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Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5-5 μM and 0.4-5 nM respectively (except for IFN-alpha1 - 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC50 values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.

We have begun examining serum levels of Type I and III interferons in normal and autoimmune donors by ELISA and a Type I reporter gene activity assay to determine if there are expression pattern signatures of these diseases. Samples were obtained from normal donors and patients with Systemic Lupus Erythematosus, Rheumatoid Arthritis or Multiple Sclerosis and assayed for IFN-α), IFN-β, IFN-ω, and IFN. Normal controls had little detectable IFN-α, IFN-ω, or Type I IFN activity. Low levels of IFN-β were detected in 15% of the normal controls. Approximately 35% of the normal controls had detectable IFN-λ. In the Lupus samples, 45% had detectable IFN-α. IFN-ω and IFN-β were detected in 20 and 40%, respectively, of the Lupus samples. Overall, greater than 80% of the Lupus samples had detectable Type-I IFN. Rheumatoid arthritis samples may have elevated IFN-β and IFN-ω relative to normal controls, but not IFN-α. Multiples sclerosis samples had a slight tendency towards elevated IFN-β. A low percentage of Lupus and multiple sclerosis samples had detectable Type-I IFN activity by the reporter gene assay. None of the samples displayed significant differences in Type III IFN expression. Multiplex ELISA was used to examine a subset of the samples for cytokine profiles and in Lupus samples IL-13 was decreased. In arthritis samples IL-8 and IL-23 were elevated, while in Multiple Sclerosis samples IL-2, IL-4 and IL-10 were elevated. The implications of these results will be discussed.

The human lymphoma cell line NK-92 has many characteristics of human natural killer cells. We have examined NK-92 activation by a full panel of human alpha Interferons (IFNs) by measuring IFN gamma secretion. We confirmed that NK-92 cells secrete IFN gamma in response to IFN alpha 2 in a dose-dependent manner. The IFN gamma response is enhanced by addition of IL-2, IL-18 or PMA, as has been seen with human donor NK cells. The EC50 for IFN alpha 2 ranges between 700–3000 pg/ml. Inclusion of IL-18 exhibited little effect on IFN potency but significant enhancement of IFN efficacy. EC50 values differ by >1000 fold between the IFN subtypes. Alpha 1 demonstrated the weakest activity while alpha 10 and beta 1a were the most potent. The relative potency of the IFNs correlates well with ability to phosphorylate STAT1 and 2. When NK-92 potency is compared with antiviral activity on A549 cells and antiproliferative activity on OVCAR3 cells, there are subtle but significant differences in the rank orders of potency and potencies relative to IFN alpha 2. These studies will be extended to examination of donor NK cells, additional signal transduction pathways, and the secretion of other cyto/chemokines