Journal of Immunogenetics (1984) 11, 149-154.

H - 2 C O N T R O L O F P O L Y A D E N Y L A T E D m R N A P R O D U C T I O N I N T H E S P L E E N O F M I C E I N J E C T E D W I T H B R U C E L L A A N T I G E N S

J . P I E R R E Z , * 0 . G U E R C l t A N D D . O T H $

*Laboratoire de Biophysique (Professor Bertrand), FacultP A de MPdecine, BPI 84, 54505 Vandoeuvre-les-Nancy,? Service de MPdecine A , CHU de Nancy-Brabois, Vandoeuvre-

les-Nancy and $ INSER M U95, Vandoeuvre-les-Nancy, France

(Received 6 Julv 1983)

S U M M A R Y

RNA was extracted from the splenocytes of Brucella abortus antigen stimulated mice and of control mice. The proportion of chromatographically separated polyadenylated 11.2s mRNA, was determined. With the technique used, only stimulated mice exhibited significant amounts of this RNA species. The highest level was reached 1 day after the stimulation, and the decay from this level presented an oscillatory form during the 4 weeks following the injection.

In two different genetic backgrounds, H-Zb mice did not respond to the stimulus, in contrast to H-2" and H-2f mice. H-2b/H-2f heterozygotes behaved roughly as intermediate between H-2b and H - 2 f mice. This genetic control seems to parallel the genetic control of some Brucella-induced, thymus- dependent events previously described.

I N T R O D U C T I O N

Stimulation of lymphoid cells with antigenic or mitogenic substances results in an increase in biochemical synthesis.

If the stimulus consists of an injection of kiiled Brucella abortus bacteria or antigenic extracts. a number of functions are modified several days after the injection (Toujas et al., 1972). In addition to specific antibodies and to 'non-specific' gammaglobulins, growth factors and other regulatory molecules are produced which affect stem cells as well as cells implicated in various immune mechanisms, both thymus-dependent and thymus- independent (Toujas, Dazord & Guelfi, 1975). The effects of such a stimulation include an increase of natural killer (NK) activity (0 th et al., unpublished), and a strong thymus- dependent adjuvant effect, which turned out to be H-2 controlled, as well as other T lymphocyte functions (0 th & Sabolovic, 1978). Another H-2 dependent consequence of treatment with Brucella is in the kinetics of splenomegaly (Oth, Sabolovic & Le Garrec, I 9 74).

Correspondence: D. 0 t h . Institut Armand-Frappier. 53 I Boulevard des Prairies. C.P. 100. Succ. L.-D.-R..

149

W e de Laval, Quebec, Canada H 7 N 423.

150 The H-2 complex has been shown to control many different functions of the lymphoid

system, implicating a number of different syntheses. As one of the key events of these syntheses is the production of new mRNAs (Wallace et al., 1979; Torelli et al., 1981; Bleackley et al., 1981), we studied the kinetics of the production of mRNA in the spleens of Brucella injected mice of different, well-defined H-2 haplotypes.

J. Pierrez, 0. Guerci and D. 0 t h

M A T E R I A L S A N D M E T H O D S

Mice All the mice used for the experiments were bred in the facilities of INSERM U95

Research Unit, Vandoeuvre-les-Nancy, France. The breeding stock of CBA/Ca were from MRC Labs, Carshalton, England, those of CBA/Br from Antoni van Leeuwenhoekhuis, Amsterdam (Dr Dux), those of A/%, A.CA and A.BY from Karolinska Institute, Stockholm (Dr E. Klein) and those of B 10.A, B 10.M and B 10 mice from H6pital St-Louis, Paris (Dr Colombani). They were used as 2-6-month-old adults.

H-2 haplotype typing

ing to Kaliss (1973), in our standard conditions (0 th & Sabolovic, 1978).

Antigen

1 : 2 in saline and injected intraperitoneally (0.5 mi per mouse).

Extraction and characterization of polyadenylated messenger R N A The method described by Pilch (1977) was used for the extraction of RNA from the

splenocytes, with some modifications which have been described in detail (Pierrez, Guerci & Oth, 1983). It was checked that contamination with DNA and protein accounted for less than 0 - 1%.

Analytical chromatography was performed according to Jones, Lundgren and Jay (1976) on a lysine-sepharose 4B (Pharmacia, Uppsala, Sweden) column (1.6 x 12 cm) as applied to eukaryotic cells. The gel was suspended in 0.02 M Tris-HCI buffer, pH 5, containing 0.01 M MgCI, and 0.05 M NaC1. The sample (approx. 2 mg) was dispersed in this buffer and loaded onto the column. Elution was performed at room temperature using the same buffer, applying a NaCl gradient of 0-05 M to 0.5 M.

The different fractions collected were precipitated in cold ethanol, dissolved in saline and dialyzed against 200 volumes of saline. The OD,,,/OD,,, ratio was always 2.1-2.2, confirming the purity of the RNA fractions. The sedimentation coefficient of each fraction was measured with a Beckmann analytical ultracentrifuge (and rotor) at 20°C and 180,000 g. Five peaks eluted from the column with sedimentation coefficients consistently found to be 4S, 5 S , l l . 2S , 18s and 28s when the spleens were harvested from antigen stimulated mice. If the spleen donors had not been stimulated, the 11.2s peak was always lacking. It was shown to be RNAse sensitive, but insensitive to DNAse or to pronase. It was therefore considered that the 1 1 .2s fraction represented messenger RNA synthesized as a consequence of the antigenic stimulus. It was found to be selectively trapped by a

(A.CA x A.BY)F, were typed for their H-2 haplotypes by haemagglutination, accord-

Brucella antigen from Bio-Merieux, Marcy I’Etoile, France, batch 0 19 10, was diluted

H-2 control of polyadenylated mRNA production 15 1 poly U-sepharose gel (Pierrez, Guerci & Oth, 1983). The relative proportion of this polyadenylated mRNA to total RNA was calculated by the formula:

OD,,, of mRNA fractions

OD,,, of non-mRNA fractions x 100,

where ‘OD,,, of mRNA’ represents the area under the elution peak of the polyadenylated mRNA, and is proportional to both mRNA quantity and to mRNA extinction coefficient. ‘OD,,, of non-mRNA’ stands for the sum of the areas under the other elution peaks, which is proportional to both quantities and extinction coefficient of other RNA species. The proportion is expressed in that form because of the difference existing between the extinction coefficient value of polyadenylated mRNA and that of the other RNAs, due to the exceptional proportion of adenine in the former.

Each determination point represents the mean? standard deviation of five different measurements from five different mice.

R E S U L T S

Kinetics of polyadenylated mRNA production For practical convenience, (CBA/Ca x CBA/Br)F, hybrid mice were used for

preliminary kinetic studies of mRNA production after the injection of Brucella antigens, up to day 30. Fig. 1 clearly depicts three waves of mRNA production on days 1. 11 and 23. The large number of mice used for this determination and the reproducibility of the results found in two separate experiments permits us to state that the production of mRNA in the

7 2 I

Number o f days following the in ject ion of Erucelio antigens

FIG. I . Relative proportions of polyadenylated m R N A in total R N A in the splenocytes of (CBA/Ca x CBA/Br)F, mice, several days after an intraperitoneal injection of Brucella antigens. The results of two independent experiments are shown.

152 J. Pierrez, 0. Guerci and D. 0 t h TABLE I. Determination of the proportion of polyadenylated mRNA in the spleens of Erucellu-injected mice

belonging to several strains (mean 4 SD)

Splenocytes donors status

Genetic H-2 background Mouse strain haplotype

A AJSn ala A.CA f!f A.BY blb

(A.CA x A.BY)F, f l b C57B1/10 BI0.A o la

B 10.M ff B10 blb

Days before assays of injection with Erucella antigens Not

treated 6 10 15 20 50

< l 9 i 2 1 5 + 4 3 + 1 7 + 2 NT' < I 6 2 1 2 7 t 6 1 0 + 2 1 4 k 1 NT < I < I < I 4 1 1 1 6 5 3 7 1 _ 2 4 2 1 NT < I 4 + _ 3 1 0 k 2 NT 6 + 3 NT < I 1 0 1 2 2 4 + 7 9 2 3 NT 3 < I < 1 . 5 <2

<1.5 <1.2 <1 .4 NT

3 k 1 7 + 2 4 5 1

* Not tested.

TABLE 2. Determination of the proportion of polyadenylated mRNA in the spleens of Erucellu injected (A.CA x A.BY)F, mice typed for their H-2 haplotype (mean

2 SD) ~~~ ~ ~~~~ ~ ~ ~~~~ ~

H 2 haplotype Number of mice used mRNA ir SD on day 10

fif 7 blb 7 fib 16

24.9 2 5 . 5 1.6 2 0.5

13.8 2 4.9

situation studied has an oscillatory decay, the maximum values always being considerably higher than the values found in the non-stimulated controls.

H-2 control of polvadenylated mRNA production Congenic resistant A/Sn, BIO.A ( H - 2 9 , A.CA, B1O.M ( H - 2 9 , A.BY and B10 (H-2')

mouse strains were injected with Brucella antigens and production of mRNA was determined 6, 10, 15, 20 and 50 days after the injection, using five mice for each determination. Table 1 shows that A.BY and B10 mice, both of H-2' haplotype, never presented large amounts of mRNA, compared to mice of the H-2" and H-2f haplotypes, on both the A and C57B1/10 genetic backgrounds. The (A.CA x A.BY)F, hybrids gave figures roughly intermediate between the parental strains. H-2 typed (A.CA x A.BY)F, mice were used for mRNA determination on day 10: the finding that H-2' mice produce high amounts of mRNA, H-2' mice low amounts, and H-2f/H-2b mice intermediate amounts was confirmed (Table 2).

D I S C U S S I O N

The kinetics of polyadenylated mRNA production, after stimulation with Brucella, has been studied using a large number of mice and the results have been reproduced in two independently performed experiments. Production of mRNA, in this case, seemed to be an oscillatory decay phenomenon. No oscillations were observed in the amount of

H-2 control of polyadenylated mR NA production 153 anti-Brucellu specific antibodies, which began to be detectable in the serum only 4 days after injection (Toujas, Dazord & Guelfi, 1975). Thus, in this case, fluctuations of the antibody titres were not observed as has been found in other situations (Stimpfling & Richardson, 1967).

The products coded for by mRNAs of in uitro stimulated T lymphocytes are numerous (Kecskemethy & Schafer, 1982) and include lymphokines such as interferon and IL-2, for which mRNA of 11-12 S species have been found in the mouse (Bleackley et ul., 1981). In the present work Brucella stimulation was given in vivo and certainly a large synthesis of mRNA-species must have been elicited, not necessarily restricted to T cell products. However, the results obtained here show that mRNA production as a whole, and under the conditions used, is a sharply controlled parameter. with a strong H-2 dependence.

After Brucella antigen injection, the H-2b haplotype was associated with absence of stimulation of thymus-dependent functions, but behaved normally in the production of anti-Brucellu specific agglutinins, a function which is not thymus-dependent (0 th & Sabolovic, 1978). These results raised the question as to whether this weak reactivity of H-2b mice was due to an active suppression mechanism, or to an absence of reaction. On the other hand, H-2b was also associated with differential kinetics of splenomegaly. after injection of Brucella antigen ( 0 t h et al., 1974).

The results obtained in the present experiments show that polyadenylated mRNA, in the spleens of mice recently injected with Brucella, may present different behaviour depending on the H-2 haplotype of the mouse strain. In H-2b mice, polyadenylated mRNA was produced either in too small amounts or in a form too labile for detection by the technique used. We can interpret these results as a whole by an action by H-2b or H-Zb-associated products on the synthesis of some products that influence immune reactivity and substances influencing the kinetics of processes like splenomegaly.

The heterozygotes between ‘high’ and ‘low’ Brucella reactive parents exhibited an intermediate response in the production of polyadenylated mRNA, which corresponds roughly to what had been found with the thymus-dependent adjuvant effect, as well as with the splenomegaly effect after such a treatment (0 th et ul., 1974). This would illustrate a regulatory role for the H-2 complex, for which molecular models have been proposed (Ivanyi, 1978).

A C K N O W L E D G M E N T S

We wish to thank Ms M. C. Llorens and Mr Georges for skilful technical assistance. This work was supported by INSERM (Paris) and by the National Cancer Institute of Canada.

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