Species Of Human Leukocyte Interferon Research Articles

Antiviral activity of natural and recombinant human leukocyte interferons in tobacco protoplasts

Several purified species of human leukocyte interferon, including recombinant interferons, inhibit the multiplication of tobacco mosaic virus (TMV) in tobacco protoplasts derived from various cultivars. Viral RNA accumulation was determined by dot-blot hybridization to specific cDNA probes, and virus antigen was determined serologically. Interferon apparently inhibited both TMV-RNA replication and its expression into coat protein. However, these effects were of limited duration. Maximum effect was obtained when interferon was applied to the cells either prior to inoculation or within the first hour after inoculation. Antibodies to interferon abolished its antiviral activity in protoplasts. Tobacco protoplasts were about 1000 times more responsive to interferon than the reference animal viral-cell system and showed an “antiviral state” at a ratio of 1 molecule of interferon per cell.

Some species of human leukocyte interferon are glycosylated

The carbohydrate content of all of the species of human leukocyte interferon (IFN-α) which have been derived from patients with chronic myelogeneous leukemia (CML) and purified to homogeneity has now been determined. Amino sugar content was measured by high-performance liquid chromatography and fluorescamine detection of acid hydrolysates of each sample. Two species showed significant amounts of glucosamine. Most of the purified species of leukocyte interferon from a myeloblast cell line were also tested, and two species were found to contain sugar residues. These forms also differed from the CML interferons in that they revealed the presence of greater amounts of galactosamine. The apparent lack of carbohydrate in some of the higher-molecular-weight species of interferon implicated factors other than glycosylation in the molecular weight differences. The results indicate that some species of IFN-α are glycosylated to various degrees.

Rapid purification of leukocyte interferons by high-performance liquid chromatography

Many species of interferon have been identified and purified from human leukocytes, and genes for these interferons have been cloned in bacterial systems. However, it is still unclear as to how the expression of each of the different leukocyte interferons is regulated and how they function in the natural system. In preparation for a study of the in vivo role of individual leukocyte interferons we have developed a rapid and convenient high-performance liquid chromatographic method for purifying several species of human leukocyte interferon. Interferon produced by the induction of leukocytes (obtained from buffy coats of normal donors) with Sendai virus and partially purified by ethanol fractionation was further purified by size-exclusion high-performance liquid chromatography (on two Waters,I-125 columns) in 0.05 M sodium phosphate buffer, pH 6, containing 0.2 M sodium chloride. The flow-rate was 2 ml/min and interferon activity (antiviral) was eluted from the columns in less than 15 min in a single fraction well resolved from high-molecular-weight contaminants. This material was then purified by reversed-phase high-performance liquid chromatography on a wide-pore diphenyl column (Whatman Protesil 300) with a gradient system at pH 2.4, consisting of (A) 0.05 M KH2PO4—methoxyethanol (19:1) and (B) 1-propanol—methoxyethanol (19:1). Several peaks of interferon activity were distinguishable in this system. Analysis of the pooled peaks of interferon activity by SDS polyacrylamide gel electrophoresis confirmed the presence of more than one form of leukocyte interferon. The specific activity of the purified leukocyte interferers was ca. 108 units/mg protein.

Effects of several species of human leukocyte interferon on cytotoxic activity of NK cells and monocytes

Ten species of purified human leukocyte interferon were tested for their ability to modulate the cytolytic activity of natural killer (NK) cells and the cytolytic and cytostatic activities of monocytes. The interferon species were tested at several antiviral titers and examined for quantitative differences in their ability to modulate immunological function. At the higher doses of interferon (i.e., greater than 500 units) all of the interferon species demonstrated significant augmentation of cytolysis and cytostasis. However, when low levels (i.e., 10-50 units) of interferon were employed, appreciable differences between the various interferon species were seen. A similar pattern of relative potency among the various species of pure leukocyte interferon was seen for augmentation of cytolysis by monocytes and NK cells. In contrast, a different pattern of relative potency was observed for augmentation of cytostasis. These results demonstrated substantial quantitative differences (as much as 100-fold) in the ability of the various species of human leukocyte interferon to induce significant levels of augmentation of these cell-mediated functions. Such results should have significant impact in choosing a specific interferon species for appropriate clinical trials.

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH 2-terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.

Amino acid sequence of a human leukocyte interferon.

The primary structures of three major species of human leukocyte interferon differ from the structure predicted from the DNA sequence of recombinants containing leukocyte interferon-coding regions. Compared to the recombinant interferon produced in bacteria, three of the purified natural proteins isolated from leukocytes lack the 10 COOH-terminal amino acids suggested by the DNA sequence.

Production of high levels of human leukocyte interferon from a continuous human myeloblast cell culture.

A myeloblast cell line has proved to be an excellent source of human leukocyte interferon. These cells, primed with interferon and induced with Sendai virus, produced optimal levels of human leukocyte interferon. The cells grew readily in spinner flasks and in medium containing horse serum. Interferon production over several months yielded an average titer of 2 X 10(5) reference units of interferon per 10(7) cells. The interferon produced by these cells appeared to be predominantly species of human leukocyte interferon. Since these cells seemed to consist of myeloblasts, it is clear that cells other than B-lymphocytes can produce leukocyte interferon.

Human leukocyte interferon: production, purification to homogeneity, and initial characterization.

A method of fractionating proteins by high-performance liquid partition chromatography has been developed and used for isolation and purification to homogeneity of one of the species of human leukocyte interferon. The homogeneous interferon exhibited a sharp peak on high-performance liquid chromatography and a single narrow band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Extraction of the gel gave a single sharp peak of antiviral activity coinciding with the protein band. The specific activity of pure interferon was found to be 2--4 X 10(8) units/mg, based on amino acid analysis. The molecular weight is 17,500--18,000.

Antigenic properties and heterospecific antiviral activities of human leukocyte interferon species.

Human interferon obtained in peripheral leukocytes was purified approximately 1000-fold by affinity chromatography on anti-leukocyte interferon globulins coupled to Sepharose 4B, and by filtration on SDS-Sephadex G-100. The interferon was subsequently resolved into two molecular species by adsorption chromatography on SDS-hydroxylapatite. The two species which were eluted at different phosphate molarities from hydroxylapatite, could also be distinguished on the basis of electric charge properties and they migrated at different rates in SDS-polyacrylamide gels. Crossneutralization tests with monospecific rabbit anti-leukocyte and anti-fibroblast interferon sera revealed that the two species possessed leukocyte interferon-specific antigenic determinants. Both were immunogenic in mice and they were neutralized to a comparable degree by antisera against either component. A variable degree of antiviral activity was expressed by both interferon components in bovine, porcine and murine cells. However, the two interferon species were equally active in this respect, and the protective effects exhibited in homologous and heterologous cell cultures were similarly susceptible to reduction by beta-mercapto-ethanol. We conclude that the two molecular species of human leukocyte interferon are biologically similar.