lmmunochemistrv 1977, Vol. 14, pp. 479M.88. Pergamon Press. Printed in Great Britain

A COMPARATIVE BIOCHEMICAL STUDY OF CELLS SYNTHESIZING IMMUNOGLOBULINS WITHOUT DETECTABLE ANTIBODY FUNCTION AND OF ANTIBODY-SYNTHESIZING CELLS CHRISTINE PETIT, JEAN-CLAUDE A N T O I N E and STRATIS AVRAMEAS Unit6 d'Immunocytochimie, D6partement de Biologie Mol6culaire, Institut Pasteur, 25, rue du Docteur Roux, 75015 Paris, France (First received 24 December 1976; in revised form 14 March 1977)

Abstract--After a primary immunization, two populations of immunoglobulin-containingcells could be detected. The first one which appeared contained in their cytoplasm Ig without detectable antibody " function against the antigen injected (IFC).* This population is progressively replaced by antibody-synthesizing cells (AFC). In the present paper, we investigated whether some features of the biosynthesis of the Ig derived from IFC could account for the absence of detectable antibody function of these Ig. We studied the amounts of Ig produced by IFC and AFC and the molecular composition of the Ig derived from each population of cells. Dissociated immune lymph nodes containing essentially either IFC or AFC were incubated for 4 hours in presence of aH-leucine. The Ig present in the supernatants and in the cellular lysates of these cultures were precipitated by anti-immunoglobulin antibodies. The rate of Ig biosynthesis and secretion of these two populations were compared. It appeared that IFC and AFC release in the supernatant the same amount of radioactive Ig and that they contain about the same amount of Ig in their cytoplasm. The anti-immunoglobulin precipitates were analyzed by SDS polyacrylamide-gel electrophoresis. It was found that IFC and AFC secreted completely associated Ig and a small percentage of free radioactive light chains (100,(,). AFC taken at day 24 or at day 28 secreted nearly exclusively IgG. IFC taken at day 4 or 5 secreted an amount of radioactivity equally divided between IgG and IgM. At either day 4/5 or day 24/28, the same distribution of the Ig between IgG and IgM was found in the cellular lysate and in supernatant. No precursor molecule of light and heavy chains was detected in IFC and AFC. In conclusion this study revealed no difference in the biosynthesis and in the molecular composition of the Ig derived from the two populations of cells which could explain the absence of detectable antibody function of the Ig contained in IFC.

INTRODUCTION

The study of the immune response of rats and mice led us to describe two populations of immunoglobulin-synthesizing cells. After a primary antigenic stimulation the first population of immunoglobulin-staining cells which appeared contained immunoglobulin without detectable function (IFC). This population was progressively replaced by antibody-staining cells (AFC) (Miller et al., 1974, 1975; Antoine & Avrameas, 1976). This observation was also noted in conven* Abbreviations used: IFA, Freund's incomplete adjuvant; IFA Po, horseradish peroxidase emulsified in Freund's incomplete adjuvant; IFC, cells containing immunoglobulins without detectable antibody function directed against the antigen injected; AFC, cells containing antibodies directed against the antigen injected; rats IFA, rats injected with IFA; rats IFA-Po, rats injected with l mg of Po emulsified in IFA; rats IFA 4/5, rats injected with IFA 4 or 5 days before; rats IFA 24/28 rats injected with IFA 24 or 28 days before; culture IFA, culture of cells derived from lymph nodes of rats IFA; culture IFA Po, culture of cells derived from lymph nodes of rats IFA-Po; S, supernatant; CL, cellular lysate; SIFA, SIFA-Po, supernatants of culture of cells derived from lymph nodes of rats injected respectively with IFA and ,IFA-Po; CLIFA, CLIFA-Po, cellular lysates of culture of cells derived from lymph node of rats injected respectively with IFA and IFA-Po. 479 IMM. 14,/7--A

tional, specific pathogen-free and germ-free rats immunized with different antigens in the presence or absence of adjuvants (Avrameas et al., 1976). Mainly, two experiments suggested that some relationships existed between these two populations. (1) In some plasma cells containing antibodies in a restricted area of their cytoplasm, immunoglobulinantigenic determinants were found to be present in the remainder of their cytoplasm (Miller et al., 1974, 1975; Avrameas et al., 1976). (2) The two populations synthesized immunoglobulins which shared some idiotypic specificities in common (Cazenave et al., 1974). In all of these results, IFC and AFC were identified by immunoenzymatic staining. In these techniques, IFC were detected by their immunoglobulin (Ig) antigenic determinants and their inability to fix the antigen, and AFC were detected by their ability to fix the antigen. Because of the techniques employed to reveal these two populations of cells, the existence of IFC could be explained by different hypothesis which have to be tested by other techniques: (a) IFC might be cells containing only antibodies but in too small an amount to be detected by their ability to fix the antigen but enough to be detected by their antigenic determinants. (b) IFC might be cells synthesizing only non-associated chains of antibodies or synthesizing molecular precursors of heavy and light

480

CHRISTINE PETIT, JEAN-CLAUDE ANTOINE and STRATIS AVRAMEAS

chains associated or not. These precursors have been described by Milstein et al. (1972), Blobel and Dobberstein (1975), and Schechter et al. (1976). To answer these questions we first compared the amounts of Ig secreted and contained by IFC and AFC after a biosynthetic radioisotopic labelling of their Ig. This comparison, performed with lymph nodes of rats taken at the beginning and at the end of the primary immune response, requires establishing previously what percentage of the Ig secreted and found in the cellular lysates has to be attributed respectively to Ig non-staining cells, i.e. some lymphocytes and to Ig-staining cells, i.e. IFC and AFC. Secondly, in order to investigate whether the IFC are distinguished from AFC by the molecular composition of their Ig, we analysed the Ig biosynthesized by these two populations of cells. In the present paper we report the results, which indicated that IFC and AFC have similar rates of Ig biosynthesis and secretion and that the molecular composition of Ig secreted and contained by IFC could not explain the absence of detectable antibody function of these Ig.

MATERIALS AND METHODS Animals and immunizations Five-month old random bred OFA rats and inbred Fischer rats of both sexes were used. They were reared in specific pathogen-free conditions until the start of their immunization. Rats were injected in each hind footpad with 500 ,ug of peroxidase (Po) (grade Rz = 3, Boerhinger, Mannheim, Germany) emulsified in a mixture composed of 100/~1 of 0.15 M NaC1 made with pyrogen-free water (Biosedra, Malakoff, France) and 100 #1 of Freund's incomplete adjuvant (IFA) (Difco Laboratories, Detroit, MI, U.S.A.). As controls, some rats received in each hind footpad 1001d of 0.15 M NaCI emulsified with IFA. Preparation of antisera and of purified antibodies Rabbit anti-rat Ig and anti-ovalbumin antisera, sheep anti-rabbit Ig and anti-rat Ig antisera were prepared as described earlier (Avrameas, 1969; Gonatas et al., 1972). Purified antibodies directed against rat 72a, 72b, ~t heavy chains and light chains of immunoglobulins were isolated by passage of rabbit anti-rat Ig antisera through rat IgG and IgM immunoadsorbents prepared with glutaraldehyde (Avrameas & Ternynck, 1969). Rat IgG were isolated as described previously (Gonatas et al., 1972) and rat IgM were prepared from sera of rats bearing IR 202 myeloma (gift from Dr. H. Bazin, Department of Experimental Medicine, Brussels, Belgium) following procedures already published (Antoine & Avrameas, 1976). Rabbit anti-ovalbumin antibodies were purified by passage of rabbit antiovalbumin antisera on polyacrylamide beads BioGel P 300 (Calbiochem. Luzern, Switzerland) coated with ovalbumin (Grade Ili, Sigma Chemical Co., St. Louis, MO, U.S.A.) by using glutaraldehyde as the cross-linking agent (Ternynck & Avrameas, 1972). Sheep anti-rabbit Ig antibodies, free of cross-reactivity with rat Ig, were prepared by first adsorbing sheep antisera on a rat IgG immunoadsorbent until the cross-reaction with this antigen disappeared; and then by isolating anti-rabbit Ig antibodies on a rabbit IgG immunoadsorbent. Labellin9 of purified antibodies Purified rabbit and sheep anti-rat Ig antibodies were labelled with peroxidase following a two-step procedure (Avrameas & Ternynck, 1971). In some experiments, peroxidase-labelled sheep Fab fragments of anti-rat Ig anti-

bodies, prepared as described previously', were also used (Antoine et al., 1974). Preparations of cells and biosynthetic labelling In the first three experiments rats immunized with peroxidase and control rats injected with adjuvant alone were killed 4 or 5 days after the injection. In the three last experiments the cells of the experimental and those of control animals injected 4 or 5 and 24 or 28 days before were simultaneously studied. For each experiment 2-3 rats of each group were used. Their lymph nodes were excised, pooled and teased at room temperature with fine forceps in Earle's medium without glucose, containing 2.5~,/, foetal calf serum and 250#M of each of the following sugars: D-glucosamine, D-galactose, D-mannose and L-fucose (Sigma Chemical Co., St. Louis, MO, U.S.A.). Foetal calf serum before use was dialysed against 1(30 volumes of Earle's medium as described by Melchers (1971). After two centrifugations at 200g for 7 min at room temperature, and washings with the Earle's medium, the cells were suspended in MEM (Gibco bio-cult, Glasgow, U.K.) not containing L-leucine and r~-glucose, and supplemented with: non-essential amino acids, L-glutamine, streptomycine and penicillin (Specia, Paris, France), dialysed foetal calf serum, D-glucosamine, D-galactose, D-mannose and L-fucose (Melchers, 1971). The viability of the cells was tested by trypan blue exclusion and in each experiment about 90°Jo of the living cells were counted. The final cellular concentration was adjusted to 5 x 10 6 living cells/ml and three ml of the suspension were put in No. 3017 Falcon tissue culture (Falcon Plastics, Oxnard, U.S.A.). After addition of 60 ~tCi of radioactive L-3H leucine (25-43 Ci/mM, CEA, Gif-sur-Yvette, France) per ml of cells, flasks were incubated at 37°C in an atmosphere of N2 + O2 + COz (83:7:10) for 4 hr under 7 cycles/min agitations on a rocker platform (Bellco Glass, Inc. Vineland, N J, U.S.A.). The radioactive L-3H leucine was diluted in cold L-leucine and the concentration of L-leucine added at the beginning of the culture was 2 × 10 -8 mole/ml. After the incubation, living and dead cells were counted. No more than 5-10~,, of dead cells was found. An aliquot of the cells was withdrawn to prepare smears for counting antibody and immunoglobulin-forming cells (see below). Lysis of cells Cells and culture medium were separated by centrifugation. Cells (2-4 x l0 T) were lysed in 1 ml of 0.5~o Nonidet NP 40 (Soci6t6 Shell, Paris, France) in TKM (10mM Tris-HC1, pH 7.4, 25 mM KC1, 5 mM MgC12) containing 10 mM iodoacetamide and 10 3 M PMSF (phenylmethylsulfonylfluoride, Sigma Chemical Co., St. Louis, MO). After 10 min at 20°C and 20 rain at 4°C the cell lysates were centrifuged at 100,000g max for 30min. Culture mediums were spun simultaneously. After centrifugation the cell homogenates and the culture mediums were dialysed overnight in phosphate-buffered saline (PBS) (10 -2 M phosphate buffer pH 7.4, 0.15 M NaC1) containing 0.02~0 NP40. This step was eventually followed by a centrifugation at 100,000.q max for 30 min (Lisowska-Bernstein et al., 1973). Each sample was concentrated by dialysis under vacuum against PBS, 0.02~o NP40, and after measuring the volume, each sample was stored at -80°C or immediately used for the immunoprecipitation experiments. Aliquots were withdrawn before and after the difference steps and counted in a scintillation counter (Spectrom6tre 5. Scintillation Liquide, SL-30. Intertechnique, Plaisir, France) for total radioactivity and for the trichloroacetic acid (TCA)precipitable radioactivity. Immunoprecipitation of secreted and synthesized immunoglobulins In 1~0 foetal calf serum-coated tubes various amounts of concentrated culture supernatants or cells lysates were

A Biochemical Study of Cells Synthesizing 'Non-specific' Immunoglobulins added, followed by 60 #g of purified rabbit antibodies directed against ), (2a + 2b),/t and L chains. Controls were done by adding 60 #g of rabbit anti-ovalbumin antibodies instead of anti-Ig antibodies. After 1 hr at 37°C and 30 min at 4°C, 250 #g of purified sheep anti-rabbit Ig free of crossreacting anti-rat Ig antibodies were added. The tubes were incubated for 1 hr at 37°C and overnight at 4°C. The medium where all the above working steps were performed was PBS containing: l mM leucine, 2~o Triton X-100 (Sigma Chemical Co., St. Louis, MO), 0.01~o NP40. The final volume of the mixture was 0.5 ml. The doses of purified antibodies used were previously determined by performing quantitative precipitation tests. The precipitates were then thoroughly washed following a procedure described by Lisowska-Bernstein et al. (1973): after centrifugation at 4100g the pellets were washed: (1) three times with 3 ml of cold PBS containing 0.05~o of NP40 and 1 mM L-leucine; (2) one time with PBS; (3) one time in 10~o TCA; (4) one time with PBS. Precipitates were dissolved in a known volume of the buffer used for gel electrophoresis [0.0625 M Tris-HC1, buffer pH 6.8 containing 10% glycerol, 3~o Na-dodecyl sulfate (SDS)] and incubated for a few minutes at 80°C. From each dissolved precipitate 10pl was withdrawn for radioactivity counting. In the results presented below the radioactivity counted in the control precipitates was deduced from that of the specific precipitates. For gel analysis experiments, half of the samples were reduced by adding 5~ of fl-mercaptoethanol to this buffer.

Gel electrophoresis The native and reduced precipitates dissolved in the sample buffer were analysed by acrylamide-gel electrophoresis in slabs by using the discontinuous SDS, Tris~lycine buffer system of Laemmli (1970), with 3~o of acrylamide in the stacking gel and 7.5, 8.75 or 16~o of acrylamide in the separating gel. The amount of radioactivity put at the top of the gel varied from 500 to 10,000 counts/ min following the sample. Various markers of known mol. wts were run simultaneously. After completion of the run, gels were stained with 0.2~ Coomassie brilliant blue for 1 hr, destained, and then sliced in 0.5 or 1 mm fractions. The content of each fraction was extracted with 5 ml of POPOP/PPO/toluene solvent containing 5~o of tissue solubiliser TSI (Koch-Light Laboratories Ltd, Colnbrook, Bucks, U.K.). After 24 hr at room temperature the fractions were counted in a scintillation spectrometer.

Detection and counting of immunoglobulin and antibodystaining cells At the end of the incubation of the cells with 3H-L-leucine (see above), some of them were cytocentrifuged. Then smears were dried and fixed with 4~o paraformaldehyde dissolved in 0.2 M cacodylate, HC1 buffer pH 7.4. The slides were then immediately incubated with 100#g/ml solution of peroxidase or a 125-250 #g/ml solution of peroxidase-labelled sheep anti-rat Ig antibodies or Fab fragments to detect, respectively, antibody and immunoglobulin-forming cells. The peroxidase activity was revealed according to the method of Graham and Karnovsky (1966). The number of cells containing immunoglobulins without detectable antibody activity (IFC) was calculated by subtracting the number of antibody-staining cells from the number of immunoglobulin-staining cells. Detailed procedures to detect and to count AFC and IFC have been already published (Antoine & Avrameas, 1976). RESULTS

(1) Development of IFC and AFC during primary im-

mune response against peroxidase The kinetics of development of Ig-staining cells, IFC and A F C during the primary immune response

481 o

g ¢5

g, 25.

9 "~--", 5 7

11

14

20

x 24 25

98

Days after antigen Fig. 1. Kinetics of development of the Ig-staining cells (© ©) of the cells synthesizing Ig without detectable antibody function [IFC] ( × × ) and of the antibodyproducing cells [AFC] (0-------0) in the popliteal lymph nodes of Fischer rats immunized in the hind footpads with 1 mg of peroxidase emulsified in Freund's incomplete adjuvant. (A A): Kinetics of development of Ig-staining cells detected in the popliteal lymph node of control Fischer rats injected with Freund's incomplete adjuvant alone. The results are expressed as the percentage of positive cells at various times after the primary injection. of Fischer rats injected with horseradish peroxidase emulsified in Freund's incomplete adjuvant (IFA-Po) or with Freund's incomplete adjuvant alone (IFA) are reported in Fig. 1. After injection of I F A - P o , we observed that the first population of Ig-staining cells which arose was nearly exclusively composed of IFC. At day 5, the percentage of Ig-staining cells was 3.15~o of the total nucleated cell population; less than 1~o of these Ig-staining cells are AFC. At day 20, IFC and AFC populations reached their greatest size. At day 24-28 we observed a decrease of the percentage of Ig-staining cells to 2.75~o, but 65~o of these were now AFC. Then, at day 5 and at day 24/28, Fischer rats I F A - P o had a similar percentage of Ig-staining cells. At day 5 however only cells containing Ig without detectable antibody function were present, while at day 24/28 the majority of Ig-staining cells were antibody-producers. So, in order to compare the rate of Ig biosynthesized and secreted by IFC and AFC we used Fischer rats I F A - P o immunized 5 and 24/28 days before. When instead of Fischer rats, O F A rats were used, a faster evolution of their immune response was noted, since at day 24/28 the Ig-staining cells represented only 1~o of the total cell population. (2) Biosynthesis and secretion of total proteins and of

ig Intracellular and secreted total proteins and Ig were determined in each culture of cells taken from rats IFA (cells IFA) and from rats I F A - P o (cells

2.2 * 1.2

IFA-PO

s CL s CL s CL s CL

C”L

S

of extracellular

Dl

(S) and

cellular

245 3580 670 3454 264 3312 454 3037

i. + + + + + + +

76 734 92 643 84 1682 228 1373

0.75 & 0.10

lk

1.3 (0.04)d 2.2 + 1.2 (1.4 + 0.8)”

+

2 4.2

0.1

1 0.4

the quantities

3.1 k 1.7

3 11.3 + 5.1

Ig-staining cells in IFA-PO

between

Ig synthesized

3.25 + 2.30

4 11 _+4

of radioactive

2.6 + 2.2

5 4.4 * 1.3

and secreted

during

for

+ * + + + f i &

0.11 0.020 0.17 0.065 0.17 0.039 0.26 0.047

and the numbers

0.9 f 0.1

6 1.3 + 0.6

4 hr of culture

0.7 * 0.1

7 0.5 + 0.23

of cells containing

1.5 6 1.5 10 1.5 5 2.5 8

of specific

+ f f * + rf f k

43 i_ 5

8 63 f 21

C3H]Ig

24k

12

9 12.5 + 3.0

synthesized

Ig

precipitates

[3H]-L-leucine

intracytoplasmic

14.0 52 4.6 34 7.0 54 4.0 47

counted in the same sample. The results are expressed in 0,), the radioactivity

0.21 0.035 0.48 0.097 0.28 0.042 0.44 0.090

c41

for 4 hr with

Non-specific precipitates Specific precipitates J

10’ cells incubated

[31 L’!Z!-Jg’~~~ C3~]-~C~ precipitable material

(CL) counted

Mean of the 6 experiments performed at day 3 5 i S.F. Mean of the 3 experiments performed at day 24/28 kS.E. ’ Numbers of Ig-staining cells detected by immunoenzymatic techniques (AFC included) in rats anti-IFA and in rats anti-IFA -PO. ‘The same number of cells was used to measure the quantities of radioactrvc Ig found in each sample. ’ The percentage of radioactive Ig which could be attributed to lymphocytes was estimated using the equation reported in Results ‘Numbers in brackets arc per cent values of AFC in the total nucleated cell population.

24128

415

Days

Ig-stain-

Table 2. Correlations

lysates

material (anti-Ig).

[‘HI-TCA precipitable material x 10m3 dis/min per IO’ cells

mediums

The results are the mean of three experiments *SE. a The percentage of immunoglobulin-containing cells included IFC and AFC. b S = extracellular medium; CL = cellular lysate. ’ Ratio between the specifically precipitated radioactive Ig and the radioactive TCA precipitable d Ratio between radioactivity of non-specific precipitates (anti-ovalbumin) and specific precipitates being taken as 100%.

0.8 f 0.2

2.9 + 0.5

IFA-PO

IFA

0.4 + 0.2

IFA

Ig (%)

415

24/28

radioactivity

Cells containing intracytoplasmic

Cl1

and Ig-associated

Culture

1. TCA precipitable

Day

Table

A Biochemical Study of Cells Synthesizing 'Non-specific' Immunoglobulins IFA-Po) immunized 4/5 or 24/28 days before. As shown in Table 1 (column 2) during 4 hr of incubation, similar amounts of (3H)leucine were incorporated into proteins of cellular lysates derived from rats IFA immunized 4/5 or 24/28 days before (respectively CL IFA4/5 and CL IFAz4/zs) and from rats I F A - P o immunized 4/5 or 24/28 days before (respectively CL IFA-Po4/5 and CL IFA-Po24/ES). For the same time of incubation it was found that the radioactivity associated with total proteins released in supernatant by cells IFA4~s or IFA24t2s and by cells IFA-Po4,s or IFA-Po24/zs (respectively SIFAa/s, SIFA24/28 and SIFA-P04/5, SIFA-Po24/ES) was smaller than the radioactivity associated with cellular proteins (Table 1, column 2). Furthermore, at each day a larger quantity of radioactive proteins was detected in SIFA-Po than in SIFA. For all experiments and in each culture, the ratios between radioactive Ig and radioactive proteins were greater in the supernatants than in the cellular lysates (Table 1, column 3). Furthermore, it was found that SIFA and CLIFA at each day contained a smaller percentage of radioactive Ig than SIFA-Po and CLIFA-Po. (3) Rate of secretion of IFC and AFC (a) Rate of secretion of lg-stainin 9 cells. In immune lymph node cell suspensions two types of Ig synthesizing cells are present: the Ig non-staining cells, i.e. lymphocytes, and the Ig-staining cells, i.e. IFC and AFC. The comparison of the rate of secretion of IFC and AFC required a knowledge of the proportion of the total radioactive Ig released in the supernatant of the culture to be attributed either to lymphocyte or to Ig-staining cells. To evaluate the part of radioactive Ig due to Ig-staining cells and released in the medium in 4 hr of culture, we used rats IFA. These rats IFA possess a much smaller percentage of lgstaining cells than rats I F A - P o (Table 2, column 3). So from the comparison between the quantities of radioactive Ig in SIFA and SIFA-Po and the numbers of Ig-staining cells counted in the cultures IFA and IFA-Po, the percentage of released radioactive Ig derived from Ig-staining cells could be estimated. At day 4/5 and at day 24/28, the mean of the ratios between the amount of radioactive Ig measured in SIFA-Po and of radioactive Ig measured in SIFA (Table 2, column 4) did not significantly differ from the mean of the ratio between the numbers of Ig-staining cells counted in the corresponding cultures (Table 2, column 3). To estimate the degree of correlation between these two ratios, their correlation coefficient was calculated and a regression curve was traced from the variations of these two ratios (Fig. 2). The correlation coefficient is 0.95. The slope of the regression curve did not significantly differ from the value 1. The Student-Fischer t-test was used for the statistic analysis (Schwartz, 1972). This striking correlation suggested: (1) that the radioactive Ig released in supernatant in 4 hr of culture were almost exclusively produced by the Ig-staining cells, and (2) that the average rate of secretion of Ig-staining cells was the same for rats anti-IFA and anti-IFA-Po. (b) Comparison of the rates of secretion of Ig-stainin 9 cells 5 and 24/28 days after the immunization. Taking into account the previous results which indicated that

15-

483

°J

/° I9-c0ntainin 9 cells in rats anti IFA. Po lg containingtells in ratsanti IFA

Fig. 2. This curve represents the variations of the ratios between the quantity of radioactive Ig detected in the supernatants of cultures anti-IFA-PO and detected in the supernatants of cultures anti-IFA in function of the variations of the ratios of the numbers of Ig-staining cells revealed in the corresponding cultures; the points derived from all experiments performed at days 4/5 and 24/28. The slope of this curve (Po) was determined using statistical analysis (Schwartz, 1972). radioactive Ig released in the supernatant was derived nearly exclusively from Ig-staining cells, a comparison of the rates of secretion of Ig-staining cells 5 and 24/28 days after the immunization was performed with Fischer rats IFA Po. These rats possessed at both these days nearly the same number of Ig-staining cells. The mean of the ratios between numbers of Igstaining cells detected at day 5 and detected at day 24/28 was 1.10_ 0.25 and the mean of the ratios between the quantities of radioactive Ig secreted in the corresponding supernatants of culture was 1.3 + 0.5. Then it can be concluded that Ig-staining cells derived from rats immunized 5 days and 24/28 days before have similar average rates of secretion. (4) Amounts of radioactive 19 contained in IFC and AFC (a) Amounts of I 9 synthesized by Ig-staining cells. In order to compare the rate of biosynthesis of lgstaining cells taken 4/5 and 24/28 days after the immunization, it was first necessary to establish what part of the radioactive Ig present in the cellular lysates could be assigned to lymphocytes and what to Ig-staining cells. For this, as previously, we compared the numbers of Ig-staining cells and the amounts of radioactive Ig found in the cellular lysates. At day 4/5 and 24/28, the ratio between the quantities of radioactive Ig precipitated in CLIFA-Po and precipitated in CLIFA (Table 2, column 5) was found not identical but smaller than the ratio between the numbers of Ig-staining cells revealed in the corresponding cultures (Table 2, column 3). This smaller percentage was considered to be due to the presence of lymphocyte Ig in the cellular lysates. To try to quantitate this part of radioactive Ig produced by the lymphocytes, the two following assumptions were made: (i) For a given day, the lymphocytes originating

484

CHRISTINE PETIT, JEAN-CLAUDE ANTOINE and STRATIS AVRAMEAS

from the cultures IFA and I F A - P o have the same rate of lg biosynthesis. (ii) For a given day, the quantity of radioactive Ig synthesized by an Ig-staining cell derived either from IFA or from I F A - P o is identical. This last assumption is strongly suggested by the previous experiments reported above on the secretion of Ig by these Ig-staining cells. For cultures I F A - P o or IFA, we can consider that the radioactive Ig detected in the cellular lysate is composed of Ig derived from Ig-staining cells and of Ig derived from Ig non-staining cells, i.e. lymphocytes. So, for each culture at a given day, the following equation (1) expresses the total radioactive Ig present in the cellular lysate. /3H Ig CL) = (a x b) + {c x d)

(1)

where (3H Ig CL) = amount of total radioactive Ig present in the cellular lysates derived from cultures I F A - P o of IFA; a = number of Ig-staining cells counted in the culture; b = average amount of radioactive Ig in one Ig-staining cell after 4 hr of culture; c = number of Ig non-staining cells in the culture; d = average amount of radioactive Ig contained in one lysed Ig non-staining cell (lymphocytes) after 4 hr of culture. The average amount of radioactive Ig present in one Ig-staining cell (value b) can be calculated using equation (2). This last equation is obtained by subtracting the amount of radioactive Ig present in CL IFA from the amount of radioactive Ig present in CL I F A - P o [each amount being expressed by equation (1)]. Taking into consideration the two assumptions made above and the observation that the percentage of lymphocytes in cultures I F A - P o and IFA are practically identical, the following equation (2) was obtained. (3H Ig CL IFA-Po) - (3H Ig CL IFA) = (all~A_Po-- alFA) b (2) where a~vA-Po and alva are respectively the number of lg-staining cells in cultures I F A - P o and IFA. When the value b has been calculated, the average amount of radioactive Ig contained in one lysed Ig non-staining cells (value d) can be estimated using the equation (1). It appeared in these different experiments that in the cellular lysates after 4 hr of incuba-

tion, the average amount of radioactive Ig attributable to one Ig non-staining cell is about 100 times fewer than the one attributable to one Ig-staining cell. The percentages of the radioactive Ig originating from lymphocytes in the cultures at day 4/5 and 24/28 are reported in Table 2 (columns 8 and 9). (b) Comparison of the amounts of radioactive ly present in lg-stainin# cells 5 and 24/28 days after the immunization. In the experiments performed with Fischer rats, immunized with I F A - P o the ratio between the numbers of lg-staining cells counted at day 5 and at day 24/28 was 1.1 ___0.25 while the ratio between the amounts of radioactive Ig detected in the corresponding cellular lysates was 1.2 + 0.1. In these experiments using the above calculations, 15-20~o of the radioactive Ig could be attributed to Ig non-staining cells. Therefore, the most simple explanation which could agree with these observations would be that at day 5 and at day 24/28 the Ig-staining cells have in their cytoplasm nearly the same amounts of radioactive Ig. Using the determination of the amount of radioactive Ig present in the Ig-staining cells at each day, it appeared that after 4 hr of culture, these cells released in the supernatant at day 4/5 and at day 24/28, respectively, 1.45 + 0.7 and 1.2 + 0.1 times the amount of radioactive Ig that they contain. (5) Class of I O synthesized and secreted at day 4/5 and 24/28 after the immunization The radioactivity associated with p and 7 heavy chains in the supernatant and in the cellular lysates derived from rats IFA and I F A - P o is presented in Table 3. All of these evaluations were done after reduction of the precipitates by fl-mercaptoethanol and electrophoresis on 8.75~o or 16% SDS polyacrylamide-gels (see Figs. 3c, 3d, 4c, 4d, 5a, 5b). In the supernatants of cultures derived from rats IFA Po at day 4/5 after the immunization 37.5~o of the radioactivity of heavy chains was associated to # chains while at day 24/28 only 5~o of this radioactivity was still associated with p chains. At each day the distribution of heavy chains between g and 7' in the cellular lysates I F A - P o was similar to that found in the corresponding supernatants. Rats injected 4/5 and 24/28 days before with IFA alone gave quite similar results to rats injected with IFA-Po, although some differ-

Table 3. Distribution of radioactive heavy chains detected in supernatants and cellular lysates between /~ and 7 chains //a

),a

Day

Immunization

Sample

(~o)

(%)

4/5

1FA

S CL S CL S CL S CL

43.0 + 10.5 52.0 + 8.5 37.5 +__8.0 36.5 + 10.0 8.5 + 4.5 19 + 5 5.0 + 0.5 19.0 ± 7.5

57.0 _ 10.5 48.0 _%_+8.5 62.5 + 8.0 63.5 +_ 10.0 91.5 + 4.5 81 + 5 95.0 _+ 0.5 81.0 + 7.5

IFA-Po 24/28

IFA 1FA Po

For 4/5 days: mean of 4 experiments ±S.E. For 24/28 days: mean of 2 experiments ±S.E. a Per cent values of radioactivity bound to /~ or to y chains, the sum of ~ plus 7 was taken o/ as 100/o.

A Biochemical Study of Cells Synthesizing 'Non-specific' Immunoglobulins

a

c

[gM 19S

2.

end

II '°

f

of

~°,q,,o°

I

'

t

1.5.

1

j

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Fig. 3. Electrophoresis on 8.75% (a and b) and on 16% (c and d) cross-linked SDS polyacrylamide gels of r3H] leucine-labelled supernatants derived from cultures anti-IFA-PO (a and c: days 4/5; b and d: days 24/28) precipitated with rabbit anti-lg antibodies and analysed before (a and b) or after reduction with ]7-mercaptoethanol (c and d). In Fig. 3d the open circles (O O) indicate the migration pattern of the radioactive materials non-specifically precipitated with anti-ovalbumin antibodies. Fractions are numbered from negative to positive electrode.

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Fig. 4. Electrophoresis on 16% cross-linked SDS polyacrylamide gels of [3HI leucine-labelled cellular lysates derived from cultures anti-IFA-PO (a and c: day 5; b and d: day 28) precipitated with anti-Ig antibodies and analysed before (a and b) or after (c and d) reduction with /7-mercaptoethanol. In Fig. 4a, the open circles (O O) indicate the migration pattern of the radioactive materials non-specifically precipitated with anti-ovalbumin antibodies. Fractions are numbered from negative to positive electrode.

485

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CHRISTINE PETIT, JEAN-CLAUDE ANTOINE and STRATIS AVRAMEAS

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A comparative biochemical study of cells synthesizing immunoglobulins without detectable antibody function and of antibody-synthesizing cells.

lmmunochemistrv 1977, Vol. 14, pp. 479M.88. Pergamon Press. Printed in Great Britain A COMPARATIVE BIOCHEMICAL STUDY OF CELLS SYNTHESIZING IMMUNOGLOB...
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