Transfer Agent of Immunity. VI. Serial Passive Transfers of Cellular Immunity to Salmonella Infection by Immune Ribonucleic Acid

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1 Japan. J. Microbiol. Vol. 15 (2), , 1971 Transfer Agent of Immunity VI. Serial Passive Transfers of Cellular Immunity to Salmonella Infection by Immune Ribonucleic Acid Kazuko SAITO, Nobutaka OSAWA, and Susumu MITSUHASHI Department of Microbiology, School of Medicine, Gunma University, Maebashi (Received for publication, September 5, 1970) ABSTRACT An immune ribonucleic acid (RNA) was extractable from the spleen cells of mice hyperimmunized with live vaccine of Salmonella enteritidis. The RNA was capable of inducing cellular immunity and developing cellular antibody in the peritoneal macrophages of mice injected with this agent. It was found that cellular immunity was detectable even 90 days after injection in the peritoneal macrophages of mice which had received an intraperitoneal injection with this agent. Results of serial passive transfers of cellular immunity through immune RNA led us to the conclusion that this agent does not contain antigen or fragment thereof and may replicate actively in the recipient cells, although the mechanism still remains to be elucidated. The development of cellular immunity by immune RNA was inhibited by puromycin but not by actinomycin D. However, serial passive transfers of cellular immunity through immune RNA was inhibited by treatment of recipient mouse with actinomycin D, implying the role of DNA-dependent RNA polymerase in the processing of immune RNA in recipient cells. Using these results, the role of immune RNA and the possible mechanisms of immune RNA replication are discussed. In preceding papers, it was indicated that mice hyperimmunized with live vaccine of Salmonella enteritidis or mice recovering from a near fatal infection with a virulent strain of S. enteritidis with the aid of antibiotics acquired high resistance against further infection with the same organism [11, 13, 20], and the mononuclear phagocytes (referred to as macrophages) of immunized mice inhibited intracellular multiplication of virulent strain in the absence of antibody in the cell culture medium [17]. A cell-associated antibody was detected in the immunized macrophages by use of immune transfer and immune adherence hemagglutination (IAHA) [10, 12]. This type of cellular antibody was detected in the immunized macrophages after absorption with killed vaccine but not detected in the macrophages of mice immunized with killed vaccine of S. enteritidis [10]. Hence, this type of macrophage immunity was referred to as cellular immunity [17]. It was also found that cellular immunity was transferable, from immune to nonimmune macrophages, through the transfer agent (TA) of ribonucleic acid (RNA) nature [15, 16, 23, 24]. When peritoneal macrophages were treated in vivo or in vitro with immune RNA, they developed cellular immunity and cellular antibody 159

2 160 K, SAITO, N. OSAWA AND S. MITSUHASHI [15, 25]. In the present article, the properties of immune RNA, extracted with phenol from the spleen cells of immunized mice, are presented, and the role of immune RNA in antibody formation is demonstrated. MATERIALS AND METHODS Experimental animals. Five-to 7-weekold ddn strain mice of both sexes (raised by the Central Animal Laboratory, Gunma University) weighing 20 to 25g were used. an equal volume of phenol solution. The RNA was then precipitated with 2 volumes of cold absolute ethanol. The RNA was collected by centrifugation at 3000 ~g for 15min, washed twice with 30min of 70% ethanol and suspended in physiological saline. Normal RNA was similarly extracted from the spleen cells of normal mice instead of immune mice. All procedures were performed in a cold room or in an ice bath. The amount of RNA extracted from a spleen of immune mouse was usually 1.5 Organism. Organisms used were a virulent strain and an attenuated strain to 2mg. SER, of Salmonella enteritidis. The bacteria were cultured on agar plates or in nutrient broth for 18 hr at 37C, aerobically. Immunization. Immunization of mice with a live vaccine was reported previously [11, 13]. Briefly, mice were injected intravenously with 10-5mg (dry weight) of a live vaccine of an attenuated strain SER and 21 days later, with 10-7mg of virulent strain of S. enteritidis. Immunized mice were used 2 months after the last injection. Preparation of transfer agent (immune RNA). The RNA was extracted with phenol from the spleen cells of immunized mice according to the method described by Kidson et al. [6]. Seven to 8 spleens of immunized mice were homogenized with homoblender (Sakuma Seisakusho Ltd., Tokyo, Japan) in 10ml of 0.005M Tris- HCl buffer at 7.4 containing 0.025M KCl, 0.005M MgC12 and 0.005M disodium naphthalene-1, 5-disulfonate (Wako Pure Chem. Industries, Osaka, Japan). The homogenized spleen cells were shaken for 15min Cellular immunity. Peritoneal macrophages were collected from the abdominal cavity of mice treated with RNA preparations 5 days before the collection. Cellular immunity was examined by infecting them in vitro with a virulent strain Tissue culture of macrophages, infection of bacteria and observation of macrophages after infection were reported previously [17, 23]. Serial passive transfers of cellular immunity. The original immune RNA was extracted with phenol from the spleen cells of mouse hyperimmunized with live vaccine of S. enteritidis and used as immune RNA for the first transfer. Cellular immunity in the peritoneal macrophages of the first recipient mouse was examined 5 days after intraperitoneal injection with 1mg of immune RNA and successively the RNA was extracted from the spleen cells of the same mouse which served as the second donor of RNA preparation. Similarly, the second recipient mouse was injected intraperitoneally with 1mg of this RNA preparation extracted from the spleen cells of the first recipient mouse (second donor of with an equal volume of 90% redistilled RNA preparation). phenol and 10% Tris-HCI buffer (ph 7.4) containing 0.1% 8-hydroxyquinoline. An aqueous phase was obtained by centrifugation at 6000 ~g for 10min. The aqueous phase was extracted 3 times for 20min with Assay of cellular antibody. Immune transfer and immune adherence hemagglutination [18, 19] were used for titration of cellular antibody. Briefly, peritoneal exudate cells were collected 5 days after treat-

3 TRANSFER AGENT OF IMMUNITY 161 ment with RNA preparation and serial two-fold dilutions of cell suspension in gelatin-veronal buffer (GVB++) [10] were prepared. Similarly, normal cell suspensions were prepared from the peritoneal cavity of normal mice instead of mice treated with the RNA preparation. Equal volumes of peritoneal cell suspension and bacterial suspension (1 ~108 organisms of S. enteritidis ) were incubated at 30C for 60min in GVB++. The incubated mixture was centrifuged at 2600 ~g for 5min to remove peritoneal exudate cells and the supernatant containing sensitized bacteria was further centrifuged for 10min at ~g. The sensitized bacteria thus obtained were washed twice by centrifugation with Tris-veronal buffer (TVB++) [10] and suspended in TVB++ to make a suspension of 1 ~108 organisms/ml. Eight tenths ml of sensitized bacteria was incubated with 0.2 ml of 10 times diluted complement (2 units of complement). After 15min of incubation at 37C with shaking, 0.1ml of human erythrocytes was added and shaken for 10 min. After 60min of incubation, the pattern of sedimented erythrocytes was read. For the determination of cellular antibody after absorption with a killed vaccine of , equal volumes of heat killed vaccines of (5 ~109 organisms/ml) and macrophages (108/ml) were mixed and kept in a water bath at 30C. After 30min of incubation, macrophages were collected by centrifugation at 2600 ~g for 5min, washed twice with GVB++ and suspended in the same buffer. In vitro treatment of peritoneal macrophages with immune RNA. Macrophages were obtained from peritoneal cavity of normal mice as described previously [17, 23]. They were washed twice with Hanks' basal salt solution by centrifugation at 335 ~g for 5min and inoculated into culture bottle containing 70% of Hanks' basal salt solution, 30% of horse serum and 10 units/ml of penicillin. After 20hr of incubation, macrophages adhered to the bottom of the culture bottle and tissue culture medium was exchanged with fresh culture medium containing immune RNA (100 ƒê g/ml). Treatment with metabolic inhibitors. Actinomycin D (AD) was obtained through the courtesy of Merck, Sharp & Dohme Research Lab., Rahway, N.J., U.S.A. It was found that LD50 of AD was 10ƒÊg for a mouse weighing about 20 to 25g. To test the effect of AD, each mouse received an intraperitoneal injection with 5ƒÊg of AD one day before the treatment with immune RNA. The macrophages prepared from the peritoneal cavity of normal mice were cultured by the techniques previously described. After 20hr of cultivation, macrophages adhered to the culture bottle. Tissue culture medium was removed and exchanged with the medium containing various concentrations of AD and cytotoxic effect was examined microscopically by observation of the rounding and exfoliation of the cells. The results indicated that the maximum concentration of AD which did not show any harmful effect on macrophages during 3 days of incubation was 0.001ƒÊg/ml. The in vitro effect of puromycin on cultured macrophages was examined by a method similar to that described above. It was indicated that 12.5ƒÊg/ml of puromycin was the maximum dose which did not show any harmful effect on cultured macrophages during 3 days of incubation. The inhibitor, i.e., AD or puromycin, was resolved in culture medium containing transfer agent and macrophages were cultured in the medium with or without inhibitor. After 3 days of incubation, cellular immunity was assayed by the method de-

4 162 K. SAITO, N. OSAWA AND S. MITSUHASHI Table 1. Dose effect of immune RNA on the development of cellular., immunity against infection with S. enteritidis Numbers indicate a mean of 3 different experiments and data in each experiment show a mean of 5 counts. a) Cellular resistance against infection was determined on day 1, 2 and 3 after infection of peritoneal macrophages in tissue culture with S. enteritidis and the results on day 3 are shown in the table. Number of bacteria in 100 infected macrophages and survival number of macrophages after infection are expressed in percent of initial number. The number indicates a mean of 5 different experiments. b) Cellular antibody was determined by immune transfer and immune adherence hemagglutination on day 5 after treatment of macrophages with RNA preparations. c) Normal macrophages in tissue culture without infection. The numbers indicate a mean of 10 different experiments. scribed previously [11, 15, 23]. RESULTS Dose Effect of Immune RNA on the Induction of Cellular Immunity An immune RNA was extracted from the spleen cells of immunized mice. Normal mice were injected intraperitoneally with 1ml of physiological saline containing several concentrations of immune RNA, As a control, normal RNA was extracted similarly from the spleen cells of normal mouse. Five days after injection with either immune or normal RNA, peritoneal macrophages of mice treated with RNA preparations were collected from abdominal cavity and cellular immunity were examined according to the method described in Materials and Methods. As shown in Table 1, 1mg of immune RNA preparation extracted from the spleen cells of immunized mice was capable of developing cellular immunity in the peritoneal macrophages of treated mice but 0.5mg was ineffective. Duration of Cellular Immunity in the Peritoneal Macrophages of Mice Treated In Vivo with Immune RNA Mice received an intraperitoneal injection with 1.5mg of immune RNA preparation extracted from the spleen cells of immunized mice. At appropriate time intervals after injection, the peritoneal macro-

5 TRANSFER AGENT OF IMMUNITY 163 phages were collected and their cellular immunity was examined. As shown in Table 2, cellular resistance against infection was still demonstrable in the peritoneal Table 2. Duration of cellular immunity in the peritoneal macrophages of mice treated in vivo with immune RNA Cellular immunity was examined by resistance of cultured macrophages against infection with S. enteritidis , which were obtained from the peritoneal cavity of mice treated with RNA preparations. a) See the footnote of Table 1. Cellular immunity was examined on day 5, 13, 21 and 90 after an intraperitoneal injection with 1.5mg of either immune or normal RNA preparation. b) See the footnote of Table 1. Table 3. Serial passive transfers of cellular immunity in macrophages against infection with S. enteritidis a) Immune RNA was extracted with phenol from the spleen cells of mice hyperimmunized with live vaccine of S. enteritidis. b) RNA was extracted similarly from the spleen cells of each recipient mouse which had received an intraperitoneal injection with the RNA 3 days prior to preparation. Number in parenthesis indicates the amount (mg) of RNA extracted from spleen cells. c) Normal RNA was extracted with phenol from the spleen cells of normal mouse instead of immune mouse. d) See the footnote a) and c) of Table 1.

6 164 K. SAITO, N. OSAWA AND S. MITSUHASHI Table 4. Serial passive transfers of cellular immunity and formation of antibody in macrophages of the 4th recipient mouse a) Peritoneal macrophages were collected on day 5 after treatment with 1mg of RNA preparation extracted with phenol from the spleen cells of the 3rd recipient mouse. b) See Materials and Methods. macrophages which were collected 90 days after treatment with an immune RNA preparation. Serial Passive Transfers of Cellular Immunity As shown in Table 1, 1mg of immune RNA preparation was found to be the minimum effective dose for the induction of cellular immunity. Usually 1.5 to 2mg of RNA preparation was extractable from a spleen of an immunized mouse. Therefore 1mg of an immune RNA preparation extracted from the spleen cells of immunized mice was used for the study of serial passive transfers of cellular immunity. As shown in Table 3, the macrophages of 4th recipient mouse showed cellular resistance against infection with virulent strain of S. enteritidis and inhibited intracellular growth of bacteria. The cellular antibody was demonstrated by JAHA reaction on the macrophages obtained from the peritoneal cavity of the 4th recipient mouse that had been serially and passively treated with RNA preparations (Table 4). If we assume that (a) the total amount of the original immune RNA extracted from the spleen cells of immunized mice (1st donor), was taken in the spleen cells of recipient mouse when injected intraperitoneally and (b) the total amount of immune RNA injected was extractable from the spleen cells of recipient mouse, the 4th recipient mouse should receive an intraperitoneal injection with 0.2mg (1/1.8 ~ 1/1.6 ~ 1/1.6 ~ 1/1.5) of the original immune RNA preparation extracted from the spleen cells of immunized mouse (1st donor). This amount of immune RNA preparation should be ineffective in the induction of cellular immunity. Effect of Actinomycin D on the Serial Transfers of Immunity To test the effect of actinomycin D on the transfer of cellular immunity through immune RNA, a mouse was injected intraperitoneally with 0.1ml of 5ƒÊg of actinomycin D one day before the treatment with immune RNA. As shown in Table 5, the 1st recipient mouse which had received an intraperitoneal injection with 1mg of immune RNA developed cellular immunity, even though the mouse had been treated with actinomycin D. By contrast, the 2nd recipient could not develop cellular immunity when the mouse was injected with the RNA preparation extracted from the spleen of 1st recipient mouse which had been treated with actinomycin D. However, it should be noted that the RNA preparation extracted from spleen of either immunized mouse or the 1st recipient of immune

7 TRANSFER AGENT OF IMMUNITY 165 Table 5. Effect of actinomycin D(AD) on the transfer of immunity by immune RNA preparation a) The 1st recipient received an intraperitoneal injection with 1000ƒÊg of immune RNA extracted from the spleen of immunized mouse. b) See Materials and Methods.c ) See the footnote of Table 1. The results on day 3 after infection are shown in the Table. RNA without actinomycin D treatment, could induce the cellular immunity in the recipient mouse even with or without treatment of actinomycin D. In Vitro Effect of Actinomycin D or Puromycin on the Development of Cellular Immunity through Immune RNA Peritoneal cells were obtained from normal mice and were cultured in the culture bottle provided with several cover slips. After 24hr of incubation, the medium was exchanged with fresh medium containing both actinomycin D (0.001ƒÊg/ml) and immune RNA (100ƒÊg/ml). After 3 days of incubation, macrophages were infected with virulent strain of S. enteritidis to examine the development of cellular immunity. As shown in Fig. 1, the macrophages treated and resisted intracellular multiplication of bacteria. The effect of puromycin, a known inhibitor of protein synthesis, was examined. When normal macrophages treated with both puromycin (12.5ƒÊg/ml) and immune RNA (100ƒÊg/ml), macrophages did not develop cellular immunity and macrophages were destroyed by intracellular growth of bacteria (Fig. 2). These results indicated that macrophages developed cellular immunity after treatment with immune RNA even in the presence of actinomycin D, but did not in the presence of puromycin. By contrast, serial passive transfers of immunity through immune RNA was found to be inhibited by treatment of recipient mouse with actinomycin D. in vitro with both actinomycin D and immune RNA, developed cellular immunity

8 166 K. SAITO, N. OSAWA AND S. MITSUHASHI Davs after infection Fig. 1. Effect of actinomycin D on the induction of cellular immunity through immune RNA. Macrophages were obtained from peritoneal cavity of normal mice and inoculated into culture chamber as described in Materials and Methods. After 20hr of incubation, culture medium was removed and exchanged with culture medium containing 100ƒÊg/ml of immune RNA or immune RNA (100ƒÊg/ml) and actinomycin D (0.001ƒÊg/ml). After 3 days of incubation, cellular immunity was determined as described in Materials and Days after infection Fig. 2. Inhibition of the development of cellular immunity through immune RNA by puromycin. Macrophages were obtained from peritoneal cavity of normal mouse and inoculated into culture chamber as described in Materials and Methods. After 20hr of incubation, culture medium was exchanged with culture medium containing immune RNA (100ƒÊg/ml) or both immune RNA (100 ƒêg/ml) and puromycin (12.5ƒÊg/ml), After 3 days of incubation, cellular immunity was determined as described in Materials and Methods. Methods. DISCUSSION It was indicated by this laboratory that cellular immunity against infection with S. enteritidis was transferable from immune to nonimmune macrophages through the transfer agent of immunity [15, 16, 21-25]. It was also found that this agent was inactivated by treatment with ribonuclease but not with deoxyribonuclease or trypsin [14, 25], indicating that this agent was of RNA nature. This agent was also demonstrated from animals immunized with red blood cells, Salmonella flagella and diphtheria toxoid [5, 7, 14]. The transfer agent is extractable from the supernatant fluid of cell culture [23], or from the ribosomal fraction of immune macrophages [25], or extracted with phenol from the macrophages or spleen cells of immunized mice [16, 17, 21, 23-25]. Fishman [1] and Fishman and Adler [2] reported that the lymphnode cells produced

9 TRANSFER AGENT OF IMMUNITY 167 antibody to T2 bacteriophage when they were incubated with an RNA preparation extracted from the macrophages which had been incubated with the antigen in question. The preliminary experiments from this laboratory have disclosed that serial passive transfer was successful in immunity against infection with S. enteritidis or in the immunity to Salmonella flagella [25]. It was also reported that immune RNA was capable of conferring the ability for the secondary response of antibody formation on recipient cells [8, 27]. An ability of immune RNA to confer secondary response was found to be serially and passively transmissible [9]. These results and those described in this article may lead us to the conclusion that the immune RNA does not contain antigen or fragment thereof and may replicate actively in a recipient cell, although the mechanisms still remain to be elucidated. Previous papers indicated that cellular immunity against infection with S. enteritidis persisted for a long time after immunization and in the peritoneal macrophages which were collected successively several months apart from an immunized mouse [26]. It should be noted that cellular immunity was detectable even after an injection of macrophages which were collected from the peritoneal cavity of mice treated with immune RNA. This fact suggested that immune RNA played a role in the spread of immunity though whole body from the site injected with antigen and the persistence of immunity for a long time. However, the inhibition of serial transfers of immunity through immune RNA by treatment of recipient mice with actinomycin D suggests that the DNA-dependent RNA polymerase is involved in the mechanism responsible for; (a) successive transfers of immunity through immune RNA, (b) persistence of immunity through immune RNA for a long time, and (c) the spread of immunity through whole body from the site injected with immune RNA. In the role of RNA in antibody formation, 2 agents have been reported; (a) RNA itself which is functional per se, and (b) an agent in which antigen is complexed with RNA, termed as superantigen [3, 4]. Detailed studies for the comparison of the role of transfer agent (immune RNA) with that of antigen in antibody formation will be described elsewhere. ACKNOWLEDGEMENT We are indebted to the courtesy of Merck Sharp & Dohme Research Laboratory, Rahway, N.J., U.S.A. for the supply of actinomycin D through T. M. Sonneborn and F. Haurowitz, Indiana University, U.S.A. REFERENCES [1] Fishman, M Antibody formation in vitro. J. Exp. Med. 114: [2] Fishman, M., and Adler, F. L Antibody formation in vitro. II. Antibody synthesis in X ray irradiated recipient of diffusion chambers containing nucleic acid from macrophages cultured with antigen. J. Exp. Med. 117: [3] Friedman, H Antibody plaque formation by normal mouse spleen cell cultures exposed in vitro to RNA from immune mice. Science 146: [4] Friedman, H. P., Stavitsku, A. B., and Solomon, J. M Induction in vitro of antibodies to phage T2: Antigen in the RNA extract employed. Science 149: [5] Kawakami, M., Kitamura, K., Mikami, H., and Mitsuhashi, S Transfer agent of immunity. II. Conversion of nonimmune spleen cells into antibody-forming cells by transfer agent in ribonucleic acid fraction of immunized mice. Japan. J. Microbiol. 13: [6] Kidson, C., and Kirby, K Isolation characteristics of rapidly labeled RNA from normal rat liver. J. Mol. Biol. 7:

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