Immunochemical characterization of the auto antibodies produced by mouse peritoneal cells in culture

lmmunochemistry, 1977, Vol. 14. pp. I 9. Pergamon Press.

Printed in Great Britain

IMMUNOCHEMICAL CHARACTERIZATION OF THE AUTO ANTIBODIES PRODUCED BY MOUSE PERITONEAL CELLS IN CULTURE* ALAIN E. BUSSARD, MARIE-ANTOINETTE VINIT and JACQUELINE M. PAGES Department of Cellular Immunology, lnstitut Pasteur, Paris 75015, France (Received l0 June 1976)

Abstract--The formation of anti-sheep red blood cells (SRBC) and anti-bromelinized mouse red blood cells (BrMRBC) plaques of hemolysis (PFC) by mouse peritoneal cells (PC) in culture has been studied. A very high number of PFC (up to 10%) could be detected after 4-5 days of culture. In most instances the same cells produced anti-SRBC and anti-BrMRBC antibodies, indicating a high degree of cross reactivity between antigenic determinants of the two types of erythrocytes. Autoradio-immunoelectrophoresis radioactively (tac leucine) tagged tissue culture fluid or cell extracts showed the PC in culture actively synthesized IgG and IgM. A certain amount of these Ig was characterized as anti-SRBC and anti-BrMRBC antibodies. It is suggested that the spontaneous formation of anti-SRBC plaques by PC from non-immunized mice is due to autoimmunization by antigen(s) present on the mouse own erythrocytes.

Cell division was not required in the recruitment of antibody forming cells but blast transformation was a constant and general feature of the cell behaviour during the culture (Pages et aL, 1976). It was concluded that PC contain a large proportion of preprogrammed cells and that culture conditions lead to a derepression of the built-in mechanism of antibody synthesis. The nature of the antigen involved in this precommitment was suggested by the following: it was known (De Heer & Edgington, 1974) that NZB mice spontaneously developed antibodies against their own erythrocytes, some of these antibodies being detected only by the use of bromelain-treated erythrocytes. In 1974, Cunningham demonstrated that the spleen cells of normal mice would produce PFC against bromelain-treated mouse red blood cells (MRBC). This activity increased if the spleen cells were cultured for 1 4 days (Lord & Dutton, 1975a, b). Mouse peritoneal cells also form PFC against bromelain-treated MRBC and in a very large proportion, as shown in our laboratory (Pages & Bussard, 1975a, b). Thus, it could logically be inferred that the postulated 'primitive' antigen which initially sensitized the PC was present on the mouse's erythrocytes, and that the anti-SRBC activity of these PC was due to a cross-reactivity between the antigenic determinants of SRBC and MRBC, these latter epitopes being revealed by bromelain treatment. In order to confirm this hypothesis, it was required to show that the same cells were implied in the production of anti-SRBC and anti-BrMRBC antibodies. The first positive data were obtained by the observation of complete plaques of hemolysis by a single PC in a mixed plating system containing SRBC + BrMRBC (Pages & Bussard, 1976b). In this article, we present the further development of this approach, in order to answer the question: are the cells which produce anti-SRBC antibody the same cells as those which produce anti-MRBC anti-

INTRODUCTION

Ten years ago, it was shown in our laboratory that mouse peritoneal cells from unstimulated donors could spontaneously develop plaques of hemolysis (PFC) against sheep red blood cells (SRBC) when placed in a proper system of detection (carboxymethyl-cellulose: CMC) (Bussard, 1966, 1967). Since then, the phenomenon has been confirmed (Bendinelli & Wedderburn, 1967), extended (Nossal et al., 1970; Bussard et al., 1970) but was still limited to the use of a very sensitive method of plaque detection: the local hemolysis in CMC (Ingraham, 1963; Ingraham & Bussard, 1964). In 1969 Raidt et al. found that, in tissue culture, PC (peritoneal cells) could develop high numbers of PFC against SRBC, detectable by a modified Jerne technique (Mishell & Dutton, 1967). In 1974, we used mouse peritoneal cell cultures to investigate the non-involvement of antigen in the development of PFC (Pages et al., 1974). PC were ideally suited for in vitro culture; their viability in culture conditions was much higher than the viability of mouse spleen cells, and their plaque forming capacity (against SRBC) increased considerably (up to 10% of the viable cells in culture). Moreover, the anti-SRBC activity of these cultured cells could be demonstrated by all available local hemolysis methods: agarose (Jerne), liquid thin layer (Cunningham) or CMC, though the latter technique, being much more sensitive, detects a much higher number of PFC (Nossal et al., 1971). The presence of antigen (SRBC) or even protein (foetal calf serum could be a potential antigen) was not required to induce the development of PFC (Gisler et al., 1975).

* Supported by a research grant from INSERM, No. 27-76-59 and from the CNRS (ER No. 119). IMM. 14/1

A

l

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ALAIN E. BUSSARD, MARIE-ANTOINETTE VINIT and JACQUELINE M. PAGES

body'? To resolve this question, we investigated the double activity of PFCs by successive micromanipulations of single cells in single plating systems (either SRBC or MRBC). A plaque inhibition technique was used to determine if the formation of double plaques (anti-SRBC and anti-MRBC) was due to the synthesis of one molecular species of antibody lysing both erythrocytes by virtue of cross-reactivity. Since the n u m b e r of P F C in culture was so large it was possible to detect, in vitro, the synthesis of immunoglobulins and specific antibodies: this was achieved by labelling the immunoglobulins synthesized during the culture period with radioactive amino acids. MATERIALS AND METHODS

Cell cultures Mouse PC from non-immunized 20-40 weeks old CBA/J or NZB mice were harvested as previously described (Bussard, 1966). Single cell suspensions were prepared and incubated without SRBC according to the technique of Mishell & Dutton (1967). One ml cultures contained 2 to 4 x 10 6 PC in RPMI 1640 medium supplemented with 10°t foetal calf serum (FCS). MEM lissue culture medium without leucine was used in the experiments designed for the radioactive tagging of synthesized antibodies. Detection of PFC Direct plaque-forming activity of cultured cells was measured by the Cunningham technique of local hemolysis (Cunningham, 1965) or by the CMC technique (Nossal et al., 1970) (counts were performed after 1 or 3 hr incubation at 37C). The cell viability was measured by the Trypan blue exclusion technique. Plaque-forming activity of the populations was expressed as the number of plaque forming cells per million viable cells scanned (PPM). Enzyme treatment of red blood cells A saturated solution of bromelain was obtained by incubating a suspension of bromelain (No. B 2252 from Sigma Chemical Co.) 10 mg/ml in PBS, at 37'C for 1 hr, with occasional shaking. After centrifugation (1000g for 10 rain), the supernatant was removed and kept at -20'~C. For enzyme treatment. 1 vol of saturated bromelain solution was mixed with 1 vol of thoroughly washed packed red cells and incubated 30 rain at 37'C with occasional shaking. The red cells were washed 3 times in MEM Hepes buffered medium and used within 48 hr. Inhibition of phtque.formation by erythrocyte stromatas Stromatas were prepared by hemolysis of erythrocytes in hypotonic phosphate buffer and isolated as reported by Dodge et al. (1963). The stromatas to be incorporated in the detection medium were counted by phase contrast microscopy in an hemocytometer after treatment with 2.5°0 glutaraldehyde in cacodylate buffer and overnight settling in the counting chamber at room temperature. Two ratios of stromatas to erythrocytes were used (5 : 1 or 10:1); the usual concentration of RBC in the detection system was 2 x 108 cells/ml. Micromanipulation of PFC Individual cells were picked from the center of a plaque (CMC technique in oil) by a micropipet attached to a Leitz micromanipulator under microscopic observation (200 magnification in phase contrast). Care was taken to pick

cells which were alone in the center of a plaque of hemolysis. The cells were transferred into new preparations containing RBC in CMC plus complement. New plaques would appear very soon after the transfer--usually within 10 rain or less--at 37cc. When a transferred cell did not develop a plaque, the viability of the cell could be checked by a further transfer into detective media containing the first type of RBC in which it had previously given a plaque. Two transfers were routinely performed and the yields generally were good.

ldentificafion of antibodies produced by the cells Antibodies were identified either at the level of the individual cell or as the result of their syntheses by a population of PFC. Both techniques require a very specific antimouse # chains produced in rabbit and absorbed (on immunoabsorbent columns) with mouse IgG. This serum was kindly given to us by Dr. Liacopoulos. Radioactive labelling techniques The proteins and antibodies synthesized by cultured PC were studied by the incorporation of 14C leucine. The tissue culture medium was RPMI 1640 which contained 3.8 × 10 -4 mole/l of leucine. Culture supernatants and cellular extracts (obtained by freezing and thawing the cells three times) were collected after 4 days of culture in the presence of radioactive leucine. The radioactivity in the total protein was counted in TCA precipitates after washing with a large excess of nonradioactive leucine. In experiments where anti-sheep and anti-mouse erythrocyte antibodies had to be absorbed on these erythrocytes, the culture supernatants or cell extracts were incubated with 1.3 x 101° RBC to 1 ml of fluid. The antibody preparations were absorbed twice, though it was found that specific fixation was accomplished by the first incubation, since the second samples of RBC absorbed a constant amount of radioactivity. This amount was similar to that which was absorbed on non-cross reacting RBC (horse RBC). Immunoelectrophoresis and autoradiography Immunoelectrophoresis (IEP) of radioactive culture media or cell extracts was carried out in 1~ agarose (in Tris buffer pH 8.25, ionic strength 0.05 M) for 1 hr in a field of 5 V/cm, 15/20 mA. Normal mouse serum, at a final concentration of 1%, was present as a carrier in the experimental sample in the central well. Anti-mouse immune serum and anti-mouse IgM immune serum were used in the upper and lower troughs to identify the proteins produced during the culture. After washing and drying, the I.E.P. slides were exposed to X-Ray films (Kodax Regulix) for 15-30 days. To identify specific antibodies, culture media or cell extracts were absorbed on the reactive RBC: SRBC, BrMRBC, or non-cross reactive horse RBC, prior to immunoelectrophoresis. RESULTS

T h e development of P F C against SRBC and bromelain-treated M R B C by peritoneal cells in culture is a massive p h e n o m e n o n which can involve 1-3% of the population as detected by the C u n n i n g h a m technique or 6-10°o as detected by the C M C technique at day 4 or 5 of culture (Table 1). These numbers represent the mean; in the case of N Z B mice or CBA retired breeders, this n u m b e r

Auto Antibodies produced by Mouse Peritoneal Cells in Culture

3

Table 1. Recruitment of PFC against SRBC and MRBC by mouse peritoneal cells in culture

Days of culture

SRBC

0 4 5 6 7

85 3670 5020 2230 630

1 2 3 4

3500 22200 28500 62501)

Phtquc-forming a c t H t y of the P(" in cuhure (in PPM)" Liquid technique BrSRBC MRBC BrMRBC 1840 8850 13150 5300 3040

N.D 0 0 0 0

N.D. 20880 28400 11760 11560

C M C technique N.D. N.D. N.D. ND. ND. N.D. N.D. ND.

11800 47100 70200 81500

"Plaques per million viable cells scored. reached 20-30% even as measured by the Cunningham technique. The use of LPS in the culture media enhanced the development of PFC (Pages et al., 1975a). 2-Mercaptoethanol at a concentration of 5 x 10-5 M, permitted the survival of PC for months with a slow decrease in PFC activity (unpublished data). The difference in sensitivity between the liquid and gel techniques of local hemolysis is probably due to the fact that the latter detects both ceils which produce small amounts of antibody (the detection of a plaque requiring the lysis of 5-7 erythrocytes in a monolayer, in a volume of 2 x 10-8 cm 3) and cells which produce antibody later than 1 hr after plating (the liquid technique involves plaque counts after 1 hr incubation; the CMC is read after 3 hr). With populations of PC from mice considered as high producers or PC activated in culture, the differences found between the plaque counts obtained by the 2 techniques diminishes. It is interesting to note that untreated MRBC gave no plaques while SRBC gave plaques without bromelain treatment. Bromelain treatment of SRBC increased the number of plaques by 2~4-fold. Cross reactivity between SRBC and M R B C Mixed platin 9. The degree of cross reactivity, or more precisely, the number of PC involved in the formation of cross reacting or non-cross reacting antibodies directed against SRBC and MRBC can be studied by counting the number of plaques obtained in single plating systems (containing SRBC or BrMRBC) or in mixed plating systems (containing SRBC and BrMRBC). When SRBC and BrMRBC were incorporated in CMC gel or liquid detection media with PC from cultures, 3 types of plaques could be seen: (1) Clear plaques, in which both types of erythrocytes were lysed from the center to the periphery with fairly sharp edges (see Fig. 1A). (2) ~Sombreros' plaques in which a clear central zone was surrounded by a second zone (corona) in which only one type of erythrocytes was lysed (Fig. 1B). These two types of plaques are considered as indicating a degree of cross reactivity between the epitopes on the two types of erythrocytes since they both exhibit a clear zone (where the two types of erythrocytes are lysed) (Cunningham, 1974).

Fig. 1. Plaque morphology. Mixed suspension of SRBC + BrMRBC, (A) clear plaque (complete); (B) 'sombrero' plaque; (C) cloudy plaque (incomplete). The experiment shown in Fig. 2 demonstrates that sombreros plaques result from the exclusive lysis of BrMRBC in the outer zone. The right half of the figure represents a zone of lysis formed in an agarose gel containing SRBC and complement; the left half shows a zone of lysis developed in a gel containing BrMRBC and complement. The center well contained supernatant from a 4-day culture of PC. It can be seen that the radius of the left zone of lysis (MRBC) was greater than the radius of the right zone of lysis (SRBC) showing that the former cells were lysed to a larger extent. (3) Incomplete plaques (Fig. IC) which consisted solely of a cloudy zone containing unlysed erythrocytes throughout the entire plaque from the center outward. These plaques could indicate a non-cross reactivity between SRBC and BrMRBC in that only one species of erythrocytes was lysed. Micromanipulation of single PFC was used to investigate this problem.

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ALAIN E. BUSSARD, MARIE-ANTOINETTE VINIT and JACQUELINE M. PAGES

Br MRBC

SRBC

Fig. 2. Spot test. The center well is filled with PC supernarant of culture at day 4. The right-half of the plate consists of SRBC in agarose, the left-half consists of BrMRBC in agarose.

Table 2 shows the results of 3 experiments on cultured PC which were placed in single or mixed suspensions of RBC. It can be seen that, while the absolute activities were different between experiments 1, 2 and 3, the high degree of cross reactivity remained similar. This cross reactivity was calculated by assuming that both clear and sombreros plaques were indicative of cross reactivity. Their numbers were added and compared to the total number of plaques. If we call S the number of SRBC plaques (single plating), M the number of (Br)MRBC plaques (single plating), C the number of clear plus sombreros plaques, and I the number of incomplete plaques: S+M=2C + 1. In any case, it is clear that a large number of cells produced an antibody which was able to lyse both sheep and bromelain-treated mouse erythrocytes. We must emphasize the fact that the apparent plaque forming activity of a given PC population against SRBC and BrMRBC varied with the experimental conditions, among which the source and age of guinea-pig complement are of paramount importance. Furthermore, the ratios of anti-SRBC and antiBrMRBC activities of the same PC population varied with the different guinea-pig complements. In Table 3, it can be seen that for a series of guineapig sera, originating from a fairly homogeneous group of animals, the number of PFC against SRBC varied around the arithmetic mean (AM)of 51,600 with a S.E. of 14.6~);, the anti BrMRBC activity had an AM of 70,800 with a S.E. of 14.2°;; and the ratio of these

o/ activities was 1.45 with a S.E. of 15.2/o. With more heterogeneous guinea-pig sera, anti-RBC activities were more widely distributed and the S.E. of the ratios would reach as high as 19~i. These findings are not limited to the use of individual sera, but can also apply to pooled guinea-pig sera. Thus, it is quite clear that the greatest care should be taken in the choice of the complement by pre-testing it. Variations in complement efficiency, added to the well-known differences in the sensitivity of the methods available, actually make rather illusive any attempt to estimate the absolute number of PFC in a cell population. Micromanipulation of PFC. The above experiments were performed by plating populations of PC and we felt that the results should be verified and extended by experiments done at the individual cell level, These micromanipulation experiments could answer two questions: (a) is the lysis of both types of RBC in complete plaques the result of the independent action of antibody molecules on each RBC ? (b) which type of RBC is lysed in incomplete plaques, inasmuch as these plaques result from the lysis of only one type of RBC ? Cells were picked from the center of clear plaques produced in mixed suspensions: a first transfer was made into sheep or Br mouse red blood cell suspensions. The second transfer was performed using the same procedure (Table 4A). The yield of the first transfer was very high in both kinds of RBC, indicating that clear plaques did not result from any interactions between the two types of erythrocytes. The second transfer also gave a very high yield of cell reactivation and it is interesting to note that these yields were as high in heterologous transfers (from SRBC to BrMRBC and vice versa) as in homologous transfers, indicating again that the cells which formed complete plaques were lysing both types of erythrocytes by the secretion of the same antibody which could act independently on each erythrocyte. In the second type of experiments (Table 4B), cells picked from the center of incomplete plaques were transferred either into a SRBC or BrMRBC suspension. The yields were very high in the latter case, while they were quite low in the former. These results established the fact that most incomplete plaques were due to BrMRBC lysis and not to SRBC lysis. In addition, a high number of plaque forming cells

Table 2. Antibodies produced by cultured peritoneal ceils, cross reacting with SRBC and (Br)MRBC

Exp. No.

System of detection

(;lear

1 2 3

Single plating (SRBC)

14610 40920 41740

1 2 3

Single plating (BrMRBCI

13791 38810 39640

1 2 3

Double plating (SRBC) + (BrMRBC)

2740 34980 7215

"(Clear + sombreros/Total No. of PFC) x 100.

Plaque forming activity (CM(') Type of plaques Sombreros

Incomplete

Degree of " cross-reactivity (in '!,,}

12330 13880 21280

3740 5020 7940

88 9I 78

Auto Antibodies produced by Mouse Peritoneal Cells in Culture Table 3. Variability in the number of PFC obtained by the same PC with different complements Exp. No.

tested

SRBC

Activities ~ BrMRBC

Ratio M/S

I 11

9 4

51600 + (14.6%) 35100 _+ (46)

b70800 ± (14.2) 51800 + (42)

1.45 ± (15.2) 1.37 ± 119)

No. of C'

"In PPM. bS.E. in percentage. Table 4. Micromanipulation of PFC (A)

From complete plaques (mix suspension) to single suspensions 47 [SRBC] 42 (89)

No. of cells transferred to: No. of plaques Yield (%) Second transfer No. of cells

42

[BrMRBC] 37 (881

11 [SR BC] 8 (72}

(PFC~

No. of plaques Yield (",3

16

13

[BrMR BC]

[SR BC]

[BrMR BC]

15 (94l

I1 (85i

12 12 (100t

From incomplete plaques (mix suspension} to single suspensions

(B)

30 [SRBC] 7 (23)

No. of cells transferred to: No. of plaques Yield (%) Second transfer PFC No. of non PFC

40

[BrMRBC] 24 (60)

2 .

6 11 [BrMRBC] 14 (82)

[SRBC] 0

No. of plaques Yield (%)

(83~o) or non-plaque forming cells (82%) against SRBC produced plaques in a second transfer into BrMRBC while a small number of PFC against BrMRBC produced plaques in a second transfer into

SRBC (2070). Inhibition of plaques by erythrocyte 9hosts. Another method of detecting the antigenic relationship between different RBC is based on inhibition experiments in which ghosts of one type of erythrocyte are incorporated in the local he.molysis detection media in addition to the same type or a cross reacting type of erythrocytes. For these experiments, we used the liquid technique of local hemolysis (Cunningham) since it provided a high level of plaque inhibition by RBC ghost s.

2O

15

1 [BrMRBC]

[SRBC] 4 (20}

3

The results of these experiments are shown in Table 5.

In homologous inhibition experiments (SRBC/ SRBC ghosts or BrMRBC/BrMRBC ghosts) it was easy to obtain a high degree of inhibition when the plaque forming activity was low. When this activity was high, the homologous inhibition mouse/mouse ghosts was strong while the homologous inhibition sheep/sheep ghosts was much less efficient. In heterologous inhibition experiments, it was always the BrMRBC stromatas which were the best inhibitors, especially at low concentrations. To summarize, we can state that for both types of plaques (SRBC or BrMRBC) the best inhibitors were the BrMRBC ghosts. This is particularly signifi-

Table 5. Inhibition of anti-SRBC or anti-(Br)MRBC formed by peritoneal cells in culture

Days

of culturc

Inhibiting ghosts SRBC Ratio G/F"

Indicator cells

5

10

5

5440

490 91 28000 50 16200 66

80 98 25000 55 4400 91

320 94 283 99 1080 98

ND ND

20600 18 157100 31 119250 52

15800 52 I 14450 50 94400 62

4200 87 22440 90 1980 92

250 99 2160 99 ND ND

4

Acti',iD b

5

Inhibition ~ Activity Inhibition

56000

Acti'dty

47800

SRBC

6

Inhibition 4

Act i~ ity

25001)

Inhibition 5

( Br)M R BC

A cti'qty

229150

Inhibition 6

Activity

Inhibition

(BrIMRBC Ratio G / F °

None

248830

"Ratio: ghosts per RBC. The C of RBC was 5 × 108 cells/ml. bMeasured in the liquid technique PPM. qn per cent. The PC were obtained from the CBA old retired breeders 12 month old.

10 50 99 0

100

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ALAIN E. BUSSARD, MARIE-ANTOINETTE VINIT and JACQUELINE M. PAGES

cant since the BrMRBC seem to lyse very easily. It can thus be concluded that the apparent affinity of the anti-erythrocyte antibodies is higher towards the BrMRBC determinants than it is towards those of SRBC.

Identification of antibodies produced in culture It has already been shown by us (Bussard, 1966) and Gisler et al. (1975) that the hemolytic substance produced by PC in culture was indeed an IgM since PFC gould be completely suppressed when a monospecific anti-mouse lgM was incorporated in the detection media. The following experiments were undertaken in order to demonstrate that this IgM was synthesized during culture, and also to investigate the type and amount of antibody produced during the culture period,

lmmunohemolysis and the +barrier' effect on immunodiffusion. In an agar layer containing SRBC (Fig. 3A) or (Br)MRBC (Fig. 3B) and complement, 3 wells were prepared: the central well was filled with anti-lgM serum, the right well was filled with cell extracts, and the left well was filled with culture supernatant. After an overnight incubation at 37'C, a zone of lysis around the two experimental wells could be seen which terminated near the central well containing the anti-IgM serum. This barrier effect indicated that the diffusing hemolytic antibody was an IgM (Milstein, 1975). In Fig. 3B, the agar layer contained BrMRBC. The zone of lysis around the right well containing the cell extract was more intense than the zone around the left well containing the tissue culture fluid, showing that the cell extract had a higher concentration of antibody than the tissue culture fluid.

Detection by immuno-electrophoresis and autoradio9raphy of radioactive 19 produced during culture. Peritoneal cells (4 x 106 cells/ml) were cultured for 4 days in the presence of radioactive I+C isoleucine, specific radioactivity, 122 mCi/mM, at 5 #Ci per ml (exper-

iments 1 and 2). At the end of the culture, 80,000 anti-SRBC PPM could be counted, i.e. 3.1 × l0 s PFC per ml of culture. In experiment 3, after three days of culture under normal conditions, the cells were washed and placed in a leucine-free MEM (in order to increase the specific radioactivity of the media) and 2.5 #Ci of laC radioactive leucine added. The cells were cultured for an additional 24 hr. At the end of this period, the immunological activity against SRBC was 53,(100 PPM (143,000PFC/ml), and against BrMRBC the response was 120,000 PPM (324,000 PFC/ml). In all experiments, ceils were cultured in presence of 10°J;i FCS. At the end of the culture period, cells were washed three times in presence of a large excess of non-radioactive isoleucine or leucine, and subjected to freezing and thawing to prepare soluble extracts. Immunoelectrophoresis was performed as described in Techniques, and after fixation and coloration (Fig. 4A), autoradiographs (Figs. 4B and C) were exposed for 15 days. It can be seen in Fig. 4B that during the culture period, two immunoglobulins, identified as lines 1 and 2 (in front of the anti-mouse serum trough), had been synthesized. Line No. 2 was identified as mouse IgM since it fused with the line obtained in front of the trough containing anti-mouse IgM antibody. Line No. 1 was identified by the use of specific anti-mouse IgG1, (IgG2 + IgG3) and IgG3 sera kindly provided by Dr Hijmans. While anti IgGl and anti IgG: sera did not precipitate any radioactive constituents of the tissue culture supernate, the anti (IgG: + IgG3) serum formed a line of precipitate which fused with the line No. 1 shown by the anti-mouse immune serum (Fig. 4C). Thus, it was concluded that at least two classes of immunoglobulins were synthesized during the tissue culture period: an IgM and an IgG~. The specificities of these Ig were studied by absorption experiments: tissue culture fluid was absorbed (1 ml by SRBC, BrMRBC and Horse RBC as a con-

Fig. 3. In both figures the center well contains anti-mouse lgM antiserum, the right well contains PC extracts, and the left well contains PC culture supernatant (at day 4}. (A) Agarose layer containing SRBC: (B) agarose layer containing BrMRBC.

Auto Antibodies produced by Mouse Peritoneal Ceils in Culture

7

"A.

B

C

Fig. 4(A). Immunoelectrophoresis (I.E.P.) of tissue culture supernatant containing mouse serum as carrier. The upper trough contains rabbit anti-total mouse serum; the lower trough contains rabbit anti-total mouse serum: the lower trough specific anti-mouse IgM serum. (B) Autoradiography of the same I.E.P. Lower trough contains anti-mouse lgM serum. (C) Autoradiography of the same I.E.P. Lower trough contains anti-mouse (IgG2 + IgG3) serum. trol for non-specific absorption). It can be seen (Figs. 5B and C) that only line No. 1 was unaffected by the absorption procedure indicating that these immunoglobulins were not anti-SRBC antibodies. In order to obtain an approximate value of the radioactivity present in the line of precipitate shown in Fig. 5, the slides were counted in a new type of gas flow counter. The relative proportions of radioactivity present in the IgM band (line 2, Figs. 5A-C) as compared to the non-absorbed sample (line 2, Fig. 4B) were computed by two methods: measurement of the surfaces of the peaks projected on a screen and recorded on polaroid, or direct calculation from the counts given channel by channel on a teletype. The non-specific absorption (on horse RBC) accounted for 40~o of the radioactivity present in IgM. BrMRBC could absorb 76~o of total radioactivity and SRBC could fix 66~o. If it is valid to deduce the non-specific absorption from these results, one can conclude that approx 36~o of the IgM produced by PC during culture is directed against BrMRBC and 26~o against SRBC. DISCUSSION We have clearly demonstrated that PC in culture develop a large number of plaque forming cells

against SRBC and bromelain-treated MRBC. It was proven by different experimental approaches that the same cells (in more than 80~ of the cases) secreted antibody able to lyse both SRBC and BrMRBC. The plaques are due to the active synthesis of IgM antibodies during the culture period. The amount of IgM antibody produced per 4 × 10 6 cells during 4 days of culture is of the order of 5 mg. Specific anti-SRBC antibody represents approx 25~o of the total lgM produced. In addition to these specific IgM antibody molecules, other immunoglobulins, of the IgG3 class but of unknown specificity, are synthesized by the cells in culture. The syntheses correlate with a profound morphological transformation of the PC population which will be described in a later publication. Briefly, while small lymphocytes constitute the majority of the PC population at day 0, by day 5 or 6, large cells with numerous microvilli and lobed nuclei are the more common cells. As we have previously shown, this transformation takes place in a population with a low mitotic index (J. Pages et al., 1976). Antibody producing cells were selected by micromanipulation from the center of plaques and examined by E.M. (Electon Microscopy). At day 0, lymphocytes and plasmocytes were found in roughly equivalent proportions, but at day 5, the PFC were

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ALAIN E. BUSSARD, MARIE-ANTOINETTE V1NIT and JACQUELINE M. PAGES

---,2

tration operating in the mouse whereby auto antibody memory cells would be preferentially accumulated in the peritoneal cavity, or the PC could be locally triggered in situ by mouse erythrocytes (more or less permanently). Both mechanisms would lead to the existence of a large population of memory cells (for anti-MRBC antibody production). This would be in agreement with the fact that PC cannot be immunized in vitro with all antigens tested. (2) All lymphoid populations (i.e. in the spleen and other organs) contain a large population of antiMRBC antibody forming cells, but only for peritoneal cells do the culture conditions lead to an optimal derepression of the mechanism of antibody production. According to this last hypothesis, it would be diffucult to visualize how these lymphoid cells would not be pluripotential, in regard to the production of antiMRBC antibody and an antibody of another specificity, if the proportion of anti-MRBC precursor cells would be as high as the one found in peritoneal cells o/ (30~o).

2

Fig. 5(A). Autoradiography of the same supernatant as used in Fig. 4, but after absorption on horse RBC. (B) As in (A), but after absorption on SRBC. (C) As in (A), but after absorption on BrMRBC.

essentially mature plasmocytes plus some histiocytic cells which look like macrophages with phagocytised RBC ghosts or debris in their cytoplasm. The observations that mouse PC in culture produce anti-mouse RBC antibodies and that it is frequently the same cell which produces antibody to lyse both types of erythrocytes (sheep and mouse) add a new dimension to the peritoneal cell phenomenon. Our data support the hypothesis of Cunningham (1974) that erythrocytes are naturally self-immunogenic in the mouse and that auto-antibodies are continuously produced, but not detected, because the corresponding epitopes are not exposed. This self-antigen is likely to be the cross reacting immunogen we postulated in 1967. Anti-SRBC antibodies developed by PC would thus be cross reacting anti-MRBC antibodies. The very large amount (up to 30~,o) of PC which are able to produce anti-sheep and anti-mouse erythrocyte antibodies is striking. Since the onset of antibody synthesis in culture is not dependent on cell division, we must assume that the precursor ceils are the same and in same number as the producer cells. Thus, what we observe in culture is a process of derepression and differentiation. Two possibilities exist: (1) there is a selective mechanism of cell concen-

It could be hypothesized, following Jerne, that self antigens are members of a different class of antigens as compared to the class of foreign antigens; nevertheless, the suicide (Jerne) or abortion theory (Nossal, 1975) of auto antibody producing cells is weakened by the fact that there is, in a larger population of lymphoid cells, a permanent potentiality to produce auto-antibodies, though it is true that these antibodies are not directed against epitopes which are readily available on the surface of the self-erythrocytes. The mechanism by which PC and other lymphoid cells are repressed in vivo should be elucidated. We have already shown that anti-0 treatment of T cells or the removal of glass adherent cells at the onset of culture does not affect the capacity of the remaining population to develop PFC against SRBC and MRBC (Gisler et al., 1975). This does not rule out the possibility that new glass adherent cells arise during culture and that they cooperate with B cells by an activation mechanism. As for T cells, the removal of 0 bearing cells at the onset of culture (Gisler, 1975) does not rule out that new 0 bearing cells could develop in culture and act as helper cells for derepression. The effect of soluble factors liberated in the medium during culture should also be investigated; if an enhancing factor were found, it could be assayed in vivo as a derepressor of antibody formation. The most important question, now, is to establish if our findings with PC apply to other lymphoid cells currently used as models in cellular immunology. That is, are some epitopes of the SRBC recognized as new by the mouse's spleen cells, or have they all been previously seen'? The fact that some epitopes on SRBC have already been 'seen' by an adult mouse, via self-immunization by identical or cross reacting epitopes present on its own erythrocytes, has already been established for spleen cells by us (Pages & Bussard, 1975b). We have some evidence (publication in preparation) that, while anti-SRBC plaques produced spontaneously by spleen cells in culture can be entirely inhibited by mouse stromatas, anti-SRBC plaques from spleen cells of mice immunized with

Auto Antibodies produced by Mouse Peritoneal Cells in Culture SRBC cannot be entirely suppressed by the presence of mouse stromatas in the detection medium. These findings favour the hypothesis that some of the epitopes of injected SRBC do not cross react with the epitopes of M R B C and that in this case, at least, we are dealing with a true primary stimulation. It remains that, whenever SRBC are used as antigen in the mouse, care should be taken to establish which part of the immune reaction relates to a recall of memory cells established by self-antigens and which part is really dealing with a true primary stimulation.

REFERENCES

Bendinelli M. & Wedderburn N. (1967) Nature, Lond. 215, 157. Bussard A. E. (1966) Science 153, 837. Bussard A. E. (1967) Cold Spring Harb. Syrup. quant. Biol. 32, 465. Bussard A. E., Nossal G., Mazie J. C. & Lewis H. (1970) J~ exp. Med. 131, 917. Cunningham A. J. (1965) Nature, Lond. 207, 1106. Cunningham A. J. (1974) Nature, Lond. 252, 749. De Heer D. H. & Edgington T. S. (1974) Clin. exp. Immunol. 16, 431.

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Dodge J. T., Mitchell C. & Hanahan D. J. (1963) Archs Bioehem. Biophys. 100, 119, Gisler R. H., Pages J. M. & Bussard A. E. (1975) Ann, lmmunol. Inst. Pasteur, Paris 126C, 231. Ingraham J. (1963) C.r. hebd. Sdanc. Acad. Sci. Paris 256, 5005. lngraham J. & Bussard A. E. (1964) J. exp. Meal. 126, 423. Lord E. & Dutton R. W. (1975a) Fedn Proc. 34, 967. Lord E. & Dutton R. W. (1975b) J. Immun, 115, 1199. Milstein C. & Kohler G. (1975) Nature, Lond. 256, 495, Mishell R. I. & Dutton R. W. (1967) J. exp. Med. 126, 423. Nossal G., Bussard A. E., Lewis H. & Mazie J. C. (1970) J. exp. Med. 131, 894. Nossal G., Lewis H. & Warner N. L. (1971) Cell. Immunol. 2, 13. Nossal G. J. V. & Pike B. L. (1975) J. exp. Med. 141, 904, Pages J., Gisler R., Arnaud D. & Bussard A. E. (1974) Ann. Immunol. Inst. Pasteur, Paris 125C, 435. Pages J. M. & Bussard A. E. (1975a) Ann. lmmunol. Inst. Pasteur 126C, 356. Pages J. M. & Bussard A. E. (1975b) Nature, Lond. 257, 316. Pages J. M., Gisler R. H., Arnaud D. & Bussard A. E. (1976) Ann. lmmunol. Inst. Pasteur, Paris 127C, 31. Raidt D. H., Dutton R. W. & Mishell R. I. (1969) Fedn Proc. 28, 553.