Faculty Bibliography

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

Human T-cell hybridomas were established by hybridization of concanavalin A-stimulated human peripheral blood lymphocytes with a 6-thioguanine-resistant mutant cell line, designated SH9, derived by irradiation from a cloned human cutaneous T lymphoma line, Hut102-B2. High levels of interferon (IFN) were demonstrated in the supernatants of hybridoma L265 and its subclones. Whereas no IFN was detected in SH9 cell cultures, up to 1,330 units of IFN per ml were produced spontaneously by the hybrids. On induction with 12-omicron-tetradecanoylphorbol 13-acetate, IFN synthesis in hybridoma cultures was enhanced 8- to 16-fold. Neutralization with specific antisera and determination of antiviral activities in human and bovine cells showed that the IFN secreted by the hybridomas was immune IFN (IFN-gamma). Analysis of DNA content, karyotype, and cell surface phenotype, including T cell specific antigens and receptors, confirmed the T cell hybrid nature of L265 clones. No correlation was found in the hybridomas between IFN production and the expression of HTLV, a retrovirus released by Hut102-B2 and SH9 cells

Highly purified human interferon-gamma (IFN-gamma) was iodinated with 125I-Bolton-Hunter reagent to a specific activity of 34 microCi/micrograms of protein. After iodination, molecular sieve chromatography was used to isolate fractions containing IFN-gamma at approximately 80-90% radioactive purity. This preparation of 125I-IFN-gamma bound specifically to high affinity binding sites on human GM-258 fibroblasts. Scatchard analysis of binding data revealed the presence of 2400 binding sites/cell, each binding with an apparent Kd of 1.5 X 10(-10) M. Binding was competitively inhibited in the presence of unlabeled IFN-gamma and, to a lesser degree, by IFN-beta, but not by IFN-alpha. Neutralizing antibodies specific for IFN-gamma efficiently inhibited the specific binding of 125I-IFN-gamma to cells. We conclude that the specific high affinity binding site is the receptor for IFN-gamma

Some immunologic parameters in homosexual patients with Kaposi's sarcoma (KS) or unexplained lymphadenopathy resemble findings in patients with autoimmune diseases such as systemic lupus erythematosus (SLE). Many patients with SLE have an unusual acid-labile form of human leukocyte interferon (HuIFN-alpha) in their serum. Sera from 91 homosexual men were tested for the presence of HuIFN. Of 27 patients with KS, 17 had significant titers of HuIFN in their serum. Ten of 35 patients with lymphadenopathy and three of four patients with other clinical symptoms also had circulating HuIFN. In contrast, only two of 25 apparently healthy subjects had serum HuIFN. All 32 samples of HuIFN had antiviral activity on bovine cells, a characteristic of HuIFN-alpha, and all of 14 representative samples tested were neutralized by antibody to HuIFN-alpha. In addition, the HuIFN-alpha in six of eight representative patients was inactivated at pH 2 and therefore appears to be similar to the HuIFN-alpha found in patients with SLE. These findings suggest that an autoimmune disorder may underly lymphadenopathy and KS in homosexual men

Human thymocytes in culture synthesized small quantities of interferon (IFN) when stimulated by the lectins concanavalin A or phytohemagglutinin. IFN production by lectin-activated thymocytes was enhanced in the presence of live B lymphoblastoid cells, irradiated B lymphoblastoid cells, or the conditioned medium from B lymphoblastoid cell cultures. The IFN synthesized in mixed cultures had characteristics of IFN-gamma, whereas the IFN synthesized by B lymphoblastoid cells alone could be identified as IFN-alpha on the basis of its neutralization with specific antisera and stability at pH 2. These findings indicate that human thymocytes in culture synthesize IFN-gamma and that B lymphoblastoid cells and their products considerably stimulate IFN-gamma synthesis by lectin-activated human thymocytes in culture. This stimulation was not diminished in the presence of antibodies to IFN-alpha, indicating that IFN-alpha production by B lymphoblastoid cells was not responsible for the stimulatory effect. Removal of adherent cells from thymocyte suspensions did not abrogate IFN-gamma production

A previously undescribed species of human leukocyte, or alpha, interferon is present in the serum of many patients with systemic lupus erythematosus. It was shown to be alpha-interferon by neutralization with specific antiserums, affinity column chromatography, and antiviral activity on bovine cells. However, 23 of 30 interferon samples tested were inactivated by incubation at pH 2, a characteristic of human 'immune,' or gamma, interferon. Multiple samples of interferon from the same patient had similar biological properties, but samples from different patients were not all identical, suggesting that several variants of this species of human alpha-interferon may exist