The interferons: 50 years after their discovery, there is much more to learn

The interferons (IFNs) and their receptors represent a subset of the class 2 alpha-helical cytokines that have been in chordates for millions of years. This brief review focuses on the discovery and purification of interferons, cloning of human IFN-alpha and IFN-beta, interferon receptors, activities and therapeutic uses of interferons, and the side effects of interferons.     

Interferon: effects on the immune response and the mechanism of activation of the cellular response.

The discovery of interferon in 1957 by Drs. Isaacs and Lindenmann led to major revisions in the concepts of man's defenses against viral infections. There are at least two types of interferon. Along with their antiviral properties, they have recently been shown to exert a suppressive effect on the humoral and cellular immune response; they affect both B and T lymphocytes. A variety of substances, including virus, polyribonucleotides, and mitogens for T lymphocytes, are good interferon inducers. T lymphocytes seem to be necessary for these inducers to exert their immunosuppressive effects. The immunosuppressive effects of interferon inducers suggests that interferons may be mediators of suppressor T lymphocyte effects. In the virus system, interferon does not exert its antiviral effects by direct action on the virus, but rather derepresses a cell gene that results in the production of an antiviral protein. This antiviral protein is probably the mediator of inhibition of virus replication. This is a complex sequence of events that results in the interaction of interferon with the cell membrane and the resulting production of the antiviral state in the cell. This review will examine the various steps of this involved process.     

Interferon: the pathways of discovery I. Molecular and cellular aspects

This historical account covers 50 years of seminal research work on interferon done since its discovery in 1957. Topics related to molecular structure, production and action of interferons are considered from the viewpoint of how our insights have expanded and deepened within the context of evolving tools and general knowledge in cellular and molecular biology. Lines of thought that linked each discovery to the next are expounded.     

Interferons as cell-regulatory molecules

Summary This review presents the current evidence for interferons as cell-regulatory molecules. Apart from inducing an antiviral state, interferon preparations are powerful inhibitors of cell growth and have selective effects on cellular protein synthesis. In addition, interferons are produced during most immune reactions and can exert positive and negative influences on these reactions. Thus interferon molecules are of interest to cell biologists, immunologists, and oncologists. Interferon as a cell regulator offers a unique approach to cancer therapy, but for its judicious use, more understanding of basic mechanisms of action is required.     

Beta and gamma interferons act synergistically to produce an antiviral state in cells resistant to both interferons individually.

We showed previously that the mouse fibroblastoid cell line Ltk-aprt- is resistant to the antiviral effects of beta interferon. This lack of response reflects a partial sensitivity to the interferon that is accompanied by a failure to activate expression of several interferon-regulated genes, although certain other genes respond in a normal manner. We show here that Ltk-aprt- cells were also unable to establish an antiviral state and to activate expression of 2,5-oligo(A) synthetase when treated with gamma interferon. Strikingly, however, treatment with a combination of beta interferon and gamma interferon provided complete protection against viral replication. Although the cells were completely insensitive to up to 250 U of the interferons per ml added singly, essentially complete protection from viral cytopathic effects was achieved when as little as 10 U of each of the interferons per ml were combined. Expression of 2,5-oligo(A) synthetase was also sensitive to this synergistic effect. Activation of an antiviral state could also be achieved by sequential treatment, first with gamma interferon and then with beta interferon. Partial protection against viral replication could be achieved by pretreatment with gamma interferon for as little as 1 h before incubation with beta interferon and could be blocked by the addition of specific antibodies or by cycloheximide, indicating that gamma interferon induces the synthesis of a protein which can act synergistically with a signal produced by the beta-interferon receptor. We suggest that Ltk-aprt- cells suffer from defects in one or more components of the gene activation pathways for both type I and type II interferons. Nonetheless, gamma interferon is able to activate the expression of a gene encoding a protein required for signal transduction. This protein acts synergistically with a transient signal produced in response to beta interferon, thereby activating the expression of a further group of genes.     

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.     

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.     

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 interferons on dengue virus multiplication in cultured monocytes/macrophages

The effects of interferons on dengue virus multiplication in cultured human and mouse monocytes/macrophages were studied. Interferon treatment before, but not after virus inoculation suppressed virus multiplication dose-dependently. Recombinant human leukocyte A interferon was as effective as ordinary human fibroblast interferon in suppressing dengue virus multiplication in cultured human monocytes. Human monocytes, a population of non-proliferating cell lineage, maintained their interferon-mediated antiviral state for a few days after removal of the interferons.     

Interferon levels in human pulmonary tumors are lower than plasma levels

It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.     

The interferon receptors

Early studies on the mode of action of interferons have indicated that a receptor system on the cell surface is involved in its action. The first direct evidence to a high-affinity binding site was found only after pure interferon was available. Two different receptors, one specific for interferons-alpha and beta, and the other for interferon-gamma were recognized. A correlation between affinity to the receptor and specific activity was established. Cross-linked complexes of labeled interferons with their receptors were visualized on gel electrophoresis and even partially purified. Internalization of interferons after binding to the receptor was reported. The role of gangliosides as helpers of interferon binding was recently investigated. Fragments of interferons which still retained binding capacity were described and helped in elucidating the binding site on the interferon molecule.     

Advances in therapy for hepatitis C infection

The first approved therapy for chronic hepatitis C virus (HCV) infection was recombinant interferon. Subsequently, controlled studies demonstrated that the combination of interferon-alpha and ribavirin leads to significantly higher virologic sustained responses in patients with chronic hepatitis C. A novel modification of the interferon molecule resulted in the formulation of pegylated interferons, which have a longer half-life than standard interferon. Two recent trials have established the superiority of pegylated interferons compared with interferon-alpha in inducing sustained virologic responses in patients with chronic HCV infection, with or without cirrhosis. Presumably, pegylated interferons will replace standard interferon in treating HCV infection. Phase 3 trials of pegylated interferons in combination with ribavirin are currently under way. Noninterferon-based therapies for the treatment of HCV infection are also in the developmental and experimental phases. Our aims in this review are to present the currently available therapeutic options for HCV infection and the evidence supporting their use in typical patients with chronic hepatitis C or in patients with special circumstances. We also briefly review novel therapeutic approaches, including noninterferon-based therapies.     

Human leukocyte interferon has no structural or biological relationship to corticotropin

In view of recent reports that certain preparations of human leukocyte interferons are structurally and biologically related to the pituitary hormones corticotropin (ACTH) and β-endorphin, we have investigated the properties of two human leukocyte interferons (IFN-α) prepared by recombinant DNA technology. The antiviral activities of purified IFN-αA and IFN-αD were not affected by a large molar excess of ACTH antiserum nor did ACTH interfere in interferon immunoassays. Neither IFN-αA, IFN-αD nor pepsin digests of these proteins were able to stimulate steroidogenesis in adrenocortical cells. There was no cross reaction between ACTH antiserum and the two leukocyte interferons or the pepsin digests of the interferons. These results cast doubt on recent proposals that some of interferon's biological effects are mediated by ACTH or β-endorphin-related fragments of the interferon molecule.     

The biochemical mechanisms of action of the interferons

The interferon family of proteins consists of a variety of antigenically distinct types that are encoded by different genes. Several of these genes have been cloned and the availability of large amounts of the recombinant interferons has greatly facilitated characterization of their biochemical and biological activities. The specificity of interaction of interferons with cell surface receptors has been investigated in binding and covalent cross-linking experiments employing 125I-labeled interferons. In addition to their antiviral activity, the interferons have effects on cell growth and differentiation. The interferons also are potent modulators of the immune response. The interferons exert their biological activities by altering the expression of several cellular proteins. Current knowledge about the mechanisms of signal transduction and the regulation of expression of interferon-induced mRNAs and proteins are discussed.     

Purification and structural analysis of interferon

Studies with crude or partly purified interferon have provided a significant amount of structural information. However, complete biochemical characterization required purification to homogeneity. Earlier work on fractionation has met with many difficulties because interferon was available only in minute quantities. A scale-up of production, adaptation of multi-step purification schemes, use of high-resolution separation techniques and highly sensitive analytical methods have yielded pure interferons and hence many structural data. Specific activities, amino-acid compositions, partial sequences and structural homologies of many interferons were determined. Finally, cloned copy DNA (cDNA) fragments derived from specific interferon mRNA, as well as isolated interferon genes, have been sequenced and the data were used to elucidate complete sequences of many interferons with a high degree of confidence.     

The biology of interferon actions

The interferons comprise a group of proteins which were first identified by their ability to protect cells against virus infections. They are synthesized and secreted by a variety of cell types in response to various inducers and exert their effects in vivo by interaction with specific cellular receptors. In this sense the interferons are analogous to polypeptide hormones. In recent years it has become clear that the interferons are capable of influencing cellular physiology and behavior in a number of ways. Their effects include antiviral actions, inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation and activation of various cell types in the immune system. This review aims to summarize the current state of biology of interferon actions with special emphasis on the hemopoetic system.     

Orally administered interferons exert their white blood cell suppressive effects via a novel mechanism.

Interferons (IFN) have been approved for a number of clinical uses. The accepted routes of administration are intramuscular, subcutaneous, and intravenous. Recently, interferons administered by the oral route have been shown to exert a systemic effect. Oral administrations of IFN-alpha, IFN-beta, and IFN-gamma have been shown to cause a suppression of the peripheral white blood cell (WBC) count in mice. This study investigates the mechanism by which this suppression occurs. The results show that, in contrast to their intraperitoneal administration, oral administration of rHuIFN-alpha A/D or rMuIFN-gamma does not result in the presence of detectable levels of interferons in the blood. In addition, although the presence of circulating specific antibody to interferon blocks the peripheral WBC suppressive effects of intraperitoneally administered MuIFN-beta or rMuIFN-gamma, the presence of those antibodies does not block the peripheral WBC suppressive effects of the orally administered interferons. The peripheral WBC suppressive effect of orally administered rHuIFN-alpha A/D and rMuIFN-gamma can be transferred by injection of blood from oral interferon-treated donor mice to recipient mice. Recipient mice receiving plasma from donor mice showed no peripheral WBC suppression. Recipient mice receiving blood cells from donor mice showed significant peripheral WBC suppression. No effect of orally administered rHuIFN-alpha A/D on the relative percentages of lymphocytes, neutrophils, and monocytes was noted. These results indicate that the mechanism by which orally administered interferons exert their WBC suppressive effect differs from that of intraperitoneally administered interferons. WBC suppression resulting from orally administered interferons may involve cell to cell transfer of the interferons' effects, rather than the systemic distribution of the interferons in the blood. These studies further suggest that there may be a role for oral administration as a new route of interferon administration and provide a glimpse into the mechanism by which the orally administered interferons exert their systemic effects.     

Perspectives on interferon research and on its therapeutic use

After briefly reviewing recent advances in the knowledge of various molecular species of interferon, some prospects of the basic research in this field are discussed: 1) interferon as a physiological hormone endowed with antiviral and immunomodulatory functions and possibly interacting with the neuroendocrine system; 2) interferon as an inducible protein, a model which allows a refined molecular analysis using the probes made available by cloning; 3) the various mechanisms of interferon action at the biochemical level. In the last section, the past and future of interferon as a therapeutic agent are examined. For its therapeutic evaluation, interferon needs to be compared with the best chemotherapeutic agents now available against viruses and tumors. Large scale clinical trials require a large production of interferons. Hence, a dilemma will be encountered: either pursue trials with natural preparations which are always a mixture of several types and subclasses of interferons in no defined proportions; wait for availability of the interferons produced by genetic recombinants which are cloned species, and try these various species, each one alone and in combination, or even more use hybrid molecular species. The availability of accurate markers for the action of interferon in man will be of great usefulness to study its therapeutic action and furthermore to define its indications. Finally, the possibility is raised that some non toxic interferon inducers, such as Poly A, Poly U, could also be used as well as interferon itself.