Melatonergic signalling instructs transcriptional inhibition of IFNGR2 to lessen interleukin-1ß-dependent inflammation.

BACKGROUND: Immunotransmitters (e.g., neurotransmitters and neuromodulators) could orchestrate diverse immune responses; however, the elaborated mechanism by which melatonergic activation governs inflammation remains less defined. METHODS: Primary macrophages, various cell lines, and Pasteurella multocida (PmCQ2)-infected mice were respectively used to illustrate the influence of melatonergic signalling on inflammation in vitro and in vivo. A series of methods (e.g., RNA-seq, metabolomics, and genetic manipulation) were conducted to reveal the mechanism whereby melatonergic signalling reduces macrophage inflammation. RESULTS: Here, we demonstrate that melatonergic activation substantially lessens interleukin (IL)-1ß-dependent inflammation. Treatment of macrophages with melatonin rewires metabolic program, as well as remodels signalling pathways which depends on interferon regulatory factor (IRF) 7. Mechanistically, melatonin acts via membrane receptor (MT) 1 to increase heat shock factor (Hsf) 1 expression through lowering the inactive glycogen synthase kinase (GSK3) ß, thereby transcriptionally inhibiting interferon (IFN)-γ receptor (IFNGR) 2 and ultimately causing defective canonical signalling events [Janus kinase (JAK) 1/2-signal transducer and activator of transcription (STAT) 1-IRF7] and lower IL-1ß production in macrophages. Moreover, we find that melatonin amplifies host protective responses to PmCQ2 infection-induced pneumonia. CONCLUSIONS: Our conceptual framework provides potential therapeutic targets to prevent and/or treat inflammatory diseases associating with excessive IL-1ß production.

Quercetagetin inhibits macrophage-derived chemokine in HaCaT human keratinocytes via the regulation of signal transducer and activator of transcription 1, suppressor of cytokine signalling 1 and transforming growth factor-ß1.

BACKGROUND: Inflammatory chemokines, such as macrophage-derived chemokine (MDC/CCL22), are elevated in the serum and lesioned skin of patients with atopic dermatitis (AD), and are ligands for C-C chemokine receptor 4, which is predominantly expressed on T helper 2 lymphocytes, basophils and natural killer cells. We have previously reported that quercetagetin has an inhibitory activity on inflammatory chemokines, which is induced by interferon (IFN)-γ and tumour necrosis factor (TNF)-α, occurring via inhibition of the signal transducer and activator of transcription 1 (STAT1) signal. OBJECTIVES: To investigate the specific mechanisms of quercetagetin on the STAT1 signal. METHODS: We confirmed the inhibitory activity of quercetagetin on MDC and STAT1 in HaCaT keratinocytes. The interaction between STAT1 and IFN-γR1 was investigated using immunoprecipitation. The small interfering RNA approach was used to investigate the role of suppressor of cytokine signalling 1 (SOCS1) and transforming growth factor (TGF)-ß1 induced by quercetagetin. RESULTS: Quercetagetin inhibited the expression of MDC at both the protein and mRNA levels in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. Moreover, quercetagetin inhibited the phosphorylation of STAT1 through upregulation of SOCS1. Increased expression of SOCS1 disrupted the binding of STAT1 to IFN-γR1. Furthermore, quercetagetin augmented the expression of TGF-ß1, which is known to modulate the immune response and inflammation. CONCLUSIONS: These results suggest that quercetagetin may be a potent inhibitor of the STAT1 signal, which could be a new molecular target for anti-inflammatory treatment, and may thus have therapeutic applications as an immune modulator in inflammatory diseases such as AD.

Miltefosine promotes IFN-gamma-dominated anti-leishmanial immune response.

Leishmania donovani, a protozoan parasite, resides and replicates as amastigotes within macrophages. The parasite inflicts the disease visceral leishmaniasis by suppressing host cell function. Neither a therapeutic vaccine nor an effective anti-leishmanial drug to reverse the immunosuppression is available. Although miltefosine (hexadecylphosphocholine or HPC) is a promising orally bioavailable anti-leishmanial drug, its efficacy is seriously compromised by contra-indications in pregnant women. Further rational redesigning of the drug requires studies on its mechanism of action, which is unknown at present. Because miltefosine is proposed to have immunomodulatory functions, we examined whether miltefosine exerts its anti-leishmanial functions by activating macrophages. We observed that miltefosine's anti-leishmanial function was significantly compromised in IFN-gamma-deficient macrophages suggesting the importance of endogenous IFN-gamma in miltefosine-induced anti-leishmanial functions of macrophages. Miltefosine induced IFN-gamma, neutralization of which reduced the anti-leishmanial functions of macrophages. IFN-gamma responsiveness is reduced in L. donovani-infected macrophages but is significantly restored by miltefosine, as it enhances IFN-gamma receptors and IFN-gamma induced STAT-1 phosphorylation but reduced activation of SHP-1, the phosphatase implicated in the down-regulation of STAT-1 phosphorylation. Miltefosine induced protein kinase C-dependent and PI3K-dependent p38MAP kinase phosphorylation and anti-leishmanial function. Miltefosine promotes p38MAP kinase-dependent anti-leishmanial functions and IL-12-dependent Th1 response. Leishmania donovani-infected macrophages induced Th2 response but miltefosine treatment reversed the response to Th1-type. Thus, our data define for the first time the mechanistic basis of host cell-dependent anti-leishmanial function of miltefosine.

Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro.

BACKGROUND/AIMS: The receptor of interferon-gamma (IFN-gammaR) consists of IFN-gammaR1 and R2. Resistance to the anti-proliferative effect of IFN-gamma is due to downregulation of IFN-gammaR2. The aim of this study was to investigate whether iron chelation could upregulate IFN-gammaR2 and enhance the anti-proliferative effect of IFN-gamma in colon cancer cell lines. METHODOLOGY: The colon cancer cell lines, SW480, COLO, and WiDr were treated with the iron chelating agent DFO, and the expression of IFN-gammaR1 and IFN-gammaR2 was evaluated by FACS. The anti-proliferative effect of IFN-gamma was investigated by MTT assay, and the proapoptotic effect was investigated by FACS with Annexin-V. RESULTS: FACS demonstrated that DFO increased the expression of IFN-gammaR2, whereas the effect on IFN-gammaR1 expression was less marked. MTT assay showed that cell growth was inhibited by DFO. Addition of DFO and IFN-gamma inhibited further, but inhibition was not observed with IFN-gamma alone. Apoptotic cells were increased by DFO, and further increased with DFO + IFN-gamma together. CONCLUSIONS: Expression of IFN-gammaR2 is restored by iron chelation, and the increased expression of IFN-gammaR2 enhances the anti-proliferative effect of IFN-gamma through induction of apoptosis in colon cancer cells.

Influence of interferon-alpha and ribavirin on the transcription of interferon-gamma and IFN-gamma receptor genes in Jurkat cells.

Interferon-alpha and ribavirin are currently the only drugs registered in the chronic hepatitis C therapy. Their actions are based on both direct antiviral activities, and their influence on genes expressions. In the presented study, using Jurkat cell line as an in vitro model for interferon-gamma synthesis, influence of interferon-alpha and ribavirin on the expressions of IFN-gamma and its receptor subunits (IFNgR1 and IFNgR2) were studied. Expressions of the studied genes were measured at the transcriptional level using Real-Time RT-PCR method. Results indicate that both drugs, IFN-alpha and ribavirin, induced changes in IFN-gamma and its receptor expressions. While IFN-alpha stimulated the expressions of the studied genes (IFN- gamma, IFNgR1, and IFNgR2), ribavirin showed the contradictory influence. The inhibitory effect of ribavirin dominated IFN-alpha action and was responsible for the decrease in the mRNA levels of IFN-gamma and the receptor of IFN-gamma. This phenomenon observed at in vitro model may be responsible for the IFN-gamma decrease during hepatitis C therapy with IFN-alpha and ribavirin which was suggested by some authors.

Delivery of interferon-beta to the monkey nervous system following intranasal administration.

We determined the nervous system targeting of interferon-beta1b (IFN-beta1b), a 20 kDa protein used to treat the relapsing-remitting form of multiple sclerosis, following intranasal administration in anesthetized, adult cynomolgus monkeys. Five animals received an intranasal bolus of [(125)I]-labeled IFN-beta1b, applied bilaterally to the upper nasal passages. Serial blood samples were collected for 45 min, after which the animals were euthanized by transcardial perfusion-fixation. High resolution phosphor imaging of tissue sections and gamma counting of microdissected tissue were used to obtain the distribution and concentration profiles of [(125)I]-IFN-beta1b in central and peripheral tissues. Intranasal administration resulted in rapid, widespread targeting of nervous tissue. The olfactory bulbs and trigeminal nerve exhibited [(125)I]-IFN-beta1b levels significantly greater than in peripheral organs and at least one order of magnitude higher than any other nervous tissue area sampled. The basal ganglia exhibited highest [(125)I]-IFN-beta1b levels among CNS regions other than the olfactory bulbs. Preferential IFN-beta1b distribution to the primate basal ganglia is a new finding of possible clinical importance. Our study suggests both IFN-beta and IFN-alpha, which share the same receptor, may be bound with relatively high affinity in these structures, possibly offering new insight into a neurovegetative syndrome induced by IFN-alpha therapy and suspected to involve altered dopamine neurotransmission in the basal ganglia. Most importantly, our results suggest intranasally applied macromolecules may bypass the blood-brain barrier and rapidly enter the primate CNS along olfactory- and trigeminal-associated extracellular pathways, as shown previously in the rat. This is the first study to finely detail the central distribution of a labeled protein after intranasal administration in non-human primates.

Interferon-gamma is up-regulated in the hippocampus in response to intermittent fasting and protects hippocampal neurons against excitotoxicity.

Dietary restriction (DR) increases the life span of many different organisms, and recent findings have demonstrated neuroprotective effects of DR in rodent and nonhuman primate models of neurodegenerative disorders. The neuroprotective mechanism of action of DR is unknown, but it may result from a mild cellular stress response involving increased production of neurotrophic factors. Because several different cytokines are known to be up-regulated in brain cells in response to stress, we determined whether DR affected cytokine expression in the rat brain. Levels of expression of interferon-gamma (IFN-gamma) and its receptor were significantly increased in the hippocampus of rats that had been maintained on an intermittent fasting DR regimen compared with rats on the ad libitum control diet. Pretreatment of embryonic rat hippocampal cell cultures with IFN-gamma protected neurons against glutamate-induced death. IFN-gamma-mediated neuroprotection was associated with an enhanced recovery of intracellular Ca(2+) concentrations following exposure to glutamate. Our data show that intermittent fasting DR stimulates IFN-gamma-mediated neuroprotective signaling in the hippocampus, suggesting a role for this cytokine in the previously reported ability of DR to protect neurons in animal models of severe epileptic seizures, stroke, and neurodegenerative disorders.

Interferon receptors and the caspase cascade regulate the antitumor effects of interferons on human pancreatic cancer cell lines.

BACKGROUND: Interferons (IFNs) have antiproliferative effects on tumor cells. The apoptotic effects and sensitization to chemotherapy conferred by IFN therapy, however, are not clearly understood. The aims of the present study were to explore the apoptotic effects of IFNs in human pancreatic cancer cell lines and to attempt to define their ability to synergistically enhance sensitivity to 5-fluorouracil (5-FU) and gemcitabine, a mechanism that depends on the expression of IFN receptors. METHODS: Human pancreatic cancer cells were cultured alone or in combination with the chemotherapeutic agents 5-FU and gemcitabine. Differential dosages of IFN-alpha, -beta, and -gamma were also added to the cell lines concomitantly during a period of 24 to 96 hours. The cell line viability and effects of treatment were examined using the methylthiazol tetrazolium assay and single-stranded DNA apoptosis assay. The expression of IFN receptors was determined using immunohistochemistry. Caspase-8 inhibitor was used to block the caspase cascade. RESULTS: The antiproliferative and apoptotic effects of IFNs were most profoundly demonstrated on those cells that expressed the respective IFN receptor. The apoptotic effects provided by the interferons, however, were blocked by caspase-8 inhibition. The addition of IFNs significantly enhanced the cytotoxic effects of 5-FU and gemcitabine in those cell lines that expressed the corresponding IFN-alpha, -beta, or -gamma receptors. CONCLUSIONS: This study on pancreatic cancer cell lines has demonstrated that IFNs mediate apoptosis through IFN receptors and the caspase cascade. Enhanced cytotoxicity occurred when IFNs were combined with 5-FU and gemcitabine.

Inquiring into the differential action of interferons (IFNs): an IFN-alpha2 mutant with enhanced affinity to IFNAR1 is functionally similar to IFN-beta.

Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-alpha2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.

Importance of signaling via the IFN-alpha/beta receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor.

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for approximately 80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for approximately 10% of IFN-alpha/betaR-/- mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-alpha/betaR-/- mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-alpha/betaR-/- mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-alpha/betaR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-alpha/betaR-/- mice were not. Thus, signaling via the IFN-alpha/betaR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge.

Natural killer cells inhibit hepatitis C virus expression.

Natural killer (NK) cells are critical in host innate defense against certain viruses. The role of NK cells in controlling hepatitis C virus (HCV) remains obscure. We examined whether NK cells are capable of inhibiting HCV expression in human hepatic cells. When NK cells are cultured with the HCV replicon-containing hepatic cells, they have no direct cytolytic effect but release soluble factor(s) suppressing HCV RNA expression. Media conditioned by NK cell lines (NK-92 and YTS) or primary NK cells isolated from healthy donors contain interferon gamma (IFN-gamma) and potently inhibit HCV RNA expression. Ligation of CD81 on NK cells inhibits IFN-gamma production and results in decreased anti-HCV activity. In addition, the antibodies to IFN-gamma or IFN-gamma receptors abolish the anti-HCV activity of NK cell-conditioned media. The role of IFN-gamma in NK cell-mediated, anti-HCV activity is supported by the observation that NK cell-conditioned media enhanced expression of signal transducer and activator of transcription-1, a nuclear factor that is essential in IFN-gamma-mediated antiviral pathways. NK cell-conditioned media have the ability to stimulate intracellular IFN-alpha expression in the hepatic cells, suggesting a mechanism responsible for NK cell-mediated, anti-HCV activity. Thus, NK cells hold the potential to play a vital role in controlling HCV replication in hepatic cells using an IFN-gamma-dependent mechanism.

Indole-3-carbinol stimulates transcription of the interferon gamma receptor 1 gene and augments interferon responsiveness in human breast cancer cells.

Indole-3-carbinol (I3C), a naturally occurring compound of Brassica vegetables, has promising anticancer properties and activates an anti-proliferative pathway that induces a G1 cell cycle arrest of human breast cancer cells. A microarray analysis of I3C treated versus untreated MCF-7 breast cancer cells revealed that I3C increased expression of the interferon gamma receptor 1 (IFNgammaR1). Western blot and RT-PCR analysis demonstrated that I3C strongly and rapidly stimulated IFNgammaR1 gene expression. Transfection of a series of 5' deletion constructs of the IFNgammaR1 reporter plasmids revealed that I3C significantly stimulates the promoter activity of the IFNgammaR1 and uncovered an I3C-responsive region between -540 and -240 bp of the IFNgammaR1 promoter. I3C stimulation of the IFNgammaR1 expression suggests that indole treatment should enhance IFNgamma responsiveness in breast cancer cells. A combination of I3C and IFNgamma synergistically activated STAT1 proteins by increasing phosphorylation at the Tyr-701 site. In addition, I3C and IFNgamma together more effectively induced a G1 cell cycle arrest and stimulated expression of the p21(Waf1/Cip1) cell cycle inhibitor, compared with the effects of either agent alone. Our results suggest that one mechanism by which I3C mediates these anticancer effects is by stimulating expression of the IFNgammaR1 and augmenting the IFNgamma response in MCF-7 human breast cancer cells.

Interferon-alpha/beta receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006.

BACKGROUND: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. RESULTS: This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-kappaB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-alpha/beta receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. CONCLUSION: This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-alpha/beta receptor-mediated paracrine pathway.

The Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits gamma interferon-regulated HLA-DR and Fc gamma R1 on human macrophages through Toll-like receptor 2.

Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4(+) T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-gamma) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-gamma receptor (IFN-gamma R) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-gamma-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-gamma-induced expression of Fc gamma RI. Thus, M. tuberculosis, through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-gamma R signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis persistence in macrophages.

Interleukin-4 and transforming growth factor beta have opposing regulatory effects on gamma interferon-mediated inhibition of Cryptosporidium parvum reproduction.

It was shown previously that enterocytes activated by gamma interferon (IFN-gamma) are efficient effector cells in the killing of Cryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor beta (TGF-beta) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-beta might modulate the effect of IFN-gamma in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-beta can abolish the inhibitory effect on C. parvum development and that neither IL-13 nor IL-10 influenced the action of IFN-gamma. In contrast, IL-4 cooperated with low concentrations of IFN-gamma (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-gamma and IL-4 was intracellular Fe(2+) deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-gamma signaling, were similar when cells were stimulated with IFN-gamma alone and with IFN-gamma and IL-4 gamma, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-beta and IL-4 on IFN-gamma function did not appear to involve any alteration in the level of expression of IFN-gamma receptors.

A peptide mimetic of human interferon (IFN)-beta.

Type I interferons (IFNs) are cytokines that are used clinically as antiviral and antitumour agents. The interaction of IFNs with their heterodimeric type I IFN receptor comprised of IFNAR1 and IFNAR2 is a first step to inducing biological actions. Here, we describe the successful mimicry of IFN-beta by a peptide isolated by phage-display screening using a neutralizing anti-IFN-beta monoclonal antibody. The 15-mer peptide, designated SYR6, was shown to compete with IFN-beta for binding to type I IFN receptor in a concentration-dependent manner, and was shown to elicit antiviral activity on cultured cells. This antiviral activity was not eliminated in the presence of neutralizing monoclonal antibodies to IFN-alpha, -beta and -gamma, and a low concentration of soluble type I IFN receptor, suggesting that it was not due to IFN contamination or the induction of endogenous IFNs by SYR6. This peptide might be a potent agonist to provide a mechanism of activating heterodimeric cytokine receptors.

Relationship between 24-hour rhythm in antiviral effect of interferon-beta and interferon-alpha/beta receptor expression in mice.

The influence of interferon-beta (IFN-beta) dosing time on antiviral activity was investigated in ICR male mice under light-dark cycle conditions (lights on at 07:00, off at 19:00) with food and water available ad libitum. There was a significant dosing time-dependent change in 2',5'-oligoadenylate synthetase (2',5'-OAS) activities, as an index of antiviral activity, in liver at 12 h after IFN-beta (15 MIU/kg, i.v.) injection. IFN-beta-induced 2',5'-OAS activity was more potent after the drug injection during the late dark phase. The higher antiviral effect of IFN-beta was observed when the interferon-alpha/beta receptor (IFNAR) expression in the liver increased, and the lower effect was observed when its expression decreased. IFN-beta-induced fever was more serious after IFN-beta injection from the late dark phase to the early light phase. A significant dosing time-dependent change was demonstrated for plasma IFN-beta concentrations, which showed a higher level during the light phase and a lower level during the dark phase. The dosing time-dependent change of plasma IFN-beta concentrations was not associated with that of the antiviral effect or fever induced by IFN-beta. These results suggest that selecting the most suitable dosing time of IFN-beta, associated with the 24-h rhythm of IFNAR expression in the liver, may be important to increase effectively the antiviral activity of the drug in experimental and clinical situations.

Interferon-alpha/beta-receptor interactions: a complex story unfolding.

The Type I interferons (IFN-alpha/beta) exhibit pleiotropic biological activities. Notably, the different IFN subtypes activate the same cell surface receptor complex to mediate variable responses. Accumulating evidence suggests that distinct differences in critical amino acid residues among the different IFN-alphas and IFN-beta determine the nature of the ligand-receptor interaction and the subsequent responses. This review focuses on IFN-receptor interactions, the key residues involved in this interaction and the potential for targeted modifications of the ligand to enhance bioactivity.

Requirement for both IL-12 and IFN-gamma signaling pathways in optimal IFN-gamma production by human T cells.

Phytohemagglutinin (PHA)-derived T lymphoblasts or T cell clones from patients genetically deficient in IL-12R beta 1 (IL-12R beta 1(-/-)) or IFN-gamma R1 (IFN-gamma R1(-/-)) produced two- to threefold reduced IFN-gamma levels compared to the corresponding cells from healthy individuals after anti-CD3 and PMA stimulation. Moderate IFN-gamma production was observed in PHA-derived T lymphoblasts or T cell clones derived from healthy subjects in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, whereas it was negligible in the presence of both mAb. However, when anti-IFN-gamma R1 and/or anti-IL-12 mAb were added during restimulation, the cells produced normal levels of IFN-gamma, indicating that both IFN-gamma and IL-12 had an effect on the priming phase. Moderate production of IFN-gamma was partially enhanced only in IFN-gamma R1(-/-) T cell clones generated in the presence of IL-12, but was almost completely abolished when IL-12R beta 1(-/-) and IFN-gamma R1(-/-) T cell clones were generated in the presence of anti-IFN-gamma R1 or anti-IL-12 mAb, respectively. IL-4 production was enhanced in T cell clones from IL-12R beta 1(-/-),but not from IFN-gamma R1(-/-) patients, whereas IL-10 and IL-2 production did not differ significantly in polyclonal T cells or clones from healthy and deficient individuals. These results indicate that IL-12R beta 1- and IFN-gamma R1-dependent signals co-ordinately regulate IFN-gamma, but not IL-2 and IL-10 production, whereas only IL-12 negatively controls IL-4 production by in vitro-generated T cell clones. Thus, although IL-12 and IFN-gamma signals are each sufficient for moderate production of IFN-gamma by human T cells, both are needed for optimal IFN-gamma production, and in the absence of both IFN-gamma production is completely abrogated.

Identification of a phorbol ester-responsive element in the interferon-gamma receptor 1 chain gene.

Human monocytic leukemia THP-1 cells differentiate into macrophage-like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). During this process, interferon-gamma (IFN-gamma)-inducible expression of human leukocyte antigen-DR alpha is markedly enhanced. The enhancement of human leukocyte antigen-DR alpha expression is at least due to the TPA-dependent induction of the IFN-gamma receptor 1 chain and IFN-gamma receptor 2 chain genes. Here we have studied the mechanism of TPA-induced up-regulation of the IFN-gamma receptor 1 chain gene. Reporter gene analyses of 5'-deletion constructs of the IFN-gamma receptor 1 gene (IFNGR1) promoter indicated that the critical region for control of transcription and the TPA-responsive element (TRE) were present in the -128 to -109 base pair (bp) region. We confirmed that this region of the IFNGR1 promoter was responsive to TPA-induced signals by using a reporter construct whose promoter consisted of the -128 to -109 bp fragment and the minimal herpes simplex virus thymidine kinase promoter. Moreover, a supershift assay indicated that Sp1 bound to this TRE in TPA-treated THP-1 cells. These results suggest that in TPA-treated cells the binding of Sp1 to the TRE of the IFNGR1 promoter causes the up-regulation of this gene.