Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Association of decreased natural and antibody-dependent cellular cytotoxicity and production of natural killer cytotoxic factor and interferon in neonates

Cord blood lymphocytes (CBL) were compared with adult peripheral blood lymphocytes (a-PBL) for their: (i) natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, (ii) target-binding capacity, (iii) ability to induce soluble natural killer cytotoxic factor (NKCF), (iv) interferon (IFN)-, interleukin 2 (IL-2)-, and lectin-induced augmentation of NK activity, and (v) ability to produce IFN against tumor targets in vitro. CBL depleted of adherent cells and Percoll-separated, NK-enriched subpopulations demonstrated significantly lower NK, ADCC, and target-binding activities compared to a-PBL. CBL produced significantly lower levels of NKCF directed against K562 tumor targets in comparison with a-PBL. Although the NK activity of CBL was not stimulated by either IFN or IL-2 to the same levels shown by a-PBL, the percentage enhancement of cytotoxicity of CBL by IFN and IL-2 was greater than that of a-PBL. Lectin-induced enhancement of cytotoxicity was significantly greater for CBL in comparison with a-PBL. Further, the ability of CBL lymphocytes to produce IFN-gamma in vitro against K562 target cells was significantly lower than that of adult PBL. These studies suggest an association between decreased NK, ADCC, and target-binding activities, induction of NKCF and IFN production by CBL, and increased susceptibility of neonates to infection.     

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.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Interferon production and tumor cell killing by human lymphocytes stimulated in mixed-lymphocyte culture

The in vitro synthesis of interferon (IFN) by human lymphocytes stimulated in mixed-lymphocyte culture (MLC) was examined. The production of IFN in MLC was restricted to T lymphocytes and maximum levels of IFN were detected in supernatants from cells incubated for 5 to 7 days. The IFN produced was identified as IFN-gamma by antibody neutralization. To identify the T cell responsible for IFN production, purified T lymphocytes were separated into subpopulations after incubation in 5 mM theophylline. Theophylline-resistant (T-res) T cells retain the ability to form sheep erythrocyte (SRBC) rosettes and are depleted in IgG Fc receptor-positive T cells (T gamma cells). Theophylline-sensitive (T-sens) T cells fail to form rosettes after theophylline treatment and are enriched in T gamma cells. In addition, analyses using monoclonal antibodies showed that T-sens cells were enriched in OKM1-, HNK-1-, and 7.2-positive cells and T-res cells contained increased numbers of 9.6- and OKT4-positive cells. Following MLC stimulation, equivalent levels of IFN-gamma were produced by T-res and T-sens cells and both subpopulations maintained natural killer (NK)-like cytotoxicity against K562 target cells. Addition of partially purified IFN-gamma to unstimulated T-res and T-sens cells resulted in the maintenance of NK-like cytotoxicity in a manner analogous to that observed after MLC. Additional experiments indicated that peripheral blood lymphocytes depleted of 9.6- or OKM1-positive cells by complement-mediated lysis were devoid of cytotoxicity against K562 cells. Furthermore, MLC stimulation of 9.6- or OKM1-depleted cells failed to restore cytotoxic activity. In summary, these experiments demonstrate that the maintenance of NK-like cytotoxicity by MLC-stimulated T cells is associated with the synthesis of IFN-gamma, that MLC stimulated T-res and T-sens T-cell subsets produce equivalent amounts of IFN, and that 9.6- or OKM1-positive cells are required for the maintenance of NK-like cytotoxicity in MLC.     

Autologous enhancement by interferon of natural killer activity of human peripheral blood lymphocytes

The in vitro action of interferon (IFN)-alpha and IFN-gamma from six healthy donors and ten patients with multiple sclerosis (MS) on natural killer (INK) activity of peripheral blood lymphocytes (PBL) was studied in an autologous system. The NK activity of PBL was detected by a cytotoxic test using (3)H-uridine human erythromyeloblast K562 cells. Autologous IFN-alpha and IFN-gamma did not augment NK activity of PBL from healthy donors in vitro, whereas in samples from MS patients the IFNs strongly stimulated NK cell cytotoxic function. This stimulation suggests the existence of an inhibitor of regulatory IFN action, that is produced in healthy donors simultaneously with IFN in response to IFN induction, but which is lacking in commercial IFN preparations. The factor-containing supernatants from healthy donors reduced the stimulatory action of autologous IFNs in patients with MS almost until complete blockade. Because this inhibitor was absent in patients with MS, deficiency of an inhibitor of IFN regulatory action in MS could open the way to treatment of this compartment of the immune system.     

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.     

Mechanism of defective natural killer cell activity in patients with AIDS is associated with defective distribution of tubulin

Previous studies have demonstrated the importance of some cytoskeleton components in killing mechanisms. In fact, a microtubule and microfilament (MF) rearrangement in the lytic sequence of CTL and NK cells has been observed. In particular, MF seem to be related to the binding phase, because MF inhibitors suppress the binding of NK cells to the target, whereas microtubule inhibitors suppress only the killing phase. In this paper, the distribution of two cytoskeleton components, actin and alpha- and beta-tubulin, has been studied in PBL from AIDS patients, who maintain the capacity to bind to the target cell line K562 but are not able to kill it. PBL were labeled with mAb to these two cytoskeleton components, and then indirect immunofluorescence was used to visualize their distribution in the conjugates. A normal polarization of actin in the effector PBL was found, whereas no tubulin rearrangement was evident in the effector and target cells. On the contrary, in conjugates of PBL or large granular lymphocytes from normal donors and K562, a polarization of actin in the effector cell and a polarization of tubulin both in the effector and in the target cells, at the site of the attachment, was evident. These data suggest that a deficiency of tubulin rearrangement may underlie the inability of the NK cells from AIDS patients to kill their target.     

Natural cytotoxicity of lymphocytes and monocytes and its augmentation by OK432 in melanoma patients

Summary Lymphocytes and monocytes from the peripheral blood of 30 patients with malignant melanoma were tested for natural cytotoxicity against K562 cells in a 3-h ⁵¹Cr-release assay, and the effects of OK432 (a streptococcal preparation) on the cytotoxicity were examined. The lymphocyte cytotoxicity of melanoma patients was similar to that of normal donors and control patients with benign skin disease. Furthermore, the lymphocyte cytotoxicity of melanoma patients was not correlated to the stage of the disease. Similarly, lysis of K562 cells by monocytes isolated by adherence to autologous serum-coated plastic dishes in melanoma patients was comparable to that of controls and not associated with the stage of the disease. Positive monocyte reactions were recorded in 10 of 30 (33%) melanoma patients, seven of 21 (33%) normal donors and three of 10 (30%) control patients. There was no correlation between lymphocyte cytotoxicity and monocyte cytotoxicity. Overnight treatment of monocytes and lymphocytes with OK432 resulted in an increase in cytotoxicity. Significant augmentation of cytotoxicity by OK432 was observed in 28% of the monocyte samples and 86% of the lymphocyte samples, while partially purified human interferon augmented cytotoxicity in 63% of the monocyte samples and all the lymphocyte samples. These results suggest that neither lymphocyte nor monocyte cytotoxicities are depressed in melanoma patients as compared with normal donors and patients with benign disease and that OK432 has a stronger stimulatory effect on lymphocytes than on monocytes.     

Natural killer cell activity in tumor-draining lymph nodes: investigations in patients with malignant melanoma and head and neck cancer

Mononuclear cells isolated from peripheral blood (PB) of patients with malignant melanoma and head and neck cancer were tested for natural killer (NK) cell activity against K562 targets. Significantly lower levels of NK activity were observed in patients with malignant melanoma with metastatic disease in comparison to stage I and control population. In head and neck cancer NK activity was normal in tumor stage 1-2 and significantly impaired in stage 3-4. Mononuclear cells (MNC) isolated from primary tumor-draining lymph nodes (LNs) had significant NK activity in 12 of 25 (48%) of melanoma and 9 of 19 (47%) of head and neck lymph nodes at E:T 40:1. Reactivity was always significantly lower than in autochthonous Preincubation (+37 degrees C, 18 h) of lymph node mononuclear cells (LNMNC) resulted in a significant increase in cytotoxicity in 36% of melanoma and 32% of head and neck LNs. Simultaneous incubation with interferon (IFN)-alpha resulted in 24% of melanoma and 44% of head and neck LNs in a significant further augmentation of NK activity. LNMNC were furthermore found to react in 2-24% (mean 7.79 +/- 2.1) with the monoclonal antibody HNK-1, which identifies a certain proportion of NK cells. No correlation was found between percentage HNK-1+ cells and the expression of NK activity. From these studies it may be concluded that tumor draining LNs contain cells which are active against K562 targets expressing low but variable levels of cytotoxicity. NK cell activity can be increased by incubation of LNMNC at 37 degrees C with or without addition of IFN. These results suggest that modulation of NK activity in tumor draining LNs might be achieved by local application of biological response modifiers, which might finally lead to an increase of local tumor defense mechanisms.     

beta-Endorphin augments the cytolytic activity and interferon production of natural killer cells

We have investigated the in vitro effects of the neurohormone beta-endorphin (b-end) on natural killer (NK) activity and interferon (IFN) production mediated by large granular lymphocytes (LGL). LGL-enriched fractions from peripheral blood mononuclear cells (PBMC) from normal human volunteers were obtained by fractionation over discontinuous Percoll gradients. LGL were preincubated with or without various concentrations of b-end or the closely related peptides alpha-endorphin (a-end), gamma-endorphin (g-end), or D-ALA2-beta-endorphin (D-ALA2-b-end), a synthetic b-end analogue. NK activity was assayed on 51Cr-labeled K562 target cells. Preincubation of LGL effectors (but not K562 targets) for 2 to 18 hr with concentrations of b-end between 10(-7) M and 10(-10) M produced significant augmentation of NK cytolytic activity (mean percentage increase: 63%). The classic opiate antagonist naloxone blocked the enhancing effect when used at a 100-fold molar excess relative to b-end. Neither a-end nor g-end could augment NK activity, whereas D-ALA2-b-end produced an enhancement comparable with that produced by b-end. In addition, incubation of LGL with b-end in the presence of phytohemagglutinin or poly I:C significantly augmented IFN production. These findings demonstrate that b-end enhances NK activity and IFN production of purified LGL, and suggests that b-end might bind to an opioid receptor on LGL that can be blocked by naloxone. These results lend support to the concepts of regulation of the immune response by neurohormones and the functional relationship between the nervous and immune systems.     

Autologous tumor killing and natural cytotoxic activity of tumor-associated macrophages in cancer patients

Summary Tumor-associated macrophages (TAM) isolated from pleural effusions and ascites fluids of cancer patients were tested for cytotoxicity against freshly isolated autologous tumor cells and K562 in a 4-h⁵¹ Cr-release assay, and in vitro effects of OK432 (a streptococcal preparation) and partially purified human leukocyte interferon (IFN) on their cytotoxicities were examined. Positive cytotoxicities against K562 were recorded for TAM samples from 2 of 23 pleural effusions and 3 of 10 ascites specimens. Tumor-associated macrophages were not cytotoxic to autologous tumor cells, while low but significant lysis was observed with tumor-associated lymphocytes (TAL) samples from 2 of 13 pleural effusions and 1 of 6 ascites specimens. In vitro treatment with OK432 resulted in an enhancement of natural cytotoxicity in 4 of 13 TAM and 10 of 15 TAL samples. An induction or augmentation of autologous tumor killing activity by OK432 was observed in 2 of 10 TAM and 8 of 11 TAL samples. In contrast, IFN failed to induce autologous tumor killing activity, although IFN-enhanced lysis of K562 was detected in 1 of 7 TAM and 2 of 9 TAL samples. These results indicated that autologous tumor killing and natural cytotoxic activities were defective in macrophages and lymphocytes at the site of the tumor growth, and both activities were strongly enhanced by OK432 rather than IFN.     

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.     

Enhancement of susceptibility of HSV-1-infected cells to natural killer lysis by interferon

The effect of interferon (IFN) on the natural killer (NK) activity of human PBL against HSV-1-infected HeLa cells was studied. Human PBL from several individuals did not consistently show a preferential lysis of HSV-1-, vaccinia-, or adenovirus type 5-infected cells with respect to uninfected HeLa cells. Treatment with IFN of effector PBL increased their lytic activity but did not alter the degree of preference on the lysis of the target cells shown by untreated PBL. Pretreatment with IFN of HSV-1-infected HeLa cells increased their susceptibility to lysis 5- to 10-fold. In contrast, identical pretreatment of the uninfected, adenovirus type 5- or vaccinia virus-infected HeLa cells before the assay decreased their susceptibility to NK lysis. This effect was not likely to be due to a block of the viral replication because other inhibitors like mitomycin C did not have the same effect. All target cells induced IFN synthesis in effector PBL cells. A similar level of IFN was induced by HSV-1-infected or uninfected HeLa cells. Pretreatment with IFN of HSV-1-infected, but not uninfected, HeLa cells induced 5 to 10 times more IFN by PBL, in good correlation with the increase in lytic activity. PBL treated with IFN, however, in conditions to give maximal stimulation of NK activity, presented the same preferential lysis of HSV-1-infected HeLa cells and synthesized similar levels of IFN as untreated PBL. In addition, HSV-1-infected HeLa cells were killed through different target structures than uninfected cells. Taken together, our results indicate an effect of IFN at the level of the NK target structures in HSV-1-infected HeLa cells by increasing either their number or, more likely, their affinity for NK cells independent of the effect of IFN in the effector cells or as an antiviral agent.     

Human lymphocyte responses to hepatitis A virus-infected cells: interferon production and lysis of infected cells

Peripheral blood mononuclear cells (PBMC) from nonimmune healthy donors who did not have antibody to hepatitis A virus lysed hepatitis A virus-infected BS-C-1 cells to a greater degree than uninfected BS-C-1 cells. The predominant effector cells were contained in the nonadherent peripheral blood lymphocyte (PBL) fraction, although some lytic activity was associated with adherent cells. Characterization of the PBL with monoclonal antibodies showed that the responsible effector lymphocytes were contained in Leu-11+ and M1+ subsets, but not in the T3+ or T4+ subsets. The phenotypes of the effector cells active in the lysis of hepatitis A virus-infected cells are similar to those of human natural killer cells that lyse K562 cells. Human PBL produced high titers of interferon-alpha (IFN-alpha) when exposed to hepatitis A virus-infected cells. These results imply that hepatitis A virus infection may be controlled by lymphocyte responses in the liver, i.e., by lymphocyte-mediated lysis of the hepatitis A virus-infected cells, and by the production of high titers of IFN-alpha by lymphocytes exposed to hepatitis A virus-infected cells. Furthermore, these results, along with the observations that hepatitis A virus infection results in a persistent noncytocidal infection in vitro, support the hypothesis that lysis of hepatocytes infected with hepatitis A virus is by lymphocyte-mediated cytotoxicity and not by virus-induced destruction of the liver cell.     

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.     

Natural killer (NK) cells as a responder to interleukin 2 (IL 2). II. IL 2-induced interferon gamma production

In the accompanying paper, we showed that natural killer (NK) cells were a major population in the naive spleens of normal mice that responded directly to a T cell growth factor, interleukin 2 (IL 2), and clonally replicated without other stimulating agents. The cloned cells growing in IL 2 showed a potent NK activity against several NK targets without addition of an NK-activating agent, interferon (IFN). In the present study, therefore, we examined whether these cloned NK cells on their own produced IFN. It was found that all NK clones growing in IL 2 produced IFN in the culture fluids. The titers of IFN produced in the IL 2-containing media correlated well with the number of growing cells. With the culture in the absence of IL 2, neither cell growth nor IFN production could be detected. Addition of Con A into the culture in the IL 2-free media showed no IFN production. The antiserum neutralizing IFN alpha and IFN beta failed to significantly neutralize IFN produced by NK clones. Treatment with either a pH of 2.0 or antiserum neutralizing mouse IFN gamma resulted in a marked reduction of IL 2-induced NK IFN, indicating that a major part of IFN produced was IFN gamma. These results indicate that IL 2 stimulates NK clones to proliferate, accompanied by IFN gamma production. The results also show that an NK clone, when stimulated with Sendai virus, produced a type 1 IFN (IFN alpha and/or IFN beta), suggesting that murine NK cells can produce both type 1 (alpha and/or beta) and type 2 (gamma) IFN, depending on inducers.     

Natural killing can be independent of interferon generated in vitro

PBL produce interferon in response to culture with the tumor cell line K562, but this production of interferon does not correlate with natural cytotoxicity. A basal level of natural killing is independent of interferon generated in vitro. We base this conclusion on the following findings: (i) natural killing and interferon level are not temporally correlated; (ii) preincubation of lymphocytes at 37 °C greatly reduces their ability to produce interferon but does not affect their lytic capacity against K562; and, (iii) addition of an anti-interferon antibody has no effect on NK. We conclude that NK against K562 is not dependent upon or correlated with the level of interferon generated during in vitro assay.     

Induction of interferon-gamma production by human natural killer cells stimulated by hydrogen peroxide

Interferon (IFN)-inducing activity of hydrogen peroxide in human peripheral mononuclear cells was investigated. Among the mononuclear cells, purified nonadherent cells produced IFN, but not B cells and monocytes. The maximal titer of IFN by purified nonadherent cells was observed after a 72-hr cultivation in the presence of 10(-2) mM H2O2 without affecting their viability. Furthermore, the purified nonadherent cells, but not the unpurified mononuclear cells, showed an augmented cytotoxicity to K562 when stimulated with hydrogen peroxide. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into the low and high density fractions for which natural killer (NK) cells and T cells were enriched, respectively. The NK-enriched low density fractions, but not the T cell-enriched high density fractions, showed IFN production by the stimulation of hydrogen peroxide. IFN production as well as large granular lymphocytes and HNK-1+, Leu-11+ cells of the NK-enriched fractions were abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1+) but not against T cells (OKT3) in the presence of complement. Moreover, hydrogen peroxide-inducing IFN production seems to be regulated by monocytes. The antiserum neutralizing IFN-alpha and IFN-beta failed to neutralize substantially IFN-produced NK cells. The treatment with either pH 2 or antiserum-neutralizing human IFN-gamma resulted in marked reduction, indicating that a major part of IFN was IFN-gamma. The purified nonadherent cells showed IFN production and augmented cytotoxicity when cultured separately from activated macrophages by opsonized zymosan; furthermore, both IFN production and enhancement of cytotoxicity were abrogated by catalase. These results suggest that both exogenous and endogenous hydrogen peroxide might be responsible for a part of immunoregulation.