Faculty Bibliography

Highly purified human interferon (IFN)-gamma was treated with a preparation of mixed glycosidases in order to evaluate the effect of carbohydrate depletion on its biological activity, isoelectric point, and molecular size. Glycosidase treatment did not reduce the antiviral activity of IFN-gamma in cultures of human fibroblasts and in bat lung cells. No antiviral activity was observed before or after treatment with glycosidases in pig, mink, bovine, murine, and monkey cells. The degree of neutralization of IFN-gamma activity with specific antibody was also not significantly affected by glycosidase treatment. Several components of IFN-gamma activity were resolved by nonequilibrium pH gradient electrophoresis, with major peaks of activity at pI 8.5 and 8.7. Glycosidase treatment of IFN-gamma resulted in a reduced charge heterogeneity and a higher pI of 9.3. 125I-labeled IFN-gamma was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two bands with molecular weights of 25,000 and 20,000. Glycosidase treatment reduced the apparent molecular weight of these bands to 18,500 and 16,000, respectively. The results suggest that both the Mr = 25,000 and 20,000 bands, thought to be monomeric forms of IFN-gamma, are glycosylated

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

Earlier studies from several laboratories showed that interferon-alpha (IFN-alpha) is present in the sera of a large percentage of patients with systemic lupus erythematosus (SLE). We now report the detection of IFN-alpha by indirect immunofluorescence in renal sections of three patients with SLE but not in six control kidneys. The immunofluorescence reaction was mediated by three hyperimmune antisera to IFN-alpha raised in three different species, but not by any preimmune serum. The reaction was specifically blocked by absorption of the anti-IFN-alpha sera with purified IFN-alpha made by recombinant DNA techniques or with IFN-alpha isolated from the serum of an SLE patient, but not by bovine serum albumin or human immunoglobulin G. In contrast, antisera to IFN-beta or IFN-gamma did not mediate immunofluorescence. The pattern of IFN-alpha deposition resembled that seen with anti-human immunoglobulin G, suggesting association with immune complexes. Immune complexes were then preparatively eluted from the homogenate of an SLE kidney by treatment with buffer at pH 2.8. Biologically active IFN was found in this eluate and was demonstrated to be IFN-alpha by specific neutralization with IFN antisera. These results extend the specific association of IFN-alpha with SLE

Interleukin-2 (IL-2), purified to apparent homogeneity, enhanced interferon (IFN) production in phytohemagglutin (PHA)-stimulated cultures of Ficoll-Hypaque-purified human mononuclear cells derived from plateletpheresis residues. Cells incubated with IL-2 in the absence of PHA did not produce detectable IFN. Neutralization with specific antisera and lack of activity in bovine cells indicated that the IFN produced in cells treated with IL-2 was IFN gamma. Addition of IL-2 to cultures stimulated IFN production in a dose-dependent fashion, with 100 U/ml of IL-2 generally producing optimal stimulation. There was considerable variability in the magnitude of the IFN response and the degree of its enhancement by IL-2 treatment in cells from different donors. However, an enhancement of IFN production after treatment with 100 U/ml of IL-2 was regularly observed in 11 experiments, with the increase ranging from 3- to 37-fold (mean 8.6-fold). The difference between IFN yields in control cultures and cultures treated with 100 U/ml of IL-2 was statistically significant (P less than 0.001). In contrast, 1000 U/ml of IL-2 strongly inhibited IFN induction by PHA. Treatment of cultures with IL-2 did not alter the kinetics of IFN production which peaked at 48-72 hr after PHA stimulation. When PHA was added only 24 to 96 hr after the establishment of cultures, rather than at the time of their seeding, both IFN production and endogenous IL-2 production were enhanced. The addition of exogenous IL-2 to such cultures caused only a modest further enhancement of IFN production. These data suggest that a threshold concentration of IL-2 (exogenously added or endogenously produced) is required for optimal IFN gamma production by human mononuclear cells

Cyclosporin A depresses the synthesis of gamma interferon by human thymocytes and T lymphocytes in vitro. This observation is of potential clinical significance because the long-term treatment of transplant patients with cyclosporin A, a widely used immunosuppressive agent, can give rise to B-cell lymphoma resulting from Epstein-Barr virus activation

Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes

Interleukin-2 (IL-2) and immune interferon (IFN-gamma) production was studied in Ficoll-hypaque purified human mononuclear cells derived from plateletpheresis residues. As previously reported, costimulation by 5 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 5 microgram/ml of phytohemagglutinin (PHA) caused a marked enhancement of IFN-gamma levels compared to the yields obtained with PHA alone. IL-2 levels were also increased 50-300 times by this induction protocol. Mezerein (MZN), a compound structurally related to TPA, was found to be similar to TPA in enhancing IFN-gamma and IL-2 levels. In addition to TPA and MZN, four other related phorbol esters caused a stimulation of IFN-gamma and IL-2, with the production of IL-2 paralleling the production of IFN-gamma. Kinetic experiments indicate that low levels of IL-2 first became detectable 6 h after induction, whereas IFN-gamma could be demonstrated only later. Furthermore, IL-2 production reached a plateau earlier than IFN-gamma production in the TPA/PHA treated cultures. Dialysis at pH 2 abrogated the IFN-gamma activity but not the IL-2 activity. A three-step purification procedure developed for IFN-gamma isolation effectively separated IFN-gamma from IL-2. Our results show that there is a close correlation between the magnitude of IL-2 and IFN-gamma production under the experimental conditions employed. Physical separation of the two lymphokines is important for future studies on the interactions between IFN-gamma and IL-2 in various cellular immune reactions