Concanavalin A-induced interferon gamma production by murine spleen cells and T cell lines. Lack of correlation with Lyt 1,2 phenotype

Concanavalin A(Con A)-induced interferon-gamma (IFN gamma) production by resting or preactivated murine spleen cells negatively selected with monoclonal antibodies specific for Lyt 1,2 antigens plus complement (C) and by interleukin 2(IL-2)-dependent T cell lines of different Lyt phenotype was studied. The data show that most of the IFN-gamma produced upon stimulation of resting spleen cells was a product of Lyt 1+2+ cells. Lyt 1-2+ cells were negative for IFN gamma production. When spleen cells that had been preactivated for 3 days with Con A were restimulated with Con A, Lyt 1+2+, Lyt 1+2- as well as Lyt 1-2+ cells produced IFN gamma in a relationship of approximately 5:3:1. In both cases the picture remained unaltered independently when the supernatants were harvested after 1, 3 or 5 days. Furthermore, two IL-2 dependent T cell lines were studied in regard to Con A-induced IFN gamma production. Line 1.3 was Thy 1+, Lyt 1-2+, whereas line 20.9. was Thy 1+, Lyt 1+2-. Both lines produced initially high titers of IFN gamma upon stimulation with Con A. After prolonged passage in vitro, however, they progressively lost the capacity to produce IFN gamma.     

Effects of natural human interferon-alpha, -beta and -gamma on interleukin 2 production in human peripheral lymphocytes

Effects of interferon (IFN) on PHA-induced interleukin 2 (IL-2) production by human peripheral mononuclear cells were studied comparatively with natural human IFN-alpha, IFN-beta and IFN-gamma, using an equivalent unit of their antiviral activity ranging from 10 to 1000 IU/ml. IL-2 activity was assessed in cultures with or without IFN by a standard bioassay using murine CTLL-2 cells. PHA-induced production of IL-2 in cultures of peripheral mononuclear cells was unaltered or slightly suppressed by the simultaneous presence of IFN-alpha and IFN-beta. The effect was the same, whether or not indomethacin was present in the cultures. In contrast, the addition of IFN-gamma to the PHA-stimulated cultures markedly enhanced IL-2 production, while IFN-gamma per se had no effect on IL-2 production in the absence of PHA. The enhancement of IL-2 production due to IFN-gamma was more marked in cultures which did not include indomethacin than in cultures which contained indomethacin (1 x 10(-6) M).     

Spontaneous production of gamma interferon and induced production of beta interferon by human T-lymphoblastoid cell lines.

Two human T-lymphoblastoid cell lines, CCRF/CEM and Molt 4, produced beta interferon (IFN-beta) upon infection with Sendai virus. Molt 4, but not CCRF/CEM, spontaneously produced up to 300 U of IFN-gamma per ml, apparently not contaminated with IFN-alpha or -beta. Phytohemagglutinin, a T-cell mitogen, did not stimulate IFN production in these lines. A third T-lymphoblastoid line, CCRF/HSB2, produced no IFN either spontaneously or after infection with Sendai virus or treatment with phytohemagglutinin. The Molt 4 cells contained an mRNA which could be translated by oocytes to give IFN-gamma. Molt 4 cells therefore provide a convenient source of human IFN-gamma and its mRNA for experimental purposes.     

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.     

Detection of altered T helper 1 and T helper 2 cytokine production by peripheral blood mononuclear cells in patients with multiple sclerosis utilizing intracellular cytokine detection by flow cytometry and surface marker analysis.

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.     

The human intracellular Mx-homologous protein is specifically induced by type I interferons

The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.     

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.     

Frontline: absence of functional STAT4 activation despite detectable tyrosine phosphorylation induced by murine IFN-alpha

We previously reported that IL-12, but not IFN-alphaA/D, induces T helper type (Th) 1 development and STAT4 phosphorylation in murine CD4+ T cells. However, a recent study reported that IFN-alphaA/D and recombinant murine IFN-alphaA can induce STAT4 phosphorylation, although more weakly than IL-12, largely in CD8+ rather than CD4+ T cells. That report did not examine Th1 development or directly demonstrate induction of IFN-gamma by IFN-alpha. To address these differences, we compared IFN-alphaA/D, murine IFN-alphaA, and IL-12 for STAT4 phosphorylation, formation of active nuclear DNA binding complexes, induction of Th1 development, and production of IFN-gamma in murine CD4+ T cells. IFN-alphaA induced detectable STAT4 phosphorylation, although at significantly lower levels than induced by IL-12. Furthermore, in contrast to IL-12, IFN-alphaA failed to induce Th1 development or the formation of DNA binding complexes or to synergize with IL-18 for induction of IFN-gamma production. STAT1-deficient CD4+ T cells showed increased IFN-alphaA-induced STAT4 phosphorylation but still exhibited significantly lower amounts of cytokine-induced IFN-gamma compared to IL-12. In summary, these results suggest that in contrast to IL-12, IFN-alphaA does not play a functionally significant role in meditating the STAT4-dependent induction of Th1 development or IFN-gamma production in CD4+ T cells.     

Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

Interferon-gamma induces HLA-DR expression by thyroid epithelium.

We recently showed that human thyroid epithelial cells, which are normally negative for HLA-DR molecules, express HLA-DR in thyroid autoimmunity. Furthermore, induction of HLA-DR on normal thyroid cells can be achieved by culture with plant lectins. We have now found that recombinant human interferon-gamma (IFN-gamma) induces expression of HLA-DR molecules on cultured human thyroid cells, whereas Namalva IFN-alpha, recombinant IFN-beta or recombinant interleukin-2 (IL-2) do not. All three IFN, but not IL-2, enhanced thyroid cell HLA-A,B,C expression. The results strongly implicate T cells (which are the source of IFN-gamma) in the aberrant induction of DR on thyroid epithelial cells which is proposed to be a central feature of the immunopathological processes leading to autoimmunity.     

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)     

IL-6 produced by type I IFN DC controls IFN-gamma production by regulating the suppressive effect of CD4+ CD25+ regulatory T cells

The dendritic cell family is composed of different subsets differentially governing the immune response. Type I interferon (IFN) dendritic cells (DC) are endowed with the ability to trigger both Th1 and Th2 type responses. In view of the pivotal role of regulatory T cells in limiting the effectiveness of effector cells, we analyzed the interactions between these cells and type I IFN DC. DC were generated from monocytes in the presence of IFN-beta and interleukin (IL)-3 (DCI3) or granulocyte macrophage-colony-stimulating factor and IL-4 (DCG4) and activated by poly(I:C). Despite the release of lower amounts of IL-12 after maturation, DCI3 were able to induce a higher IFN-gamma production by T lymphocytes during the mixed leucocyte reaction (MLR) as compared with DCG4. mRNA analysis disclosed that DCI3 overtranscribed the IL-6 gene and secreted high amounts of the protein. Neutralization of IL-6 revealed that this cytokine specifically contributed to the IFN-gamma release induced by DCI3. Finally, depletion of CD25+ T cells before the MLR identified these cells as a target for IL-6. We conclude that DCI3 are endowed with the property of regulating the suppressive effect of regulatory T cells through high IL-6 production. This novel mechanism of T cell control is relevant for the use of DCI3 in vaccination strategies.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells.

Interleukin-27 (IL-27) supports proliferation of naive CD4(+) T cells and enhances interferon-gamma (IFN-gamma) production by activated T cells and natural killer (NK) cells. We report here that IL-27 induces Stat1 and Stat3 phosphorylation and activation in human and murine cell lines and primary human T cells. IL-27 also induces T-Bet, a Stat1-dependent gene crucial to Th1 cell commitment. Similarly, IFN-alpha activates Stat1 and Stat3 and T-Bet expression in naive T cells. Induction of T-Bet results in upregulation of IL-12Rbeta2 on naive T cells, which is essential for responsiveness to IL-12 and differentiation to a Th1 phenotype. Both IL-27 and IFN-alpha induce expression of IL-12Rbeta2 in T cells. In contrast, IFN-gamma, which activates Stat1 but not Stat3, induces expression of T-Bet but not IL-12Rbeta2 in naive T cells. We propose that IL-27 and IFN-alpha are important for early Th1 commitment and act upstream of IL-12 and IFN-gamma in this pathway.     

Induction of interferon in murine bone marrow-derived macrophage cultures by 10-carboxymethyl-9-acridanone

10-Carboxymethyl-9-acridanone (CMA) induced high titers of interferon (IFN) in murine leukocyte cultures. Thymocytes, lymph node, spleen and peritoneal exudate cells responded to CMA with IFN production. Pure macrophages derived from the bone marrow were the most efficient producers of CMA-induced IFN. The yields of IFN-α β in the macrophage supernatants depended on the concentration of the inducer and titers up to 3000 IU/ml were measured after exposure to the optimal dose (500 μg/ml). CMA was found to be the first low molecular weight compound that induced in vitro titers of IFN nearly as high as obtained after exposure to Newcastle disease virus, which is one of the most potent interferon inducers.     

Antigen-specific lymphocyte transformation induced by oocyst antigens of Eimeria bovis.

Lymphoproliferative responses against a preparation of Eimeria bovis antigens (EBAg) were measured in E. bovis-immune and naive animals. Optimal lymphocyte responsiveness could be measured after 7 days of culture in the presence of antigen at a cell concentration of 2 X 10(5) cells per well. The specificity of the reaction was confirmed by limiting dilution analysis. Whereas immune peripheral blood mononuclear cells responded to EBAg (f = 1/18,824), naive cells did not (f = 0). The helper function of cells proliferating in response to EBAg was investigated by raising T-cell lines and a clonal population derived from a line. The T-cell line showed an enhanced reactivity to EBAg by limiting dilution analysis (f = 1/256) and was interleukin-2 dependent. Limiting dilution analyses indicated at least two populations of cells: one that was interleukin-2 restricted and antigen dependent and another that was antigen independent. Supernatants from T-cell lines and the clone were analyzed for the production of lymphokines after antigen stimulation. Minimal amounts of interleukin-2 were produced. The T-cell line produced both gamma interferon (IFN-gamma) (750 U) and IFN-alpha (1,250 U), whereas the clone produced IFN-gamma (1,250 U) only. Short-term (4-day) stimulation of immune cells by EBAg induced the production of IFN-gamma (600 U) and a non-IFN macrophage-activating lymphokine. We conclude that this macrophage-activating lymphokine is only produced after short-term culture and that further culture of T cells results in the proliferation of other clones producing other factors (such as IFN).     

Interferon-gamma is not an antiviral, but a growth-promoting factor for T lymphocytes

The effects of interferon (IFN)-gamma or IFN-alpha/beta on virus yield, (2'-5')oligo(A) synthetase activation, H-2 antigen expression and proliferation of T lymphocytes have been investigated. Under the culture conditions used, vesicular stomatitis virus or Semliki Forest virus replication in T cells was not impaired by the addition of IFN-gamma, whereas it was completely inhibited by the addition of IFN-alpha/beta. In contrast, B cell lines, macrophage-transformed cell lines and fibroblasts were fully protected by both IFN-gamma as well as IFN-alpha/beta following virus infection. The lack of sensitivity of T lymphocytes to the antiviral effects of IFN-gamma was not due to absence of specific membrane receptors, since in saturation binding experiments with 125I-labeled murine IFN-gamma most T cell lines displayed a number of binding sites and a degree of affinity comparable to those found on B cells, which are fully sensitive to IFN-gamma antiviral activity. Analysis of IFN-induced dsRNA-dependent (2'-5')oligo(A) synthetase activity, one of the biochemical markers for cellular responses to IFN, showed that it was not induced in T lymphocytes after IFN-gamma treatment, whereas IFN-alpha/beta induced high levels. Both IFN-gamma and IFN-alpha/beta enhanced H-2 antigen expression on T cells as well as on cells of different histological type. Moreover, when IFN-gamma was tested for its antiproliferative activity on T cells, it was found to consistently potentiate the response of these cells to mitogens or growth factors, rather than inhibit their proliferation. Taken as a whole these results suggest that on T lymphocytes IFN-gamma should not be regarded as an antiviral agent, but rather as a modulator of T cell growth and functional differentiation, transducing intracellular signals dissimilar to those observed with target cells of different origin.