Eur. J. Immunol. 1977. 7 151-153

Surface Ig on activated B cells

A. burgois*, K. Kitajima, I.R. Hunter and Brigitte A. Askonas

Surface immunoglobulins of lipopolysaccharidestimulated spleen cells

National Institute for Medical Research, London

The behavior of IgM, IgD and IgG

151

The nature of Ig receptors carried by lipopolysaccharide (LPS)-stimulated mouse spleen B cells was analyzed by surface iodination, direct antiserum precipitation and polyacrylamide gel electrophoresis. LPS activation led t o a rapid decrease of surface IgD to 20 % and 80 % of the original level on days three and five of culture, respectively. Efficiency of iodination of cells doubled after culture but the proportional radioactivity in IgM remained constant. We could definitively identify y-chains on the surface of cells, but only in small amounts (1 of total surface Ig) and this expression of IgG remained constant during 5 days of culture.

1. Introduction

2. Materials and methods

A high proportion of B lymphocytes in mouse spleen express on their surface IgM and "1gD"-like molecules; the latter share many properties with human IgD and hence are referred t o as IgD [ 1-61. However, there remain some uncertainties concerning the expression of Ig receptors by mouse B cells, and the role of the different Ig classes in the developmental stages and maturation of the B cell lineage. IgG for example has not been definitively detected by surface labeling, although 1-3 % of mouse B lymphocytes have been reported t o be IgG or IgA-positive by fluorescent staining [ 5, 7, 81.

2.1. Spleen cell culture

We wished to analyze the nature of Ig receptors on B cells after activation with lipopolysaccharide (LPS) while cells are undergoing proliferative cycles and maturing. In our culture system, US leads to proliferation and regeneration of small B lymphocytes in the majority of the stimulated population, while the early wave of IgM synthesis and secretion is restricted to a small proportion of cells [9]. Late in culture, maturation into IgG as well as IgM secretion occurs, but this is preceded by several proliferative phases [lo]. External surface labeling followed by immunochemical techniques and polyacrylamide gel electrophoresis, show a rapid disappearance of surface IgD by LPS activation and definitively a very small amount of surface IgG, which remains constant, but does not increase during five days of culture.

[I 15791

* Char& de recherche au Centre National de la Recherche Scientifiiue, Fellow of the European Molecular Biology Organization. Resent address: Centre d'lmmunobgie de Marseille-Luminy, F-13288Marseille-Cedex 2, France

Conespondence: Brigitte A. Askonas,Division of Immunobgy and

Experimental Biology, National Institute for Medical Research, Mill

Spleen cell suspensions (CBA/H mice, 3-4 months of age) were cultured at 37 OC as described by Zauderer and Askonas [lo]. In brief, 2 x 106 - 3 x 106 cells/ml were treated with 10 pg/ml LPS in 60 mm Falcon dishes, in 5 ml of medium M [RPMI 1640, antibiotics, 10 % fetal calf serum, 5 x 2-mercaptoethanol(2-ME)], in an atmosphere of 10 % C02, 7 % 02 and 83 % Nz, with a medium change every three days. 2.2. Radiolabeling of surface Ig molecules

To eliminate red blood cells and dead cells, viable cells were centrifuged for 20 min a t 20 OC and 2000 x g, on a FicollIsopaque gradient [ 1 I ] and then washed three times with PBS. 2 x 10' cells were labeled with 4 mCi of lZ5lby the lactoperoxidase-catalyzed procedure [ 1 21 and washed once in phosphate buffered saline (PBS), surface proteins were solubilized by cell lysis (1 0 min at 0 "C) in 1 % (w/v) NonidetP40 (Shell) in PBS containing 1 mM phenylmethylsulfonyl fluoride and I00 mM recrystallized iodoacetamide [ 1 1. This lysate was passed over Sephadex G-25 equilibrated with the same solution and centrifuged for 10 min at 1 2 000 x g.

2.3. Radioactivity of Ig chains Each cell lysate was divided into three samples and the lzsIlabeled surface Ig precipitated by various antisera in the presence of 20 pg carrier Ig as follows: (a) with a polyvalent rabbit anti-mouse-Ig antiserum; (b) rabbit antibody reacting specifically with mouse IgG. Antiserum to Fc fragments of IgGz, 5563 myeloma protein was absorbed by insolubilized mouse IgM, IgA, 5563 Fab and K-chains isolated from the serum of mice carrying plasmacytomas. Specificity for mouse IgG was established by testing cross-reaction with [3H]leucine labeled myeloma proteins of different classes; (c) as control, 25 pg of rabbit Ig were precipitated with goat ant i-rabbit-Ig.

Hill, London NW7 IAA, England Abbreviatbm Lps:E COP lipopolysaccharide PBS: Phosphate

buffered saline 2-ME2-Mercaptoethanol SDS: Sodium dodecyl sulfate

The antibody precipitates were washed three times with icecold 0.5 % (w/v) Nonidet-P40 in PBS, once with PBS, dispersed in 50 m M sodium phosphate pH 7 and reduced in

Eur. J. Immunol. 1977. 7: 151-153

A. Bourgois, K. Kitajima, I.R. Hunter and B.A. Askonas

152

2 r n M dithioerythritol (Sigma Chemical Co., St. Louis, Mo.) 2 "; sodium dodecyl sulfate (SDS) at 100 OC for 10 min a n d alkylated with 100 mM iodoacetamide. Gel analysis ( 1 / 3 or 1 /6 of samples) was performed in 7.5 % (w/v) polyacrylamide gels, 0.1 % SDS f o r 4 h a t 1 8 m A [ 131. Various 1311labeled markers [ 141 were a d d e d as indicated. Radioactivity was determined o n 1 m m slices; values were corrected for cross-channel spill and plotted with t h e t o p of t h e gel o n the left hand side of t h e figures.

i n 6Ip-chains d r o p s f r o m 0.6 d a y 0 t o 0.1 a n d 0.04 o n days three a n d five o f culture, respectively (Table I). Only trace a m o u n t s of 6-chain are detectable after 1 1 days o f culture ( n o t illustrated). T h e disappearance of 1gD cannot be attributed t o interaction between LPS a n d t h e cell surface; some batches of 2-ME also activate B lymphoid cells early in the M), and a simicultures a t very low concentrations(5 x lar early loss in surface IgD expression could be observed (not illustrated).

3. Results

Table 1. Surface expression of Ig receptors following LPS stimulation of spleen c e W )

3.1. Efficiency of iodination of spleen cells in culture T h e uptake of 12s1 per unit nilrnber of cells i n t o protein precipitable with trichloroacetic acid is increased during t h e course of culture f r o m 2.5 5% in a normal spleen cell suspension to 5 70 after five days o f culture with LPS. T h e reason for this is n o t clear; it may be d u e t o culture alone or t o t h e many changes which take place during cell activation a n d division (such a s increase in microvilli [ 151 or increase in cell size, etc.). T h e proportion of 1251-labeledIgM t o t o t a l surface protein and thus the Ig density, remains constant (not shown).

3.2. Surface IgD on B cells in LPS culture Surface iodination of cultured cells a n d analysis of the Ig receptors by direct precipitation with antiserum in the presence of carrier Ig a n d gel electrophoresis, shows that LPS stimulation of t h e B cells leads t o a rapid disappearance of lgD receptors relative t o IgM Fig. 1). This decrease is already significant on day o n e a n d the ratio of radioactivity

15[

8 0 0 A

II I

II

V i a h k celk/rnl x 10"

Day

Radioactivity ratio

1x1 radioactivity of heavy chains

cpm x 10-3 0 1

2.2 I .n

3 5

1.5 4.4

P

6

Y

6/P

66 63 78 70

40

0.6

26 8

1. I

0.61 0.4 1 0.1 0.04

3

0.7 0.6

a) 2 x 106 spleen ceIls/ml cultured with 10 /.fg/ml LPS. Radioactivity of heavy chains estimated following radioiodination of cells, precipitation of Ig with a polyvalent antiserum, reduction and analysis on polyacrylamide gels (see Fig. l). Each analysis was done with a similar amount of radioactive Ig, representing a different number of cells on different days of culture.

3.3. IgG on t h e surface of splenic lymphocytes By direct precipitation with antiserum specific foi IgG a small a m o u n t of 1gG receptor is detectable o n t h e surface of freshly prepared mouse spleen cells (about 1 % of total

b

B

C

D

B

C

D

80

Fraction No.

Figure 1. Analysis of surface immunoglobulins on splenic B cells after LPS stimulation.

Spleen cells before or after culture were surface-labeled with l Z 5 l , lysed and 1251-labeledsurface Ig recipitated with various antisera, reduced and analyzed by polyacrylamide gel electrophoresis in SDS (see Section 2.3.). Internal 1311-labeled~14] markers were included with the test sample, and their positions on the gel are indicated on the figure by the arrows [(A) unreduced mouse IgG, mol. wt. 150 000;(B)reduced bovine serum albumin, mol. wt. 68 000; (C) reduced creatine phosphokinase, mol. wt. 40 000; (D)reduced human myeloma h-chain, mol. wt. 24 0001. Gcls a to c: 12sl-labelcd Ip precipitated with polyvalent antiserum to lg in cell lysate (see Section 2.3.). (a) Spleen cell suspension before culture; (b) 1 day of culture with 10 pg LPS/ml; (c) 5 days of culture with 10 pg LPS/ml. Gel d: 1 day of culture with LPS. (--) Ig precipitated with antiserum specific for mouse y-chains; (- - - - - ) control precipitate (rabbit IgG and goat anti-rabbit IgG). For gels a, band d, 1/3 of the cell lysates were analyzed, whereas gel c contained 1/6 of the cell lysate (5 day culture), so that each sample, contained a similar level of radioactivity.

Eur. J. Immunol. 1917.7: 151-153 Ig receptors) (Fig. Id). This small amount of IgG remained constant during the five days of culture, although no detectable IgG is secreted during this time.

4. Discussion Technically, it is essential in this type of analysis to co-precipitate Ig receptors directly with antiserum in the presence of pure carrier Ig; this yields small antigen-antibody precipitates (about 80 p g ) and clean controls (Fig, 1 d). On the other hand. indirect precipitation using low titer, specific rabbit antisera and anti-rabbit lg sera, yields large precipitates with high background contamination. Some contaminating peaks o n the gels coincide with the position of Ig chains, in particular ychain; these misleading radioactive peaks most likely represent Fc receptors and their degradation products (Bourgois, A., t o be published). By direct precipitation technique, we could demonstrate the presence of surface IgG o n normal spleen cells, but in very small amounts, i.e. about 1 % of total receptor Ig (Fig. 1 d ; Table 1). The fact that IgG can be detected during several days of culture makes it unlikely that cytophilic IgG is responsible for its presence; for the first 7-1 0 days spleen cells in this high density culture do not secrete detectable IgG and medium is changed every three days. The presence of IgG surface receptors on mouse spleen cells has been a somewhat controversial topic using fluorescent labeled reagents (antiserum specific for IgG). However, low percentage (1 -2 %) of cells carrying surface IgG has been observed by such techniques [7, 8, Roelants, G.E. and Mayor-Whitley, K.S.,t o be published), and our present experiments directly confirm the presence of such small amounts of surface IgG. The disappearance of IgD during LPS stimulation of B cells starts early (35 % and 92 % losses after one a n d five days). Disappearance of 1gD in stimulated cultures wasalready observed by fluorescent staining [ 71.IgD does not build up again during any of the days tested (days 1 , 3 , 5 and 11). LPS in these cultures stimulates about 40 % of the B cells t o divide, and a major proportion of these cells then are restimulated into further proliferative cycles [ 9, lo]. Culture conditions per se arc unlikely t o account for disappearance of IgD since in continuous culture lymphocytes appear t o maintain IgM and IgD surface expression [ 161. IgD has not been found

Surface Ig on activated B cells

15 3

on IgG secreting myeloma cells [ 71 and therefore appears lost on final maturation. It is unlikely that IgD-bearing cells are not stimulated and simply losi in the culture which tends t o select stimulated cells. More than 80 % of B cells in spleen carry both IgD and IgM 161 and 40 % of B cells are stimulated in these cultures [9]; t h e actual role of IgD remains indefinite, however, the results are in line with the suggestion that proliferation and maturation may be linked with a suppression o f IgD expression or a release or cleavage of the surface IgD during the triggering process [4,51. Received November 4,1976;in revised form January 27,1977.

5. References 1 Abney, E.R. and Parkhouse, R.M.E., Nature 1974.252:600.

2 Abney, E.R., Hunter, I.R. and Parkhouse, R.M.E., Nature 1976. 2.59:404. 3 Melcher, U., Vitetta, E.S., McWilliams, M., Lamm, M.E., PhillipsQuagliata, J.M.and Uhr, J.W., J. Exp. Med. 1974,140:1427. 4 Bourgois, A., Abney, E.R. and Parkhouse, R.M.E., Eur. J. Immunol. 1977,in press. 5 Vitetta, E.S. and Uhr, J.W., Science 1915. 189: 964. 6 Goding, J.W. and Layton, J.E., J. Exp. Med. 1976. 144: 852. I Parkhouse, R.M.E., Abney, E.R., Bourgois, A. and Willcox, H.N.A., Cold Spring Harbor p l a n t . BioL Symp. 1977. 41, in press. 8 Cooper, M.D., Kearney, J.F., Lawton, A.R., Abney, E.R., Parkhouse, R.M.E., Preud'homme, J.L. and SeUgmann, M., Ann. Immunol. (Inst. Pasteur) 1976..1 2 7 C 513. B.A., Roeknts,G.E., Mayor-Withey, K.S. and Welstead, 9 AS~OMS, J.L., Eur. J. Immunol.1976.6: 250. 10 Zauderer, M. and Askonas, B.A., Nature 1976.260:61 1. 1 1 Parish,C.R., Kirov, S.M., Bowern, N. and Blanden, R.V., Eur. J. Immunol. 1914.4: 808. 12 Marchalonis, J.J., Cone, R.E. and Sauter, V.,Biochem. J. 1971. 124: 921. 13 Della Corte, E. and Parkhouse, R.M.E., Biochem. J. 1973.136: 589. 14 Hunter, W.M. and Greenwood, F.C., Nature 1962.194:495. 15 Pasternak,C.A., J. 7Reor. Biol. 1976.58: 365. 16 Remkumar-Reddy. E., Rice, P.J., Chung, K-C. and Sarma, P.S., Cell 1976.8: 397.