Eur. J. Immunol. 1975.5: 47-53
R.H. Stevens, Brigitte A. Askonas and Jacqueline L. Welstead National Institute for Medical Research, London
H-chain mRNA in LPS-treated cells
47
Immunoglobulin heavy chain mRNA in mitogen-stimulated B cells This paper relates the synthesis of DNA, immunoglobulin and heavy chain (H) mRNA in murine spleen cells following activation of B cells with lipopolysaccharide from E. coli (LPS). Spleen cells (CBA/H mice) were cultivated with 10 % FCS and 10 pg LPS/ml. 4 h pulses with [ 3H]thymidine showed that DNA synthesis was stimulated within the first day following LPS activation and exhibited a sharp peak at 24 h. The shape of the DNA synthesis curve suggests that the cells susceptible to LPS stimulation are activated in a synchronous manner. Stimulation of Hchain mRNA (H-mRNA) synthesis proceeded rapidly (within 6 h of LPS addition) and peaked around 24 h, in parallel to DNA synthesis. The H-mRNA was isolated and quantitated by making use of its interaction with IgG [ 1, 21. The actual level of H-mRNA in the culture increased threefold during the first 24 h and then doubled within the next 48 h. Estimates of the actual number of H-mRNA were approximately 200 molecules H-mRNA/cell on day 0 rising to I800/cell on day 3. In such a mixed cell population these figures will be accurate only within a factor of 2-3 (at least 35 % B cells in spleen cell suspensions at the commencement of the culture, with up to 35-60 % of plasma blasts by day 3 and 4 of LPS treatment). Translation of the lymphoid cell mRNA in oocytes from Xenopus laeuis demonstrated that stimulation of H-mRNA synthesis was restricted to p-mRNA, although some 7-mRNA was present in the original spleen cells. High levels of synthesis of immunoglobulin followed after a lag period of about 24 h following LPS addition peaking after 48 and 7 2 h; the proportional lg production relative to total protein synthesis reached 26 % on days 3 and 4. Stimulation of Ig production was limited to IgM. Rapid stimulation of mitosis and H-mRNA synthesis thus precedes the maximum synthesis of Ig molecules, suggesting a translational block on H-mRNA during cell maturation. There was no apparent block on the transport of H-mRNA from the nucleus during early stages of activation.
1 . Introduction Antigenic challenge results in proliferation and differentiation of antigen-sensitive lymphocytes which recognize a particular antigen. This leads to the development of blast cells and then plasma cells with an organized endoplasmic reticulum and high levels of immunoglobulin (Ig) production and secretion. Although production of Ig at the stage of the mature plasma cell has been analyzed extensively [3, 41, we know little of the events in induction of Ig production and plasma cell maturation. Biochemical studies with antigenstimulated lymphoid cells have been difficult, since lymphoid tissue is made up of a mixed cell population. Responses to individual antigens are multiclonal, but only a small proportion of lymphoid cells respond to individual antigens. Induction of high levels of Ig production is therefore restricted to a small percentage of the spleen cell population.
rI 7661 ~~~
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* Present address: Department of Microbiologyand Immunology, School of Medicine, U.C.L.A., Los Angeles, Calif. 90024, USA. Correspondence: B.A. Askonas, National Institute for Medical Research, Mill Hill, London NW7 lAA, GB Abbreviations: LPS: Lipopolysaccharidefrom E. coli PBS: Phosphate buffered saline F a : Fetal calf serum PWM: Pokeweed mitogen Con A: Concanavalin A mRNA: Messenger RNA H-mRNA: H-chain mRNA T cells: Thymus-derived lymphocytes B cells: Non-thymusdependent lymphocytes PFC: Plaque-forming cells TCA: Trichloroacetic acid TRF: Thymus cell-replacingfactor SDS: Sodium dodecyl sulfate
In an attempt to study the relation between cell division, transcription of Ig genes, translation of Ig mRNA molecules and cellular maturation, we therefore turned to mitogen-activated B cells as preliminary model system. I t has been reported previously that various substances such as pokeweed mitogen (PWM) [5], insolubilized concanavalin A (Con A), lipopolysaccharide (LPS) from gram-negative bacteria and various polysaccharides [ 6 , 7 ] activate a high proportion of mouse B cells. This results in spleen cell cultures in uitro, in mitosis, blast cell formation and subsequent maturation of B cells with high levels of Ig production..Ig formation of up t o 20-30 % of total protein synthesis has been observed after 3-4 days of tissue culture [8, 91. Lipopolysaccharide (LPS) from E. coli appears to be the most reproducibly powerful B cell activator with the strongest enhancement of IgM synthesis, whereas PWM and Con A also stimulate T cells. For these reasons LPS treatment of mouse spleen cultures has been used for this study. The observation that heavy chain (H) messenger RNA (HmRNA) interacts with Ig has enabled the isolation of heavy chain message quantitatively in multiple samples [2]. The type of Ig coded for WBS then assayed in frog oocytes following injection of mRNA and its subsequent translation [ 12, 131. Quantitative assays of immunoglobulin H-mRNA have proved possible utilizing the interaction between H-mRNA and IgG [ 21 and a modification of the nitrocellulose binding assay for measuring RNA-protein interaction [ 14, 151. In this paper we report detailed kinetics of DNA, Ig, and H-mRNA synthesis following LPS stimulation of B cells in spleen.
48
R.H. Stevens, B.A. Askonas and J.L. Welstead
2. Materials and methods 2.1. Spleen cell suspension Spleens from CBA/H mice ( 3 - 4 months old) were suspended in RPMI 1640 medium (Flow Labs., Irvine, Scotland), containing 10 % FCF serum (Biocult Labs., Glasgow, Scotland) by teasing with forceps. Clumps of cells were permitted to settle 1-2 min, She cell suspension removed and centrifuged 8 min at 200 x g. The cells were washed once with medium, and cultured for 4 days at 5 x 1 O6 cells/ml in the above medium in petri dishes ( 4 or 12 ml in Falcon dishes 3002 or 3003 respectively) in 5 % Co2/95 % air at 37 "C. Cell viability was estimated in 0.02 % trypan blue in PBS. LPS from E. coli (Difco 055BS, Detroit, Mich.) was used at 10 pg/ml. At appropriate times all cells were suspended with a silicone rubber policeman; DNA and protein synthesis, and mRNA were analyzed on suitable aliquots.
2.2. [3H]thymidine incorporation At various time intervals, 1.5 x 1 O6 viable cells were pulsed with 1 pCi [3H]thymidine (I Cilrnmol) for 4 h at 37 "C in 1 ml of the above medium (Falcon dishes 3001). The cells were harvested on Whatman glass fiber filters (GF/A), washed three times each with PBS, 7 % trichloroacetic acid (TCA) and ethanol, dried with ether and counted in Kinard's [ 161 scintillation f h i d .
Eur. J. Immunol. 1975.5: 47-53
washed with diethyl ether-ethanol ( 3 : I ) , air dried and resup pended in 1 ml binding buffer (500 mM KCI, 10 mM TrisHCI, I mM MgCI2, pH 7.4) and applied to a 5 ml cellulose column equilibrated with binding buffer [ 181. More than 95 % of ribosomal RNA was removed by washing with 20 ml binding buffer and the mRNA fraction eluted by washing with 10 mM Tris-HCI, pH 7.6. This mRNA fraction was used for all assays. Recovery of mRNA by this procedure was consistently between 75 and 80 % of total mRNA; this was determined from the recovery of a measured amount of purified radioactive H-mRNA (from 5563 myeloma cells) following phenol extraction with nonradioactive spleen cells.
2.5. Polyacrylamide gel analysis of immune precipitates Washed immune precipitates were dissolved by boiling in 2 % SDS for 2 min. For analysis of unreduced immune precipitates the samples were made t o 2 % with iodoacetamide and loaded directly on top of 7.5 7% acrylamide gels containing SDS. Reduced immune precipitates were prepared by dissolving in 2 % SDS as above and adding 2-mercaptoethanol to 1 %. The samples were incubated for 1 h at room temperature, iodoacetamide added (to 2 %) and the samples loaded onto the polyacrylamide gels containing SDS. Electrophoresis was conducted at I0 mA/gel for 5 h at room temperature. Following electrophoresis the gels were sliced and dissolved in NH40H. Scintillation fluid [ 171 was added and the radioactivity determined in a Beckman LS-I 5 0 scintillation counter.
2.3. [''Slmethionine incorporation 1.5 x 1 O6 viable spleen cells were pulsed for I h at 3 7 O C with 10 pCi/ml of [35S]methionine (Radiochemical Centre, Amersham, I60 Ci/mmol) in EDM medium lacking methionine. The cells were pelleted by centrifugation, lysed in 0.1 ml 0.5 % deoxycholate, adjusted to 1.5 ml with PBS and centrifuged 3 0 min at 35 000 x g. Methionine incorporation was analyzed (a) into total cellular protein precipitated with 7 % TCA, (b) Ig precipitated at equivalence with specific rabbit antiserum to mouse IgM and IgG in the presence of carrier Ig, (c) control antibody/antigen precipitates (rabbit IgG and goat anti-rabbit IgG). After three washes with PBS at 4 O C , the precipitates dissolved in 0. I ml 0.1 N NaOH were counted in Bray's [ 171 scintillation fluid. The radioactivity of the control precipitates was subtracted from the radioactivity of specific Ig precipitates to give the incorporation into Ig. 2.4. Extraction of lymphocyte mRNA Total cellular RNA was prepared from lymphoid cells as described by Stevens [2]. Briefly, 10' cells were washed with PBS ( I 50 mM NaCI, 1 mM MgCI2, 1 mM CaCI2, 10 mM phosphate buffer, pH 7.6) and resuspended in 1 5 ml RNA extraction buffer (100 mM sodium acetate, 2 0 0 mM NaCI, 2 mM EDTA, 0.1 % sodium dodecyl sulfate (SDS), pH 5.0 with acetic acid) and mixed with two volumes of a phenol (redistilled)-chloroform-isoarnylalcohol mixture (3: 1 : 0:0.05). The samples were shaken vigorously for 5 min in a 4 5 "C water bath and then chilled on ice. The samples were then centrifuged (2000 x g for 5 min) and the aqueous layer removed. An additional 10 ml of extracting buffer was added to the original phenol and the extraction repeated. The two aqueous layers were combined and extracted with an equal volume of fresh phenol-chloroform-isoamyl alcohol mixture. The final aqueous layer was mixed with 2 vol of ethanol and the RNA allowed to precipitate overnight at - 20 O C . The RNA was recovered by centrifugation ( I 5 000 x g for 10 min),
Ig produced by the spleen cells was analyzed as described above except that 5 % SDS polyacrylamide gels [ 191 were utilized and electrophoresis was for 3 1/2 h. The gel slices were solubilized as described by Choules and Zimm [ 201 and counted in Kinard's fluid [ 161.
2.6. Oocyte translation of lymphocyte mRNA Mature oocytes were obtained from Xenopus Iaevis and were injected with mRNA as described previously [ 12, 131. Following 24 h incubation at room temperature in the presence of [3'S] methionine, the oocytes (50 per sample) were homogenized in 2 ml PBS containing 1 % NP-40 (Shell Chemicals). The cellular debris was removed by centrifugation (I0 000 x g for IS min) and the mousespecific immunoglobulins in the supernatant fluids were isolated by direct antibody precipitation [ 131.
2.7. Quantitative estimation of H-mRNA Quantitative determinations of H-mRNA were performed by the nitrocellulose filter assay [2]. Samples of total mRNA ( 3 H or 3zP-labeled) and IgG (purified rabbit anti-DNP antibody with high affinity [21], kindly provided by Dr. N.M. Green), were mixed at room temperature ( I 00 p1 total volume) and allowed to come to equilibrium ( 7 min). The reaction conditions were 150 mM NaCI, 1 mM MgCI2, 1 mM CaC12, 10 mM phosphate buffer, pH 7 h . DNP ovalbumin ( 5 pg) was added and duplicate 50 pl samples were filtered through nitrocellulose filters (Millipore, Type HA, 0.45 p pore size) and washed with 0.4 ml PBS. The samples were air dried and the radioactivity determined. The DNPovalbumin does not participate in the reaction, but combination with the anti-DNP antibody allows firmer binding of the protein-RNA complex to the filter and provides more reliable resu It s.
Eur. J. Immunol. 1975.5: 47-53
3.Results 3.1. General remarks For detailed examination of mRNA production, 0.5 x 10' - 1 x 10' cells were required for each sample and cultures were therefore plated at a density of 5 x lo6 cells/ml, scaled up to a volume of 12 ml in petri dishes.
Optimal stimulation of Ig formation in the 3-4 day cultures occurred with doses of 10 pg LPS/ml. Higher concentrations of LPS led to increased cell death and lower doses resulted in lower levels of DNA and Ig stimulation. We first estab lished the kinetics of [ 3H]thymidine incorporation and Ig production relative to total protein synthesis in these particular cultures, which showed somewhat lower cell viability than the small scale cultures. Treatment of normal spleen cells with LPS led to a rapid increase in DNA synthesis after the first 6 h of culture with a peak around 24 h (Fig. 1 , 4 h pulses). By 48 h, DNA synthesis had dropped, but was still severalfold higher than in the control cultures. Days 3 and 4 showed a further general decrease in thymidine incorporation. The control cultures showed some stimulation of DNA synthesis which probably is attributable FCS, as different batches of FCS give rise to different levels of thymidine incorporation. This stimulation in the control cultures could result from the stimulation of either T or B cells in the mixed spleen cell population. The DNA stimulation over and above the levels found in control cultures can be assigned t o B cell mitosis, since LPS does not stimulate T cells [22].
H-chain mRNA in LPStreated cells
49
duction (26 % of total protein by day 3) indicates that if half of the cells are highly differentiated, such individual cells would produce about 50 % of Ig. This is higher than in IgM-producing myeloma cells. This high Ig production in the culture may also reflect loss of small lymphocytes and selective preservation of LPS activated cells. The actual cell number decreases in these cultures. However, recent studies with fluorescent markers show that B cells increase whereas unstimulated T cells are lost (Askonas + Roelants, unpublished). These results demonstrate that LPS causes a rapid and uniform activation of a large proportion of the B cell population, with DNA synthesis preceding maturation or differentiation into cells producing high levels of Ig. Although these cultures were rather short term, they were appropriate for studying the early relationships between mitosis, Ig gene transcription and the translation of Ig mRNA. Table 1. Cell viability and protein synthesis in LPStreated spleen cell culturesa) Cell#
Radioactivity
Vmble
auna Day x 104 0 1 2 3
1.2 0.95 0.68 0.41
%all % viability Recovery 93 79 62 56
79 57 39
(cpn/loS cells) Total protein
lg
89500 99500 61000
5850 18100 16150
a) Normal spleen cells (5 x 06/ml) in Falcon petri dishes culture1 in 10 % FCS (see Section 2 .). 10 pg/ml LPS were. added on day . 1.5 x 106viable cells pulsed 1 h with [35S]methionine (10 pCi/ml) at various time intervals.
3.2. Nature of Ig formed by LPS-stimulated spleen cells
Oays of culture
Figure 1. LPS stimulation of normal spleen cells (CBA/H). [3H]thymidine incorporation (4 h pulse) by 1.5 x 106 spleen cells at various times of tissue culture.
Spleen cell stimulation with 10 pg LPS/ml; ( X ) control spleen cells. ( 1 ) Brackets indicate period of thymidine pulse. % Ig produo tion refers to [35S]methionine incorporation into Ig relative to total protein synthesis (1.5 x 106 spleen cells pulsed for 1 hat 37 O C with 10 pCi[35S]methionine/rnl). ( 0 ) LPSstimulated spleen cells.
(0)
Ig production only started to increase significantly after the first 24 h in such cultures, rates of Ig synthesis are high at
48 and 72 h; there was a considerable increase in the proportional Ig production relative to total protein synthesis on days 2, 3 and 4. (Table 1 and Fig. 1). In our control cultures, Ig production only rose by 10-20 %. The cells were pulsed with [35S]methionine for 1 h, and since secretion of Ig during such short pulses is low, the values reflect rate of total Ig synthesis. The analysis in the table apply to the actual cells which were used for mRNA extraction. In parallel to Ig production blast cells increased in frequency with time and by days 3 and 4 we observed 40 and 60 % of the cells to be Large and pyroniphilic. It is not possible to estimate the exact proportion of cells activated by LPS. The very high Ig pro-
Mouse spleen cell suspensions form mainly IgM [8, 231; analysis by SDS polyacrylamide gel electrophoresis of pulse-labeled Ig formed by our LPS-stimulated spleen cells at the height of Ig production (day 3) showed the production of p and light chains (Fig. 2). The formation of trace amounts of 7chain cannot be excluded by this method, but from the radioactive profile this could not represent more than 2 % of total Ig production. The original spleen cell suspension similarly produced mainly p and L-chains (Fig. 2a) with a very small amount of radioactivity in the y region. LPS results in a considerable increase in p/L ratio. This enhancement of purely IgM production was also observed in experiments with primed spleen cells which might be expected to contain a greater number of y precursor cells. In our hands LPS treatment did not enhance IgG production under any culture conditions. This is in agreement with other work using B cell mitogens, such as pokeweed mitogen [8], Con A [9] and LPS [6].
3.3. Translation of p-mRNA The stimulation of Ig production following the addition of LPS to the spleen cell suspension could arise from cytoplasmic activation of preformed mRNA for the p and L-chain proteins or from the synthesis of increased amounts of the p and Lchain mRNA or a combination of both. The relative amounts of Ig-mRNA in resting and stimulated lymphocytes can be determined by the translation of purified lymphocyte mRNA
50
R.H. Stevens, B.A. Askonas and J.L. Welstead
n 7
Eur. J. Immunol. 1975.5: 47-53
The Ig produced in oocytes injected with mRNA from day 0 lymphocytes consisted o f y2L2, y and L-chain proteins with a minor p component. Three days following the activation of the spleen cell suspension with LPS, the lymphocyte mRNA contained over 15-fold more p-mRNA relative t o y-mRNA than the day 0 population of lymphocytes. There was also an increased amount o f the rnRNA for L-chain protein. Due to the heterogeneity of the cell suspension, it is difficult to determine whether the y-mRNA resides in the same cells which are producing the p-mRNA or whether the two types of mRNA are produced in separate cells.
-X
1
r .-
3.4. Quantitation of lymphoid cell H-mRNA
20 30 Distance from origin lmml
10
Although the oocyte assay for mRNA activity allows relative amounts of mRNA to be determined, it is nevertheless limited in that quantitative assays are not possible. Quantitative measurements of H-mRNA can, however, be obtained by utilization of the interaction between H-mRNA and irnrnunoglobulin [ 1, 101.
LO
Figure 2. Polyacrylamide gel analysis of Ig produced by spleen cells. Intracellular Ig (1 h pulse with [35S]methionine) precipitated with rabbit antibody to mouse IgC and IgM, reduced with mercaptoethanol and dissociated in SDS (see Section 2.5.). The Ig chains are analyzed by SDS polyacrylamide gel electrophoresis[ 191. Reduced lgCza myeloma protein 5563, labeled with [3H]leucine served as markers for -y and K-chains (a) 0 time spleen cells; (b) day 3 of culture of spleen cells with 10 pg LPS/ml.
in a system which equally translates exogenous mRNA molecules. When injected with mRNA the oocytes from Xenopus luevis have been shown to efficiently and equally translate many mRNA molecules [ 121 including Ig [ 131. Equal amounts of the mRNA obtained from lymphocytes on days 0 and 3 following stimulation with LPS were injected into oocytes from Xenopus Iaevis. After overnight incubation, the mouse-specific Ig synthesized from the injected lymphocyte mRNA was removed from the oocyte homogenate with specific antiserum. Polyacrylamide gel analysis o f the resulting unreduced antibody precipitates is shown in Fig. 3. Identification of the translated products is based o n parallel gels with marker proteins. L
n 1-
m
I
I
10
20
1
I
30 LO Oistance from origin l m m l
When H-mRNA and IgC are mixed at concentrations greater than M , an essentially stoichometric formation o f a (H-mRNA-lgG) complex occurs [ 21. The protein complex is retained by nitrocellulose filters while uncomplexed mRNA passes through the filter. The concentration of H-mRNA in a total mixture of mRNA can therefore be determined by titration of various amounts of radioactive mRNA against a fixed concentration of IgG. From the resulting saturation curve, the concentration of H-mRNA can be determined. Fig. 4 shows the titration of nP-labeled 5563 Gza myeloma mRNA vs rabbit anti-DNP antibody ( 6 x 1 0 - 1 2 M ) . One-half saturation of the IgG occurred when 1 1 p1 of the total mRNA preparation was added. Thus, the concentration of 5563 H-mRNA in the total mRNA sample was 2.7 x lo-" M. From the total number of 5563 cells ( 2 x lo7)and from the total volume of mRNA (8.0 ml), the total number o f H-mRNA molecules was 6000/5563 myeloma cell. This value is in good agreement with the amount of H-mRNA required to synthesize the Ig produced by exponentially growing 5 563 cells (600 mol/cell/ sec) [ 2 ] .This suggests that the retention of H-mRNA by the filter is quantitative. Determination of the amount o f H-mRNA in a sample of nonradioactive mRNA can be determined by the ability of the nonradioactive H-rnRNA to compete with the radioactive H-mRNA for a constant amount of IgG. Such an 500 1
2
10 20 30 LO
50 60 70 5563 mANA l k l l
I
50
Figure 3. Translation of lymphoid cell mRNA in oocytes from Xenopus laevis. The mRNA was extracted and purified from lymphoid cells ( 2 x 1 0 8 cells) on day 0 (c3-0) or day 3 ( 0 - 0 ) following stimulation with LPS ( 1 0 pglml). The mRNA samples were resuspended to a final concentration of lOpg/ml in 66 % L-15 medium [ 2 5 ] containing 1 mCi [%]methionine; for each RNA preparation 50 oocytes were injected and incubated 18 h at 22 O C . Following incubation the oocytes were homogenized and the mouse specific Ig isolated and analyzed by e k e trophoresis on 7.5 76 polyacrylamide gels containing SDS as described in Section 2.5.
Figure 4. Quantitationof H-mRNA from 5563 myeloma cells. Exponentially growing 5563 myeloma cells (2 x 1 0 5 cells/ml) were incubated with 100 ~Ci/ml[32P]orthophosphate for 6 h in phosphatedeficient L-15 medium. Total RNA was extracted from 2 x 1 0 7 cells and the mRNA fraction prepared as in Section 2.4. The mRNA was adjusted to a concentration of 0.66 pg/ml with PBS to a volume of 8 ml and increasing amounts of mRNA were added to a fixed concentration of rabbit anti-DNP Ig (6 x 10-12 M ) in a total volume of 100 pl. After 7 min at room temperature, DNP-ovalbumin was added (5 p g ) and two 50 pl aliquots were removed for filtration through nitrocellulose filters (Millipore, type HA). Following a PBS wash (0.4 ml) the samples were air dried and the radioactivity determined. The specific activity of the RNA was around 1.2 x 106 cpm/pg and the rctcntion on the filters about 2 %.
Eur. J. Immunol. 1975.5: 47-53
H-chain mRNA in LPS-treated cells
51
assay requires that the different H-mRNA molecules react equally well with the rabbit IgG, and preliminary results suggest that this is indeed true (Stevens, manuscript in preparation).
spleen cell suspension it is apparent that this calculation provides only a very approximate estimate of the actual quantity of H-mRNA per activated cell, and may vary by a factor of 2-3 on different days of tissue culture.
Saturation-competition assays for the quantitative determination of H-mRNA from lymphoid cells at various times following LPS stimulation are shown in Fig. 5. Radioactive 5563 H-mRNA was titrated against fixed amounts of rabbit antiDNP IgG as in Fig. 4 and the concentration of 5563 H-mRNA determined. Increasing amounts of unlabeled spleen cell mRNA were then added t o equivalent amounts of 32P-labeled 5563 H-mRNA and IgG, and the radioactivity o f 5563 H-mRNA retained in the RNA-IgG complex was determined. From the competition curves, it is apparent that the spleen cell suspension on day 0 contains H-mRNA. Since recovery of mRNA was found to be 75-80 %, the quantity of H-mRNA on different days of tissue culture can be compared. Between days 0 and 1 following LPS stimulation there is a greater than threefold increase in the amount of H-chain mRNA in the cell culture.
3.5. Intracellular distribution of H-chain m R N A
Between days 1 and 3, there is once again a doubling of the total quantity of H-mRNA, despite the loss in the number of cells.
The mRNA competition curves suggest that considerable synthesis of H-mRNA occurs within 2 4 h following LPS activation of the spleen cell suspension. There is, however, little increase in Ig synthesis until between 24 and 28 h (Fig. 1). Under somewhat abnormal conditions in mouse myeloma cells, H-mRNA is synthesized normally, but transport of the mRNA to the cytoplasm is considerably reduced [24]. Whether a similar phenomenon was responsible for the discrepancy between the amount of cellular H-mRNA and the production of IgM on day 1 was tested by measuring the synthesis and transport of H-mRNA at various times following LPS stimulation. At selected intervals following stimulation, cell suspensions were labeled for 4 h with [3H]uridine and the nuclear and cytoplasmic mRNA isolated. The amount o f H-mRNA in each sample was determined by a modification of the nitrocellulose filter binding assay in which a large excess of IgG was added in order t o effect quantitative binding of the HmRNA to the filter.
Ibl
L o-o-o-
o
-o-o
-0
-
\
I
30
6
The time course for the synthesis of H-mRNA and its trana port into the cytoplasm is shown in Fig. 6. Synthesis of HmRNA commences rapidly following the addition o f LPS t o the spleen cell suspension and reaches a peak at day 1. With continued incubation, the synthesis rapidly declins, reaching the day 0 level by day 4. There is no evidence of storage of H-mRNA within the nucleus at any stage during the LPS stimulation.
12 18 2L 30
Lymphocyte mANA Ipg x lO*l
Figure 5. Quantitation of H-mRNA in LPS-treated cells. (a) Radioactive mRNA from 5563 myeloma cells was extracted and M. the concentration of 5563 H-mRNA was determined to be 18x In 100 pl total volume was added 5563 mRNA (3 x M), rabbit anti-DNP IgG (3 x 10-12 M) and increasing amounts of purified mRNA from lymphoid cells at day 0 (1 x 108 cells, 16 pg total mRNA) and day 1 (8 x 107 cells, 21 pg total mRNA) following LPS stimulation.
After 7 min incubation at room temperature, DNP-OA was added (5 pg) and duplicate 50 rl aliquots were removed, filtered through nitrocellulose filters and the radioactivity determined as in Section 2.7. (0-0) day 0, (x-x) day 1. (b) As in Fig. 5a but the concentrationsof 5563 H-mRNA and IgG were 8 x 10-12 M and the lymphoid cell mRNA was isolated at days 1 (8 x lo7 cells, 15 pg total mRNA) or 3 (4 x 107 cells, 20 pg total day 1 , (0-0) day 3, mRNA) following LPS stimulation. (x-x) (0-0) yeast RNA control. From the molar concentrations of the H-mRNA and the number of cells from which the RNA was extracted it can be calculated that on day 0 there were approximately 200 H-mRNA molecules per cell in the tissue culture. By days 1 and 3, the amount of H-mRNA was approximately 6 4 0 and 1800 molecules per cell respectively*. Due to the heterogeneity of the
* Sample calculation: From Fig. 5a the day 0 lymphocyte mRNA gave 50 % competition with 32P-labeledH-mRNA when 0.13 pg lymphocyte mRNA was added to the reaction mixture. The reaction mixture contained 3 x 10-16molesof 5563 H-mRNA and therefore 0.13 pg of unlabeled lymphoid cell mRNA contained 3 x 10-16 moles H-mRNA. The total number of moles of H-mRNA in the total lym moles. This is phoid cell mRNA preparation (16 pg) is 3.7 x equal to 2.2 x 1010 molecules/l x 108 lymphocytes, or 220 H-mRNA molecules per cell.
BC
I3CL
\
11)(((1 bys following LPS stimulation Figure 6 . Intracellular distribution of H-mRNA in LPSstimulated
lymphoid cells. Normal spleen cells (5 x lo6 cells/ml) were stimulated with 10 pg/ml LPS on day 0 and at selected time intervals, 2 x 107 cells were labeled for 4 h with 100 pCi/ml[3H]uridine. The nuclear and cytoplasmic fractions were prepared with 1 % NP-40 at 1 O C [ 131 and the nuclear and cytoplasmicmRNA isolated. The ethanokprecipitated mRNA fractionswere resuspended in 200 pl PBS. For each time point, three 50 pl samples were mixed with 3 x 10-10 M rabbit anti-DNP Ig in a total volume of 100 pl. After incubation for 6 min at room temperature, duplicate 50 pl fractions were filtered and washed as in Section 2.7. The filters were air dried and the radioactivity determined. (0-0) nuclear H-mRNA;(0-0) cytoplasmic H-mRNA.
4. Discussion It has been observed previously that some lectins (such as PWM and Con A) as well as bacterial lipopolysaccharides and polysaccharides act as B cell mitogens in vitro in murine spleen cell cultures with concomitant enhancement of IgM synthesis and secretion [ 5-81. Blast cells and plasma blasts
52
R.H.Stevens, B.A. Askonas and J.L. Welstead
appear in large numbers, and by day 3 and 4 of culture make up a large proportion of the surviving cells [ 5 , 261. Analysis o f spleen cell cultures 7 2 h after PWM addition showed that the cells formed 15 % of IgM relative to total protein production, a threefold increase over the control culture [ 81. Such levels o f IgM production compare favorably with IgM formation by mouse plasma cell tumors, [ 271 and in conjunction with the high proportion of plasmablasts would suggest that a high proportion of the spleen cells left in culture were activated into differentiation and IgM production. We are, o f course, aware that the rapid stimulation of a large number o f B cells in vitro is not identical to the time course o f antibody induction in vivo following antigen challenge. This involves gradual recruitment o f cells and their proliferation into antigen-reactive cells in which antibody production and cell maturation is induced by complex control mechanisms (antigen, as well as cell-cell interactions) within lymphoid tissues. However, the high IgM production following B cell mitogens should provide some indication of the molecular events during antibody induction and the relation between mitosis, Ig-mRNA transcription and translation during maturation o f B cells. LPS treatment of our spleen cell culture (CBA/H mice) in agreement with others resulted in considerable enhancement of IgM production on days 2 and 3, and by day 3 and 4 of culture IgM formation represented 2 6 % o f total protein synthesized. A high proportion (about 5 0 - 6 0 %) of the viable cells were large and pyroniphilic. However, we observed a peak o f DNA synthesis (using 4 h pulses with [3H]thymidine) around 2 4 h of culture with 10 % FCS. This contrasts with the linear increase in [ 3H]thymidine incorporation for 7 2 h after a preliminary lag period o f 24 h in spleen cell cultures from athymic nude mice to which either Con A or LPS had been added [9, 281. The cause for the difference between the two types of culture are not clear. Tissue culture conditions could affect the kinetics, but, in fact, they were similar (except for our larger scale requirements and different batches of FCS). Our kinetics of thymidine incorporation are more in line with those reported by Coutinho and Moller [7]. Normal spleen cells activated by type Ill pneumococcal polysaccharide showed a peak in DNA synthesis on day 2 of culture ( 2 4 h thymidine pulse). It is possible that spleen cells from nude mice d o behave differently from normal cells in these cultures. T cells might cause enhanced B cell activation and eventually possible suppression. There was an additional difference between the two types of cultures stimulated with LPS. Andersson and Melchers observed that after a lag of 24 h, DNA synthesis, IgM production and the appearance of PFC-secreting, antibodybinding NNP hapten increased in parallel [ 91 and, thus, there seemed to be simultaneous cell division and differentiation. LPS treatment of spleen cells in our hands showed a rapid wave of mitosis, with a lag of about 2 4 h before significantly increased Ig production was observed. Transcription of H-chain message increased rapidly (within 6 h of stimulation), with a peak in H-mRNA production around 2 4 h. Our results are in line with induction of antibody production in spleen cell cultures stimulated with SRBC. DNA synthesis and the susceptibility of cells t o suicide with high specific activity thymidine precedes cellular maturation and the appearance of antibody-secreting cells (plaque-forming cells) by 24-48 h [29]. There is additional evidence that division precedes differentiation. In cultures o f spleen cells from athymic mice (nulnu), thymus cell-replacing factor (TRF) restores the response to SRBC [30]. Inhibition o f DNA synthesis by a tem-
Eur. J . Immunol. 1975.5: 47-53
perature shift to 35 OC during the first two days inhibits restoration o f the anti-SRBC response, whereas the same shift at day 2 at the time o f TRF additifin permits TRF action and the development o f PFC [ 231. The presence of y-mRNA in the spleen cell suspension is intriguing considering that little, if any, of this mRNA is translated into protein (Fig. 2). Evidence for y-mRNA in lymphoid cells resides in the fact that oocytes injected with total mRNA produced an antibody precipitable protein migrating with an apparent molecular weight o f 5 5 000. Furthermore, a proportion o f this material is assembled within the oocytes into whole 7 S molecules of 150 000 molecular weight in a manner analogous t o myeloma 5563 lgGza [ I ] . As the spleen cell suspension is a heterogeneous mixture of cells, it is not possible at this time t o determine whether the y and p-mRNA exist within the same cells with a translational block o n y-mRNA or whether the y-mRNA resides in cells refractive t o LPS stimulation. About 18 % of adult murine spleen cells have IgG receptors [3 1 ] and therefore this lymphocyte population can produce IgG in small amounts. However, in confirmation o f work by others [6, 81 we found that mainly IgM production is stimulated by LPS and only a subpopulation o f B cells in spleen appear to be susceptible t o LPS stimulation. The oocyte translation system does not permit exact quantitation of mRNA - it is possible that ymRNA is translated preferentially to p-mRNA - this point has not been tested. However, the assay shows that both and p-mRNA are present in the spleen cells and the low levels of IgG production d o suggest a translational block o n y-mRNA. The possibility of p-mRNA degradation is unlikely t o account for our results, since you can reextract the purified mRNA and obtain very similar ratios of y and p-chain production in oocytes. Estimates of H-mRNA in lymphocytes following LPS stimulation have revealed that the average number of H-mRNA molecules per cell increases from 2 0 0 on day 0 to 1800 o n day 3. Considering the heterogeneity of the cell suspension, the actual number of H-mRNA molecules in any particular cell will be different from the average by a factor o f 2 or 3. I t is apparent, however, that despite extensive blast formation and differentiation o f B lymphocytes during LPS activation, the H-mRNA content of the cells is considerably reduced compared to mouse IgGza myeloma cells (SO00 H-mRNA molecules per cell [ 21). This suggests that although LPS initiates differentiation o f B lymphocytes into plasma cells, full differentiation does not occur in the in vitro system. Alternatively, there may be a difference in the extent o f differentiation between IgM and IgG-producing cells. The binding assay for H-mRNA is not capable of resolving and p-mRNA molecules. From Pig. 3, however, it is clear that the majority o f H-mRNA synthesis following LPS stimulation is p-mRNA. Although the synthesis of mRNA is rapid following LPS stimulation, increased production of immunoglobulin proceeds only following a lag o f about 24 h . This lag does not appear to result from hindered transport of the mRNA into the cytoplasm (Fig. 6 ) and must, presumably, result from a translational block on the p-mRNA. At early times following mitogenic stimulation there is little structural organization o f the cytoplasm [ 261. The synthesis of H and L-chain proteins has been shown to normally occur on membrane-bound polyribosomes [ 31, although it is not clear what role membranes play in the regulation of Ig synthesis or, indeed, whether membranes are an absolute requirement for Ig synthesis.
Eur. I. Immunol. 1975.5: 53-58
Immunochemical study of x-irradiated DNA
The kinetics of Ig production are consistent with the morphology of mouse spleen cells stimulated with LPS analyzed by electromicroscopy [26]. After 16-28 h, B cells become large lymphoblasts with cytoplasmic polyribosomes. Development of endoplasmic reticulum commences only after 28 h and gradually continues. By 2 1/2 days many plasmablasts are found. it has been shown in mouse myeloma cells that under conditions where Ig can interact with H-mRNA, a feed back repression on H-chain protein synthesis occurs. It is conceivable that under conditions of a deficit of membranes, the Ig produced in lymphocytes is not secreted, but allowed t o interact with H-mRNA, resulting in a reduction of H-chain synthesis. At later times following LPS stimulation, when there is increased membrane organization, the mRNA and Ig would become separated and maximum H-chain protein synthesis would proceed. This hypothesis is under further consideration.
53
8 Parkhouse, R.M.E., Janossy, G. and Greaves, M.F., Nature 1972. 235: 21. 9 Andersson, J. and Melchers, F., Proc. Nut. Acad. Sci. US 1973. 70: 416. 10 Stevens, R.H. and Williamson, A.R., J. Mol. Biol. 1973. 78: 515. 11 Stevens, R.H. and Williamson, A.R., J. Mol. Biol. 1973. 78: 505. 12 Lane, C., Marbaix, G. and Gurdon, I., J. Mol. Biol. 197 1. 6 I : 7 3. 13 Stevens, R.H. and Williamson, A.R., Nature 1972. 239: 143.
14 Jones, O.W. and Berg, P., J. Mol. Biol. 1966. 22: 199. 15 Rigs, A.D., Suzuki, H. and Bourgeois, S., J. Mol. Biol. 1970. 48: 67. 16 Kinard, F.E., Rev. Sci. Instrum. 1957. 28: 293. 17 Bray, G., Anal. Biochem. 1960. I : 279. 18 Kitos, P., Saxon, G. and Amos, H., Biochem. Biophys. Res. Comm. 1972.47: 1426. 19 Summers, D.F., Maizel, J.V. and Darnell, J.E., Roc. Nut. Acad. Sci. US 1965.54: 5 0 5 . 20 Choules, G.L. and Zimm, B.H., Anal. Biochem. 1965. 13: 366. 21 Valentine, R.C. and Green, N.M., J. Mol. Biol. 1967.27: 615.
Received February 19, 1974;in revised form July 10, 1974.
22 Andersson, J., Moller, G. and Sjoberg, 0..Cell. Immunol. 1972. 4: 381. 23 Askonas, B.A., Schimpl, A. and Wecker, E., Eur. J. Immunol. 1974.4: 164.
5. References
24 Stevens, R.H. and Williamson, A.R., Nature-New Biol. 1973. 245: 101.
1 Stevens, R.H. and Williamson, A.R., Proc. Nut. Acad. Sci. US 1973. 70: 1127.
25 Leibovitz, A., Amer. J. Hyg. 1963. 78: 173.
2 Stevens, R.H., in preparation.
26 Shohat, M., Janossy, G. and Dourmashkin, R.R., Eur. J. Immunol. 1973.3: 680.
3 Bevan, M.J., Parkhouse, R.M.E., Williamson, A.R. and Askonas, B.A., Progr. Biophys. Mol. Biol., Pergamon Press 1972. 5: 133. 4 Scharff, M.D. and Laskov, R., Progr. Allergy 1970.14: 37.
5 Greaves, M. and Janossy, G., Transplant. Rev. 1972. 1 1 : 87. 6 Andersson, J., Sjoberg, 0. and Moller, G., Transplant. Rev. 1972. 11: 131. 7 Coutinho, A. and Moller, G., Eur. J. Immunol. 1973. 3: 608.
Pier G. Natali Immunology Laboratory, Regina Elena Institute for Cancer Research, Rome
27 Parkhouse, R.M.E. and Askonas, B.A., Biochem. J. 1969.115: 163. 28 Melchers, F. and Andersson, J., Transplant. Rev. 1973. 14: 76. 29 Dutton, R.W. and Mishell, R.I., J. Exp. Med. 1967. 126: 443. 30 Schimpl, A. and Wecker, E., Nature 1972.237: 15. 31 Owen, J.T., Cooper, M.D. and Raff, M.C., Nature 1974.249: 361.
Denaturing effect of x-irradiation on deoxyribonucleic acid aqueous solutions An immunochemical study The denaturation of calf thymus DNA aqueous solutions following x-ray radiation (x-DNA) has been investigated with the help of a rabbit antidenatured DNA antiserum. These antibodies were shown t o recognize exclusively single-stranded DNA determinants on DNA molecule in double immunodiffusion analysis. By means of this method it was possible t o demonstrate that increasing numbers of single-stranded DNA determinants were formed in DNA solutions exposed t o increasing doses of radiation. The rabbit serum appeared t o be a highly sensitive reagent (0.5 pg/ml of singlestranded DNA) capable of detecting DNA denaturation when low ionizing doses were used and when only minor changes in DNA physico-chemical parameters were measurable. Antigenic comparison between x-DNA and heatdenatured DNA demonstrated that antigenic determinants are shared by the two molecules. The possible application of immunochemical methods to the study of nucleic acid denaturation by different physical and chemical agents is discussed. [I 8121
Correspondence: Pier G. Natali, Istituto Regina Elena, V. Regina Elena 291, Rome 00161, Italy Abbreviations: X-DNA X-ray irradiated DNA H D N A Heatdenatured DNA PBS: Phosphate buffered saline MAK: Methylated albumin kieselguhr MBSA Methylated bovine serum albumin
1. Introduction The serologic demonstration of an ti-DNA antibodies in the sera of patients with autoimmune disease [ 11 has stimulated numerous attempts t o induce anti-nucleic acid-specific antibodies in experimental animals.
R.H. Stevens, Brigitte A. Askonas and Jacqueline L. Welstead National Institute for Medical Research, London
H-chain mRNA in LPS-treated cells
47
Immunoglobulin heavy chain mRNA in mitogen-stimulated B cells This paper relates the synthesis of DNA, immunoglobulin and heavy chain (H) mRNA in murine spleen cells following activation of B cells with lipopolysaccharide from E. coli (LPS). Spleen cells (CBA/H mice) were cultivated with 10 % FCS and 10 pg LPS/ml. 4 h pulses with [ 3H]thymidine showed that DNA synthesis was stimulated within the first day following LPS activation and exhibited a sharp peak at 24 h. The shape of the DNA synthesis curve suggests that the cells susceptible to LPS stimulation are activated in a synchronous manner. Stimulation of Hchain mRNA (H-mRNA) synthesis proceeded rapidly (within 6 h of LPS addition) and peaked around 24 h, in parallel to DNA synthesis. The H-mRNA was isolated and quantitated by making use of its interaction with IgG [ 1, 21. The actual level of H-mRNA in the culture increased threefold during the first 24 h and then doubled within the next 48 h. Estimates of the actual number of H-mRNA were approximately 200 molecules H-mRNA/cell on day 0 rising to I800/cell on day 3. In such a mixed cell population these figures will be accurate only within a factor of 2-3 (at least 35 % B cells in spleen cell suspensions at the commencement of the culture, with up to 35-60 % of plasma blasts by day 3 and 4 of LPS treatment). Translation of the lymphoid cell mRNA in oocytes from Xenopus laeuis demonstrated that stimulation of H-mRNA synthesis was restricted to p-mRNA, although some 7-mRNA was present in the original spleen cells. High levels of synthesis of immunoglobulin followed after a lag period of about 24 h following LPS addition peaking after 48 and 7 2 h; the proportional lg production relative to total protein synthesis reached 26 % on days 3 and 4. Stimulation of Ig production was limited to IgM. Rapid stimulation of mitosis and H-mRNA synthesis thus precedes the maximum synthesis of Ig molecules, suggesting a translational block on H-mRNA during cell maturation. There was no apparent block on the transport of H-mRNA from the nucleus during early stages of activation.
1 . Introduction Antigenic challenge results in proliferation and differentiation of antigen-sensitive lymphocytes which recognize a particular antigen. This leads to the development of blast cells and then plasma cells with an organized endoplasmic reticulum and high levels of immunoglobulin (Ig) production and secretion. Although production of Ig at the stage of the mature plasma cell has been analyzed extensively [3, 41, we know little of the events in induction of Ig production and plasma cell maturation. Biochemical studies with antigenstimulated lymphoid cells have been difficult, since lymphoid tissue is made up of a mixed cell population. Responses to individual antigens are multiclonal, but only a small proportion of lymphoid cells respond to individual antigens. Induction of high levels of Ig production is therefore restricted to a small percentage of the spleen cell population.
rI 7661 ~~~
~
* Present address: Department of Microbiologyand Immunology, School of Medicine, U.C.L.A., Los Angeles, Calif. 90024, USA. Correspondence: B.A. Askonas, National Institute for Medical Research, Mill Hill, London NW7 lAA, GB Abbreviations: LPS: Lipopolysaccharidefrom E. coli PBS: Phosphate buffered saline F a : Fetal calf serum PWM: Pokeweed mitogen Con A: Concanavalin A mRNA: Messenger RNA H-mRNA: H-chain mRNA T cells: Thymus-derived lymphocytes B cells: Non-thymusdependent lymphocytes PFC: Plaque-forming cells TCA: Trichloroacetic acid TRF: Thymus cell-replacingfactor SDS: Sodium dodecyl sulfate
In an attempt to study the relation between cell division, transcription of Ig genes, translation of Ig mRNA molecules and cellular maturation, we therefore turned to mitogen-activated B cells as preliminary model system. I t has been reported previously that various substances such as pokeweed mitogen (PWM) [5], insolubilized concanavalin A (Con A), lipopolysaccharide (LPS) from gram-negative bacteria and various polysaccharides [ 6 , 7 ] activate a high proportion of mouse B cells. This results in spleen cell cultures in uitro, in mitosis, blast cell formation and subsequent maturation of B cells with high levels of Ig production..Ig formation of up t o 20-30 % of total protein synthesis has been observed after 3-4 days of tissue culture [8, 91. Lipopolysaccharide (LPS) from E. coli appears to be the most reproducibly powerful B cell activator with the strongest enhancement of IgM synthesis, whereas PWM and Con A also stimulate T cells. For these reasons LPS treatment of mouse spleen cultures has been used for this study. The observation that heavy chain (H) messenger RNA (HmRNA) interacts with Ig has enabled the isolation of heavy chain message quantitatively in multiple samples [2]. The type of Ig coded for WBS then assayed in frog oocytes following injection of mRNA and its subsequent translation [ 12, 131. Quantitative assays of immunoglobulin H-mRNA have proved possible utilizing the interaction between H-mRNA and IgG [ 21 and a modification of the nitrocellulose binding assay for measuring RNA-protein interaction [ 14, 151. In this paper we report detailed kinetics of DNA, Ig, and H-mRNA synthesis following LPS stimulation of B cells in spleen.
48
R.H. Stevens, B.A. Askonas and J.L. Welstead
2. Materials and methods 2.1. Spleen cell suspension Spleens from CBA/H mice ( 3 - 4 months old) were suspended in RPMI 1640 medium (Flow Labs., Irvine, Scotland), containing 10 % FCF serum (Biocult Labs., Glasgow, Scotland) by teasing with forceps. Clumps of cells were permitted to settle 1-2 min, She cell suspension removed and centrifuged 8 min at 200 x g. The cells were washed once with medium, and cultured for 4 days at 5 x 1 O6 cells/ml in the above medium in petri dishes ( 4 or 12 ml in Falcon dishes 3002 or 3003 respectively) in 5 % Co2/95 % air at 37 "C. Cell viability was estimated in 0.02 % trypan blue in PBS. LPS from E. coli (Difco 055BS, Detroit, Mich.) was used at 10 pg/ml. At appropriate times all cells were suspended with a silicone rubber policeman; DNA and protein synthesis, and mRNA were analyzed on suitable aliquots.
2.2. [3H]thymidine incorporation At various time intervals, 1.5 x 1 O6 viable cells were pulsed with 1 pCi [3H]thymidine (I Cilrnmol) for 4 h at 37 "C in 1 ml of the above medium (Falcon dishes 3001). The cells were harvested on Whatman glass fiber filters (GF/A), washed three times each with PBS, 7 % trichloroacetic acid (TCA) and ethanol, dried with ether and counted in Kinard's [ 161 scintillation f h i d .
Eur. J. Immunol. 1975.5: 47-53
washed with diethyl ether-ethanol ( 3 : I ) , air dried and resup pended in 1 ml binding buffer (500 mM KCI, 10 mM TrisHCI, I mM MgCI2, pH 7.4) and applied to a 5 ml cellulose column equilibrated with binding buffer [ 181. More than 95 % of ribosomal RNA was removed by washing with 20 ml binding buffer and the mRNA fraction eluted by washing with 10 mM Tris-HCI, pH 7.6. This mRNA fraction was used for all assays. Recovery of mRNA by this procedure was consistently between 75 and 80 % of total mRNA; this was determined from the recovery of a measured amount of purified radioactive H-mRNA (from 5563 myeloma cells) following phenol extraction with nonradioactive spleen cells.
2.5. Polyacrylamide gel analysis of immune precipitates Washed immune precipitates were dissolved by boiling in 2 % SDS for 2 min. For analysis of unreduced immune precipitates the samples were made t o 2 % with iodoacetamide and loaded directly on top of 7.5 7% acrylamide gels containing SDS. Reduced immune precipitates were prepared by dissolving in 2 % SDS as above and adding 2-mercaptoethanol to 1 %. The samples were incubated for 1 h at room temperature, iodoacetamide added (to 2 %) and the samples loaded onto the polyacrylamide gels containing SDS. Electrophoresis was conducted at I0 mA/gel for 5 h at room temperature. Following electrophoresis the gels were sliced and dissolved in NH40H. Scintillation fluid [ 171 was added and the radioactivity determined in a Beckman LS-I 5 0 scintillation counter.
2.3. [''Slmethionine incorporation 1.5 x 1 O6 viable spleen cells were pulsed for I h at 3 7 O C with 10 pCi/ml of [35S]methionine (Radiochemical Centre, Amersham, I60 Ci/mmol) in EDM medium lacking methionine. The cells were pelleted by centrifugation, lysed in 0.1 ml 0.5 % deoxycholate, adjusted to 1.5 ml with PBS and centrifuged 3 0 min at 35 000 x g. Methionine incorporation was analyzed (a) into total cellular protein precipitated with 7 % TCA, (b) Ig precipitated at equivalence with specific rabbit antiserum to mouse IgM and IgG in the presence of carrier Ig, (c) control antibody/antigen precipitates (rabbit IgG and goat anti-rabbit IgG). After three washes with PBS at 4 O C , the precipitates dissolved in 0. I ml 0.1 N NaOH were counted in Bray's [ 171 scintillation fluid. The radioactivity of the control precipitates was subtracted from the radioactivity of specific Ig precipitates to give the incorporation into Ig. 2.4. Extraction of lymphocyte mRNA Total cellular RNA was prepared from lymphoid cells as described by Stevens [2]. Briefly, 10' cells were washed with PBS ( I 50 mM NaCI, 1 mM MgCI2, 1 mM CaCI2, 10 mM phosphate buffer, pH 7.6) and resuspended in 1 5 ml RNA extraction buffer (100 mM sodium acetate, 2 0 0 mM NaCI, 2 mM EDTA, 0.1 % sodium dodecyl sulfate (SDS), pH 5.0 with acetic acid) and mixed with two volumes of a phenol (redistilled)-chloroform-isoarnylalcohol mixture (3: 1 : 0:0.05). The samples were shaken vigorously for 5 min in a 4 5 "C water bath and then chilled on ice. The samples were then centrifuged (2000 x g for 5 min) and the aqueous layer removed. An additional 10 ml of extracting buffer was added to the original phenol and the extraction repeated. The two aqueous layers were combined and extracted with an equal volume of fresh phenol-chloroform-isoamyl alcohol mixture. The final aqueous layer was mixed with 2 vol of ethanol and the RNA allowed to precipitate overnight at - 20 O C . The RNA was recovered by centrifugation ( I 5 000 x g for 10 min),
Ig produced by the spleen cells was analyzed as described above except that 5 % SDS polyacrylamide gels [ 191 were utilized and electrophoresis was for 3 1/2 h. The gel slices were solubilized as described by Choules and Zimm [ 201 and counted in Kinard's fluid [ 161.
2.6. Oocyte translation of lymphocyte mRNA Mature oocytes were obtained from Xenopus Iaevis and were injected with mRNA as described previously [ 12, 131. Following 24 h incubation at room temperature in the presence of [3'S] methionine, the oocytes (50 per sample) were homogenized in 2 ml PBS containing 1 % NP-40 (Shell Chemicals). The cellular debris was removed by centrifugation (I0 000 x g for IS min) and the mousespecific immunoglobulins in the supernatant fluids were isolated by direct antibody precipitation [ 131.
2.7. Quantitative estimation of H-mRNA Quantitative determinations of H-mRNA were performed by the nitrocellulose filter assay [2]. Samples of total mRNA ( 3 H or 3zP-labeled) and IgG (purified rabbit anti-DNP antibody with high affinity [21], kindly provided by Dr. N.M. Green), were mixed at room temperature ( I 00 p1 total volume) and allowed to come to equilibrium ( 7 min). The reaction conditions were 150 mM NaCI, 1 mM MgCI2, 1 mM CaC12, 10 mM phosphate buffer, pH 7 h . DNP ovalbumin ( 5 pg) was added and duplicate 50 pl samples were filtered through nitrocellulose filters (Millipore, Type HA, 0.45 p pore size) and washed with 0.4 ml PBS. The samples were air dried and the radioactivity determined. The DNPovalbumin does not participate in the reaction, but combination with the anti-DNP antibody allows firmer binding of the protein-RNA complex to the filter and provides more reliable resu It s.
Eur. J. Immunol. 1975.5: 47-53
3.Results 3.1. General remarks For detailed examination of mRNA production, 0.5 x 10' - 1 x 10' cells were required for each sample and cultures were therefore plated at a density of 5 x lo6 cells/ml, scaled up to a volume of 12 ml in petri dishes.
Optimal stimulation of Ig formation in the 3-4 day cultures occurred with doses of 10 pg LPS/ml. Higher concentrations of LPS led to increased cell death and lower doses resulted in lower levels of DNA and Ig stimulation. We first estab lished the kinetics of [ 3H]thymidine incorporation and Ig production relative to total protein synthesis in these particular cultures, which showed somewhat lower cell viability than the small scale cultures. Treatment of normal spleen cells with LPS led to a rapid increase in DNA synthesis after the first 6 h of culture with a peak around 24 h (Fig. 1 , 4 h pulses). By 48 h, DNA synthesis had dropped, but was still severalfold higher than in the control cultures. Days 3 and 4 showed a further general decrease in thymidine incorporation. The control cultures showed some stimulation of DNA synthesis which probably is attributable FCS, as different batches of FCS give rise to different levels of thymidine incorporation. This stimulation in the control cultures could result from the stimulation of either T or B cells in the mixed spleen cell population. The DNA stimulation over and above the levels found in control cultures can be assigned t o B cell mitosis, since LPS does not stimulate T cells [22].
H-chain mRNA in LPStreated cells
49
duction (26 % of total protein by day 3) indicates that if half of the cells are highly differentiated, such individual cells would produce about 50 % of Ig. This is higher than in IgM-producing myeloma cells. This high Ig production in the culture may also reflect loss of small lymphocytes and selective preservation of LPS activated cells. The actual cell number decreases in these cultures. However, recent studies with fluorescent markers show that B cells increase whereas unstimulated T cells are lost (Askonas + Roelants, unpublished). These results demonstrate that LPS causes a rapid and uniform activation of a large proportion of the B cell population, with DNA synthesis preceding maturation or differentiation into cells producing high levels of Ig. Although these cultures were rather short term, they were appropriate for studying the early relationships between mitosis, Ig gene transcription and the translation of Ig mRNA. Table 1. Cell viability and protein synthesis in LPStreated spleen cell culturesa) Cell#
Radioactivity
Vmble
auna Day x 104 0 1 2 3
1.2 0.95 0.68 0.41
%all % viability Recovery 93 79 62 56
79 57 39
(cpn/loS cells) Total protein
lg
89500 99500 61000
5850 18100 16150
a) Normal spleen cells (5 x 06/ml) in Falcon petri dishes culture1 in 10 % FCS (see Section 2 .). 10 pg/ml LPS were. added on day . 1.5 x 106viable cells pulsed 1 h with [35S]methionine (10 pCi/ml) at various time intervals.
3.2. Nature of Ig formed by LPS-stimulated spleen cells
Oays of culture
Figure 1. LPS stimulation of normal spleen cells (CBA/H). [3H]thymidine incorporation (4 h pulse) by 1.5 x 106 spleen cells at various times of tissue culture.
Spleen cell stimulation with 10 pg LPS/ml; ( X ) control spleen cells. ( 1 ) Brackets indicate period of thymidine pulse. % Ig produo tion refers to [35S]methionine incorporation into Ig relative to total protein synthesis (1.5 x 106 spleen cells pulsed for 1 hat 37 O C with 10 pCi[35S]methionine/rnl). ( 0 ) LPSstimulated spleen cells.
(0)
Ig production only started to increase significantly after the first 24 h in such cultures, rates of Ig synthesis are high at
48 and 72 h; there was a considerable increase in the proportional Ig production relative to total protein synthesis on days 2, 3 and 4. (Table 1 and Fig. 1). In our control cultures, Ig production only rose by 10-20 %. The cells were pulsed with [35S]methionine for 1 h, and since secretion of Ig during such short pulses is low, the values reflect rate of total Ig synthesis. The analysis in the table apply to the actual cells which were used for mRNA extraction. In parallel to Ig production blast cells increased in frequency with time and by days 3 and 4 we observed 40 and 60 % of the cells to be Large and pyroniphilic. It is not possible to estimate the exact proportion of cells activated by LPS. The very high Ig pro-
Mouse spleen cell suspensions form mainly IgM [8, 231; analysis by SDS polyacrylamide gel electrophoresis of pulse-labeled Ig formed by our LPS-stimulated spleen cells at the height of Ig production (day 3) showed the production of p and light chains (Fig. 2). The formation of trace amounts of 7chain cannot be excluded by this method, but from the radioactive profile this could not represent more than 2 % of total Ig production. The original spleen cell suspension similarly produced mainly p and L-chains (Fig. 2a) with a very small amount of radioactivity in the y region. LPS results in a considerable increase in p/L ratio. This enhancement of purely IgM production was also observed in experiments with primed spleen cells which might be expected to contain a greater number of y precursor cells. In our hands LPS treatment did not enhance IgG production under any culture conditions. This is in agreement with other work using B cell mitogens, such as pokeweed mitogen [8], Con A [9] and LPS [6].
3.3. Translation of p-mRNA The stimulation of Ig production following the addition of LPS to the spleen cell suspension could arise from cytoplasmic activation of preformed mRNA for the p and L-chain proteins or from the synthesis of increased amounts of the p and Lchain mRNA or a combination of both. The relative amounts of Ig-mRNA in resting and stimulated lymphocytes can be determined by the translation of purified lymphocyte mRNA
50
R.H. Stevens, B.A. Askonas and J.L. Welstead
n 7
Eur. J. Immunol. 1975.5: 47-53
The Ig produced in oocytes injected with mRNA from day 0 lymphocytes consisted o f y2L2, y and L-chain proteins with a minor p component. Three days following the activation of the spleen cell suspension with LPS, the lymphocyte mRNA contained over 15-fold more p-mRNA relative t o y-mRNA than the day 0 population of lymphocytes. There was also an increased amount o f the rnRNA for L-chain protein. Due to the heterogeneity of the cell suspension, it is difficult to determine whether the y-mRNA resides in the same cells which are producing the p-mRNA or whether the two types of mRNA are produced in separate cells.
-X
1
r .-
3.4. Quantitation of lymphoid cell H-mRNA
20 30 Distance from origin lmml
10
Although the oocyte assay for mRNA activity allows relative amounts of mRNA to be determined, it is nevertheless limited in that quantitative assays are not possible. Quantitative measurements of H-mRNA can, however, be obtained by utilization of the interaction between H-mRNA and irnrnunoglobulin [ 1, 101.
LO
Figure 2. Polyacrylamide gel analysis of Ig produced by spleen cells. Intracellular Ig (1 h pulse with [35S]methionine) precipitated with rabbit antibody to mouse IgC and IgM, reduced with mercaptoethanol and dissociated in SDS (see Section 2.5.). The Ig chains are analyzed by SDS polyacrylamide gel electrophoresis[ 191. Reduced lgCza myeloma protein 5563, labeled with [3H]leucine served as markers for -y and K-chains (a) 0 time spleen cells; (b) day 3 of culture of spleen cells with 10 pg LPS/ml.
in a system which equally translates exogenous mRNA molecules. When injected with mRNA the oocytes from Xenopus luevis have been shown to efficiently and equally translate many mRNA molecules [ 121 including Ig [ 131. Equal amounts of the mRNA obtained from lymphocytes on days 0 and 3 following stimulation with LPS were injected into oocytes from Xenopus Iaevis. After overnight incubation, the mouse-specific Ig synthesized from the injected lymphocyte mRNA was removed from the oocyte homogenate with specific antiserum. Polyacrylamide gel analysis o f the resulting unreduced antibody precipitates is shown in Fig. 3. Identification of the translated products is based o n parallel gels with marker proteins. L
n 1-
m
I
I
10
20
1
I
30 LO Oistance from origin l m m l
When H-mRNA and IgC are mixed at concentrations greater than M , an essentially stoichometric formation o f a (H-mRNA-lgG) complex occurs [ 21. The protein complex is retained by nitrocellulose filters while uncomplexed mRNA passes through the filter. The concentration of H-mRNA in a total mixture of mRNA can therefore be determined by titration of various amounts of radioactive mRNA against a fixed concentration of IgG. From the resulting saturation curve, the concentration of H-mRNA can be determined. Fig. 4 shows the titration of nP-labeled 5563 Gza myeloma mRNA vs rabbit anti-DNP antibody ( 6 x 1 0 - 1 2 M ) . One-half saturation of the IgG occurred when 1 1 p1 of the total mRNA preparation was added. Thus, the concentration of 5563 H-mRNA in the total mRNA sample was 2.7 x lo-" M. From the total number of 5563 cells ( 2 x lo7)and from the total volume of mRNA (8.0 ml), the total number o f H-mRNA molecules was 6000/5563 myeloma cell. This value is in good agreement with the amount of H-mRNA required to synthesize the Ig produced by exponentially growing 5 563 cells (600 mol/cell/ sec) [ 2 ] .This suggests that the retention of H-mRNA by the filter is quantitative. Determination of the amount o f H-mRNA in a sample of nonradioactive mRNA can be determined by the ability of the nonradioactive H-rnRNA to compete with the radioactive H-mRNA for a constant amount of IgG. Such an 500 1
2
10 20 30 LO
50 60 70 5563 mANA l k l l
I
50
Figure 3. Translation of lymphoid cell mRNA in oocytes from Xenopus laevis. The mRNA was extracted and purified from lymphoid cells ( 2 x 1 0 8 cells) on day 0 (c3-0) or day 3 ( 0 - 0 ) following stimulation with LPS ( 1 0 pglml). The mRNA samples were resuspended to a final concentration of lOpg/ml in 66 % L-15 medium [ 2 5 ] containing 1 mCi [%]methionine; for each RNA preparation 50 oocytes were injected and incubated 18 h at 22 O C . Following incubation the oocytes were homogenized and the mouse specific Ig isolated and analyzed by e k e trophoresis on 7.5 76 polyacrylamide gels containing SDS as described in Section 2.5.
Figure 4. Quantitationof H-mRNA from 5563 myeloma cells. Exponentially growing 5563 myeloma cells (2 x 1 0 5 cells/ml) were incubated with 100 ~Ci/ml[32P]orthophosphate for 6 h in phosphatedeficient L-15 medium. Total RNA was extracted from 2 x 1 0 7 cells and the mRNA fraction prepared as in Section 2.4. The mRNA was adjusted to a concentration of 0.66 pg/ml with PBS to a volume of 8 ml and increasing amounts of mRNA were added to a fixed concentration of rabbit anti-DNP Ig (6 x 10-12 M ) in a total volume of 100 pl. After 7 min at room temperature, DNP-ovalbumin was added (5 p g ) and two 50 pl aliquots were removed for filtration through nitrocellulose filters (Millipore, type HA). Following a PBS wash (0.4 ml) the samples were air dried and the radioactivity determined. The specific activity of the RNA was around 1.2 x 106 cpm/pg and the rctcntion on the filters about 2 %.
Eur. J. Immunol. 1975.5: 47-53
H-chain mRNA in LPS-treated cells
51
assay requires that the different H-mRNA molecules react equally well with the rabbit IgG, and preliminary results suggest that this is indeed true (Stevens, manuscript in preparation).
spleen cell suspension it is apparent that this calculation provides only a very approximate estimate of the actual quantity of H-mRNA per activated cell, and may vary by a factor of 2-3 on different days of tissue culture.
Saturation-competition assays for the quantitative determination of H-mRNA from lymphoid cells at various times following LPS stimulation are shown in Fig. 5. Radioactive 5563 H-mRNA was titrated against fixed amounts of rabbit antiDNP IgG as in Fig. 4 and the concentration of 5563 H-mRNA determined. Increasing amounts of unlabeled spleen cell mRNA were then added t o equivalent amounts of 32P-labeled 5563 H-mRNA and IgG, and the radioactivity o f 5563 H-mRNA retained in the RNA-IgG complex was determined. From the competition curves, it is apparent that the spleen cell suspension on day 0 contains H-mRNA. Since recovery of mRNA was found to be 75-80 %, the quantity of H-mRNA on different days of tissue culture can be compared. Between days 0 and 1 following LPS stimulation there is a greater than threefold increase in the amount of H-chain mRNA in the cell culture.
3.5. Intracellular distribution of H-chain m R N A
Between days 1 and 3, there is once again a doubling of the total quantity of H-mRNA, despite the loss in the number of cells.
The mRNA competition curves suggest that considerable synthesis of H-mRNA occurs within 2 4 h following LPS activation of the spleen cell suspension. There is, however, little increase in Ig synthesis until between 24 and 28 h (Fig. 1). Under somewhat abnormal conditions in mouse myeloma cells, H-mRNA is synthesized normally, but transport of the mRNA to the cytoplasm is considerably reduced [24]. Whether a similar phenomenon was responsible for the discrepancy between the amount of cellular H-mRNA and the production of IgM on day 1 was tested by measuring the synthesis and transport of H-mRNA at various times following LPS stimulation. At selected intervals following stimulation, cell suspensions were labeled for 4 h with [3H]uridine and the nuclear and cytoplasmic mRNA isolated. The amount o f H-mRNA in each sample was determined by a modification of the nitrocellulose filter binding assay in which a large excess of IgG was added in order t o effect quantitative binding of the HmRNA to the filter.
Ibl
L o-o-o-
o
-o-o
-0
-
\
I
30
6
The time course for the synthesis of H-mRNA and its trana port into the cytoplasm is shown in Fig. 6. Synthesis of HmRNA commences rapidly following the addition o f LPS t o the spleen cell suspension and reaches a peak at day 1. With continued incubation, the synthesis rapidly declins, reaching the day 0 level by day 4. There is no evidence of storage of H-mRNA within the nucleus at any stage during the LPS stimulation.
12 18 2L 30
Lymphocyte mANA Ipg x lO*l
Figure 5. Quantitation of H-mRNA in LPS-treated cells. (a) Radioactive mRNA from 5563 myeloma cells was extracted and M. the concentration of 5563 H-mRNA was determined to be 18x In 100 pl total volume was added 5563 mRNA (3 x M), rabbit anti-DNP IgG (3 x 10-12 M) and increasing amounts of purified mRNA from lymphoid cells at day 0 (1 x 108 cells, 16 pg total mRNA) and day 1 (8 x 107 cells, 21 pg total mRNA) following LPS stimulation.
After 7 min incubation at room temperature, DNP-OA was added (5 pg) and duplicate 50 rl aliquots were removed, filtered through nitrocellulose filters and the radioactivity determined as in Section 2.7. (0-0) day 0, (x-x) day 1. (b) As in Fig. 5a but the concentrationsof 5563 H-mRNA and IgG were 8 x 10-12 M and the lymphoid cell mRNA was isolated at days 1 (8 x lo7 cells, 15 pg total mRNA) or 3 (4 x 107 cells, 20 pg total day 1 , (0-0) day 3, mRNA) following LPS stimulation. (x-x) (0-0) yeast RNA control. From the molar concentrations of the H-mRNA and the number of cells from which the RNA was extracted it can be calculated that on day 0 there were approximately 200 H-mRNA molecules per cell in the tissue culture. By days 1 and 3, the amount of H-mRNA was approximately 6 4 0 and 1800 molecules per cell respectively*. Due to the heterogeneity of the
* Sample calculation: From Fig. 5a the day 0 lymphocyte mRNA gave 50 % competition with 32P-labeledH-mRNA when 0.13 pg lymphocyte mRNA was added to the reaction mixture. The reaction mixture contained 3 x 10-16molesof 5563 H-mRNA and therefore 0.13 pg of unlabeled lymphoid cell mRNA contained 3 x 10-16 moles H-mRNA. The total number of moles of H-mRNA in the total lym moles. This is phoid cell mRNA preparation (16 pg) is 3.7 x equal to 2.2 x 1010 molecules/l x 108 lymphocytes, or 220 H-mRNA molecules per cell.
BC
I3CL
\
11)(((1 bys following LPS stimulation Figure 6 . Intracellular distribution of H-mRNA in LPSstimulated
lymphoid cells. Normal spleen cells (5 x lo6 cells/ml) were stimulated with 10 pg/ml LPS on day 0 and at selected time intervals, 2 x 107 cells were labeled for 4 h with 100 pCi/ml[3H]uridine. The nuclear and cytoplasmic fractions were prepared with 1 % NP-40 at 1 O C [ 131 and the nuclear and cytoplasmicmRNA isolated. The ethanokprecipitated mRNA fractionswere resuspended in 200 pl PBS. For each time point, three 50 pl samples were mixed with 3 x 10-10 M rabbit anti-DNP Ig in a total volume of 100 pl. After incubation for 6 min at room temperature, duplicate 50 pl fractions were filtered and washed as in Section 2.7. The filters were air dried and the radioactivity determined. (0-0) nuclear H-mRNA;(0-0) cytoplasmic H-mRNA.
4. Discussion It has been observed previously that some lectins (such as PWM and Con A) as well as bacterial lipopolysaccharides and polysaccharides act as B cell mitogens in vitro in murine spleen cell cultures with concomitant enhancement of IgM synthesis and secretion [ 5-81. Blast cells and plasma blasts
52
R.H.Stevens, B.A. Askonas and J.L. Welstead
appear in large numbers, and by day 3 and 4 of culture make up a large proportion of the surviving cells [ 5 , 261. Analysis o f spleen cell cultures 7 2 h after PWM addition showed that the cells formed 15 % of IgM relative to total protein production, a threefold increase over the control culture [ 81. Such levels o f IgM production compare favorably with IgM formation by mouse plasma cell tumors, [ 271 and in conjunction with the high proportion of plasmablasts would suggest that a high proportion of the spleen cells left in culture were activated into differentiation and IgM production. We are, o f course, aware that the rapid stimulation of a large number o f B cells in vitro is not identical to the time course o f antibody induction in vivo following antigen challenge. This involves gradual recruitment o f cells and their proliferation into antigen-reactive cells in which antibody production and cell maturation is induced by complex control mechanisms (antigen, as well as cell-cell interactions) within lymphoid tissues. However, the high IgM production following B cell mitogens should provide some indication of the molecular events during antibody induction and the relation between mitosis, Ig-mRNA transcription and translation during maturation o f B cells. LPS treatment of our spleen cell culture (CBA/H mice) in agreement with others resulted in considerable enhancement of IgM production on days 2 and 3, and by day 3 and 4 of culture IgM formation represented 2 6 % o f total protein synthesized. A high proportion (about 5 0 - 6 0 %) of the viable cells were large and pyroniphilic. However, we observed a peak o f DNA synthesis (using 4 h pulses with [3H]thymidine) around 2 4 h of culture with 10 % FCS. This contrasts with the linear increase in [ 3H]thymidine incorporation for 7 2 h after a preliminary lag period o f 24 h in spleen cell cultures from athymic nude mice to which either Con A or LPS had been added [9, 281. The cause for the difference between the two types of culture are not clear. Tissue culture conditions could affect the kinetics, but, in fact, they were similar (except for our larger scale requirements and different batches of FCS). Our kinetics of thymidine incorporation are more in line with those reported by Coutinho and Moller [7]. Normal spleen cells activated by type Ill pneumococcal polysaccharide showed a peak in DNA synthesis on day 2 of culture ( 2 4 h thymidine pulse). It is possible that spleen cells from nude mice d o behave differently from normal cells in these cultures. T cells might cause enhanced B cell activation and eventually possible suppression. There was an additional difference between the two types of cultures stimulated with LPS. Andersson and Melchers observed that after a lag of 24 h, DNA synthesis, IgM production and the appearance of PFC-secreting, antibodybinding NNP hapten increased in parallel [ 91 and, thus, there seemed to be simultaneous cell division and differentiation. LPS treatment of spleen cells in our hands showed a rapid wave of mitosis, with a lag of about 2 4 h before significantly increased Ig production was observed. Transcription of H-chain message increased rapidly (within 6 h of stimulation), with a peak in H-mRNA production around 2 4 h. Our results are in line with induction of antibody production in spleen cell cultures stimulated with SRBC. DNA synthesis and the susceptibility of cells t o suicide with high specific activity thymidine precedes cellular maturation and the appearance of antibody-secreting cells (plaque-forming cells) by 24-48 h [29]. There is additional evidence that division precedes differentiation. In cultures o f spleen cells from athymic mice (nulnu), thymus cell-replacing factor (TRF) restores the response to SRBC [30]. Inhibition o f DNA synthesis by a tem-
Eur. J . Immunol. 1975.5: 47-53
perature shift to 35 OC during the first two days inhibits restoration o f the anti-SRBC response, whereas the same shift at day 2 at the time o f TRF additifin permits TRF action and the development o f PFC [ 231. The presence of y-mRNA in the spleen cell suspension is intriguing considering that little, if any, of this mRNA is translated into protein (Fig. 2). Evidence for y-mRNA in lymphoid cells resides in the fact that oocytes injected with total mRNA produced an antibody precipitable protein migrating with an apparent molecular weight o f 5 5 000. Furthermore, a proportion o f this material is assembled within the oocytes into whole 7 S molecules of 150 000 molecular weight in a manner analogous t o myeloma 5563 lgGza [ I ] . As the spleen cell suspension is a heterogeneous mixture of cells, it is not possible at this time t o determine whether the y and p-mRNA exist within the same cells with a translational block o n y-mRNA or whether the y-mRNA resides in cells refractive t o LPS stimulation. About 18 % of adult murine spleen cells have IgG receptors [3 1 ] and therefore this lymphocyte population can produce IgG in small amounts. However, in confirmation o f work by others [6, 81 we found that mainly IgM production is stimulated by LPS and only a subpopulation o f B cells in spleen appear to be susceptible t o LPS stimulation. The oocyte translation system does not permit exact quantitation of mRNA - it is possible that ymRNA is translated preferentially to p-mRNA - this point has not been tested. However, the assay shows that both and p-mRNA are present in the spleen cells and the low levels of IgG production d o suggest a translational block o n y-mRNA. The possibility of p-mRNA degradation is unlikely t o account for our results, since you can reextract the purified mRNA and obtain very similar ratios of y and p-chain production in oocytes. Estimates of H-mRNA in lymphocytes following LPS stimulation have revealed that the average number of H-mRNA molecules per cell increases from 2 0 0 on day 0 to 1800 o n day 3. Considering the heterogeneity of the cell suspension, the actual number of H-mRNA molecules in any particular cell will be different from the average by a factor o f 2 or 3. I t is apparent, however, that despite extensive blast formation and differentiation o f B lymphocytes during LPS activation, the H-mRNA content of the cells is considerably reduced compared to mouse IgGza myeloma cells (SO00 H-mRNA molecules per cell [ 21). This suggests that although LPS initiates differentiation o f B lymphocytes into plasma cells, full differentiation does not occur in the in vitro system. Alternatively, there may be a difference in the extent o f differentiation between IgM and IgG-producing cells. The binding assay for H-mRNA is not capable of resolving and p-mRNA molecules. From Pig. 3, however, it is clear that the majority o f H-mRNA synthesis following LPS stimulation is p-mRNA. Although the synthesis of mRNA is rapid following LPS stimulation, increased production of immunoglobulin proceeds only following a lag o f about 24 h . This lag does not appear to result from hindered transport of the mRNA into the cytoplasm (Fig. 6 ) and must, presumably, result from a translational block on the p-mRNA. At early times following mitogenic stimulation there is little structural organization o f the cytoplasm [ 261. The synthesis of H and L-chain proteins has been shown to normally occur on membrane-bound polyribosomes [ 31, although it is not clear what role membranes play in the regulation of Ig synthesis or, indeed, whether membranes are an absolute requirement for Ig synthesis.
Eur. I. Immunol. 1975.5: 53-58
Immunochemical study of x-irradiated DNA
The kinetics of Ig production are consistent with the morphology of mouse spleen cells stimulated with LPS analyzed by electromicroscopy [26]. After 16-28 h, B cells become large lymphoblasts with cytoplasmic polyribosomes. Development of endoplasmic reticulum commences only after 28 h and gradually continues. By 2 1/2 days many plasmablasts are found. it has been shown in mouse myeloma cells that under conditions where Ig can interact with H-mRNA, a feed back repression on H-chain protein synthesis occurs. It is conceivable that under conditions of a deficit of membranes, the Ig produced in lymphocytes is not secreted, but allowed t o interact with H-mRNA, resulting in a reduction of H-chain synthesis. At later times following LPS stimulation, when there is increased membrane organization, the mRNA and Ig would become separated and maximum H-chain protein synthesis would proceed. This hypothesis is under further consideration.
53
8 Parkhouse, R.M.E., Janossy, G. and Greaves, M.F., Nature 1972. 235: 21. 9 Andersson, J. and Melchers, F., Proc. Nut. Acad. Sci. US 1973. 70: 416. 10 Stevens, R.H. and Williamson, A.R., J. Mol. Biol. 1973. 78: 515. 11 Stevens, R.H. and Williamson, A.R., J. Mol. Biol. 1973. 78: 505. 12 Lane, C., Marbaix, G. and Gurdon, I., J. Mol. Biol. 197 1. 6 I : 7 3. 13 Stevens, R.H. and Williamson, A.R., Nature 1972. 239: 143.
14 Jones, O.W. and Berg, P., J. Mol. Biol. 1966. 22: 199. 15 Rigs, A.D., Suzuki, H. and Bourgeois, S., J. Mol. Biol. 1970. 48: 67. 16 Kinard, F.E., Rev. Sci. Instrum. 1957. 28: 293. 17 Bray, G., Anal. Biochem. 1960. I : 279. 18 Kitos, P., Saxon, G. and Amos, H., Biochem. Biophys. Res. Comm. 1972.47: 1426. 19 Summers, D.F., Maizel, J.V. and Darnell, J.E., Roc. Nut. Acad. Sci. US 1965.54: 5 0 5 . 20 Choules, G.L. and Zimm, B.H., Anal. Biochem. 1965. 13: 366. 21 Valentine, R.C. and Green, N.M., J. Mol. Biol. 1967.27: 615.
Received February 19, 1974;in revised form July 10, 1974.
22 Andersson, J., Moller, G. and Sjoberg, 0..Cell. Immunol. 1972. 4: 381. 23 Askonas, B.A., Schimpl, A. and Wecker, E., Eur. J. Immunol. 1974.4: 164.
5. References
24 Stevens, R.H. and Williamson, A.R., Nature-New Biol. 1973. 245: 101.
1 Stevens, R.H. and Williamson, A.R., Proc. Nut. Acad. Sci. US 1973. 70: 1127.
25 Leibovitz, A., Amer. J. Hyg. 1963. 78: 173.
2 Stevens, R.H., in preparation.
26 Shohat, M., Janossy, G. and Dourmashkin, R.R., Eur. J. Immunol. 1973.3: 680.
3 Bevan, M.J., Parkhouse, R.M.E., Williamson, A.R. and Askonas, B.A., Progr. Biophys. Mol. Biol., Pergamon Press 1972. 5: 133. 4 Scharff, M.D. and Laskov, R., Progr. Allergy 1970.14: 37.
5 Greaves, M. and Janossy, G., Transplant. Rev. 1972. 1 1 : 87. 6 Andersson, J., Sjoberg, 0. and Moller, G., Transplant. Rev. 1972. 11: 131. 7 Coutinho, A. and Moller, G., Eur. J. Immunol. 1973. 3: 608.
Pier G. Natali Immunology Laboratory, Regina Elena Institute for Cancer Research, Rome
27 Parkhouse, R.M.E. and Askonas, B.A., Biochem. J. 1969.115: 163. 28 Melchers, F. and Andersson, J., Transplant. Rev. 1973. 14: 76. 29 Dutton, R.W. and Mishell, R.I., J. Exp. Med. 1967. 126: 443. 30 Schimpl, A. and Wecker, E., Nature 1972.237: 15. 31 Owen, J.T., Cooper, M.D. and Raff, M.C., Nature 1974.249: 361.
Denaturing effect of x-irradiation on deoxyribonucleic acid aqueous solutions An immunochemical study The denaturation of calf thymus DNA aqueous solutions following x-ray radiation (x-DNA) has been investigated with the help of a rabbit antidenatured DNA antiserum. These antibodies were shown t o recognize exclusively single-stranded DNA determinants on DNA molecule in double immunodiffusion analysis. By means of this method it was possible t o demonstrate that increasing numbers of single-stranded DNA determinants were formed in DNA solutions exposed t o increasing doses of radiation. The rabbit serum appeared t o be a highly sensitive reagent (0.5 pg/ml of singlestranded DNA) capable of detecting DNA denaturation when low ionizing doses were used and when only minor changes in DNA physico-chemical parameters were measurable. Antigenic comparison between x-DNA and heatdenatured DNA demonstrated that antigenic determinants are shared by the two molecules. The possible application of immunochemical methods to the study of nucleic acid denaturation by different physical and chemical agents is discussed. [I 8121
Correspondence: Pier G. Natali, Istituto Regina Elena, V. Regina Elena 291, Rome 00161, Italy Abbreviations: X-DNA X-ray irradiated DNA H D N A Heatdenatured DNA PBS: Phosphate buffered saline MAK: Methylated albumin kieselguhr MBSA Methylated bovine serum albumin
1. Introduction The serologic demonstration of an ti-DNA antibodies in the sera of patients with autoimmune disease [ 11 has stimulated numerous attempts t o induce anti-nucleic acid-specific antibodies in experimental animals.
