A new IFN-like cytokine, limitin, modulates the immune response without influencing thymocyte development.

A novel IFN-like molecule, limitin, was recently identified and revealed to suppress B lymphopoiesis through the IFN-alphabeta receptor, although it lacked growth suppression on myeloid and erythroid progenitors. Here we have studied diverse effects of limitin on T lymphocytes and compared limitin with previously known IFNs. Like IFN-alpha and -beta, limitin modified immunity in the following responses. It suppressed mitogen- and Ag-induced T cell proliferation through inhibiting the responsiveness to exogenous IL-2 rather than suppressing the production of IL-2. In contrast, limitin enhanced cytotoxic T lymphocyte activity associated with the perforin-granzyme pathway. To evaluate the effect of limitin in vivo, a lethal graft-versus-host disease assay was established. Limitin-treatment of host mice resulted in the enhancement of graft-versus-host disease. Limitin did not influence thymocyte development either in fetal thymus organ cultures or in newborn mice injected with limitin-Ig, suggesting that limitin is distinguishable from IFN-alpha and -beta. From these findings, it can be speculated that the human homolog of limitin may be applicable for clinical usage because of its IFN-like activities with low adverse effects on, for example, T lymphopoiesis, erythropoiesis, and myelopoiesis.     

IFN-zeta/ limitin: a member of type I IFN with mild lympho-myelosuppression

Interferon (IFN)-Zeta/limitin has been considered as a novel type I IFN by the Nomenclature Committee of the International Society for Interferon and Cytokine Research. IFN-Zeta/limitin shows some sequence homology with IFN-alpha and IFN-beta, has a globular structure with five alpha-helices and four loops, and recognizes IFN-alpha/beta receptor. Although IFN-zeta/limitin displays antiviral, immunomodulatory, and antitumor effects, it has much less lympho-myelosuppressive activities than IFN-alpha. Treatment of cells with type I IFNs induces and/or activates a number of molecules, which regulate cell cycle and apoptosis. It is noteworthy that IFN-zeta/limitin activates the Tyk2-Daxx and Tyk2-Crk pathways weaker than IFN-alpha. Because experiments using antisense oligonucleotides have revealed their essential role in type I IFN-related suppression of lympho-hematopoiesis, little ability of IFN-zeta/limitin to activate the Tyk2-dependent signaling pathway may explain its uniquely narrow range of biological activities. Further analysis of structure-function relationship of type I IFNs will establish an engineered cytokine with useful features of IFN-zeta/limitin.     

Type I interferons and limitin: a comparison of structures, receptors, and functions

The type I interferon (IFN) family includes IFN-, IFN-β, IFN-, and IFN-τ. These molecules are clustered according to sequence homologies, use of the same cell surface receptor, and similar functions. IFN- and IFN-β have a globular structure composed of five a-helices. Their receptors, IFNAR1 and IFNAR2, belong to the class II cytokine receptor family for a-helical cytokines. Information about structure-function relationships between these and other IFNs is being provided by comparative sequence analysis, reference to a prototypic three-dimensional structure, analysis with monoclonal antibodies, construction of hybrid molecules and site directed mutagenesis. While much remains to be done, it should someday be possible to understand differences among IFNs in terms of how they interact with their corresponding receptors. Our recently identified IFN-like molecule, limitin, has weak sequence homology to IFN-, IFN-β, and IFN-ω and displays its biological functions through the same IFN-/β receptors. While limitin has antiproliferative, immunomodulatory, and antiviral effects like IFN- and IFN-β, it is unique in lacking influence on myeloid and erythroid progenitors. Further analysis of this functionally unique cytokine should be informative about complex IFN–receptor interactions. Furthermore, a human homologue or synthetic variant might be superior for clinical applications as an IFN without myelosuppressive properties.     

Effect of interferon on growth and division cycle of Friend erythroleukemic murine cells in vitro

The administration of appropriate doses of interferon to cultures of Friend leukemia cells causes a pronounced inhibition of cell growth. Several lines of evidence indicate that this effect is due to interferon itself, rather than to unknown contaminants of interferon preparations. Autoradiograph analysis of growth parameters of Friend leukemia cells during treatment with interferon demonstrates that the rate of entry into the S phase, the percent decline of unlabeled mitoses, and the mitotic indexes are significantly lower in interferon- treated cell cultures than in control untreated cultures when tritiated thymidine was added 12 h after the administration of interferon. These data indicate that fractions of interferon-treated cell population are delayed in both G1 and in G2 phases of the cell cycle. This was confirmed by exact measurements of the length of the various phases of the cycle. The interferon-induced inhibition of growth of Friend leukemia cells is reversible after removal of the compound. Autoradiograph data obtained from control cultures and from cultures previously treated with interferon that had been washed free of interferon and reseeded in interferon-free medium, demonstrate that during the first 12 h after removal of interferon, a large majority of the cells previously treated with interferon had a deranged flow into the S phase, a high number of unlabeled mitoses, and a low mitotic index. These data provide further evidence for the above-mentioned prolongations of G1 and G2 phases of the cell cycle. All growth parameters tested reverted to normal values within 12 h after washing out interferon.     

Characterization of the murine alpha interferon gene family

Mouse and human genomes carry more than a dozen genes coding for closely related alpha interferon (IFN-alpha) subtypes. IFN-alpha, as well as IFN-beta, IFN-kappa, IFN-epsilon, and limitin, are thought to bind the same receptor, raising the question of whether different IFN subtypes possess specific functions. As some confusion existed in the identity and characteristics of mouse IFN-alpha subtypes, the availability of data from the mouse genome sequence prompted us to characterize the murine IFN-alpha family. A total of 14 IFN-alpha genes were detected in the mouse genome, in addition to three IFN-alpha pseudogenes. Four IFN-alpha genes (IFN-alpha1, IFN-alpha7/10, IFN-alpha8/6, and IFN-alpha11) exhibited surprising allelic divergence between 129/Sv and C57BL/6 mice. All IFN-alpha subtypes were found to be stable at pH 2 and to exhibit antiviral activity. Interestingly, some IFN subtypes (IFN-alpha4, IFN-alpha11, IFN-alpha12, IFN-beta, and limitin) showed higher biological activity levels than others, whereas IFN-alpha7/10 exhibited lower activity. Most murine IFN-alpha turned out to be N-glycosylated. However, no correlation was found between N-glycosylation and activity. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies, suggesting that IFN-alpha subtypes did not diverge primarily to acquire specific biological activities but probably evolved to acquire specific expression patterns. In L929 cells, IFN genes activated in response to poly(I*C) transfection or to viral infection were, however, similar.     

Atopic manifestations in the acquired immune deficiency syndrome: response to recombinant interferon gamma.

Six patients with the acquired immune deficiency syndrome (AIDS) had exacerbations or recurrences of previously quiescent atopic disease when they developed immunodeficiency. Four developed a different atopic illness from that suffered previously. Atopic symptoms developed within three months after the patients developed AIDS or during prodromal illness. Two of the patients were treated with recombinant interferon gamma: both showed a striking improvement in symptoms and cellular immunity. These results indicate that cellular immunity, through interferon gamma, may have a role in regulating atopic disease.     

Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy.

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.     

Ficolins and infectious diseases

Ficolins are serum complement lectins, with a structure similar to mannose-binding lectin (MBL) and lung surfactant protein (SP)-A and SP-D. Ficolins activate the lectin complement system and play important roles in host innate immunity. Ficolins are members of the collectin family of proteins, which act as pattern recognition receptors (PRRs). They are soluble oligomeric defense proteins with lectin-like activity, and are able to recognize pathogen-associated molecular patterns (PAMPs), which are carbohydrate molecules on the surface of pathogens, and of apoptotic, necrotic, and malignant cells. Upon binding to their specific PAMPs, ficolins may trigger activation of the immune system either (1) by initiating activation of complement via the lectin pathway, (2) by a primitive type of opsonophagocytosis, or (3) by stimulating secretion of the inflammatory cytokines interferon (IFN)-Γ, interleukin (IL)-17, IL-6, and tumor necrosis factor (TNF)-α, and production of nitric oxide (NO) by macrophages, thus limiting the infection and concurrently orchestrating the subsequent adaptive immune response. Recently, a number of reports have shown that dysfunction or abnormal expression of ficolins may play crucial roles in viral and bacterial diseases and in inflammation. This review summarizes the reports on the roles of ficolins in the infectious diseases, and provides insight into ficolins as novel innate immune therapeutic options to treat these diseases.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.     

Long-term effect of interferon on keratinocytes that maintain human papillomavirus type 31

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.     

The antitumor effects of interferon: a personal history

Early experiments showed that administration of mouse interferon preparations inhibited the development of viral-induced or spontaneous viral associated leukemias in mice. Interferon alpha/beta was also shown to inhibit the growth of transplantable tumors of different origins in all strains of mice tested. The finding that interferon alpha/beta inhibited the growth of sublines of tumors selected for resistance to interferon alpha/beta indicated the role of interferon induced host mechanisms in the antitumor effects observed. The different host antitumor mechanisms and especially the interaction of interferon alpha/beta with the immune system have been briefly discussed. Injection of mice with a neutralizing antibody to interferon alpha/beta demonstrated the essential role of endogenous interferon alpha/beta in the defense of the mouse against the development of syngeneic, allogeneic and xenogeneic tumors.     

Interferon- and sarcolectin-dependent cellular regulatory interactions

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.     

A functional cyclooxygenase enzyme is not required for mediation of the pleiotropic effects of human alpha or beta interferon

Aspirin, indomethacin, and phenbutazone at 50 μM concentration inhibit cyclooxygenase in cultured human foreskin fibroblasts as evidence by the suppression of the major prostaglandin species which aaccumulate in the culture medium. In contrast to data reported for mouse interferon on target mouse cells, these agents have no effect on the introduction of antiviral activity by human α and β interferons. Similarly, these agents have no effect on interferon induced inhibition of cell growth $\text{in vitro}$ or on interferon induced natural killer cell activity.     

Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway

LIGHT was recently described as a member of the tumor necrosis factor (TNF) 'superfamily'. We have isolated a mouse homolog of human LIGHT and investigated its immunoregulatory functions in vitro and in vivo. LIGHT has potent, CD28-independent co-stimulatory activity leading to T-cell growth and secretion of gamma interferon and granulocyte-macrophage colony-stimulating factor. Gene transfer of LIGHT induced an antigen-specific cytolytic T-cell response and therapeutic immunity against established mouse P815 tumor. In contrast, blockade of LIGHT by administration of soluble receptor or antibody led to decreased cell-mediated immunity and ameliorated graft-versus-host disease. Our studies identify a previously unknown T-cell co-stimulatory pathway as a potential therapeutic target.     

Regulation of 2'',5''-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.     

Interferons as antivirals in man

Interferons have now been used in both prophylaxis and treatment of a number of human viral infections. The major action has been as a prophylactic for sites within the body that are not yet involved by disease. Such a prophylactic effect can be obtained early in the treatment of acute viral infection or even during chronic viral disease. Both local and systemic prophylaxis have been achieved with regard to both respiratory and herpesviral illness. In addition, Dane particle suppression can be achieved consistently with dosages of 10(6) units or greater daily to patients with chronic hepatitis B virus infection. In certain cases with prolonged therapy there can be permanent eradication. With leucocyte-derived material of approximately 10(6) or 10(7) units per milligram protein, the major side effects have been an initial febrile response, fatigue, malaise, marrow suppression, and inhibition of hair growth. So far, side effects have been rapidly reversible on lowering of dosage. Present studies with the use of lymphoblastoid interferon and bacterial-derived interferon employ materials of significantly greater specific activity. Such experience suggests that the same general side effects that were limiting with leucocyte interferon are present with interferon produced from recombinant DNA by bacterial as well as with lymphoblastoid interferon.     

Chemical Carcinogenesis in Mice inhibited by Interferon

SEVERAL experiments have demonstrated that the anti-viral substance, interferon, can inhibit the growth of spontaneous^1,2, transplanted³ and virus-induced neoplasms in mice^4–7. Lieberman et al.⁵ reported that interferon treatment partially suppressed X-radiation-induced leukaemia in C57B1/6 mice. As they pointed out, the inhibitory effect provided additional evidence for the theory that X-rays cause lymphoma through the activation of a leukaemogenic type C RNA viral intermediate. In this communication, we report studies with CF-1 mice (Carworth Farms, New York City) to determine the effects of interferon on SC tumour induction by 3-methylcholanthrene (3-MC). These mice were previously shown to harbour high levels of type C RNA gs antigen and.infectious virus in normal spleens and in induced tumours, while spontaneous tumours rarely develop until late in life^8–10.     

Origin and Possible Mode of Action of a Tissue Antagonist of Interferon

WE have presented data¹ supporting the hypothesis proposed by Taylor² and by Friedman and Sonnabend³ that interferon acts through a protein which confers the antiviral state on the cell. We postulated two separate regulatory mechanisms: one governing interferon synthesis and the other the production and action of the antiviral protein in the cell⁴. Our studies on the regulation of the antiviral state led us to the demonstration of a substance in human amniotic and chorionic membranes which, when added to the cell 4–18 h after interferon, decreased the antiviral state⁵. The extraction and purification of this tissue antagonist of interferon (TAI) have been previously described. TAI was resistant to proteases (trypsin, pepsin) and to nucleases (RNAase, DNAase). It was also resistant to heating at 56° C or 75° C for 1 h and was only inactivated at 95° C. Although the biologically active component was not chemically defined, its properties were reminiscent of mucopolysaccharides. It is unknown at present whether the TAI represents a substance or a group of substances.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.