Transfer of cellular immunity in vivo with immune RNA in an allogeneic murine model

CLINICAL

IMMUNOLOGY

Transfer

SUSANA

AND

of Cellular

SERRATE,’

Servicio de It?rnu,lohiologia.

IMMUNOPATHOLOGY

Immunity Allogeneic

22, 75-82 (1982)

in Vivo with Immune Murine Model

M. L. SATZ,~ M. B. SZTEIN,~

AND MARTA

RNA in an

BRAUN~

Instiruto de Oncologia “A. H. Roffo, ” Universidad de Buenos Aires. Buerws Aires. Argentina

Immune RNA (I-RNA) was extracted from lymphoid organs of BALBlc mice immunized with AKR lymphoid cells. Normal BALB/c mice injected with I-RNA (but not with normal RNA) acquired the capacity to respond to the specific antigen as measured by leukocyte migration inhibition reaction. Positive responses appeared 2 days after I-RNA injection, lasted for 2 months, and were negative by 7 months. This transference was specific and dependent on I-RNA molecular integrity. The minimal amount to effect a transference was 0.05 mg I-RNA/mouse. None of the mice injected once or twice with I-RNA developed specific alloantibodies. However, one injection of I-RNA, followed by the administration of the specific antigen, induced a secondary antibody response.

INTRODUCTION

Many laboratories have reported that RNA extracted from the lymphoid organs of immune animals (“immune RNA,” I-RNA) is able to transfer specific sensitivity to normal animals or lymphoid cells. In previous work, some of us extended this phenomenon to an autoimmune model, e.g., experimental allergic orchitis (1 - 3). Immune responses against histocompatibility antigens have also been successfully transferred by I-RNA. Mannick and Edghal (4) reported accelerated graft rejection in rabbits treated with I-RNA extracted from the lymph nodes of animals bearing skin allografts. Furthermore, similar results were obtained by Ramming and Pilch (5). Two hypothesis have been proposed to explain how I-RNA may transfer immunity: the first one postulates that I-RNA is a “super antigen” composed of small fractions of antigenic proteins with the RNA molecules acting as carrier or adjuvant. The second hypothesis maintains that I-RNA acts as an informational molecule and that it is able to induce the synthesis of active antigen receptors on the surface of noncommitted cells. The latter is supported by work of our laboratory that shows that normal BALB/c spleen cells treated with I-RNA extracted from BALB/c mice immunized against AKR cells, specifically acquire the capacity to respond in a leukocyte migration inhibition reaction (LMIR) against AKR ’ Present address: Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20205. r Present address: Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20205. a Present address: Laboratory of Microbiology and Immunology, National Institute of Dental Research. National Institutes of Health, Bethesda Md. 20205. A Present address: Unidad de Inmunofarmacologia, CEFAPRIN, CONICET, Serrano 665, 1414 Buenos Aires, Argentina. 75 0090-1229/82/010075-08$01.00/O Copyright All rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

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extracts (6). Moreover, we have also demonstrated that I-RNA-converted cells bear on their surface alloantigen receptors with idiotypic determinants (6). These idiotypic determinants are similar or identical to those that have been shown to be present on the immune lymphocytes of the I-RNA donors (7). The present work was carried out to further study the mechanism of action of I-RNA. We here report that I-RNA is able to transfer sensitivity against alloantigens when injected into normal mice. We also show that this transfer is resistant to the treatment of the I-RNA with an extremely active proteolytic enzyme, proteinase K (8, 9) and that in viva administration of I-RNA does not seem to act as a triggering mechanism for antibody formation but that it primes the recipient to subsequently have an enhanced LMIR and secondary antibody response. MATERIALS

AND METHODS

Mice. BALB/c, AKR, (BALB/c x AKR)F,, and C57BL/6 were bred in our colony. Two- to six-month-old mice were used throughout. Immunizations. All alloimmunizations were done between animals of the same sex. Pooled lymphoid cells from spleen, lymph nodes, and thymus were obtained by pressing the organs through a stainless-steel mesh; cells were resuspended in TCM 199 containing antibiotics. Mice received one SC injection of lo7 cells, followed by two to four ip injections of 2-4 x lo7 cells. at weekly intervals. In order to obtain I-RNA with double specificity, animals were immunized with one id injection of 10’ cells emulsified in Freund’s complete adjuvant (FCA) (Difco), followed by two to four ip injections of cells alone, as above. Control animals received saline emulsified in FCA or saline alone. RNA extraction. Mice were killed 3-7 days after the last injection, their lymph nodes and spleen were immediately removed and placed in chilled ethanol at -70°C. Organs were stored at this temperature or used immediately for RNA extraction. This was done by the phenol-chloroform method of Perry et al. (10) with minor modifications, as described in detail previously (2, 3). RNA was precipitated and stored in absolute ethanol at -20°C. RNA was collected by centrifugation at 22,000g for 10 min, solubilized in sterile saline and its concentration was calculated by absorption at 260 nm, considering that 1 OD = 0.04 mg. Normal RNA (N-RNA) was prepared in the same way from the lymphoid organs of nonimmunized animals. The presence of contaminating nucleases and/or the presence of hidden breaks in RNA molecules was revealed by incubating 0.2 mg RNA in 0.1 ml of sterile saline at 25°C for 1 hr. After this, up to 0.1% SDS and 1 mM EDTA were added and the sample was heated at 60°C for 5 min (11). The preparations were then analyzed either by sucrose density gradient fractionation (1 - 3) or by 2.4% polyacrylamide gel electrophoresis (12). Enzyme treatment. Aliquots of some samples of I-RNA were treated with proteinase K by the method of Flanegan et al. (13). Briefly, 1 mg of RNA was digested for 1 hr at 37°C in 0.2 ml of a solution containing 0.5% SDS, 0.1 M NaCl, 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA and 0.2 mg/ml proteinase K (Boheringer-Mannheim, New York). RNAase treatment was done as previously described (l-3, 6). After this treatment, RNA showed a single 4 S peak when

IN

WV0

TRANSFER

OF ALLOREACTIVITY

WITH

I-RNA

77

analyzed by sucrose density gradient fractionation. The RNA was then extracted twice with phenol-chloroform and precipitated with chilled ethanol. In vivo treatment with RNA. Mice were injected ip with 0.2 ml of saline containing different amounts of N-RNA or I-RNA. Alternatively, normal BALB/c spleen cells were incubated with RNA (0.5 mg/lO* cells) for 20 min at 37”C, and each mouse was then injected with 2-3 x lo7 treated cells. All animals were tested for LMIR and serum alloantibodies at different times after RNA treatment. Antigens. Partially purified extracts of AKR and C57BU6 lymphoid cells were prepared as described before (7, 14). Protein content was determined by Lowry’s method (15). Preservative free purified protein derivative (PPD) was a gift of Dr I. Kantor (Panamerican Zoonosis Center, WHO, Ramos Mejia, Argentina). Leukocyte migration inhibition reaction. LMIR was done as previously described (7): spleen cells were packed into capillary tubes and placed in plastic chambers to assess migration. TCM 199 containing antibiotics was used as culture medium. Saline or one of the following antigens were added: AKR extracts, C57BW6 extracts (both at 0.10-o. 15 mg/ml) or PPD (0.01 mg/ml). After 20-24 hr, migration areas were projected onto a paper, cut out, and weighed. Mean areas were obtained from seven to nine replicative capillaries and migration indices (MI) were calculated as described before (7). Sera. The presence of alloantibodies was investigated by complementdependent cytotoxicity using AKR thymocytes as target cells and trypan blue exclusion as the viability test, or by direct hemmaglutination of AKR erythrocytes; both methods were carried out by routine techniques described elsewhere DAY

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1. In l+vct transfer of LMIR against AKR extracts. Normal BP B/c mice were injected ip either with: (1) 0.2 mg N-RNA (A) or I-RNA (0) solubilized in saline, or (2) syngeneic spleen cells preincubated in vi/t-o with N-RNA (a) or I-RNA (0). Mice were killed after different periods of time and tested for LMIR against AKR extracts. Dotted line represents 2 SD below the mean migration index of N-RNA-treated mice. Efficiency of I-RNA transfer with both techniques is depicted at the bottom of the figure. Migration index was calculated as described under Materials and Methods. FIG.

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AL..

(16, 17). All the BALB/c anti-AKR immune mice used as I-RNA donors developed serum alloantibodies with cytotoxic titers ranging from 1:40 to 1:640. These same sera, when tested for haemmaglutinating antibodies, showed titers that were 30 to 50 times greater than their corresponding cytotoxic titers. RESULTS In Vil’o Transfer

of LMIR

with I-RNA

As may be seen in Fig. 1, normal BALBic mice injected with I-RNA acquired the capacity to recognize AKR antigens when tested in vitro in a LMIR. Positive results appeared after 48 hr of I-RNA injection and lasted for at least 60 days. The injection of I-RNA in saline was more efficient that the injection of syngeneic cells previously incubated in vitro with I-RNA. N-RNA had no effects in either form when compared with untreated animals (not shown). Dose-response experiments showed that the minima1 dose necessary to effect a transference was 0.05 mg of I-RNA per mouse, provided it was given together with 0.45 mg of N-RNA as carrier (Fig. 2). Doses above 0.3 mg of I-RNA/mouse no longer achieved any increases in I-RNA transfer efficiency. The specificity of I-RNA is shown in Fig. 3: the sensitivity was always restricted to the antigen(s) used to immunize the donors. Active

Molecules

it1 I-RNA

Preparutions

Previous works have shown that in the allogeneic in vitro model the effect of I-RNA is resistant to Pronase treatment but sensitive to RNAase (6). In order to further investigate the role of any contaminating protein antigen (that could be DO! Mug I-RNA

au9 RNA &g .RNA

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FIG. 2. Ability of different doses of I-RNA to sensitize normal mice. Normal BALBic mice were injected with different amounts of I-RNA alone or mixed with N-RNA as carrier. After 7 days. animals were tested for LMIR against AKR extracts. Dotted line, as in Fig. I.

IN VW0

TRANSFER

OF

ALLOREACTIVITY

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FIG. 3. Specificity of the transference with 0.2 mg of N-RNA or RNA extracted cells plus FCA or saline plus FCA. Mice C57BLi6 (A) extracts, or PPD (0).

L -mediated by I-RNA. Normal BALB/c mice were injected ip from mice immunized with: AKR cells, C57BL/6 cells, AKR were killed 7 days later and tested for LMIR against AKR (m),

resistant to pronase degradation) in the RNA preparations, I-RNA was treated with proteinase K, a very potent proteolytic enzyme (8,9). Two out of three mice injected with proteinase K-digested I-RNA showed positive LMIR when tested 8 days later (MI: 61, 120, and 63). This efficiency of the transfer is comparable with that obtained with untreated I-RNA (Fig. 1). On the contrary we consistently failed to confer reactivity with I-RNA preparations after extensive degradation after RNAase treatment (Table 1; Refs. (1-3, 6)). Moreover, I-RNA preparations that had hidden breaks in their primary structure, only revealed by reversible denaturation at 60°C (Fig. 4) were also inactive: two out of the nine pools of IRNA used in this work had hidden breaks and did not transfer immunity (Table 1). The other seven pools used had the same molecular pattern before and after heating and all of them transferred immunity. TABLE 1 HIDDEN BREAKSCONTAINING I-RNA PREPARATIONS TO TRANSFER ALLOREACTIVITY IN VIVO~

RNAaSe-TREATEDOR

MI against

I-RNA Nondegraded RNAase-treated With hidden

breaks

” Normal BALBlc mice were injected ip with 0.2 mglmouse scribed under Materials and Methods. Seven days later animals antigens.

ARE NOR ABLE

AKR

56, 81, 68 101, 95, 98 121, 105.98 of each I-RNA preparation, as dewere tested for LMIR against AKR

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ET AL.

e n

2

4

8

6

2

ELECTAOPHOAETIC

4

6

MOBILITY

8

(cm)

FIG. 4. Detection of hidden breaks in RNA preparations. RNA preparations were analyzed by polyacrylamide gel electrophoresis. (A) Profile obtained with a sample of I-RNA incubated for 2 hr at 37°C. (B) Profile obtained after the same preparation was further treated for 5 min at 60°C. Extensive degradation can be observed as indicated by the presence of a prominent 4 S peak.

Humoral

Immune

Responses

Induced

by I-RNA

None of the sera of the I-RNA injected animals contained antibodies detectable either by complement-dependent cytotoxicity or by direct hemagglutination (Table 2, group I). In order to study if I-RNA had any ability to trigger alloantibody synthesis, the following experiment was done. Different groups of mice received a first ip injection of I-RNA: 7 days later they received an ip booster of I-RNA (group II) or lo7 AKR cells (group III) and 5 days later blood samples were obtained by retroorbital puncture. Control animals received N-RNA and AKR cells (group IV). Only the animals in groups III and IV developed hemagglutinating antibodies; no alloantibodies could be detected by this method nor by complement-dependent cytotoxicity in the sera of mice injected twice with I-RNA. Mice belonging to group III developed significantly higher titers than those of group IV. These results suggest that I-RNA is not able to trigger antibody formation.

HUMORAL Group I II III IV

N 32 5 5 5

IMMUNE First

injection I-RNA I-RNA I-RNA N-RNA

TABLE 2 RESFQNSES INDUCED Booster none I-RNA AKR cells AKR cells

BY

I-RNA” Hemmaglutination 0 0 3.7 c 0.5 (40-640) 2.5 c 0.2 (20- 160)

’ Group I was composed of all animals tested for LMIR. Groups I1 to IV received two injections at 7-day intervals and mice were killed 5 days later. Doses given: RNA, 0.2 mg/mouse: cells, IO’, both by ip route. Results are expressed as log, of reciprocal titer/IO -+ SD and (range). P between groups III and IV < 0.025.

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TRANSFER

OF

ALLOREACTIVITY

WITH

I-RNA

81

DISCUSSION Al-Askari and Lawrence (18) have demonstrated that in allogeneic murine models immune, but not normal lymphocytes give positive LMIR upon in vitro stimulation with alloantigens. This has been confirmed in our previous report (7). Here we show that spleen cells from normal BALBfc mice injected with I-RNA acquire the capacity to recognize allogeneic antigens when tested in vitro in a LMIR. Positive results appeared 2 days after RNA injection, lasted for 2 months and were negative by 7 months (Fig. 1). This transfer was always specific for the antigen used for the I-RNA donor immunization (Fig. 3). Our results agree with those of Bhoopalam et al. (19) who reported that peripheral blood lymphocytes from BALB/c mice injected with I-RNA extracted from a syngeneic plasmocytoma, acquire specific plasmocytoma immunoglobulins on their surface 48 hr after RNA injection and that this “cell conversion” lasts for 6-.8 weeks. They also found that the biologically active fraction is sensitive to RNAse but resistant to trypsin and it is restricted to molecules between 12 S and 23 S. These latter results are also in agreement with our previous findings (6). The strong proteolytic activity of proteinase K on native as well as on denatured proteins and its broad spectrum of action are well documented (8). Proteinase K has been very useful in the elimination of small peptides from viral RNA preparations (9) and in the isolation of undegraded mRNA from polysomes (2). After proteinase K treatment, I-RNA preparations were still able to confer immune reactivity to normal cells; on the contrary, this ability was always dependent on I-RNA primary molecular integrity, suggesting an active role for RNA itself. In previous work we showed that I-RNA, acting as mRNA, is able to induce antigen receptors on the surface of normal cells and that these receptors are very similar or identical to those present on immune cells of the I-RNA donors. However, the possibility that I-RNA may trigger other differentiation signals as well, could not be resolved. We here show that I-RNA per se is not able to induce ahoantibody production when injected into normal mice, although it is able to prime or prepare the recipient cells, since animals preinjected with I-RNA had better responses to one injection of allogeneic cells than controls. Kurashige and Mitsuhashi (21) have shown that mice injected twice with I-RNA extracted from Salmonella tennesee-immunized donors, do not develop agglutinins to flagellin; however, when the mice were primed with I-RNA and subsequently challenged with the specific antigen, a secondary type antibody response was observed. These results disagree with those of other authors who reported antibody formation by normal animals or their lymphoid cells when treated with I-RNA (22-25). It is worth mentioning that in those models, I-RNA preparations were found to contain antigen fragments and this fact could account for the ability of such preparations to induce antibody production. Our failure to induce antibody synthesis in normal animals would favor the interpretation that I-RNA’s effect is mediated only by mRNA. In conclusion, our results show that I-RNA is able to induce a state of sensitivity against a specific alloantigen when injected into normal mice. This capacity is dependent on the integrity of RNA molecules and is resistant to an active proteolytic enzyme. The state of sensitivity induced by I-RNA is expressed both, by the development of a LMIR as well as by a secondary type of antibody production.

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ACKNOWLEDGEMENTS We thank Dr. Joost Oppenheim for his critical review of the manuscript. We are also very grateful to Drs. Mario Lebendiker and Eduardo Scodller for their help with the polyacrylamide gel electrophoresis technique. Marta Braum is Member of the Research Career of the Consejo National de Investigaciones Cientificas y Tecnicas (CONICET). Argentina. S. Serrate. M. L. Satz, and M. B. Sztein are Research Fellows of CONICET. These studies were supported by grants from the CONICET and from the Secretaria de Estado de Ciencia y Technologia.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. I I. 12. 13. 14. IS. 16. 17. IS. 19. 20. 21. 22. 23. 24. 25.

Fainboim, L.. Sztein. M.. Serrate. S.. and Mancini, R. E.. C/;U. t\-p. /I,I,JI~u~J/. 34, 92, 197X. Fainboim, L., Sztein, M., Serrate. S.. and Satz, L., /,,~IIIuu~&~,~J 39, 3 I I, 1979. Sztein. M. B.. Satz, M. L., and Serrate, S.. ./. C/;/I. f.rrh. fm/rrrrn~,/. 3. 39, 1980. Mannick, J. A., and Edgahl, R. H.. ./. C/ill. /,t,~c~st. 43, 2166, 1964. Ramming, K. P., and Pilch. Y. H.. T,-cr,?.sp/lr~rttrri,,ll 7. 296. 1969. Satz. M. L., Sztein, M. B.. Serrate, S., and Braun, M., Mol. C-P//. 61;~~~/1e,~r. 33. 105. 1980. Braun. M., Sztein. M. B., Satz, M. L.. Jasnis, M. A,. and Saal, F.. .Sc,n,~~/. ./. /~~IIuu)I~~/. 10. 25. 1979. Ebeling, W.. Hennrich. N., Klockow. M., Metz. H.. Orth, H. D.. and Lang, H.. tlrr. J. Bioc~/~c~/,~. 47. 91, 1974. Hewlett. M. J.. and Florkiewicz. R. Z., Pr,,c,. .Ytrt. A~,cld. .Yc,;. USA. 77. 303. 1980. Perry. R.. LaTorre, J. L.. Kelley. D., and Greenberg. J.. Bio~./zi~~. B;op/~y\. .4crtr 262. 720. 1971. Denoya. C. D.. Scodller, E. A. and Latorre, J. L., F‘ERS Lcrr. 106, 97. 1979. Weinberg, R., Loening. V. L.. Willems. M.. and Penman, S.. P~(Jc. ,‘l’trr i\c,~(/ .SC,;. i!SA 58. 1088, 1967. Flanegan. J. B., Petterson, R. F., Ambros. V.. Hewlett, M. J.. and Baltimore. D.. Prr,(. Vtri Acrrd. Sri. USA 74, 961, 1977. Braun. M.. Sen, L., Bachmann, A. E.. and Pavlovsky, A. S.. R/(xu/ 39, 368. 1972. Lowry, 0. R.. Rosebrough, R.. Farr, R., and Randall, A., J Biol. C/IC,,U. 193. 265. 1951. Braun, M.. and Saal. F., Cell. I,n,nrln,,/. 30. 254, 1977. Welsh. K. I.. and Batchelor, J. R., 1~ “Handbook of Experimental Immunology“ (D. M. Weir. Ed.), p. 35. I, Blackwell. London, 1978. Al Askari. S.. and Lawrence, H. S., Cell. /IU,,~UI~O/. 5. 402. 1972. Bhoopalan. N.. Yakulis. V.. Costea, N.. and Heller, P., B/
Received

June

IO. 1981: accepted

with

revisions

August

6. 1981.