- Email: [email protected]
Effect of rabbit interferon on immune responses
CELLULAR
12, 290-295 (1974)
IMMUNOLOGY
Effect G. J. Deportments
of Rabbit
A. E.
THORBECKE,
of Pathology,
Interferon
Dermatology, Medicine,
on Immune
FRIEDMAN-KIEN,
AND
and Microbiology, New York, New
Received October
Responses’
New York
J. York
VILEEK
University
School
of
30, 1973
Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes ~VZz&o nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon. It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.
INTRODUCTION Interferons have long been known as selective inhibitors of viral multiplication. More recently, it has become evident that interferons exert a number of additional biological activities. Of particular interest are the effects on various functions of lymphoid cells. Thus, Lindahl-Magnuson et al. (1) showed that interferon suppressed DNA synthesis induced in mouse lymphocytes by phytohemagglutinin or by allogeneic cells. It was also demonstrated that interferon enhanced the cytotoxic effect on target cells of normal (2, 3) or previously sensitized lymphocytes (4). On the other hand, the administration of interferon was recently shown to suppress the graft-vs.-host reaction (5) , and to prolong allograft survival in mice (6). In an older study, Mazzur and Paucker (7) failed to demonstrate an inhibitory effect of interferon on the ongoing antibody production in mouse spleen cells isolated from animals immunized in z&o with sheep red blood cells or a bacterial antigen. With the availability of highly potent preparations of rabbit interferon (8) and of a method of induction of antibody formation in rabbit lymph node cells in culture (9), it seemed worthwhile to reinvestigate the effect of interferon on antibody formation. The experiments described in the present communication show a lack of suppressionof antibody production, contrasting with the partial inhibition of DNA synthesis afforded by the same interferon preparation on rabbit lymph node cells stimulated with antigen. MATERIALS
AND
METHODS
Rabbit interferon was prepared in cultures of secondary rabbit kidney cells stimulated with polyinosinate-polycytidylate (poly 1.~01~ C) and “superinduced” 1 This investigation was supported AI-07057 from the USPHS.
in part by Grants AI-03076,
290 Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
CA-14462,
AI-10999,
and
INTERFERON
AND
IMMUNE
RESPONSES
291
with cycloheximide and actinomycin D (8). To remove poly I ’ poly C and the metabolic inhibitors, the cells were thoroughly washed prior to the addition of the interferon-production medium which consisted of serum-free Eagle’s minimum essential medium (MEM). Crude interferon was concentrated ZO-fold by ultrafiltration through a membrane filter (Diaflo UM 10, Amicon Corp.). The concentrate was dialyzed against MEM and sterilized by filtration through a 0.45-pm membrane filter (Millipore Corp.). The final preparation contained 1.5 X lo6 reference interferon units and 560 pg of protein per ml. A preparation of “mock” interferon was employed as control material. This material was prepared in rabbit kidney cell cultures that were treated in the same way as for the production of interferon, except that they had not been exposed to poly I*poly C. After concentration and dialysis, the mock interferon preparation contained less than 100 reference interferon units and 580 pg of protein per ml, A semi-micro-method described in detail elsewhere (10) was employed for all interferon titrations. Animals. Adult male New Zealand white rabbits were used. For studies on the secondary humoral immune response in vitro, animals were immunized with a single dose of 120 Lf diphtheria toxoid (DT) injected into the hind footpads (11). DT was obtained as a concentrated purified stock containing 1050 LF/ml through the courtesy of W. S. Hammond of Lederle Laboratories, Pearl River, NY (lot #60D-11). The popliteal lymph nodes were used for tissue culture 5-12 mo later. For studies on the proliferative response, rabbits were immunized with 0.5 mg dinitrophenylated bovine y globulin (DNP-BGG, 50 DNP groups per molecule) in complete Freund’s adjuvant into the four footpads and subcutaneously (12). Lymph node cells from these rabbits were used 3-7 mo later. Antibody production agahst sheep red blood cells in rabbits. Female rabbits weighing approximately 4 kg each were used. The rabbits were bled from the central ear artery prior to the onset of the experiment. All rabbits were injected intravenously with 0.5 ml of a 20% suspension of sheep erythrocytes (SE). Immediately following this injection, groups of three rabbits were injected iv with 750,000 reference units of interferon (in 0.5 ml) or with 0.5 ml of mock interferon. The same treatment was repeated daily for a period of 10 days. A third group of four immunized rabbits received neither interferon nor mock interferon. All rabbits were bled from the ear at the indicated intervals and individual serum samples were titrated for hemagglutinating antibodies. Tissue culture wethods for antibody production (9, 11). The popliteal lymph nodes were cut into l-Z-mm fragments and approximately 15 fragments of tissue were distributed over the walls of 16 X 125-mm screw-cap roller-type culture tubes, previously coated with normal rabbit plasma. To each culture tube was added 1 ml of modified MEM containing 25% normal rabbit serum. To tubes receiving antigen, 0.05 ml Hanks’ balanced salt solution containing 1 Lf DT was added and left with the tissue for 16 hr. Interferon was added in a volume of 0.050.1 ml to make final dilutions recorded in the tables. Within each experiment, duplicate or triplicate tubes were prepared for each assay. Culture tubes were kept in a 37°C incubator in a roller drum. Medium was replaced every l-3 days. The total culture period was 6-16 days. Aseptic technique was carefully observed throughout these studies. Preparation of cultures Ior proliferative response (12). Cell suspensions were prepared by teasing the popliteal lymph nodes and filtering the suspension through
292
THORBECKE,
FRIEDMAN-KIEN
AND
VILEEK
gauze. Cells were cultured at 3-4 x 10” cell per 1 ml of Dubecco’s medium, containing 10% normal, heat-inactivated rabbit serum and either “mock” interferon or interferon in the desired concentration, under a 5% CO,--957; air atmosphere. Antigen additions to the culture consisted of 100 pg DNP-BGG. Phytohemagglutinin-P (PHA) from Difco Laboratories was added at 1 ~1 per culture. One microcurie of tritiated thymidine (Schwartz Bio-Research, Inc., Orangeburg, NY ; specific activity 0.36 Ci/mM) was added after 24 or 48 hr of culture and the cells were harvested 24 hr later. Thymidine incorporation was measured as previously described ( 12). Antibody titrations. Culture fluids and sera were stored at -20°C until they were titrated for antibody. Anti-DT agglutination titers were determined using sensitized tanned SE (13)) and anti-SE antibodies were measured by simple hemagglutination (doubling dilutions). RESULTS Effect of interferon on the antibody production against sheep red blood cells. Daily administration of 750,000 units of interferon had no striking effect on the average peak antibody titers against SE in the rabbit sera. The initial antibody rise was somewhat faster in the interferon-treated animals than in the “mock” interferon-treated or control groups. However, the maximum levels attained and the rates of decline of the antibody titers were similar in all three groups (Fig. 1). The reason for the earlier appearance of antibodies in the group of rabbits treated with interferon is not clear. It could have been caused by interferon itself, but it cannot be ruled out that this effect was due to the presence of very small quantities of poly I *poly C which might have been present in the interferon preparation. It is known that poly 1.~01~ C can enhance antibody production (14). Contamination with poly 1.~01~ C might have resulted from the release of very small quantities of the polynucleotide inducer from the cells into the production medium (see Materials and Methods), although it is known that only a small fraction (about 1%) of the polynucleotide added to cultures becomescell-associated and that most of the cell-bound poly I *poly C is rapidly degraded (15, 16).
FIG. 1. Anti-sheep erythrocyte antibody formation in rabbits treated with interferon. Animals received 10 consecutive daily injections of interferon (0), “mock” interferon (O), or no treatment (A). ‘For details of procedure see Materials and Methods.
INTERFERON
AND
IMMUNE
TABLE LACK
OF EFFECT
OF INTERFERON
RESPONSE
OF RABBIT
1
(IF)
ON THE SECONDARY
LYMPH-NODE
Expt.
IF
FRAGMENTS
Log,
Additive to culture medium
None “Mock”
ANTIBODY in vitro”
reciprocal antibody in culture fluidsb Expt.
1
14.5 15.2 14.2 16.3
IF l/24 l/100 l/24 l/100 1/1000
293
RESPONSES
titers
2
Expt.
18.8 19.0
13.5 13.3
17.6 17.8
13.8
3
5 Immunization of rabbits was by footpad injection of 200Lf DT on Days -95 (Expt. l), -150 (Expt. Z), and -205 (Expt. 3). Reexposure to 1Lf DT/ml for 16 hr was used to induce secondary response in e&o. IF or “mock” IF was added to the medium immediately after removal of DT (Expts. 1 and 2) or together with DT (Expt. 3). IF or “mock” IF was also added to each medium change and kept in the cultures throughout the duration of the experiments. b Given as mean of 6 passive hemagglutinin titers of three successive medium changes taken from duplicate cultures at intervals of 1 (Expt. 3) or 3 (Expts. 1 and 2) days.
E$ect of interferon on antibody forwzeti5n dwing the secondary response in vitro. The results in Table 1 represent three separate experiments in which various concentrations of interferon were added at each medium change. “Mock” interferon as well as the interferon preparation itself showed nonspecific toxicity when used at dilutions lower than l/24. In one experiment, not represented in the table, a l/5 dilution of both the “mock” interferon and interferon preparations reduced the control log2 peak titer in media taken on Days 6 and 9 from 20.0 to 9.5 and 9.8, respectively. It can be seen from the table that, at final dilutions of l/24 or higher, no significant effect was observed. Cultures to which antigen had not been added initially had log2 peak titers of 3.8-5.0, demonstrating that most of the antibody in the culture fluids had indeed been formed in response to the secondary exposure to diphtheria toxoid. Figure 2 illustrates that the rate of antibody production in the cultures was also unaffected by the presence of interferon throughout the culture period. In this
FIG. 2. Anti-diphtheria toxoid titers tissue producing a secondary response preparation of interferon (0).
Days
of Culture
in culture in vitro
fluids in the
removed presence
daily from of interferon
rabbit (0)
lymph node or “mock”
294
THORBECKE,
FRIEDMAN-KIEN
TABLE
AND
VILtEK
2
DITPKESSIVE EFFECT OF INTERFERON (IF) ON THE PROLIIWI~ATIW RESPONSE OF RABBIT LYMPH-NODE CELLS TO DNP-BGG Additives to culture mediuma
CPM Expt.
Antigen + “Mock” IF No antigen + “Mock” IF Antigen + IF No antigen + IF
22,893 f 2889 f 11,283 f 2671 f
4 1708 358 917 466
per cultureb Expt. 49,762 3540 32,720 2902
5 I!= f f f
3132 626 4376 476
a IF and “mock” IF dilutions were l/100 in Expt. 4 and l/20 in Expt. 5. The antigen was 100 fig DNP-BGG per ml. b Thymidine, 1 pc per ml, added after 24 hr (Expt. 4) or 48 hr (Expt. 5) of culture period. Cells were harvested 24 hr later. Means were calculated from three to four values; P value for difference between “mock” IF and IF in Expt. 4 is
experiment (Expt 3, Table 1) the interferon titers were determined in the culture fluids collected from tubes to which interferon had been added 24 hr previously in a concentration of 15,000 units/ml. Between 5000 and 17,000 units/ml were recovered in the culture media, indicating that little or no inactivation of interferon had occurred in the cultures during 24 hr of incubation. Antigen addition to this amount of lymph node tissue in vitro did not induce a detectable interferon titer in the medium. Effect of interferon on. the proliferative response in vitro. The results in Table 2 demonstrate that interferon in final concentrations of l/20-1/100 partially suppressed the proliferative response to DNP-BGG. In Expt 4 there was approximately 50% (P < 0.01) inhibition and in Expt 5 about 35% (P < 0.05). The response to PHA was examined simultaneously in Expt 4 and was found to be inhibited to only a very slight degree (14%, not in the table). A low titer of interferon-like activity was induced in the cell suspensionsby exposure to DNP-BGG which was not found in supernatants of cells cultured without antigen. DISCUSSION The inhibitory effect of interferon on the proliferative response to antigen in vitro seen in the present results with rabbit lymph node cells is in agreement with the findings of others, showing depression of mouse spleen cell responsesto transplantation antigens and PHA (1) and to concanavalin A (17). The degree of inhibition in our experiments was much less than that seen by Lindahl-Magnusson et al. (1). This was not due to a greater resistance to inhibition of the responseto antigen, since in an experiment in which the responsesto antigen and PHA were compared, the response to PHA was even less inhibited than the one to antigen. Preliminary findings suggest that a very high concentration of human interferon, when added to similar rabbit lymph-node cultures, produced a comparable degree of depression of antigen-induced DNA synthesis. Human interferon is known to have antiviral activity in rabbit cells (18). The complete lack of an inhibitory effect on both the primary antibody response in vivo and the secondary response in vitro is in apparent contradiction with the
INTERFERON
AND
IMMUNE
RESPONSES
29.5
effect on the proliferative response. While the interferon was present throughout the whole response in vitro, including the initial phase in which cellular proliferation is known to be required (19, 20)) not even a delay in appearance of antibody in the culture fluids was seen. The responseswhich have been shown to be inhibited by interferon are known to be primarily T lymphocyte functions. The secondary response in vitro, however, while probably requiring some T cell activity, is likely to be relatively independent of T cell proliferation and, therefore, a partial inhibition of this activity may well go completely unnoticed in this system. These findings do not rule out the possibility that a strongly T-dependent humoral immune response might be subject to inhibition by interferon. It has been shown that proliferation of a number of different cell types, including nonlymphoid cells (21, 22), is partially inhibited by interferon. Thus, it may well be that the action of interferon on the proliferation of lymphoid cells seen in the present studies is an expression of its general ability to depress cell growth. ACKNOWLEDGMENTS The excellent technical assistance of M. Bell, P. Sanchez, and E. Brady is gratefully knowledged.
ac-
REFERENCES 1. Lindahl-Magnusson, P., Leary, P., and Gresser, I., Nature (New BioZ.) 237, 120, 1972. ‘2. Svet-Moldavsky, G. J., and Chernyakovskaya, I. Yu., Nature (London) 215, 1299, 1967. 3. Boreckjr, L., Lackovie, V., and Waschke, K., In “Developmental Aspects of Antibody Formation” (J. Sterzl and I. Riha, Eds.), p. 745. Academia Publishing House, Prague, 1970. 4. Lindahl, P., Leary, P., and Gresser, I., Proc. Nab. Acad. Sci. USA 69, 721, 1972. 5. Hirsch, M. S., Ellis, D. A., Proffitt, M. R., Black, P. H., and Chirigos, M. A., Nature (New Biol.) 244, 102, 1973. 6. Mobraaten, L. E., DeMaeyer, E., and De Maeyer-Guignard, J., personal communication, August, 1973. 7. Mazzur, S. R., and Paucker, K., J. Immunol. 98, 689, 1967. 8. VilEek, J., and Ng, M. H., J. Viral. 7, 588, 1971. 9. Michaelides, M. C., and Coons, A. H., J. Exp. Med. 117, 1035, 1963. 10. Vilfek, J., Barmak, S. L., and Have& E. A., J. Vkol. 10, 614, 1972. 11. Jacobson,E. B., and Thorbecke,G. J., J. Exp. Med. 130, 287, 1969. 12. Thorbecke, G. J., and Siskind, G. W., 1. Immsnol. 110, 648, 1973. 13. Stavitsky, A. B., J. Zmmunol. 72, 360, 1954. 14. Woodhour, A. F., Friedman, A., Tytell, A. A., and Hilleman, M. R., Proc. Sot. Exp. Biol. Med. 131, 809, 1969. 15. Bausek, G. H., and Merigan, T. C., Virology 39, 491, 1969. 16. Kelly, T. A., and Levy, H. B., personal communication, June 1973. 17. Lee, S. H. S., Ngan, J., O’Shaugnessy, M. V., and Rozee, K. R., personal communication, December 1972. 18. Desmyter, J., Rawls, W. E., and Melnick, J. L., Proc. Nat. Acad. Sci. USA 59, 69, 1968. 19. O’Brien, T. F., and Coons, A. H., J. Exp. Med. 117, 1063, 1963. 20. Stecher, V. J., and Thorbecke, G. J., J. Exp. Med. 125, 33, 1967. 21. Paucker, K., Cantell, K., and Henle, W., Virology 17, 324, 1962. 22. Gresser, I., Brouty-Boy&, D., Thomas, M. T., and Macieira-Coelho, A., Proc. Nat. Actrd. Sci. USA 66, 1052, 1970.
12, 290-295 (1974)
IMMUNOLOGY
Effect G. J. Deportments
of Rabbit
A. E.
THORBECKE,
of Pathology,
Interferon
Dermatology, Medicine,
on Immune
FRIEDMAN-KIEN,
AND
and Microbiology, New York, New
Received October
Responses’
New York
J. York
VILEEK
University
School
of
30, 1973
Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes ~VZz&o nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon. It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.
INTRODUCTION Interferons have long been known as selective inhibitors of viral multiplication. More recently, it has become evident that interferons exert a number of additional biological activities. Of particular interest are the effects on various functions of lymphoid cells. Thus, Lindahl-Magnuson et al. (1) showed that interferon suppressed DNA synthesis induced in mouse lymphocytes by phytohemagglutinin or by allogeneic cells. It was also demonstrated that interferon enhanced the cytotoxic effect on target cells of normal (2, 3) or previously sensitized lymphocytes (4). On the other hand, the administration of interferon was recently shown to suppress the graft-vs.-host reaction (5) , and to prolong allograft survival in mice (6). In an older study, Mazzur and Paucker (7) failed to demonstrate an inhibitory effect of interferon on the ongoing antibody production in mouse spleen cells isolated from animals immunized in z&o with sheep red blood cells or a bacterial antigen. With the availability of highly potent preparations of rabbit interferon (8) and of a method of induction of antibody formation in rabbit lymph node cells in culture (9), it seemed worthwhile to reinvestigate the effect of interferon on antibody formation. The experiments described in the present communication show a lack of suppressionof antibody production, contrasting with the partial inhibition of DNA synthesis afforded by the same interferon preparation on rabbit lymph node cells stimulated with antigen. MATERIALS
AND
METHODS
Rabbit interferon was prepared in cultures of secondary rabbit kidney cells stimulated with polyinosinate-polycytidylate (poly 1.~01~ C) and “superinduced” 1 This investigation was supported AI-07057 from the USPHS.
in part by Grants AI-03076,
290 Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
Inc. reserved.
CA-14462,
AI-10999,
and
INTERFERON
AND
IMMUNE
RESPONSES
291
with cycloheximide and actinomycin D (8). To remove poly I ’ poly C and the metabolic inhibitors, the cells were thoroughly washed prior to the addition of the interferon-production medium which consisted of serum-free Eagle’s minimum essential medium (MEM). Crude interferon was concentrated ZO-fold by ultrafiltration through a membrane filter (Diaflo UM 10, Amicon Corp.). The concentrate was dialyzed against MEM and sterilized by filtration through a 0.45-pm membrane filter (Millipore Corp.). The final preparation contained 1.5 X lo6 reference interferon units and 560 pg of protein per ml. A preparation of “mock” interferon was employed as control material. This material was prepared in rabbit kidney cell cultures that were treated in the same way as for the production of interferon, except that they had not been exposed to poly I*poly C. After concentration and dialysis, the mock interferon preparation contained less than 100 reference interferon units and 580 pg of protein per ml, A semi-micro-method described in detail elsewhere (10) was employed for all interferon titrations. Animals. Adult male New Zealand white rabbits were used. For studies on the secondary humoral immune response in vitro, animals were immunized with a single dose of 120 Lf diphtheria toxoid (DT) injected into the hind footpads (11). DT was obtained as a concentrated purified stock containing 1050 LF/ml through the courtesy of W. S. Hammond of Lederle Laboratories, Pearl River, NY (lot #60D-11). The popliteal lymph nodes were used for tissue culture 5-12 mo later. For studies on the proliferative response, rabbits were immunized with 0.5 mg dinitrophenylated bovine y globulin (DNP-BGG, 50 DNP groups per molecule) in complete Freund’s adjuvant into the four footpads and subcutaneously (12). Lymph node cells from these rabbits were used 3-7 mo later. Antibody production agahst sheep red blood cells in rabbits. Female rabbits weighing approximately 4 kg each were used. The rabbits were bled from the central ear artery prior to the onset of the experiment. All rabbits were injected intravenously with 0.5 ml of a 20% suspension of sheep erythrocytes (SE). Immediately following this injection, groups of three rabbits were injected iv with 750,000 reference units of interferon (in 0.5 ml) or with 0.5 ml of mock interferon. The same treatment was repeated daily for a period of 10 days. A third group of four immunized rabbits received neither interferon nor mock interferon. All rabbits were bled from the ear at the indicated intervals and individual serum samples were titrated for hemagglutinating antibodies. Tissue culture wethods for antibody production (9, 11). The popliteal lymph nodes were cut into l-Z-mm fragments and approximately 15 fragments of tissue were distributed over the walls of 16 X 125-mm screw-cap roller-type culture tubes, previously coated with normal rabbit plasma. To each culture tube was added 1 ml of modified MEM containing 25% normal rabbit serum. To tubes receiving antigen, 0.05 ml Hanks’ balanced salt solution containing 1 Lf DT was added and left with the tissue for 16 hr. Interferon was added in a volume of 0.050.1 ml to make final dilutions recorded in the tables. Within each experiment, duplicate or triplicate tubes were prepared for each assay. Culture tubes were kept in a 37°C incubator in a roller drum. Medium was replaced every l-3 days. The total culture period was 6-16 days. Aseptic technique was carefully observed throughout these studies. Preparation of cultures Ior proliferative response (12). Cell suspensions were prepared by teasing the popliteal lymph nodes and filtering the suspension through
292
THORBECKE,
FRIEDMAN-KIEN
AND
VILEEK
gauze. Cells were cultured at 3-4 x 10” cell per 1 ml of Dubecco’s medium, containing 10% normal, heat-inactivated rabbit serum and either “mock” interferon or interferon in the desired concentration, under a 5% CO,--957; air atmosphere. Antigen additions to the culture consisted of 100 pg DNP-BGG. Phytohemagglutinin-P (PHA) from Difco Laboratories was added at 1 ~1 per culture. One microcurie of tritiated thymidine (Schwartz Bio-Research, Inc., Orangeburg, NY ; specific activity 0.36 Ci/mM) was added after 24 or 48 hr of culture and the cells were harvested 24 hr later. Thymidine incorporation was measured as previously described ( 12). Antibody titrations. Culture fluids and sera were stored at -20°C until they were titrated for antibody. Anti-DT agglutination titers were determined using sensitized tanned SE (13)) and anti-SE antibodies were measured by simple hemagglutination (doubling dilutions). RESULTS Effect of interferon on the antibody production against sheep red blood cells. Daily administration of 750,000 units of interferon had no striking effect on the average peak antibody titers against SE in the rabbit sera. The initial antibody rise was somewhat faster in the interferon-treated animals than in the “mock” interferon-treated or control groups. However, the maximum levels attained and the rates of decline of the antibody titers were similar in all three groups (Fig. 1). The reason for the earlier appearance of antibodies in the group of rabbits treated with interferon is not clear. It could have been caused by interferon itself, but it cannot be ruled out that this effect was due to the presence of very small quantities of poly I *poly C which might have been present in the interferon preparation. It is known that poly 1.~01~ C can enhance antibody production (14). Contamination with poly 1.~01~ C might have resulted from the release of very small quantities of the polynucleotide inducer from the cells into the production medium (see Materials and Methods), although it is known that only a small fraction (about 1%) of the polynucleotide added to cultures becomescell-associated and that most of the cell-bound poly I *poly C is rapidly degraded (15, 16).
FIG. 1. Anti-sheep erythrocyte antibody formation in rabbits treated with interferon. Animals received 10 consecutive daily injections of interferon (0), “mock” interferon (O), or no treatment (A). ‘For details of procedure see Materials and Methods.
INTERFERON
AND
IMMUNE
TABLE LACK
OF EFFECT
OF INTERFERON
RESPONSE
OF RABBIT
1
(IF)
ON THE SECONDARY
LYMPH-NODE
Expt.
IF
FRAGMENTS
Log,
Additive to culture medium
None “Mock”
ANTIBODY in vitro”
reciprocal antibody in culture fluidsb Expt.
1
14.5 15.2 14.2 16.3
IF l/24 l/100 l/24 l/100 1/1000
293
RESPONSES
titers
2
Expt.
18.8 19.0
13.5 13.3
17.6 17.8
13.8
3
5 Immunization of rabbits was by footpad injection of 200Lf DT on Days -95 (Expt. l), -150 (Expt. Z), and -205 (Expt. 3). Reexposure to 1Lf DT/ml for 16 hr was used to induce secondary response in e&o. IF or “mock” IF was added to the medium immediately after removal of DT (Expts. 1 and 2) or together with DT (Expt. 3). IF or “mock” IF was also added to each medium change and kept in the cultures throughout the duration of the experiments. b Given as mean of 6 passive hemagglutinin titers of three successive medium changes taken from duplicate cultures at intervals of 1 (Expt. 3) or 3 (Expts. 1 and 2) days.
E$ect of interferon on antibody forwzeti5n dwing the secondary response in vitro. The results in Table 1 represent three separate experiments in which various concentrations of interferon were added at each medium change. “Mock” interferon as well as the interferon preparation itself showed nonspecific toxicity when used at dilutions lower than l/24. In one experiment, not represented in the table, a l/5 dilution of both the “mock” interferon and interferon preparations reduced the control log2 peak titer in media taken on Days 6 and 9 from 20.0 to 9.5 and 9.8, respectively. It can be seen from the table that, at final dilutions of l/24 or higher, no significant effect was observed. Cultures to which antigen had not been added initially had log2 peak titers of 3.8-5.0, demonstrating that most of the antibody in the culture fluids had indeed been formed in response to the secondary exposure to diphtheria toxoid. Figure 2 illustrates that the rate of antibody production in the cultures was also unaffected by the presence of interferon throughout the culture period. In this
FIG. 2. Anti-diphtheria toxoid titers tissue producing a secondary response preparation of interferon (0).
Days
of Culture
in culture in vitro
fluids in the
removed presence
daily from of interferon
rabbit (0)
lymph node or “mock”
294
THORBECKE,
FRIEDMAN-KIEN
TABLE
AND
VILtEK
2
DITPKESSIVE EFFECT OF INTERFERON (IF) ON THE PROLIIWI~ATIW RESPONSE OF RABBIT LYMPH-NODE CELLS TO DNP-BGG Additives to culture mediuma
CPM Expt.
Antigen + “Mock” IF No antigen + “Mock” IF Antigen + IF No antigen + IF
22,893 f 2889 f 11,283 f 2671 f
4 1708 358 917 466
per cultureb Expt. 49,762 3540 32,720 2902
5 I!= f f f
3132 626 4376 476
a IF and “mock” IF dilutions were l/100 in Expt. 4 and l/20 in Expt. 5. The antigen was 100 fig DNP-BGG per ml. b Thymidine, 1 pc per ml, added after 24 hr (Expt. 4) or 48 hr (Expt. 5) of culture period. Cells were harvested 24 hr later. Means were calculated from three to four values; P value for difference between “mock” IF and IF in Expt. 4 is
experiment (Expt 3, Table 1) the interferon titers were determined in the culture fluids collected from tubes to which interferon had been added 24 hr previously in a concentration of 15,000 units/ml. Between 5000 and 17,000 units/ml were recovered in the culture media, indicating that little or no inactivation of interferon had occurred in the cultures during 24 hr of incubation. Antigen addition to this amount of lymph node tissue in vitro did not induce a detectable interferon titer in the medium. Effect of interferon on. the proliferative response in vitro. The results in Table 2 demonstrate that interferon in final concentrations of l/20-1/100 partially suppressed the proliferative response to DNP-BGG. In Expt 4 there was approximately 50% (P < 0.01) inhibition and in Expt 5 about 35% (P < 0.05). The response to PHA was examined simultaneously in Expt 4 and was found to be inhibited to only a very slight degree (14%, not in the table). A low titer of interferon-like activity was induced in the cell suspensionsby exposure to DNP-BGG which was not found in supernatants of cells cultured without antigen. DISCUSSION The inhibitory effect of interferon on the proliferative response to antigen in vitro seen in the present results with rabbit lymph node cells is in agreement with the findings of others, showing depression of mouse spleen cell responsesto transplantation antigens and PHA (1) and to concanavalin A (17). The degree of inhibition in our experiments was much less than that seen by Lindahl-Magnusson et al. (1). This was not due to a greater resistance to inhibition of the responseto antigen, since in an experiment in which the responsesto antigen and PHA were compared, the response to PHA was even less inhibited than the one to antigen. Preliminary findings suggest that a very high concentration of human interferon, when added to similar rabbit lymph-node cultures, produced a comparable degree of depression of antigen-induced DNA synthesis. Human interferon is known to have antiviral activity in rabbit cells (18). The complete lack of an inhibitory effect on both the primary antibody response in vivo and the secondary response in vitro is in apparent contradiction with the
INTERFERON
AND
IMMUNE
RESPONSES
29.5
effect on the proliferative response. While the interferon was present throughout the whole response in vitro, including the initial phase in which cellular proliferation is known to be required (19, 20)) not even a delay in appearance of antibody in the culture fluids was seen. The responseswhich have been shown to be inhibited by interferon are known to be primarily T lymphocyte functions. The secondary response in vitro, however, while probably requiring some T cell activity, is likely to be relatively independent of T cell proliferation and, therefore, a partial inhibition of this activity may well go completely unnoticed in this system. These findings do not rule out the possibility that a strongly T-dependent humoral immune response might be subject to inhibition by interferon. It has been shown that proliferation of a number of different cell types, including nonlymphoid cells (21, 22), is partially inhibited by interferon. Thus, it may well be that the action of interferon on the proliferation of lymphoid cells seen in the present studies is an expression of its general ability to depress cell growth. ACKNOWLEDGMENTS The excellent technical assistance of M. Bell, P. Sanchez, and E. Brady is gratefully knowledged.
ac-
REFERENCES 1. Lindahl-Magnusson, P., Leary, P., and Gresser, I., Nature (New BioZ.) 237, 120, 1972. ‘2. Svet-Moldavsky, G. J., and Chernyakovskaya, I. Yu., Nature (London) 215, 1299, 1967. 3. Boreckjr, L., Lackovie, V., and Waschke, K., In “Developmental Aspects of Antibody Formation” (J. Sterzl and I. Riha, Eds.), p. 745. Academia Publishing House, Prague, 1970. 4. Lindahl, P., Leary, P., and Gresser, I., Proc. Nab. Acad. Sci. USA 69, 721, 1972. 5. Hirsch, M. S., Ellis, D. A., Proffitt, M. R., Black, P. H., and Chirigos, M. A., Nature (New Biol.) 244, 102, 1973. 6. Mobraaten, L. E., DeMaeyer, E., and De Maeyer-Guignard, J., personal communication, August, 1973. 7. Mazzur, S. R., and Paucker, K., J. Immunol. 98, 689, 1967. 8. VilEek, J., and Ng, M. H., J. Viral. 7, 588, 1971. 9. Michaelides, M. C., and Coons, A. H., J. Exp. Med. 117, 1035, 1963. 10. Vilfek, J., Barmak, S. L., and Have& E. A., J. Vkol. 10, 614, 1972. 11. Jacobson,E. B., and Thorbecke,G. J., J. Exp. Med. 130, 287, 1969. 12. Thorbecke, G. J., and Siskind, G. W., 1. Immsnol. 110, 648, 1973. 13. Stavitsky, A. B., J. Zmmunol. 72, 360, 1954. 14. Woodhour, A. F., Friedman, A., Tytell, A. A., and Hilleman, M. R., Proc. Sot. Exp. Biol. Med. 131, 809, 1969. 15. Bausek, G. H., and Merigan, T. C., Virology 39, 491, 1969. 16. Kelly, T. A., and Levy, H. B., personal communication, June 1973. 17. Lee, S. H. S., Ngan, J., O’Shaugnessy, M. V., and Rozee, K. R., personal communication, December 1972. 18. Desmyter, J., Rawls, W. E., and Melnick, J. L., Proc. Nat. Acad. Sci. USA 59, 69, 1968. 19. O’Brien, T. F., and Coons, A. H., J. Exp. Med. 117, 1063, 1963. 20. Stecher, V. J., and Thorbecke, G. J., J. Exp. Med. 125, 33, 1967. 21. Paucker, K., Cantell, K., and Henle, W., Virology 17, 324, 1962. 22. Gresser, I., Brouty-Boy&, D., Thomas, M. T., and Macieira-Coelho, A., Proc. Nat. Actrd. Sci. USA 66, 1052, 1970.