Mechanisms of depression of splenic natural killer cell function in C57BL/6 mice infected with Leishmania donovani

C57BL/6 mice chronically infected with the protozoan parasite Leishmania donovani exhibit profoundly depressed splenic natural killer (NK) cell activity as measured by in vitro cytolysis of lymphoma target cells. Injection of infected mice with an interferon (IFN) inducer or in vitro treatment of infected splenocytes with IFN, a phorbol ester, or indomethacin failed to restore their NK activity to the degree shown by age-matched, uninfected mice. Fractionation of infected splenocytes by nylon wool, Sephadex G-10, or carbonyl iron and magnetism treatments was also unable to effect an increase in NK activity. Addition of infected splenocytes to uninfected ones in in vitro NK assays suppressed the NK activity of the latter, and the suppression could be partially or wholly abrogated by prior fractionation of infected splenocytes by the methods noted above. In vitro treatment of infected splenocytes with concanavalin A revealed the presence of NK activity in these cell populations. The results indicate that splenocytes in L. donovani-infected mice become insensitive to IFN stimulation; and the impairment of another, possibly IFN-independent pathway of NK-cell activation may also contribute to the observed L. donovani-induced depression in splenic NK activity in C57BL/6 mice.     

Induction of natural killer cell activity of thoracic duct lymphocytes by polyinosinic-polycytidylic acid (poly(I:C)) or interferon

Natural killer (NK) cell activity of thoracic duct lymphocytes (TDL) was examined in normal mice and in mice treated with polyinosinic-polycytidylic acid (poly(I:C) and interferon (IFN). TDL from mice treated with phosphate-buffered saline (PBS) expressed little or no NK cell activity against YAC-1 target cells at effector-to-target ratios of up to 200:1, even after in vitro treatment with murine L-cell IFN. In contrast, TDL from poly(I:C)- or IFN-treated mice expressed significant NK activity, which correlated with the significantly higher NK activity of splenocytes from these mice compared to the NK activity of splenocytes from PBS-treated mice. These data indicate that although TDL from normal mice express no detectable NK cell activity, NK cell activity can be induced in TDL by in vivo treatment with poly(I:C) or IFN.     

Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.     

Oligodeoxynucleotides containing palindrome sequences with internal 5'-CpG-3' act directly on human NK and activated T cells to induce IFN-gamma production in vitro.

Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-gamma is mediated mostly by monocytes/macrophages activated by olig-DNA. However, its action on human IFN-gamma-producing cells has not been well investigated. In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells. Bacillus Calmette-Guérin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-gamma with an increased CD69 expression, and the autocrine IFN-gamma enhanced their cytotoxicity. The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab. T cells did not produce IFN-gamma in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab. In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5'-CpG-3' motif(s) appeared to play an important role in the IFN-gamma-producing ability of NK cells. The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-gamma inducer for NK cells. The CG palindrome was also important for activated NK and T cells in their IFN-gamma production, although certain nonpalindromes acted on them. Among the sequences tested, cell activation- or cell lineage-specific sequences were likely; i.e., palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells. These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-gamma production.     

IFN-beta production by macrophages obtained from mice undergoing graft vs host disease

We have previously shown that when splenocytes are obtained from mice undergoing graft vs host disease (GVHD), and then placed in culture, IFN-beta is produced spontaneously in supernatants without any stimulus. Thus, when B10.D2 spleen cells are injected into sublethally irradiated BALB/c recipient mice, in vitro splenic IFN-beta production is readily apparent between days 10 to 20 post-transplantation. In the present study, experiments were carried out to characterize the cell population(s) responsible for spontaneous IFN-beta production from GVHD spleen cells. Specific antibody and C lysis of selected cell types, including T cells, B cells, and NK cells, failed to abrogate in vitro IFN production. However, IFN-producing cells from GVHD splenocytes were nylon wool adherent and could be isolated using discontinuous Percoll gradient centrifugation from the upper most large cell fractions. IFN production was also partially resistant to irradiation. Using indirect immunofluorescence and flow cytometry, IFN production was shown to be associated with MAC-1 positive cells. MAC-1 negative splenocytes demonstrated no spontaneous IFN production. Treatment of GVHD spleen cells with silica, a selective toxin for phagocytic cells, resulted in a complete inhibition of IFN production. Thus, in vitro IFN production from GVHD splenocytes appeared to come from cells having a phenotype associated with macrophages. This in vitro IFN production by macrophages represents a unique aspect of GVHD which is not typically found in tissues of normal mice.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Different functional sites on rIFN-alpha 2 and their relation to the cellular receptor binding site

Functional domains on the recombinant interferon-alpha 2 (rIFN-alpha 2) molecule, which are involved in antiviral and NK enhancing activities, have been defined by immunochemical mapping with MAb, and their relationship with the IFN cellular receptor binding site has been studied. With 20 different anti-IFN-alpha 2 MAb selected by their binding to 125I-labeled IFN and by immunoprecipitation of the 20 Kd IFN molecule, we have defined three spatially separated epitopes (designated as sites A, B, and C) and two partially overlapping antigenic determinants on the IFN-alpha 2 molecule. Functional relation of IFN-alpha 2 A, B, and C epitopes have been determined by assaying the effect of various anti-IFN MAb on IFN-mediated biologic activities. MAb directed to sites A and B neutralized the antiviral activity of IFN. Furthermore, the MAb specific for site B displayed a neutralizing potency threefold higher than MAb directed to site A. Site B was also involved in the enhancing activity of IFN on NK-mediated cell cytotoxicity, whereas site A was not. MAb directed to site C partially affected the IFN-boosted NK activity but did not neutralize the IFN antiviral activity. Inhibition studies of 125I-IFN binding to human U-937 myelomonocytic cells by anti-IFN MAb demonstrated that MAb directed to site B blocked different IFN biologic functions by preventing its binding to the cellular receptor, whereas MAb directed to sites A and C caused no inhibition and partial inhibition of this binding, respectively.     

Natural killer (NK) cell activating factor released from murine thymocytes stimulated with an anti-tumor streptococcal preparation, OK-432

Natural killer (NK) activity of mouse splenocytes was significantly augmented when the splenocytes were incubated for 3 to 4 hr with culture supernatants of mouse thymocytes stimulated by OK-432, an antitumor preparation from the Streptococcus pyogenes SU-strain. Antiviral activity was also detected in the culture supernatants, but IL 2 activity was not. When the culture supernatants of thymocytes stimulated by OK-432 were fractionated on a column of Blue Sepharose CL-6B, NK enhancing activity and antiviral activity were observed in partly overlapping fractions that bound to the column. However, the antiviral activity in the Blue Sepharose-bound fraction was neutralized completely by treatment with anti-IFN (alpha, beta) antiserum, whereas significant NK cell enhancing activity was still observed after treatment with anti-IFN (alpha, beta) antiserum. When the Blue Sepharose-bound fraction was subjected to gel filtration, the NK cell enhancing activity was detected in the 25,000 to 35,000 and 40,000 to 67,000 m.w. regions, but antiviral activity was observed in the over 67,000 m.w. region. These results indicate that a new kind of lymphokine, called natural killer cell activating factor (NKAF), distinct from IFN and IL 2, was found. The NKAF was found to have the following properties: its pI value is between pH 5.5 and 6.5, it binds to concanavalin A- and lentil agglutinin-Sepharose, and it is stable with pH 2-24 hr treatment. In addition, NKAF-producing cells were peanut agglutinin (PNA)-thymocytes when thymocytes were fractionated by the agglutination-sedimentation method with the use of PNA.     

The role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1

The relative roles of interferon (IFN) and natural killer (NK) cells in herpes simplex virus type 1 (HSV-1) infection of mice were examined. Adoptive transfer of adult mouse leukocytes into 4- to 6-day-old suckling mice protected the recipients from HSV-1 infection, as judged by viral titers in the spleen 2 days postinfection. Protection was mediated by several classes of leukocytes, including those depleted of NK cell activity by antibody to asialo GM1 and those depleted of macrophages by size separation. Mice receiving these leukocytes produced significantly higher levels of IFN 6 hr postinfection (early IFN) than did HSV-1-infected mice not receiving donor leukocytes. Antibody to IFN, under conditions that blocked early but not late IFN synthesis, greatly enhanced HSV-1 synthesis in mice receiving leukocytes and completely removed the protective effect mediated by leukocytes. High doses of anti-asialo GM1 blocked both NK cell activity and early IFN production and resulted in high titers of HSV-1. This effect on virus synthesis was not seen if mice were given antibody 1 day postinfection. Lower doses of anti-asialo GM1, which still depleted NK cell activity but had no effect on early IFN production, did not enhance HSV-1 synthesis. Depletion of NK cell activity with a low dose of antibody had no effect on the reduced HSV-1 synthesis resulting from prophylactic IFN treatment or on the enhanced HSV-1 synthesis resulting from antibody to IFN treatment. Thus, resistance to acute HSV-1 infection in mice correlates with early IFN production but not with NK cell activity, suggesting that NK cells are not major mediators of natural resistance in this model and that the antiviral effect of IFN is not mediated by NK cells.     

Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.     

Effects of diethyldithiocarbamate, an inhibitor of interferon antiviral activity, upon human natural killer cells

In support of a postulated role of the Cu++-dependent enzyme, superoxide dismutase (SOD), in antiviral effects of interferon (IFN), a close correspondence was previously shown to exist between inactivation of cellular SOD and concomitant blockade of IFN antiviral activity in fibroblasts by the Cu++-chelating agent, diethyldithiocarbamate (DDC). To further define the extent of "anti-IFN" activity, we initiated studies of DDC effects on IFN stimulation in the NK cell system. Unexpectedly, DDC directly inhibited cytotoxicity mediated by unstimulated NK cells. Pronounced inactivation occurred rapidly (less than 30 min), but was spontaneously reversible in the absence of DDC. Neither cell viability nor lymphocyte binding to target cells was detectably affected. Preincubation of DDC with Cu++ or Zn++ failed to neutralize its inhibitory effects nor could function be restored in DDC-pretreated NK cells by subsequent addition of Cu++, Zn++, Mg++, or Ca++. DDC treatment that inactivated NK cells did not detectably alter lymphocyte SOD activity. Thus, inhibition was probably not attributable to chelating properties of DDC. N-ethyl maleimide (NEM) and para-( hydroxymercuri ) benzoic acid ( PMBA ), enzyme inhibitors that preferentially react with sulfhydryl groups, both inactivated NK cells in a time- and dose-dependent manner similar to that of DDC. Preincubation with the sulfhydryl compound, cysteine, neutralized in parallel fashion the capacity of NEM, PMBA , and DDC to inhibit NK cell activity. Thus, a previously unreported reactivity of DDC with sulfhydryl groups appeared to be the basis of inhibition. NK cells incubated 1 hr with IFN and subsequently cultured 17 to 23 hr without IFN were activated to an extent comparable to cells continuously incubated 18 to 24 hr with IFN. Exposure to IFN for 1 hr was therefore sufficient to commit NK cells to acquisition of a fully activated state. Whether preactivated by a 1-hr or 18- to 24-hr IFN treatment, activated NK cells retained the DDC-sensitive phenotype characteristic of fresh unstimulated NK cells. Thus, prolonged IFN treatment did not render NK cells resistant to DDC or preferentially activate a DDC-sensitive NK cell subset. An 18- to 24-hr incubation of DDC-pretreated cells in the continual presence of IFN resulted in the boosting of NK cell activity. However, the 1-hr IFN pulse treatment protocol was consistently ineffective in boosting when IFN was added just after DDC-pretreatment. These results strongly suggested that DDC temporarily rendered NK cells unresponsive to what, under normal circumstances, approximated an optimally potentiating IFN stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.     

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3-) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-gamma, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-gamma, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-gamma and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-gammaR knockout mice demonstrated that IFN-gamma was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.     

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.     

Elevated natural killer cell-mediated cytotoxicity, plasma interferon, and tumor cell rejection in mice persistently infected with lymphocytic choriomeningitis virus

To assess the effects of chronic virus infection on NK cells, the related phenomena of interferon (IFN) production, NK cell activation, and resistance to tumor implants were studied in mice persistently infected with lymphocytic choriomeningitis virus (LCMV). NK cells from these LCMV-carrier mice displayed augmented killing of the NK-sensitive YAC-1 target cell. They did not lyse the more resistant targets L-929 and P815, whereas NK cells from acutely infected mice efficiently lysed all three cell types. The plasma from LCMV-carrier mice contained an antiviral substance identified as IFN type I, based on species specificity, virus nonspecificity, resistance to pH 2, and sensitivity to antibody to type I IFN. IFN titers in plasma from LCMV-carrier mice were 32 to 64 U/ml, about 20-fold less than those in acutely infected mice. Both the IFN and NK cell levels continuously remained elevated in the LCMV carrier mice up to at least 6 months of age. IFN is known to activate NK cells and to induce their blastogenesis in vivo. As determined by centrifugal elutriation, large NK blast-size cells were isolated from the spleens of acutely infected mice, but not from either normal or LCMV-carrier mice, suggesting augmented NK cell-mediated lysis in the absence of enhanced proliferation. Poly inosinic-cytidylic acid induced high levels of NK cell-mediated cytotoxicity and blastogenesis in both control and LCMV-carrier mice, but IFN was induced to lower levels in carriers as compared with controls. Coincidental with augmented NK cell activity, the LCMV-carrier mice rejected intravenously injected 125IUdR-labeled tumor cells more efficiently than did normal mice. Thus, LCMV carrier mice have low levels of type I IFN, moderately augmented NK cell activity lasting for at least 6 months, and increased resistance to tumor cell implants. This indicates that augmented NK cell-mediated cytotoxicity can be maintained in vivo over prolonged periods of time in the presence of chronic low-level IFN stimulation.     

Role of interferon in human natural killer activity against target cells infected with HSV-1

Human natural killer (NK) cells show high cytotoxic activity against target cells infected with herpes simplex virus type 1 (HSV-1). Substantial amounts of interferon (IFN) were generated in co-cultures of NK effector cells and infected target cells; however, the cytotoxic activity seen against a specific infected cell target did not correlate with the amount of IFN induced. The production of IFN increased steadily from 4 to 18 hr of co-culture, as did NK activity; however, IFN production peaked 4 hr later than NK activity. Pretreatment of NK effector cells with exogenous IFN increased cytotoxic activity against all targets tested, but the differential pattern of reactivity against cells infected with wild type and mutant viruses was unaltered. When effector cells were treated with the RNA synthesis inhibitor actinomycin D before co-culture with virus-infected targets, IFN production was markedly reduced, without a concomitant reduction in cytotoxicity. Similarly, the addition of anti-IFN antiserum to co-cultures greatly decreased the available IFN present, but had no effect on NK activity. We conclude that the induction of cytotoxic activity in co-cultures of NK effector cells and HSV-1-infected target cells is independent of the induction of IFN.     

In vivo significance of NK cell on resistance against virus (HSV-1) infections in mice

Susceptibility to infection with herpes simplex virus type 1 (HSV-1) was examined in euthymic as well as athymic nude mice which were continuously depleted of natural killer (NK) cell activity by i.v. injection of anti-asialo GM1. In those NK cell activity-depleted mice, the mortality rate of infection with HSV-1 and the virus titers in the brain, liver, and spleen were notably higher than in the control mice. The enhanced susceptibility was demonstrated only in the mice receiving anti-asialo GM1 and HSV-1 simultaneously, but not in the mice in which NK cell deletion was postponed by injecting the antisera 5 days after the virus inoculation. Interferon (IFN) production of peritoneal exudate cells was also reduced in the anti-asialo GM1-injected mice. The decline of resistance against HSV-1 infection proved to be primarily due to deletion of NK cells, but not due to the inability to produce IFN, because repeated injections of IFN increased the NK cell activity and prolonged the life of HSV-1-infected mice with an intact NK cell activity. In the NK cell activity-depleted mice, however, neither the NK cell activity nor the life span was improved by the administration of IFN.     

Anti-hepatoma activity in mice of a polysaccharide from the rhizome of Anemone raddeana

A neutral polysaccharide fraction (ARP) prepared from the rhizome of Anemone raddeana was tested for its anticancer activity in H22 tumor-bearing mice by oral administration. ARP could not only significantly inhibit the growth of H22 transplantable tumor, but also remarkably promote splenocytes proliferation, NK cell and CTL activity, as well as serum IL-2 and TNF-α production in tumor-bearing mice. In addition, ARP treatment to tumor bearing mice had no toxicity to body weight, the liver and kidney. Moreover it could reverse the hematological parameters induced by 5-fluorouracil (5-FU) to near normal. The above results suggested that the antitumor activity of ARP might be achieved by improving immune response, and they could act as antitumor agent with immunomodulatory activity.     

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.