Shared receptor components but distinct complexes for alpha and beta interferons.

The type I interferon family includes 13 alpha, one omega and one beta subtypes recognized by a complex containing the receptor subunits ifnar1 and ifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1. To investigate the reported differences in the way that alpha and beta interferons signal through the receptor, we introduced alanine-substitutions in the ifnar2 extracellular domain, and expressed the mutants in U5A cells, lacking endogenous ifnar2. A selection, designed to recover mutants that responded preferentially to alpha or beta interferon yielded three groups: I, neutral; II, sensitive to alpha interferon, partially resistant to beta interferon; III, resistant to alpha interferon, partially sensitive to beta interferon. A mutant clone, TMK, fully resistant to alpha interferon with good sensitivity to beta interferon, was characterized in detail and compared with U5A cells complemented with wild-type ifnar2 and also with Tyk2-deficient 11.1 cells, which exhibit a similar alpha-unresponsive phenotype with a partial beta interferon response. Using anti-receptor antibodies and mutant forms of beta interferon, three distinct modes of ligand interaction could be discerned: (i) alpha interferon with ifnar1 and ifnar2; (ii) beta interferon with ifnar1 and ifnar2; (iii) beta interferon with ifnar2 alone. We conclude that alpha and beta interferons signal differently through their receptors because the two ligand subtypes interact with the receptor subunits ifnar 1 and ifnar2 in entirely different ways.

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

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.

Specific antiviral activities of the human alpha interferons are determined at the level of receptor (IFNAR) structure.

Differences in activity among the family of human IFNs alpha are much reduced if these ligands are assayed on bovine cells. In particular, the activity of IFN alpha D is much higher on bovine than on human cells. To examine these differences, the bovine counterpart of the human IFNAR has been cloned and expressed in a human cell line. The transfected cell line now recognizes the human IFN alpha D as a high-specific-activity IFN subtype, indicating that the differences in sensitivity between the bovine and human cells to the human IFN alpha lie in the structure of the IFNAR chain rather than in the other components of the functional receptor.

The type I interferon receptor: structure, function, and evolution of a family business.

Recent results indicate that coherent models of how multiple interferons (IFN) are recognized and signal selectively through a common receptor are now feasible. A proposal is made that the IFN receptor, with its subunits IFNAR-1 and IFNAR-2, presents two separate ligand binding sites, and this double structure is both necessary and sufficient to ensure that the different IFN are recognized and can act selectively. The key feature is the duplication of the extracellular domain of the IFNAR-1 subunit and the configurational geometry that this imposes on the intracellular domains of the receptor subunits and their associated tyrosine kinases.

Pulmonary catabolism of interferons: alveolar absorption of 125I-labeled human interferon alpha is accompanied by partial loss of biological activity.

The catabolism of interferon was examined in isolated rabbit lungs which were ventilated and perfused with homologous blood. Natural human interferon-alpha (HuIFN-alpha) from lymphoblastoid Namalwa cells or recombinant DNA-derived HuIFN-alpha 2 were labeled with 125I, mixed with an excess of the respective cold interferons and added to the perfusion blood. Protein-bound and acid-soluble radioactivity, as well as antiviral activity, were measured at regular time intervals. During the first 3 h of perfusion, only very small fractions of the interferons disappeared from the perfusate, irrespective of whether lungs were inserted in the perfusion system. This indicated that catabolism of interferons in the pulmonary circulation was negligible. On the other hand, when the interferons were instilled into the bronchial-alveolar tree, absorption of antiviral activity differed from that of acid-precipitable protein-associated radioactivity. While most of the radioactivity was transferred into the perfusate, only 2% of antiviral activity of natural HuIFN-alpha and 30% of that of HuIFN-alpha 2 were recovered in the perfusate. In both cases acid-soluble radioactivity in the system reached about 10%. Since radioiodide, instilled in the bronchial-alveolar tree, was transported rapidly into the perfusate, this type of analysis did not help in locating the site(s) of degradation. Alveolar macrophages did not catabolize or inactivate interferons in vitro.

Highly purified leucocyte interferons for renal transplant recipients.

The effect of alternate day administration of four human interferons alpha on temperature, leucocyte and thrombocyte counts was evaluated in renal transplant recipients. These preparations were strikingly different in nature and concentration of impurities. No differences were found in side effects suggesting that they are not due to contaminants but to an intrinsic property of interferon alpha molecules.

Estimates of normal binding of a human recombinant alpha interferon to peripheral blood mononuclear cells from a study matching healthy subjects to subjects with insulin dependent diabetes.

This study compares the binding of a human recombinant alpha-interferon to peripheral blood mononuclear cells (PBMC) from patients with insulin dependent diabetes (IDDM) mellitus and control subjects. Diurnal and longer term of variation, feeding, fasting and haemoglobin glycosylation were examinated for their influence on interferon binding to PBMC. No gross differences in binding were demonstrated, in particular no effect of glucose levels was seen on the binding of interferon alpha-2 to PBMC.

Non-specific adsorption of interferon to immobilized serum immunoglobulin.

Human leukocyte interferon attaches to hyperimmune serum immunoglobulins, coupled to agarose, even though the antibodies are not specific for interferon. Unless it is first washed off with ethylene glycol, the attached interferon elutes together with immune-specific antigens during immuno-affinity chromatography.

[Immunization against interferon in man]. / Immunisation anti-interféron chez l'homme.

We have summarized 14 human cases with antibodies to interferon (anti-IFN) reported in the literature. In 4 cases, the patients developed auto-antibodies without any injection of exogenous interferon (IFN). In most cases, antibodies were directed against alpha IFN (13/14) and especially against recombinant alpha 2 IFN (8/14). When tested, antibodies to IFN were always IgG. In a few subjects, the IFN system has been studied more completely with the following results: the competent cells are able, after induction, to produce IFN; the endogenous IFN is neutralized by the antibodies to IFN; the antibodies to IFN are able to inhibit the interaction of IFN with its specific cellular receptors. Clinically, the raising of an anti-IFN immunization does not seem to impair the antiviral or antitumoral effects of IFN. These data can be related to the relative low neutralizing titres of antibodies to IFN with regard to levels of IFN produced at the site of the viral infection. We discuss the putative benefit of antibodies on the availability of circulating IFN and the possible role of antibodies to IFN in autoimmune diseases.

Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells.

A single species of human interferon alpha (IFN alpha) was labelled with 125I to high incorporation for binding studies on the B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination. Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same initial culture of cells. 125I-labelled IFN alpha 2 (IFN alpha prepared from Escherichia coli carrying human alpha 2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 +/- 0.07 days-1) at a mean concentration of 235000 +/- 20000 cells ml-1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the 'steady-state', appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occurring consecutively over the whole of the dose range studied (1-100 U/ml; 0.25-25 pM IFN). In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e. in analogy to Michaelis-Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited. We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.

Production of human lymphoblastoid interferon.

The interferon response of 21 lines of human lymphoblasts varied greatly. Interferon from the best producer (11,000 U/ml) resembled human leukocyte interferon.

Raising antibodies to human leukocyte interferon.

Human leukocyte interferon proved to be a good immunogen in sheep, rabbits and guinea pigs. Sera from immunized animals neutralized the antiviral action of leukocyte interferon at high dilution. The highest anti-interferon titres obtained were 1:1,200,000 for sheep, 1:150,000 for rabbits, 1:30,000 for guinea pigs. Partial purification of human leukocyte interferon, by ethanol precipitation, improved its qualities as an immunogen. While it appeared more efficient to give initial injections without adjuvant, the inclusion in booster injections, of Freund's complete adjuvant produced a markedly superior response and, in one sheep, maintained high levels of circulating antibody for several months.

Effects of long-term treatment of human carcinoma cells with interferon alpha.

Three tumor cell lines derived from human colon, urinary bladder and ovarian carcinoma were serially passaged in the continuous presence of human interferon alpha for extended periods of time. Phenotypic changes induced by interferon differed among these three cell lines. Thus interferon enhanced colon tumor cell aggregation but inhibited the aggregation of bladder tumor cells. The antiproliferative activity of interferon was more pronounced in bladder and ovarian cells than in colonic cells. However, the tumorigenicity of parental and cloned colon tumor cells injected i.p. or s.c. was markedly reduced by passage of the cells with interferon. Interferon treatment reduced the tumorigenicity of ovarian tumor cells when these cells were injected i.p. but not when injected s.c. The tumorigenicity of bladder tumor cells was not affected by interferon.

The cellular receptor of the alpha-beta interferons.

This is a selective review of recent trends in research on the cellular receptor for the alpha-beta interferons. It deals mainly with work published in the last three years (1985-88), and therefore mainly with receptors for the human interferons. The binding characteristics of several human alpha interferons are examined, and the importance of in vitro experimental models for establishing the relationship between receptor binding and the cellular response is emphasized.

Receptor dynamics of closely related ligands: "fast' and "slow' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.