Role of serotonin in the regulation of human natural killer cell cytotoxicity

Addition of serotonin to mixtures of target cells and natural killer (NK)-enriched human mononuclear cells (MNC) in a 4-hr 51Cr-release assay strongly augmented NK cell cytotoxicity (NKCC) vs K562, Chang, or Molt-4 target cells. The effect was dose dependent at serotonin concentrations of 10(-4) to 10(-7) M, expressed at several effector to target cell ratios, and required the presence of accessory monocytes. A 5-HT1-specific receptor agonist, 8-OH-DPAT, mimicked the enhancing properties of serotonin with similar potency. Equimolar concentrations of the mixed 5-HT1/5-HT2 receptor antagonist cyproheptadine, but not the 5-HT2-specific antagonist ketanserin, completely blocked the serotonin-induced NKCC enhancement. Monocyte/NK cell mixtures incubated with serotonin for 1 hr produced a soluble factor that could enhance the cytotoxicity of autologous, NK-enriched cells depleted of monocytes, which did not respond to serotonin alone. The factor displayed no IFN or IL 2 activity as judged by the lack of antiviral activity and inability to support the growth of an IL 2-dependent cell line. In the presence of monocytes, serotonin (10(-5) M) was considerably more effective than human IFN-alpha or IFN-gamma at optimal concentrations and was about equally effective as IL 2 at a final concentration of 50 U/ml in a short-term NK assay. The potency and efficacy for serotonin were similar to that earlier reported for histamine in monocyte-containing effector cells. The NKCC-enhancing effect of serotonin was additive to that induced by IFN-alpha, IFN-gamma, or IL 2, but not to histamine. The presented data suggest an earlier unrecognized, serotonin receptor-mediated regulation of human NK cells.     

Histamine H2-receptor-mediated regulation of human natural killer cell activity

We have investigated effects of histamine on the spontaneous cytotoxic activity of human natural killer (NK) cells in vitro. Addition of histamine (10(-3) to 10(-7) M) to assay cultures of Percoll-fractionated mononuclear cells (MNC) and erythroleukemic K 562 target cells resulted in a strong enhancement of the cytotoxicity of low-density MNC, enriched for NK cell cytotoxicity (NKCC). No enhancing or suppressing effects of histamine could be detected after removal of monocytes/adherent cells from the effector cell suspensions. When unfractionated MNC were used as NK effectors, similar results were obtained, i.e., dose-dependent enhancement of NKCC by histamine in the presence of monocytes and lack of effect in nonadherent effector cells. Freshly isolated monocytes displayed low spontaneous cytotoxicity against K 562 targets and were not induced by histamine. The histamine-induced enhancement was mimicked by dimaprit, a specific histamine H2-receptor agonist, but not by N-methyldimaprit, a chemical control for H2-receptor agonist activity of dimaprit. Furthermore, the enhancement was completely antagonized by the specific histamine H2-receptor antagonists cimetidine and ranitidine. The effect of histamine could not be ascribed to endogenous interferon (IFN) production, since no IFN activity could be detected in histamine-treated MNC effectors. Also, the enhancing effects of histamine and human leukocyte IFN-alpha were clearly additive. On the basis of these findings, we suggest that histamine, via specific activation of H2 receptors, may be an important regulator of human NK cell activity.     

Augmentation of human natural cytotoxic cell activity by leukotriene B4 mediated by enhanced effector-target cell binding and increased lytic efficiency

The addition of leukotriene B4 (LTB4) to cytotoxicity assays measuring natural killer (NK) or natural cytotoxic (NC) cell activities resulted in significantly augmented killing of K562 or herpes simplex virus (HSV)-infected target cells, respectively. Since the mechanism of cytotoxicity implies several steps, including the binding of effectors to targets which is Mg2+-dependent and the programming of lysis of the target which is Ca2+-dependent, we undertook to define the step(s) at which LTB4 acted in augmenting cytotoxicity. Our results showed that LTB4 significantly increased the percentage of effector-target conjugates when K562- or HSV-infected targets were incubated with lymphocytes. Maximal binding occurred at a concentration of LTB4 of 1 X 10(-10) M. Preincubation of lymphocytes and not target cells with LTB4 was sufficient to observe the increased binding. PBML binding to and killing of the NK-resistant target clone I, derived from K562, was not enhanced by LTB4. In the absence of Ca2+, cytotoxicity was impaired and LTB4 could not restore it. Use of a single cell lytic assay demonstrated augmented efficiency of lysis of both K562 and HSV-infected targets in the presence of LTB4. These findings suggest that LTB4 may augment natural cytotoxicity by enhancing target cell recognition by cytotoxic effector cells and subsequently by augmenting their lytic efficiency.     

The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.

Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

Role of interferon in natural kill of HSV-1-infected fibroblasts

The production of interferon during natural killer (NK) assays against HSV-1-infected fibroblasts (NK(HSV-1)) was studied to determine whether this interferon was responsible for inducing the preferential lysis of herpes-virus-infected target cells over uninfected target cells. The interferon produced during NK(HSV-1) assays was analyzed and found to have the properties of HU-IFN-alpha. Little or no IFN was produced during NK assays against uninfected fibroblasts (NK(FS)) or K562 (NK(K562)) cells. Although the appearance of interferon in the culture supernatants seemed to parallel the development of cytotoxicity during NK(HSV-1) assays, the levels of cytotoxicity and IFN generated did not correlate, arguing against a strict quantitative dependence of cytotoxicity upon IFN production. NK(K562) and NK(FS) cytotoxicity developed with little or no production of IFN. When IFN-pretreated effector cells were used, there was still a preferential lysis of infected over uninfected target cells. This preferential lysis by IFN-treated effector cells of infected over uninfected targets was seen as early as 2 hr into the assay. Anti-IFN antibodies added to the NK assays, although neutralizing all the IFN produced during the assays, had no effect on NK(FS) or NK(K562) cytotoxic activity and caused a slightly reduction of NK(HSV-1) activity only in one of three experiments. We conclude that although IFN is generated during NK(HSV-1) assays, this IFN cannot solely account for the increased lysis of infected over uninfected cells and that NK(HSV-1) activity is in some other way dependent on the virus infection.     

Cytotoxic studies in human newborns: lessened allogeneic cell-induced (augmented) cytotoxicity but strong lymphokine-activated cytotoxicity of cord mononuclear cells

Nonspecific cytotoxic responses such as natural killer activity can be increased in vitro by incubating effector cells with soluble factors or allogeneic cells. We sought to determine if newborn cells, known to be deficient in most cytotoxic responses, including resting NK activity, could develop enhanced cytotoxic responses following incubation with allogeneic cells (augmented cytotoxicity) or with lymphokines (lymphokine-activated cytotoxicity). Cord whole mononuclear cells (WMC) incubated with irradiated Raji cells for 5 days develop lower levels of cytotoxicity toward K562 targets at both a 20:1 effector:target (E:T) ratio (39 +/- 2.7% vs 49 +/- 3.6%) and a 10:1 E:T ratio (29 +/- 2.6% vs 40 +/- 3.6%) than do adult cells. Lessened specific cytotoxicity of cord cells developed toward the sensitizing Raji cells was also observed at both E:T ratios. Attempts to enhance the induced cytotoxicity by incubation with interferon or isoprinosine were unsuccessful. In contrast, lymphokine (i.e., interleukin 2)-activated killer (LAK) cytotoxicity is not deficient in cord WMC. Indeed, the level of LAK cytotoxicity is equivalent to that observed with similarly treated adult cells despite a lower baseline level of cytotoxicity toward the target cells. In the presence of purified IL-2 for 5 days, cord WMC cytotoxicity against K562 cells increased from 12 +/- 2.6 to 71 +/- 4.2% and against Raji cells increased from 9.6 +/- 2.5 to 48 +/- 6.7%. Similarly treated adult cells increased their killing against K562 from 23 +/- 4.2 to 61 +/- 4.5% and against Raji from 12 +/- 3.0 to 36 +/- 5.3%. This substantial lymphokine-activated cytotoxicity of newborn cells suggests the possibility of therapeutic intervention with purified lymphokines in neonatal infections or neoplasms.     

Mechanism of cell contact-mediated inhibition of natural killer activity

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.     

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.     

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.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Detection of double target binders at the single cell level: an evaluation of the specificity of NK and MLC-induced NK-like cells

MLC-generated cells were tested on 7 consecutive days in the single cell cytotoxicity assay to determine the kinetics of natural and allospecific killing. Maximum cytotoxicity to the NK-sensitive target, K562, was found on Day 3 of MLC with an increase at that time in both the number of cells binding and the number of cells killing K562. The maximum allospecific response was found on Days 6 and 7 with an increase in cells able to bind and kill the alloantigen-bearing target. To determine whether the anti-K562 and allospecific killing were mediated by the same effector cells or different cell populations, both targets were tested simultaneously in the single cell assay. At no time during the 7 days were cells detected capable of simultaneously binding both K562 and allospecific targets. These data indicate that there are two different cell populations responsible for allospecific cytotoxicity and MLC-induced NK-like cytotoxicity. The cytotoxic specificity of unstimulated and MLC-generated NK-like cells was also investigated. When two different NK-sensitive targets (e.g., K562 and MOLT-4) were tested together in the single cell assay, there was no concurrent binding of targets by either fresh PBL prior to MLC stimulation or Day 3 MLC-generated cells. When unstimulated effector cells were enriched for NK activity by Percoll density gradient centrifugation, only a small number of effector cells simultaneously binding two different NK-sensitive targets was detected in the single cell assay. These results imply that the NK cell population is heterogeneous and composed of subpopulations recognizing diverse target specificities.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Monocyte and NK cell cytotoxic activity in human adherent cell preparations: discriminating effects of interferon and lactoferrin

The comparative cytotoxic specificities of freshly isolated human adherent and nonadherent blood mononuclear cells were examined against seven established target cell lines in 4 and 18 hr chromium release assays. The relative sensitivity of each target cell line to the cytotoxic effects of both adherent and nonadherent effector cells in cultures was identical. Moreover, the relative enhancing effects of interferon on cytotoxicity by both effector cell types were also identical. These adherent cell preparations were contaminated with up to 6% NK cells, as demonstrated by OKM1 staining and flow microfluorometry. These NK cells were loosely adherent and could be removed by vigorous wash procedures. The remaining tightly adherent monocytes also had the capacity to kill K562 cells and Chang cells, but these cytotoxic effects could not be increased by interferon. Enhancement by lactoferrin, however, was consistently observed. Treatment of mononuclear cells with Leu-lla, a monoclonal antibody that reacts with all NK cells, also abolished the enhancing effects of interferon, but not lactoferrin. These studies suggest that caution must be exercised in attributing all cytotoxic activities in adherent cell preparations to monocytes, and that lactoferrin and interferon can be used as functional probes to detect two distinct blood mononuclear cell subsets with natural cytotoxicity.     

Natural killer cell activity correlates with the amount of beta 2-microglobulin on human peripheral blood lymphocytes

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.     

Decay-accelerating factor expression on either effector or target cells inhibits cytotoxicity by human natural killer cells.

Previous studies have shown that freshly isolated CD16+ NK cells are deficient in the expression of decay-accelerating factor (DAF), or CD55, a membrane regulator of C3 activation. In this study we investigated the significance, for NK cell-mediated lysis, of DAF expression on the target and effector cells. The effect of DAF expression on the susceptibility of NK cell targets was investigated by several means: first, DAF- cell lines were cloned from K562; second, the cloned DAF- cells were reconstituted with exogenous purified DAF; and third, anti-DAF F(ab')2 was used to block DAF function on the DAF+ K562 cells. Consistently, the presence of DAF in the target cell membrane, either naturally occurring or experimentally incorporated, afforded the target cell protection against lysis, and this protection could be blocked with anti-DAF. Similarly, targets for antibody-dependent cell-mediated cytotoxicity with exogenous DAF incorporated in their plasma membrane became less sensitive to antibody-dependent cell-mediated cytotoxicity by NK cells compared with the same target cells without incorporated DAF in their membranes. DAF incorporated in the plasma membranes of the effector NK cells made the NK cells less effective at killing K562 targets. The known function of DAF is to regulate C3 activation, and we were able to demonstrate that the isolated NK cell is capable of releasing C3. It is also possible that the participation of DAF in NK cell function represents a new, noncomplement-dependent function for DAF.     

Augmentation of natural killer cell activity by ImuVert: a biological response modifier derived from Serratia marcescens

The natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) from healthy human volunteers was studied following in vitro incubation with ImuVert, a biological response modifier derived from the bacterium Serratia marcescens. Exposure of these cells to ImuVert for as little as 10 minutes followed by an additional incubation in vitro of at least 12 hours and optimally 18 hours resulted in a substantial, consistent, and dose-dependent augmentation of NK cell activity against K562 tumor cells. Additional studies indicate that the augmented cell expressed the leu 11 cell surface marker and that peripheral blood monocytes were essential in the induction of augmented NK cell activity but were not the effector cell of NK activity.     

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.