Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Interferon induces tryptophanyl-tRNA synthetase expression in human fibroblasts.

A cDNA clone complementary to an interferon (IFN)-induced mRNA was isolated and used to characterize the regulation of expression of its RNA by the IFNs and to identify the protein its RNA encodes. This cDNA hybridizes to IFN-induced 3.1- and 2.3-kilobase mRNAs that are synthesized in response to both IFN-alpha and IFN-gamma. IFN-gamma induces the sustained accumulation of these mRNAs while IFN-alpha induces their transient accumulation. Cycloheximide (50 micrograms/ml) failed to inhibit the induction of these mRNAs by either IFN-alpha or IFN-gamma, suggesting that their induction does not require de novo protein synthesis. DNA sequence analysis of this cDNA reveals that it encodes a protein of Mr 53,168 that has sequence homology with and the biological activity of a tryptophanyl-tRNA synthetase, an enzymatic activity that has been demonstrated to play a role in and be modulated by the growth of cells. Elevated levels of this enzyme may be involved in the cell growth inhibitory activity of the IFNs.     

Decreased DNA repair capacity of UV-irradiated cells following interferon treatment

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-alpha, IFN-beta and IFN-gamma on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-alpha had a stronger inhibitory effect than IFN-gamma. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.     

Regulation of macrophage growth and antiviral activity by interferon-gamma

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.     

Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-alpha/beta, IFN-gamma, and TNF-alpha at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-alpha/beta or IFN-gamma, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-gamma, but not IFN-alpha/beta, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-alpha. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.     

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.     

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.     

Expression of gamma-interferon receptor in murine bone marrow-derived macrophages associated with macrophage differentiation: evidence of gamma-interferon receptors in the regulation of macrophage proliferation

The effect of purified, recombinant murine gamma interferon (IFN-gamma) on the regulation of macrophage proliferation induced by colony-stimulating factor 1 (CSF-1) was investigated. Although both hemopoietic stem cells (GM-CFC) and tissue-derived peritoneal exudate macrophages (PEM) proliferated in response to CSF-1, the more mature PEM were much more sensitive to an antiproliferative effect of IFN-gamma. The role of IFN-gamma receptor expression and its relationship to growth inhibition was examined. Bone marrow cells as a whole did not exhibit an appreciable amount of IFN-gamma receptor binding activity. Likewise, nonadherent (NA) cells derived from CSF-1-stimulated bone marrow cultures displayed low levels of IFN-gamma receptor binding activity. On the contrary, more mature adherent (AD) cells (monocytes/macrophages) from the same culture exhibited high levels of IFN-gamma receptor binding activity, which continued to increase with culture time. The elevated IFN-gamma binding activity is due to an increase in total receptor number rather than the binding affinity as judged by Scatchard analysis. Similar to the relationship between PEM and GM-CFC, more mature AD cells were also more susceptible to the inhibitory effect of IFN-gamma on CSF-1-induced proliferation than their less mature NA counterparts. The fact that the sensitivity to IFN-gamma correlated well with the expression of existing IFN-gamma receptors strongly suggests that the inhibitory effect is mediated through IFN-gamma receptors. This study shows that the expression of IFN-gamma receptors in mononuclear phagocytes may not only represent one of the phenotypic parameters acquired by the growing macrophages during the process of differentiation, but may play some role in controlling proliferation.     

Differential effects of type I IFN and IFN-gamma on the binding of tumor necrosis factor to receptors in two human cell lines

The effect of IFN-alpha and IFN-beta on the expression of cell surface receptors for tumor necrosis factor (TNF) was examined in two human cell lines. In HeLa cells, IFN-alpha and IFN-beta increased 125I-TNF binding, whereas in HT-29 cells these two IFN either slightly decreased or had no effect on 125I-TNF binding. In contrast, IFN-gamma increased 125I-TNF binding in both cell lines. Both IFN-alpha and IFN-beta exerted an antagonistic effect on IFN-gamma-induced stimulation of TNF receptor expression in HT-29 cells, but did not inhibit TNF receptor induction by IFN-gamma in HeLa cells. IFN-gamma and, to a lesser extent, IFN-beta were synergistic with TNF in producing cytotoxic/cytostatic activity in HT-29 cells. Despite the inhibitory effect of IFN-beta on the IFN-gamma-induced stimulation of TNF receptor expression, IFN-beta did not inhibit the synergistic enhancement of TNF cytotoxicity by IFN-gamma in HT-29 cells. The dissociation between the effects of IFN-beta on TNF receptor expression and on the cytotoxic activity of TNF in HT-29 cells suggests that TNF receptor modulation is not a major mechanism of synergism between IFN and TNF.     

Interferon gamma inhibits proliferation and hyaluronic acid adhesion of human malignant glioma cells in vitro

Malignant gliomas are frequent and the prognosis is poor. The cytokine interferon gamma (IFN-gamma) enhances several immune phenomena and may be used in immunotherapy of tumours. Therefore we investigated the influence of IFN-gamma on human cell lines T98G, U87MG, 86HG39 and 85HG66, measuring cell viability (MTT-test) and proliferation (3H-thymidine uptake). IFN-gamma markedly decreased viability and proliferation of all investigated cell lines. Expression of CD44 and adhesion to hyaluronic acid (HA) are involved in glioma invasion. Influence of IFN-gamma on these two features has also been investigated. IFN-gamma markedly decreased HA-adhesion in all three investigated cell lines, whereas CD44 expression remained uninfluenced. To summarise, IFN-gamma strongly decreased cell growth and HA-adhesion of malignant glioma cell lines in vitro. We suggest further investigations to characterise better the role of IFN-gamma as a treatment opportunity for malignant gliomas.     

Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon.

The susceptibility to alpha/beta interferon (IFN-alpha/beta) or to gamma interferon (IFN-gamma) of various lymphocytic choriomeningitis virus (LCMV) strains was evaluated in C57BL/6 mice and in various cell lines. Anti-IFN-gamma treatment in vivo revealed that the LCMV strains Armstrong, Aggressive, and WE were most susceptible to IFN-gamma whereas Traub, Cl 13-Armstrong, and Docile were resistant. The same pattern of susceptibility to recombinant IFN-gamma was observed in vitro. In vivo treatment with anti-IFN-alpha/beta showed a sizeable increase in replication of Aggressive, Armstrong, and WE; effects were less pronounced for Docile, Cl 13-Armstrong, or Traub. Correspondingly, WE, Armstrong, and Aggressive were all relatively sensitive to purified IFN-alpha/beta in vitro, and Cl 13-Armstrong, Docile, and Traub were more resistant. Overall, there was a good correlation between the capacity of LCMV strains to establish a persistent infection in adult immunocompetent mice and their relative resistance to IFN-gamma and IFN-alpha/beta.     

Enhancement of monocyte-mediated tumoricidal activity by multiple interferon-alpha species

Twenty-one interferon (IFN)-alpha species were evaluated for their ability to enhance monocyte-mediated lysis of the human melanoma cell line, A375. A wide variation in the potency of the different species in inducing monocyte tumoricidal action was observed. In addition, many IFN-alpha species were found to induce as much or more tumoricidal activity than recombinant IFN-gamma. The degree of monocyte activation induced by the various species generally correlated with their antiviral activity. Those which were better at inducing monocyte tumoricidal action also gave the highest antiviral specific activities. Studies were conducted to determine if the relative potency of the IFN-alpha species could be changed by altering certain parameters of the cytotoxicity assay. All IFN-alpha species tested required only 30 min in culture with the monocytes to induce activation. There were no changes in the relative potency of the species when cytotoxicity was measured at different times, nor when the effector:target ratio was altered. Competitive binding studies revealed that those IFN-alpha species which induced little activity in the bioassays were also generally poor in their ability to bind the IFN-alpha receptor on human monocytes, while the IFN-alpha species which induced relatively more activity in the bioassays were better able to bind to the IFN-alpha receptor. These data indicate that there are dramatic differences in activities among the IFN-alpha species which may, in part, be explained by different binding affinities. In addition, the differences observed among the IFN-alpha species demonstrate the need for further functional and structural characterization of the individual IFN-alpha species which could lead to a more effective clinical application of IFN-alpha.     

Degradation of STAT1 and STAT2 by the V proteins of simian virus 5 and human parainfluenza virus type 2, respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons

Human cell lines were isolated that express the V protein of either simian virus 5 (SV5) or human parainfluenza virus type 2 (hPIV2); the cell lines were termed 2f/SV5-V and 2f/PIV2-V, respectively. STAT1 was not detectable in 2f/SV5-V cells, and the cells failed to signal in response to either alpha/beta interferons (IFN-alpha and IFN-beta, or IFN-alpha/beta) or gamma interferon (IFN-gamma). In contrast, STAT2 was absent from 2f/PIV2-V cells, and IFN-alpha/beta but not IFN-gamma signaling was blocked in these cells. Treatment of both 2f/SV5-V and 2f/PIV2-V cells with a proteasome inhibitor allowed the respective STAT levels to accumulate at rates similar to those seen in 2fTGH cells, indicating that the V proteins target the STATs for proteasomal degradation. Infection with SV5 can lead to a complete loss of both phosphorylated and nonphosphorylated forms of STAT1 by 6 h postinfection. Since the turnover of STAT1 in uninfected cells is longer than 24 h, we conclude that degradation of STAT1 is the main mechanism by which SV5 blocks interferon (IFN) signaling. Pretreatment of 2fTGH cells with IFN-alpha severely inhibited both SV5 and hPIV2 protein synthesis. However, and in marked contrast, pretreatment of 2fTGH cells with IFN-gamma had little obvious effect on SV5 protein synthesis but did significantly reduce the replication of hPIV2. Pretreament with IFN-alpha or IFN-gamma did not induce an antiviral state in 2f/SV5-V cells, indicating either that the induction of an antiviral state is completely dependent on STAT signaling or that the V protein interferes with other, STAT-independent cell signaling pathways that may be induced by IFNs. Even though SV5 blocked IFN signaling, the addition of exogenous IFN-alpha to the culture medium of 2fTGH cells 12 h after a low-multiplicity infection with SV5 significantly reduced the subsequent cell-to-cell spread of virus. The significance of the results in terms of the strategy that these viruses have evolved to circumvent the IFN response is discussed.     

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Combination of tumor necrosis factor alpha and interferon alpha induces apoptotic cell death through a c-myc-dependent pathway in p53 mutant H226br non-small-cell lung cancer cell line.

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-alpha) and natural human interferon alpha (IFN-alpha). Studies were performed with two human non-small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-alpha and TNF-alpha significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-alpha or TNF-alpha alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-alpha/TNF-alpha induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-alpha/TNF-alpha. Antisense c-myc inhibited IFN-alpha/TNF-alpha cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-alpha/IFN-alpha induce apoptosis through a c-myc-dependent pathway rather than a p53-dependent pathway. (c)2001 Elsevier Science.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

Effect of interferon-gamma on myelin basic protein-specific T cell line proliferation in response to antigen-pulsed accessory cells.

We have shown previously that treatment of SJL/J mice with anti-interferon-gamma monoclonal antibody (mAb) exacerbated experimental allergic encephalomyelitis (EAE) only if administered at the time of encephalitogenic challenge. Here we investigate the role of interferon-gamma (IFN-gamma) and anti-IFN-gamma mAb in the early events of T cell activation in vitro. Pretreatment of murine peritoneal exudate cells (PEC) with IFN-gamma led to a significant increase in their ability to activate myelin basic protein (MBP)-specific, short-term T cell lines. When exogenous IFN-gamma was added to cocultures of T cells and MBP-pulsed PEC, the antigen-specific T cell proliferation was considerably reduced. Anti-IFN-gamma mAb added to these cultures neutralized the inhibitory effect of the exogenous IFN-gamma on T cell proliferation but had no visible effect on class II MHC expression by the antigen-pulsed PEC present in the same cultures. A reduction in T cell proliferation was also observed when the T cells were treated with IFN-gamma prior to coculture with the MBP-pulsed PEC. These results demonstrate that, on one hand, IFN-gamma enhances the ability of PEC to induce antigen-specific T cell proliferation but, on the other hand, acts on the T cells themselves by inhibiting their proliferation in response to the antigen-pulsed PEC. This may explain why treatment with anti-IFN-gamma antibody in vivo induces EAE exacerbation.