Faculty Bibliography

Highly purified natural or recombinant human immune interferon (IFN-gamma) was found to be directly cytolytic to certain tumor cell lines in vitro. Out of 5 human tumor cell lines and one normal fibroblast line tested, the colon adenocarcinoma line HT-29 and the rhabdomyosarcoma line A673 were highly sensitive to cytolysis by interferon, as determined by 125I-iododeoxyuridine release in a 72 h microcytotoxicity assay. Cytolysis was marked at IFN-gamma concentrations of less than I U/ml, and it reached a near-maximal level at 6.4 U/ml. A synergistic cytolysis on HT-29 cells of IFN-gamma and 5-fluorouracil (5-FU) was observed at 5-FU concentrations ranging from 64 to 640 micrograms/ml. In contrast, no synergism was observed between IFN-gamma and mitomycin C. The direct cytolytic activity and synergistic cytolysis with 5-FU of the IFN-gamma preparations used in the present study were abolished completely by treatment with a neutralizing monoclonal antibody specific for human IFN-gamma

Antisera raised against a human X mouse hybrid cell line containing human chromosome 21 as its only human chromosome, block induction of an antiviral state by human alpha interferon (IFN-alpha), block induction of (2'-5')oligoisoadenylate synthetase [2'-5')A synthetase), and block binding of 125I-labeled and 35S-labeled recombinant, human IFN-alpha A, but not 125I-labeled IFN-gamma, to cell surface receptors. The data presented clearly demonstrate that the cell surface receptors for IFN-alpha and IFN-gamma are different, and provide independent evidence of the role of a chromosome 21 coded cell surface molecule in the pathway to the generation of the antiviral state.

Mouse monoclonal antibodies B1 and B3 are specific for natural and Escherichia coli-derived recombinant human gamma-interferon (IFN-gamma). The two antibodies recognize different epitopes of the IFN-gamma molecule and do not compete with each other's binding. We have used these two antibodies to construct a solid-phase, sandwich immunoradiometric assay for human IFN-gamma. Purified antibody B1 was coated on polystyrene beads (0.64 cm in diameter) and used as the solid-phase immunoadsorbent and antibody B3 was labeled with 125I and used as tracer. This assay can be completed in about 4 hr and is capable of detecting IFN-gamma levels in human serum or tissue culture fluids as low as 0.1 NIH reference unit/ml. Recombinant human IFN-gamma derived from E. coli was detectable at a concentration of 0.02 ng/ml. The assay appears to be specific for the biologically active forms of IFN-gamma, since after exposure to pH 2, 37 degrees C, or 56 degrees C, biological activity and reactivity in the immunoradiometric assay decreased in parallel. The immunoradiometric assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule

Enhanced natural killer (NK) activity was detected in the spleens of mice as early as 24 hr after single i.v. inoculation with gamma-irradiated Plasmodium berghei sporozoites. The activity peaked at 48 hr post-injection, and declined below baseline level by day 8. Reinoculation of mice with irradiated sporozoites produced an increased NK activity significantly smaller than the original activity. Spleen cells sensitized in vivo as well as nonsensitized spleen cells stimulated in vitro with sporozoites produced high levels of interferon (IFN) and displayed enhanced NK activity. Characterization of the IFN through the use of specific antibodies revealed that it was mainly IFN-gamma. The cellular basis for IFN-gamma induction was linked to the mitogenicity of P. berghei sporozoites for T cells. The possibility exists that IFN-gamma may have a regulatory effect on antibody production against P. berghei sporozoites

Purified natural and recombinant human immune interferon (IFN-gamma) were found to activate human monocytes from peripheral blood to exert enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells. A marked monocyte activation was observed at low concentrations (1 and 10 U/ml) of IFN-gamma. Marked monocyte activation was also obtained with two lymphokine preparations, produced in peripheral blood mononuclear cell (PBM) cultures induced with phytohemagglutinin (PHA) or by combined stimulation with PHA and 12-O-tetradecanoylphorbol 13-acetate (TPA). The component responsible for macrophage activation in such lymphokine preparations in the past was considered to be 'macrophage-activating factor' (MAF). When monoclonal antibody specifically neutralizing IFN-gamma was added to these lymphokine preparations, all MAF activity disappeared, indicating that IFN-gamma is the sole protein showing MAF activity in these preparations

Two IgG1/kappa class monoclonal antibodies specific for human immune interferon (IFN-gamma), designated B1 and B3, were developed. Specific binding of both monoclonal antibodies to natural or Escherichia coli-derived recombinant human IFN-gamma was demonstrated in a solid-phase radioimmunoassay or by immunoprecipitation. Antibody B3 showed potent neutralizing activity against both natural and recombinant IFN-gamma. Antibody B1, which showed neutralizing activity only when very high concentrations were employed, was used for preparing immunosorbents for affinity chromatography of IFN-gamma. When a highly purified preparation of 125I-labeled natural IFN-gamma was loaded onto the affinity column, all of the biological activity was retained on the column. The bulk of 125I-labeled IFN-gamma bound to the affinity column be eluted in biologically active form, suggesting that antibody B1 could be used for the purification of human IFN-gamma. Analysis of IFN-gamma eluted from the column by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE) indicated that both of the known molecular weight subspecies of IFN-gamma (25,000 and 20,000 MW), as well as the presumed dimer of 45,000 MW, were retained by the B1 antibody affinity column

Immune interferon (IFN-gamma), endogenously labeled with [35S]methionine, was produced in human peripheral blood lymphocyte cultures stimulated with 12-O-tetradecanoylphorbol-13-acetate and phytohemagglutinin. 35S-IFN-gamma, immunoprecipitated from the crude culture fluid with a monoclonal antibody, was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three monomeric forms with molecular weights of 25,000, 20,000, and 15,500, which we designate IFN-gamma I, II, and III, respectively. IFN-gamma I was the most, and IFN-gamma III the least, abundant in both immunoprecipitated 35S-IFN-gamma and chromatographically purified IFN-gamma preparations. Changes in the molecular size of the monomeric forms after glycosidase treatment suggested that IFN-gamma I contains more carbohydrate than IFN-gamma II, and that IFN-gamma III may not be glycosylated at all. Hence, the differences in the carbohydrate contents are likely to be the major cause of the molecular size heterogeneity of IFN-gamma I, II, and III

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells

Monoclonal antibody GIF-1 was found to neutralize human natural immune interferon (IFN-gamma), but not Escherichia coli-derived recombinant IFN-gamma. In addition, GIF-1 antibody failed to immunoprecipitate 125I-labeled recombinant IFN-gamma, whereas it precipitated natural IFN-gamma in a concentration-dependent manner. The lack of recognition of recombinant IFN-gamma by antibody GIF-1 may not be due to the absence of the oligosaccharide moiety in the molecules of recombinant IFN-gamma alone, because removal of carbohydrate from natural IFN-gamma by treatment with a mixture of glycosidases did not alter the selective binding of antibody, i.e., deglycosylated and untreated natural IFN-gamma were equally neutralized and immunoprecipitated by GIF-1 antibody. In addition, a minor monomeric component of natural IFN-gamma with the m.w. of 15,500, which apparently lacks carbohydrate, was also recognized by antibody GIF-1. These results suggest that the discriminative recognition of natural and recombinant IFN-gamma by monoclonal antibody GIF-1 may be due to a conformational difference at or near the active regions of natural and recombinant human IFN-gamma molecules

Mixed cell cultures of syngeneic lymph node (LN), spleen, or thymus and gamma-irradiated, syngeneic lymphoma cells of transplantable reticulum cell sarcomas (gamma-RCS) produced within 24 hours high titers of interleukin 2 (IL-2) and immune interferon in their supernatant (SN). These lymphokine titers were much higher than those seen after stimulation with allogeneic cells. SN also had marked enhancing activity for antibody production by anti-T-cell serum plus complement-treated spleen cells to trinitrophenylated polyacrylamide in vitro. This activity could be removed by absorption with cells of an IL-2-dependent cytotoxic cell line. Mixtures of gamma-RCS and LN cells from SJL/J F1 hybrid mice produced these lymphokines only when the non-SJL parent contributed H-2s or H-2b, but not H-2k or H-2d, in the I-region. These I-region restrictions were similar to those observed previously with respect to the ability of T-cells from SJL F1 hybrids to give proliferative responses to gamma-RCS in vitro and of these mice to support tumor growth in vivo. gamma-RCS also induced rapid interferon production in vivo, but serum titers 24 hours after injection consisted primarily of interferon resistant to pH 2 and neutralized by antibody to virally induced interferon (IFN-alpha/beta), and the production of IFN-alpha/beta was not subject to the same genetic restrictions. Although reticulum cell sarcoma cell extracts had no detectable effect in vitro, they were capable of inducing transient IFN production in vivo.