Interferons Mouse Characterization

Neither the alpha, beta or alpha + beta interferons has significant antiviral activity when assayed in human diploid fibroblast, aneuploid human or rabbit kidney cell strains. When assayed in RtK rat kidney cell cultures challenged with vesicular stomatitis virus, the cross- reactivities (as percentage of homologous titer, absolute units) are: alpha, 5- 10%; beta, 0.02- 0.2%; alpha + beta, 1.2- 3.5%.

There is slight synergy (200- 300%) in antiviral action between the alpha and beta interferons. There is marked synergy (10- to 100- fold) in the antiviral actions of either the alpha or beta interferons with mouse gamma as reported by Fleischmann et al. [19].

In one mouse cell strain we have observed that the alpha interferon is significantly more cytostatic per unit antiviral activity than is the beta interferon, and this cytostatic activity of alpha is antagonized by small amounts of beta. We suggest that cooperativity (synergy or antagonism) should be regarded as a general feature of the actions of interferons and other cytokines.

Although a good deal of what is known about the interferon system derives from the mouse, little is known about how the individual interferon gene products differ in the many biochemical and physiological activities which have been attributed to interferons [4]. Because of the diversity of in vitro and in vivo mouse models for malignant, viral and immune pathogenesis, the mouse presents an ideal system to approach the question why are there multiple interferons in mammalian species?

For these reasons, Access BioMedical Research provides three types of mouse interferon preparations for research use: unfractionated alpha + beta as well as the individual alpha and beta interferon species. The alpha + beta interferons (Cat. 200XX) consist of the unfractionated mixture in native proportions typically 5- 15% alpha and 85- 95% beta. The alpha and beta preparations (Cat. 220XX and 201XX respectively) contain <0.5% of the opposite species. All are provided in sterile lyophilized form and are stable indefinitely in the dry form; the rehydrated reagents are stable for months to years at 4 degrees C if sterility is maintained.

The individual interferon species are useful as distinct positive controls and in comparative studies, as in- house activity standards, as immunochemical standards, and in mixing experiments with other interferons or cytokines in studies of cooperativity of action. Both the individual interferons and their mixture should be regarded as distinct entities in studies of cellular physiology in vitro and pathogenesis, prophylaxis and therapy in vivo

Alpha and beta interferons are completely stable in extended incubation at pH 2 at 4 degrees C. In contrast, gamma is completely inactivated in 12 hr at pH 2 at 4 degrees C. The absence of any gamma interferon in our mouse alpha, beta or alpha + beta interferons further can be stated confidently since (1) gamma is not produced by fibroblastoid cell cultures, (2) gamma synthesis is not induced by viral or double- stranded RNA inducers and (3) all of our mouse interferon products are typically held at pH 2 at 4 degrees C for at least seven days prior to further handling.

Following published methods, the alpha and beta interferons are resolved at appropriate pH and ionic strength by adsorption to immobilized Cibachron blue, controlled- pore glass beads, specific immunoaffinity sorbents and other ligands, as well as by proprietary methods using a variety of inorganic sorbents.

The antiviral action of alpha is not neutralized by rabbit antiserum raised against purified beta interferon; the antiserum has <0.02% cross- reactivity toward mouse alpha and gamma interferons. Fig. 4 shows the lack of neutralization of two alpha preparations (Fig. 4A and 4B) and two control titrations demonstrating the expected linearity when beta interferon is titrated against anti- beta interferon serum (Fig. 4C) and when alpha interferon is titrated against anti- (alpha + beta) interferon serum (Fig. 4D). Titration of mouse gamma interferon against the anti- beta or anti- (alpha + beta) interferon sera shows no detectable neutralization (< 0.02%) of activity (not shown).

Discontinuous gel electrophoresis in 8- 18% polyacrylamide gradients is shown in Fig. 5. The alpha interferon activity has an apparent molecular weight of 27- 28 Kd. The beta interferon activity has an apparent molecular weight of 35- 36 Kd and the two are completely resolved. In gels containing 1% SDS and 1% 2- mercaptoethanol, the activity of alpha is >95% eliminated whereas the activity of beta is preserved if not marginally increased (not shown).

At the request of the Expert Committee on Biological Standardization of the World Health Organization, murine alpha, beta and unfractionated alpha + beta interferons produced by Access BioMedical Research were provided to Dr. S. Grossberg (Medical College of Wisconsin) for incorporation into three new mouse interferon activity standards distributed by the NIH (USA) and NIBSC (UK) (Table 1, p. 6). The alpha and beta interferons were radio iodinated and analyzed by SDS- PAGE by Drs. S. Grossberg and J. Sedmak, who found no evidence of alpha in the beta preparation [28] and no evidence of beta in the alpha preparation [personal communication].