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A natural IgM antibody does inhibit polyclonal and antigen-specific IgM but not IgG B-cell responses
Immunology Letters, 39 (1994) 235-241 0165 2478 / 94 / $ 7.00 ~, 1994 Elsevier Science B.V. All rights reserved
IMLET 02083
A natural IgM antibody does inhibit polyclonal and antigenspecific IgM but not IgG B-cell responses K a t a l i n Kiss, Ferenc Uher* a n d J~mos Gergely Department of Immunology, Lor6nd E6tv6s University, GOd, J6vorka S. u. 14, H-213l, Hungary (Received 26 July 1993; accepted 6 December 1993)
1.
Summary
Since a B-cell growth-inhibitory natural IgM antibody was identified in the culture supernatants of LPS-stimulated murine splenic B lymphocytes [11], attempts have been made to define other possible functional role(s) of this antibody. Here we show that this regulatory IgM is able to inhibit not only the proliferation of splenic B cells, but also their IgM secretion during LPS-induced polyclonal, as well as antigen (FITCKLH)-specific antibody responses. In contrast, IgG1 production of hapten (FITC)-specific B cells neither during restimulation with LPS nor in the presence of carrier-specific T lymphocytes in vitro was affected by regulatory IgM. Therefore, whereas newly emerging naive B cells are highly susceptible, IgG-secreting B cells appear to be completely resistant to inactivation by the regulatory IgM autoantibody. 2.
Introduction
Humoral immune responses generated upon antigen rechallenge differ significantly from the primary response observed after initial antigen exKey words: B cell; Natural IgM; Memory response; (Mouse) *Corresponding author: Ferenc Uher, Ph.D., Dept. of Immunology, Lor~md E6tv6s University, G6d, J~.vorka S. u. 14, H2131, Hungary. Abbreviations: B-SN, culture supernatants of murine splenic B cells stimulated with LPS for 96 h. SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) E 0 1 7 4 - B
posure in that memory responses display a more rapid appearance of dramatically increased levels of serum antibody comprised of higher affinity antibodies generally bearing isotypes other than IgM [l~t]. Several theories have been proposed to account for simultaneous generation of antibody-forming cell clones and secondary B cells during the course of primary immune response. The most widely accepted view is that primary B cells, upon stimulation, undergo unequal division giving rise to both antibody-forming cells and secondary B cells. Alternatively, a precursor cell subpopulation separate from that responsible for the generation of primary antibody-forming cells may give rise to secondary B cells [reviewed in 5]. It was found that the J l l D monoclonal antibody (mAb) binds well to most primary B cells and poorly to most secondary B cells [6]. Conversely, the heat-stable antigen recognized by mAb J11D is expressed at high levels on the surface of a majority of virgin B cells and at low levels on memory cells [7-9]. Furthermore, it was also suggested that the primary progenitors to secondary B cells are distinct from the primary antibody-forming cell precursors [10]. Our question in this field stems from previous studies in which we have demonstrated that a culture supernatant of LPS-stimulated murine splenic B lymphocytes (B-SN) is able to inhibit the growth of freshly isolated B cells via an IgM antibody [11]. The binding specificity of this IgM is not yet defined but appears to be a lymphocyte surface structure distinct from membrane immunoglobulin, MHC II antigen, B220, transferrin 235
and Fcz receptors [12]. The regulatory autoantibody allows the normal progression of early (GO to GO*, and GO* to G1A) but not late (G1B, S, G2 and M) steps in the cycle of polyclonally stimulated B lymphocytes and does not affect the increased antigen-presenting capacity of LPS-activated B cells [13,14]. On the basis of the above results the question emerged whether regulatory IgM is able to affect the differentiation of B cells during polyclonal and/or antigen-specific primary and secondary immune responses. We found that this IgM inhibits the polyclonal, as well as hapten-specific IgM, but not IgG (mainly IgG1) antibody production in response to LPS, or FITC-KLH, or FITC-BSA complexes, respectively. 3.
3.1.
Materials and Methods
Animals
DBA/2 mice, 2-3 months old, were obtained from the LATI (G6d6116, Hungary).
mM) of phosphate buffer. After centrifugation of the solution at 20,000 × g for 15 min, the plasma membrane-rich pellet was washed 3 times with phosphate buffer and finally resuspended in BSS. Protein was quantitated as described by Lowry et al. [15]. Mouse erythrocytes were treated with a saturated solution of bromelain in PBS for 30 min at 37°C then washed 3 times in PBS. Oxasolone was coupled to BSA. ExtrAvidin-peroxidase was purchased from Sigma. FITC was coupled to both KLH and BSA. 3.3.
Mice were immunized by i.p. injection of 50/~g of FITC-KLH in complete Freund's adjuvant. Eight weeks later the animals were boosted with 50 #g of antigen in incomplete Freund's adjuvant i.p. Another group of animals was immunized by injection of 100 /~g of BSA in complete Freund's adjuvant into hind footpads. 3.4.
3.2.
Cell preparation
Antibodies and reagents
LPS (Escherichia coli 055;B5) prepared by the Westphal technique, bromelain from pineapple stem, crystallized OVA (type XIII), and BSA were all obtained from Sigma (St. Louis, MO). KLH was purchased from Calbiochem (San Diego, CA). Human dsDNA was the kind gift of Katalin Takfics (OHVI, Budapest, Hungary). Mouse IgG was purified from pooled normal DBA/2 serum by ammonium sulfate precipitation and ionexchange chromatography on DE-52 (Whatman Chemical Separation, Clifton, N J). Normal IgM was prepared from the same serum sample by precipitation in low ionic strength buffer followed by gel filtration on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden). Affinity purified biotin-conjugated goat anti-mouse IgM (/~-chain specific) was purchased from Sigma. Affinity-purified biotin-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 subclass-specific antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL). Erythrocyte membranes were prepared by lysis of 1 ml packed volume of red blood cells in 40 ml (50 236
Immunization
Single mononuclear cell suspension of spleen was prepared on Ficoll-Uromiro (Pharmacia, and Bracco, Milan, Italy) gradient by the method of B6yum [16]. Spleen cells were then depleted of T lymphocytes by antibody and complementmediated cytotoxicity as previously described [17]. Lymph node cells were prepared from the popliteal and inguinal nodes of primed animals 7 days after injection. T cell-enriched populations were obtained by the method of Julius et al. [18]. 3.5.
Culture conditions
The usual procedure consisted of 2-stage in vitro culture as previously described [11]. Briefly, B lymphocytes were cultured at 2 x 10 6 cells/ml density in tissue culture flasks with 20 #g/ml LPS at 37°C in 5% CO2-in-air incubator for 96 h in RPMI-1640 medium supplemented with 10% FCS (v/v), L-glutamine (2 mM), sodium pyruvate (1 raM), non-essential amino acids, 2-mercaptoethanol (5 × 10 5 M), Hepes buffer (20 mM), penicillin (100 IU/ml) and streptomycin (100 #g/ ml). The cells were then sedimented and the cul-
ture supernatants (B-SN) or their affinity-purified IgM fractions assayed for the inhibitor. In the second stage, various cell populations and reagents (see Results for details) were cultured in 1 ml final volume in 24-well fiat-bottomed plates (Costar 3524, Costar Data Packaging, Cambridge, MA) in RPMI-1640 medium supplemented as described above. Antibody production was measured using ELISA assays at the peaks of the various responses as determined in preliminary experiments.
3.6.
Measurement of polyclonal lg secretion
The total amounts of IgM and IgG antibodies in the culture supernatants were assessed by indirect competitive ELISA. Plates were coated with 100 pl of 10 pg/ml purified normal mouse IgM or IgG, respectively. Antigen-coated plates were saturated with 1% BSA in PBS and reacted both with 50 pl dilution of culture supernatants (1:5 to 1:625) and 50 #1 of biotin-coupled anti-mouse IgM or I g G for 1 h at 37°C. Then peroxidasecoupled ExtrAvidin was added and after 1 h incubation at 37°C enzymatic activities revealed by the addition of O P D in phosphate/citrate buffer containing 5-10% H202 (v/v). The reaction was read at 492 nm by a MR700 Microplate Reader (Dynatech Laboratories, Chantilly, VA). Positive and negative reference samples were used to draw standard curves. All tests were done in triplicate and results were calculated by interpolating from the standard curves.
3.7.
Measurement of self- and non-self specific IgM antibody production by indirect ELISA
Plates were coated with 50 #l/well (10 /~g/ml) solutions of self- (membrane fraction of BrMRBC, dsDNA, aggregated IgG) and non-self (BSA, K L H , membrane fraction of SRBC) antigens in PBS. Antigen-coated plates were saturated with 1% BSA in PBS and reacted with 50 /al dilution of culture supernatants (1:5 to 1:625) for 1 h at 37°C. Subsequently, they were incubated for 1 h at 37°C with biotin-labeled antimouse IgM. Then peroxidase-coupled ExtrAvidin was added, and after an additional 1 h incubation at 37°C, enzymatic activities were revealed as de-
scribed above (section 3.6).
3.8. Hapten-specific antibody responses Measurement of hapten (FITC)-specific primary (IgM), and secondary (IgG1, IgG2a, IgG2b, and IgG3) antibody levels in the culture supernatants were also performed by indirect ELISA. Plates were coated with F I T C - K L H , or FITC-BSA, or K L H , or BSA, respectively. Culture supernatants were diluted 1:5 to 1:625. Biotin-coupled anti-mouse lgM, I g G l , IgG2a, IgG2b, IgG3 and peroxidase-coupled ExtrAvidin were used as described in section 3.7. 4.
Results
4.1.
Effect of the regulator)' lgM on polyclonal immunoglobulin secretion in vitro
Results of experiments designed to assess the W.
N
!, 0
| D
i
,,
ill //
11
1
\\
$
D~
4
5
I
in oulture
Fig. 1. Ability of B-SN to inhibit LPS-induced polyclonal lgM secretion in vitro. T-cell-depleted spleen cells were cultured at 2 × 1 0 6 cells/well in I ml of complete medium either alone (0- -O), or in presenceof I_PS(10 ,ug/ml) (m-m), or LPS plus B-SN (12% v/v) (0- -0), during the indicated periods. Culture supernatants were always replaced with fresh medium 24 h before determination of the IgM level by an isotype-specificcompetitive ELISA. One representativeexperiment out of five. ")2"7
TABLE 1 THE INHIBITION OF THE LPS-INDUCED POLYCLON A L lgM S E C R E T I O N IS M E D I A T E D BY AN lgM ANTIBODY ~ Reagents LPS (10 #g/ml) -
+ + +
+
+
Secreted IgM b (/~g/ml) B-SN (12% v/v) -
Untreated Adsorbed on anti-/~ antibody-coated Sepharose 4B e Adsorbed on and eluted from anti-p antibodycoated Sepharose 4B d Dialyzed for 48 h ~
B-cell cultures had a similar inhibitory effect on the polyclonal IgM secretion of the cells (Table 1). A significant amount of IgG was never detected in the culture supernatants o f LPS-stimulated B cells (data not shown).
Regulatory IgM inhibits both self- and nonself-specific antibody production in vitro
4.2.
4
55 5
44
Next we investigated the specificity of B cell-secreted IgM antibodies at different times after stimulation with LPS in the presence or absence of B-SN. We used a panel of self- (membrane frac-
11 5
aCulture conditions and ELISA assay were identical as described in Fig. 1. barter 4 days of culture. CSamples of B-SN were adsorbed on F(ab')2 goat anti-mouse #antibody coupled to Sepharose 4B. aRegulatory molecules were specifically eluted from the anti-# antibody-coated beads using high amounts (2 mg/ml in complete medium) of normal mouse lgM. eSamples of B-SN were dialyzed against complete medium for 48 h at 4°C. lOne representative experiment out of three.
=-
.o .Q C m
~g 30
effect of B-SN on LPS-induced polyclonal antibody secretion in time are shown in Fig. 1. Splenic B cells were cultured in complete medium either alone or in the presence of LPS or LPS plus B-SN for 6 days. The amounts of secreted IgM in the culture supernatants were determined daily by ELISA. We found that the LPS-induced IgM secretion was greatest on the 4th day of culture (on an average 90/~g/ml). When LPS and B-SN were added together, a marked (96%) inhibition of polyclonal antibody production was observed. In contrast, no measurable inhibition was found on day 3, probably due to in vivo preactivated cells in the splenic B-cell preparations. Thus, B-SN is able to inhibit polydonal antibody (IgM) secretion by LPS-stimulated B lymphocytes. Furthermore, the addition of affinity-purified IgM fraction of B-SN or extensively dialyzed B-SN to LPS-stimulated 238
20
//
10 -
O*
/'~/
,.//.' ~ / // " / '
/ Days in culture
Fig. 2. The regulatory IgM inhibits LPS-induced self-, as we]] as non-self-antigen specific IgM antibody production in vitro. T cell-depleted DBA/2 spleen cells were placed in culture at 2 x l06 cells/well in 24-well plates and stimulated with LPS (10 #g/ ml), or with LPS plus B-SN (12% v/v) during the indicated period. Culture supernatants were collected as described in Fig. 1. Antigen-specific lgM antibodies were detected by indirect ELISA (dilution of supernatants l:5) and the percentage of the B-SN-mediated inhibition calculated. Coats were aggregated IgG ( , - , ) , membrane fraction o f BrMRB C ( O - O ) , dsDNA ( i - i ) , BSA ( [ ] - [ ] ) , KLH ((3 ©), and membrane fraction of SRBC (@-@), respectively. One representative experiment out of three.
tions of BrMRBC, dsDNA, aggregated IgG) and non-self (membrane fractions of SRBC, BSA, KLH) antigens in an indirect ELISA (see Materials and Methods) to detect antigen-specific antibodies. As shown in Fig. 2, the percentage of BSN-mediated inhibition of antibody production was similar for self-, as well as for non-self-reactive IgM antibodies throughout the entire culture period tested.
4.3.
Effect of the regulatory IgM on antigenspecific primary and secondary antibody responses
Induction of antibody responses to most antigens (soluble proteins, hapten-carrier complexes) requires the participation of both helper T cells and antigen-presenting cells. Generally, primary antigen exposure results in an initial antibody response and the formation of memory cells [4]. Therefore, we compared the effect of the regulatory IgM on primary and on secondary antibody responses.
B-SN inhibits hapten-specific primat 3, antibody response To determine whether the regulatory IgM could influence the thymus-dependent primary antibody response, mice were injected with FITC-KLH i.p. and 1 week later with splenic B cells prepared from immunized animals. Cells were then cultured either in complete medium alone, or restimulated with LPS, or with LPS plus B-SN in vitro. Culture supernatants were always replaced with fresh medium 24 h before ELISA performed on FITC-BSA- and BSAcoated plates. Maximal hapten (FITC)-specific IgM secretion was observed after 3 days of culture in the presence of LPS. As shown in Fig. 3, B-SN was able to inhibit the hapten-specific lgM response at this time. Control experiments were accomplished with B cells isolated from unimmunized animals. However, no significant FITC-specific antibody production was found in the control cultures (data not shown).
4.3.2.
B-SN does not inhibit hapten-specific secondary antibody response A basic feature of thymus-dependent antibody responses is the generation of memory: on second contact with an antigen a secondary response is produced in which somatically mutated IgG antibodies with increased affinity are synthesized [14]. In order to examine the effects of the regulatory IgM for hapten-specific IgG secretion, mice were primed and boosted with FITC-KLH i.p. One week after the secondary antigen challenge splenic B cells were isolated and restimulated in vitro with LPS in the presence or absence of BSN. To assess the amount of hapten (FITC)-specific antibodies, secreted immunoglobulins reactive with FITC-BSA and/or BSA were measured in the culture supernatants daily by isotype-speciB 0.7.
G
0.7
(I.6
4.3.1.
(
0.5
0.4 O4
03: p
I
e",
o
uL
Fig. 3. FITC-specific primary antibody response in the presence or absence of the regulatory IgM. Splenic B cells from mice immunized with 50/~g FITC-KLH i.p. were cultured at 2 x 106 cells/well in complete medium alone (l~), or with LPS (10 ~g/ml) (m), or with LPS plus B-SN (12% v/v) (m) for 3 days in vitro. Culture supernatants were collected as described in Fig. 1. The level of specific IgM antibodies were determined by indirect ELISA using (A) FITC-BSA- and (B) BSA-coated plates. One representative experiment out of three.
239
A
TABLE 2
IJ
IN VITRO HAPTEN (FITC)-SPECIFIC B CELL M E M O R Y RESPONSE a B-SN
Anti-FITC IgG1 response (OD492) b
+
0.340 0.401
a2 x l06 FITC-KLH specific secondary B cells and 4 × 105 BSA primed T cells were incubated with 5 #g/ml FITC-BSA in 1 ml final volume in the presence or absence of B-SN (12% v/v) for 7 days. bAnti-FITC-specific lgGl antibody levels were determined by indirect ELISA using FITC-BSA-coated plates. CAll negative controls (cells without antigen, or naive cells with antigen) gave OD492 less than 0.05. dOne representative experiment out of four.
0.4-
°F
0
02-
0.1.
0.0'
B
o2-
i
O.I-
IoM
IgG3
IgG 1
IgG2a
Fig. 4. FlTC-specific secondary antibody response in the presence or absence of the regulatory IgM. Mice were immunized with 50/~g of FITC-KLH and a secondary challenge added 8 weeks later. Splenic B cells were then isolated and cultured at 2 x 106 cells/well in 24-well plates in complete medium alone (lq), or with LPS (10/~g/ml) (11), or with LPS plus B-SN (12 v/v) (m) for 3 days in vitro. Culture supernatants were collected as described in Fig. 1. The level of specific lgM, lgG3, lgG1, and IgG2a antibodies were determined by isotype-specific indirect ELISA using (A) FITC-BSA- and (B) BSA-coated plates. One representative experiment out of five.
tic ELISA. Fig. 4 shows that on day 3, at the peak of the FITC-KLH-specific antibody response, no signifcant amounts of anti-BSA antibodies were detected (Fig. 4B). On the other hand, the marked anti-FITC response was biased towards IgGl antibodies. Smaller amounts of anti-FITC IgM, IgG2a, and IgG3 antibodies were also found in the culture supernatants at this time (Fig. 4A). B-SN, however, was only able to inhibit the IgM, and slightly enhance the IgG3 component, of the hapten-specific antibody production. To further evaluate this question, splenic B cells were isolated from FITC-KLH-immunized 240
mice and co-cultured in vitro with BSA-primed T lymphocytes. FITC-BSA was added as antigen with or without B-SN to the culture wells. After 7 days of incubation the FITC-specific IgG1 response was measured by ELISA (Table 2). Again, no inhibition of anti-FITC IgG1 response by B-SN was found. 5.
Discussion
The present results provide evidence for a novel regulatory pathway of humoral immune responses. We found that a natural IgM antibody secreted by LPS-stimulated murine splenic B cells is able to inhibit polyclonal, as well as hapten-specific, IgM antibody responses. In contrast, this regulatory IgM did not affect hapten-specific IgG antibody (mainly IgGl) production. This difference may indicate distinct signaling characteristics among B cells involved in primary versus secondary antibody responses. Alternatively, this might reflect heterogeneity within splenic B-cell populations. For example, it was suggested that HSA expression distinguishes lineages corresponding to primary versus memory B-cell progenitors, since the 3040% of MHC II- and B220-positive cells with the highest levels of HSA appear incapable of generating memory clones [5]. Regardless of its basis, the lack of IgM-mediated inhibition during anti-hapten IgGl response might show an important new feature of memory
B cells. However, because using LPS during the restimulation o f pre-committed B cells in vitro, there remains another explanation. O u r data can also be interpreted as if the regulatory IgM inhibits Bcell differentiation towards I g M secretion, regardless o f whether the cells are naive or m e m o r y (see the inhibition o f I g M secretion in Fig. 4). On the other hand, it m a y not affect differentiation towards IgG1 secretion, which does not exist in the first place a m o n g primary B cells. At this point, it must also be noted that the regulatory I g M allows the normal progression o f early (GO*, G1A), but not late (G1B, S, G2, and M) steps in the cycle o f polyclonally stimulated B lymphocytes [13]. Moreover, it does not affect the increased antigen-presenting capacity o f activated B cells [12 and Uher and Gergely, manuscript in preparation]. Thus, regulatory I g M has the unique feature o f permitting all early steps o f B-cell activation, including effective antigen presentation, whereas unnecessary polyclonal B-cell expansion and differentiation seem to be excluded by this antibody. Taken together these data might suggest a possible antibody-mediated p a t h w a y in the regulation o f h u m o r a l immune response against thymus-dependent antigens, which allows the production o f high- but not low-affinity I g G and often polyreactive I g M antibodies in the antigen-specific responses o f B lymphocytes. Extension o f these findings m a y have practical importance for therapies o f some h u m o r a l immunodeficiency and B cell malignancies.
References [1] [2] [3] [4] [5]
Eisen, H.N. and Siskind, G.W. (1964) Biochemistry 3,996. Mitchison, N.A. (1971) Eur. J. Immunol. 1, 18. Klinman, N.R. (1972) J. Exp. Med. 136, 241. Nossal, G.J.V. (1992) Cell 68, 1. Linton, P.-J. and Klinman, N.R. (1992) Sem. lmmunol. 4, 3. [6] Bruce, J., Symington, F.W., McKearn, T.J. and Sprent, J. (1981) J. Immunol. 127, 2496. [7] Bruce, J., Symington, F.W., McKearn, T.J. and Sprent, J. (1981) J. Immunol. 127, 2496. [8] Symington, F.W. and Hakomori, S.I. (1984) Mol. Immunol. 21,507. [9] Raychaudhuri, S. and Cancro, M.P. (1985) J. Exp. Med. 161,816. [10] Linton, P.-J., Decker, D.J. and Klinman, N.R. (1989) Cell 59, 1049. [11] Alonso, M.E., Uher, F. and Gergely, J. (1991) Int. Immunol. 3, 1283. [12] Uher, F., Alonso, E.M., Mihalik, R., Balogh, 1~. and Gergely, J. (1992) Immunobiology 185, 292. [13] Uher, F., Mihalik, R., Alonso, M.E. and Gergely, J. (1992) Immunol. Lett. 33, 255. [14] Uher, F., Rajnav61gi, 1~. and Erdei, A. (1992) Immunol. Today 13, A4. [15] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265. [16] B6yum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 31. [17] Uher, F., Lamers, M.C. and Dickler, H.B. (1985) Cell. lmmunol. 95, 368. [18] Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) Eur. J. Immunol. 3, 645.
Acknowledgements The authors wish to t h a n k Drs. A n n a Erdei and Zsuzsa Kert6sz for critical c o m m e n t s and helpful discussion. This work was supported by G r a n t T5323 from O T K A .
241
IMLET 02083
A natural IgM antibody does inhibit polyclonal and antigenspecific IgM but not IgG B-cell responses K a t a l i n Kiss, Ferenc Uher* a n d J~mos Gergely Department of Immunology, Lor6nd E6tv6s University, GOd, J6vorka S. u. 14, H-213l, Hungary (Received 26 July 1993; accepted 6 December 1993)
1.
Summary
Since a B-cell growth-inhibitory natural IgM antibody was identified in the culture supernatants of LPS-stimulated murine splenic B lymphocytes [11], attempts have been made to define other possible functional role(s) of this antibody. Here we show that this regulatory IgM is able to inhibit not only the proliferation of splenic B cells, but also their IgM secretion during LPS-induced polyclonal, as well as antigen (FITCKLH)-specific antibody responses. In contrast, IgG1 production of hapten (FITC)-specific B cells neither during restimulation with LPS nor in the presence of carrier-specific T lymphocytes in vitro was affected by regulatory IgM. Therefore, whereas newly emerging naive B cells are highly susceptible, IgG-secreting B cells appear to be completely resistant to inactivation by the regulatory IgM autoantibody. 2.
Introduction
Humoral immune responses generated upon antigen rechallenge differ significantly from the primary response observed after initial antigen exKey words: B cell; Natural IgM; Memory response; (Mouse) *Corresponding author: Ferenc Uher, Ph.D., Dept. of Immunology, Lor~md E6tv6s University, G6d, J~.vorka S. u. 14, H2131, Hungary. Abbreviations: B-SN, culture supernatants of murine splenic B cells stimulated with LPS for 96 h. SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) E 0 1 7 4 - B
posure in that memory responses display a more rapid appearance of dramatically increased levels of serum antibody comprised of higher affinity antibodies generally bearing isotypes other than IgM [l~t]. Several theories have been proposed to account for simultaneous generation of antibody-forming cell clones and secondary B cells during the course of primary immune response. The most widely accepted view is that primary B cells, upon stimulation, undergo unequal division giving rise to both antibody-forming cells and secondary B cells. Alternatively, a precursor cell subpopulation separate from that responsible for the generation of primary antibody-forming cells may give rise to secondary B cells [reviewed in 5]. It was found that the J l l D monoclonal antibody (mAb) binds well to most primary B cells and poorly to most secondary B cells [6]. Conversely, the heat-stable antigen recognized by mAb J11D is expressed at high levels on the surface of a majority of virgin B cells and at low levels on memory cells [7-9]. Furthermore, it was also suggested that the primary progenitors to secondary B cells are distinct from the primary antibody-forming cell precursors [10]. Our question in this field stems from previous studies in which we have demonstrated that a culture supernatant of LPS-stimulated murine splenic B lymphocytes (B-SN) is able to inhibit the growth of freshly isolated B cells via an IgM antibody [11]. The binding specificity of this IgM is not yet defined but appears to be a lymphocyte surface structure distinct from membrane immunoglobulin, MHC II antigen, B220, transferrin 235
and Fcz receptors [12]. The regulatory autoantibody allows the normal progression of early (GO to GO*, and GO* to G1A) but not late (G1B, S, G2 and M) steps in the cycle of polyclonally stimulated B lymphocytes and does not affect the increased antigen-presenting capacity of LPS-activated B cells [13,14]. On the basis of the above results the question emerged whether regulatory IgM is able to affect the differentiation of B cells during polyclonal and/or antigen-specific primary and secondary immune responses. We found that this IgM inhibits the polyclonal, as well as hapten-specific IgM, but not IgG (mainly IgG1) antibody production in response to LPS, or FITC-KLH, or FITC-BSA complexes, respectively. 3.
3.1.
Materials and Methods
Animals
DBA/2 mice, 2-3 months old, were obtained from the LATI (G6d6116, Hungary).
mM) of phosphate buffer. After centrifugation of the solution at 20,000 × g for 15 min, the plasma membrane-rich pellet was washed 3 times with phosphate buffer and finally resuspended in BSS. Protein was quantitated as described by Lowry et al. [15]. Mouse erythrocytes were treated with a saturated solution of bromelain in PBS for 30 min at 37°C then washed 3 times in PBS. Oxasolone was coupled to BSA. ExtrAvidin-peroxidase was purchased from Sigma. FITC was coupled to both KLH and BSA. 3.3.
Mice were immunized by i.p. injection of 50/~g of FITC-KLH in complete Freund's adjuvant. Eight weeks later the animals were boosted with 50 #g of antigen in incomplete Freund's adjuvant i.p. Another group of animals was immunized by injection of 100 /~g of BSA in complete Freund's adjuvant into hind footpads. 3.4.
3.2.
Cell preparation
Antibodies and reagents
LPS (Escherichia coli 055;B5) prepared by the Westphal technique, bromelain from pineapple stem, crystallized OVA (type XIII), and BSA were all obtained from Sigma (St. Louis, MO). KLH was purchased from Calbiochem (San Diego, CA). Human dsDNA was the kind gift of Katalin Takfics (OHVI, Budapest, Hungary). Mouse IgG was purified from pooled normal DBA/2 serum by ammonium sulfate precipitation and ionexchange chromatography on DE-52 (Whatman Chemical Separation, Clifton, N J). Normal IgM was prepared from the same serum sample by precipitation in low ionic strength buffer followed by gel filtration on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala, Sweden). Affinity purified biotin-conjugated goat anti-mouse IgM (/~-chain specific) was purchased from Sigma. Affinity-purified biotin-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 subclass-specific antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL). Erythrocyte membranes were prepared by lysis of 1 ml packed volume of red blood cells in 40 ml (50 236
Immunization
Single mononuclear cell suspension of spleen was prepared on Ficoll-Uromiro (Pharmacia, and Bracco, Milan, Italy) gradient by the method of B6yum [16]. Spleen cells were then depleted of T lymphocytes by antibody and complementmediated cytotoxicity as previously described [17]. Lymph node cells were prepared from the popliteal and inguinal nodes of primed animals 7 days after injection. T cell-enriched populations were obtained by the method of Julius et al. [18]. 3.5.
Culture conditions
The usual procedure consisted of 2-stage in vitro culture as previously described [11]. Briefly, B lymphocytes were cultured at 2 x 10 6 cells/ml density in tissue culture flasks with 20 #g/ml LPS at 37°C in 5% CO2-in-air incubator for 96 h in RPMI-1640 medium supplemented with 10% FCS (v/v), L-glutamine (2 mM), sodium pyruvate (1 raM), non-essential amino acids, 2-mercaptoethanol (5 × 10 5 M), Hepes buffer (20 mM), penicillin (100 IU/ml) and streptomycin (100 #g/ ml). The cells were then sedimented and the cul-
ture supernatants (B-SN) or their affinity-purified IgM fractions assayed for the inhibitor. In the second stage, various cell populations and reagents (see Results for details) were cultured in 1 ml final volume in 24-well fiat-bottomed plates (Costar 3524, Costar Data Packaging, Cambridge, MA) in RPMI-1640 medium supplemented as described above. Antibody production was measured using ELISA assays at the peaks of the various responses as determined in preliminary experiments.
3.6.
Measurement of polyclonal lg secretion
The total amounts of IgM and IgG antibodies in the culture supernatants were assessed by indirect competitive ELISA. Plates were coated with 100 pl of 10 pg/ml purified normal mouse IgM or IgG, respectively. Antigen-coated plates were saturated with 1% BSA in PBS and reacted both with 50 pl dilution of culture supernatants (1:5 to 1:625) and 50 #1 of biotin-coupled anti-mouse IgM or I g G for 1 h at 37°C. Then peroxidasecoupled ExtrAvidin was added and after 1 h incubation at 37°C enzymatic activities revealed by the addition of O P D in phosphate/citrate buffer containing 5-10% H202 (v/v). The reaction was read at 492 nm by a MR700 Microplate Reader (Dynatech Laboratories, Chantilly, VA). Positive and negative reference samples were used to draw standard curves. All tests were done in triplicate and results were calculated by interpolating from the standard curves.
3.7.
Measurement of self- and non-self specific IgM antibody production by indirect ELISA
Plates were coated with 50 #l/well (10 /~g/ml) solutions of self- (membrane fraction of BrMRBC, dsDNA, aggregated IgG) and non-self (BSA, K L H , membrane fraction of SRBC) antigens in PBS. Antigen-coated plates were saturated with 1% BSA in PBS and reacted with 50 /al dilution of culture supernatants (1:5 to 1:625) for 1 h at 37°C. Subsequently, they were incubated for 1 h at 37°C with biotin-labeled antimouse IgM. Then peroxidase-coupled ExtrAvidin was added, and after an additional 1 h incubation at 37°C, enzymatic activities were revealed as de-
scribed above (section 3.6).
3.8. Hapten-specific antibody responses Measurement of hapten (FITC)-specific primary (IgM), and secondary (IgG1, IgG2a, IgG2b, and IgG3) antibody levels in the culture supernatants were also performed by indirect ELISA. Plates were coated with F I T C - K L H , or FITC-BSA, or K L H , or BSA, respectively. Culture supernatants were diluted 1:5 to 1:625. Biotin-coupled anti-mouse lgM, I g G l , IgG2a, IgG2b, IgG3 and peroxidase-coupled ExtrAvidin were used as described in section 3.7. 4.
Results
4.1.
Effect of the regulator)' lgM on polyclonal immunoglobulin secretion in vitro
Results of experiments designed to assess the W.
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Fig. 1. Ability of B-SN to inhibit LPS-induced polyclonal lgM secretion in vitro. T-cell-depleted spleen cells were cultured at 2 × 1 0 6 cells/well in I ml of complete medium either alone (0- -O), or in presenceof I_PS(10 ,ug/ml) (m-m), or LPS plus B-SN (12% v/v) (0- -0), during the indicated periods. Culture supernatants were always replaced with fresh medium 24 h before determination of the IgM level by an isotype-specificcompetitive ELISA. One representativeexperiment out of five. ")2"7
TABLE 1 THE INHIBITION OF THE LPS-INDUCED POLYCLON A L lgM S E C R E T I O N IS M E D I A T E D BY AN lgM ANTIBODY ~ Reagents LPS (10 #g/ml) -
+ + +
+
+
Secreted IgM b (/~g/ml) B-SN (12% v/v) -
Untreated Adsorbed on anti-/~ antibody-coated Sepharose 4B e Adsorbed on and eluted from anti-p antibodycoated Sepharose 4B d Dialyzed for 48 h ~
B-cell cultures had a similar inhibitory effect on the polyclonal IgM secretion of the cells (Table 1). A significant amount of IgG was never detected in the culture supernatants o f LPS-stimulated B cells (data not shown).
Regulatory IgM inhibits both self- and nonself-specific antibody production in vitro
4.2.
4
55 5
44
Next we investigated the specificity of B cell-secreted IgM antibodies at different times after stimulation with LPS in the presence or absence of B-SN. We used a panel of self- (membrane frac-
11 5
aCulture conditions and ELISA assay were identical as described in Fig. 1. barter 4 days of culture. CSamples of B-SN were adsorbed on F(ab')2 goat anti-mouse #antibody coupled to Sepharose 4B. aRegulatory molecules were specifically eluted from the anti-# antibody-coated beads using high amounts (2 mg/ml in complete medium) of normal mouse lgM. eSamples of B-SN were dialyzed against complete medium for 48 h at 4°C. lOne representative experiment out of three.
=-
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~g 30
effect of B-SN on LPS-induced polyclonal antibody secretion in time are shown in Fig. 1. Splenic B cells were cultured in complete medium either alone or in the presence of LPS or LPS plus B-SN for 6 days. The amounts of secreted IgM in the culture supernatants were determined daily by ELISA. We found that the LPS-induced IgM secretion was greatest on the 4th day of culture (on an average 90/~g/ml). When LPS and B-SN were added together, a marked (96%) inhibition of polyclonal antibody production was observed. In contrast, no measurable inhibition was found on day 3, probably due to in vivo preactivated cells in the splenic B-cell preparations. Thus, B-SN is able to inhibit polydonal antibody (IgM) secretion by LPS-stimulated B lymphocytes. Furthermore, the addition of affinity-purified IgM fraction of B-SN or extensively dialyzed B-SN to LPS-stimulated 238
20
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/ Days in culture
Fig. 2. The regulatory IgM inhibits LPS-induced self-, as we]] as non-self-antigen specific IgM antibody production in vitro. T cell-depleted DBA/2 spleen cells were placed in culture at 2 x l06 cells/well in 24-well plates and stimulated with LPS (10 #g/ ml), or with LPS plus B-SN (12% v/v) during the indicated period. Culture supernatants were collected as described in Fig. 1. Antigen-specific lgM antibodies were detected by indirect ELISA (dilution of supernatants l:5) and the percentage of the B-SN-mediated inhibition calculated. Coats were aggregated IgG ( , - , ) , membrane fraction o f BrMRB C ( O - O ) , dsDNA ( i - i ) , BSA ( [ ] - [ ] ) , KLH ((3 ©), and membrane fraction of SRBC (@-@), respectively. One representative experiment out of three.
tions of BrMRBC, dsDNA, aggregated IgG) and non-self (membrane fractions of SRBC, BSA, KLH) antigens in an indirect ELISA (see Materials and Methods) to detect antigen-specific antibodies. As shown in Fig. 2, the percentage of BSN-mediated inhibition of antibody production was similar for self-, as well as for non-self-reactive IgM antibodies throughout the entire culture period tested.
4.3.
Effect of the regulatory IgM on antigenspecific primary and secondary antibody responses
Induction of antibody responses to most antigens (soluble proteins, hapten-carrier complexes) requires the participation of both helper T cells and antigen-presenting cells. Generally, primary antigen exposure results in an initial antibody response and the formation of memory cells [4]. Therefore, we compared the effect of the regulatory IgM on primary and on secondary antibody responses.
B-SN inhibits hapten-specific primat 3, antibody response To determine whether the regulatory IgM could influence the thymus-dependent primary antibody response, mice were injected with FITC-KLH i.p. and 1 week later with splenic B cells prepared from immunized animals. Cells were then cultured either in complete medium alone, or restimulated with LPS, or with LPS plus B-SN in vitro. Culture supernatants were always replaced with fresh medium 24 h before ELISA performed on FITC-BSA- and BSAcoated plates. Maximal hapten (FITC)-specific IgM secretion was observed after 3 days of culture in the presence of LPS. As shown in Fig. 3, B-SN was able to inhibit the hapten-specific lgM response at this time. Control experiments were accomplished with B cells isolated from unimmunized animals. However, no significant FITC-specific antibody production was found in the control cultures (data not shown).
4.3.2.
B-SN does not inhibit hapten-specific secondary antibody response A basic feature of thymus-dependent antibody responses is the generation of memory: on second contact with an antigen a secondary response is produced in which somatically mutated IgG antibodies with increased affinity are synthesized [14]. In order to examine the effects of the regulatory IgM for hapten-specific IgG secretion, mice were primed and boosted with FITC-KLH i.p. One week after the secondary antigen challenge splenic B cells were isolated and restimulated in vitro with LPS in the presence or absence of BSN. To assess the amount of hapten (FITC)-specific antibodies, secreted immunoglobulins reactive with FITC-BSA and/or BSA were measured in the culture supernatants daily by isotype-speciB 0.7.
G
0.7
(I.6
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0.4 O4
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Fig. 3. FITC-specific primary antibody response in the presence or absence of the regulatory IgM. Splenic B cells from mice immunized with 50/~g FITC-KLH i.p. were cultured at 2 x 106 cells/well in complete medium alone (l~), or with LPS (10 ~g/ml) (m), or with LPS plus B-SN (12% v/v) (m) for 3 days in vitro. Culture supernatants were collected as described in Fig. 1. The level of specific IgM antibodies were determined by indirect ELISA using (A) FITC-BSA- and (B) BSA-coated plates. One representative experiment out of three.
239
A
TABLE 2
IJ
IN VITRO HAPTEN (FITC)-SPECIFIC B CELL M E M O R Y RESPONSE a B-SN
Anti-FITC IgG1 response (OD492) b
+
0.340 0.401
a2 x l06 FITC-KLH specific secondary B cells and 4 × 105 BSA primed T cells were incubated with 5 #g/ml FITC-BSA in 1 ml final volume in the presence or absence of B-SN (12% v/v) for 7 days. bAnti-FITC-specific lgGl antibody levels were determined by indirect ELISA using FITC-BSA-coated plates. CAll negative controls (cells without antigen, or naive cells with antigen) gave OD492 less than 0.05. dOne representative experiment out of four.
0.4-
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0
02-
0.1.
0.0'
B
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i
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IoM
IgG3
IgG 1
IgG2a
Fig. 4. FlTC-specific secondary antibody response in the presence or absence of the regulatory IgM. Mice were immunized with 50/~g of FITC-KLH and a secondary challenge added 8 weeks later. Splenic B cells were then isolated and cultured at 2 x 106 cells/well in 24-well plates in complete medium alone (lq), or with LPS (10/~g/ml) (11), or with LPS plus B-SN (12 v/v) (m) for 3 days in vitro. Culture supernatants were collected as described in Fig. 1. The level of specific lgM, lgG3, lgG1, and IgG2a antibodies were determined by isotype-specific indirect ELISA using (A) FITC-BSA- and (B) BSA-coated plates. One representative experiment out of five.
tic ELISA. Fig. 4 shows that on day 3, at the peak of the FITC-KLH-specific antibody response, no signifcant amounts of anti-BSA antibodies were detected (Fig. 4B). On the other hand, the marked anti-FITC response was biased towards IgGl antibodies. Smaller amounts of anti-FITC IgM, IgG2a, and IgG3 antibodies were also found in the culture supernatants at this time (Fig. 4A). B-SN, however, was only able to inhibit the IgM, and slightly enhance the IgG3 component, of the hapten-specific antibody production. To further evaluate this question, splenic B cells were isolated from FITC-KLH-immunized 240
mice and co-cultured in vitro with BSA-primed T lymphocytes. FITC-BSA was added as antigen with or without B-SN to the culture wells. After 7 days of incubation the FITC-specific IgG1 response was measured by ELISA (Table 2). Again, no inhibition of anti-FITC IgG1 response by B-SN was found. 5.
Discussion
The present results provide evidence for a novel regulatory pathway of humoral immune responses. We found that a natural IgM antibody secreted by LPS-stimulated murine splenic B cells is able to inhibit polyclonal, as well as hapten-specific, IgM antibody responses. In contrast, this regulatory IgM did not affect hapten-specific IgG antibody (mainly IgGl) production. This difference may indicate distinct signaling characteristics among B cells involved in primary versus secondary antibody responses. Alternatively, this might reflect heterogeneity within splenic B-cell populations. For example, it was suggested that HSA expression distinguishes lineages corresponding to primary versus memory B-cell progenitors, since the 3040% of MHC II- and B220-positive cells with the highest levels of HSA appear incapable of generating memory clones [5]. Regardless of its basis, the lack of IgM-mediated inhibition during anti-hapten IgGl response might show an important new feature of memory
B cells. However, because using LPS during the restimulation o f pre-committed B cells in vitro, there remains another explanation. O u r data can also be interpreted as if the regulatory IgM inhibits Bcell differentiation towards I g M secretion, regardless o f whether the cells are naive or m e m o r y (see the inhibition o f I g M secretion in Fig. 4). On the other hand, it m a y not affect differentiation towards IgG1 secretion, which does not exist in the first place a m o n g primary B cells. At this point, it must also be noted that the regulatory I g M allows the normal progression o f early (GO*, G1A), but not late (G1B, S, G2, and M) steps in the cycle o f polyclonally stimulated B lymphocytes [13]. Moreover, it does not affect the increased antigen-presenting capacity o f activated B cells [12 and Uher and Gergely, manuscript in preparation]. Thus, regulatory I g M has the unique feature o f permitting all early steps o f B-cell activation, including effective antigen presentation, whereas unnecessary polyclonal B-cell expansion and differentiation seem to be excluded by this antibody. Taken together these data might suggest a possible antibody-mediated p a t h w a y in the regulation o f h u m o r a l immune response against thymus-dependent antigens, which allows the production o f high- but not low-affinity I g G and often polyreactive I g M antibodies in the antigen-specific responses o f B lymphocytes. Extension o f these findings m a y have practical importance for therapies o f some h u m o r a l immunodeficiency and B cell malignancies.
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Eisen, H.N. and Siskind, G.W. (1964) Biochemistry 3,996. Mitchison, N.A. (1971) Eur. J. Immunol. 1, 18. Klinman, N.R. (1972) J. Exp. Med. 136, 241. Nossal, G.J.V. (1992) Cell 68, 1. Linton, P.-J. and Klinman, N.R. (1992) Sem. lmmunol. 4, 3. [6] Bruce, J., Symington, F.W., McKearn, T.J. and Sprent, J. (1981) J. Immunol. 127, 2496. [7] Bruce, J., Symington, F.W., McKearn, T.J. and Sprent, J. (1981) J. Immunol. 127, 2496. [8] Symington, F.W. and Hakomori, S.I. (1984) Mol. Immunol. 21,507. [9] Raychaudhuri, S. and Cancro, M.P. (1985) J. Exp. Med. 161,816. [10] Linton, P.-J., Decker, D.J. and Klinman, N.R. (1989) Cell 59, 1049. [11] Alonso, M.E., Uher, F. and Gergely, J. (1991) Int. Immunol. 3, 1283. [12] Uher, F., Alonso, E.M., Mihalik, R., Balogh, 1~. and Gergely, J. (1992) Immunobiology 185, 292. [13] Uher, F., Mihalik, R., Alonso, M.E. and Gergely, J. (1992) Immunol. Lett. 33, 255. [14] Uher, F., Rajnav61gi, 1~. and Erdei, A. (1992) Immunol. Today 13, A4. [15] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265. [16] B6yum, A. (1968) Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 31. [17] Uher, F., Lamers, M.C. and Dickler, H.B. (1985) Cell. lmmunol. 95, 368. [18] Julius, M.H., Simpson, E. and Herzenberg, L.A. (1973) Eur. J. Immunol. 3, 645.
Acknowledgements The authors wish to t h a n k Drs. A n n a Erdei and Zsuzsa Kert6sz for critical c o m m e n t s and helpful discussion. This work was supported by G r a n t T5323 from O T K A .
241