Human interferon omega (omega) binds to the alpha/beta receptor.

It was proposed that human interferon omega (omega) binds to the interferon alpha/beta receptor but not to the interferon gamma receptor. However, since no studies were performed to provide direct evidence for this hypothesis, we carried out cross-linking experiments and saturation binding assays between a 32P-labeled human interferon-alpha (Hu-IFN-alpha) and unlabeled Hu-IFN-alpha A, -beta, -gamma, and -omega. These assays demonstrated that Hu-IFN-alpha A, -beta, and -omega, but not Hu-IFN-gamma, were able to block binding of 32P-labeled Hu-IFN-alpha A to human cells. These results indicate that Hu-IFN-omega binds to the alpha/beta receptor.     

Evolution of a cytokine using DNA family shuffling.

DNA shuffling of a family of over 20 human interferon-alpha (Hu-IFN-alpha) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-alpha2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-alpha2a and 185-fold relative to Hu-IFN-alpha1. Remarkably, the three most active clones were more active than the native murine IFN-alphas. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.     

Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.     

Treatment of recurrent common warts with one low dose of intralesional natural human leukocyte interferon alpha

Nine patients with recurrent and long lasting common warts were treated with intralesional Hu-IFN-alpha. The schedule was a single dose per wart, ranged between 10(5) and 2 x 10(5) IU. Placebo was also administered in 3 of these patients. Complete remission was observed in 7 of the 9 patients. The pattern of warts involution and the possible interferon mechanism of action are discussed. A significant pain relief, produced by interferon injection was observed in the patients with plantar warts and in one patient with subungueal wart.     

Induction of interferon in man by vaccines.

The purpose of this study was to extend the spectrum of vaccines with interferon-inducing potential in man. The vaccines selected for study were the commercially available attenuated poliomyelitis vaccine type 2 (Sabin strain) and the new live attenuated influenza A/England/42/72 (H3N2) vaccine ("Alice" strain). Five subjects, two of whom had low or undetectable polio type 2 neutralizing antibody levels were given the type 2 vaccine (10-4.7 TCID50) in the standard manner orally. Even though the two individuals with low titers experienced a fourfold or greater antibody rise and one of them shed the virus in his stool, neither they nor the remaining three volunteers developed detectable levels of interferon in their sera obtained at very closely spaced intervals from day 0 to day 25 following immunization. Fifteen subjects were given approximately 10-7.5 TCID50 of influenza A/England/42/72 (H3N2) by nasal drops. Specimens consisting of sera and nasal washings were obtained at closely timed intervals for 23 days, starting with day 3 following immunization. Interferon could be detected in three of nine (33.3%) subjects who had fourfold or greater HI antibody rises. No interferon was detected in nasal washings, however. It is concluded that poliomyelitis is not a good interferon inducers in man. Live attenuated influenza vaccine does induce an interferon response in subjects with low initial serum antibody titers. This response is at best modest. The latter finding also suggests that the attenuation of the Alice strain of influenza A vaccine is not dependent on its interferon inducing potential.     

Histamine-induced suppressor factor inhibition of NK cells: reversal with interferon and interleukin 2

Culture supernatants of lymphocytes stimulated with 10(-3) to 10(-8) M histamine contain histamine-induced soluble suppressor factor (HISSF) that significantly inhibits the natural killer (NK) cell functions of allogeneic lymphocytes. Lymphocytes precultured with increasing concentrations of HISSF showed a dose-dependent suppressive effect on their NK activity. HISSF was not cytotoxic itself and produced suppressive effects on PBL, NK-enriched large granular lymphocytes (LGL), and isolated T cells. Suppression was evident throughout a range of effector:target cell ratios. Production of HISSF was specifically blocked by the H2 antagonist cimetidine, but not by the H1 antagonist clemastine fumarate. Furthermore, H1 and H2 antagonists themselves do not induce production of HISSF. Although HISSF could inhibit the cytotoxicity of LGL, LGL themselves do not produce HISSF. HISSF inhibition of NK activity could be completely reversed by treating effector lymphocytes with recombinant interferon-alpha (IFN) for 1 or 2 hr or culturing them with purified interleukin 2(IL 2) for 36 hr. Our data suggest that exogenous IFN and IL 2 may have therapeutic potential in the treatment of immunological diseases associated with histamine-induced suppressor cell activity.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.     

Parasite-mediated upregulation of NK cell-derived gamma interferon protects against severe highly pathogenic H5N1 influenza virus infection

Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection.     

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 g/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F₁ and F₂ generations, and from backcrosses of F₁ hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.     

Andes and Prospect Hill hantaviruses differ in early induction of interferon although both can downregulate interferon signaling

Hantavirus pulmonary syndrome (HPS) is a severe respiratory disease which is thought to result from a dysregulated immune response to infection with pathogenic hantaviruses, such as Sin Nombre virus or Andes virus (ANDV). Other New World hantaviruses, such as Prospect Hill virus (PHV), have not been associated with human disease. Activation of an antiviral state and cell signaling in response to hantavirus infection were examined using human primary lung endothelial cells, the main target cell infected in HPS patients. PHV, but not ANDV, was found to induce a robust beta interferon (IFN-beta) response early after infection of primary lung endothelial cells. The level of IFN induction correlated with IFN regulatory factor 3 (IRF-3) activation, in that IRF-3 dimerization and nuclear translocation were detected in PHV but not ANDV infection. In addition, phosphorylated Stat-1/2 levels were significantly lower in the ANDV-infected cells relative to PHV. Presumably, this reflects the lower level of IRF-3 activation and initial IFN induced by ANDV relative to PHV. To determine whether, in addition, ANDV interference with IFN signaling also contributed to the low Stat-1/2 activation seen in ANDV infection, the levels of exogenous IFN-beta-induced Stat-1/2 activation detectable in uninfected versus ANDV- or PHV-infected Vero-E6 cells were examined. Surprisingly, both viruses were found to downregulate IFN-induced Stat-1/2 activation. Analysis of cells transiently expressing only ANDV or PHV glycoproteins implicated these proteins in this downregulation. In conclusion, while both viruses can interfere with IFN signaling, there is a major difference in the initial interferon induction via IRF-3 activation between ANDV and PHV in infected primary endothelial cells, and this correlates with the reported differences in pathogenicity of these viruses.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Critical role of constitutive type I interferon response in bronchial epithelial cell to influenza infection

Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.     

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.     

Failure to induce alopecia areata in C3H/HeJ mice with exogenous interferon gamma

Alopecia areata is a cell mediated autoimmune disease that targets actively growing, anagen stage hair follicles in several mammalian species. Upregulation of MHC I due to interferon gamma is considered to be one of the initiating steps. To test this hypothesis we used the spontaneous C3H/HeJ mouse model, induced anagen by wax stripping the skin, and injected recombinant murine interferon gamma. Alopecia areata is a complex polygenic trait with low penetrance in these mice. Injection of interferon gamma did not change the frequency or time of onset of alopecia in these mice suggesting this protein alone is not sufficient to initiate disease.     

Protective role of beta interferon in host defense against influenza A virus

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.     

A point mutation of human interferon gamma abolishes receptor recognition.

We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma. The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity. The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification. However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity. Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D). Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity. Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma. Models of the X-ray crystal structure of human IFN gamma [Ealick, P.E., W.J. Cook, S. Vijay-Kumar, M. Carson, T.L. Nagabhushan, P.P. Trotta and C.E. Bugg (1991) Science, 252, 698-702] suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix. We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma.     

Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

Kinetics of internalisation and degradation of surface-bound interferon in human lymphoblastoid cells

The binding of iodinated human interferon-alpha 2 (IFN-alpha 2) was studied on the human T cell line, Molt 4. After its initial binding to cells, the IFN is transferred to a trypsin-resistant compartment before appearing in the medium as TCA-soluble material, while the total cell-associated IFN declines to one-third of its maximum value after 3 h incubation. The Na+/H+ ionophore monensin did not prevent intracellular accumulation of IFN but did completely inhibit its breakdown. We interpret our results as evidence for receptor-mediated internalisation of IFN followed by intracellular breakdown.     

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay

BackgroundIn a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides.AimsTo identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test.MethodsWe surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides.ResultsSeven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a “brute force” approach for peptide design.ConclusionsThe H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.