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.     

Evidence for an early calcium-independent event in the activation of the human natural killer cell cytolytic mechanism

In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium.     

Role of lipoxygenation in human natural killer cell activation

Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC.     

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.     

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.     

Cholera Toxin Inhibits Lethal Hit Stage of Natural Killer Cell-Mediated Cytotoxicity

Cytolytic process which was affected by cholera toxin (CT) resulting in the loss of natural killer (NK) cell activity was analyzed. Conjugate formation assay, membrane phospholipid methylation assay and serine esterase (granzyme A) release assay were used to determine the stage of the CT-induced inhibition of NK cell-mediated cytotoxicity. A human NK cell line YT cell-mediated cytotoxicity was completely abolished by CT pretreatment or addition of CT to the assay system. The conjugate formation assay revealed that the binding between YT cells and target cells was not affected by CT. The defined triggering stage which is coupled with membrane phospholipid methylation was not affected by CT treatment, either. On the other hand, the lethal hit stage which is represented by serine esterase (SE) release was completely inhibited by CT treatment of YT cells. Therefore, CT inhibits the stage after binding and triggering—i.e., lethal hit stage of NK cell-mediated cytotoxicity. The results also suggest that there exists a CT-sensitive negative cytotoxic signal transduction pathway as well as usual positive signal transduction pathway and these pathways might cross talk each other in the NK cell cytotoxic process.     

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.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Target cell-directed inactivation and IL-2-dependent reactivation of LAK cells

In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2.     

Inhibition of lymphokine-activated killer cell-mediated cytotoxicity by phorbol ester

As previously reported, the culture of mouse spleen cells in the presence of high amounts of human rIL-2 for 4 days caused proliferation and generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. However, an addition of PMA to the culture resulted in a striking inhibition of the generation of LAK cells. In contrast, IL-2-induced cell proliferation, IL-2R expression, and LFA-1 expression were enhanced by the addition of PMA. Kinetic studies revealed that the addition of PMA during the final 24 h, but not 4 h, of the culture was sufficient to inhibit the generation of LAK cells. The same inhibition of LAK activity was observed when 4-day cultured LAK cells were pretreated with PMA for over 12 h before cytotoxicity assay. Flow cytometry analysis showed that PMA pretreatment had no effect on the binding of LAK cells to target cells. PMA pretreatment of LAK cells caused total disappearance of protein kinase C (PKC) activity from LAK cells concomitant with the loss of LAK activity. However, PMA-pretreated LAK cells cultured for another 24 h in the absence of PMA revealed levels of PKC activity and cytotoxicity identical with untreated LAK cells. These results strongly suggest that PMA-induced down-regulation of LAK cell-mediated cytotoxicity is due to the inactivation of PKC-dependent transduction systems that are essential post LAK cell-target cell binding.     

Induction of cytotoxic effector activity in the HL-60 promyelocytic cell line by incubation with phorbol myristate acetate: a model system of human spontaneous monocyte-mediated cytotoxicity

In an attempt to develop a constant and reproducible in vitro system for a detailed analysis of cytotoxic effector mechanisms of nonimmune mononuclear phagocytes, the HL-60 promyelocytic cell line was studied for its cytotoxic action on chicken erythrocyte target cells. HL-60 cells cultured in complete medium were found to be noncytotoxic for chicken erythrocytes in an 18-hr 51Cr-release assay. These cells have been shown to acquire several characteristics of mature macrophages upon incubation with phorbol myristate acetate (PMA), and when PMA was included in the medium during the assay, the HL-60 cells became strongly cytotoxic to the target cells in the absence of exogenous antibody, lectin, or serum complement. Freshly isolated peripheral blood monocytes also became cytotoxic in the presence of PMA, whereas peripheral blood lymphocytes and the U937 histiocytic cell line did not. Detectable target lysis was observed between 4 and 8 hr after HL-60 stimulation with PMA, and HL-60 cells prestimulated with PMA for 24 hr retained their cytotoxic activity following washing and assay in PMA-free medium. Cytotoxic HL-60 cells developed after exposure to 10(-6) to 10(-9) M PMA, and significant target cell lysis occurred at effector:target cell ratios as low as 0.5:1. The PMA-induced HL-60-mediated cytotoxic response was markedly inhibited by blockers of protein synthesis, inhibition of microfilament function, and depletion of cellular superoxide and hydrogen peroxide. Interestingly, cytotoxicity of HL-60 cells for chicken erythrocyte targets was modulated by the direct addition of certain simple saccharides to the assay in a fashion similar to that observed with spontaneously cytotoxic mononuclear cells from several vertebrate and invertebrate species. Thus, the cytolytic effector function induced in HL-60 cells by incubation with PMA presents a useful model for the study of cellular cytotoxic mechanisms as well as the mechanisms utilized by nonimmune cells in the recognition of non-self.     

Inhibition of postbinding target cell lysis and of lymphokine-induced enhancement of human natural killer cell activity by in vitro exposure to ultraviolet B radiation

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to ultraviolet B (uvB) radiation has been shown to inhibit natural killer (NK) cell-mediated cytotoxicity in a dose-dependent fashion. The purpose of this study was to examine the manner by which uvB produced these deleterious effects. Inhibition of NK activity was not due to lethal injury to NK cells since the viability of cell populations enriched for NK activity was greater than 90% with the uvB doses employed. uvB appeared to directly affect NK cells since procedures which removed suppressor mechanisms, such as removal of monocytes and pharmacologic inhibition of the cyclooxygenase pathway, failed to reverse the response. Furthermore, no suppression of activity of unirradiated NK cells could be produced by coincubation of unirradiated NK cells with uv-irradiated NK cells. When the single cell assay for binding and killing was employed to determine at which stage in the lytic sequence inhibition occurred, it was found that binding was normal but lysis of bound targets and the recycling capacity of active NK cells were markedly reduced. At uvB doses above 50 J/m2, both interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were ineffective in augmenting NK cell-mediated cytotoxic reactions after cells had been irradiated with uvB. Furthermore, incubation of NK cells with IFN-alpha prior to irradiation failed to protect against the inhibitory effects. These studies provide evidence that in vitro exposure of NK cells to uvB radiation inhibits their function by a direct nonlethal effect and that this inhibition occurs selectively at the postbinding stage of target cell lysis.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

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.     

Phosphatidylinositol metabolism accompanies early activation events in tumor target cell-stimulated human natural killer cells

This study examined the role of phospholipid metabolism in human natural killer (NK) cells upon activation by tumor target cells(TC). The effector cell (EC) population consisted of peripheral blood lymphocytes enriched for NK cells. Upon a 5-min exposure of EC to the NK-sensitive tumor TC K562 and U937, nearly four- and threefold increases in the incorporation of 32P into phosphatidylinositol (PI) occurred, respectively. In contrast, no increase in 32P incorporation into PI was seen when two NK-resistant TC were used. In addition, little or no change in the incorporation of 32P into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine took place with any of the above TC. Depletion of Leu 11b-positive cells abolished the increase in 32P incorporation into PI when K562 were used in the phospholipid assay. Furthermore, labeling kinetics of this phospholipid turnover showed that it occurred less than 5 min following exposure to NK-sensitive TC and that phosphatidic acid, a breakdown product of phosphoinositides, was produced during this 5-min period. These results indicated that metabolism of a phosphoinositide took place and that it occurred in association with early activation events in NK cells. Quercetin and dibutyryladenosine-cyclic monophosphate (dbcAMP) plus theophylline exerted profound inhibitory effects on both NK activity and PI metabolism, suggesting a linkage between the two events. The inhibitors had no effect on target cell-binding capacity, indicating that the inhibition occurred postbinding. PI metabolism took place in the absence of extracellular calcium even though NK activity was completely abolished under the same conditions. Thus, we have shown PI metabolism, but not other phospholipids, to occur in human NK cells upon exposure to NK-sensitive TC, in association with early activation events. This event was independent of extracellular calcium and could be inhibited by quercetin or dbcAMP plus theophylline.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Murine trophoblast resists cell-mediated lysis. II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.     

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity

Impaired cell-mediated immunity predisposes individuals to severe systemic HSV infections. A potential approach for enhancing antiviral immunity is to alter the specificity of T cells and NK cells so that they become cytotoxic against HSV. We describe here the use of heteroconjugate antibodies to augment the killing of HSV-infected cells. Two different types of heteroconjugate antibodies were used: 1) CD3-specific mAb, covalently linked to HSV-specific mAb (e.g., anti-CD3 x anti-HSV-1 glycoprotein C); 2) FcR-specific mAb linked to HSV-specific mAb (e.g., anti-Fc gamma RIII x anti-HSV-1 glycoprotein D). Whereas freshly isolated, PBL were not cytotoxic against HSV-infected target cells in a 5-h 51Cr-release assay, co-incubation with either heteroconjugate resulted in significant cytotoxicity. In vitro activated PBL (anti-CD3 + IL-2) also became more potent killers of HSV-infected cells in the presence of each heteroconjugate. The specificity of anti-CD3 x anti-HSV-1 and anti-Fc gamma RIII x anti-HSV-1 gD for enhancing T cell and NK cell immunity, respectively, was confirmed by using cloned, homogeneous human T cell and NK cell lines as effectors. Kinetic analysis demonstrated that as soon as the infected cells began to express HSV glycoproteins on their surface they became susceptible to this enhanced killing. Prolonged culture of HSV-infected cells with heteroconjugate antibodies and effector cells also decreased the amount of viral replication that occurred, as measured in a plaque inhibition assay. These results suggest that heteroconjugate antibodies are potent immunotherapeutic tools that enhance anti-HSV immunity.     

The effect of unphosphorylated and phosphorylated sugar moieties on human and mouse natural killer cell activity: is there selective inhibition at the level of target recognition and lytic acceptor site?

A number of different sugars were investigated for their effect on human and mouse natural killer cell (NK)-mediated cytolysis. From the pool of nonphosphorylated sugars, D-mannose, N-acetyl-D-glucosamine (NAcGlc), D-glucose, and, to a lesser extent, beta-gentiobiose were found to inhibit human NK cytolysis. Mouse NK activity against YAC-1 target cells was reduced consistently in the presence of D-mannose and NAcGlc only. The sugars, NAcGlc, D-glucose, and beta-gentiobiose, were specifically inhibitory against NK-mediated cytolysis with no inhibitory effects being observed against ADCC, monocyte-mediated cytolysis, or CTL activity. Pretreatment and washing at either the target or effector cell level as well as direct target binding assays using Percoll-purified NK cells indicated that at least NAcGlc and beta-gentiobiose function at the recognition stage of NK cytolysis. D-Mannose, which was the most effective nonphosphorylated sugar inhibitor, was capable of inhibiting all cell-mediated cytotoxic mechanisms tested (NK, ADCC, monocyte, and CTL) and its action did not appear to be solely due to an impairment in the recognition event. All the phosphorylated sugars caused significant inhibition of human and mouse NK-mediated cytolysis, although repeated analyses of sugar titration curves consistently showed mannose-6-phosphate (Man-6-P) to be the most effective inhibitor. Inhibition with the phosphorylated sugars was apparent against all cytotoxic mechanisms investigated. It is possible that these sugars may function as general metabolic inhibitors or may activate a common signal which negatively regulates cell-mediated cytotoxic mechanisms. Nevertheless, the relative degree of inhibition with the majority of these sugars (particularly Man-6-P) was greater against NK and ADCC activity than against monocyte and CTL activity. Furthermore, studies with selected well-characterized human and mouse NK-resistant target cells strongly indicated that these sugars, particularly Man-6-P, compete at an acceptor site responsible for the uptake of the NK lytic factor, which is independent of the recognition structure(s).