The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.

Sera from 61 patients with systemic lupus erythematosus (SLE) were serially screened over a period of at least 2 years for IFN and anti-IFN antibodies. IFN concentrations were measured both with a cytopathic effect assay and a more sensitive radioimmunoassay. Of the patients 15% (9/61) had IFN in their serum at one or more occasions as measured in the bioassay (greater than or equal to 6 IU/ml); employing a RIA (greater than or equal to 1 IU/ml) 28% (17/61) of the patients studied were positive for IFN-alpha. Fifteen patients had a measurable interferonemia over 2-16 months; only two patients had detectable IFN in their serum at only one occasion. In five patients, hourly and daily variations of the IFN titer as measured by RIA were found to amount to less than 80%. The IFN activity found in these sera was characterized as IFN-alpha by means of acid stability, cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous and heterologous antisera. IFN antibodies were quantified with a neutralization bioassay, an ELISA, and a radioimmunoassay. Of the 61 patients 5% (3) possessed high titers of anti-IFN antibodies which persisted over 2 years. The IFN-alpha antibody positive patients had an inactive form of the disease over years without visceral involvement but decreased serum complement levels (C4, C3, CH50) and repeated episodes of Quincke-like edema.

Mx protein, an intracellular protein induced by type I interferons (IFNs), is useful as a marker for the IFN-induced state. It is detectable, for example, in leukocytes of patients undergoing IFN-alpha treatment as well as in patients suffering from viral or autoimmune diseases. For immunizations and standardizations, recombinant human MxA protein was expressed in Escherichia coli and purified from inclusion bodies by several steps of chromatography. Two monoclonal antibodies against nonoverlapping epitopes and specific for human Mx protein were selected to establish a simple two-site immunometric enzyme assay. In addition, a monoclonal antibody also reacting with Mx proteins of other species was identified. Prior to assay, whole blood samples were lysed with a nonionic detergent. The sample was incubated on wells coated with a first monoclonal antibody (1304.5.32) together with a second biotinylated monoclonal (1302.34.16), which, after washing, was revealed by an avidin-alkaline phosphatase system. Limit of detection was 5 ng/ml. In two-thirds of normal blood samples (n = 87), Mx protein levels were below 5 ng/ml; 25 samples (29%) had Mx levels between 5 and 50 ng/ml; and 4 samples (5%) were above 50 ng/ml. No Mx was found in plasma, and the mononuclear cell fraction accounted for the bulk of Mx in blood. In vitro, as determined by flow cytometry, monocytes and lymphocytes accumulated Mx protein for 24 h with similar kinetics and remained at plateau levels for more than 70 h. Monocytes contained around eight times more Mx than lymphocytes. The immunoassay was also suitable for detecting Mx after IFN induction in heparinized blood.

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