Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.     

The effect of age of cattle on the in vitro production of interferon by peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from normal cattle of different ages and from specific pathogen-free (SPF) calves, 2 to 4 weeks old, were cultured with bovine herpes virus type 1 (BHV1), parainfluenza-3 virus (PI3) and phytohaemagglutinin (PHA). The interferon (IFN) produced was characterized by acid stability and neutralizing antisera to recombinant bovine interferons. The virus preparations were presented either live or inactivated and as cell-bound virus or free virions. PBMC from cattle of all ages produced IFN-alpha when stimulated with live BHV1 and PI3 viruses. IFN-alpha was also produced with inactivated BHV1, even in cell cultures from SPF calves. However, inactivated PI3 virus failed to induce IFN in PBMC cultures from normal cattle, but approximately half of the animals, mostly calves, produced IFN-gamma spontaneously in 48 h cultures in the absence of added antigen. PHA induced IFN-gamma at an optimal concentration of 20 micrograms per ml after 3 days in culture. An age-related maturation of the IFN response was observed as PBMC from calves less than 2 weeks old produced little or no IFN when induced with either PHA or inactivated BHV1, although some IFN-alpha was produced in cultures containing live virus. Both adherent and non-adherent cells from adults and calves over 2 weeks old produced IFN on induction with inactivated BHV1 but only the non-adherent cell population produced IFN spontaneously or in response to inactivated PI3.     

Impaired antiviral response in human hepatoma cells.

Hepatitis B, C, and D viruses can infect liver cells and in some individuals establish a chronic phase of infection. Presently, relatively little information is available on the antiviral mechanisms in liver cells. Because no good in vitro model infection systems for hepatitis viruses are available, we have used influenza A, Sendai, and vesicular stomatitis (VSV) viruses to characterize interferon (IFN) responses and IFN-induced antiviral mechanisms in human hepatoma cell lines. HepG2 or HuH7 cells did not show any detectable IFN-alpha/beta production in response to influenza A or Sendai virus infections. Treatment of cells with IFN-alpha resulted in upregulation of IFN-alpha-inducible Mx, 2',5'-oligoadenylate synthetase (OAS) and HLA class I gene expression but only with exceptionally high levels of IFN-alpha (>/=100 IU/ml). Accordingly, high pretreatment levels of IFN-alpha, 1000 IU/ml for influenza A and VSV and 100 IU/ml for Sendai virus, were required before any detectable antiviral activity against these viruses was seen. IFN-gamma had some antiviral effect against influenza A virus but appeared to be ineffective against VSV and Sendai virus. IFN-gamma upregulated HLA class I protein expression, whereas Mx or OAS expression levels were not increased. There was a modest upregulation of HLA class I expression during Sendai virus infection, whereas influenza A virus infection resulted, after an initial weak upregulation, in a clear decrease in HLA class I expression at late times of infection. The results suggest that hepatoma cells may have intrinsically poor ability to produce and respond to type I IFNs, which may contribute to their inability to efficiently resist viral infections.     

Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production.

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.     

Production of type I (alpha/beta) interferon after virus infection of cloned, alloantigen-sensitized mouse T lymphocytes

Mouse T lymphocytes sensitized to alloantigens were cloned by limiting dilution in the presence of interleukin 2. Clones were tested for surface markers Thy-1, Lyt-1 and Lyt-2, and for cytotoxic function. Production of interferon (IFN) by clones either (a) stimulated with allogeneic cells; (b) activated with concanavalin A (Con A); or (c) infected with Semliki Forest virus or Newcastle disease virus were assayed. All clones produced IFN upon Con A stimulation and most after virus infection. Analysis of the IFN produced by a single clone, using anti-IFN antisera, showed that while Con A stimulation induced production of type II IFN (IFN-gamma), the IFN produced after virus infection was type I IFN (IFN-alpha/beta).     

Interferon production and response to exogenous interferon in two cell lines of mouse brain origin persistently infected with Sendai virus

Summary Two persistently infected cell lines established from C3H mouse brain cells infectedin vivo with Sendai virus were shown to differ with respect to interferon (IF) production and response to exogenous IF. MB/Sen carrier cells contained 1–5 per cent antigen positive cells when examined by immunofluorescence, and virus was occasionally recovered from the culture medium. MB/Sen_AS carrier cells were maintained with 0.16 per cent Sendai antiserum in the supernatant medium. All MB/Sen_AS cells contained viral antigen and infectious virus was present in the culture medium.MB/Sen released IF spontaneously into the culture medium. Further IF production could be stimulated in MB/Sen by superinfection with Newcastle disease virus (NDV) or vesicular stomatitis virus (VSV). Exogenous IF provided good protection against VSV challenge.In contrast, MB/Sen_AS produced no IF spontaneously but could be stimulated by NDV and VSV to produce IF. Exogenous IF failed to reduce the amount of VSV released into the supernatant fluid. Replication of VSV was restricted in MB/Sen_AS as shown by a 2.3 log₁₀ lower virus yield compared to MB/Sen.     

Lymphokines produced by herpesvirus-transformed marmoset monkey lymphoid cell lines. I. Characterization of a constitutively produced interferon

Conditioned media from cultures of marmoset monkey T-lymphoid cell lines transformed by Herpesvirus saimiri or Herpesvirus ateles were found to contain interferon (IFN) activity. Titers between individual cell lines varied by a factor of 100; large amounts (up to 10(5) units/ml, assayed on human cells) were produced in one of the cell lines. IFN production was enhanced by the diterpene tumor promoters, TPA and mezerein, but not by classical T-cell mitogens. The IFN resembles human IFN-gamma by the following criteria: lability at pH 2, stability against 2-mercaptoethanol, cross-species activity, shape of dose-response curves, and molecular weight determined by size-exclusion chromatography (50,000-55,000). Its activity was not inhibited, however, by antiserum against human IFN-gamma or antisera against human IFN-alpha or IFN-beta.     

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.     

Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology.     

The immunosuppressive effects of measles virus on T cell function--failure to affect IL-2 release or cytotoxic T cell activity in vitro.

Measles virus (MV) is known to depress T cell function. In order to determine whether this results from alteration in the production of, or response to, interleukin-2 (IL-2) we studied the effect of in vitro infection with MV on human IL-2 dependent T cell lines. MV produced a cytopathic productive infection in these cells. Class I allospecific cytotoxic T cells retained their cytotoxic activity 48 h after infection. Both cytotoxic and Leu 3a/4a positive T cell lines continued to respond to IL-2 by proliferation up to 26 h after infection. The ability of human tonsillar lymphocytes to generate IL-2 in response to phytohaemagglutinin following MV infection was then studied. In early measles infection (up to 48 h) there was no suppression of IL-2 production: in fact measles infected cells spontaneously released low levels of IL-2 in the absence of lectin. Similarly, IL-2 release was not affected by Herpes simplex virus infection of such cultures, although lymphocytes infected with Sendai or respiratory syncytial viruses produced considerably less IL-2. These observations suggest that MV-induced immunosuppression is not a result of inhibition of differentiated T cell function, IL-2 generation or responsiveness, but may be more directly related to virus-induced cytopathic effects in activated T cells.     

Type I interferon production by plasmacytoid dendritic cells and monocytes is triggered by viruses, but the level of production is controlled by distinct cytokines.

The principal interferon-alpha/beta (IFN-I)-producing cells are plasmacytoid dendritic cell (PDC) precursors belonging to the lymphoid lineage. Monocytes that can differentiate into dendritic cells (DC) also produce IFN-I, although much less than PDC, after interaction with infectious agents. We show that whereas viruses trigger these cells to produce IFN-I, the amount of IFN is tightly controlled by cytokines. Monocytes produced IFN-I in response to Sendai virus (SV) infection, and PDC responded to both SV and herpes simplex virus (HSV). All cytokines tested failed to induce production of IFN-I in the absence of infection. However, among 18 relevant cytokines, incubation of PDC with interleukin-4 (IL-4), IL-15, and IL-7 alone or in combination with IL-3 before infection, enhanced IFN-I secretion. At variance, IL-12 alone or in synergy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was active on SV-infected but not on HSV-infected monocytes. Tumor necrosis factor-alpha (TNF-alpha) and IL-4 inhibited IFN-I production by PDC and monocytes, respectively, and IL-10 strongly inhibited IFN-I production in both cell lineages. The response of PDC to IL-7 and IL-15, which also activate natural killer (NK) cell maturation, further emphasizes the cooperation between these two cell subsets in the control of innate immunity.     

Gamma interferon production in endotoxin-responder and -nonresponder mice during infection.

The production of gamma interferon (IFN-gamma) in response to infection and to a number of other agents was compared in Lpsn (C3H/HeN and C57BL/10ScSn) and Lpsd (C3H/HeJ and C57BL/10ScCr) mouse strains. Large differences in IFN-gamma production were observed between C57BL/10ScCr mice and the other mouse strains. With the exception of C57BL/10ScCr, all mouse strains, including C3H/HeJ, exhibited transient levels of IFN-gamma during infection with Salmonella typhimurium. Spleen cells of these mice, explanted on day 3 of infection, produced in vitro IFN-gamma spontaneously; this production was enhanced considerably by heat-killed S. typhimurium, heat-killed Propionibacterium acnes, concanavalin A (ConA), or lipopolysaccharide (LPS). These stimuli, except for LPS, also induced IFN-gamma production in cultures of normal spleen cells from noninfected animals. In contrast, C57BL/10ScCr mice produced no IFN-gamma following infection with S. typhimurium. Also, spleen cells of these mice, explanted on day 3 of infection, exhibited no spontaneous IFN-gamma production. A marginal response was obtained by additional stimulation of the cells with killed S. typhimurium, and a moderate response was obtained with ConA. Normal spleen cells from noninfected C57BL/10ScCr mice showed no IFN-gamma response to killed S. typhimurium, killed P. acnes, or LPS and only a low response to ConA. Impaired IFN-gamma production in C57BL/10ScCr mice was also evident during infection with Plasmodium chabaudi chabaudi, with which a low IFN-gamma response was seen only occasionally. Also, spleen cells from infected animals (days 2 to 8 after infection) exhibited only a very low level of IFN-gamma production in vitro; however, this production could be enhanced further by ConA. In comparison, C57BL/10ScSn mice infected with P. chabaudi chabaudi produced significant amounts of IFN-gamma. Spleen cells explanted from infected animals produced IFN-gamma spontaneously in vitro; this production was enhanced further by killed P. acnes and ConA. The results showed that in addition to the defect in LPS responsiveness, C57BL/10ScCr mice possess a defect in IFN-gamma production in response to different stimuli. During infection, IFN-gamma production and sensitization to LPS occurred in parallel. Infected Lpsn mice exhibited enhanced sensitivity and infected Lpsd C3H/HeJ mice exhibited reasonable sensitivity to the lethal effects of LPS. Lpsd C57BL/10ScCr mice remained resistant to LPS when infected with S. typhimurium and exhibited only marginal sensitivity when infected with P. chabaudi chabaudi.     

Effect of interferon inductors on infection induced by hepatitis C virus and activity of mRNA cytokines in cell cultures SW-13 and MT-4

An experimental model of hepatitis C virus (HCV) infection in cell culture in vitro was used to study the influence of interferon (IFN) inducers on HCV infection activity. In combination with the RT-PCR method, this model was also used to study the dynamics of cytokine mRNA activity for IFN-alpha, IFN-gamma, IL-I beta, IL-2, IL-4, IL-6, IL-8, BL-10, IL-12, IL-18, and TNF-alpha. The research was carried out using long-term cell cultures SW-13 (human paradrenal adenocarcinoma cells) and MT-4 (human cells of lymphoblastoid origin) inoculated with HCV under conditions of acute infection. The obtained data showed that cell cultures SW-13 and MT-4 were sensitive to replication of HCV (cytopathogenic variant). The addition of IFN inducers Savratz, Kagocel, and Cycloferon to infected cell cultures usually resulted in suppression of HCV reproduction in these cultures. Cycloferon had the greatest antiviral activity (virus titer level decreased by a factor of 2.51 g and 5.51 g TCD50 in cell cultures SW-13 and MT-4, respectively). It was suggested that induction of IFN-gamma, IL-4 and IL-8 plays a certain role in HCV reproduction suppression. The results of this work provide an opportunity for more efficacious use of IFN inducers in therapy of HCV infection.     

Relationship between natural killer (NK) cells and interferon (IFN) alpha-producing cells in human peripheral blood. Studies with a monoclonal antibody with specificity for human natural killer cells

The relationship between NK active and IFN alpha-producing cells in human peripheral blood was studied with a monoclonal antibody with specificity for NK cells (anti-Leu11b). Removal of Leu11b antigen expressing leukocytes with antibody-mediated complement-dependent lysis resulted in a marked reduction of NK activity. In contrast, the depletion of Leu11b positive cells did not affect the production of IFN alpha in response to influenza A/X31 virus, Corynebacterium parvum, or Molt 4 human leukemic cells. The results indicate that NK activity and synthesis of IFN alpha are mediated by different leukocyte subpopulations. The findings further suggest that the augmentation of the cytotoxic activity of NK cells by IFN may not be the consequence of positive self-regulation, but rather of cellular cooperation.     

Interferons in rheumatoid arthritis: alterations in production and response related to disease activity

Various markers associated with the production of and response to interferons (IFNs) were studied in patients with either inactive rheumatoid arthritis (RA) or active RA, and in healthy subjects. The IFN markers assessed were serum and synovial fluid (SF) levels, the activity in peripheral blood leukocytes (PBL) of (2'-5') oligoadenylate synthetase (OAS), and the production in vitro by PBL of IFN-alpha/beta in response to Sendai virus or Poly(I):Poly(C) as inducers, and of IFN-gamma using PHA or Con A as inducers. IFN activity, tested by antiviral assays using two different cell lines, was not demonstrable in the serum of any patient with RA. The activity of (2'-5') OAS in PBL, which may indirectly indicate exposure of leukocytes to IFN, was increased in RA compared with healthy subjects, more so in patients with inactive RA. The production of IFN-alpha/beta by PBL in response to Sendai virus was low in active RA but high in inactive RA, relative to production in healthy subjects. The production of IFN-gamma by PBL in RA was lower than in healthy subjects, more so in active RA. Thus inactive RA (remission status) is marked by evidence of PBL having been influenced by interferon and being a state of augmented inducibility to an IFN-alpha/beta stimulus, whereas active RA is associated with low inducibility of PBL to an IFN stimulus, but no evidence of IFN production in vivo. Our findings underscore the relevance of interferon to remission/activity in rheumatoid arthritis.     

Differential effect of type I and type II interferons on neopterin production and amino acid metabolism in human astrocyte-derived cells

Intrathecal production of neopterin, a pteridine produced by interferon (IFN)-gamma-stimulated monocyte-derived macrophages, is associated with neurological disorders and infections. We investigated whether IFN-alpha/beta, IFN-gamma, or human immunodeficiency virus (HIV) induce neopterin production by human astroglioma cells. IFN-alpha/beta and IFN-gamma, but not HIV, induced neopterin. Interestingly, IFN-gamma, but not IFN-alpha/beta, increased expression and activity of the tryptophan-catabolizing enzyme indoleamine (2,3)-dioxygenase. In contrast, IFN-alpha/beta, but not IFN-gamma, reduced the uptake of three aromatic amino acids in U87MG and U138 astroglioma cells. Thus type I and type II IFN stimulate astrocyte-derived cells to produce neopterin and exert differential effects on amino acid metabolism.     

Association of human leucocyte low responsiveness to inducers of interferon alpha with HLA-DR 2

The influence of antigens of the major histocompatibility complex (MHC) on the production of interferon (IFN) alpha or IFN gamma by human peripheral blood leucocytes (PBL) in vitro has been studied. Synthesis of IFN gamma by PBL stimulated with purified phytohaemagglutinin (PHA-P) or protein A of Staphylococcus aureus (SpA) appeared not to be controlled by MHC antigens. The production of IFN alpha, however, was influenced by the HLA type of the donor. Low responsiveness of PBL to inducers of IFN alpha (influenza virus, Molt 4 cells) was associated with HLA-DR 2. Implications of these observations for studies of IFN production and natural killer (NK) cell activity are discussed.     

In vitro augmentation of human natural cytotoxic activity.

Stimulation of human blood lymphocyte preparations with mitomycin C-treated lymphoid cell lines produced increased levels of cytotoxicity against both NK-susceptible and NK-resistant target cell lines. The greatest effect was seen following stimulation by the B lymphocyte-derived lines, Bri8 and raji. K562 also stimulated high levels of activity while the T lymphocyte-derived lines, CCRF/CEM and MOLT 4, produced smaller increases activity was also found in PHA- and MLC-stimulated populations. Stimulation by lymphoid cell lines gave increased cytotoxic activity against all five cell lines when used as target cells and the pattern of target cell susceptibility was maintained, with K562, CCRF/CEM and MOLT 4 being more susceptible than Bri8 and Raji. No direct correlation was found between the level of cytotoxic activity and the level of 3H-thymidine uptake in stimulated effector cell populations. The B cell lines stimulated high levels of isotopic uptake, while the T cell lines gave no significant stimulation. Similarly, the level of 3H-thymidine incorporation following PHA and MLC stimulation showed no direct correlation with the level of cytotoxic activity. Stimulation of lymphocyte transformation did not appear to be necessary for the induction of cytotoxic activity, although the largest increases in cytotoxicity occurred in populations showing high isotope incorporation. No correlation was found between the target cell susceptibility of the different cell lines and their ability to stimulate cytotoxicity.