IFN-alpha/beta Research Articles

Superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha in primary ATL cells occurs downstream of STAT1 signaling

Adult T-cell leukemia (ATL) is an aggressive CD4+CD25+ leukemia with poor prognosis, which usually develops several decades after HTLV-1 infection. In contrast to HIV-infection, the treatment of HTLV-1-associated diseases rely on a limited number of drugs. For ATL, combination therapy with IFN-alpha+AZT has shown clinical benefit in the non-lymphoma subtypes. Type I IFNs (IFN-alpha/beta) are essential cytokines with proved anti-cancer and antiviral action in vitro and in vivo. Nonetheless, their mechanisms of action in HTLV-1 infection remain unclear and a side-by-side comparison of both type I IFNs has not been performed in ATL. We show, in short-term culture of primary mononuclear cells from ATL patients, that both IFNs cause increased apoptosis, exert an anti-proliferative and antiviral effect, and decrease pro-inflammatory cytokine levels. However, IFN-beta treatment was significantly more effective in inhibiting viral p19 protein levels and lymphoproliferation, as compared to IFN-alpha. This pronounced effect of IFN-beta was explained by an induction of a higher number of known IFN-stimulated genes and antiviral genes by microarray analysis (76 vs. 26 genes were selected with p 2-fold difference vs. control). In PBMCs from healthy donors, ATL patients as well as in HTLV-1-infected cell lines, both IFNs have comparable activity in phosphorylating STATs 1 through 5 (PhosFlow), although phospho-STAT1 levels were up to tenfold higher than phospho-STAT2 through 5. This predominant STAT1-mediated antiviral gene signature was confirmed by Ingenuity Pathway analysis. In conclusion, our data suggest the superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha occurs downstream of STAT1 signaling.

Interferon-Lambda: A New Addition to an Old Family

The discovery and initial description of the interferon-lambda (IFN-lambda) family in early 2003 opened an exciting new chapter in the field of IFN research. There are 3 IFN-lambda genes that encode 3 distinct but highly related proteins denoted IFN-lambda1, -lambda2, and -lambda3. These proteins are also known as interleukin-29 (IL-29), IL-28A, and IL-28B, respectively. Collectively, these 3 cytokines comprise the type III subset of IFNs. They are distinct from both type I and type II IFNs for a number of reasons, including the fact that they signal through a heterodimeric receptor complex that is different from the receptors used by type I or type II IFNs. Although type I IFNs (IFN-alpha/beta) and type III IFNs (IFN-lambda) signal via distinct receptor complexes, they activate the same intracellular signaling pathway and many of the same biological activities, including antiviral activity, in a wide variety of target cells. Consistent with their antiviral activity, expression of the IFN-lambda genes and their corresponding proteins is inducible by infection with many types of viruses. Therefore, expression of the type III IFNs (IFN-lambdas) and their primary biological activity are very similar to the type I IFNs. However, unlike IFN-alpha receptors which are broadly expressed on most cell types, including leukocytes, IFN-lambda receptors are largely restricted to cells of epithelial origin. The potential clinical importance of IFN-lambda as a novel antiviral therapeutic agent is already apparent. In addition, preclinical studies by several groups indicate that IFN-lambda may also be useful as a potential therapeutic agent for other clinical indications, including certain types of cancer.

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN-alpha/beta) are major actors of the innate anti-viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN-inducing TLR3 agonist. PolyI:C plus IL-2/IL-12 induced IFN-beta (but not IFN-alpha) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti-IFNAR1 or anti-IFN-beta Ab prevented the production of IFN-gamma induced by polyI:C plus IL-2/IL-12. Similarly, IFN-gamma production induced by polyI:C plus IL-12 was reduced in NK cells isolated from IFNAR1(-/-) compared with WT mice. The ability of polyI:C plus IL-12 to induce IFN-gamma production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL-2/IL-12, produce IFN-beta that induce, in an autocrine manner, the production of IFN-gamma and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.

Excessive CpG 1668 stimulation triggers IL‐10 production by cDC that inhibits IFN‐α responses by pDC

Upon stimulation with a wide range of concentrations of CpG oligodeoxynucleotide 2216 (CpG 2216), plasmacytoid DC are induced to produce type I IFN (IFN-alpha/beta). In contrast, CpG 1668 shows a bell-shaped dose-response correlation, i.e. only intermediate but not high doses of CpG 1668 induce IFN-alpha/beta. Interestingly, high-dose CpG 1668 completely inhibited IFN-alpha responses induced by CpG 2216. Experiments using supernatant of high-dose CpG-1668-treated cells indicated that secreted inhibitor(s) mediated the IFN-alpha shut-off. Among modulating cytokines, IL-10 turned out to be one important negative regulator. In line with this, supernatants of IL-10-deficient DC cultures stimulated with high-dose CpG 1668 did not inhibit IFN-alpha production. Interestingly, high-dose CpG 1668 also inhibited IFN-alpha responses induced by the DNA-encoded mouse cytomegalovirus, whereas IFN-alpha responses induced by negative-strand RNA-encoded vesicular stomatitis virus were only marginally affected. Experiments with DC cultures devoid of TLR9 indicated that TLR9 was critically required to mediate stimulatory and modulatory signals by low and high concentrations of CpG 1668, respectively. Analysis of purified DC subsets showed that conventional DC were the main IL-10 producers, whereas plasmacytoid DC hardly produced any IL-10.

TLR pathways and IFN‐regulatory factors: To each its own

TLR trigger the induction of type I IFN (IFN-alpha/beta), providing a crucial mechanism of anti-viral defense. Until recently, TLR were thought to induce type I IFN responses by activating two transcription factors which belong to the IFN-regulatory factor (IRF) family, IRF-3 and IRF-7. TLR-3 and TLR-4 induce IFN-beta by activating IRF-3; TLR-9 induces IFN-alpha and IFN-beta through IRF-7, at least when engaged by type A CpG oligonucleotides (CpG-A) in plasmacytoid DC (pDC). In this issue of the European Journal of Immunology, it is demonstrated that TLR-9 induces IFN-beta when engaged by type B CpG oligonucleotides (CpG-B) in myeloid DC and macrophages. Remarkably, this response is independent of IRF-3/7 and, in fact, requires another IRF family member, IRF-1. IRF-1 is recruited by TLR-9 through the adaptor MyD88. Deficiency of the TLR-9-->IRF-1-->IFN-beta pathway results in impaired anti-viral responses not only in vitro but also in vivo. These results demonstrate that TLR induce IFN-alpha or IFN-beta responses by activating distinct IRF, depending on the TLR ligand and the cell type. These distinct TLR-IRF pathways may allow the immune system to tailor its responses to viral pathogens.

Stat1-mediated cytoplasmic attenuation in osteoimmunology

Signal transducer and activator of transcription 1 (Stat1) is a critical mediator of gene transcription in type I interferon (IFN-alpha/beta) signaling that is essential for host defense against viruses. In the skeletal system, type I IFNs (IFN-alpha/beta) also play an important physiological role in the inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation and bone resorption: mice deficient in IFN signaling exhibit decreased bone mass accompanied by the activation of osteoclastogenesis. On the other hand, an unexpected increase in bone mass was observed in Stat1-deficient mice, indicating that Stat1 has a hitherto unknown function in the regulation of bone formation. Indeed, Stat1 was found to have a unique, non-canonical function as a cytoplasmic attenuator of Runx2, a key transcription factor for osteoblast differentiation. Thus, the loss of Stat1 results in excessive activation of Runx2 and osteoblast differentiation, thereby tipping the balance in favor of bone formation over bone resorption. This is an interesting example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a novel regulatory mechanism of bone remodeling by immunomodulatory molecules. Here, we summarize recent advances in the study of Stat1 and IFNs in the context of osteoimmunology, including latest reports that question whether the inhibitory function of Stat1 in chondrocytes is responsible for dwarfism in achondroplasia.

Type I interferons and IL-12: convergence and cross-regulation among mediators of cellular immunity.

Therapeutic use of type I IFN (IFN-alpha/beta) has become common. Many of the diverse diseases targeted are marked by pathogenetic abnormalities in cell-mediated immunity (CMI), these cellular immune responses either causing injury to the host, lacking sufficient vigor for virus or tumor clearance, or both. In general, therapeutic efficacy is limited. It is thus notable that the pleiotropic effects of type I IFN on CMI remain poorly understood. We characterized the effects of type I IFN on the production of IL-12, the central immunoregulatory cytokine of the CD4(+) T cell arm of CMI. We show that type I IFN are potent inhibitors of IL-12 production by human monocytes/macrophages. The underlying mechanism involves transcriptional inhibition of the IL-12p40 gene, marked by down-regulation of PU.1 binding activity at the upstream Ets site of the IL-12p40 promoter. Type I IFN have previously been shown to be able to substitute for IL-12 in driving IFN-gamma production from T and NK cells. The ability of IFN-alpha/beta to suppress IL-12 production while up-regulating IFN-gamma production suggests a possible mechanistic basis for the difficulties of employing these cytokines in diseases involving abnormalities of CMI.

Inhibition of angiogenesis by type I interferons in models of Kaposi's sarcoma.

Kaposi's Sarcoma (KS) is a pathology which occurs with increased frequency and in a particularly aggressive form in AIDS patients. The HIV-1 Tat protein appears to be an important co-factor in the induction of the extensive neo-vascularization associated with AIDS-KS. Tat acts as a chemoattractant for endothelial cells in vitro, inducing both chemotactic and invasive responses. Several clinical trials have been performed testing the effectiveness of diverse biological agents in therapy of KS, among these the type I interferons. Type I IFNs have diverse biological functions besides their anti-viral activity, including anti-angiogenic properties. We have shown that IFN alpha and IFN beta are potent inhibitors of both primary and immortalized endothelial cell migration and morphogenesis in vitro as well as neo-angiogenesis induced by HIV-1 Tat in vivo. The inhibitory effect of IFN class I on HIV-Tat associated angiogenesis further supports its use as a therapy for epidemic Kaposi's sarcoma. The use of recombinant IFNs at the levels required to obtain a therapeutic effect are associated with side effects and toxicity, therefore we are now developing a gene therapy approach for constant and local delivery type I IFNs.

Autoimmunity is a type I interferon-deficiency syndrome corrected by ingested type I IFN via the GALT system.

Type I interferons (IFN-alpha/beta), products of the innate immune system, can modulate immune function whereas proinflammatory IFN-gamma (type II IFN), a product of the acquired immune system upregulates inflammation and enhances cell mediated immunity. We have proposed a unifying hypothesis of the origin of autoimmunity as a type I IFN immunodeficiency syndrome involving inadequate regulation of the acquired immune system product IFN-gamma by the IFN-alpha/beta innate immune system. The common theme of ingested type I IFNs in autoimmunity is inhibition of proinflammatory type II IFN systemically or at the target organ. In multiple sclerosis (MS) and insulin-dependent diabetes mellitus (IDDM) at the target organ, and in rheumatoid arthritis (RA) as a regulator of other proinflammatory cytokines, IFN-gamma is the nexus of inflammation in autoimmunity. Ingested type I IFNs counteract type II IFN, overcome the relative lack of type I IFN activity, and ameliorate autoimmunity. The administration of type I IFNs (IFN-alpha/beta) via the gut offers an exciting alternative to systemic application for overcoming the type I IFN immunodeficiency in autoimmunity. Successful use of ingested type I IFN in three separate prototypical autoimmune diseases suggests a broad antiinflammatory therapeutic profile for this technology.

Interferon-α/β Inhibits the Apoptosis Induced by Lipopolysaccharide and Interferon-γ in Murine Peritoneal Macrophages

Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-alpha/beta) down-regulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-gamma. Moreover, IFN-alpha/beta also decreased cell death in LPS/IFN-gamma-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-alpha/beta) against the apoptosis induced by treatment with LPS and IFN-gamma showed that type I IFNs were very effective when added up to 1 h after IFN-gamma/LPS stimulation. Addition of IFN-alpha/beta 4 h after stimulation with IFN-gamma/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-alpha/beta suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections.

Overexpression of c-Fos induces apoptosis of CD43+ pro-B cells.

The proto-oncogene product c-Fos, a component of the transcription factor AP-1, is induced in early B lineage cells. To investigate a role of c-Fos in early B cell development, fetal liver (FL) cells from transgenic mice carrying an IFN-alphabeta (IFN)-inducible c-fos gene (Mx-c-fosD) were cultured on a stromal cell layer with IL-7. Although B lineage cells normally developed in the Mx-c-fosD FL cell culture, the development was perturbed by the addition of IFN at the beginning of culture. When IFN was added in the FL culture after B lineage cells developed, pro-B (B220+,CD43+) cells were selectively dying by apoptosis within 48 h after IFN stimulation. This apoptosis was intrinsically induced in the pro-B cells that overexpressed c-fos when the Mx-c-fosD FL (H-2Kb) cells were cocultured with the normal C3H FL (H-2Kk) cells. The molecular basis of the apoptosis was investigated by examining expression of the genes that regulate apoptosis. The IFN stimulation did not modulate expression of Bcl-2 and Fas in early B lineage cells from the Mx-c-fosD FL culture. However, Rag-2 was down-regulated in these cells within 12 h after IFN stimulation. These results suggest that the c-Fos plays a causal role in deletion of pro-B cells with nonfunctional Ag receptor.

NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection.

Studies were undertaken to characterize mechanisms for NK cell cytokine delivery in vivo. Conditions of systemic IFN-beta expression elicited by polyinosinic-polycytidylic acid (poly(I:C)) treatment or IFN-alpha beta production during lymphocytic choriomeningitis virus or murine cytomegalovirus infections resulted in profound splenic histologic changes, with relocalization of nucleated cells from red to white pulp regions. Cell-trafficking experiments, with fluorescently labeled populations, showed that poly(I:C) induced T/B cell-dependent leukocyte migration into white pulp regions. Splenic leukocytes prepared from severe combined immunodeficient (SCID) and bone marrow cells prepared from SCID or normal C57BL/6 mice revealed a unique poly(I:C)-induced accumulation of non-T/non-B cells along splenic red and white pulp region borders characteristic of marginal zones. Lymphocytic choriomeningitis virus and murine cytomegalovirus infections also induced this trafficking pattern. Ab treatments of normal and SCID cell donor mice to eliminate specific cell subsets demonstrated that the unique migration of non-T/non-B cells was dependent upon cells with the NK phenotype, NK1.1+ AGM1+F4/80-. In situ hybridization showed that, at early times after infections, small proportions of cells along red and white pulp divisions expressed high levels of IFN-gamma mRNA, and that these cells were depleted by protocols eliminating NK cells. The results suggest an exciting model for induction of NK cell trafficking from bone marrow to secondary compartments. This migratory pattern may localize NK cells to receive additional activation signals and/or to precisely deliver NK cell-produced cytokines.

Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA.

We have recently shown that oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG motif) can induce B cells to proliferate, differentiate, and secrete cytokines. In this study we demonstrate that CpG motifs contained in ODN as short as 15 bases in length were quite effective at inducing NK cell lytic activity in vitro in both human and murine lymphocytes. Such ODN were also effective at inducing NK lytic activity, in vivo, in mice. Experiments designed to determine the cellular and cytokine requirements for NK cell induction revealed that B and T cells are not necessary, that the ODN do not augment the activity of highly purified NK cells, and that the ODN augment NK cell activity indirectly by inducing the secretion of IL-12, IFN-alpha beta, and TNF-alpha. Various ODN sequences were prepared to determine the optimal ODN length, motif, palindrome, backbone modification, and dose requirements. We found no requirement for a palindromic sequence but a definite requirement for an unmethylated CpG motif. While necessary, however, a CpG motif was not sufficient for NK cell induction. Instead, there appeared to be stringent requirements for the immediate flanking bases at the 5' and 3' ends as well as for flanking sequences outside the immediate 5' and 3' bases. In particular poly(G) ends seemed to exert a complex qualitative and quantitative effect which could be up- or down-modulating depending on whether the ODN backbone was phosphorothioate modified or not.

IFN-alpha beta reconstitutes the deficiency in lipid A-activated AKR macrophages for nitric oxide synthase.

AKR mouse peritoneal macrophages (PM) were previously found to have a defect in their response to lipid A for nitric oxide (NO)-mediated tumor cytotoxicity, which was related to a lower level of C1q synthesis and reconstituted by exogenous IFN-gamma or C1q. We used AKR-PM as a model to further define the role of IFN-alpha beta in modulation of induction of macrophage nitric oxide synthase (NOS) in response to lipid A. Studies have revealed that AKR-PM produced a significantly lower level of IFN-alpha beta than responsive C3H-PM in response to lipid A. AKR-PM failed to increase NOS mRNA synthesis and NO generation when exposed to lipid A, although they had normal levels of TNF-alpha bioactivity and mRNA expression. This partial deficiency of AKR-PM to lipid A stimulation was reconstituted completely by exogenous IFN-alpha beta for both synthesis of NOS mRNA and release of NO. The failure of AKR-PM to produce NOS to lipid A stimulation appears to be related to reduced secretion of IFN-alpha beta and the resultant failure to express TNF-alpha type II receptor (TNF-RII) mRNA, which in turn decreases TNF-alpha binding to its receptor for autocrine induction of NOS. Insufficient synthesis and secretion of endogenous IFN-alpha beta may be the primary reason for AKR-PM refractoriness to induction of NOS in response to lipid A. furthermore, the close correlation between lack of IFN-alpha beta secretion and decreased TNF-RII mRNA synthesis may implicate a critical role for IFN-alpha beta in the upregulation of macrophage TNF-RII receptor expression for autocrine induction of NOS during lipid A stimulation.

Endogenous IFN-alpha beta suppresses colony-stimulating factor (CSF)-1-stimulated macrophage DNA synthesis and mediates inhibitory effects of lipopolysaccharide and TNF-alpha.

Murine bone marrow-derived macrophages (BMM) are widely used as a suitable model to study the proliferative response to macrophage-CSF or CSF-1. We report here that the amount of DNA synthesis observed in BMM cultures in response to CSF-1 can be masked quite significantly by low levels of IFN-alpha beta produced in the cultures. It was found that Ab to IFN-alpha beta could enhance the proliferative response in CSF-treated BMM that were able to respond to endogenous IFN-alpha beta; however, BMM from mice lacking a component of the type I IFN receptor did not show any enhancement of CSF-1-dependent DNA synthesis on addition of the Ab. While DNA synthesis in CSF-1-stimulated BMM from normal mice was also very sensitive to the inhibitory actions of very low concentrations of added IFN-alpha beta, DNA synthesis in BMM from the "knockout" mice was not, indicating that the type I IFN receptor component containing the null mutation was essential for signal transduction. Previously it was shown that bacterial LPS, TNF-alpha, IFN-gamma, and cAMP could all inhibit CSF-1-stimulated BMM DNA synthesis and proliferation. Using the combined approach of blocking IFN-alpha beta Ab and the IFN receptor "knockout" mice, it was found here that the growth-inhibitory effects of LPS and TNF-alpha are due, to a significant extent, to endogenous IFN-alpha beta, whereas those of IFN-gamma and cAMP occur by a different mechanism. it is proposed that the type I IFN receptor (IFNAR 1) "knockout" mice may be useful in delineating some of the in vivo actions of CSF-1, LPS, TNF-alpha, and possibly other agents.

Immune defence in mice lacking type I and/or type II interferon receptors.

Mice lacking the receptor for type I interferon (IFN-alpha beta, A129 mice), for type II interferon (IFN-gamma, G129 mice) or for both receptors (AG129 mice) have been generated by embryonic stem cell mediated gene targeting and inter-crossing A129 x G129, respectively. The role of the two IFN systems in controlling a range of infections has been studied using these mice. Type I IFN is shown to be responsible for the immune defence against most viral infections tested (Lymphocytic Choriomeningitis Virus, Semliki Forest Virus, Theiler's Virus, Vesicular Stomatitis Virus), type II IFN seems to be of little importance. In Vaccinia Virus and Theiler's Virus infection, however, both IFN systems were found to play a nonredundant role. IFN-gamma was critical for the defence against intracellular bacteria (Mycobacterium, Listeria) and parasites (Leishmania), whereas IFN-alpha beta was not. IFN-alpha beta is produced by virus-infected cells within hours and plays an important role in preventing virus spread early. Production of IFN-gamma on the other hand needs activation of the immune system and plays a major role later, i.e. mostly during the immune response. Data obtained with the mice described here show that both IFN systems seem to have evolved to complement each other in the host defence against a wide variety of infectious agents.

Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.

The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.

Enhancement of immunostimulatory activity by dual substitution of C8-substituted guanine ribonucleosides: correlation with increased cytokine secretion.

Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine). All of the compounds enhance natural killer (NK) activity, lymphocyte proliferation, and antibody production in dose-dependent fashion. However, the potency and maximal activity of the disubstituted analogues are considerably greater than those of the monosubstituted analogues. Spleen cells stimulated for 48 h with the disubstituted compounds produce immunoreactive interleukin (IL) 1 alpha, IL-6, tumor necrosis factor-alpha (TNF alpha), and interferon-gamma (IFN gamma). Monosubstituted analogues induce lower quantities of IL-6, TNF alpha, and IFN gamma and fail to induce detectable levels of IL-1 alpha. Total IFN activity, assessed by viral inhibition assay, is also lower for the monosubstituted analogues. Augmentation of antibody secretion by B cells is diminished for neither mono- nor disubstituted compounds upon incubation with anti-cytokine antibodies. In contrast, anti-IFN alpha beta markedly reduces the effects of monosubstituted analogues on NK activity but has less marked effects on NK induction by the disubstituted compounds. A similar pattern of differences is seen for lymphocyte proliferation. Thus, although the analogues induce synthesis of several cytokines, to date only IFN alpha beta appears directly involved in enhancement of NK activity and lymphocyte proliferation. The present data do not, however, exclude the existence of an autocrine stimulatory mechanism not susceptible to inhibition by anti-cytokine antibodies.

Potential requirement of a functional double-stranded RNA-dependent protein kinase (PKR) for the tumoricidal activation of macrophages by lipopolysaccharide or IFN-alpha beta, but not IFN-gamma.

We analyzed the expression of the dsRNA-dependent protein kinase (PKR) during the activation of murine macrophages to the tumoricidal state by LPS and/or IFNs. LPS induced PKR expression in a dose-dependent manner at levels that were comparable with those observed in response to IFNs. By using the PKR inhibitor 2-aminopurine (2-AP), we have shown that the pathways of macrophage tumoricidal activation elicited by LPS and IFN-alpha beta, but not by IFN-gamma, included a 2-AP-sensitive step. In fact, LPS- and IFN-alpha beta-induced activation was inhibited by 2-AP, whereas the activation by IFN-gamma was not affected by the presence of the inhibitor. 2-AP did not affect the activation of protein kinase C or protein kinase A in intact cells. In the presence of 2-AP the up-regulation of IFN-beta mRNA by LPS was specifically inhibited, whereas the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA or the induction of PKR remained unchanged, thereby demonstrating that 2-AP inhibited selective macrophage genes. The differential sensitivity to 2-AP suggested that the expression of a functional PKR may be required for the macrophage tumoricidal response triggered by LPS and IFN-alpha beta but not IFN-gamma.

Evidence for glycosphingolipid modification of the type 1 IFN receptor.

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.