Mutational analysis of the IFNAR1 binding site on IFNalpha2 reveals the architecture of a weak ligand-receptor binding-site

Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFNalpha2 by creating a panel of 21 single alanine mutant proteins, and determined their binding affinities. The IFNAR1 binding site on IFNalpha2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNAR1, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFNalpha2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed.     

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth.

Human fibroblast and leukocyte interferons were found to suppress lymphocyte mitogenesis induced by optimal doses of phytohemagglutinin and concanavalin A. In certain situations (low doses of mitogen and/or low doses of interferon), however, interferon significantly enhanced mitogenesis. In experiments using varying concentrations of interferon, dose-response curves with different slopes were obtained for fibroblast and leukocyte interferons. The effect of interferon was apparently exerted during early stages of the lymphocyte cell cycle. There was no inhibitory effect of interferon if the lymphocytes were washed with medium before being exposed to mitogen. Interferon increased the binding of radiolabeled mitogens to cells. The results suggest that the immunological effects of interferon are consequences of actions on lymphoid cells. Fibroblast and leukocyte interferons seem to have different modes of action, or to bind differently to target cells. Possible mechanisms for the suppressive and enhancing effects of interferons on lymphoid cells are discussed.     

Comparative antiproliferative and receptor binding activities of interferons alpha and beta on lymphoblastoid and melanoma cells

The relative antiproliferative and receptor binding characteristics of the hitherto little-characterized interferon alpha 4a on cells of lymphoid and epithelial origin are compared with two other type I interferons, alpha 2 a and beta. Using the lymphoblastoid cell line, Daudi, interferons alpha 4 a and alpha 2 b had similar antiproliferative activity, and were about 10-fold more active than IFN beta. By contrast, using the melanoma cell line Sk-Mel-28, IFN beta was the most active, whereas IFN alpha 2b and IFN alpha 4a were respectively 60-fold and greater than 1000-fold less active than on Daudi cells. Receptor binding did not correlate with antiproliferative sensitivities, but confirmed a shared receptor component for these three interferons. These results indicate that the antiproliferative activities of three type I IFNs differs markedly on different cell types and that this is unlikely to be due to receptor binding, but more likely a post receptor binding event.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.     

Human leukocyte interferon (HuIFN-alpha): potent endorphin-like opioid activity

Human leukocyte interferon, but not fibroblast or immune interferons, binds to opiate receptor $\text{in}\text{,}\text{vitro}$. When injected intracerebrally into mice, human leukocyte, but not fibroblast or immune interferon, caused potent endorphin-like opioid effects. These effects include analgesia, lack of spontaneous locomotion and catalepsy. All of these actions of human leukocyte interferon were preventable and reversible by the opiate antagonist naloxone. The findings suggest that some of the side effects of leukocyte interferon therapy may be mediated by opiate receptor binding. They also provide evidence for a regulatory circuit between the immune and neuroendocrine system. This putative circuit could be an etiologic site for certain psychopathological states.     

Determination of the human type I interferon receptor binding site on human interferon-alpha2 by cross saturation and an NMR-based model of the complex

Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFNalpha2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNalpha2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNalpha2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Angstrom, and its well-defined orientation between IFNalpha2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand-receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules.     

Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.     

Enrichment and initial characterization of the solubilized receptor for mouse gamma interferon

The work reported here constitutes a first step in characterizing the receptor for mouse gamma interferon at the biochemical level. The myelomonocytic cell line, WEHI-3, was the source of starting material. Iodinated recombinant mouse gamma interferon incubated with WEHI-3 cells, as well as membranes prepared from them, bound specifically to a single class of sites with a Kd of 7 x 10(-9)M. Membranes were solubilized with the non-ionic detergent octyl-beta-D-glucopyranoside. As solubilization proceeded, binding activity could be assayed by precipitating the receptor with acetone in the presence of egg phosphatidylcholine liposomes. The Kd of the receptor in association with liposomes was 13 nM. Again here, only a single class of binding activity was found, and specificity for gamma, compared to other interferons, was maintained. This is the first time that the receptor for mouse gamma interferon has been solubilized and recovered in functional form. Further characterization included at least a 200-fold enrichment of binding activity by ligand affinity chromatography, resulting in the identification of a 95 kDa protein as the most likely candidate for either the receptor or a binding subunit thereof.     

Isolated interferon alpha-receptor complexes stabilized in vitro

We describe the extraction and stabilization in vitro of discrete complexes of interferon and cellular receptor proteins. A homogeneous complex of Mr 230 000 was extracted at the time of peak receptor binding (30 min). Complex formation was specific for human interferon. At later times a second complex could also be extracted suggesting transfer of interferon to a second site.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Effect of mouse interferons on the development of Ehrlich ascitic carcinoma

Intraperitoneal injection of various preparations of mouse interferons (L cell tissue culture interferons, concentrated or partly purified, and also serum interferon) significantly inhibited the development of Ehrlich's ascites carcinoma in randombred mice. In view of comparatively low activity of serum interferon, the effect of normal mouse serum on the tumour development and its action on L cell tissue culture interferon was investigated. It was shown that normal mouse serum inhibits the action of L cell tissue culture interferon and promotes the development of Ehrlich's ascites carcinoma.     

The effect of natural and recombinant leukocyte interferons on DNA repair and the formation of chromosome aberrations induced by N-methyl-N'-nitro-N-nitrosoguanidine

The anticlastogenic action of natural leukocyte and recombinant (alpha 2) interferons was studied in human lymphocyte cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine. The criteria of cell viability, proliferation, chromosome aberrations, frequency of micronucleus formation, formation and repair of DNA breaks were used for estimation of interferons activity. Reduction of the induced chromosomal aberrations was obtained in cells pretreated with interferons. The protective effect of natural leukocytic interferon was more expressed as compared with the effect of recombinant (alpha 2) interferon. The natural interferon was also more efficient than the recombinant one in DNA breaks formation and repair.     

Interferon: pharmacokinetics and toxicity

Interferons disappear rapidly from the serum of animals and man, and the kidney may be the major site of interferon destruction. The relevance of serum levels of interferons to their therapeutic activity has not been clearly established, particularly as the stimulation of host defence mechanisms by interferons may be important. Relatively low serum levels of antiviral activity are seen after intramuscular injections of fibroblast interferon compared with those after the same dose of leucocyte interferon. Injections of very pure leucocyte and lymphoblastoid interferons from several sources cause fever, headaches, malaise and myalgia associated with a corticosteroid response and probably with inflammatory prostaglandin synthesis. These reactions become less with repeated dosing but very large doses of lymphoblastoid interferon have been shown to cause liver damage and serious metabolic disturbances. Treatment with moderate doses of exogenous interferons may occasionally be associated with the development of neutralizing antibodies.     

Construction of a novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain and enhancement of its antitumor activity

A novel fusion protein harboring mouse interferon γ and epidermal growth factor receptor binding domain was constructed with the method of genetic and protein engineering. The fusion protein kept complete antiviral activity with the titer of 10⁸ IU per liter of culture. The EGF-RBD of the fusion protein exhibited competitive binding activity against¹²⁵I-mEGF for mEGF receptors on A431 cells. The fusion protein was shown to be more potent in inhibiting the growth of cultured mouse breast carcinoma cells than interferon γ. Experimental data on mouse B16 malignant melanoma model indicated that the tumor weight of fusion protein-treated group was statistically significantly smaller than that of interferon γ -treated group. The work here provides a necessarily reliable clue for the upcoming clinical employment of a novel class of targeting interferons.     

Optimization of protective properties of interferons in human cells exposed to mutagens estimated by the DNA repair activity

Protective properties of human interferons against physical and chemical mutagens have been described earlier. This work was aimed at detecting an optimum of protective action of interferons in human fibroblasts using two criteria: the number of single-strand DNA breaks formed and the index of DNA repair synthesis. The protective ability of interferon was shown to be expressed starting after 4 h of cells' pretreatment and proceeding through 40 h in experiments with N-methyl-N-nitro-N-nitrosoquanidin. The phenomenon of stimulation of DNA repair synthesis in human cells pretreated with interferon proceeded even after replating cells during 8 h in the experiments with UV irradiation.     

Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.     

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.     

The influence of type I interferons on immune cells can be mediated through regulation of estrogen receptor alpha level

Autoimmune disorders are connected with the actions of sex hormones. Clinical observations have shown that especially estrogens are involved in these phenomena. In some cases the administration of estrogens can increase the pathological symptoms of a disorder, while in others they can cause disease remission. In multiple autoimmune diseases, type I interferons, a family of cytokines acting through the common receptor IFNAR1/IFNAR2, seem to have action convergent with that of estrogens. We hypothesize that this coincidence is not accidental and type I interferons can regulate the level of estrogen receptor alpha (ERα) and consequently change the sensitivity of immune cells to estrogen's action. There is evidence that ERα is responsible for the effects exerted by estrogens and that this phenomenon mainly involves antigen-presenting cells. On the other hand, research on IFN-tau, a type I interferon family members, showed that this cytokine can modulate ERα levels in ovine endometrium. Because of the common receptor for these interferons, we suspect that other type I interferons can act in this way not only in endometrial cells, but also in immune cells. If there is such a mechanism, it can be exploited in the therapy of immune disorders, especially autoimmune disease, for example through simultaneous administration of less toxic interferons and estrogens.