Classification of interferons with antibody to immune interferon

CELLULAR

IMMUNOLOGY

53,65-70

(1980)

Classification of lnterferons with Antibody to Immune Interferon1 LLOYD C. OSBORNE, JERZY A. GEORGIADES, AND HOWARD M. JOHNSON* The University

of Texas Medical

Branch, Department

of Microbiology,

Galveston,

Texas 77550

Received July 27, 1979

Mouse immune interferon, induced by the T-cell mitogen staphylococcal enterotoxin A (SEA), was partially purified and used to immunize rabbits. The resulting antiserum neutralized all immune interferon preparations tested, including interferon induced in vitro by SEA, concanavalin A, phytohemagglutinin P, and pokeweed mitogen, and in mixed lymphocyte cultures. Interferon produced in vivo with specific antigen was also neutralized. The antiserum was equally potent against all these interferon preparations. The serum did not neutralize any virus-type interferon preparation tested, but immune interferon induced by SEA in athymic nude mouse spleen cells was neutralized. The neutralizing activity was precipitable by 33% ammonium sulfate, and was not removed by absorption of the serum with mouse cells. The data suggest that immune intetferons produced under diverse conditions are antigenicaliy the same or closely related.

INTRODUCTION Based on current knowledge, two distinct groups of interferons are recognized in the human and mouse systems. One group, the virus-type (type I) interferons, are classically induced by viruses or synthetic polynucleotides and are stable at pH 2. Within this group in humans, two antigenic types have been described (1). The same may hold for the mouse system, but this has not been clearly established (2). They are called fibroblast and leukocyte interferons to indicate their cellular sources. The other group of interferons, the immune (type II) interferons, are induced in primed lymphocytes with specific antigens or in unprimed lymphocytes with T-cell mitogens (3-5). These interferons are labile at pH 2 and are not neutralized by antibodies to fibroblast or leukocyte interferons (3-5). There is no information on the antigenic relationship of antigen-induced and mitogen-induced immune interferons. If antigen-induced and T-cell mitogen-induced interferons are the same, then the rationale for studying the effects of T-cell mitogen-induced interferon on immune functions as a model for interferon-induced immune modulation would be clearly established. It would also suggest that other lymphokines induced by mitogens may be the same as those produced in viva during an immune response. Further, antisera to immune interferon would allow selective deletion of functions such as suppressor cell activity since data have suggested that immune interferon may mediate such activity (6). We present here the first report of production and use of specific 1 Supported by American Cancer Society Grant IM-148. L.C.O. was supported by a James W. McLaughlin Predoctoral Fellowship. 2 To whom correspondence should be addressed. 65 0008-8749/80/090065-06$02.00/O

Copyright 0 1980by AcademicPress,Inc. AU rights of reproduction

in any form reserved.

66

OSBORNE,

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AND JOHNSON

antibodies to mouse immune interferon to classify interferons. The neutralization data strongly suggest that antigen-induced and mitogen-induced immune interferons are antigenically the same or closely related. In addition, we show that antiserum to immune interferon has no neutralizing activity against virus-type interferons induced under various conditions. MATERIALS

AND METHODS

Large-scale production and puri$cation of interferon. Methods for induction of immune interferon in mouse spleen cell cultures with the T-cell mitogen staphylococcal enterotoxin A (SEA) have been described previously (7). The interferon was concentrated by differential ammonium sulfate precipitation which resulted in modest (threefold) purification. The interferon was then purified approximately 300-fold by bovine serum albumin (BSA)/Affi-Gel 10 hydrophobic chromatography to a specific activity of approximately lo5 units/mg protein. Rabbit immunization. Rabbits received an initial injection with salt-precipitated immune interferon and six immunizations with chromatographed interferon of high specific activity. The partially purified interferon averaged 14,000 units per immunization. All interferons were administered intramuscularly and intradermally at monthly intervals and at multiple sites in Freund’s complete adjuvant. Serum was obtained 10 days after the seventh inoculation. Absorption of antiserum. Prior to testing in neutralization tests serum was absorbed with mouse spleen and/or L cells. The cells were added to the serum to a final concentration of lo8 spleen cells/ml and 2 x IO6 L cells/ml. After 1 hr incubation at room temperature, the cells were removed by centrifugation. Production of other interferon preparations. Antigen-induced interferon was produced according to a previously described technique (3). Mice infected with Mycobacterium bovis, strain BCG, were challenged with old tuberculin; serum collected 3 hr later was used in the assay. Mixed lymphocyte culture (MLC) interferon was produced as follows: BALB/c mouse spleen cells were treated with 25 &ml mitomycin C for 1 hr at 37°C and washed three times to remove the drug. These cells were mixed with an equal amount of C57BU6 mouse spleen cells (10’ total lymphocytes/ml) and incubated 4 days at 37°C under culture conditions that were previously described (7). The supernatant from these cultures contained the interferon. Concanavalin A (Con A), pokeweed mitogen (PWM), and phytohemagglutinin P (PHA-P) were also used to produce immune interferon in the in vitro system as described (7). Lipopolysaccharide (LPS)-induced interferon was produced as previously described (8). Swiss/Webster mice were inoculated intravenously with LPS prepared from Escherichia coli 0127 B8 (Difco Laboratories, Detroit, Mich.) and bled 2 hr later. Serum collected from the blood contained the interferon. Interferon assays. A microplaque reduction assay for interferon was performed as described using vesicular stomatitis virus (40 PFU/well) (9). Neutralization tests. Two types of neutralization tests were performed. In one, half-log dilutions of interferon were mixed with equal volumes (0.2 ml) of (a) culture medium, (b) a 1:30 dilution of serum collected from a rabbit prior to first immunization (preimmunization serum), (c) a 1:30 dilution of anti-immune interferon from the same rabbit, or (d) a 1:60 dilution of anti-mouse L-cell

ANTIBODY

67

TO IMMUNE INTERFERON

interferon, obtained from the NIAID, NIH, Bethesda, Maryland. (One milliliter of anti-L-cell interferon, undiluted, neutralizes 60,000 units of interferon.) After 1 hr incubation at room temperature, the mixtures were individually added to L cells for interferon assay, and titers of interferon were calculated. In the second type of assay, twofold dilutions (beginning with 1:20) of either preimmunization serum or anti-immune interferon were mixed with equal volumes (0.2 ml) of interferon. The final concentration of interferon was 6 to 16 units/ml, as calculated by back-titration done on the same day. These mixtures were similarly incubated and assayed. In this case, the endpoint of the neutralization was defined as the highest dilution of serum which neutralized the given amount of interferon. Interferon was considered neutralized if greater than 50% of a control level of virus replicated after the incubation with the interferon. (In this system, as little as 6 units/ml interferon completely blocked virus plaque formation.) RESULTS AND DISCUSSION Data on antibody neutralization of interferons induced under various conditions and with different inducers are presented in Table 1. Newcastle disease virus (NDV)-induced L-cell interferon is probably fibroblast interferon (2), while

TABLE 1 Antibody Neutralization of Various Mouse Interferon+’ Interferon titer after incubation with Experiment No. 1

Source of interferon

Inducer

Medium

Preimmunization serum

Anti-immune interferon

Anti-L-cell interferon

L cells Peripheral leukocytes* Mouse serum Spleen cells’

NDV NDV

5000 60

5000 60

5ooo 55

300
LPS SEA

800 580

710 280

720 27


2

Spleen Spleen Spleen Spleen Spleen

SEA Con A PWM PHA Antigen

1055 520 520 400 2100

720 360 350 200 1620

40 40 33
Not done Not done 430 Not done Not done

3

Spleen cells Spleen cells from athymic nude mice

22 400

9 300

<2 25

10 500

cells cells cells cells cells

MLC SEA

a Interferon dilutions were mixed with equal volumes (0.2 ml) of (a) culture medium (Eagle’s medium with 2% fetal calf serum), (b) a 1:30 dilution of serum collected from a rabbit prior to first immunization, (c) a 1:30 dilution of anti-immune interferon from the same rabbit, or (d) a 160 dilution of anti-mouse L-cell interferon, obtained from NIAID, NIH, Bethesda Md. (One milliliter anti-L-cell interferon, undiluted, neutralizes 60,000 units of interferon.) After an hour of incubation at room temperature, the mixtures were added to mouse L 929 cells for interferon assay. b Peripheral leukocytes were from Swiss/Webster mice. c Unless otherwise specified, spleen cells were from C57Bl/6 mice.

68

OSBORNE,

GEORGIADES,

AND JOHNSON

interferon induced in mouse peripheral leukocytes stimulated with NDV may be leukocyte interferon, based on data from the human system (1). More definitive work needs to be done to clarify this issue. Interferon induced in mice by LPS and collected 2 hr after injection, as described in Table 1, is probably fibroblast interferon (8). Anti-L-cell interferon neutralized all three of these virw+type interferon preparations. The anti-immune interferon serum did not affect these interferons. Interferons induced in spleen cell cultures by SEA, Con A, PWM, and PHA-P were all specifically neutralized approximately IO-fold by a 1:30 dilution of anti-immune interferon (Table 1). Thus all of the mitogen-induced immune interferons were antigenically related. PWM-induced immune interferon was not neutralized by antibodies to L-cell interferon in our system, suggesting that essentially all of the interferon activity was immune. Earlier studies established that immune interferons induced with SEA, Con A, PHA-P, and specific antigen were not neutralized by antibodies to L-cell interferon (3, 6, 10). Immune interferon induced in mice infected with Mycobacterium bovis, strain BCG, and then challenged with old tuberculin was similarly neutralized by antibody to immune interferon. Antigen-induced immune interferon, then, is antigenically related to mitogen-induced immune interferon. Finally, interferon induced in a one-way MLC also appears to be antigenically related to these immune interferons since it was neutralized by anti-immune interferon. This confirms an earlier report which claimed that MLC-induced interferon was immune interferon based on other criteria (11). In an effort to determine whether immune interferons induced in various ways were neutralized equally well by anti-immune interferon, relatively constant amounts of various interferons were mixed with ditferent dilutions of the antiserum. The results are presented in Table 2. A I:20 dilution of preimmunization serum was unable to neutralize the 6 to 16 units/ml of interferon used in this test. The endpoint dilution of the anti-immune interferon which neutralized 6 to 8 units/ml of interferon was 1:80 for all interferons tested, whether induced in vitro with SEA, Con A, PWM, or PHA-P, or in vivo with specific antigen. When the level of interferon in the system was doubled, the endpoint of the antiserum was halved, thus establishing a linear relationship between the concentration of interferon and the amount of antiserum required to neutralize it. By extrapolation, we calculated that 1 ml of undiluted antiserum should neutralize 1070to 1280units of the various interferons. Since the neutralizing capacity of the serum was virtually the same for all immune interferons tested, it appears that all immune interferons are antigenically the same or at least very closely related. Interferon induced by SEA in spleen cells of athymic nude mice was neutralized by antibody to immune interferon (Table 1). It was shown previously that athymic nude mouse spleen cells were capable of producing immune interferon as effectively as spleen cells from thymus-bearing mice (10). We show here that this interferon is antigenically very similar if not the same as that from normal mice. Immune interferon produced by athymic nudes required lymphocytes with Thy-l markers on their surfaces, since treatment with anti-Thy- 1 and complement blocked PHA-P induction of interferon (10). It has recently been shown that the bone marrow and spleen of athymic nude mice contain a T cell that is an accessory (helper) cell required for development of cytotoxic lymphocytes (12). Certainly, more work

ANTIBODY

69

TO IMMUNE INTERFERON TABLE 2

Quantitation of Neutralization of Immune Interferon from Various Sources by Anti-Immune Interferon” Reciprocal of dilution at endpoint Interferon inducer

Units interferon/ml

Preimmunization serum

Anti-immune interferon

Units of interferon neutralized/ml of antiserum*

Antigen

8.0 16.0

<20 <20

80 40

1280 1280

SEA

7.5 15.0

<20 <20

80 40

1200 1200

PWM

8.0 16.0

<20 <20

80 40

1280 1280

PHA

6.8 13.5

<20 <20

80 40

1080

6.7 13.3

(20 <20

80 40

1070 1070

Con A

a Twofold dilutions (beginning with 1:20) of either preimmunization serum or anti-immune interferon were mixed with equal volumes (0.2 ml) of interferon. The final concentration of interferon was 6.7 to 16 units/ml, as calculated by back-titration done on the same day. The endpoint recorded is the reciprocal of the highest dilution of serum which neutralized the given amount of interferon. Interferon was considered neutralized if greater than 50% of a control level of virus replicated after incubation with the interferon. (In this system, no virus was capable of replicating in cells treated with as little as 6 units/ml interferon.) b Values are calculated by the formula: (interferon concentration x interferon volume x antiserum endpoint)/volume of antiserum added. The final volume of interferon in all experiments was 0.4 ml, and the volume of antiserum added to the system was 0.2 ml.

needs to be done to determine the cellular source of immune interferon in athymic nude mice. Because the interferon used to immunize the rabbits was not absolutely pure, there are undoubtedly antibodies to other molecules present. We know, however, that over 90% of the macrophage migration inhibitory factor activity had been depleted from the purified interferon used for immunization (13), and preliminary studies on antilymphotoxin activity in our antiserum suggest a lack of correlation between anti-immune interferon activity and antikilling activity. A 1:30 dilution of the antiserum which reduced the interferon titer from 1000 to only 40 units/ml reduced lymphotoxin activity from 186 to 136 units/ml (G. Trivers, J. A. Georgiades, L. C. Osborne, and H. M. Johnson, in preparation). If the antiserum contained antibodies to mouse cell receptors, it is conceivable that these antibodies could have blocked the action of interferon during the incubation of the interferon assay without neutralizing the interferon itself. The interferon used to immunize the rabbits was produced in mouse spleen cells, so it is possible that the rabbits received mouse cell receptors along with the interferon during the immunization. To rule out the presence of antireceptor antibodies, antiserum was incubated with mouse spleen cells and/or mouse L cells (the cells used in the interferon assay). After removal of the cells, the serum was used in a

70

OSBORNE,

GEORGIADES,

AND JOHNSON

neutralization test against SEA-induced interferon. The results with the absorbed antiserum were identical with those of Table 1. Therefore the data presented in Table 1 were not due to blockage of interferon action by antibody to interferon receptors. Treatment of serum with 33% saturated ammonium sulfate yields a precipitate which is composed primarily of immunoglobulins. The neutralizing activity of anti-immune interferon was completely precipitated by 33% saturated ammonium sulfate salt (data not shown). Although preimmunization serum had slight (twofold in some cases) neutralizing activity against immune interferon (Table l), a 33% salt precipitate of normal rabbit serum had no neutralizing activity. The salt precipitation data, then, distinguish between the specific neutralization by anti-immune interferon serum and the slight nonspecific inhibition by normal rabbit serum. This is the first report of antibody to mouse immune interferon. The data suggest that immune interferons produced under various conditions are antigenically the same or are closely related. We have confirmed the antigenic distinction of immune and virus-type interferons since antiserum to either type was unable to neutralize the other type. Data from athymic nude mice suggest that a T helper cell population is capable of producing immune interferon. This does not preclude the possibility that other T-cell populations are capable of producing immune interferon. These results should be relevant to elucidation of mechanisms of (a) protection against virus infection, (b) immunoregulation, and (c) antitumor effects, possibly mediated through interferon-induced natural killer cell mechanisms. REFERENCES 1. Havell, E. A., Berman, B., Ogbum, C. A., Berg, K., Paucker, K., and Vilcek, J., Proc. Nat. Acad. Sci. USA 72, 2185, 1975. 2. Maehara, N., Ho, M., and Armstrong, J. A., Infec. Immunity 17, 572, 1977. 3. Youngner, J. S., and Salvin, S. B., J. Immunol. 111, 1914, 1973. 4. Wheelock, E. F., Science 149, 310, 1965. 5. Johnson, H. M., Stanton, G. J., and Baron, S., Proc. Sot. hp. Biol. Med. 154, 138, 1977. 6. Johnson, H. M., and Baron, S., Cell. Immunol. 25, 106, 1976. 7. -Osborne, L. C., Georgiades, J. A., and Johnson, H. M., Znfec. Immunity 23, 80, 1979. 8. Maehara, N., and Ho, M., Infec. Immunity 15, 78, 1977. 9. Campbell, J. B., Gmnberger, T., Kochman, M. A., and White, S. L., Canad. J. Microbial. 21,1247, 1975.

10. Wietzerbin, J., Stefanos, S., Falcoff, R., Lucero, M., Catinot, L., and Falcoff, E., Idec. Immunity 21, %6, 1978.

11. Virelizier, J. L., Allison, A. C., and DeMaeyer, E., Znfec. Immunity 17, 282, 1977. 12. Jacobs, S. W., and Miller, R. B., J. Immunol. 122, 582, 1979. 13. Georgiades, J. A., Osborne, L. C., Moulton, R. G., and Johnson, H. M., Proc. Sot. Exp. Biol. Med. 161, 167, 1979.