suggesting an additional requirement for IFN-alpha in the production of NKCF. The possible role of IFN-alpha in the generation of NKCF and expression of its lytic activity is discussed.