CELLULAR
IMMUNOLOGY
23, 309-319
Evidence That PATRICIA
BILELLO,~
(1976)
Immune ~~ARVIN
RNA Is Messenger FISF1hlAN,3
AND
GEBHARD
RNA1 Kocn
2 Roche Institute of Molecular Biology, hrutley, New Jersey 07110, and Biology Dcjartment, New York University, New York, New York, and s St. Jzrde Children’s Research Hospital, Memphis, Tennessee 38101 Received January
14,1976
Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19s proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.
INTRODUCTION When rabbit peritoneal exudate (PE) cells are incubated with bacteriophage, the cell extracts yield RNA capable of inducing the formation of phage specific neutralizing antibodies in rabbit spleen cell cultures (1). The cells involved as donors in these experiments were rabbit peritoneal exudate cells, consisting of 55-95 % macrophages (2, 3). The immunogenic activity of the PE cell homogenates resides in two RNA fractions. One of these fractions contains a biologically active RNA designated informational RNA (i-RNA), the other contains RNA-antigen complexes (2, 4). The i-RNA induces the formation of 19s immunoglobulins which contain the allotypic markers of the RNA donor rabbit (2). Informational RNA free of detectable antigen has been shown to induce humoral antibody production (5, 6, 7, S), and to transfer allotypic markers (9, 10). The ability of RNA to induce cellular immunity has also been demonstrated (11, 12, 13). The cell population responsible for the production of i-RNA and RNA-antigen complex has been separated from other PE cells by centrifugation on discontinuous bovine serum albumin (BSA) gradients (3). Immunogenic activity remains confined to approximately 15% of the total cells. It has been reported that synthesis of i-RNA is observed when PE cells are incubated with a low input of phage antigen to macrophage (1: 100) (14). Th us a small subpopulation of cells capable of recognizing the antigen are involved in the synthesis of i-RNA. Thus, while there appears to be evidence that i-RNA functions as mRNA, it has not yet been clearly established that i-RNA is capable of directing synthesis of both the light (L) and heavy (H) chains of immunoglobulins. These studies were undertaken in order to further define the role of i-RNA in initiating an immune response. The ability of i-RNA to function as a messenger RNA was 1 Supported in part by National Institutes of Health Grant AI 13180. 309 Copyright 0 1976 by Academic Press, Inc. AU rights of reproduction in any form reserved.
310
BILELLO,
FISHMAN
AND
KOCH
investigated by in vitro translation of i-RNA in cell-free protein synthesizing systems prepared from mouse L cells. Proteins synthesized in response to i-RNA were then characterized in neutralization and antigen binding assays and by sedimentation on sucrose gradients. MATERIALS Preparation of Inforvnational
AND
METHODS
RNA
Peritoneal exudate (PE) cells from rabbits homozygous at the b locus were obtained as previously described (2). The cells were suspended in solution B (0.15 J4 NaCl, 6 x 10m3M KCl, 15 ml phosphate buffer, distilled water to one liter, final pH 7.6). Ten ml of solution B containing 1.5 X 10” PE cells and 1.5 x 1O1l phage particles (high input) or 1.5 X lo7 phage particles (low input), either Escherichia coli phage T2 or Bacillus subtilis phage SP82, were incubated 30 min at 37°C without stirring. The RNA was extracted from PE cells by the hot phenol method (15) and was designated T2 i-RNA. Normal RNA refers to RNA extracted from PE cells not exposed to phage. The ethanol precipitated RNA was dissolved in sodium acetate buffer containing 0.14 M LiCl and 10e3 M MgSO+ Prior to use in in vitro translation studies, the RNA was extracted one more time with water-saturated phenol for 5 min at room temperature with intermittant shaking. The RNA from the aqueous phase was precipitated twice with ethanol, and dissolved in 1O-3 M EDTA pH 7.2. RNA concentration was determined from the absorbance measured spectrophotometrically using as the conversion factor the ratio of 40 pg RNA/ml for an ODzGo of 1.0 (1 cm light path). Spleen Cell Cultures The procedure used to prepare splenic cell cultures was that of Dutton and Mishell (16) with the major modification that normal rabbit serum (previously determined to be free of anti-T2 activity) replaced the fetal calf serum. Cultures (in 35 mm petri plates) consist of 2 X lo7 dispersed spleen cells in 1.0 ml medium (containing the RNA). These were incubated in an atmosphere of 7% 02, 10% COZ, 83% Na. Addition of the serum was delayed for 2 hr after initiation of the culture. Preparation
of Polysomal RNA
After incubation of the PE cells with T2 phage for 30 min at 37”C, the harvested cells were suspended in 5 ml MSB buffer (0.25 M KCl, 0.01 M MgC&, 0.01 M Tris-HCl pH 7.4). PE cell suspensions were disrupted in a Dounce homogenizer with 10 strokes in 3 ml MSB buffer. Ten percent Nonidet was added to give a final concentration of 1% Nonidet, the mixture was then incubated for 3 min at 4°C. The supernatant was recovered by centrifugation for 10 min at 12,OOOgand applied to a discontinuous sucrose gradient consisting of 5 ml 60% sucrose in MSB buffer and 15 ml 30% sucrose in MSB and the gradient was centrifuged in an SW25 rotor, at 100,OOOgfor 2.5 hr. Polysomes were removed from the 30/60 interface and the RNA extracted with 10 ml Tris-SDS, pH 9 (0.1 M Tris, 10 mM KCl, 0.2 mM MgClz, 0.5% SDS) and 10 ml phenol 10 min at 4°C with stirring. The aqueous phase was recovered by centrifugation for 10 min
IMMUNE
RNA
IS
MESSENGER
RNA
311
at 12,000g and the phenol phase was re-extracted with Tris buffer pH 9.0 (no SDS). The aqueous phases were combined and the RNA precipitated overnight with 2.5 volumes e,thyl alcohol and 0.1 volume 1% NaCl. The RNA was collected by centrifugation at 5009 for 10 min, washed with 6670 alcohol and 0.03% NaCl and resuspended in Solution B. Preparation of Cell-Free En-tracts The procedure used was analogous to that of McDowell et al. (17). Mouse L cell fibroblasts were maintained in suspension culture in Spinner Minimum Essential Medium supplemented with 5% fetal calf serum (Grand Island Biological Co., Grand Island, New York). The L cells at a concentration of 5 x lo” cells/ml were collected by centrifugation in a Sorvall RC-3 at 2000 rpm for 5 min, washed in buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N-‘2ethanesulfonic acid, Calbiochem, LaJolla, California 92037)) 0.15 M NaCl at pH 7.6, then suspended in 2 packed cell volumes of swelling buffer (10 mM HEPES, 10 mM KCl, 0.15 mM MgS04 6 mM ,f%-mercaptoethanol pH 7.6) for 10 min at 0°C. The cells were lysed with 1.5 strokes of the tight-fitting, B pestle in a Dounce homogenizer, and centrifuged at 10,OOOg for 10 min at 1°C in a Sorvall RC-2B. The resultant S10 supernatant was restored to isotonicity by the addition of 0.1 volume 10 x buffer B (see below) and 5% glycerol was added to stablize the extract. The S10 supernatants were passed over a 1.7 cm X 30 cm Sephadex G-25 (coarse) column previously equilibrated with buffer B (20 mM HEPES, 5 mM MgCIZ, 120 mM KCl, 6 mM ,8-mercaptoethanol, 6 mM dithiothreitol (DTT) pH 7.6 containing 5% glycerol). The most turbid fractions (O.D. at 260 nm and 280 nm ‘of 22 and 16) were pooled, aliquoted, and stored in liquid nitrogen. Translation
of i-RNA
in L-Cell-Free
Extracts
Reaction mixtures consisted of a total volume of 50 ~1: 10 ~1 cell-free extract, 100 mlM KCl, 12 m&1 HEPES pH 7.6, 3.0 mM Mg”, 8 mM DTT, 3.6 mM /?-mercaptoethanol, 5 pg creatine phosphokinase, 220 PM creatine phosphate, 1 mM ATP, 0.2 mM GTP, and 10 &i %-methionine (New England Nuclear, specific activity 300 Ci/mmole). The cell-free extracts were not pre-incubated and protein synthesis in the cell-free extracts was allowed to continue for 30 min at 25”. The effective dose range was 20-600 pg/ml of total RNA or 2-20 pg/ml of dT-cellulose purified RNA. Aliquots were removed at designated times and spotted on Whatman #3 paper discs (2.3 cm), presoaked with 50 ~1 of MEM 50 X essential amino acids (Grand Island Biological Co., Grand Island, N.Y.). TCA precipitable counts were cletermined by the method of Mans and Novelli (18). Briefly, the filters were precipitated in 10% tricholoracetic acid TCA for 30 min, washed 2~ with 5% TCA, and heated at 90-95” for 15 min, washed 2X again with ether for 5 min. The dried filters were counted in LSC Complete (Yorktown Research, Hackensack, N. J.) in a Beckman LS-200 liquid scintillation counter. Binding of Antibody
to T2 Plaage
The i-RNA and the endogenous translation products were precipitated with neutralized 50% (NHa)&Os at O”, and centrifuged at 120009 20 min at 1°C.
312
BILELLO,
FISHMAN
AND
KOCH
The precipitate was resuspended in an amount of isotonic buffer (0.15 M NaCl, 10e3 1M Tris pH 7.5) resulting in a 4- to S-fold concentration of the translation product. Then, 100 ~1 of translation product concentrates were incubated with 200 ~1 T2 phage 2 X 1Ol2 plaque forming units/ml for one hr at 37”C, then overnight at 4”. Antigen-antibody complexes were separated by gel filtration on a Sepharose 2B column 0.8 X 15 cm previously equilibrated with a buffer containing 10e3 M Tris, 10m3M MgClz and 1.0 M NaCl at pH 7.5. Phenol red was used as a marker. Neutralization
Assays
Serial 2-fold dilutions of the fluids to be tested were made in Solution B containing 0.1% fetal calf serum. After the addition of a constant amount of phage (400 PFU/tube) the mixture was incubated 30 min at 37°C. Duplicate or triplicate aliquots, each containing 100 PFU/tube if no neutralization has occurred were then plated on E. coli B. (for T2 phage) or on the indicator strain of B. subtilis (for SP82 phage). Plaques were counted after overnight incubation at 37°C. Where complement was employed to amplify neutralization (19), serum from normal rats (selected for low background anti-T2 neutralizing antibodies) at a final dilution of 1 : 20, was added to the incubation mixture. Sera used as a source of complement were never heat inactivated. Allotype amplification (19) of neutralization was performed as follows: 400 PFU of phage were added to the test extracts and incubated for 30 min at 37” followed by a second incubation for 90 min at 4” ; anti-allotype serum (1 : 10 dilution) was added and after an incubation for 30 min at 37” aliquots were plated as outlined above. Reagents used in the allotype amplification of neutralization were prepared by intensive immunization of appropriate rabbits with rabbit (Yglobulin. The anti-4 reagent was made in al, a1/b5, b5 rabbits by injection of al, a1/b4, b4 globulin, and the anti-5 serum by immunization of al, a1/b4, b4 rabbits with a’, a1/b5, b5 globulin. Reduction of Antigen-Antibody
Complexes
35S-methionine-labeled translation products were incubated with T2 or SP82 phage as decribed. After separation of the antigen-antibody complexes on Sepharose 2B, the complexes were reduced by 100 mM p-mercaptoethanol in the presence of 2 x 10m3M EDTA for 30 min at room temperature (20). Dissociation of Antigen-Antibody
Complexes at Low pH
Fractions containing the 35S-methionine label bound to T2 phage after reduction with p-mercaptoethanol were adjusted to pH 2.4 by the addition of the appropriate amounts of 10 N HCl. After incubation for 30 min at 20”, the fractions were subjected to gel filtration in Sepharose 2B. Sucrose Density Gradient Centrifugation Translation products synthesized in the L cell-free system were fractionated on a 10-37s linear surcose gradient (1) by centrifugation at 33,000 rpm in an SW39 rotor for 16 hr. The position of normal rabbit serum 19s immunoglobulins was determined spectrophotometrically at 280 nm.
313 RESULTS Induction of Antibody Formation in Spleen Cell Cultures by InzvlcMnogenic RNA in the Presence of Actinowzycin D When T2 antigens, solubilized in 0.5% SDS or T2 i-RNA, were added to dispersed spleen cell cultures antibody synthesis was induced and antibodies were secreted into the tissue culture fluid (1). Ninety-six hours after stimulation the fluids from 23 plates of cell cultures were pooled, and the cells and debris were removed by centrifugation. The tissue culture fluids were assayed for antibody activity in the complement enhanced neutralization test, (19). A neutralization of 25% of the test dose of phage by a given dilution of tissue culture fluid was highly significant (21, 22). The synthesis of phage neutralizing activities by spleen cell cultures in the presence or absence of Actinomycin D is shown in Table 1. Synthesis of phage neutralizing activities in spleen cell cultures following the addition of 10e4 pg protein nitrogen SDS-solublized T2 was inhibited by the presence of as little as 1 pg/ml of Actinomycin D. In marked contrast, the induction of T2 neutralizing activity by T2 i-RNA was found to be unaffected by actinomycin D which provides strong experimental evidence suggesting that i-RNA itself can act as mRNA for the synthesis of T2 phage antibodies in the spleen cells. Induction
of Antibodies
by RNA
Isolated
from
the Polysome Fractions
If i-RNA functions as mRT\‘A in peritoneal exudate cells, then this RNA should be present on the polysomes. RNA isolated from polysome fractions was added to spleen cell cultures. Figure 1 illustrates that 20 PLg polysomal RNA/ culture is sufficient to induce an immune response as measured in the neutralization assay. This antibody response is equivalent to one produced by lo&200 pg unfractionated RNA (total). Normal RNA was added to the polysomal RNA so that each culture received a total of 100 pg of RNA. The response increases significantly with increasing doses of i-RNA. These results provide further support that i-RNA acts as an mRNA. TABLE
1
Effect of Actinomycin D on the Immunogenicity and T2 i-RNA in Spleen Culture Addition
T2 i-RNA
to spleen cells
300 ~g
10m4 pg T2 solubilized
Actinomycin 1 P kc/ml
t +
D
of Solubilized Cells
7ONeutralization
T2
of T2
1:4
1:8
1:16
50 48 45 6
41 35 43 10
35 28 27 7
T2 i-RNA was obtained from cells exposed to T2 phage at a ratio of 1 phage particle per 100 cells. A constant amount of phage and complement at a final dilution of 1: 20 was added to serial 2-fold dilutions of the fluid to be tested, then incubated 30 min at 37”. Duplicate or triplicate aliquots were plated on E. coli B. Neutralization values of 20”’,jOor less do not represent significant antibody.
314
BILELLO,
I:16
FISHMAN
AND
KOCH
r
0
I 20
II
I 40
I 60
I
I, 80
1 100
pg POLYSOMAL RNA FIG. 1. Antibody induction by polysomal RNA. Polysomal RNA isolated from PE cells previously stimulated with T2 antigen was added to spleen cell cultures. Each culture received a total of 100 pg of RNA, that is, normal RNA was added to the polysomal RNA in appropriate quantities. Neutralization is expressed as 30% neutralization titers of a test dose of phage at the indicated dilution of the tissue culture fluid.
Synthesis of Antibody in a Cell-Free System To determine if i-RNA prepared from allotype specific rabbit PE cells contained all the coding information to direct the synthesis of proteins which possessed T2 neutralizing activity, T2 i-RNA was added to S1Ocell-free extracts prepared from mouse L cells. Synthesis of protein in the SIO extracts was followed by monitoring the incorporation of 36S-methionine. Addition of T2 i-RNA stimulated the incorporation of 35S-methionine 2-4 fold above the incorporation of the endogenous reaction (23). The specificity of the T2 i-RNA directed protein synthesis in the cell-free extracts was analyzed by an allotype amplified T2 phage neutralization assay. As shown in Table 2 neutralization assays performed with the in vitro translation products in the presence of anti-allotype serum give TABLE Amplification L-cell-free Translation
of Neutralization
2
of T2 Phage with Anti-Allotype-4
‘rONeutralization
system product
anti-4 1:4
1,1/4,4 T2 i-RNA (high)* 1,1/4,4 T2 i-RNA (low)* 1,1/4,4 Normal RNA Endogenous 2,3/5,5 i-RNA Control (Anti-4 or -5 only)
Serum of T2 anti-5
1:8
1:8
19
37
0
39
32
9
12
13
0
19
16 2
0 25
10 10-12
2-10
* RNA was extracted from cells previously exposed to phage at a ratio of: high = 100 phage particles/cell low = 1 phage particle/100 cells Serial dilutions of the fluids to be tested were incubated with a constant amount of phage 30 min at 37”, then 90 min at 4”. Antiallotype serum at 1: 10 dilution was added and incubated 30 min at 37”. Duplicate or triplicate aliquots were plated on E. roli B. Neutralization values of 20% or less do not represent significant antibody.
IMMIJNE
RNA
IS
MESSENGER
FRACTION
RNA
315
NUMBER
FIG. 2. Separation of antigen-antibody complexes. %S-mcthionine labeled products from the cell-free translation were precipitated with 50% (NH,)&O,, concentrated 5-fold, incubated with T2 phage as described in Materials and Methods and applied to Q Sepharose 2B column equilibrated with buffer containing 10m3M Tris, 10m3M MgCL, and 1.0 M NaCl at pH 7.5. Phenol red was used as a size marker. l --endogenous product; n--T2 i-RNA directed translation product. Total counts refer to the radioactivity eluted from the column.
specific neutralization of T2 phage. Using T2 i-RNA from rabbits with 4,4 allotype, the neutralizing activity was amplified by anti-allotype 4, but not with antiallotype 5 serum. Titers of about 40% neutralization at a 1 : 8 dilution were obtained. Likewise, the translation products with allotype 5,s i-RNA gave significant neutralization titers, and exhibited amplification of the neutralizing activity by anti-allotype 5 serum, but not with anti-allotype 4. No signficant neutralization was observed with the normal macrophage RNA translation product or the endogenous product. The data strongly suggest that the newly synthesized protein contains the allotype markers of the RNA donor rabbits immunoglobulins. Comjlex
Formation
of T2 Phage with i-RNA
Specified Proteins
Binding of phage with the labeled translation products from the cell-free system were carried out in an effort to further characterize the i-RNA specified proteins. The newly synthesized proteins from the cell-free extracts, were first fractionated by precipitation with 50% ammonium sulfate and then incubated with phage as described earlier. Separation of the T2 antigen-translation product complexes formed during this incubation from unbound proteins was carried out by Sepharose 2B gel filtration. Phage T2 and SP 52 eluted in the void volume of Sepharose 2B, whereas 19s immunoglobulins (IgM) appear in later fractions, clearly separated from both phage and 7S immunoglobulins (IgG). The elution patterns of both i-RNA and endogenous 3”S-labeled proteins were compared (Fig. 2). The bulk of the label is low molecular weight material (fractions 16 to 25). The label eluting in fractions 7-9 represents proteins bound to T2 or unspecific complexes of high molecular weight. While only 2.2% of the ammonium sulfate precipitated labeled proteins from the endogenous reaction eluted in fractions 8-9, S.6% of the i-RNA directed proteins eluted with T2 phage. The product from the endog-
316
BILELLO,
FISHMAN
FRACTION
FIG.
labeled in Fig. 30 min Phenol
.4XD
KOCH
NUMBER
3. Reduction of antigen-antibody complexes with ,%mercaptoethanol. ‘5-methionine T.2 i-RNA cell-free translation products were prepared and incubated with antigen as 2, subjected to reducing conditions, i.e., 100 mM P-merca’ptoethanol, 2 X lo-” M EDTA, at room temperature, then appled to a Sepharose 2B column as described in Fig. 2. red was used as a size marker.
enous reaction contained mostly low molecular weight proteins. In the elution pattern of the i-RNA directed translation product however, unbound product, still of high molecular weight, eluted after the T2 phage complex at the 19s position (Fig. 2, frac. 11-13). Gel filtration on Sephadex G-200 of this high molecular weight product yields a single peak of 800,000 molecular weight, the size of 19s immoglobulin. Some i-RNA directed translation product eluted in fractions 17 and 18 (7s position) and the bulk of the label in fractions 19 and 20 (Fig. 2). At this time these proteins have not been further characterized ; they may also carry T2 specificity. The 19s antibodies can be dissociated into 7S subunits by reduction with 100 mM P-mercaptoethanol as described. The mixture was again subjected to gel filtration on Sepharose ZB (Fig. 3). Forty-two percent of the labeled proteins synthesized in the presence of i-RNA remained specifically
FRACTION
NUMBER
FIG. 4. Dissociation of the antigen-antibody complexes. Fracti’ons 7 to 9, Fig. 3, were pooled, adjusted to pH 2.4 by addition of appropriate amounts of 10 N HCI. After incubation for 30 min at 37”, the fractions were rechromatographed in Sepharose 2B, 10e3M Tris, 1O-3M MgCL, 1.0 M NaCl pH 7.5. Phenol red was used as a size marker.
IMMUNE
2 4
RiKA
IS
6
IO 12 14 16 I8 20
8
FRACTION
hfESSENGER
RiTA
317
’
NUMBER
FIG. 5. Sucrose gradient analysis of the i-RNA directed translsation product. The i-RNA translation product was fractionated in a 10-37’5~ linear sucrose gradient in an SW-39 rotor at 33,000 rpm 16 hr at 4°C in the presence of normal rabbit serum. 0-0 percent neutralization of a test dose of T2 phage, 0-O optical density of normal rabbit serum determined spectrophotometrically at 280 nm.
bound to the T2 phage. No labeled proteins from the endogenous reaction remained T2 bound after exposure to p-mercaptoethanol. Complete dissociation of the complex between the i-RNA specified 7s proteins and T2 phage was achieved upon exposure of the complex to pH 2.4 and separation by Sepharose 2B column chromatography (Fig. 4). In control experiments, T2 i-RNA specified proteins and SP82 phage incubated mixtures were subjected to gel filtration of Sepharose 2B. Here 3--S% of the ammonium sulfate precipitated label eluted together with the SP82 phage. In contrast to the binding experiments with T2 phage, where 42% of the bound counts remained firmly bound to the T2 phage, no labeled proteins remained bound to SP82 phage after reduction with 100 mM p-mercaptoethanol. These results suggest that the translation product from T2 i-RNA stimulated reaction forms 19s antibodies which consist of 7s subunits with T2 specificity. Sucrose Gradient Analysis
of the Neutralizing
Activity
To determine the size of the in vitro product responsible for specific neutralization of T2, the T2 i-RNA stimulated reactions were subjected to sucrose gradient analysis (Fig. 5). The neutralization assays of the gradient fractions showed the neutralizing activity to be coincident with normal rabbit serum proteins having the 19s size of IgM. DISCUSSION Previous investigators have concluded that i-RNA functions as mRNA in cells in vitro (1, 5, 6) and in cell-free extracts (24, 25). Our results support this conclusion. Informational RNA induces an immune response in spleen cell cultures even in the presence of actinomycin D (Table 1) which blocks transcription of new RNA (26). Therefore, the production of specific antibodies in spleen cell
318
BILELLO,
FISHMAN
AND
KOCH
cultures after exposure to i-RNA is not dependent on newly synthesized RNA. In addition, informational RNA was extracted from polysomal fractions of macrophages 30 min after stimulation with antigen. Both these observations indicate the mRNA nature of i-RNA. That macrophages may indeed be antibody forming cells has been recently reported (27). If the i-RNA serves as an mRNA one should be able to assess its function in protein synthesis by demonstrating that it can be translated in cell-free systems. Several investigators have taken this approach, using cell-free extracts prepared from spleen cells (24, 25), however, the data obtained with the spleen cell system are subject to one severe criticism-the cell-free system may itself contain information required for antibody synthesis. In order to avoid this criticism, mouse L cells were selected as the source of the cell-free system assuming that they normally are not involved in antibody production. Consequently, de novo synthesis of specific neutralizing activity could be effected only by exogenous messenger activity of the i-RNA. Addition of rabbit T2 i-RNA to L cell-free extracts resulted in a 2-4 fold stimulation of incorporation of YY-methionine into proteins (23). The product synthesized in the cell-free extracts in response to i-RNA was analyzed in an allotype amplified phage neutralization assay. Only the RNA isolated from rabbit macrophages exposed to T2 phage was able to direct the synthesis of T2 phage specific neutralizing antibodies in the cell-free system. The specific product synthesized in the cell-free extracts in response to the i-RNA was found to sediment in sucrose gradients with 19s and to elute from Sepharose and Sephadex columns at the 19s position similar to that observed in tissue culture. The product of the cell-free system was heterogenous in size indicating the formation of proteins both smaller and larger than pentameric molecules of authentic IgM with the subunits consisting of 7s proteins only. The T2 i-RNA translation product formed a complex with T2 phage (Fig. 2). Exposure of the complex to 100 mM p-mercaptoethanol resulted in the release of about 607~ of the labeled proteins in the form of 7s proteins (Fig. 3). One explanation for the lack of complete resistance to mercaptoethanol is that not all of the 7s subunits carry antigen specificity. Under these same reducing conditions, however, none of the labeled proteins remained with the unrelated SP82 phage. The residual label bound to T2 dissociated upon exposure of the complex to pH 2.4. These results strongly indicate that i-RNA encodes and directs the synthesis of complete antibody molecules. The size of the i-RNA as determined by glycerol gradient centrifugation is 16 to 1% which is equavalent to 4 to 7.5 X 10” daltons of nucleic acid, enough to code for 40,000 to 75,000 daltons of proteins and could thus code for immunoglobulin heavy and light chains which are 55,000 and 22,000 daltons respectively (28). The studies of Fishman and Adler (14) indicate that macrophages, donors of immune RNA, are multifunctional and consist of functionally distinct subpopulations. In fact, experiments on cell separations on gradients or Ficoll (29) and on BSA gradients (3) demonstrated the existence of at least 5 subpopulations of macrophages. Evidence is accumulating that macrophages belonging to one of these subclasses of light density are the source of immunogenic RNA (3). The evidence presented here and other experimental data showing rapid incorporation of radioactive uridine into this RNA (25) support the view that
IMMUNE
RNA
IS
MESSENGER
RNA
319
i-RNA is newly synthesized by peritoneal macrophages when these cells are exposed to antigen. The close morphological association of macrophages with lymphocytes observed in vitro and in viva suggests that the i-RNA could be transferred, via cytoplasmic bridges, from macrophages to lympocytes (14). Such a mechanism would facilitate a rapid lateral spread of antibody formation and could account for the often observed rapid increase in IgM antibody producing cells which cannot readily be explained by cell division (16). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Meiss, H. K., and Fishman, M., J. Imwzu~tol. 108, 1172, 1971. Adler, F. L., Fishman, M., and Dray, S., J. Zmnazmol.97, 554, 1966. Rice, S. G., and Fishman, M., Cellular Imnzzcnol. 11, 130, 1974. Gottlieb, A. A., Science 165, 592, 1969. Bell, C., and Dray, S., Science 171, 199, 1971. Duke, L. J., Miller, C., and Harshman, S., Natzfre New Rio/. 235, 180, 1972. Jacherts, D., 2. Med. Mikrobiol. 152, 262, 1966. Mitsukashi, S., Kurashige, S., Kawakami, M., and Nojima, T., Jnfi. J. Microbial. 12, 261, 1968. 9. Jacherts, D., 2. Med. Mikrobiol. 152, 1, 1968. 10. Bell, C., and Dray, S., J. Zmmunol. 107, 83, 1971. 11. Thor, D. E., and Dray, S., J. Imnmnol. 101, 469, 1968. 12. Paque, R. E., and Dray, S., Cellular I~tanzzrnol.5, 30, 1972. 13. Likhite, V., and Sehon, A., Science 175, 204, 1972. 14. Fishman, M., Irt “The Role of RNA in Reproduction and Development” (Niu and Segal, Eds.), p. 127. North Holland Publ. Co., 1973. 15. Scherrer, K., and Darnell, J. E., Biocltcm. Bioq’fhys. Res. Cowwmn. 7, 486, 1962. 16. Mishell, R. I., and Dutton, R. W., J. E.rfi. Med. 126, 423, 1967. 17. McDowell, M. J., Joklik, W. K., Villa-Komaroff, L., and Lodish, H. F., Proc. Nat. Acad. Sci. USA 60, 2649, 1972 18. Mans, R. J., and Novelli, G. D., Arch. Biochcm. Biophgs. 94, 48, 1961. 19. Adler, F. L., Walker, W. S., and Fishman, M., Virology 46, 797, 1971. 20. Beale, D., and Feinstein, A., J. Biochent. 112, 187, 1969. 21. Schaefer, A. E., Fishman, M., and Adler, F. L., J. Inznzz~~zol.112, 1981, 1974. 22. Adler, L. T., Curley, D. M., and Fishman, M., J. Immzorol. 110, 811, 1973. 23. Bilello, P. A., Koch, G., and Fishman, M., Fed. Proc. 34, Abstract No. 4600. 24. Jachertz, D., Am. N.Y. Acad. Sci. 207, 122, 1973. 25. Mittlestaedt, R., and Koch, G. (manuscript in preparation). 26 Brockman, R. W., and Anderson, E. P., Am Rezl. Biochcm. 32, 463, 1962. 27. Lowy, I., Teplitz, R. L., and Bussard, A. E., Cellzllar Ztnmztnol. 16, 25, 1975. 28. Laskov, R., and Scharff, M. D., J. Exp. Med. 131, 515, 1970. 29. Walker, W. S., Nature New Biol. 229, 211, 1971.
IMMUNOLOGY
23, 309-319
Evidence That PATRICIA
BILELLO,~
(1976)
Immune ~~ARVIN
RNA Is Messenger FISF1hlAN,3
AND
GEBHARD
RNA1 Kocn
2 Roche Institute of Molecular Biology, hrutley, New Jersey 07110, and Biology Dcjartment, New York University, New York, New York, and s St. Jzrde Children’s Research Hospital, Memphis, Tennessee 38101 Received January
14,1976
Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19s proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.
INTRODUCTION When rabbit peritoneal exudate (PE) cells are incubated with bacteriophage, the cell extracts yield RNA capable of inducing the formation of phage specific neutralizing antibodies in rabbit spleen cell cultures (1). The cells involved as donors in these experiments were rabbit peritoneal exudate cells, consisting of 55-95 % macrophages (2, 3). The immunogenic activity of the PE cell homogenates resides in two RNA fractions. One of these fractions contains a biologically active RNA designated informational RNA (i-RNA), the other contains RNA-antigen complexes (2, 4). The i-RNA induces the formation of 19s immunoglobulins which contain the allotypic markers of the RNA donor rabbit (2). Informational RNA free of detectable antigen has been shown to induce humoral antibody production (5, 6, 7, S), and to transfer allotypic markers (9, 10). The ability of RNA to induce cellular immunity has also been demonstrated (11, 12, 13). The cell population responsible for the production of i-RNA and RNA-antigen complex has been separated from other PE cells by centrifugation on discontinuous bovine serum albumin (BSA) gradients (3). Immunogenic activity remains confined to approximately 15% of the total cells. It has been reported that synthesis of i-RNA is observed when PE cells are incubated with a low input of phage antigen to macrophage (1: 100) (14). Th us a small subpopulation of cells capable of recognizing the antigen are involved in the synthesis of i-RNA. Thus, while there appears to be evidence that i-RNA functions as mRNA, it has not yet been clearly established that i-RNA is capable of directing synthesis of both the light (L) and heavy (H) chains of immunoglobulins. These studies were undertaken in order to further define the role of i-RNA in initiating an immune response. The ability of i-RNA to function as a messenger RNA was 1 Supported in part by National Institutes of Health Grant AI 13180. 309 Copyright 0 1976 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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investigated by in vitro translation of i-RNA in cell-free protein synthesizing systems prepared from mouse L cells. Proteins synthesized in response to i-RNA were then characterized in neutralization and antigen binding assays and by sedimentation on sucrose gradients. MATERIALS Preparation of Inforvnational
AND
METHODS
RNA
Peritoneal exudate (PE) cells from rabbits homozygous at the b locus were obtained as previously described (2). The cells were suspended in solution B (0.15 J4 NaCl, 6 x 10m3M KCl, 15 ml phosphate buffer, distilled water to one liter, final pH 7.6). Ten ml of solution B containing 1.5 X 10” PE cells and 1.5 x 1O1l phage particles (high input) or 1.5 X lo7 phage particles (low input), either Escherichia coli phage T2 or Bacillus subtilis phage SP82, were incubated 30 min at 37°C without stirring. The RNA was extracted from PE cells by the hot phenol method (15) and was designated T2 i-RNA. Normal RNA refers to RNA extracted from PE cells not exposed to phage. The ethanol precipitated RNA was dissolved in sodium acetate buffer containing 0.14 M LiCl and 10e3 M MgSO+ Prior to use in in vitro translation studies, the RNA was extracted one more time with water-saturated phenol for 5 min at room temperature with intermittant shaking. The RNA from the aqueous phase was precipitated twice with ethanol, and dissolved in 1O-3 M EDTA pH 7.2. RNA concentration was determined from the absorbance measured spectrophotometrically using as the conversion factor the ratio of 40 pg RNA/ml for an ODzGo of 1.0 (1 cm light path). Spleen Cell Cultures The procedure used to prepare splenic cell cultures was that of Dutton and Mishell (16) with the major modification that normal rabbit serum (previously determined to be free of anti-T2 activity) replaced the fetal calf serum. Cultures (in 35 mm petri plates) consist of 2 X lo7 dispersed spleen cells in 1.0 ml medium (containing the RNA). These were incubated in an atmosphere of 7% 02, 10% COZ, 83% Na. Addition of the serum was delayed for 2 hr after initiation of the culture. Preparation
of Polysomal RNA
After incubation of the PE cells with T2 phage for 30 min at 37”C, the harvested cells were suspended in 5 ml MSB buffer (0.25 M KCl, 0.01 M MgC&, 0.01 M Tris-HCl pH 7.4). PE cell suspensions were disrupted in a Dounce homogenizer with 10 strokes in 3 ml MSB buffer. Ten percent Nonidet was added to give a final concentration of 1% Nonidet, the mixture was then incubated for 3 min at 4°C. The supernatant was recovered by centrifugation for 10 min at 12,OOOgand applied to a discontinuous sucrose gradient consisting of 5 ml 60% sucrose in MSB buffer and 15 ml 30% sucrose in MSB and the gradient was centrifuged in an SW25 rotor, at 100,OOOgfor 2.5 hr. Polysomes were removed from the 30/60 interface and the RNA extracted with 10 ml Tris-SDS, pH 9 (0.1 M Tris, 10 mM KCl, 0.2 mM MgClz, 0.5% SDS) and 10 ml phenol 10 min at 4°C with stirring. The aqueous phase was recovered by centrifugation for 10 min
IMMUNE
RNA
IS
MESSENGER
RNA
311
at 12,000g and the phenol phase was re-extracted with Tris buffer pH 9.0 (no SDS). The aqueous phases were combined and the RNA precipitated overnight with 2.5 volumes e,thyl alcohol and 0.1 volume 1% NaCl. The RNA was collected by centrifugation at 5009 for 10 min, washed with 6670 alcohol and 0.03% NaCl and resuspended in Solution B. Preparation of Cell-Free En-tracts The procedure used was analogous to that of McDowell et al. (17). Mouse L cell fibroblasts were maintained in suspension culture in Spinner Minimum Essential Medium supplemented with 5% fetal calf serum (Grand Island Biological Co., Grand Island, New York). The L cells at a concentration of 5 x lo” cells/ml were collected by centrifugation in a Sorvall RC-3 at 2000 rpm for 5 min, washed in buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N-‘2ethanesulfonic acid, Calbiochem, LaJolla, California 92037)) 0.15 M NaCl at pH 7.6, then suspended in 2 packed cell volumes of swelling buffer (10 mM HEPES, 10 mM KCl, 0.15 mM MgS04 6 mM ,f%-mercaptoethanol pH 7.6) for 10 min at 0°C. The cells were lysed with 1.5 strokes of the tight-fitting, B pestle in a Dounce homogenizer, and centrifuged at 10,OOOg for 10 min at 1°C in a Sorvall RC-2B. The resultant S10 supernatant was restored to isotonicity by the addition of 0.1 volume 10 x buffer B (see below) and 5% glycerol was added to stablize the extract. The S10 supernatants were passed over a 1.7 cm X 30 cm Sephadex G-25 (coarse) column previously equilibrated with buffer B (20 mM HEPES, 5 mM MgCIZ, 120 mM KCl, 6 mM ,8-mercaptoethanol, 6 mM dithiothreitol (DTT) pH 7.6 containing 5% glycerol). The most turbid fractions (O.D. at 260 nm and 280 nm ‘of 22 and 16) were pooled, aliquoted, and stored in liquid nitrogen. Translation
of i-RNA
in L-Cell-Free
Extracts
Reaction mixtures consisted of a total volume of 50 ~1: 10 ~1 cell-free extract, 100 mlM KCl, 12 m&1 HEPES pH 7.6, 3.0 mM Mg”, 8 mM DTT, 3.6 mM /?-mercaptoethanol, 5 pg creatine phosphokinase, 220 PM creatine phosphate, 1 mM ATP, 0.2 mM GTP, and 10 &i %-methionine (New England Nuclear, specific activity 300 Ci/mmole). The cell-free extracts were not pre-incubated and protein synthesis in the cell-free extracts was allowed to continue for 30 min at 25”. The effective dose range was 20-600 pg/ml of total RNA or 2-20 pg/ml of dT-cellulose purified RNA. Aliquots were removed at designated times and spotted on Whatman #3 paper discs (2.3 cm), presoaked with 50 ~1 of MEM 50 X essential amino acids (Grand Island Biological Co., Grand Island, N.Y.). TCA precipitable counts were cletermined by the method of Mans and Novelli (18). Briefly, the filters were precipitated in 10% tricholoracetic acid TCA for 30 min, washed 2~ with 5% TCA, and heated at 90-95” for 15 min, washed 2X again with ether for 5 min. The dried filters were counted in LSC Complete (Yorktown Research, Hackensack, N. J.) in a Beckman LS-200 liquid scintillation counter. Binding of Antibody
to T2 Plaage
The i-RNA and the endogenous translation products were precipitated with neutralized 50% (NHa)&Os at O”, and centrifuged at 120009 20 min at 1°C.
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BILELLO,
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The precipitate was resuspended in an amount of isotonic buffer (0.15 M NaCl, 10e3 1M Tris pH 7.5) resulting in a 4- to S-fold concentration of the translation product. Then, 100 ~1 of translation product concentrates were incubated with 200 ~1 T2 phage 2 X 1Ol2 plaque forming units/ml for one hr at 37”C, then overnight at 4”. Antigen-antibody complexes were separated by gel filtration on a Sepharose 2B column 0.8 X 15 cm previously equilibrated with a buffer containing 10e3 M Tris, 10m3M MgClz and 1.0 M NaCl at pH 7.5. Phenol red was used as a marker. Neutralization
Assays
Serial 2-fold dilutions of the fluids to be tested were made in Solution B containing 0.1% fetal calf serum. After the addition of a constant amount of phage (400 PFU/tube) the mixture was incubated 30 min at 37°C. Duplicate or triplicate aliquots, each containing 100 PFU/tube if no neutralization has occurred were then plated on E. coli B. (for T2 phage) or on the indicator strain of B. subtilis (for SP82 phage). Plaques were counted after overnight incubation at 37°C. Where complement was employed to amplify neutralization (19), serum from normal rats (selected for low background anti-T2 neutralizing antibodies) at a final dilution of 1 : 20, was added to the incubation mixture. Sera used as a source of complement were never heat inactivated. Allotype amplification (19) of neutralization was performed as follows: 400 PFU of phage were added to the test extracts and incubated for 30 min at 37” followed by a second incubation for 90 min at 4” ; anti-allotype serum (1 : 10 dilution) was added and after an incubation for 30 min at 37” aliquots were plated as outlined above. Reagents used in the allotype amplification of neutralization were prepared by intensive immunization of appropriate rabbits with rabbit (Yglobulin. The anti-4 reagent was made in al, a1/b5, b5 rabbits by injection of al, a1/b4, b4 globulin, and the anti-5 serum by immunization of al, a1/b4, b4 rabbits with a’, a1/b5, b5 globulin. Reduction of Antigen-Antibody
Complexes
35S-methionine-labeled translation products were incubated with T2 or SP82 phage as decribed. After separation of the antigen-antibody complexes on Sepharose 2B, the complexes were reduced by 100 mM p-mercaptoethanol in the presence of 2 x 10m3M EDTA for 30 min at room temperature (20). Dissociation of Antigen-Antibody
Complexes at Low pH
Fractions containing the 35S-methionine label bound to T2 phage after reduction with p-mercaptoethanol were adjusted to pH 2.4 by the addition of the appropriate amounts of 10 N HCl. After incubation for 30 min at 20”, the fractions were subjected to gel filtration in Sepharose 2B. Sucrose Density Gradient Centrifugation Translation products synthesized in the L cell-free system were fractionated on a 10-37s linear surcose gradient (1) by centrifugation at 33,000 rpm in an SW39 rotor for 16 hr. The position of normal rabbit serum 19s immunoglobulins was determined spectrophotometrically at 280 nm.
313 RESULTS Induction of Antibody Formation in Spleen Cell Cultures by InzvlcMnogenic RNA in the Presence of Actinowzycin D When T2 antigens, solubilized in 0.5% SDS or T2 i-RNA, were added to dispersed spleen cell cultures antibody synthesis was induced and antibodies were secreted into the tissue culture fluid (1). Ninety-six hours after stimulation the fluids from 23 plates of cell cultures were pooled, and the cells and debris were removed by centrifugation. The tissue culture fluids were assayed for antibody activity in the complement enhanced neutralization test, (19). A neutralization of 25% of the test dose of phage by a given dilution of tissue culture fluid was highly significant (21, 22). The synthesis of phage neutralizing activities by spleen cell cultures in the presence or absence of Actinomycin D is shown in Table 1. Synthesis of phage neutralizing activities in spleen cell cultures following the addition of 10e4 pg protein nitrogen SDS-solublized T2 was inhibited by the presence of as little as 1 pg/ml of Actinomycin D. In marked contrast, the induction of T2 neutralizing activity by T2 i-RNA was found to be unaffected by actinomycin D which provides strong experimental evidence suggesting that i-RNA itself can act as mRNA for the synthesis of T2 phage antibodies in the spleen cells. Induction
of Antibodies
by RNA
Isolated
from
the Polysome Fractions
If i-RNA functions as mRT\‘A in peritoneal exudate cells, then this RNA should be present on the polysomes. RNA isolated from polysome fractions was added to spleen cell cultures. Figure 1 illustrates that 20 PLg polysomal RNA/ culture is sufficient to induce an immune response as measured in the neutralization assay. This antibody response is equivalent to one produced by lo&200 pg unfractionated RNA (total). Normal RNA was added to the polysomal RNA so that each culture received a total of 100 pg of RNA. The response increases significantly with increasing doses of i-RNA. These results provide further support that i-RNA acts as an mRNA. TABLE
1
Effect of Actinomycin D on the Immunogenicity and T2 i-RNA in Spleen Culture Addition
T2 i-RNA
to spleen cells
300 ~g
10m4 pg T2 solubilized
Actinomycin 1 P kc/ml
t +
D
of Solubilized Cells
7ONeutralization
T2
of T2
1:4
1:8
1:16
50 48 45 6
41 35 43 10
35 28 27 7
T2 i-RNA was obtained from cells exposed to T2 phage at a ratio of 1 phage particle per 100 cells. A constant amount of phage and complement at a final dilution of 1: 20 was added to serial 2-fold dilutions of the fluid to be tested, then incubated 30 min at 37”. Duplicate or triplicate aliquots were plated on E. coli B. Neutralization values of 20”’,jOor less do not represent significant antibody.
314
BILELLO,
I:16
FISHMAN
AND
KOCH
r
0
I 20
II
I 40
I 60
I
I, 80
1 100
pg POLYSOMAL RNA FIG. 1. Antibody induction by polysomal RNA. Polysomal RNA isolated from PE cells previously stimulated with T2 antigen was added to spleen cell cultures. Each culture received a total of 100 pg of RNA, that is, normal RNA was added to the polysomal RNA in appropriate quantities. Neutralization is expressed as 30% neutralization titers of a test dose of phage at the indicated dilution of the tissue culture fluid.
Synthesis of Antibody in a Cell-Free System To determine if i-RNA prepared from allotype specific rabbit PE cells contained all the coding information to direct the synthesis of proteins which possessed T2 neutralizing activity, T2 i-RNA was added to S1Ocell-free extracts prepared from mouse L cells. Synthesis of protein in the SIO extracts was followed by monitoring the incorporation of 36S-methionine. Addition of T2 i-RNA stimulated the incorporation of 35S-methionine 2-4 fold above the incorporation of the endogenous reaction (23). The specificity of the T2 i-RNA directed protein synthesis in the cell-free extracts was analyzed by an allotype amplified T2 phage neutralization assay. As shown in Table 2 neutralization assays performed with the in vitro translation products in the presence of anti-allotype serum give TABLE Amplification L-cell-free Translation
of Neutralization
2
of T2 Phage with Anti-Allotype-4
‘rONeutralization
system product
anti-4 1:4
1,1/4,4 T2 i-RNA (high)* 1,1/4,4 T2 i-RNA (low)* 1,1/4,4 Normal RNA Endogenous 2,3/5,5 i-RNA Control (Anti-4 or -5 only)
Serum of T2 anti-5
1:8
1:8
19
37
0
39
32
9
12
13
0
19
16 2
0 25
10 10-12
2-10
* RNA was extracted from cells previously exposed to phage at a ratio of: high = 100 phage particles/cell low = 1 phage particle/100 cells Serial dilutions of the fluids to be tested were incubated with a constant amount of phage 30 min at 37”, then 90 min at 4”. Antiallotype serum at 1: 10 dilution was added and incubated 30 min at 37”. Duplicate or triplicate aliquots were plated on E. roli B. Neutralization values of 20% or less do not represent significant antibody.
IMMIJNE
RNA
IS
MESSENGER
FRACTION
RNA
315
NUMBER
FIG. 2. Separation of antigen-antibody complexes. %S-mcthionine labeled products from the cell-free translation were precipitated with 50% (NH,)&O,, concentrated 5-fold, incubated with T2 phage as described in Materials and Methods and applied to Q Sepharose 2B column equilibrated with buffer containing 10m3M Tris, 10m3M MgCL, and 1.0 M NaCl at pH 7.5. Phenol red was used as a size marker. l --endogenous product; n--T2 i-RNA directed translation product. Total counts refer to the radioactivity eluted from the column.
specific neutralization of T2 phage. Using T2 i-RNA from rabbits with 4,4 allotype, the neutralizing activity was amplified by anti-allotype 4, but not with antiallotype 5 serum. Titers of about 40% neutralization at a 1 : 8 dilution were obtained. Likewise, the translation products with allotype 5,s i-RNA gave significant neutralization titers, and exhibited amplification of the neutralizing activity by anti-allotype 5 serum, but not with anti-allotype 4. No signficant neutralization was observed with the normal macrophage RNA translation product or the endogenous product. The data strongly suggest that the newly synthesized protein contains the allotype markers of the RNA donor rabbits immunoglobulins. Comjlex
Formation
of T2 Phage with i-RNA
Specified Proteins
Binding of phage with the labeled translation products from the cell-free system were carried out in an effort to further characterize the i-RNA specified proteins. The newly synthesized proteins from the cell-free extracts, were first fractionated by precipitation with 50% ammonium sulfate and then incubated with phage as described earlier. Separation of the T2 antigen-translation product complexes formed during this incubation from unbound proteins was carried out by Sepharose 2B gel filtration. Phage T2 and SP 52 eluted in the void volume of Sepharose 2B, whereas 19s immunoglobulins (IgM) appear in later fractions, clearly separated from both phage and 7S immunoglobulins (IgG). The elution patterns of both i-RNA and endogenous 3”S-labeled proteins were compared (Fig. 2). The bulk of the label is low molecular weight material (fractions 16 to 25). The label eluting in fractions 7-9 represents proteins bound to T2 or unspecific complexes of high molecular weight. While only 2.2% of the ammonium sulfate precipitated labeled proteins from the endogenous reaction eluted in fractions 8-9, S.6% of the i-RNA directed proteins eluted with T2 phage. The product from the endog-
316
BILELLO,
FISHMAN
FRACTION
FIG.
labeled in Fig. 30 min Phenol
.4XD
KOCH
NUMBER
3. Reduction of antigen-antibody complexes with ,%mercaptoethanol. ‘5-methionine T.2 i-RNA cell-free translation products were prepared and incubated with antigen as 2, subjected to reducing conditions, i.e., 100 mM P-merca’ptoethanol, 2 X lo-” M EDTA, at room temperature, then appled to a Sepharose 2B column as described in Fig. 2. red was used as a size marker.
enous reaction contained mostly low molecular weight proteins. In the elution pattern of the i-RNA directed translation product however, unbound product, still of high molecular weight, eluted after the T2 phage complex at the 19s position (Fig. 2, frac. 11-13). Gel filtration on Sephadex G-200 of this high molecular weight product yields a single peak of 800,000 molecular weight, the size of 19s immoglobulin. Some i-RNA directed translation product eluted in fractions 17 and 18 (7s position) and the bulk of the label in fractions 19 and 20 (Fig. 2). At this time these proteins have not been further characterized ; they may also carry T2 specificity. The 19s antibodies can be dissociated into 7S subunits by reduction with 100 mM P-mercaptoethanol as described. The mixture was again subjected to gel filtration on Sepharose ZB (Fig. 3). Forty-two percent of the labeled proteins synthesized in the presence of i-RNA remained specifically
FRACTION
NUMBER
FIG. 4. Dissociation of the antigen-antibody complexes. Fracti’ons 7 to 9, Fig. 3, were pooled, adjusted to pH 2.4 by addition of appropriate amounts of 10 N HCI. After incubation for 30 min at 37”, the fractions were rechromatographed in Sepharose 2B, 10e3M Tris, 1O-3M MgCL, 1.0 M NaCl pH 7.5. Phenol red was used as a size marker.
IMMUNE
2 4
RiKA
IS
6
IO 12 14 16 I8 20
8
FRACTION
hfESSENGER
RiTA
317
’
NUMBER
FIG. 5. Sucrose gradient analysis of the i-RNA directed translsation product. The i-RNA translation product was fractionated in a 10-37’5~ linear sucrose gradient in an SW-39 rotor at 33,000 rpm 16 hr at 4°C in the presence of normal rabbit serum. 0-0 percent neutralization of a test dose of T2 phage, 0-O optical density of normal rabbit serum determined spectrophotometrically at 280 nm.
bound to the T2 phage. No labeled proteins from the endogenous reaction remained T2 bound after exposure to p-mercaptoethanol. Complete dissociation of the complex between the i-RNA specified 7s proteins and T2 phage was achieved upon exposure of the complex to pH 2.4 and separation by Sepharose 2B column chromatography (Fig. 4). In control experiments, T2 i-RNA specified proteins and SP82 phage incubated mixtures were subjected to gel filtration of Sepharose 2B. Here 3--S% of the ammonium sulfate precipitated label eluted together with the SP82 phage. In contrast to the binding experiments with T2 phage, where 42% of the bound counts remained firmly bound to the T2 phage, no labeled proteins remained bound to SP82 phage after reduction with 100 mM p-mercaptoethanol. These results suggest that the translation product from T2 i-RNA stimulated reaction forms 19s antibodies which consist of 7s subunits with T2 specificity. Sucrose Gradient Analysis
of the Neutralizing
Activity
To determine the size of the in vitro product responsible for specific neutralization of T2, the T2 i-RNA stimulated reactions were subjected to sucrose gradient analysis (Fig. 5). The neutralization assays of the gradient fractions showed the neutralizing activity to be coincident with normal rabbit serum proteins having the 19s size of IgM. DISCUSSION Previous investigators have concluded that i-RNA functions as mRNA in cells in vitro (1, 5, 6) and in cell-free extracts (24, 25). Our results support this conclusion. Informational RNA induces an immune response in spleen cell cultures even in the presence of actinomycin D (Table 1) which blocks transcription of new RNA (26). Therefore, the production of specific antibodies in spleen cell
318
BILELLO,
FISHMAN
AND
KOCH
cultures after exposure to i-RNA is not dependent on newly synthesized RNA. In addition, informational RNA was extracted from polysomal fractions of macrophages 30 min after stimulation with antigen. Both these observations indicate the mRNA nature of i-RNA. That macrophages may indeed be antibody forming cells has been recently reported (27). If the i-RNA serves as an mRNA one should be able to assess its function in protein synthesis by demonstrating that it can be translated in cell-free systems. Several investigators have taken this approach, using cell-free extracts prepared from spleen cells (24, 25), however, the data obtained with the spleen cell system are subject to one severe criticism-the cell-free system may itself contain information required for antibody synthesis. In order to avoid this criticism, mouse L cells were selected as the source of the cell-free system assuming that they normally are not involved in antibody production. Consequently, de novo synthesis of specific neutralizing activity could be effected only by exogenous messenger activity of the i-RNA. Addition of rabbit T2 i-RNA to L cell-free extracts resulted in a 2-4 fold stimulation of incorporation of YY-methionine into proteins (23). The product synthesized in the cell-free extracts in response to i-RNA was analyzed in an allotype amplified phage neutralization assay. Only the RNA isolated from rabbit macrophages exposed to T2 phage was able to direct the synthesis of T2 phage specific neutralizing antibodies in the cell-free system. The specific product synthesized in the cell-free extracts in response to the i-RNA was found to sediment in sucrose gradients with 19s and to elute from Sepharose and Sephadex columns at the 19s position similar to that observed in tissue culture. The product of the cell-free system was heterogenous in size indicating the formation of proteins both smaller and larger than pentameric molecules of authentic IgM with the subunits consisting of 7s proteins only. The T2 i-RNA translation product formed a complex with T2 phage (Fig. 2). Exposure of the complex to 100 mM p-mercaptoethanol resulted in the release of about 607~ of the labeled proteins in the form of 7s proteins (Fig. 3). One explanation for the lack of complete resistance to mercaptoethanol is that not all of the 7s subunits carry antigen specificity. Under these same reducing conditions, however, none of the labeled proteins remained with the unrelated SP82 phage. The residual label bound to T2 dissociated upon exposure of the complex to pH 2.4. These results strongly indicate that i-RNA encodes and directs the synthesis of complete antibody molecules. The size of the i-RNA as determined by glycerol gradient centrifugation is 16 to 1% which is equavalent to 4 to 7.5 X 10” daltons of nucleic acid, enough to code for 40,000 to 75,000 daltons of proteins and could thus code for immunoglobulin heavy and light chains which are 55,000 and 22,000 daltons respectively (28). The studies of Fishman and Adler (14) indicate that macrophages, donors of immune RNA, are multifunctional and consist of functionally distinct subpopulations. In fact, experiments on cell separations on gradients or Ficoll (29) and on BSA gradients (3) demonstrated the existence of at least 5 subpopulations of macrophages. Evidence is accumulating that macrophages belonging to one of these subclasses of light density are the source of immunogenic RNA (3). The evidence presented here and other experimental data showing rapid incorporation of radioactive uridine into this RNA (25) support the view that
IMMUNE
RNA
IS
MESSENGER
RNA
319
i-RNA is newly synthesized by peritoneal macrophages when these cells are exposed to antigen. The close morphological association of macrophages with lymphocytes observed in vitro and in viva suggests that the i-RNA could be transferred, via cytoplasmic bridges, from macrophages to lympocytes (14). Such a mechanism would facilitate a rapid lateral spread of antibody formation and could account for the often observed rapid increase in IgM antibody producing cells which cannot readily be explained by cell division (16). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Meiss, H. K., and Fishman, M., J. Imwzu~tol. 108, 1172, 1971. Adler, F. L., Fishman, M., and Dray, S., J. Zmnazmol.97, 554, 1966. Rice, S. G., and Fishman, M., Cellular Imnzzcnol. 11, 130, 1974. Gottlieb, A. A., Science 165, 592, 1969. Bell, C., and Dray, S., Science 171, 199, 1971. Duke, L. J., Miller, C., and Harshman, S., Natzfre New Rio/. 235, 180, 1972. Jacherts, D., 2. Med. Mikrobiol. 152, 262, 1966. Mitsukashi, S., Kurashige, S., Kawakami, M., and Nojima, T., Jnfi. J. Microbial. 12, 261, 1968. 9. Jacherts, D., 2. Med. Mikrobiol. 152, 1, 1968. 10. Bell, C., and Dray, S., J. Zmmunol. 107, 83, 1971. 11. Thor, D. E., and Dray, S., J. Imnmnol. 101, 469, 1968. 12. Paque, R. E., and Dray, S., Cellular I~tanzzrnol.5, 30, 1972. 13. Likhite, V., and Sehon, A., Science 175, 204, 1972. 14. Fishman, M., Irt “The Role of RNA in Reproduction and Development” (Niu and Segal, Eds.), p. 127. North Holland Publ. Co., 1973. 15. Scherrer, K., and Darnell, J. E., Biocltcm. Bioq’fhys. Res. Cowwmn. 7, 486, 1962. 16. Mishell, R. I., and Dutton, R. W., J. E.rfi. Med. 126, 423, 1967. 17. McDowell, M. J., Joklik, W. K., Villa-Komaroff, L., and Lodish, H. F., Proc. Nat. Acad. Sci. USA 60, 2649, 1972 18. Mans, R. J., and Novelli, G. D., Arch. Biochcm. Biophgs. 94, 48, 1961. 19. Adler, F. L., Walker, W. S., and Fishman, M., Virology 46, 797, 1971. 20. Beale, D., and Feinstein, A., J. Biochent. 112, 187, 1969. 21. Schaefer, A. E., Fishman, M., and Adler, F. L., J. Inznzz~~zol.112, 1981, 1974. 22. Adler, L. T., Curley, D. M., and Fishman, M., J. Immzorol. 110, 811, 1973. 23. Bilello, P. A., Koch, G., and Fishman, M., Fed. Proc. 34, Abstract No. 4600. 24. Jachertz, D., Am. N.Y. Acad. Sci. 207, 122, 1973. 25. Mittlestaedt, R., and Koch, G. (manuscript in preparation). 26 Brockman, R. W., and Anderson, E. P., Am Rezl. Biochcm. 32, 463, 1962. 27. Lowy, I., Teplitz, R. L., and Bussard, A. E., Cellzllar Ztnmztnol. 16, 25, 1975. 28. Laskov, R., and Scharff, M. D., J. Exp. Med. 131, 515, 1970. 29. Walker, W. S., Nature New Biol. 229, 211, 1971.
