Activation of a glioma-specific immune response by oncolytic parvovirus Minute Virus of Mice infection.

Rodent autonomous parvoviruses (PVs) are endowed with oncotropic properties and represent virotherapeutics with inherent oncolytic features. This work aimed to evaluate the capacity of Minute Virus of Mice (MVMp) to act as an adjuvant stimulating a mouse glioblastoma-specific immune response. MVMp was shown to induce cell death through apoptosis in glioma GL261 cells. Antigen-presenting cells (APCs) provide the initial cue for innate and adaptive immune responses, and thus MVMp-infected GL261 cells were tested for their ability to activate dendritic cells (DCs) and microglia (MG), two distinct cell types that are able to act as APCs. MG and discrete DC subsets were activated after co-culture with MVMp-infected glioma GL261 cells, as evidenced by upregulation of specific activation markers (CD80, CD86) and release of proinflammatory cytokines (tumor necrosis factor-α and interleukin-6). The in vivo analysis of immunodeficient and immunocompetent mice revealed a clear difference in their susceptibility to MVMp-mediated tumor suppression. Immunocompetent mice were fully protected from tumor outgrowth of GL261 cells infected ex vivo with MVMp. In contrast, immunodeficient animals were less competent for MVMp-dependent tumor inhibition, with only 20% of the recipients being protected, arguing for an additional immune component to allow full tumor suppression. In keeping with this conclusion, immunocompetent mice engrafted with MVMp-infected glioma cells developed a level of anti-tumor immunity with isolated splenocytes producing elevated levels of interferon-γ. In rechallenge experiments using uninfected GL261 cells, we could show complete protection against the tumor, arguing for the induction of a T-cell-mediated, tumor-specific, long-term memory response. These findings indicate that the anticancer effect of PVs can be traced back not only for their direct oncolytic effect, but also to their ability to break tumor tolerance.

Antimetastatic effect of CpG DNA mediated by type I IFN.

The mechanisms involved in the antimetastatic effect of CpG-containing DNA were investigated in a mouse model of experimental metastasis. Tumor cell colony formation in lungs or livers of mice after i.v. inoculation with syngeneic fibrosarcoma or thymoma cells was determined. The i.v. injection of plasmid DNA or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs before tumor cell application strongly inhibited metastasis. Because synthetic CpG-ODN was not directly tumor cytotoxic, the target cells for this CpG-ODN effect were determined. The cytotoxic activity on standard natural killer (NK) targets as well as on fibrosarcoma cells of splenic NK cells and NKT cell-containing liver mononuclear cells derived from CpG-ODN-treated mice was strongly enhanced. Participation of NK/NKT cells in the CpG-induced antimetastatic effect was demonstrated by reduction of the antimetastatic effect in mice depleted of NK/NKT cells and beta2-microglobulin-deficient mice. Neutralization of interleukin 12, interleukin 18, or IFN-gamma did not interfere with the CpG-induced antimetastatic effect. However, in sera of CpG-ODN-treated mice, high levels of IFN-alpha were detected, and in IFN-alpha/beta receptor-deficient mice, the CpG-ODN-induced antimetastatic effect was strongly reduced. These data indicate that CpG-ODNs activate NK/NKT cells for antimetastatic activity indirectly via IFN-alpha/beta receptor activation. The exploitation of the stimulatory activity of CpG-ODN for the innate immune system might be a useful strategy for antimetastatic therapy.

Identification of a novel dendritic cell-like subset of CD64(+) / CD16(+) blood monocytes.

Human monocytes (Mo) consist of a major subset of Fcgamma-receptor I (CD64)-positive typical low accessory phagocytes, and a minor CD64(-) DC-like subset with high T cell-accessory and IFN-alpha-releasing activity. Both populations also differentially express CD16 (Fcgamma-receptor III). Double labeling with anti-CD64 and anti-CD16 mAb, as performed here, identified four different subsets. The CD64(-) subset consists of CD64(-) / 16(+) cells with high antigen-presenting cell (APC) function and macrophage-like phenotype, and a CD64(-) / 16(-) subset of less active APC but which exhibits a higher mixed lymphocyte reaction (MLR) stimulating and IFN-alpha-producing capacity, possibly resembling plasmacytoid dendritic cell type II (DC2) blood precursors. As well as the majority of CD64(+) cells that appeared CD64(+) / 16(-) and represent typical low-accessory, CD14(high) Mo, we could identify and describe a novel minor subset of CD64(+) / 16(+) cells which is unique in combining typical DC and Mo characteristics in the same cell. These are high IL-12 production, high accessory capacity for antigen- or allogen-activated lymphocytes, and high expression of HLA-DR, CD86, and CD11c.

Interferon-gamma (IFN-gamma) levels finally become stable with increasing age as revealed by using an ELISA corresponding to the bioactivity.

The immune status is a parameter of the capacity of the immune system to fend off microorganisms. The use of leukocyte subtyping to define the immune status is clinically established. IFN-gamma is a key cytokine directing the immune response. In this study, we investigated whether IFN-gamma is a more sensitive parameter of the immune status. All persons tested showed stable quantities of white blood cell counts over the whole study. Analyses of the lymphocyte subpopulations of two time points resulted in a strong correlation with high statistical significance for the percentage of CD3, CD4, CD8, CD19, CD45 -subtypes and CD56/16 positive cells. IFN-gamma production by the individuals correlates between these time points also, but only if an ELISA strongly correlating to IFN-gamma bioactivity was used instead of other commercial IFN-gamma ELISAs. The IFN-gamma production by males was less variable than by females. Furthermore, intraindividual differences in IFN-gamma secretion were minimal after the age of 46. In conclusion, our data demonstrate that IFN-gamma is a more sensitive parameter for the actual status of the immune system, since its titer shows alterations faster than leukocyte subtyping. Furthermore, leukocyte subtypes do not correspond to the production capacity of the regulatory cytokine IFN-gamma in healthy individuals.

Failure to detect antiviral activity in serum and plasma of healthy individuals displaying high activity in ELISA for IFN-alpha and IFN-beta.

The presence of constitutively produced interferon (IFN)-alpha in the blood of healthy individuals has been the subject of contradictory discussions for years. Immunologic as well as biologic test procedures have demonstrated striking differences regarding serum IFN-alpha under physiologic conditions. We investigated the presence of immunoreactive IFN-alpha in serum samples of 923 healthy blood donors by means of a widely used commercially available ELISA. Of these, 254 (27.5%) exhibited detectable serum IFN-alpha levels. The sera of 85.1% of these people also contained IFN-beta. Both IFN were also demonstrated in EDTA-anticoagulated plasma. However, none of these samples exhibited any antiviral effect on human A549 lung carcinoma cells challenged with encephalomyocarditis virus. Samples with high IFN-alpha ELISA activity did not abolish the antiviral action of added natural IFN-alpha, thus excluding IFN-alpha inhibitory factors. The experiments suggest that the detected compounds probably did not represent IFN-alpha but were the result of a cross-reaction with unknown serum components. A variety of disorders has been associated with elevated serum IFN-alpha levels that in most cases were detected by ELISA. In view of our data, these findings need to be carefully reevaluated. For the purpose of monitoring IFN-alpha levels in therapy of atopic, autoimmune, or malignant disorders, an appropriate detection system for IFN-alpha is advisable.

Molecular cloning of a macrophage-derived, interferon-inducible secreted immunoglobulin-binding protein.

We describe the molecular cloning of a 1803-bp cDNA coding for a product termed interferon-induced immunoglobulin-binding protein (IIBP) from a library of IFN-alpha-induced primary bone marrow macrophages. The open reading frame encodes a protein of 26-kDa containing two immunoglobulin-like and one Fc receptor-like domain. Due to the constitutive release of low amounts of IFN-beta, the IIBP mRNA is already present in macrophage-colony-stimulating factor-cultured macrophages. Its expression could be blocked in the presence of either anti-IFN-beta or interleukin-4, which down-regulates the endogenous IFN-beta production. Upon addition of rIFN-alpha4 a 3-5-fold superinduction of IIBP mRNA was observed. Rat monoclonal antibodies detected a protein of the predicted size exclusively in cell culture supernatants of primary bone marrow macrophages and a B-cell line. In immunoprecipitation experiments an unknown 30-kDa protein co-precipitated. The secreted IIBP showed considerable binding to nonspecific rat IgG2a and could be precipitated using mouse IgG2a, IgG2b and IgG3 antibodies of irrelevant specificities, indicating that this gene product is a novel secreted immunoglobulin-binding protein with a new IgG isotype binding pattern that differs to that of known Fc receptors.

Tumor stimulator cell modification by infection with Newcastle Disease Virus: analysis of effects and mechanism in MLTC-CML cultures.

Effects of tumor stimulator cell modification by infection with Newcastle Disease Virus (NDV) are described as analysed in vitro in mixed lymphocyte tumor cell cultures (MLTC). Direct antitumor effects were seen with human melanoma or colon-carcinoma cells in a dose- and time-dependent manner when using live but not UV inactivated virus. When T cell stimulation was measured by [3H]-thymidine uptake, NDV infected tumor stimulator cells did not show an augmentation but rather an inhibitory effect in comparison to non-infected stimulator cells. Virus infected tumor stimulator cells were, however, capable of augmenting the induction of tumor specific cytotoxic T cells in MLTC-CML assays when using murine ESb lymphoma immune cells and syngeneic NDV modified ESb cells as stimulators. A CML stimulatory effect was also shown for NDV modified third party cells and thereof derived conditioned medium. These effects are most likely explained by interferon- which is induced in tumor cells by NDV infection and by interferon-á which is induced in responder cells when stimulated with NDV infected stimulator cells.

Interferon-induced expression of If-1h and If-1l alleles in Newcastle disease virus-infected mouse macrophages is associated with specific differences in viral gene transcription.

We have studied the expression of cytokines and viral genes induced by Newcastle disease virus (NDV) and Sendai virus in bone marrow-derived macrophages (BMM) and lymphocytes from C57BL/6 mice and the congenic line B6.C-H-28c. These mice carry the loci If-1h (high) or If-1l (low), respectively, that are responsible for up to tenfold differences in the interferon (IFN)-alpha, IFN-beta, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) response to NDV but not to Sendai virus. Only BMM but not spleen lymphocytes showed allele-specific differences in NDV-induced cytokine levels, indicating cell-specific If-1 expression. The If-1 locus harbors IFN-inducible gene(s) whose expression is prevented in the presence of cycloheximide. Our data provide evidence that the If-1l allele acts by specifically suppressing the cytokine response to NDV. Cytokine production was dependent on infectious virions, and kinetic analyses revealed a close correlation between the amount of viral transcripts and individual cytokine mRNA. BMM from lf-1l mice strongly restricted transcription of the NDV nucleoprotein (NP) gene, whereas BMM from If-1h mice supported NP transcription. Following treatment with IL-4, which inhibited constitutive IFN-beta gene expression, however, If-1l BMM became highly permissive for transcription of the viral NP gene and released high amounts of cytokines. We conclude that If-1l gene products are responsible for the low producer phenotype by efficiently interfering with NDV transcription, leading to strongly reduced intracellular levels of cytokine inducing viral dsRNA intermediates.

Inhibition by interleukin-4 of constitutive beta interferon synthesis in mouse macrophages.

We have found that interleukin-4 (IL-4) abrogated constitutive beta interferon (IFN-beta) synthesis in mouse macrophages. Analysis of endogenous IFN mRNA by reverse transcription-PCR clearly documented the presence of IFN-beta but not of IFN-alpha transcripts in RNA from bone marrow-derived macrophages (BMM). Culture of bone marrow cells for 7 days in the presence of macrophage colony-stimulating factor plus IL-4 or treatment of mature BMM with IL-4 for 3 to 4 days resulted in pronounced inhibition of IFN-beta transcript levels. As a consequence, BMM became highly permissive to viral infection and induction of the IFN-responsive 2',5'-oligoadenylate synthetase was inhibited. In view of the evidence for low-level constitutive secretion of IFN in vivo, it is possible that IL-4 influences the host's antiviral defense system.

Differential regulation of the JE gene encoding the monocyte chemoattractant protein (MCP-1) in cervical carcinoma cells and derived hybrids.

Malignant human papillomavirus type 18 (HPV18)-positive cervical carcinoma cells can be reverted to a nonmalignant phenotype by generation of somatic cell hybrids with normal human fibroblasts. Although nontumorigenic hybrids, their tumorigenic segregants, and the parental HeLa cells have similar in vitro properties, inoculation only of nontumorigenic cells into nude mice results in a selective suppression of HPV18 transcription which precedes cessation of cellular growth. Our present study, aimed at understanding the differential regulation in vitro and in vivo, shows that the JE gene, encoding the monocyte chemoattractant protein (MCP-1), is expressed only in nontumorigenic hybrids. Although the gene, including its regulatory region, is intact, no JE (MCP-1) mRNA is detected in the tumorigenic segregants and in other malignant HPV-positive cervical carcinoma cell lines. Tests of several monocyte-derived cytokines showed that only tumor necrosis factor alpha strongly induces the JE (MCP-1) gene in nontumorigenic cells and that this is accompanied by a dose-dependent reduction of HPV transcription. The JE (MCP-1) up-regulation occurs within 2 h and does not require de novo protein synthesis. The response to tumor necrosis factor alpha seems to be mediated by an NF-kappa B-related mechanism, since the induction can be completely abrogated by pretreating the cells with an antioxidant such as pyrrolidine dithiocarbamate. Interestingly, cocultivation of nonmalignant hybrids with monocyte-enriched fractions from human peripheral blood also results in an induction of the JE (MCP-1) gene and a concomitant suppression of HPV18 transcription. Neither effect is observed in malignant cells. These data suggest that JE (MCP-1) may play a pivotal role in the intercellular communication by triggering an intracellular pathway which negatively interferes with viral transcription in HPV-positive nontumorigenic cells.

In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells.

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability > 0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR alpha beta (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon gamma and tumour necrosis factor alpha was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.

Downregulation of Newcastle disease virus (NDV)-dependent IFN-alpha/beta production in macrophages by IFN-induced gene products of the locus If-1.

We have found that in cultured mouse bone-marrow-derived macrophages (BMDM), endogenous IFN-beta specifically regulates Newcastle disease virus (NDV) induced interferon (IFN)-alpha/beta synthesis, possibly by influencing the activity of genes within the regulatory locus If-1. Comparison of anti-IFN-beta-treated BMDM from C57BL/6 mice and the congenic line B.6C-H28c carrying the high (h) or low (l) producer allele of If-1, respectively, revealed a much stronger response of the If-1l allele to exogenous IFN-alpha treatment. Twenty IU rIFN-alpha 4 were sufficient to induce nearly complete suppression of NDV-induced IFN-alpha and IFN-beta production in BMDM from B6.C-H28c mice, but had no effect on the IFN-alpha/beta response induced by Sendai virus, another member of the paramyxovirus group. Simultaneous treatment of BMDM with cycloheximide inhibited the suppressive effect of rIFN-alpha 4, indicating that IFN induced the expression of one or several new proteins encoded by gene(s) within the If-1l locus which are responsible for the NDV-specific downregulation of IFN-alpha/beta production. A time course analysis indicated that the suppressive activity of IFN-induced If-1l gene products took 12 h to develop. It was preceded by an opposite priming effect, leading to enhancement of the early IFN-alpha/beta response to NDV measured 5 h after infection. This priming effect in BMDM was, however, only visible during an 8-h period of IFN-alpha treatment, whereas in the continued presence of IFN for 12 h or longer, priming was superimposed by the inhibitory action of the If-1l gene products.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous interferon specifically regulates Newcastle disease virus-induced cytokine gene expression in mouse macrophages.

In macrophages from inbred mice, the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the If-1 locus, which carries the allele for either high (h) or low (l) IFN production. Here, we report that the activity of genes within the If-1 locus is influenced by macrophage-derived endogenous IFN. In addition to various other biological effects, we observed that endogenous IFN specifically downregulated NDV-induced IFN and interleukin 6 production. Preculture of bone marrow-derived macrophages (BMM) from BALB/c (If-1l) mice in macrophage colony-stimulating factor plus anti-IFN-beta provoked a 30- to 50-fold increase in NDV-induced cytokine production compared with induced control cultures in macrophage colony-stimulating factor alone, whereas only a 4- to 6-fold increase was observed in anti-IFN-beta-treated BMM from C57BL/6 (If-1h) mice. This resulted in nearly complete abrogation of the genetically determined difference in the response to NDV. The increase was specific for NDV and was marked by strong additional activation of IFN-alpha genes. Studies using BMM from B6.C-H28c If-1l congenic mice gave results identical to those obtained with BALB/c BMM. Addition of 20 IU of recombinant IFN-alpha 4 to anti IFN-beta-treated macrophages from B6.C-H28c mice 20 h prior to NDV infection strongly downregulated the IFN-alpha, IFN-beta, and interleukin 6 responses. The genetic difference between macrophages from If-1h and If-1l mice was thus reestablished, since the same treatment caused only weak reduction of NDV-induced cytokine gene expression in BMM from C57BL/6 mice. These data suggest that the If-1h and If-1l alleles harbor IFN-inducible genes that, following activation, specifically suppress subsequent cytokine gene expression in response to NDV.

Regulation by endogenous interferon of virus-induced cytokine gene expression in mouse macrophages.

In macrophages from inbred mice the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the locus If-1, with C57BL/6 carrying the 'high-producer' allele If-1h whereas BALB/c have the 'low-producer' allele If-1l. The IFN produced consists of 90% IFN-beta and there are 10-fold differences between macrophages from If-1h and If-1l mice. Recently, we observed that interleukin-6 (IL-6) is coinduced by NDV in macrophages and seems to be under the same genetic control. Noninduced macrophages have been shown to secrete low amounts of antiviral activity endogenously when cultured in the presence of the macrophage-colony-stimulating factor (M-CSF). Here, we report that the amount of this endogenous IFN varies between macrophages from different mouse strains. Macrophages from BALB/c were found to secrete 5-10 times more endogenous IFN compared to C57BL/6. The antiviral activity could be identified as IFN-beta. Interestingly, we observed that endogenous IFN specifically down-regulates NDV-induced IFN and IL-6 production. Preculture of BALB/c macrophages in M-CSF plus anti-IFN-beta to neutralize the biological effects of the endogenous IFN provoked a 30- to 50-fold increase in NDV-induced cytokine production, resulting in a nearly complete abrogation of the genetically determined difference since the same treatment only caused a 6-fold increase in C57BL/6 macrophages following NDV infection. This increase in cytokine gene expression was specific for NDV and marked by a strong additional activation of IFN-alpha genes. Addition of mouse recombinant IFN-alpha 4 to anti-IFN-beta-treated macrophages for 18 h prior to NDV infection down-regulated again IFN gene expression and reestablished the genetic differences between macrophages from If-1h and If-1l mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus-vs endotoxin-induced activation of liver macrophages.

The response of liver macrophages (Kupffer cells) to distinct pathogenic material was investigated by comparing virus- and endotoxin-induced macrophage activation. Endotoxin-induced stimulation and induction with Newcastle disease virus (NDV) or Sendai virus led to the release of the same pattern of prostanoids characterized by a predominant production of prostaglandin E2 (PGE2). With respect to peptide mediators, hepatic macrophages secreted tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 after viral induction and endotoxin treatment, respectively. In response to viruses, however, much more interleukin-6 and TNF-alpha was detected than after endotoxin stimulation. Interferon type I (interferon-alpha/beta), on the other hand, was only detected in the supernatants of macrophages infected with viruses, but not of those exposed to endotoxin. This study also revealed that rat TNF-alpha exists in several soluble species, some of which are glycosylated.

Modification of tumor cells by a low dose of Newcastle disease virus. III. Potentiation of tumor-specific cytolytic T cell activity via induction of interferon-alpha/beta.

To investigate possibilities of augmenting tumor-specific immune responses against the highly metastatic murine lymphoma ESb, we tested the effects of the interferon inducer newcastle disease virus (NDV) or of interferon-alpha/beta as costimulator in mixed lymphocyte-tumor cell cultures (MLTC) on the tumor-specific cytolytic T cell (CTL) response. Both approaches, namely stimulation of ESb immune spleen cells with NDV-modified stimulator cells or with ESb stimulator cells and exogenous IFN-alpha/beta, led to a selective potentiation of tumor-specific CTL activity. The potent activation of tumor-specific CTL precursor (CTLP) required the simultaneous presence of the specific ESb tumor antigen--possibly to mediate a signal via the corresponding T cell receptor--and costimulators--possibly to mediate second activation signals. Increased CTL activity required only very low amounts of NDV or IFN-alpha/beta. The generation of CTL activity in the MLTC cultures could be blocked by antisera to IFN-alpha/beta, not, however by control sera. Similar effects were observed in vivo, suggesting that IFN-alpha/beta not only caused an increase in CTL activity, but was essential for the generation of CTL activity. The reduction of the generation of CTL by antiserum to IFN-alpha/beta could be overcome by excess interferon, especially when using ESb-NDV as stimulator cells.

In vitro development of bone-marrow-derived macrophages. Influence of mouse genotype on response to colony-stimulating factors and autocrine interferon induction.

Responsiveness to granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) of bone marrow cells derived from different mouse strains was investigated. There were great variations in proliferation between different strains of inbred mice. Bone marrow cells from mouse strains with a high rate of proliferation in response to GM-CSF also had a high proliferating capacity to M-CSF. The response to either CSF did not correlate with a certain H-2 haplotype. GM-CSF induced consistently higher proliferation than M-CSF. Proliferation in response to M-CSF, but not to GM-CSF, could be enhanced by the addition of antibodies against interferon (IFN). IFN is the only known inducer of (2'-5') oligoadenylate (oligo (A] synthetase. This enzyme was induced in macrophages grown in the presence of M-CSF, but not in GM-CSF promoted cells. Enzyme induction was completely abrogated by simultaneous treatment with anti-IFN alpha/beta. Infection of macrophages with herpes simplex virus type 1 (HSV) and vesicular stomatitis virus (VSV) revealed that GM-CSF-promoted cells were highly susceptible to lytic infection by these viruses. In contrast, virus titres in M-CSF-cultured cells were 100-fold lower. We conclude that, contrary to M-CSF, GM-CSF does not induce autocrine IFN during haematopoiesis. As judged from data with BALB/c mice, the sensitivity to the anti-proliferative effect of the autocrine IFN may be a factor which influences M-CSF-promoted proliferation.

Cell type specific expression and regulation of murine interferon alpha and beta genes.

The differential and cell type specific expression of various murine IFN alpha genes and IFN beta was examined by S1 nuclease protection assays in M-CSF cultured C57BL/6 mouse bone marrow macrophages and L929 fibroblasts. In Newcastle disease virus (NDV) induced macrophages, IFN beta, alpha 2, alpha 4, and alpha 1 mRNAs were the predominant species, whereas IFN alpha 6 and alpha 9 transcripts accounted for only 5% of total IFN alpha mRNAs. In L929 cells, only IFN beta, alpha 2, and alpha 4 genes were expressed efficiently following NDV induction and IFN alpha 9 mRNA was always below detectable level. Induction of macrophages with the synthetic inducer 10-carboxymethyl-9-acridanone resulted in small amounts of IFN alpha 2, alpha 4, and alpha 6 mRNAs and the IFN beta mRNA level was about 100-fold higher. Macrophages and L929 cells especially differed in the kinetics of IFN gene induction in that macrophages showed a much earlier transient expression of all IFN mRNA species. Additionally, IFN transcripts were degraded much faster in macrophage cultures than in L929cells. The IFN response of macrophages is thus characterized by a highly efficient control, providing a rapid onset and a rapid decline of IFN production, which limits release of IFN to a short time interval.

Differential expression of interferon alpha and beta induced with Newcastle disease virus in mouse macrophage cultures.

Stimulation of mouse macrophages with Newcastle disease virus (NDV) leads to a rapid and high interferon (IFN) response. The magnitude of this response is influenced by the mouse genotype. We have analysed NDV-induced IFN production at the protein and mRNA levels in two different populations of macrophages derived from 'high producer' C57BL/6 and 'low producer' BALB/c mice in vitro. The data indicate that bone marrow and peritoneal macrophages from both strains grown in the presence of L cell conditioned medium (CM) as a source of macrophage colony-stimulating factor 1 (M-CSF) or purified murine M-CSF produce 10- to 50-fold more IFN on a per cell basis than cultures of resident peritoneal macrophages. These differences were also found when steady state levels of IFN mRNA were analysed. Differential analysis for the ratios of IFN-alpha and IFN-beta showed that CM- or M-CSF-cultured macrophages produced equal amounts of both IFN species as determined by specific monoclonal antibodies and hybridization experiments using IFN-alpha and IFN-beta DNA probes, whereas resident peritoneal macrophages induced under identical conditions produced almost exclusively IFN-beta. This suggests a stimulating effect of M-CSF on IFN synthesis in NDV-induced cultures of mouse macrophages, which is in part due to additional activation of IFN-alpha gene expression.

Contamination of a monoclonal antibody with LDH-virus causes interferon induction.

Interferon induction occurred unexpectedly during an in vivo study using a mouse monoclonal antibody. The interferon was typed alpha/beta and the titer reached a maximum at 24 hours in contrast with other inducers. Similar results were obtained with a virus pool derived from the antibody and with a LDV reference strain. MAP-testing of the monoclonal antibody revealed contamination with lactate dehydrogenase virus (LDV). The production of IFN seems to be controlled genetically. This experimental error demonstrates the importance of an appropriate quality control of biological materials.