Effect of interferon-γ on antigen processing in human monocytes

The effect of interferon-γ (IFN-γ) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fcγ receptor (FcγR) was followed for various times after treatment of cells with IFN-γ. THP-1 cells that had been treated with IFN-γ for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-γ treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48–72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-γ, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-γ-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-γ modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.     

Divergent effects of FcγRIIIA ligands on the functional activities of human natural killer cells in vitro

The Fcγ receptor (R)IIIA (CD16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via FcγRIIIA, purified NK cells were cultured for 2–3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to FcγRIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2Rα, ICAM-1), cytotoxicity, cytokine production (IL-1β, IFN-γ and TNF-α) and release of soluble IL-2R. Thus, mIgG binding to FcγRIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2Rα expression, cell proliferation and cytokine release.     

Comparison of the effects of interleukin-1α, interleukin-lβ and interferon-γ-inducing factor on the production of interferon-γ by natural killer

Interferon-γ inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-γ (IFN-γ) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1γ. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1α, IL-1β and IGIF on NK cell production of IFN-γ. All three IL-1 types enhanced NK cell production of IFN-γ when induced by IL-2 or IL-12, although at high concentrations (>10 ng/ml), IGIF was five- to tenfold more potent than IL-1α or IL-1β. This effect correlated with enhanced levels of mRNA for IFN-γ when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-γ was not increased by IL-1α or IL-1β. The ability of IGIF to enhance IFN-γ production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-γ and demonstrate that the effect of IGIF on NK cell production of IFN-γ is similar to that of IL-1α and IL-1β but distinct from that of IL-12.     

Anti-parasitic effector mechanisms in human brain tumor cells: Role of interferon-γ and tumor necrosis factor-α

Toxoplasma gondii, an obligate intracellular parasite, is able to replicate in human brain cells. We recently showed that interferon (IFN)-γ-activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN-γ-mediated activation of indolamine 2,3-dioxygenase (IDO), resulting in the degradation of the essential amino acid tryptophan. In contrast, glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN-γ activation, and IFN-γ-mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)-α alone is unable to activate IDO or to induce toxoplasmostasis in any glioblastoma cell line tested. Interestingly, we found that TNF-α and IFN-γ were synergistic in the activation of IDO in glioblastoma cells 87HG31, 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF-α + IFN-γ activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF-α or IFN-γ were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore, it was found that the addition of L -tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF-α and IFN-γ may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells.     

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-α and interferon-γ

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), we investigated the effects of these two cytokines on death of luteal cells in vitro. Methods: Mouse luteal cells were cultured in serum-free medium with TNF-α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN-γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. Results:On day 3 of culture, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-γ (1,000 U/ml) and TNF-α (5,000 U/ml) did. On day 6, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-α alone at 5,000 U/ml did, and combinations of IFN-γ and TNF-α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-α. On days 3–6 of culture, combinations of IFN-γ and TNF-α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. Conclusions: The presence of IFN-γ modulates the ability of TNF-α to induce a reduction in the number of viable cells, although TNF-α alone at high concentrations can induce a reduction in the number of viable cells. © 1995 Wiley-Liss, Inc.     

IFN-γ-induced TNF-α is a prerequisite for in vitro production of nitric oxide generated in murine peritoneal macrophages by IFN-γ

The effect of IFN-γ to stimulate formation of nitric oxide (NO) by normal murine peritoneal macrophages (Mϕ) has been found to be completely dependent on the ability of IFN-γ to activate secretion of TNF-α. The NO-stimulatory effect of IFN-γ was abolished by anti-TNF-α antibodies, the inhibitory intervention of which could be fully reversed by exogenously supplied TNF-α. Accordingly, the failure of Mϕ from C3H/HeJ mice to secrete TNF-α upon stimulation with IFN-γ was associated with their complete incapability to generate NO, unless they were simultaneously treated with IFN-γ + TNF-α. Collectively, the data document that similar to the NO up-regulatory action of other cytokines, the effect of IFN-γ is not independent, but depends on a synergistic cooperation with the self-produced TNF-α. The findings thus indicate that a widespread opinion claiming that IFN-γ per se is able to stimulate biosynthesis of NO needs revision.     

Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) on human renal cell carcinoma

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20 68%. To enhance the growth inhibitory effect of IL-4. we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-γ caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-α was more limited (0 20% inhibition). The addition of IL-4 to IFN-γ on RCC cells sensitive to lL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-γ rendered RCC cells more responsive to the inhibitory effect mediated by TNF-α, The combination of TNF-Q with IL-4 and IFN-γ induced an optimal growth inhibition (up lo 90 98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic, All RCC lines tested expressed class 1 antigens, but not class II antigens. IFN-γ induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-7. lL-4 minimally affected class I expression, whereas TNF-(v up-regulated class I antigen expression. IL-4 or TNF-α did not induce class II expression. The combination of The three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-γ alone. These observations confirm the direct interaction of IL-4, IFN-γ and TNF-a with RCC tumour cells. both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal ceil carcinoma.     

Interleukin-12 is required for interferon-γ production and lethality in lipopolysaccharide-induced shock in mice

Several cytokines, in particular tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 γg/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-γ production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-γ production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1–3 ng/ml at 3–6 h after LPS injection, whereas IFN-γ production was observed only in BCG-primed mice. The priming effect of BCG on IFN-γ production appears to be mostly due to its ability to increase TNF-α production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-γ production, as shown by the ability of injection of TNF-α and LPS (1 γg/mouse), but not LPS alone, to induce IFN-γ production. However, in addition to TNF-α, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-γ production, because co-injection of TNF-α and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-γ production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-γ production, also completely protect BCG-primed mice injected with up to 10 μg of LPS from shock-induced death. Thus, IL-12 is required for IFN-γ production and lethality in an endotoxic shock model in mice.     

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)

The anti-mycobacterial activities of IFN-γ and TNF-α-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-γ or TNF-α for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0–3) was strongly suppressed in IFN-γ-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-α treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-γ-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-α treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-γ but not TNF-α enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-γ and TNF-α potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.     

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody‐dependent cellular cytotoxicity towards cancer cells

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti‐cancer antibodies act through different mechanisms, including antibody‐dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47‐SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab‐coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa‐131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa‐131R. Furthermore, ADCC was consistently enhanced by targeting CD47‐SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47‐SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47‐SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.     

Human and rodent interferon-γ as a growth factor for Trypanosoma brucei

An example for the bidirectional exchange of activating signals between a pathogen and immunocompetent cells in the host is presented. Trypanosoma brucei, which include subspecies that cause African sleeping sickness, secrete a molecule that triggers lymphocytes to produce interferon (IFN)-γ. We now report that proliferation of T. brucei is stimulated in axenic cultures by IFN-γ. The growth-enhancing effect on the pathogen is inhibited by anti-IFN-γ receptor (R) antibodies and does not occur after exposure to other cytokines, i.e. IFN-α, IFN-β and tumor necrosis factor (TNF)-α. While rodent-pathogenic T. brucei strains are stimulated by rat IFN-γ, human pathogenic strains are more potently stimulated by human IFN-γ. Rat and human IFN-γ can partially block each others effects. Mice with disrupted IFN-γ genes have reduced parasitemia and prolonged survival, while the outcome is reversed in mice that lack the IFN-γR gene.     

Chimeric Fc receptors identify immunoglobulin-binding regions in human FcγRII and FcϵRI

FcγRII and Fc?RI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas Fc?RI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig-like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in Fc?RI. Firstly, a chimeric form of the Fc?RI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high-affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high-affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser¹³⁶ to Val¹⁶⁹, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp⁸⁷ to Lys¹²⁸, Tyr¹²⁹ to Asp¹⁴⁵, and Ser¹⁴⁶ to Val¹⁶⁹.     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Interferon-γ confers resistance to experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), T cells infiltrate the central nervous system (CNS) and induce inflammation. These CD4⁺ T cells secrete interferon (IFN)-γ, levels of which correlate with disease severity, and which is proposed to play a key role in disease induction. Many strains of mice are resistant to EAE. We have studied the effect of deletion of IFN-γ on the ability to induce EAE in resistant BALB/c-backcrossed mice. As expected, only 0–6 % of BALB/c or BALB/c-backcrossed mice developed EAE when immunized with myelin basic protein in adjuvant. Strikingly, abrogation of IFN-γ expression by targeted disruption of the IFN-γ gene (GKO mice) converted them to a susceptible phenotype. As many as 71 % of these IFN-γ-deficient mice developed EAE, a frequency comparable to that seen with the susceptible SJL/J strain. In addition, EAE was of unusually high severity in mice lacking IFN-γ. Immunological characteristics of disease in IFN-γ-deficient mice were comparable to those seen in susceptible (SJL/J) mice with EAE, including perivascular infiltration in the CNS and order-of-magnitude increases for both CD3 γ chain and TNF-α mRNA levels in the spinal cord. We thus demonstrate that lack of IFN-γ converts an otherwise EAE-resistant mouse strain to become susceptible to disease. Therefore, in BALB/c mice, IFN-γ confers resistance to EAE.