Comparative study of various immunomodulators for macrophage and natural killer cell activation and antiviral efficacy against exotic RNA viruses

Several immunomodulators were compared for immunomodulatory and antiviral activity in B6C3F1 female mice. Our results demonstrate that murine recombinant gamma interferon (rIFN-G), human recombinant alpha A/D interferon (rIFN-A), ampligen (a polyribonucleotide) and CL246,738 modulate nonspecific immunity and are effective antiviral agents in vivo. Administration of each of these agents 1 day before cell harvest induced high levels of splenic natural killer (NK) cell activity against YAC-1 target cells. rIFN-G was also a potent activator of peritoneal macrophages (M phi), as evidenced by high levels of antitumor activity and changes in ectoenzyme phenotype that is characteristic of tumoricidal M phi. rIFN-A, ampligen and CL246,738 induced moderate to low levels of M phi activation by these criteria. In vivo protection experiments showed that repeated therapeutic treatment with rIFN-A protected mice against i.p. infection with Venezuelan equine encephalitis (an alpha togavirus, VEE), Banzi (a flavivirus) and herpes simplex virus type 2 (HSV-2). Similar treatment with rIFN-G was effective against VEE and HSV-2, but ineffective against Banzi virus. A single prophylactic i.p. dose of ampligen 1 day before virus challenge was very effective against Banzi virus, moderately effective against HSV-2, and ineffective against VEE and Caraparu (a bunyavirus) infection. A single prophylactic oral dose of CL246,738 provided almost complete protection of mice against VEE, Banzi, and HSV-2, and also increased the mean survival time for Caraparu infected mice. Collectively, these results indicate that rIFN-A, r-IFN-G, ampligen and CL246,738 may be useful in prophylactic or early therapeutic treatment of several serious virus infections. Since these agents stimulate NK cells and M phi, their antiviral activity may result, in part, from the alterations they induce in the natural immune system.     

The effect of 3,6-bis(2-piperidinoethoxy) acridine trihydrochloride (CL 246, 738) on acute graft vs host reactions in mice

3,6-Bis (piperidinoethoxy) acridine trihydrochloride (CL 246, 738) prevented the development of graft vs host (GVH) disease in normal BDF1 mice injected with C57BL/6 parental spleen cells. A single oral dose (50 mg/kg) given on day 0 or day -1 of GVH induction prevented the day 10 GVH-associated suppression of mitogen responsiveness and IL-2 production. The drug was ineffective if given later (days 3-7) in the reaction. The protective effect of CL 246,738 was neutralized by injecting drug-treated GVH mice with antibody to asialo GM-1 (ASGM-1). This suggested that the protective mechanism was not due to a direct effect of the drug on donor cells but rather was achieved indirectly through the activation of host ASGM-1+ cells which then rejected donor lymphocytes. This hypothesis was supported by immunofluorescence which showed that the donor-host chimerism seen in control GVH mice was not found in drug-treated GVH mice. Direct verification of this hypothesis was provided by data which showed that the transfer of CL 246, 738-activated large granular lymphocytes from normal F1 mice can prevent donor-induced immunosuppression in GVH mice. The results suggest that CL 246,738 is a potent immunostimulant which can boost natural resistance of normal unirradiated mice.     

Cellular immunosenescence in F344 rats: decreased natural killer (NK) cell activity involves changes in regulatory interactions between NK cells, interferon, prostaglandin and macrophages

Natural killer (NK) activity of F344 rat spleen cells remained constant between 1 and 18 months of age under specific pathogen-free (SPF) conditions. Between 18 and 24 months of age, however, there was a dramatic decline in activity which remained at a low baseline throughout the normal lifespan. Removal of adherent cells on G-10 Sephadex columns revealed age-related changes in adherent cell regulation of NK activity. Young (4-6 week) NK activity was consistently decreased by adherent cell removal while old (24-30 month) NK activity was slightly but reproducibly increased. Moreover, splenic macrophages from old rats purified by adherence to microexudate-coated surfaces were highly suppressive to young nonadherent NK activity. A role for endogenous prostaglandin (PG) in suppressed old rat NK activity was suggested by the effectiveness of anti-PGE2 in vivo to boost old NK activity. Although old rat NK activity was boosted to a relatively greater extent by interferon (IFN) in vitro than was young NK activity, IFN-boosted NK activity of old rats was much more sensitive to PGE2 inhibition than was IFN-boosted young rat NK activity. IFN treatment in vitro or poly(I:C) treatment in vivo induced protection against PGE2 inhibition of NK activity in young rats, while no resistance to PGE2 inhibition was induced in old rat NK cells by similar treatments. In vivo, the same protocol of IFN administration which boosted young rat NK activity further suppressed old rat activity. These results support the hypothesis that immunosuppression related to aging, which supersedes the boosting effect of IFN, involves the combined effects of suppressor macrophages (via PGE2) and intrinsic changes in effector (NK) cells which render them more sensitive to PGE2 inhibition.     

Absence of a role for natural killer cells in the control of acute infection by Toxoplasma gondii oocysts.

The active phase of primary and challenge oral infections of Toxoplasma gondii was investigated with respect to natural killer (NK) activity against YAC-1 tumour cell targets in vitro and serum interferon (IFN) titres. Primary (non-lethal) oral infection of BALB/c mice with Me49 oocysts resulted in a rapid increase of serum IFN titres, followed by augmented NK activity. NK levels became depressed, rising again by 15 days after infection to normal levels, again preceded by elevated IFN titres. In challenge infections NK was not augmented and IFN titres rose only if a high dose of oocysts was given. IFN activity was pH2-labile in all cases and considered to be due to IFN-gamma. Cold target inhibition studies indicated that T. gondii did not bind to NK cells. A bioassay for the effects of NK cells on T. gondii tachyzoites was developed and there was no evidence of killing in vitro by cells with NK function; T. gondii survived better when cultured with NK cells than when cultured alone. Studies using C57BL/6bg/bg,bg/+ and +/+ mice showed that there was no difference in mean time to death after administration of a lethal ME49 oocyst infection by mouth. Cytotoxicity against YAC-1 in both spleen and mesenteric lymph node (MLN) cell populations was highly augmented in bg/+ and +/+, but not in bg/bg mice. Genetic deficiency of NK activity had no effect on survival of mice after infection. Therefore NK has at best a minimal role to play in protection during the acute phase of Toxoplasma infection.     

Opposing effects of the interferon inducer, avridine: enhancement or suppression of tumor growth depending on treatment regimen

Tumor growth and regression was studied in C57BL/6J mice injected with Moloney sarcoma virus (MSV) and treated with the interferon (IFN)-inducing drug, avridine. Avridine decreased the persistence of tumors when given one or five days after virus, but shortened the prepatent period and increased persistence if given one day prior to virus. Additional studies were undertaken to study the role that serum interferon and natural killer (NK) cell activity might have in this phenomenon. Interferon levels were greatly enhanced (over that induced by virus alone or avridine alone) when avridine was given one day after, but not one day before, virus. Six days after viral infection, interferon titers had returned to near zero but could be boosted by injecting avridine at day 5. Multiple injections of avridine before and after virus resulted in refractoriness to interferon induction and tumor persistence. NK activity was greatly increased by virus at two days post-infection, and avridine given one day after infection significantly enhanced cytotoxicity of these splenic cells against tumor cells. By six days after infection, NK activity had returned to normal but could be increased by avridine given at five days post-infection. It appeared that high levels of interferon induced by avridine given at one or five days after infection increased NK activity and may have been responsible for enhanced regression. Pre-treatment by avridine had little effect on interferon levels over that induced by virus alone, but that did not explain the enhancement of tumor growth since NK activity was increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immunomodulatory potentials of the water-soluble yam (Dioscorea opposita Thunb) polysaccharides for the normal and cyclophosphamide-suppressed mice

Immunomodulatory potentials of water-soluble yam (Dioscorea opposita Thunb) polysaccharides (WYPs) for the normal and immuno-suppressed mice were described in this study. For the normal mice, the WYPs at 500?mg?kg^?1 elevated spleen and thymus indices by 22–42%; promoted macrophages' phagocytosis; lymphocytes' proliferation and natural killer (NK) cell activity by 18–53%; enhanced IL-2 and IFN-γ levels in the splenocytes by 42–45%; raised IL-1β, IL-6, TNF-α, iNOS and lysozyme levels in the macrophages by 40–219%; and increased serum IgM, IgA and IgG levels by 44–51%. The WYPs could restore and improve immune status of the cyclophosphamide-treated mice, as they at 500?mg?kg^?1 elevated spleen and thymus indices by 85–172%; promoted macrophages' phagocytosis, lymphocytes' proliferation and NK cell activity by 24–98%; enhanced IL-2 and IFN-γ levels in the splenocytes by 44–109% or IL-1β, IL-6, TNF-α, iNOS and lysozyme levels in the macrophages by 53–287%; and increased the three immunoglobulins by 24–69%.     

NK activity in carrageenan-treated mice

NK activity was determined by measuring 51chromium released from Yac-1 target cells incubated with spleen cells from normal or carrageenan (Car)-treated mice. Intraperitoneal administration of a single dose of i-Car (3 mg) provoked splenomegaly in mice. This splenomegaly accompanied during the first days (2-3), a marked increase of NK activity, then a decrease of this activity at day 8-9. It was returned to normal level at day 30. The modulation of NK activity in Car-treated mice is not due to the variation of the number of NK cells, since the frequency of target-binding cells (TBC) was not modified. The increase in NK activity during the first days may be due to the presence of interferon induced by carrageenan. Concomitant injection of an anti-mouse interferon globulin with carrageenan abolished the boosting of NK activity. NK activity of spleen cells from Car-treated mice at day 8 could not be stimulated by interferon in vitro as it could with the normal spleen cells. No decrease of NK activity was observed in Car-treated mice at day 8, when indomethacin was administered. Hence the decrease of this activity in Car-8 mice might be partially due to the alteration of NK effector cells induced by prostaglandins.     

Cascade of fever production in mice infected with influenza virus

The cascade of fever production in influenza was studied. To analyse fever production in a murine model, we selected DBA/2 mice that have the highest susceptibility in fibrile responses among seven mouse strains. Intranasal influenza infection- and interferon (IFN)-induced fever production was studied in this mouse model. Fever was induced prominently on day 2 after influenza infection and IFN activity was also increased in serum. Only the level of interleukin (IL)-1α, an endogenous pyrogen, rose markedly in serum among cytokines (IL-1α, IL-2, IFN-γ, and tumor necrosis factor-α) examined. Fever was induced 14 hr after intraperitoneal IFN-α treatment and IL-1α level rose significantly in the serum of the IFN-α-treated mice as compared with that of untreated mice. Fever production was significantly suppressed by treatment with anti-IFN-α/β or anti-IL-1α antibody in infected mice and the former signficantly suppressed responsive IL-1α production, indicating that elevated IFN activity induced IL-1α production and subsequently fever production in infected mice. The activity of cyclooxygenase (COX) that produces prostaglandin (PG)E₂ was significantly augmented in the brain of infected mice on day 2 after infection. Fever production was suppressed by the inhibition of COX activity with aspirin, although IL-1α level was maintained at the elevated level. Therefore, influenza infection in mice turned on the following cascade for fever induction: IFN production, IL-1α production, elevated COX activity, and PGE₂ production. We elucidated the relationship among IFN activity, IL-1α production and COX activity and demonstrated the cascade of fever production in influenza infection. © 1996 Wiley-Liss, Inc.     

Synergistic effect of thymosin alpha 1 and alpha beta-interferon on NK activity in tumor-bearing mice

We have investigated the possibility of thymosin alpha 1 (TH) cooperating with alpha beta-interferon (IFN) in boosting natural killer (NK) activity in tumor-bearing, immunosuppressed mice in vivo. Treatment with a single injection of 30,000 IU of IFN 24 h before testing enhanced NK activity in tumor-bearing mice if the IFN was administered 9 days after tumor inoculation, when the animals have normal NK responsiveness. On the other hand, the same treatment led to lower or no improvement of NK responses if the treatment was given 13 or 17 days after tumor inoculation, at a time when tumor growth causes immunosuppression. However, combination treatment with TH (200 micrograms/kg) for 4 days, followed by IFN was found to restore normal NK cell activity. Selective depletion of antigen-positive cells showed that killer cells stimulated by combination treatment with TH and IFN seem to bear phenotypic characteristics of NK cells. These studies provide the first documentation of a novel combination approach to reconstitution of immunosuppressed tumor-bearing mice using TH and IFN. We hypothesize that TH restores NK boosting activity by IFN by effecting the differentiation/induction of precursor populations of IFN-responsive cells.     

Natural killer cell activity in multiple sclerosis patients treated with recombinant interferon-alpha 2

Natural killer (NK) cell activity was evaluated in multiple sclerosis (MS) patients during a phase II trial of recombinant interferon-alpha 2 (IFN). Spontaneous NK activity against the K562 myeloid target cell increased significantly during the first week of treatment in the IFN treatment group. However, NK activity was also increased in the placebo treatment group. Long-term administration of IFN caused a decline of NK activity, below the pretreatment values. This decline was not paralleled by a decline in the percentage of Leu-7 cells in the peripheral blood. The ability of IFN to enhance NK activity during treatment was also evaluated. Enhancement of NK activity by IFN was depressed for the duration of the study in the IFN treatment group. After treatment was stopped, IFN enhancement of NK activity returned to the pre-study value. These studies demonstrate that spontaneous and IFN enhanced NK activity are profoundly affected by the administration of recombinant IFN-alpha 2 in MS patients.     

NK cell activity and skin test antigen stimulation of NK like CMC in vitro are decreased to different degrees in pregnancy and sarcoidosis.

Peripheral blood mononuclear cells (PBMNC) isolated from normal subjects, pregnant women and patients with sarcoidosis were assayed for natural killer (NK) cell activity on day 0 and for NK like cell-mediated cytolysis (CMC) after 5 days of exposure, in vitro to Candida antigen, purified protein derivative (PPD), and human leucocyte interferon (IFN). Pregnant women and women with sarcoidosis had significantly decreased levels of NK cell activity compared to normal women. Pregnant women had the lowest mean NK cell activity. Cells from women with sarcoidosis and from pregnant women also had lower levels of killing than those from the normal women after in vitro stimulation of NK like CMC with Candida antigen, PPD and IFN. The lowest stimulations of NK like killing occurred in the cells from women with sarcoidosis. Skin test antigen stimulation of NK like CMC in vitro and the DTH response in vivo were strongly correlated for both Candida antigen and PPD in the sarcoidosis patients. There was no correlation between the level of NK cell activity in the PBMNC of sarcoid patients on day 0 and the amount of NK like CMC that was present in cells from those patients after 5 days of culture with Candida antigen, PPD or IFN. A significant correlation was found, however, between Candida antigen stimulation of NK like CMC and IFN stimulation of NK like CMC in both pregnant and sarcoid groups. Reduced NK cell activity on day 0 in a given patient thus did not necessarily indicate that skin test antigen or IFN stimulation of NK like CMC on day 5 would also be depressed. In addition, NK cell activity was often noted to be normal in patients with depressed in vitro stimulation of NK like CMC.     

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。     

Dissociation of natural killing and luminol-dependent chemiluminescence

A long lasting luminol-dependent chemiluminescence was seen when mouse NK cell preparations or human NK cell preparations from Percoll gradients were mixed with NK susceptible targets. This CL could be abolished by extensive removal of adherent cells though normal NK cell activity remained. In addition, the NK cell line HY 3-Ag3 did not show any CL; although it expressed a very strong cytolytic activity. Thus, we conclude that there are two cells reacting with NK susceptible targets. First, the NK cell whose cytolytic activity does not necessarily depend on the formation of oxygen metabolites detectable by CL, and second, a more adherent cell population, found in NK cell preparations obtained after Percoll gradients, that reacts with NK-sensitive targets and leads to luminol-dependent CL. The observed CL paralleled the cytotoxicity measured by 51Cr release. The time course of the CL signal was similar in human and mouse. The maximal CL-signal obtained was about 10(5) cpm at 37 degrees C when 10(6) human effector cells were used at a ratio of 5:1 effector to target cells. The CL was shown to be highly temperature dependent.     

CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.     

Antiviral activity of biological response modifiers in a murine model of aids. requirement for augmentation of natural killer cell activity and synergy with oral AZT

We employed the Rauscher murine leukemia virus (RMuLV) as a murine retrovirus model of AIDS, to test biological response modifiers (BRM) and antiviral agents for potential therapeutic activity against the human immunodeficiency virus (HIV). We examined the relationship between the augmentation of natural killer (NK) cell activity and antiviral efficacy of a series of BRM, most of which are known inducers of interferon, in this model. Poly [I,C]-LC, MVE-2, and CL 246,738, but not Ampligen, soluble glucan, or 7-thia-8-oxoguanosine, consistently produced antiviral activity. In addition, the combination of suboptimal doses of oral 3'-azido-3'-deoxythymidine (AZT) (in drinking water) and poly [1,C]-LC produced a synergistic antiviral effect. With all the BRM tested, a consistent pattern emerged, namely that antiviral activity always correlated with the augmentation of splenic NK cell activity in infected animals. For instance, poly [I,C]-LC boosted NK activity much more in infected mice treated therapeutically (treatment initiated after infection) than prophylactically (treatment initiated before infection), and it had greater antiviral activity therapeutically than prophylactically. For the BRM tested, antiviral activity did not occur without augmentation of NK activity in infected mice. In contrast, augmentation of NK activity in uninfected mice bore no relationship to antiviral activity. Furthermore, elimination of NK cells by treating mice with anti-asialo GM₁ abolished the antiviral activity of poly [I,C]-LC. Although splenic NK activity was ablated by anti-asialo GM, serum interferon levels were not affected by this treatment. These results point to a causal connection between the augmentation of NK cell activity and the antiviral efficacy of these BRM in this murine AIDS model. NK cells thus appear to play a key role in resistance to this retrovirus, as has been suggested for HIV.     

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.     

Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.     

Decreased natural killer cell activity after prolonged administration of interferon in cancer patients

Two patients with metastatic neoplastic disease received 2-3 X 10(6) IU alpha recombinant interferon (IFN) 3 times/wk, every other week, for 3-6 mth. The natural killer (NK) activity of their peripheral blood leukocytes, was followed during the course of the treatment. A significant decrease was observed in the NK activity, which returned to normal values at the end of IFN administration. The treatment did not modify the evolution of metastasis.