Immunoferon (AM3) enhances the activities of early-type interferon inducers and natural killer cells

The "in vivo" effect of Immunoferon (AM3) on the production of interferon (IFN) and natural killer (NK) activity in young and old mice was studied. Although AM3 is not an IFN inducer by itself, enhancements in the serum IFN levels were produced when drug was associated to Newcastle disease virus or bacterial lipopolysaccharides as IFN inducers. This effect appeared to be dependent on the time lapsed between the inducer agent and drug. In addition, a significant stimulating effect on NK cell activity was also produced by AM3 treatments. This effect could be a consequence of a marked IFN induction and/or a modifying effect in prostaglandin synthesis.     

Inmunoferon (Am3) Enhances the Activities of Early–Type Interferon Inducers and Natural Killer Cells.

Abstract

The “in vivo” effect of Inmunoferon (AM3) on the production of interferon (IFN) and natural killer (NK) activity in young and old mice was studied. Although AM3 is not an IFN inducer by itself, enhancements in the serum IFN levels were produced when drug was associated to Newcastle disease virus or bacterial lipopo-lysaccharides as IFN inducers. This effect appeared to be dependent on the time lapsed between the inducer agent and drug. In addition, a significant stimulating effect on NK cell activity was also produced by AM3 treatments. This effect could be a consequence of a marked IFN induction and/or a modifying effect in prostaglandin synthesis.     

Decreased natural and antibody-dependent cellular cytotoxic activities in intravenous drug abusers

Peripheral blood lymphocytes from 14 adult male patients admitted to the hospital with complications of intravenous drug abuse (IDA) were examined for natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, lectin-dependent cellular cytotoxicity, and interferon (IFN)- and interleukin 2 (IL-2)-induced NK activity. Serum was also assayed for circulating interferon levels and soluble factor(s) capable of suppressing the cytotoxic potential of allogeneic lymphocytes from healthy donors. IDA patients demonstrated significantly decreased levels of NK and ADCC activities compared to age- and sex-matched healthy controls. The lectin, phytohemagglutinin, could significantly enhance the cytotoxicity of IDA lymphocytes; however, activity was not completely restored to normal levels. IDA sera demonstrated a significant inhibitory effect on the NK and ADCC activities of normal allogeneic lymphocytes, and these sera contained negligible levels of circulating IFN. Although the NK activity of IDA lymphocytes could not be restored completely to normal levels by either IFN-alpha or IL-2, the percentage enhancement of cytotoxicity was remarkably higher in IDA patients with significantly reduced NK activity than that observed using PBL from patients with near normal NK activity. The ability of IFN or IL-2 to enhance the decreased cytotoxicity of PBL from drug abusers suggests a novel therapeutic approach to the management of the complications of IDA.     

In Vitro Combined Effects of a Triazene Compound and Interferon-Beta on Natural Immunity Against Lymphoblastoid Cells: Studies at Effector and Target Cell Level

It was shown that Dacarbazine and other triazene compounds render murine leukemias highly immunogenic and susceptible to natural immunity (NI). In addition a pilot clinical study revealed that Dacarbazine can be cytotoxic for bone marrow blasts in patients with acute non-lymphoblastic leukemias through a mechanism that could be, at least in part, of immunological origin. However triazenes depress antigen-dependent responses and NI, whereas interferons, including interferon-beta (IFN), antagonize drug-induced impairment of NI. Therefore the complex interaction between triazenes and IFN on NI effector (i.e. NK) lymphocytes and human target lymphoblastoid cells has been investigated. the results show that: (a) EFN increases NK activity and antagonizes the depressive effects of methyl-triazene-benzoic acid (MTBA, an in vitro active triazene compound) on the NK function; (b) a lymphoblastoid cell line exposed to multiple in vitro treatments with MTBA, shows increased growth rate, augmented chemoresistance to MTBA, and higher susceptibility to NI than parental cells; (c) as expected IFN pretreatment down-regulates the susceptibility of lymphoblastoid cells to NK effectors; (d) however a net “therapeutic gain” was found if the overall influence of MTBA + IFN on target and effector cells is considered.     

The mechanism of action of a synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738), in natural killer cell activation in animals.

CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246,738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(alpha + beta) but not to IFN-(beta), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(alpha) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of natural killer cell activity of human lymphocytes in vitro: the participation of interferon in stimulation with Staphylococcus aureus Cowan I bacteria but not with protein A.

In the previous paper we reported that human natural killer (NK) cell activity was augmented greatly by preincubation with Staphylococcus aureus Cowan I bacteria (SpA CoI) or its Protein A. We examined here whether the augmentation with these stimulants is ascribable to the direct activation of NK cells or mediated by some soluble factors produced by the stimulants. It was found that a significant amount of interferon (IFN) was produced by the SpA CoI-stimulation but not by the Protein A-stimulation, although the latter usually induced augmentation of NK-cell activity not less than SpA CoI-stimulation. IFN produced by SpA CoI was considered to belong to alpha-type IFN, because it was stable at pH 2.0 and could be neutralized effectively by anti-IFN alpha antibody. Kinetics of NK-cell activation by SpA CoI (but not by Protein A) were very similar to those by IFN alpha. Furthermore, augmentation of NK-cell activity with SpA CoI-stimulated supernatant was inhibited almost completely by diluted anti-IFN alpha antibody, whereas augmentation with Protein A-stimulated supernatant could not be abolished by the same treatment. It was, therefore, suggested that augmentation of NK-cell activity with SpA CoI might be ascribable in most part to the IFN induced, whereas Protein A can stimulated NK or T cells directly or soluble factors other than IFN might work as well.     

黄芪和干扰素合剂抗单纯疱疹病毒的作用

目的研究中药黄芪乙醇提取物与重组人α２ｂ型干扰素组成栓剂或软膏的抗单纯疱疹病毒作用。方法采用２ＢＳ细胞，在９６孔板上进行栓剂或软膏水溶物对Ｉ型单纯疱疹病毒的抑制作用试验；干扰素的效价测定采用ＷＩＳＨ／ＶＳＶ法。结果黄芪与干扰素联合应用在２ＢＳ细胞培养上抑制单纯疱疹病毒的繁殖有明显的协同作用；组成复方干扰素软膏、栓剂的辅形成分对干扰素的测定系统无明显影响。结论本结果与前文报道的黄芪与干扰素联合应用对抑制１４、３９型鼻病毒有协同作用的结果是一致的     

Modulation of Lung-Associated Natural Killer Activity by Resident and Activated Alveolar Macrophages

Alveolar macrophages (AM) freshly obtained by bronchoalveolar lavage suppressed significantly, In a dose-dependent fashion, lung interstitial lymphocytes cytotoxicity against the NK-sensitive target cells, YAC-1. Kinetic experiments revealed that AM-mediated suppression of NK activity was seen following short-term incubation of AM with lymphocytes (4 h) and was unchanged after a 24 h co-culture period. Freshly obtained lung lymphocytes and lymphocytes incubated for 24 h were similarly inhibited by AM. In addition, incubation of AM for 24 h did not abrogate their suppressive effect on lung NK activity. Interestingly, AM-conditioned media, also caused a significant inhibition of lung NK activity. Furthermore, in vitro activation of AM with lipopolysaccharide (LPS, 5 μg/ml) and muramyl dipeptide (MDP, 20 μg/ml) significantly enhanced the Inhibitory effect of AM on lung NK activity. Similarly, in vivo activation of AM locally by intratracheal instillation of attapulgite, an inflammatory agent, resulted in greater AM-mediated down regulation. Taken together, these data indicate that lung NK activity is modulated by locally derived factors and suggest that pharmacologic manipulation of AM may play a determining role in the activation of lung NK activity by biological response modifiers (BRM).     

Lack of Correlation between Mycoplasma Induced IFN-γ Production in vitro and Natural Killer Cell Activity against FLD-3 Cells

Interferon (IFN) stimulates natural killer (NK) cell-mediated lysis of tumor cells. However, it is not clear whether IFN production is essential for NK cells to lyse their target cells in vitro, especially in long-term (> 18 hrs) assays. To investigate this, 0.5 x 106 normal mouse spleen cells were cocultured in RPMI 1640 medium with Friend erythroleukemia cells (FLD-3) (1 x 104) for 24 hours under conditions which cause lysis of FLD-3 cells. Supernatant fluid from such cultures demonstrated antiviral activity (100-200 units) which could be identified as IFNy. Prior filtration of spleen cells over nylon wool, and pretreatment with anti-Thy-1.2 + C' abrogated their ability to generate IFN-y without affecting their NK (FLD-3) activity. The IFN-y producing cell which could also be detected in spleens of nu/nu BALB/c mice lacked cell surface, Lyt-1, Lyt-2, and NK-1.2 antigens. The stimulus for IFN-y induction appeared to be Mycoplasma arginini carried in the FLD-3 tumor cells. Although mycoplasma-free FLD-3 cells failed to induce IFN in vitro, they retained their susceptibility to NK cell-mediated lysis. We conclude that IFN induction is not essential for NK(FLD-3) cell-mediated lysis; indeed IFN detected in NK cell assays may be produced in response to mycoplasma infection of the tumor cells. The Thy-1.2 positive cells stimulated by mycoplasma to produce IFN-γ lack several characteristics of T-cells or NK cells.     

Human mononuclear cells which produce interferon-alpha during NK(HSV-FS) assays are HLA-DR positive cells distinct from cytolytic natural killer effectors

Human mononuclear cells were previously shown to produce interferon-alpha (IFN) during 14 hr assays using herpes simplex virus type-1 infected fibroblasts [NK(HSV-FS)]. In this study, we have compared the effectors responsible for mediating NK(HSV-FS) cytolytic activity to those which produce IFN-alpha. Both activities were found to reside in non-adherent fractions, negative for non-specific esterase-staining cells. Like cells mediating NK cytolytic activity, IFN-alpha producing cells were found in light density Percoll gradient fractions. However, although NK(HSV-FS) and IFN production were largely overlapping, peak IFN production was consistently found in fractions slightly less dense than peak NK(HSV-FS) activity. IFN production was greatly augmented in fractions enriched for dendritic cells on hypertonic metrizamide gradients. The cells which produce IFN-alpha were phenotypically distinct from cytolytic NK effector cells: they lacked the Leu-11, Leu-7 and NKH1 cell surface markers shown to be present on both NK(HSV-FS) and NK(K562) effector cells. In addition, the IFN-alpha producing cells were found to be negative for a number of other markers characteristic of T cells, B cells or macrophages but were positive for Ia and HLA. The cells which produced IFN in response to UV-inactivated HSV antigen and to HSV-infected Raji cells were also found to be Leu-11 negative, and Ia positive. We conclude that the cells which produce IFN in response to HSV are a light density, Ia positive population which are distinct from NK cytolytic effector cells and co-purify with cells bearing a dendritic morphology. These results support our earlier findings that NK(HSV-FS) activity and IFN production are independent of one another and can segregate independently in vivo.     

Abnormal macrophages and NK cell cytotoxicity in human systemic lupus erythematosus and the role of interferon and serum factors

Macrophage (MO) and natural killer (NK) cell mediated cytotoxicity to K562 target cells were strikingly decreased in patients with systemic lupus erythematosus (SLE). SLE NK cells failed to release soluble factor(s) for lysing the targets. IFN-induced enhancement of both types of cytotoxicity was impaired. NK cells from healthy subjects kept their activity in culture with or without IFN for more than six days whereas SLE NK cell activity declined to zero at day 3. So, the increased IFN level of many SLE patients and a possible prior IFN priming effect seemed unrelated to the insensitivity to exogenous IFN in vitro. Inhibition factor(s) of SLE serum suppressed NK cytotoxicity in the presence of IFN whereas IFN sensitivity of MO remained unaffected indicating the complex regulation by serum components of immune reactions.     

参冬心宝口服液对柯萨奇病毒B3病毒性心肌炎小鼠的 …

目的 探讨参冬心宝口服液在小鼠体内免疫促进作用，为该药的临床应用提供理论依据。方法 以柯萨奇病毒Ｂ３型病毒感染１０日龄乳鼠为模型，观察了参冬心宝口服液对病毒感染急性期不同阶段心肌炎小鼠自然杀伤细胞活性及干扰素（ＩＦＮ）水平的影响。     

Interleukin 2 enhances natural killer cell activity through induction of gamma interferon

Highly purified interleukin 2 (IL 2), free of interferon activity, enhanced natural killer (NK) cell activity against tumor cells in mouse spleen cell cultures and in human peripheral lymphocyte cultures in a manner similar to that of interferon (IFN). We determined that IL 2 enhanced NK activity indirectly in a cascade manner by the induction of gamma IFN (IFN-gamma) in the cultures, which actually mediated the enhanced killing. Accordingly, lymphocyte cultures treated with IL 2 alone produced 10 to 100 U of IFN per ml in 6 to 24 h of culture. The IFN was typed as IFN-gamma by specific antibodies. Specific antibodies either to natural IFN-gamma or to a synthetic peptide corresponding to the human IFN-gamma N-terminal amino acids, when added to cultures treated with IL 2, completely blocked IL 2 enhancement of NK cell activity for both the mouse and human systems. IL 2-induced proliferation was not affected by the antibodies. Thus, the enhancement of NK cell activity by IL 2 is completely mediated by IL 2-induced IFN-gamma. The findings clearly indicate a cascade effect whereby one lymphokine (IL 2) induces the production of another. The latter lymphokine (IFN-gamma) then mediates an important biological effect (natural killing).     

Inhibition of induction of natural killer activity in mice by general anesthesia (Avertin): role of interferon.

We have previously reported that general anesthesia (Avertin) inhibits the induction of natural killer (NK) cell activity following administration of Poly I:C in vivo. Since Poly I:C has been shown to increase NK activity through the induction of interferon (IFN), the current study examines the role of IFN in this inhibition. The data suggest that (i) anesthesia does not affect the ability of Poly I:C to induce endogenous IFN synthesis (serum IFN levels are unaltered by anesthesia) and (ii) anesthesia is capable of inhibiting stimulation of NK activity induced directly by treatment with IFN either in vivo or in vitro. The duration of the former effect was at least 10 days, with complete recovery by Day 14 after anesthesia. Interestingly, NK activity stimulated by IFN prior to anesthesia was not significantly altered. In view of the increasingly evident role of NK cells in anti-tumor and anti-infectious host defenses, their inertness to stimulation in the post-anesthesia period may be a significant contributing factor to the clinically observed postoperative morbidity. Thus, preanesthesia stimulation of NK activity with IFN may be of therapeutic value.     

Activation of human natural killer cells by the protein-bound polysaccharide PSK independently of interferon and interleukin 2.

The protein-bound polysaccharide PSK was tested for the ability to activate human natural killer (NK) cells. When blood lymphocytes and purified CD3-CD16+ large granular lymphocytes (LGL) were treated in vitro overnight with PSK, they demonstrated enhanced NK cell activity against K562. The PSK-activated killer cells also lysed NK-resistant targets and freshly isolated autologous and allogeneic tumor cells. The PSK effect was observed with concentrations that could be obtained in the blood of cancer patients receiving oral administration of PSK. PSK-induced enhancement of NK activity was not abrogated by monoclonal antibodies (mAb) that neutralized interferon (IFN) alpha, IFN gamma, or interleukin-2 (IL-2). In addition, mAb reactive with p55 (alpha chain) or p75 (beta chain) glycoproteins of IL-2 receptors had no effects on PSK-enhanced NK activity even when used simultaneously. These results indicate that the PSK could activate human NK cells independently of IFN and IL-2/IL-2R systems.     

Abnormal interferon production and NK cell responses to interferon in children with Down's syndrome

Peripheral blood mononuclear leucocytes (PBL) of 11 children with Down's syndrome (DS, trisomy 21) and 13 healthy control children were examined for interferon (IFN) producing ability and for proliferation after in vitro exposure to various stimulants. Natural killer (NK) activity and in vitro sensitivity to IFN-α were also studied. Cells from DS patients showed a higher IFN production than normal cells, after stimulation with the IFN-γ inducers, concanavalin A and Lens culinaris lectin (LCL). Sendai Virus and β-haemolytic Streptococci group G also induced a higher IFN-α production in DS cells compared to normal PBL. Stimulation of DS PBL with Staphylococcus aureus Cowan I, which is an inducer of acid labile IFN-α in null lymphocytes, caused less IFN production in DS PBL than in normal cells. Basal NK activity was similar in DS and control PBL, but the NK activity of DS cells was more enhanced than in control cells by equivalent IFN concentrations in vitro, i.e. DS cells were more sensitive to IFN. There was no difference between DS children with and without a history of increased incidence of severe bacterial infections in any of the analysed parameters.     

Target-effector interactions in the rat NK cell system. II. Effects of interferon on lytic efficiency and on pre-NK cells in various organs, rat strains and during ontogeny.

Interferon (IFN) rapidly stimulates an inactive population of cells (pre-NK) to become lytic and this response to IFN has been used to measure pre-NK cells in the rat. Optimum conditions of IFN dose and time of in vitro exposure were chosen to measure the size of the pool of pre-NK cells in various lymphoid organs, rat strains and during ontogeny using a single cell agarose assay. With increasing dose of IFN there was a small increase in target binding cells, but a marked and similar dose-dependent increase in the number and lytic efficiency of cytotoxic cells. Following enhancement of natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity by IFN, the activity of both functions declined to baseline levels when IFN was removed, but could be restimulated following a second exposure to IFN 24 h later. It was found that the pre-NK cells exhibited the same rat strain and organ distribution as NK cells and had similar physical properties and target cell preferences, suggesting that pre-NK cells and NK cells belong to the same or related cell lineages or are similarly regulated. Cycloheximide has been shown to inhibit the stimulatory effect of IFN on human and murine NK cells whilst not influencing endogenous cytotoxicity. In contrast, we found that both the stimulatory effect of IFN on rat NK cells and endogenous activity were equally inhibited by cycloheximide. Both NK and pre-NK cells could be detected by at least 6 days of age and the pool size of both cell types increased rapidly with age. However, there were comparatively more pre-NK than NK cells in very young rats, although the balance was reversed in adult rats. The implications of these findings are discussed.     

Inverse relationship between interferon production by mononuclear leucocytes and oxidative metabolism of neutrophil granulocytes in infection prone children.

The ability of neutrophil granulocytes (PMN) from 15 infection prone children to produce luminol enhanced chemiluminescence (CL) was disturbed to varying extents in eight patients, two of whom were diagnosed as having chronic granulomatous disease (CGD). The ability of peripheral blood mononuclear leucocytes (PBL) to produce interferons (IFN) was normal, as tested with the inducers Sendai virus, E. coli, concanavalin A and L. culinaris lectin. However, the IFN response to the inducer S. aureus Cowan I (SACoI) was decreased in patients with normal CL and tended to be increased in those with decreased CL. There was a significant inverse relation between the CL of PMN and the SACoI-induced IFN responses by PBL of the patients. A regulatory effect of products of oxidative metabolism on SACoI-induced IFN production is therefore suggested. Patients, including those with CGD and controls showed similar basal and in vitro IFN enhanced natural killer (NK) activity of PBL against K-562 erythroleukaemia cells.     

Selective impairment of alpha-interferon-mediated natural killer augmentation in Sjögren''s syndrome: differential effects of alpha-interferon, gamma-interferon, and interleukin 2 on cytolytic activity.

Natural killer (NK) function has been shown to be impaired in several autoimmune diseases including Sjögren''s syndrome (SS). In the present study, in vitro effects of alpha-interferon (alpha-IFN), gamma-IFN and interleukin 2 (IL-2) on the NK cell activity were examined to analyse the regulatory system of NK-augmentation in patients with SS. The responsiveness of NK cell activity to alpha-IFN was markedly depressed in SS patients compared with normal controls, whereas the responsiveness to gamma-IFN was within normal limits. This is the first demonstration of the selective hyporesponsiveness of NK cell activity to one type of IFN in a certain disease. In addition, the kinetics study of NK-augmentation in normal donors revealed that alpha-IFN enhanced NK cell activity with a faster profile than gamma-IFN. These findings imply substantial differences between the two types of IFN in their mechanisms for enhancing NK cell activity, which deserve attention in evaluating the effects of IFNs. The present study also demonstrated that IL-2 could induce significantly higher levels of NK cell activity than alpha-IFN or gamma-IFN in SS and that this enhancing effect was almost comparable to that in normal controls. Thus, there seem to be multiple regulatory mechanisms for enhancement of NK cell activity, and a portion of the mechanisms may be selectively impaired in certain human diseases such as SS. The selective hyporesponsiveness to alpha-IFN could be relevant to the idea of viral participation in pathogenesis of SS.     

The Immunosenescent Phenotype in Mice and Humans Can Be Defined by Alterations in the Natural Immunity Reversal by Immunomodulation with Oral Am3

SUMMARY: The reactivities of monocyte/macrophages and natural killer (NK) cells (natural immunity) were evaluated following the administration of the biological response modifier AM3. The lower number of macrophages and NK cells in middle-aged mice (MAM) compared to young adult mice (YAM) were significantly elevated following AM3 treatment to equal or greater than YAM values. Both macrophage and NK cell cytotoxicity peaked at two days following AM3 treatment and remained elevated over control values for up to 8 days following a four days treatment regimen by the oral route. Of particular interest was the clinical effect of AM3 treatment in chronic bronchitis (CB) patients and various aged volunteers. In middle-aged patients with chronic bronchitis (MACBpts) AM3 treatment resulted in significant increases in the number of monocytes as well as their phagocytic and chemotactic activity. Differential NK cell cytotoxicities were observed in MACBpts compared to middle-aged healthy adults (MAHA) and young healthy adults (YHA). Cytotoxicity in YHA was 2-fold higher than MAHA and 5-fold higher than MACBpts. The depressed number of NK cells in MACBpts was reversed following the AM3 treatment to near NK cell levels in YHA. These observations help to explain how AM3 aids in the restoration of natural cellular immunity and its possible application as an adjuvant to bacterial & viral vaccines as well as in the treatment CB.