Immunology 1979 37 731

Dependence of lymphocyte surface Ig on continuous polyclonal activation*

DONNA M. BUCHHOLZt, S. DRAY, & M. TEODORESCU$ Department of Microbiology and Immunology, University ofIllinois at the Medical Center, Chicago, Illinois, U.S.A.

Acceptedfor publication 17 January 1979

Summary. We have previously shown that most rabbit splenic B cells cultured in a medium supplemented with 5% autologous serum require continuous polyclonal stimulation to maintain detectable amounts of surface Ig. In the absence of this stimulation B cells shed but do not replace their surface Ig. Here, we investigated the mechanism responsible for the loss or maintenance of surface Ig. We showed that the addition of inhibitors of mRNA and protein synthesis to the cell cultures completely abolished the Ig maintenance effect provided by the mitogen thereby sug-

gesting that it did not act by 'freezing' the membrane Ig but rather by continuously stimulating resynthesis. Moreover, by labelling the surface Ig with '251-labelled Fab anti-allotype antibody we showed that the maintenance of surface Ig by mitogen stimulation was due to the turnover of surface Ig. The cells shed and replaced their surface Ig with a half-life of about 2 h only when mitogen was present but shed without replacing the surface Ig in the absence of mitogen. Also, the B-cell mitogens, SM and LPS, were able to maintain surface Ig even at extremely small concentrations while the T-cell mitogens, Con A and PHA, failed to do so at any concentration, suggesting that direct stimulation of B cells was needed to maintain surface Ig. When spleen cells were cultured in 'crowded' conditions in the absence of mitogen they did not lose their surface Ig; under these conditions it appeared that a factor associated with the macroglobulin fraction is induced and acts in the same manner as a B-cell polyclonal activator to maintain the turnover of surface Ig. Such a factor may actually function in vivo since lymphocytes are in very close contact in the lymphoid organs. We concluded that rabbit B lymphocytes shed and replace their surface Ig with a half-life of about 2 h and that the replacement, but not the shedding of surface Ig, is dependent on continuous exogenous or endogenous polyclonal activation.

Abbreviations: B (Ig+) cell, bursa equivalent-derived, immunoglobulin bearing cells; T (Ig-) cell, thymus-derived, non-immunoglobulin bearing cells; SM, streptococcal mitogen; LPS, lipopolysaccharide; PWM, pokeweed mitogen; Ig, immunoglobulin; Con A, concanavalin A; PHA, phytohaemagglutinin; HBSS, Hanks's balanced salt solution; BSB, buffered saline borate; SRBC, sheep red blood cells; Ab-E, antibody coated erythrocytes; RFC, rosette forming cell; FCS, foetal calf serum; anti-T Ab, guinea-pig anti-rabbit thymus cell antiserum. * Presented in part at the 60th and 61st meeting of the Federation of American Societies for Experimental Biology, April, 1976 and 1977. t In partial fulfilment of the requirements for the Doctor of Philosophy degree in the Graduate College, University of Illinois at the Medical Center, Chicago, Illinois. Present address: Argonne National Laboratory, Division of Biological and Medical Research, Argonne, IL, 60439, U.S.A. t Correspondence: Professor M. Teodorescu, Department of Microbiology and Immunology, University of Illinois at the Medical Center, Chicago, Illinois, 60680, U.S.A.

INTRODUCTION

00 19-2805/79/0800-0731$02.00

B lymphocytes have surface Ig which is continuously

© 1979 Blackwell Scientific Publications 731

732

Donna M. Buchholz, S. Dray & M. Teodorescu

shed and replaced. Beginning immediately upon incubation in vitro (within minutes), the surface Ig has been shown to be released linearly and completely turned over within a few hours of incubation (Milton & Mowbray, 1972; Wilson, Nossal & Lewis, 1972). The halflife of mouse B-cell suface Ig in medium supplemented with foetal calf serum (FCS) was estimated to be about 2 h (Wilson et al., 1972) or from 2 to 8 h (Marchalonis, Cone & Alwell, 1972; Vitetta & Uhr, 1972). Some work with nude mice in serum-free medium (Anderson, Lafleur & Melchers, 1974) suggested that the majority of lymphocytes, namely the small lymphocytes, have a very slow turnover of their surface Ig (20-80 h). The addition of a mitogen to lymphocyte cultures results in blast transformation and proliferation (Greaves & Janossy, 1972). We had shown previously (Teodorescu, Buchholz & Dray, 1975) that mitogens may have another effect on lymphocytes, i.e. most rabbit splenic B cells in culture required immediate and continuous stimulation by mitogen in order to maintain detectable amounts of surface Ig. In the absence of a mitogenic stimulus, the surface Ig of most B cells was lost irreversibly within the first 4 h of culture. Although it appeared that mitogen might have promoted a continuous replacement of surface Ig which was being shed, i.e. the turnover, the actual mechanism of mitogen action in vitro and the in vivo consequences of this observation have not yet been elucidated. In the present work, we investigated the mechanism by which the surface Ig is maintained by mitogen. We investigated whether (1) the maintenance of surface Ig is an active process, i.e. it reflects the turnover of surface Ig; (2) T- and B-cell polyclonal activators function in a different manner; and (3) another signal or factor more likely to exist in vivo has the same effect as the polyclonal B-cell activators. MATERIALS AND METHODS Preparation of cell cultures Cells were obtained from New Zealand white rabbits which were homozygous at the b locus for the kappa light chain allotype (b4 or b5) as follows. The animals were exsanguinated and their spleens were excised, trimmed and washed in Hanks's balanced salt solution (HBSS, GIBCO, Grand Island, NY). Cell suspensions were prepared by pressing the tissue through a stainless steel screen, filtering through sterile cotton, and

washing four times in the cold. After the final wash, the cell pellet was resuspended in 5-10 ml RPMI-1640 medium (Gibco). The cell suspension was adjusted to contain either 106 cells per ml or a different number as specified in the Results. The cells were cultured in RPMI-1640 supplemented with 5% autologous or homologous serum in 25 cm2 Corning polystyrene tissue culture flasks (Scientific Products, McGaw Park, Ill.) in a standing or tilted position (as indicated in the Results) at 390 in 5% CO2 in air. Each flask contained a total volume of 10 ml. Viability counts, as determined by trypan blue dye exclusion and total cell count, were done both before the cells were cultured and at the termination of the cultures. Mitogens The mitogens used in these experiments were: streptolysin 0-associated mitogen (Abe, 1971) (SM, streptococcal mitogen, which was provided by Dr Riaz-ul Haque, University of Illinois at the Medical Center); concanavalin A (Con A, Worthington Biochemicals, Freehold, N.J.); phytohaemagglutinin (PHA, Gibco); lipopolysaccharide (LPS-B, Difco Laboratories, Detroit, MI); and pokeweed mitogen (PWM, Gibco).

Inhibitors Metabolic inhibitors added to the lymphocyte cell cultures in various concentrations as described in the Results were: cycloheximide (Gibco), actinomycin D (Gibco) and puromycin (Aldrich Chemical Company, Milwaukee, WI). Preparation ofpurified anti-allotype antibody Rabbit purified antibody, either anti-b4 or anti-b5, was prepared as follows. The IgG was purified by salt precipitation of rabbit serum or antiserum sequentially with 18% and with 14% Na2SO4 followed by chromatography on DEAE-cellulose equilibrated with 0 02 M phosphate buffer, pH 7 5. The IgG of the corresponding allotype was coupled to cyanogen bromide-activated Sepharose 4B columns (Pharmacia, Piscataway, NJ). The column was extensively washed with buffered saline borate (BSB) until the OD280 nm of the effluent was less than 0 01. The IgG preparation of rabbit anti-allotype antiserum was applied to the column and the purified IgG antibodies were eluted with 0-1 M glycine/H2SO4 buffer, pH 2-3. The eluate was neutralized immediately with 1 M Tris base (Tris(hydroxymethyl)aminomethane) and dialysed against 0 15 M NaCl and the purified anti-allotype antibody was adjusted to a concentration of 1 mg/ml (OD280nm =1 5).

733

Lymphocyte surface Ig

Radioiodination ofFab Fab fragments of IgG with anti-b4 or anti-5 antibody activity were obtained by papain digestion according to the method of Porter (1959). The Fab preparations were iodinated using the chloramine T technique (McConahey & Dixon, 1966). Either anti-b4 or anti-b5 Fab were dialysed against 0-1 M phosphate buffer, pH 5-5, prior to iodination. The Fab were mixed with equal volumes of radioactive iodine (1251) and chloramine T (at a concentration of 1 mg/ml). After allowing the reaction to proceed for 30 min at room temperature, sodium metabisulphite (I12 mg/ml) was added to stop the reaction. The nonbound iodine was removed by dialysis against saline to which KI had been added, with several changes over 18-24 h. The precipitable radioactivity with trichloracetic acid (TCA) was greater than 90% for each sample. In addition, the radiolabelled Fab preparations were tested to determine the amount of contamination by Fc or undigested Ig by radio-immunoprecipitation. The Fab were treated with a goat anti-rabbit Fc reagent; after a 30 min incubation a guinea-pig anti-goat Ig reagent was added. After incubation overnight the precipitates were washed four times with BSB and the supernatants and precipitates were counted in the Nuclear Chicago gamma counter (Model No. 1085, Searle Laboratories, Skokie, Ill.). Less than 5% precipitation was observed for each sample. The specificity of these reagents was determined on b4-Ig or b5-Ig-bearing cells. For example under saturation conditions the anti-b4 Fab bound fifteen times more to b4-Ig-bearing cells than to b5-Ig-bearing cells. Antibody coated erythrocytes (Ab-E) Purified antibodies were coupled to sheep red blood cells (SRBC) by using the chromic chloride method (Gold & Fudenberg, 1967). The SRBC were washed extensively with freshly prepared 0- 15 M NaCl. To one volume of washed, packed red cells one volume of purified antibody and one volume of chromic chloride ide solution (0-1% w/v in 0-15 M NaCI) was added. This was allowed to react for 5 min at 250, after which the Ab-E were washed at least four times with 0-15 M NaCl. Determination of rosette-forming cells (RFC) The percentage of lymphocytes bearing b locus allotypes was estimated by a direct rosette technique (Molinaro & Dray, 1974). The indicator cells, Ab-E,

were added to the lymphocyte suspension at a ratio of twenty to forty erythrocytes per lymphocyte. This mixture was centrifuged at 1000 g for 2 min; the cell pellet was resuspended by pipetting and stained with toluidine blue. The percentage RFC was determined microscopically by counting duplicates or triplicates of 100 cells per sample. Cells bearing thymus cell antigens (T cells) were rosetted by an indirect rosette technique using a guinea-pig anti-T cell antiserum, characterized as previously described (Wilson, Teodorescu & Dray, 1975). Lymphocytes (1 x 107 cells/ml) were sensitized with equal volumes of guinea-pig anti-T-cell antiserum at 40 for 30 min, then washed in HBSS four times. The washed cells were mixed with rabbit anti-guinea-pig IgG Ab-E and were centrifuged at 1000 g for 2 min. The pellet was resuspended and the lymphocytes were stained with toluidine blue. The percentage of RFC was determined microscopically as above.

Fractionation of 'crowded' cell supernatant Crowded cell supernatants were obtained from 24 h spleen cell cultures to which no mitogen was added and in which either the cell density was increased or the area of culturing decreased as described in the Results. The supernatants were concentrated ten-fold using a UM-10 ultrafiltration membrane (Amicon Corporation, Lexington, Mass.). The concentrated supernatant was applied to a Sephadex G-200 column (1 5 x 150 cm) (Pharmacia) which had been equilibrated with Tris-saline. Fractions were eluted from the column using a flow rate of 6 ml/h and the fractions were pooled as described in the Results, concentrated, and dialysed against saline. Pools were sterilized by filtration through 0-22 pm Millipore membranes. RESULTS Effect of different mitogens on-maintaining surface Ig of B cells in spleen cell cultures Our basic observation, which has been previously reported (Teodorescu et al., 1975), is that culturing rabbit lymphoid cells in vitro in medium supplemented with rabbit serum at a cell density of 106 cells/ml in standing plastic bottles results in a significant reduction in Ig-bearing cells within 4 h of cultivation. This loss is prevented when certain mitogens are added. The loss of surface Ig is estimated based on the reduction in the percentage of lymphocytes which form rosettes with sheep erythrocytes coated with anti-Ig allotype

antibody (Ab-E).

734

L a.

Donna M. Buchholz, S. Dray & M. Teodorescu

4020-

0O 3

4

5

6

8

7

9 10 11 12 13 14 15 Dilution of Mitogen (-IogJ

No

Mitogen

Figure 1. The effect of adding decreasing amoun of SM(.) or LPS (A) on the percentage of cells bearing munoglobulin in spleen cell cultures. LMD: I;ast concentration which induces blast transformation and Iproliferation as measured by [3H]-TdR uptake in 3 day cultur-es. The data are means from three individual experiments an d the vertical bars represent the range. The 1 00% represents th e percentage of RFC at the initiation of the culture.

sts

__

I-3

0-2

.0

100-

Dilution of PHA 10-5 10-6 0

1-4

I0

10-7

10

I0

NoMfogen

8060-

activator in most animal species, was evaluated and compared to SM which stimulates both B and T lymphocytes (unpublished observations). In a previous study, we showed that SM was able to maintain surface Ig of B cells (Teodorescu et al., 1975), i.e. the percentage of lymphocytes that form rosettes with Ab-E remained high only when the mitogen was present. When decreasing concentrations of mitogens were added to the spleen cell cultures we found that the presence of LPS, like SM, even in submitogenic concentrations (from Io-5 mg/ ml to 10-12 mg/ml) maintained a high level of rosette forming cells (RFC). The dose-response curves of SM and LPS were quite similar and the cultures not receiving these mitogens had a relatively low percentage of RFC (Fig. 1). Other polyclonal B-cell activators tested, such as pneumococcal polysaccharide III, dextran sulphate, Ficoll, have shown a similar effect (Buchholz, Chang, Dray & Teodorescu, 1979). T-cell mitogens Con A (1 0 mg/ml to 10O- mg/ml) and PHA (10-2 to 10-8 dilution) were used in the same type of experiment. Neither Con A nor PHA were effective in maintaining the initial percentage of RFC at any of the concentrations used (Fig. 2), including optimal mitogen concentrations (5 x 10-3 mg/ml for Con A and a 10-2 dilution of PHA). Thus, B-cell mitogens but not T-cell mitogens maintained a relatively high percentage of Ig-bearing cells in 24 h cultures.

0

LMD 40 - LMD

The lymphocyte population after incubation in the presence or absence of mitogens In our previous report (Teodorescu et al., 1975), we

20LMD 10°

5x

2.5X 1.2X 6X

o-'

o1

10-1 o-

o-2

3 lo-2

12-2

30

10

10

10

Dilution of Con A (mg/mI)

10

Mitogen

Figure 2. The effect of adding decreasing amoui ) on the percentage of cells bearing (-- -) or PHA ( surface Ig in spleen cell cultures. LMD: last c oncentration which induces significant blast transformation and proliferation as determined by [3H]-TdR incorporatiion in 3 day spleen cultures. -

Maintenance of surface Ig was investigatLed by treating lymphocytes with B-cell or T-cell mittogens. The rationale of this experiment was as foll ows. If the mitogen acted directly on B cells, any B-c,ell mitogen would be able to provide the signal for the maintenance of surface Ig while T-cell mitogeris would be ineffective. Thus, the effect of LPS, a polyc-lonal B-cell

cultivation the reduction in the percentage of Ig-bearing cells was not due to a preferential loss of these cells in the absence of B cell mitogens. Here, we re-examined this possibility further, i.e. we looked to see whethqr some B cells were attached to the bottom of the plastic bottle only in cultures not stimulated by SM. The lymphocytes were labelled with 5'Cr and cultured overnight with or without SM. The 5'Cr associated with the cell fraction was determined both in suspensions and on the plastic bottle. Only about 1-8% of the cells were attached either in stimulated or unstimulated cultures (Table 1). The preferential loss of B cells by lysis in culture was also investigated. First, the cell concentration decreased similarly in stimulated and non-stimulated cultures on the average by 12 5% (maximum 30%). In all experiments, however, the

735

Lymphocyte surface Ig same reduction in the absence or maintenance in the presence of mitogen of the percentage RFC was observed. Second, when T cells were counted by using the indirect rosette method (Wilson et al., 1976) the percentage of T cells remained unchanged (data not shown), i.e. the T/B cell ratio was not affected by the lack of mitogen stimulation. Third, the viability as determined by trypan blue dye exclusion was also similar (85-92%) in stimulated and unstimulated cultures after 24 h of activation (Fig. 3). The 5"Cr results presented above (Table 1) also show that the viability was not affected by the lack of mitogen for 24 h. When the cells were cultured for 4 days, however, the gradual decrease in viability in the unstimulated cultures was greater than in stimulated cultures (Fig. 3). This suggested that in the absence of mitogen the Ig-bearing Table 1. Recovery of 5ICr-labelled spleen cells stimulated or not by streptococcal mitogen in 24 h cultures

Sample

Streptococcal mitogen

c.p.m.

Supernatant Supernatant Cell pellet Cell pellet Plastic plate Plastic plate

+ + + -

27,325 +

SD

671

28,615 + 1291 46,285 + 3905 38,666 + 2497 862 + 280 (1-8%) 731 + 56 (18%)

The cells attached to the bottom of the plastic bottle were detached by SDS (Sodium dodecyl sulphate). The number in parenthesis represents the percentage radioactivity associated with the plastic surface SD, standard deviation. 100 90 c

80

-

The effect of inhibitors of mRNA and protein synthesis on maintaining surface Ig of B cells in spleen cell cultures To investigate whether mitogens act passively by 'freezing' or stabilizing (Cunningham, 1975) the Ig on the B-cell membrane, by passively adsorbing it from the culture medium, or by actively promoting a continuous replacement of shed surface Ig, metabolic inhibitors were added to the medium upon the initiation of cultures. If mitogen merely 'froze' the surface Ig or adsorbed it from the medium, inhibitors of mRNA and protein synthesis should not affect the action of the mitogen. We found that actinomycin D, even in small concentrations (0-001-2O0 ig/ml), reduced significantly the percentage of RFC even within the first 4 h of culture in the presence of mitogen (SM). The addition of puromycin (0-005-5 0, Mg/ml) or cycloheximide (0-01-7-5 pg/ml) also interfered with the effect of SM. At the higher concentrations of inhibitor, the percentage of RFC after 24 h was even less than that observed in the control cultures, i.e. those to which neither the SM nor the inhibitor were added. On the other hand, cells cultured with mitogen but without inhibitors maintained their surface Ig (Fig. 4). At various concentrations of actinomycin D, puromycin, or cycloheximide (5 pg, 0 5 Mg or 0 05 Mg/ml) with the

I

8 70

-8 60 5050

cells first lost their surface Ig and then died. Thus, we concluded that within 24 h of cultivation the reduction in the percentage of RFC, i.e. in cells bearing surface Ig was due to the loss of surface Ig in the absence of mitogenic stimulation and not to a preferential loss of Ig-bearing cells.

r-I1 -

LU,

> 40

ir

Z

830

T1

20

Is

a.

10 Il

0

+SM -SM +SM -SM +SM -SM +SM -SM 24 48 72 96

Time (h)

Figure 3. Changes in the viability of splenic lymphocytes cultured in the presence or absence of SM for 4 days. The viability was determined by the exclusion of trypan blue dye, and for each sample a minimum of 100 cells was counted three times. The data presented here represent mean values obtained from two experiments; the vertical bars represent the standard error of the mean.

0 OOI

0QOI Q05 0.10 Q20 Q25 0.50 1.0 2.0 25 50 7.5 Inhibitor Concentration

(,%gtml)

No Mitogen

or Inhibitor

Figure 4. The effect of adding increasing amounts of actinomycin D (-), puromycin (A), or cycloheximide (.) to SM stimulated spleen cell cultures on the maintenance of surface Ig. The 100% represents the percentage of RFC in cultures stimulated with SM to which no inhibitor was added. The results are from three different experiments and the vertical bars represent the range.

736

Donna M. Buchholz, S. Dray & M. Teodorescu Table 2. The effect of inhibitors on the maintenance of surface Ig when added 24 h after the cultures were initiated with streptococcal mitogen

Percentage RFC (% ± SEM) determined at time*

Streptococcal mitogen added (+) or not (-) at time 0

Inhibitors added at time 24 h

28 h

48 h

+ + + -

None Actinomycin D (2 5 Mg/ml) Puromycin (2 5ug/ml) None

87+2.0 41 + 2.0 28 + 7.6 28+0 0

61+2 0 2 + 1.5 18 + 0.0 17+2-3

* The data were calculated considering the percentage RFC at the initiation of the cultures as 100%.

spleen cell viability after 24 h of cultivation was over

85% as in the absence of the inhibitor. Thus, the inhibition of the mitogen effect on the maintenance of surface Ig was apparently not due to the toxic effects of the inhibitors. To establish whether the inhibitors were able to block the maintenance effect already started by the addition of the mitogen, either actinomycin D or puromycin was added to mitogen-stimulated spleen cell cultures after 24 h (Table 2). Even after the first 4 h in culture after the addition of either actinomycin D or puromycin, the maintenance effect provided by the mitogen was significantly reduced, and after 24 h the effect was abolished (Table 2). Thus, it appears that the effect of the mitogen on the maintenance of surface Ig does not involve a 'freezing' mechanism or passive attachment of surface Ig but rather an active process of stimulation which may be arrested by inhibitors of mRNA and protein synthesis. Another conclusion that may be drawn from the effect of these inhibitors is that the shedding of surface Ig is not, but the replacement is, dependent on active protein synthesis. The turnover of surface Ig determined by 1251-labelled anti-allotype monovalent Fab fragments The effect of inhibitors showed indirectly that the turnover of surface Ig took place only on lymphocytes stimulated by mitogen, while the unstimulated lymphocytes shed their surface Ig without replacing it. To obtain direct evidence, the spleen cell suspension from b4 rabbits were first treated with '25l-labelled anti-b4 monovalent Fab fragments for 1 h at 40 (under saturation conditions). The cells were carefully washed, and then incubated for 4 h at 390 in medium supplemented with serum from a rabbit homozygous for the

b5 Ig allotype. A control represented by Sepharose beads coated with b4 IgG was also set up. These beads were substituted for cells in order to determine whether the release in the medium of the radiolabelled Fab was not due to dissociation of antigen-antibody complexes. Both SM-stimulated and unstimulated cultures were set up. After 4 h of incubation, the radioactivity associated with the cells (or the Sepharose beads) and that released in the supernatant was determined. The radioactivity associated with the cells (Fig. 5) was reduced similarly in all cell cultures whether the mitogen was added or not. The radioactivity, however, remained essentially associated with the b4 Ig-coated Sepharose beads, i.e. only about 5% of the radioactivity was lost from the beads. Thus, it appears that the 1251-labelled Fab was released together with surface Ig and not by the simple dissociation of antigen-antibody complexes. The cells were relabelled after 4 h of incubation with anti-b4 or anti-bS Fab fragments (Fig. 5). The cells which were incubated in the absence of mitogen did not acquire any more labelled material, demonstrating that they lost most of their surface Ig. The cells incubated with the mitogen, however, acquired almost the same level of '251-Fab as the original population, indicating that these cells replaced their surface Ig. The cells relabelled after 4 h of incubation were again incubated at 390. After 24 h, the same phenomenon was noticed: the stimulated cells lost the radioactive 1251 but could be labelled again by the monovalent Fab, while the unstimulated cells remained incapable of binding anti-b4 Fab. The slightly higher counts in stimulated cells compared with the unstimulated cell population may be explained as a result ofdissociation of some of the free complexes and re-association of

Lymphocyte surface Ig Mitogen

[I90 _

100 _ 80 70 Q 60 CL 50 0) a-.a) 40 30 _ 20

737 No Mitogen

FI.

KB

10/ml

0l/mi 10/mi

1Omi 0l/ml

0

10 _ 0

f'1. A

LL

c) (D)

it@ 4

B A 24 B Time (h) Figure 5. The turnover of surface Ig in spleen cell suspensions in the presence or absence of mitogen (SM). The A bars represent radioactivity found associated with the cells after the respective incubation time (4 or 24 h). The B bars represent the radioactivity acquired after the cells were again treated with 1251-Fab anti-b4 antibody. The values on the ordinate represent the percentage of radioactivity associated with the same number of living cells compared with that obtained by labelling the initial cell suspension, considered as 100% (extreme left bar) (± standard deviation) for three individual experiments. Open bars, SM added; hatched bars, no SM added.

1251-labelled Fab with the newly synthesized Ig on the membrane. The loss of surface Ig determined by this procedure paralleled the determination of RFC by Ab-E (data not shown). To establish whether some cells acquire some surface Ig from the culture medium binding of 1251-labelled anti-b5 Fab was also determined (the activity of this Fab preparation was tested on b5-Ig bearing lymphocytes) on spleen cells cultured for 4 or 24 h. We found that almost the same low amount of anti-b5 Fab bound to the spleen cells as at 0 time, confirming our observations reported elsewhere (Buchholz et al., 1979) that the spleen cells did not attach any significant amount of Ig from the culture medium. Since about 80% of the surface Ig was released during the 4 h of cultivation (Fig. 5), when the percentage of unstimulated Ig bearing cells was also close to the lowest level (Teodorescu et al., 1978), we estimated that the half life of the surface Ig was about 2 h. Thus, we concluded that in the absence of mitogenic stimulation the surface Ig was shed but not replaced, while in the prcsence of the mitogen the surface Ig was undergoing a turnover with a half life of approximately 2 h.

Figure 6. The effect of cell crowding or cell concentration on the maintenance of surface Ig in 24 h spleen cell cultures. 100% represents the percentage of RFC at the initiation of the cultures. The mitogen used was SM.

The effect of cell crowding on maintaining surface Ig While it appears that the maintenance of surface Ig in vitro was dependent on the presence of a direct polyclonal activation of B cells, the mechanism involved in the maintenance of surface.Ig in vivo is less clear. It would not seem likely that a continuous exogenous mitogenic stimulus would be provided in vivo. Experiments were designed to simulate conditions in vivo, that is, to force the cells to be in close physical contact with each other as they exist in the lymphoid organs. The cell number was maintained at 106 cells/ml but the culture flasks were incubated either in the usual upright position or in a tilted position (Fig. 6). In standard upright cultures when the cells were distributed over a relatively wide area, the percentage of RFC dropped in the absence of mitogen but was maintained almost at the initial level when SM was present. When the culture flasks were tilted so the cells were in close contact and distributed over a very narrow area, the percentage of RFC in the absence of mitogen was similar to that in the presence of mitogen. Also, when the cells were cultured in the standard upright position at 107 or 108 cells/ml, the percentage of RFC was maintained in the absence of mitogen (Fig. 6). Thus, in crowded conditions, when the cells were in close physical contact, the B cells maintained their surface Ig in the absence of added mitogens.

738

Donna M. Buchholz, S. Dray & M. Teodorescu

Maintenance of surface Ig by factors produced by crowded cells The maintenance of surface Ig in the crowded cell population in the absence of added mitogen might have been due to cell-cell stimulation of B cells or to the induction by such stimulation of a factor with the same property as the B-cell polyclonal activators. If such a factor existed it should be present in the supernatant of crowded cell cultures. This supernatant was collected after 24 h of cultivation and added in decreasing amounts to freshly started cultures from a different rabbit. We found that this supernatant had almost the same effect as the addition of SM, i.e. the percentage of RFC was maintained close to the original levels. The supernatant from standing cultures, however, had no effect in maintaining the surface Ig

(Table 3). Even when diluted 1/40 in the culture medium, the crowded cell supernatant was active in maintaining a level of RFC similar to that observed at 0 time. The supernatant from 24 h standing cultures stimulated by Con A was as active as the supernatant from crowded cultures, although the Con A reconstituted control was inactive (Table 3). Thus, these results suggested that the crowded cells released a factor able to maintain the surface Ig of B cells and that a factor with similar activity may be induced by stimulation with Con A. To characterize the factor produced, i.e. to obtain a rough estimate of the molecular size of this factor, we fractionated the supernatant by Sephadex G-200 chromatography and tested the effect of major peaks in maintaining surface Ig (Fig. 7). The composition of each fraction was determined by immunoelectrophoresis. As expected, since the medium contained rabbit serum, the first peak contained the macroglobulins, the second was predominantly IgG, and the third, albumin. Various doses of these peaks were added to standard standing cultures to determine their activity in maintaining surface Ig. The maintenance effect was almost exclusively associated with the macroglobulin peak (Fig. 7). In another series of experiments we investigated whether the factor is also active on autologous cells. Crowded cultures were set up for 24 h and the cells were resuspended and incubated for an additional 24 h period either crowded or standing. At both time intervals, after 24 and 48 h the spleen cell population contained a relatively high percentage of RFC. (In these experiments the percentages of RFC in standard standing cultures was below 15% and those that maintained the surface Ig ranged from 43 to 50%.) Therefore, it appears that during the first 24 h the factor was produced in the supernatant in high enough concent-

Table 3. Maintenance of surface Ig by various cell-culture supernatants

Spleen cells cultured 24 h with

%RFC + SEM

Crowded cell supernatant 91.0 Crowded cell supernatant+ SM* 103.0 Standing Sm*-activated cell supernatant 106-0 83 2 Con At-activated cell supernatant 32 6 Standing culture supernatant Standing culture supernatant + Con At 30.0 SM* 93-2 None 350 *

+ +

42 6.6

+ + + + + +

8-6 12 60 52 1.2 1.2

A mitogenic concentration of SM was added.

t Con A (5 pg/ml) was added at the initiation of standing

cultures. $ Con A (5 pg/ml) was added at the termination of the standing cultures. The data were calculated considering the percentage of RFC at time 0 as 100% (actual values 45-50%). SEM, standard error of the mean. 100-

PEAK

j

75-

i

PEAK 2

RFC

PAK 3

50-

25--r- T

0

20

40

60

80

7/

/-

I

100

120

140

160

240

9g /culIure

Figure 7. The effect of the fractionated supernatant from crowded spleen cell cultures on non-crowded 24 h spleen cell cultures. The results are expressed as the means of four individual experiments; the vertical error bars represent the standard error of the means.

Lymphocyte surface Ig

ration to maintain surface Ig and that this factor acted on the cell population that produced it. Thus, a factor associated with the macroglobulin fraction was present in the supernatant of crowded cell cultures and this factor appeared to have the activity of a B-cell polyclonal activator and to be responsible for the maintenance of surface Ig.

DISCUSSION We showed here that the majority of B cells required direct polyclonal stimulation to maintain their surface Ig in vitro. This maintenance effect was shown to be an active process which could be blocked by inhibitors of protein synthesis; it reflected the turnover of surface Ig. The polyclonal activators were not required to maintain surface Ig when the cells were cultured in conditions which provided close intercellular contacts. The supernatants of crowded cultures contained a factor associated with the macroglobulin fraction which had the same effect as the polyclonal B-cell activators. The loss of rosette-forming cells in the absence of mitogen was due to a loss of surface Ig from B cells but not to a selective loss of B cells by non-specific cell sticking to plastic or by lysis. The surface Ig of most B cells, as determined by two methods, rosette formation with Ab-E and '25I-labelled Fab anti-b4, was maintained only when polyclonal B-cell activators were present in the culture medium. The anti-b4 Fab antibody bound only to the surface Ig since it had allotype specificity. The antibody-coated erythrocytes bound only as a result of the interaction between the antibody combining site and the surface Ig. The binding by a different mechanism such as the binding to Fc receptors is excluded since coating a wrong anti-allotype anti-b5 for b4-Ig bearing cells) does not result in rosette formation (Teodorescu et al., 1975). The specificity of binding of antibody-coated erythrocytes to Ig-bearing cells in another system has also been demonstrated by Haegert, Hurd & Coombs (1978). Moreover, the rosette formation with antibody-coated erythrocytes appears to be a more sensitive assay than immunofluorescence (Molinaro, Bessinger, GilmanSachs & Dray, 1975; Haegert et al., 1978). Nonetheless, we did some experiments in which the surface Ig was identified by fluorescent antibody. To get the percentage of Ig-bearing cells obtained by rosette formation we had to use two fluorescent antibody preparations, an anti-b4 antibody made in b5 rabbits and an

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anti-b5 antibody made in b4 rabbits. With this fluorescent assay, however, the same phenomenon was observed: loss of surface Ig in the absence and maintenance in the presence of mitogens (data not published). Since it was possible that in our experiments the mitogen might have simply 'frozen' the membrane, i.e. prevented the shedding of surface Ig (Cunningham, 1975), we determined the effect of inhibitors of mRNA and protein synthesis on the maintenance of surface Ig. The inhibitors of protein synthesis, puromycin and cycloheximide, abolished the maintenance effect of the mitogen in 4 h cultures. The effects of actinomycin D are difficult to interpret due to the various side effects of this drug, but still showed that an undisturbed cellular activity was required. Thus, it appeared that the loss of surface Ig was due to a continuous shedding and to a lack of replacement in the absence of active protein synthesis. This was verified in experiments designed to examine the turnover of surface Ig (Fig. 5). It is unlikely that the radiolabelled Fab attached to the surface Ig influenced in any way the release or turnover of surface Ig since the release occurred whether the cells were labelled or not in this study as well as in previously reported experiments (Teodorescu et al., 1975; Buchholz et al., 1979). Since the Ig-allotype in the culture medium was different from that on the cells the possibility of passive adsorption of the Ig was also excluded (Buchholz et al., 1979). Therefore, these data support the hypothesis that the polyclonal activators in lymphocyte cultures stimulate the B cells to synthesize replacement Ig for that which is continuously shed. As mentioned above, the half-life of surface Ig of mouse lymphocytes was estimated to be about 2 h (Wilson et al., 1972) or from 2 to 8 h (Marchalonis et al., 1972; Vitetta & Uhr, 1972), observations in agreement with the present findings on rabbit lymphocytes. We are not in agreement with the reports of Anderson et al. (1974), however, in which small B lymphocytes of nude mice were shown by other methods to have a very slow turnover. This discrepancy may be due to several factors. First, the observation that purified small B cells from nude mice has a very slow turnover (Anderson et al., 1974) (20-80 h half-life) was based only on whole populations; the fate of individual cells was not followed by a direct determination of surface Ig. Second, their small lymphocytes were purified by a procedure capable of yielding cells with most of the surface Ig already lost before the experiment started. Third, the cells were cultured in serum-free medium,

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Donna M. Buchholz, S. Dray & M. Teodorescu

devoid of mitogens; when cultured in medium supplemented with foetal calf serum (which contains mitogens) others have found, like us, a 2 h half-life (Wilson et al., 1972). Even the macrophages, which may be able to provide polyclonal B cell activators (Unanue, 1978), were removed by the purification procedure for small lymphocytes. Fourth, their methodology, used for the study of turnover, is mostly indirect, based on complex calculations. Finally, a species difference is not excluded, an aspect which is now being investigated. Here, as well as in our previous experiments (Teodorescu et al., 1975; Buchholz et al., 1979), we only followed the fate of the surface Ig of the cells in their 'natural' environment with a minimum handling of the cells. In our experiments the loss of surface Ig determined by' '251-labelled Fab, which measures the total amount of surface Ig, paralleled that of the percentage of Ig-bearing cells determined by antibody-coated erythrocytes, which take into account individual cells. Thus, since the majority of spleen or lymph node cells are small lymphocytes (over 95%), it is obvious that a fast turnover of surface Ig takes place in all lymphocytes small or large, and that this turnover is controlled by polyclonal activators. The classical T-cell mitogens, Con A and PHA, failed to maintain the surface Ig of B cells over a wide range ofconcentrations. This might be due to the short time (4 h) in which the surface Ig of most B cells is irreversibly lost as we have previously reported (Teodorescu et al., 1975). Even if mitogen activated T cells may stimulate B cells via a mitogenic factor, by the time such a factor is produced (a few hours), the surface Ig has already been lost. Only polyclonal B-cell activators (or T-independent antigens), such as LPS, pneumococcal polysaccharide III, Ficoll, dextran sulphate etc. (Buchholz et al., 1979) or even FCS (Teodorescu et al., 1975), maintained surface Ig of B cells probably by directly stimulating them. It is worth noting that even very small concentrations of LPS or SM are sufficient to maintain surface Ig. In our previous study the use of only one concentration (10 jg/ml) of Con A was misleading since impure materials might have caused the maintenance effect observed. Also, we noticed that different batches of PWM give different results, i.e. some do and some do not maintain surface Ig at various concentrations (data not shown). Thus, it appears that only selected mitogens maintain surface Ig and even very small submitogenic concentrations are efficient. It may be speculated that B cells are dependent on an outside stimulation in a way similar to hormone-

cell interactions in order to maintain their basic membrane activity, and the blast transformation and proliferation are only the consequences of an increase in the concentration of mitogenic units per cell. According to Coutinho & Moller (1975) B cells are triggered by T-independent antigens due to a concentration mechanism for B-cell mitogen provided by Ig receptors. For T-dependent antigens such mitogenic agents would be provided by T cells. The factor produced in the culture medium of crowded cells may be such a B-cell mitogen produced by T cells or by macrophages (Unanue, 1978), an aspect under investigation. Evidence presented here (Table 3) suggest that Con A-activated spleen cells produced the factor capable of maintaining the surface Ig. This factor may be similar to that described by Smith & Barker (1972) in cultures stimulated with Con A and therefore may be a T-cell product. It may be considered that mitogens are provided to the B cells in vivo, particularly from the intestinal flora (Teodorescu et al., 1975) but the presence of large percentages (60-80%) of Ig-bearing cells in the spleens of germ-free or antigen-free mice (Teodorescu & Webb, unpublished observations) made such a speculation unlikely. Therefore, we tested the hypothesis that the simple cell-cell close contacts are sufficient to provide the B cells with the stimulus required to maintain a normal turnover of their surface Ig. Indeed, when the spleen cells were cultured in conditions providing close intercellular contacts, i.e. crowded in the tilted flasks (Fig. 6), the surface Ig was maintained as if the mitogen were present. Since lymphocytes are normally packed in spleen and lymph nodes, such a mechanism is possible in vivo. Crowded cells do not lose their surface 1g. Therefore, it may be argued that the B-cell mitogens are only bringing the cells into close contact by an agglutination process. The lack of this maintenance effect by two agglutinating agents, PHA and Con A, which can bind to both B and T cells, makes such an assumption unlikely. It may be speculated that B cells stimulate other cells (maybe T cells or macrophages) by close intercellular contact to produce the macroglobulin-associated factor, which in turn acts as a polyclonal B-cell activator. Our observations may explain the success of using Marbrook's culture vessels (which provide close intercellular contacts) to obtain a primary antibody response in vitro in the presence of autologous serum and in the absence of any detectable 'outside' mitogens (Metzger, Hendricks, Teodorescu & Dray, 1977). The existence of a stimulatory interaction between autolo-

Lymphocyte surface Ig gous B and T cells has been demonstrated in mice and humans and is known as autologous or syngeneic mixed lymphocyte reaction (Opelz, Kiuchi, Takasugi & Terasaki, 1975; Von Boehmer, 1974; Smith & Pasternak, 1978). It is possible that such recognition signals play a regulatory role, i.e. some cells stimulate other cells to produce factors that act on other cells as submitogenic polyclonal activators. It is interesting to note that lymphokines appear to be associated with a-macroglobulin from the serum (Rocklin, 1975) as is the factor described here. A practical consequence of this study is that the incubation of the human peripheral blood lymphocytes at 370 as suggested by others (Lobo, Westervelt & Horwitz, 1975) supposedly to remove passively adsorbed surface Ig generates artifactually lower percentages of Ig-bearing cells (Haegert et al., 1978). The understanding of the factors controlling the turnover of surface Ig may provide new ways of manipulating the immune system. The rapid and irreversible (Teodorescu et al., 1975) loss of membrane receptors may lead to the inactivation of a particular lymphocyte subpopulation if the required stimulus is only temporarily blocked. The same may happen with malignant lymphocytes or with lymphocytes in autoimmune diseases. Also, the basic understanding of lymphocyte biology and lymphocyte interactions is dependent upon the clarification of how the turnover of the membrane components of lymphocytes in general and of small lymphocytes in particular is regulated. The very sensitive assays used here may be employed to study any polyclonal B-cell activator, exogenous or endogenous and to differentiate between T-dependent and independent antigens (Buchholz et al., 1979). The generalization derived from the present investigation may be that lymphocytes are always stimulated, but at a very low level, and that the blast transformation induced by mitogens and antigens represents only a quantitative change leading to differentiation and antibody secretion.

ACKNOWLEDGMENTS This investigation was supported by Public Health Service Grant No. Al 07043 and by the grant AM 19414 from the National Institute of Arthritis, Metabolism and Digestive Diseases. REFERENCES ABE Y. (1971) Lymphocyte stimulation with streptolysin 0

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