Human IFN Research Articles

Expression of a synthetic human interferon-α 1 gene with modified nucleotide sequence in mammalian cells

We have constructed a recombinant interferon (IFN) gene whose coding region is a chemically synthesized human (Hu)IFN-α 1 gene [Edge et al., Nature 292 (1981) 756–762] encoding the same protein as its natural counterpart while possessing important modifications. These modifications were introduced in order to: (1) change codon usage, (2) destroy integrity of repeated and complementary oligodeoxynucleotides in the coding region, (3) introduce an intron, (4) substitute other sequences for the IFN specific 3'- and 5'-flanking sequences and (5) remove the leader sequence (which encodes for the signal peptide). We report here on the transfection and isolation of murine cells which contain low-copy-number insertions of our recombinant gene. These lines maintain a high steady-state level of biologically active HuIFN-α in the cytoplasm, produced from mRNAs of M r expected for correctly processed transcripts. These results show directly that the leader sequence is essential for secretion of IFN proteins. They also argue against a crucial role of the particular features of the transcribed sequence of the natural HuIFN-α 1 gene in regulating its expression.

53] A procedure for isolation of alpha interferon genes with short oligonucleotide probes

The chapter describes a procedure for the isolation of alpha interferon genes with short oligonucleotide probes. A method has been developed, which permitted the isolation of several human alpha interferon (IFN-α) genes from a human genomic library in Charon 4A bacteriophages with 17-base synthetic oligonucleotide probes. Previously, human IFN-α genes are isolated from complementary DNA (cDNA) libraries or genomic libraries with cDNA probes. The use of select, short, synthetic probes permits the isolation of the subsets of genes within a gene family represented in a genomic library. The screening procedure with 17-base probes described in the chapter is useful for the isolation of most genes from a human genomic library, although some conditions will vary according to the sequence of the probes. Procedure yielded three IFN-α genes, which had perfect homology with the two probes utilized. Even genes containing introns could be isolated from a genomic library with short and mixed probes provided the intron does not split the sequences of the probes to a size unable to maintain a stable hybrid. Changes at positions 42 and 133 result in the replacement of acidic residues with hydrophobic residues.

Natural and antibody-dependent cellular cytotoxicity in chronic myeloid leukemia patients in remission

Non-adherent peripheral blood mononuclear cells (NA-PBMNC) from 67 chronic myeloid leukemia (CML) patients in the first and two subsequent remissions, and 23 normal healthy donors were tested for NK and ADCC activities in short term chromium release assays using K562 and antibody-coated chicken RBCs as respective targets. CML patients in remission exhibited significantly reduced NK cytotoxicity (16.1–19.7%) compared to normal healthy donors (47.4%). Of the patients tested, 55% exhibited NK levels below the mean percent cytotoxicity — 2SD (12.5%) of normal donors (low responders), while 45% exhibited NK cytotoxicity above the 12.5% level (normal responders). On the other hand, CML patients in remission showed ADCC activity comparable to that of normal healthy donors (53.3%) irrespective of whether they belonged to normal NK responder group (55.5–65.0% ADCC) or low NK responder group (39.4–48.3% ADCC). The low or normal NK responder status of CML patients was not found to be related to either progression on the disease, or the type of drug used to bring about remission, or to the period in remission at the time of testing. In-vitro treatment of effector lymphocytes with recombinant human IFN α resulted in augmented of NK activity in both low and normal NK responder patients. The IFN-augmented NK activity in low responder patients however remained below the normal levels.

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events: Studies with vascular smooth muscle cells and endothelial cells

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [ 3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFNα, recombinant human IFNα2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.

Human lymphokine preparations which generate tumoricidal properties of human monocytes in vitro may be distinct from gamma interferon.

The purpose of these studies was to determine whether stimulated human lymphocytes produce lymphokines distinct from IFN gamma, that can activate human blood monocytes to lyse tumor cells. We undertook this investigation because of the controversy concerning whether MAF and IFN gamma are the same molecule. Crude lymphokine preparations prepared from normal human mononuclear cells incubated with Con A and rich in MAF activity also contained 1000 U/ml IFN gamma as measured by the virus neutralization assay. However, the induction of tumoricidal activity in monocytes by the lymphokine preparation could be dissociated from the IFN gamma activity, based on the following data: (1) Heat treatment (100 degrees C for 2 min) removed the antiviral activity of the lymphokine yet did not diminish its MAF-like activity when measured in a 72 h cytotoxicity assay against 125I IUdR-labeled human A375 melanoma cells. (2) Likewise, treatment of this lymphokine preparation with a twofold excess of anti-IFN gamma antibody neutralized antiviral activity but once again had no effect on its ability to activate monocyte tumoricidal function. In contrast, both heat treatment and anti-IFN gamma antibody abolished monocyte activation by equivalent units of human recombinant IFN gamma. Taken together, these data suggest that there is a molecule(s) distinct from IFN gamma which can activate human monocytes for tumoricidal function. Furthermore, this dissociation of MAF and IFN gamma activity was dependent on the use of a long-term (72 h) assay, since activation of tumoricidal activity in an 18-24 h assay appeared to be attributable solely to IFN gamma.

Human monocyte activation for cytotoxicity against intracellular Leishmania donovani amastigotes: Induction of microbicidal activity by Interferon-γ

Macrophages are pivotal cells in interactions of man and leishmania. Leishmanial disease results from intracellular infection of macrophages: parasitized cells are seen in smears or biopsy specimens of lesions; macrophages cultured in vitro support replication of parasites. Paradoxically, parasite destruction is also mediated by macrophages, which become highly cytotoxic after exposure to immune lymphocytes or their lymphokine (LK) products. The precise molecular mechanisms by which lymphocytes or LK induce macrophage activation for leishmanicidal activity, however, are not yet known. We analyzed interactions of leishmania amastigotes with human monocytes cultured in vitro as a nonadherent cell pellet. Leishmania donovani and L. major replicated in freshly isolated monocytes. Monocytes treated with >200 IU/ml of the LK, human Interferon-γ (IFN-γ), destroyed tumor cells and L. donovani, but not L. major. Phorbol myristate acetate, endotoxic bacterial lipopolysaccharide, and recombinant human IFN-α and IFN-β did not induce cytotoxicity. The time course for induction of cytotoxicity contrasted sharply with that of previously described monocyte antileishmanial activity: (i) IFN-γ induced cytotoxicity even when added after infection with L. donovani; (ii) induction of cytotoxicity did not require that IFN-γ be present throughout the period of culture after infection: a 30-min preinfection pulse of IFN-γ was sufficient to induce 70% of maximal activity; and (iii) freshly isolated monocytes and cells cultured for up to 4 days in vitro prior to infection and IFN-γ treatment were equally responsive to IFN-γ. These studies provide convincing evidence for intracellular cytotoxicity for L. donovani by freshly isolated human monocytes. This system provides an important base for further analysis of induction and expression of cytotoxic mechanisms against leishmania and other intracellular organisms that cause human disease.

Gamma interferon is the lymphokine and beta interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation.

Human peripheral blood mononuclear cells activated with concanavalin A (Con A) or lipopolysaccharide (LPS) produce, respectively, lymphokines (LK) of 50,000 Mr or monokines (MK) of 20,000 Mr that inhibit the growth and collagen production of cultured human dermal fibroblasts. Because antigenic typing of the antiviral activity of these LK and MK preparations revealed that LK contained mainly gamma interferon (IFN-gamma), and MK, primarily IFN-beta, we investigated if any of the fibroblast-inhibiting activities could be attributed to human IFN. Unlike LK and MK, which act within 24 h to inhibit the growth of subconfluent foreskin and adult dermal fibroblasts, samples of purified, natural derived IFN-alpha, -beta, and -gamma and recombinant DNA-derived IFN-alpha 2 and -gamma were ineffective inhibitors at 24 h and required 48-72 h to significantly inhibit growth. However, all IFN did mimic LK/MK action in causing concentration-dependent inhibition of collagen production by confluent fibroblast microcultures. Furthermore, the collagen production-inhibiting activity of Con A-induced LK supernatant and its 50,000 Mr fraction was completely suppressed by 10(3) neutralizing U/ml of either polyclonal or monoclonal antibody to IFN-gamma, while polyclonal antibodies to IFN-alpha and -beta had no effect. Similarly, the collagen production-inhibiting activity of LPS-induced MK supernatant and its 20,000 Mr fraction was suppressed by polyclonal anti-IFN-beta but not by anti-IFN-alpha or -gamma. Anti-IFN failed to reverse early-acting LK or MK growth-inhibiting activities. These data suggest collagen production-inhibiting LK and MK are IFN-gamma and IFN-beta, respectively, and that early acting, growth-inhibiting LK and MK are not IFN.

Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor.

Recombinant murine gamma interferon (rMuIFN-gamma) was found to bind reversibly to a specific high-affinity surface receptor on L929 cells; neither murine alpha or beta nor human gamma IFN competed for receptor binding. Encapsulation of the rMuIFN-gamma in either negatively or positively charged liposomes reduced its immediate ability to bind to this surface receptor. Disruption of liposome integrity with detergent resulted in full ability of the rMuIFN-gamma to bind to the membrane receptor. Incubation of the liposomal IFN in serum-containing medium resulted in significant leakage so that the IFN was able to bind to its surface receptor. Assessment of the biological activity of the rMuIFN-gamma preparations revealed that full antiviral activity was observed in vitro with the liposomal IFN preparations without their prior disruption by detergent. The antiviral activity observed with either free or liposomal IFN was neutralized completely by antibodies against rMuIFN-gamma. Both free and liposomal rMuIFN-gamma, in conjunction with bacterial lipopolysaccharide, were also able to activate murine peritoneal macrophages to the tumoricidal state. Again, this activity of both free and liposomal IFN could be neutralized completely by antibody. These results indicate that although rMuIFN-gamma can be effectively incorporated into liposomes, it must ultimately leak out of the liposome in order to mediate its biological effects; these effects are triggered after the IFN binds to its cell surface receptors.

Human interferons alpha and beta have more potent priming activities than interferon gamma.

The priming activities of human IFN-alpha, IFN-beta and IFN-gamma were compared on the same antiviral basis using human buffy coat leukocytes stimulated with Sendai virus or concanavalin A (Con A) to produce IFN-alpha or IFN-gamma, respectively. Pretreatment of leukocytes with any type of IFN enhanced their IFN-alpha and IFN-gamma production, but IFN-alpha and IFN-beta had more potent priming activities than IFN-gamma. IFN-alpha and IFN-gamma did not potentiate the priming activity of each other in either the IFN-alpha or the IFN-gamma producing system. Pretreatment of leukocytes with relatively high doses of IFN-alpha or IFN-beta (1000 to 3000 IU/ml) resulted in a 40- to 50-fold increase in the IFN-gamma production of Con A-stimulated leukocytes. This observation will be of use in producing IFN-gamma with a high titre.

Gamma interferon enhances mitogenic responses induced by pokeweed mitogen

Interferon (IFN)-induced suppression of the lectin-stimulated lymphoproliferative response was studied comparatively with human IFN-α, IFN-β and IFN-γ, using an equal unit of their antiviral activity ranging from 31.25 to 1000 IU/ml. Both IFN-α and IFN-β inhibited phytohemagglutinin (PHA) and pokeweed mitogen (PWM)-stimulated lymphoblastogenic response similarly in a dose-related fashion, but the IFN-γ effect was far less. Indeed, the PWM-stimulated lymphocyte blastogenesis in the cultures incubated for 7 days was enhanced in the presence of IFN-γ at a concentration of 62.5 IU/ml. The enhancing effect was found to be highest at the lowest concentration of IFN-γ examined. The IFN-γ induced enhancement of lectin-stimulated blastogenesis was found mainly in the PWM cultures incubated for 7 days but less in cultures incubated for 5 days or in PHA cultures incubated for 3 days, suggesting that the observed effect might be caused by the activation of interleukin production.

Plasmid pFCE4: A new system of Escherichia coli expression-modification vectors

Two versatile expression-modification vectors were obtained by inserting the origin of replication ( ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4 + and pFCE4 −) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN α-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Combination therapy of recombinant human alpha 2 interferon and acyclovir in the treatment of herpes simplex keratitis

No statistical difference was found in both partial and complete healing time in acute epithelial herpetic keratitis between the ACV-recombinant human alpha 2 IFN (both "eyedrops" and "eyerods") and Buffy coat human leucocyte alpha IFN. A highly significant difference was found in both partial and complete healing time between the ACV-IFN combination versus the ACV-Placebo combination. The combination of ACV and recombinant human alpha 2 arg IFN is a potent anti-herpetic treatment.

Pseudogene IFN-αL: Removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10 6–10 7 units/liter) was produced in Escherichia coli from modified human α interferon (IFN-α) pseudogene L. IFN-α pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-α gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-α pseudogene L. Hence, the IFN-αL mature interferon coding sequence, which is not identical to any other α-interferon gene, has been conserved for active interferon coding information.

Lymphokines produced by Herpesvirus-transformed marmoset monkey lymphoid cell lines I. Characterization of a constitutively produced interferon

Conditioned media from cultures of marmoset monkey T-lymphoid cell lines transformed by Herpesvirus saimiri or Herpesvirus ateles were found to contain interferon (IFN) activity. Titers between individual cell lines varied by a factor of 100; large amounts (up to 10 5 units/ml, assayed on human cells) were produced in one of the cell lines. IFN production was enhanced by the diterpene tumor promoters, TPA and mezerein, but not by classical T-cell mitogens. The IFN resembles human IFN-γ by the following criteria: lability at pH 2, stability against 2-mercaptoethanol, cross-species activity, shape of dose-response curves, and molecular weight determined by size-exclusion chromatography (50,000–55,000). Its activity was not inhibited, however, by antiserum against human IFN-γ or antisera against human IFN-α or IFN-β.

A human IFN-beta 1 gene deleted of promoter sequences upstream from the TATA box is controlled post-transcriptionally by dsRNA.

Induction of IFN-beta 1 RNA was studied in the mouse cell line SR117-21E transformed by a BPV episome containing the human IFN-beta 1 gene deleted of promoter sequences upstream from position -40. Nuclei isolated from these cells synthesize constitutively IFN-beta 1 RNA from the partially deleted promoter. The IFN-beta 1 RNA synthesized by nuclei of uninduced SR117-21E cells is similar to that made by nuclei of poly(rI):(rC)-induced cells, but does not accumulate and hence no IFN is produced unless the cells have been treated either by ds RNA or by cycloheximide. We conclude that the IFN-beta 1 gene has, in addition to the transcription control due to upstream promoter sequences, an additional post-transcriptional control acting on mRNA accumulation and linked to sequences close to the TATA box and RNA start site. Both controls are relieved by ds RNA.

Studies on Purification of Human Gamma Interferon: Chromatographic Behavior of Accompanying IL2 and B-Cell Helper Activity

Gamma interferon (gamma IFN) was produced in human lymphocyte cultures stimulated by PHA. Titers were in the range of 10,000-30,000 U/ml. Crude gamma IFN was adsorbed on silicic acid, from which the antiviral activity was eluted by a buffer containing a high salt concentration and ethylene glycol. This treatment allowed quantitative recovery of gamma IFN with a specific activity of 5 X 10(5)-1 X 10(6) U/mg of proteins. IL2 and B-cell helper activities were adsorbed and eluted from silicic acid together with the antiviral activity. This finding might be of practical interest for the purification of these lymphokines, particularly IL2. Gamma interferon was further purified on Blue Sepharose to a specific activity of 2 X 10(7) U/mg. The resulting preparations still contained IL2 and B cell helper activities. However, taking advantage of the differences in apparent hydrophobicity and in isoelectric point, we were able to dissociate antiviral activity from lymphokines. Such dissociation should facilitate the study of the biological properties of human natural gamma IFN.

Antibodies to a synthetic peptide corresponding to the N-terminal end of mouse gamma interferon (IFNγ)

Antibodies to an N-terminal synthetic peptide of mouse γ interferon (MoIFNγ) neutralized the antiviral activity of MoIFNγ but not MoIFNα β , human IFN α (HuIFNα), HuIFNβ or cynomologus monkey IFNγ (CynIFNγ). Comparatively, antibodies to mouse N-terminal synthetic peptide showed only 10% reciprocal cross-reactivity in neutralization tests against heterologous HuIFNγ and 13% cross-reactivity in the ELISA test against Hu N-terminal peptide. The predetermined specificity of these antibodies make them powerful tools for studying antigenic relatedness and biological properties of IFNγs.

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

Immune interferon and leukocyte-conditioned medium induce normal and leukemic myeloid cells to differentiate along the monocytic pathway.

Conditioned medium from phytohemagglutinin-stimulated human leukocytes contains a factor that can induce promyelocytic cell lines and certain acute myelogenous leukemia cells to differentiate along the monocytic pathway. In this report, we show that immature myeloid cells from normal bone marrow or the peripheral blood of patients with chronic myelogenous leukemia can be induced to differentiate to monocyte-like cells by immune gamma interferon (IFN gamma). We have identified IFN gamma as the predominant differentiation factor contained in the conditioned medium. Purified or recombinant IFN gamma, but not various preparations of IFN alpha or beta, can induce monocytic differentiation in myeloid cells. In cultures containing conditioned medium, the cells fail to continue myeloid maturation, and are induced to express monocyte markers and functions, such as monocyte-specific surface antigens, HLA-DR antigens, Fc receptors for monomeric immunoglobulins, nonspecific esterase, and the ability to mediate antibody-dependent, cell-mediated cytotoxicity. Even myeloid cells as mature as metamyelocytes or band cells can be induced by IFN gamma to undergo monocyte differentiation, but monocyte-specific or HLA-DR antigens are not induced in mature neutrophils. These findings reveal a previously unknown, specific function of human IFN gamma and offer new insights to the regulation of monocyte recruitment and differentiation during a virus infection or immune response.

Suppressor T cell activation by human leukocyte interferon.

Murine fibroblast interferon (IFN beta) activates murine suppressor T lymphocytes in vitro, which suppress plaque-forming cell responses by spleen cells. Suppression of human in vitro immune responses by IFN was investigated to determine whether human IFN also activates suppressor T cells. Human leukocyte IFN (IFN alpha) suppressed pokeweed mitogen-induced polyclonal immunoglobulin production by human peripheral blood mononuclear cells (PBMC) by 80 to 90% at doses of 200 to 350 U/ml. Responses by IFN alpha-treated PBMC were suppressed in a dose-dependent manner; control cultures had maximal responses on day 7. PBMC incubated with 10,000 U/ml of IFN alpha contained activated suppressor cells that decreased pokeweed mitogen-stimulated, polyclonal immunoglobulin production by autologous cells by 70 to 80%. Suppression mediated by these cells was prevented by catalase, ascorbic acid, and 2-mercaptoethanol (2-ME). In murine systems, these reagents interfere with expression of suppressor T cell activity by preventing activation of soluble immune response suppressor. Selection procedures with monoclonal antibodies identified the suppressor cell as an OKT8+ (suppressor/cytotoxic) T lymphocyte. Selected OKT8+ cells required less IFN alpha (1000 U/ml) for activation and were effective in smaller numbers than unfractionated activated PBMC. IFN alpha-activated suppressor cells also inhibited proliferation in mixed lymphocyte and mitogen-stimulated PBMC cultures; again, catalase and 2-ME blocked suppression. These results indicate that IFN alpha activates suppressor T cells in human PBMC cultures; the ability of catalase, 2-ME, and ascorbic acid to block suppression suggests that these suppressor T cells have certain similarities to IFN beta or to concanavalin A-activated murine suppressor T cells.