Effect of altered membrane fluidity on NK cell-mediated cytotoxicity. I. Selective inhibition of the recognition or post recognition events in the cytolytic pathway of NK cells

NK cell-mediated cytotoxicity results from membrane interactions between NK effector and target cells. The role of membrane fluidity in these events is not known. The present study was undertaken to investigate the effect of changes in membrane lipid fluidity of NK effector and NK-sensitive target cells on the lytic pathway of NK cell-mediated cytotoxicity. Fluidity was modulated by various lipids and measured by fluorescence polarization. NK effector cells treated with phosphatidylcholine complexed with polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA) showed increased membrane fluidity. This fluidization of the effector cell membrane resulted in a significant inhibition of cytotoxic activity in the 51Cr-release assay. Single cell analysis revealed that the inhibition was due to a decrease in the frequency of NK target conjugates and reduced killing of conjugated targets. Rigidification of the NK effector cell membranes by treatment with cholesteryl hemisuccinate complexed with PVP and BSA also resulted in inhibition of cytotoxicity. This inhibition was post binding, because binding was increased and lysis was abrogated. Fluidization of K562 target cell membranes caused a slight but insignificant increase in their lysis by NK cells without affecting the binding step. On the other hand, rigidification of K562 membranes decreased the sensitivity of these target cells to lysis. Single cell analysis revealed that this inhibition of NK lysis is post binding, because the frequency of killers was significantly decreased. It was also shown that membrane rigidification of target cells that were programmed for lysis during the lethal hit stage and subsequently separated from effector cells, rendered the programmed cells resistant to killing during the killer cell-independent lysis step. These results demonstrate that fluidization or rigidification of the plasma membrane of either effector or target cells affect different stages of the NK cell-mediated cytolytic events.     

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 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.     

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.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. V. Lack of NK specificity at the level of induction of natural killer cytotoxic factors in cultures of human, murine, or rat effector cells stimulated with mycoplasma-free cell lines

We have proposed that lysis of target cells by NK cells is mediated by NK cytotoxic factors (NKCF). According to our model, for a target cell to be NK-sensitive, it must be recognized by the NK cell, it must stimulate the release of NKCF, and it must be sensitive to lysis by these factors. This report examines whether the ability to stimulate release of NKCF is a characteristic restricted to NK-sensitive tumor cells or whether it is also a property of NK-resistant target cells. Many different types of cell lines were tested for their ability to stimulate release of NKCF in the human, rat, and murine systems. It was found that mycoplasma-free NK-sensitive cell lines, resistant cell lines, and Con A could stimulate the release of NKCF. Many different types of cell lines grown in suspension or in monolayers were found to be effective stimulators, including T or B lymphoid, myeloid, and those of histiocytic origin. Cells cultured in the absence of serum stimulated NKCF release, thus ruling out the possible involvement of serum components in stimulation. NKCF was also produced by xenogeneic combinations of effector and stimulator cells, demonstrating lack of species specificity in NKCF production. Factors stimulated by NK-resistant cell lines or by Con A exhibited the same NK target specificity as supernatants stimulated by NK-sensitive tumor cells. The finding that many different NK-resistant cell lines can stimulate the release of NKCF indicates that there is no apparent NK specificity at the level of induction of NKCF release from human, rat, or murine effector cells. Therefore, the NK specificity of a target cell is determined ultimately by its sensitivity to lysis by NKCF.     

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.     

An inducer of NKCF (NK cytotoxic factor) release: localization on target-cell membrane and initial characterization.

Natural killer (NK) cells are probably involved in the elimination of virus-infected cells and of certain tumor cells. NK cell-mediated cytotoxicity (NK-CMC) was extensively studied and was found to consist of several steps. Following recognition and conjugation between the effector and the target cell, the latter one induces release of NK cytotoxic factor (NKCF) from the effector cells. The NKCF binds to the target cell which is subsequently killed. None of the molecules involved in these steps was completely characterized. In the present study it is demonstrated that isolated membranes of target cells can effectively induce the release of NKCF. Furthermore, the activity of such isolated membranes was found to be modulated by interferon (IFN) treatment of the cells prior to membrane isolation. It was therefore concluded that an NKCF-inducing structure (NKIS) is present on plasma membranes and is distinct from the NK-recognition structure. Similarly, the sensitivity to NK-CMC could be transferred from sensitive cells to IFN-gamma-treated (NK-resistant) cells by membrane fusion with the aid of Sendai virus envelope glycoproteins. It is proposed that transfer of NKIS is responsible for the acquired sensitivity to NK-CMC. In addition, it is shown that NKIS activity was recovered following membrane solubilization and reconstitution. Its level on cell surface was modulated by treatment of cells with tunicamycin, thus indicating that NKIS was probably a cell surface glycoprotein.     

Mechanisms involved in NK resistance induced by interferon-gamma.

Human tumor cell lines were treated with interferon-gamma (IFN-gamma) and then used as target cells in NK assays to measure their ability to form conjugates and stimulate the production of NK cytotoxic factors (NKCF) and to determine their susceptibility to NKCF lysis. K562 and cell lines RS1, RS3, RS7, CAC, and CAP2, obtained from solid brain tumors, were used as targets, and peripheral blood lymphocytes (PBL) from normal donors were used as effector cells. IFN-gamma-treated cell lines had a decreased susceptibility to NKCF lysis and a decreased ability to induce the release of these factors without affecting target-effector cell binding. These results were not due to changes in HLA class I antigen expression, given that the level of HLA class I antigens on the tumor cell lines was not affected, the only exception being K562. In an attempt to further clarify the possible influence of HLA class I expression on K562, IFN-gamma-pretreated K562 cells were separated into HLA class I positive and HLA class I negative subsets for the NK assays. The results showed that both populations behaved similarly upon target-effector conjugate formation, whereas the HLA class I positive population showed a reduced susceptibility to lysis by NK cells and NKCF. Thus, these results establish that NK resistance induced by IFN-gamma is mediated by blocking the target cell's ability to activate NK cell triggering and release of NKCF and by blocking its susceptibility to lysis by these factors. This analysis helps to clarify not only the NK process but also the controversial regulatory effect of IFN in NK lysis.     

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.     

Analysis of the mechanisms involved in NK resistance induced by a new tumor factor NK-RIF

The mechanisms involved in susceptibility or resistance of neoplasic cells to lysis by NK cells are not well known. We have recently described a 12-kDa factor (NK-RIF), produced and released by different tumor cell lines, making K562 resistant to NK lysis without affecting the cytotoxic function of NK effector cells. In this paper we further study the mechanism involved in NK resistance of K562 mediated by NK-RIF and its biological implications. The results show that NK-RIF does not affect the binding capacity of target and effector cells nor the levels of HLA class I antigen expression on the target cells, as a proof that resistance to NK-mediated lysis is not always associated with a defect in target effector binding or with an increased MHC class I antigen expression. However NK-RIF-treated K562 loses its capacity to induce NK cell activation and the subsequent capacity to release NKCF and makes K562 resistant to lysis by NKCF. Therefore our results show that induction of resistance to NK cytotoxicity can be the result of the modulation of target structures responsible for inducing effector cell activation without affecting target/effector binding molecules. This indicates that the structures involved in adherence and activation of NK cells have a different nature and that molecules other than HLA participate in NK resistance.     

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.     

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.     

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.     

Mechanism of inhibition of natural killing by a glycopeptide isolated from the K562 plasma membrane

The mechanism of lysis by cytotoxic T lymphocytes, K cells, and natural killer (NK) cells is imperfectly understood at this point. In this report, material (glycopeptide) isolated from the plasma membranes of K562 cells and fractionated on lectin affinity adsorbents which has been shown to inhibit NK lysis, was used in several specific NK assays to ascertain what stages of the NK-lytic sequence is inhibited by this substance. Results indicate that this glycopeptide (a) does not inhibit initial binding, but dissociates conjugates following initial effector target interactions; (b) inhibits NK lysis beyond Ca-dependent programming, and (c) inhibits lysis induced by NK cell-derived soluble cytotoxic factors (NKCF) in a soluble factor assay. These results suggest that this glycopeptide can effect the lethal hit stage of NK lysis and may represent structures which can associate directly with NKCF.     

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.     

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.     

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.     

Liposomes can function as targets for natural killer cytotoxic factor but not for tumor necrosis factor

NK cells exert their lytic action through the release of NK cytotoxic factors (NKCF) after stimulation by the bound target cell. NKCF may be related to granule-derived perforin/cytolysin on one hand and to the pleiotropic cytokine TNF on the other hand. In the present study, we show that NKCF can also lyse artificial lipid vesicles, as had been reported previously for cytotoxic granules and cytolysin. The lysis of large unilamellar vesicles was monitored by measuring the release of the encapsulated fluorescent dye carboxyfluorescein. NKCF-induced lysis was only observed with liposomes composed of a complex mixture of lipids including acidic phospholipids. No lysis could be demonstrated if the liposomes contained phosphatidylcholine as the only phospholipid, suggesting some kind of lipid specificity for the action of NKCF. A remarkable finding was that neither recombinant nor natural TNF were able to lyse large unilamellar vesicles, irrespective of their lipid composition, indicating different ways of interaction of NKCF and TNF with artificial (and presumably also biological) membranes.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Interleukin-1 alpha and tumor necrosis factor-alpha protect cells against natural killer cell-mediated cytotoxicity and natural killer cytotoxic factor

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6.