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Anti-lgM antibodies down modulate mu-enhancer activity and OTF2 levels in LPS-stimulated mouse splenic B-cells Una Chen*, Richard H.Scheuermann, Thomas Wirth+, Thomas Gerster1, Robert G.Roeder1, Keith Harshman2 and Christoph Berger§ Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland, 'The Rockefeller University, Laboratory of Biochemistry and Molecular Biology, 1230 York Avenue, New York, NY 10021-6399, USA and 2Institute for Molecularbiology 11, University of Zurich, Honggerberg, CH-8093 Zurich, Switzerland Received June 26, 1991; Revised and Accepted September 25, 1991

ABSTRACT Stimulation of small, resting, splenic B cells with bacterial lipopolysaccharide (LPS) induces proliferation, differentiation to plasma cell formation, and the expression of immunoglobulin heavy chain (IgH). When this is combined with agents which crosslink surface Ig, differentiation and the induction of surface immunoglobulin are suppressed even though proliferation proceeds. We find that anti-mu antibodies suppresses Ig gene expression of transfected mu constructs, even if either the membrane or secretory segments have been deleted. We examined the effects of anti-mu treatment on the IgH enhancer (IgHE) attached to a heterologous test gene (CAT). Indeed the IgH enhancer alone was subject to anti-mu suppression, while the SV40 enhancer was insensitive. To determine what was responsible for suppression of enhancer function by anti-mu we examined nuclear extracts from stimulated splenic B cells for the presence of sequence-specific DNA binding activities to various sites within the enhancer. We found two specific differences-an induction in 1,E5 binding activity, and a reduction in octamer transcription factor 2 (OTF2) binding activity, after anti-mu treatment. Analysis of these cells by in situ immunofluorescence with anti-OTF2 antibodies suggests that the nuclear localization of OTF2 in antimu treated cells may change, as well as its absolute level. INTRODUCTION The specific role of antigen in the activation of B-cells has been studied through crosslinking the antigen receptor (immunoglobulin, Ig) on the surface of these cells with anti-Ig (usually anti-mu) antibodies. The interaction of anti-mu with the receptors induces resting B-cells to proliferate, but they do not differentiate

into Ig secreting plasma cells. The differentiation event requires additional help from T-cells (see review by Chen 1990b). Thus, anti-mu is thought to partially mimic the activation role of Tcell dependent antigens. However, resting B-cells can also be polyclonally induced to proliferate and to differentiate via a nonantigen-specific pathway with bacterial lipopolysaccharide (LPS). When the two activation treatments are combined-adding antimu to LPS-activated B-cells-the proliferation pathway is augmented (Chen-Bettecken, et al., 1985; Seyschab, et al., 1989), while the LPS-mediated differentiation pathway normally seen with LPS treatment is overridden (Andersson et al., 1974; Kearney et al., 1978; Webb et al., 1983). The percentage of cells activated by these reagents are-anti-mu, 30-40%; LPS, 20-30%; LPS+anti-mu, 80-90% (Seyschab, et al., 1989; Chen, 1990b). Marcuzzi et al. (1989) has described an inhibition of B-cell proliferation and a down regulation of Ig transcription when intact anti-IgM was included with LPS or LPS and dextransulfate. However, this effect is mainly due to the influence of binding to Fc receptors on the surface of these B-cells. The anti-differentiative effect of F(ab')2 form of anti-IgM can be measured as a specific inhibition in the accumulation of the secretory form of mu-mRNA in an early phase of culture, as well as a specific down modulation of overall Ig-mRNA synthesis in a later phase (Chen-Bettecken et al., 1985; Leanderson and Hsu, 1985; Chen, 1988; D. Yuan, 1987 and personal communication). Vigorous Ig-transcription and the accumulation of the secretory form of mu-mRNA are considered to be the hallmarks of differentiation into the plasma cell state (Koshland, 1983; Yuan and Tucker, 1984; Chen-Bettecken et al., 1987; Chen, 1990a). Study of the molecular mechanisms that underlie the specific blockage of these events by anti-mu treatment should help to unravel the molecular processes that mediate B-cell differentiation. Here we report the study of Ig heavy chain expression and enhancer function in this LPS + anti-mu treated B-cell system and

To whom correspondence should be addressed Present addresses: + Heidelberg Center for Molecular Biology (ZMBH), Im Neuenheimerfeld 282, D-6900 Heidelberg, FRG and § Department of Pediatrics, University of California, HSW Lab 1421, San Francisco, CA 94143, USA *

5982 Nucleic Acids Research, Vol. 19, No. 21 show that on every doubly treated B-blast there is a downregulation of endogenous mu-mRNA, and that the inhibitory effect can also be demonstrated with transiently transfected mu heavy chain genes (either wild type or 3'-end variants). The fact that the heavy chain intron enhancer alone is subject to anti-mu mediated suppression, at the late stages of culture, provides evidences that the inhibitory effect is exerted at the transcriptional level. The downregulation of the OTF2 DNA-binding protein, which interacts with the enhancer, may provide the predominant molecular mechanism for enhancer inhibition. These activated, non-Ig secreting B-blasts may represent the precursors of memory B-cells.

MATERIALS AND METHODS Cell culture, mitogen, antibodies and in situ hybridization The tissue culture conditions have been described (ChenBettecken et al., 1985). Resting B-cells (ca= 1.085) are isolated by treating C57BI/6 splenic cells with a cocktail of two monoclonal anti-Thy 1.2 antibodies [HO 13.4-9 (MarshakRothstein et al., 1979), and T-24 (Dennert et al., 1980) which were kindly provided by U. Staerz and H. von Boehmer, Basel Institute for Immunology, Basel, Switzerland], and rabbit complement of low toxicity (Cederlane, Hornby, Ontario) before isolation based on cell density (Chen-Bettecken et al., 1985). Cells with a density c 1.085 were separated on a Percoll step-gradient, washed, and adjusted to 2 x 106 cells per ml in RPMI 1640 medium containing 5 % fetal calf serum and supplements. Bacterial LPS from Salmonella typhimurium (Sigma) is used at a final concentration of 20 ,ug/ml. Anti-IgM antibodies (designated anti-mu), used at a 10 ag/ml final concentration, are either goat anti-mouse mu F(ab')2 (Jackson Labs) or rabbit antimouse Ig F(ab')2 prepared according to Nisonoff et al. (1980) and adsorbed with protein A Sepharose (Pharmacia). The in situ hybridization of activated B-cells with a mu-constant region specific probe is according to Berger (1986). Primary Bcells cultured with LPS alone or LPS + anti-mu for 3-4 days were harvested. Ten thousand cells were seeded onto a glass slide, fixed, treated with pronase, prehybridized and hybridized with 10 ,1l of M-13 single-strand 35S-labeled 586 base negative strand mu-constant region (Cv) DNA probe for 3 days at 35°C. The slides were washed, exposed for 2 weeks and developed as described. Immunotyping: Polyclonal rabbit antibodies against specific Octamer binding proteins were used in this study (T. Gerster, unpublished). Anti-OTF- 1 was described by Pruijn et al., (1989) and was kindly provided by Alessandra Pierani (Rockefeller University, New York, USA). Anti-OTF-2 (serum # 2432) and anti-POU domain (serum #2435) were raised against fusion proteins. Serum #2432 was raised against a fusion protein containing the N-terminal of OTF-2. It is designated as 02 in this study. Serum #2435 was raised against a fusion protein containing the POU-specific region of OTF-2. It is designated as 03 in this study. They were used to stain the nuclei of either resting B-cells or primed B-cells after LPS and LPS + anti-mu activation For immunotyping, 104 nuclei from resting B cells or LPS and LPS + anti-mu treated B-cells from day 1 to day 4 of activation were cytocentrifuged onto microscopic slides, acetone fixed and stained first with specific rabbit antibodies and then FITC-labelled mouse anti-rabbit IgG antibody as the second

antibody. The nuclei were washed for 30 min. with PBS after each antibody staining and mounted. The intensity of immunofluorescence was examined and quantified using UV illumination on a Zeiss Axciovent 35M microscope and the Confocal Imaging System MRC-500 (Bio-Rad, Glattbrug, Switzerland).

Transfection experiment: Transient expression of transcriptional activities was measured using cell transfection techniques. Our transfection experiments were performed using DEAE-Dextran in a method modified from that of Banerji, et al. (1983) involving a DMSO shock (Picard and Schaffner, 1985). B-cells cultured with LPS or LPS + anti-mu for two days were harvested, washed once with TBS and 5 x 107 cells were transfected with 40 ,tg of plasmids. After allowing 15 minutes for transfection at room temperature, the cells were subjected to a treatment of 20% dimethylsulphoxide in an ice cold Tris-buffered saline solution for exactly 4 minutes, then harvested, washed and resuspended in medium with stimuli. Two days after transfection the cells were harvested, and two assays were performed. a) CAT assay: The plasmids used for transfection were recombinant DNAs containing either the SV40 enhancer or the 1.0kb immunoglobulin heavy-chain enhancer linked to the CAT gene, and CAT assays were performed as described (Kohrer et al., 1985, Scheuermann and Chen, 1989), and quantified by scanning laser densitometry. b) SI mwpping of the 5 '-end initiation sites of genomic Ig genes after transient expression: Three Ig genomic mu genes with 3'-end variations were used for transfection experiments and S1 mapping in order to study their effects on the 5'-end initiation sites. The clones constructed are (1) Plasmid Py contains the entire genomic Ig heavy chain gene as well as the histone H4 gene and polyoma early region and origin of replication (Grosschedl and Baltimore, 1985). (2) Plasmid Pym contains a complete mu heavy chain with only the membrane exons; the end of C,u4 and the intron were removed. Pjm was constructed by replacing the genomic sequences from a BstE 11 site in C,t4 to an XbaI site in M2 with the respective mu-membrane cDNA sequences. Otherwise the clone is identical to Py. (3) Plasmid Pys is similar to Pltm but lacks the membrane exons. PAs was constructed by converting an EcoRV site in the mu-secretory to mu-membrane intron (next to the KpnI site mentioned above) into an XhoI site and removing the sequence between this site and the XhoI site at the end of the mu-insert. The transfection protocol is as described above. Two days after transfection cells were harvested and total cellular RNAs were isolated and S1 nuclease mapping was performed as described (Grosschedl and Baltimore, 1985; Weaver et al., 1985), and quantified by scanning laser densitometry. Gel retardation experiment Gel retardation assays were performed essentially as described (Scheuermann and Chen, 1989; Scheuermann, 1990; Gamer and Revzin, 1981; Fried and Crothers, 1981). Briefly, nuclear extracts were added to a reaction mix (20i1) containing 10% glycerol, 20mM Tris-HCL pH 8.0, 10mM NaCl, 0.5mM EDTA, 5mM MgCl2, 1mM ZnCl2, 5/g BSA, Iltg poly dI-dC and -5 fmoles 32P-labelled test DNA. Following a 15 minute incubation at room temperature the samples are loaded directly onto a 4 % polyacrylamide gel in Tris-borate-EDTA Buffer and electrophoresed 1.5 hours at 10 volts/cm. The gels are then fixed in 10% methanol, 10% acetic acid, dried and exposed to autoradiography.

Nucleic Acids Research, Vol. 19, No. 21 5983 Nuclear extracts are the soluble fraction of 0.3M extracts of isolated nuclei. Nuclei are separated from cytoplasmic and membrane fraction by centrifugation through a sucrose cushion following NP-40 mediated cell lysis (Schibler et al., 1983). Test DNA's were isolated by electroelution from polyacrylamide gels following PCR amplification using primers homologous to plasmid polylinker sequences, and were labelled with 'y32P-ATP and T4 polynucleotide kinase. Test DNA's correspond to sequences numbered from the JH proximal XbaI site of the enhancer as follows: ME1, 344-364; pE2, 379-392; ME3, 396-412; gE4, 528-541; AE5, 371-380; MB, 412 -427; OCTA, 540-549, and NF-1sNR binding site, 773-827. Nuclear extracts from several cell lines are also used in this study as controls. These extraction methods are similar to those described above (for J558L and BW5147 lymphomas see Scheuermann and Chen, 1989; and for Hela cells and BJAB lymphomas see Gerster and Roeder, 1988).

RESULTS Anti-mu suppression affects both endogenous and transfected IgM genes Cellular analysis from several investigators (Anderson et al., 1974; Kearney et al., 1978; Webb et al., 1983) indicates that anti-mu antibody treatment inhibits plasma cell development normaly occuring after LPS treatment of resting, splenic B-cells. Molecular analysis indicated that one manifestation of anti-mu treatment was the downregulation of Ig heavy chain expression at both the transcriptional and post-transcriptional levels (ChenBettecken et al., 1985; Leanderson and Hsu, 1985; Chen, 1988; Hbgbom et al., 1987; D. Yuan, 1987 and personal communication). In all of these studies, the effect of anti-mu treatment on LPS activated splenic B-cells had been analyzed in bulk cell populations. Those studies could not distinguish between a downregulation in all cells or only in a proportion of the total population. Using an in situ hybridization technique, we find the inhibition of mu-mRNA expression by anti-mu in LPS activated B-cells at the single cell level (Fig. 1). We found that unstimulated resting B-cells did not have any detectable mu-mRNA (data not A

shown) and that 20-35% of splenic B-cells stimulated with LPS for 3-4 days expressed high levels of mu-mRNA (Fig. lA). These results strongly suggest a transcriptional activation of mumRNA in splenic B-cells upon LPS activation and are consistent with previous data (Berger, 1986). In addition, we found that none of the cells (>50,000 cells examined) stimulated with LPS + anti-mu at various stages of culture showed detectable amounts of mu-mRNA (Fig. 1B). We conclude that anti-mu inhibits mu-mRNA expression in all LPS activated B-blast cells. In nuclear transcriptional run-on experiments, it was previously shown that anti-mu does not affect the transcriptional rate of LPS activated B-cells during the first two days of culture but causes a significant reduction from day 3 to day 4 (Chen-Bettecken et al, 1985; Leanderson and Hsu, 1985). We were now interested in determining whether this decrease in transcription rate could also be obtained with transiently transfected genes in order to delineate the elements responsible for this downregulation. The constructs used were (1) clone Pg, which contains a rearranged NP-specific (VH 17.2.25) mu-gene in the genomic configuration (Grosschedl and Baltimore, 1985), (2) clone PMum, a derivative of PM containing only the membrane exons, and (3) clone PMs, a derivative of PM lacking the membrane exons (clone constructions are shown in the Materials and Methods section). All constructs also contain a modified mouse histone H4 gene, which allows us to control for variations in transfection efficiency (Grosschedl and Baltimore, 1985). Splenic B-cells were cultured for two days with either LPS alone or LPS+anti-mu. Both sets of activated B-cells were transfected with 40 Mg of each of these 3 plasmid constructs, restimulated with the corresponding stimulus in culture, and harvested two days later. RNA was extracted and subjected to L LU L LU - - UUmUs UUmUs _-

-Probe

4-

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I

L

S

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1 2 3 4

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Fig. 1. Detection of CA-specific mRNA molecules in B-cells stimulated with LPS or with LPS+anti-mu. Small, resting, splenic B-cells were treated with LPS (L, panel A) or LPS+anti-mu (Lg, panel B) and examined for CAs expression by in situ hybridization. Radioactive exposure of silver grains are seen as black circles, and are a measure of transcript quantitation. Bars = 1O0M.

5 6 7 8 _ _ -

H4

Fig. 2. S1-nuclease analysis of RNA from B-cells transfected with rearranged heavy chain constructs. B-cells were stimulated with either LPS alone (designated L, lanes 1, 3-5) or LPS+anti-mu (designated LU, lanes 2, 6-8) and transfected with plasmids Pt (U, lanes 3&6), P;&m (Um, lanes 4&7), or Pps (Us, lanes 5&8). Correctly initiated mu-transcripts resulted in the protection of a 57 base DNA fragment. Histone H4 transcripts protect a 99 base fragment serving as an internal control.

5984 Nucleic Acids Research, Vol. 19, No. 21 quantitative SI nuclease analysis. There was a 10 to 20 fold reduction of NP-specific mu-transcripts in the RNA prepared from cells cultured in LPS+anti-mu as compared to the RNA prepared from cells cultured with LPS alone (Fig. 2 lanes 3, 4, 5 vs. lanes 6, 7, 8). All three transfected Ig genes gave correct initiation, as shown by nuclease SI mapping. The reduction is specific for the mu-transcripts, because the mouse histone H4 gene was expressed about equally in both cultures (Fig. 2, lanes 3 to 8). Interestingly the Ig 3'-end variations, whether the transfected genes contain either the whole region (Fig. 2, lane 3 vs lane 6), mu-membrane alone (Fig. 2, lane 4 vs lane 7), or mu-secretory alone (Fig. 2, lane 5 vs lane 8), did not affect the level of Ig RNA expression in LPS or LPS+anti-mu treated splenic B-cells at this late stage of culturing. We conclude that the variations at the 3'end of the mu-genes do not affect the differential stability of expressed mu-mRNA, and that the there are no target sequences for suppression located within the deleted fragments. This conclusion is also supported by mu-RNA metabolism studies of these B-cells (Chen, 1988).

IgH enhancer is also subject to anti-mu suppression In order to determine whether this reduction could be attributed to the heavy chain enhancer, we used constructs in which the heavy chain enhancer drives the bacterial chloramphenicol acetyltransferase (CAT) gene as an indicator gene (Mosthaf et al., 1985; Scheuermann and Chen, 1989). Primary B-cells were cultured with either LPS or LPS + anti-mu for two days and then transfected with the IghE-CAT construct and restimulated with either LPS alone or LPS + anti-mu for two more days. The CAT activity measured in cells treated with LPS + anti-mu was about 4 to 8 fold lower than in cells treated with LPS alone (Fig. 3, lanes 1 and 2). When a control plasmid in which the CAT indicator gene was under SV40 enhancer regulation was used, no significant difference between the two treatments could be observed (lanes 3 and 4). This rules out any non-specific effects of anti-mu treatment on CAT expression, transfectability, or general transcriptional machinery. Therefore, we conclude that one response to the anti-mu treatment is a specific downmodulation of the mu-enhancer.

Anti-mu suppression down regulates OTF2 levels Recent gel retardation experiments with DNA fragments from the Ig promoter region (Singh et al., 1986), the Ig heavy chain enhancer core region (Gerster et al., 1987; Sen and Baltimore, 1986; Peterson and Calame, 1986 and 1987; Nelson, et al., 1990; Libermann et al., 1990) and regions flanking the enhancer (Scheuermann and Chen, 1989) have led to the identification of several nuclear factors which bind to specific DNA sequences in these regions. We used gel retardation assays to determine whether the observed transcriptional inhibition was due to qualitative or quantitative changes in the presence of such enhancer-binding factors. The level of various IgH enhancer binding proteins was measured by gel retardation assays using oligonucleotides subtrates corresponding to DNA binding sites previously identified (EE1, IE2, ItE3, IE4, /tE5, 1B, octamer, and NF-,iNR binding sites). The binding activities of nuclear extracts isolated from stimulated and unstimulated splenic B cells, as well as two lymphoid cell lines (J558L and BW5147), were measured. Treatment of resting B cells with LPS results in the induction of DNA binding activities to all of the motifs tested (data not shown). Presumably the induction of some of these DNA binding activities is responsible for the induction of heavy chain expression following LPS treatment. Since the addition of anti-mu antibodies to B cells during LPS induction suppressed both heavy chain expression and enhancer activity, the level of the LPS-induced DNA binding activities was compared with or without anti-mu treatment (Figure 4, compare lanes 4 and 5 in each panel). In most cases, little or no change

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Fig. 3. Transcriptional enhancer activity as determined by CAT assays. B-cells activated for two days with LPS (L) or LPS+anti-mu (LU) were transfected with an Ig heavy chain enhancer-CAT plasmid (IghE-CAT) (lanes 1 & 2) or an SV40 enhancer-CAT (pSV-CAT) (lanes 3 & 4) and recultured with the corresponding stimulus for two more days. CAT enzyme activity was determined as described (Kohrer et al., 1985; Scheuermann and Chen, 1989). The intensities of the spots marked 3-AcCAM and 1-AcCAM following thin layer chromatography measure the CAT enzyme activity and are an indication of enhancer strength.

Fig. 4. Gel retardation assay measuring nuclear factor binding to various Igh enhancer-specific sequences. Nuclear extracts used in standard gel retardation assays were from the BW5147 T-cell line (first lane in each panel), the J558L B-lineage cell line (second lane in each panel),small splenic B-cells stimulated with LPS (fourth lanes) or with LPS+anti-mu (fifth lanes). All extracts were made by treating isolated nuclei with 0.3M NaCl except in mixtures marked '6' (lanes 6 & 8) which were extracted at 0.6M NaCl. In mixtures marked '0' (third lanes) DNA probes were incubated without nuclear extracts. The DNA fragment used in each gel retardation assay is indicated above each panel. Bands 1 and 2 in panel OCTA represent binding of OTFI and OTF2 respectively, based on their characteristic migration and cell-type distibution (see Kemler & Schaffner, 1990).

Nucleic Acids Research, Vol. 19, No. 21 5985

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5986 Nucleic Acids Research, Vol. 19, No. 21

CA

Nucleic Acids Research, Vol. 19, No. 21 5987 Table 1

Percentage of fluorescence intensity of OTF-2 positive nucleus of activated B-cells U'S (L) treatment

color Nucleus No. Channel No. 1 2 3 4 5 6 7 8 9

mean+S.D. a

LPS+anti mu treatment

Green 65-110

Blue 110-175

Red 175-255

34.8a 25.6 21.6 37.5 25.4 45.0 23.9 16.5 12.0

30.4 26.9 30.9 34.1 35.2 31.2 36.8 30.2 35.2

20.6 33.4 24.5 16.8 24.6 13.4 29.8 40.4 34.9

26.9+10.4 31.7+3.0

26.4+8.8

color

Nucleus No. Channel No. 1' 2' 3' 4'

5' 6' 7'

Green

Blue

Red

65-110 68.7 52.9 65.3 68.0 77.1

110-175 2.2 14.1 8.4 5.6

175-255 0.0

48.2 50.8 46.6 46.4

8' 9'

0.2 1.8 12.6

1.5 19.8

mean+S.D. 58.2+11.5 7.3+6.8

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

percent of pixel with fluorescent intensity falling within the indicated channel number range.

was seen in the level of DNA binding activity with the addition of anti-mu (Fig. 4 panel El; binding to sites E2, E3, E4, AB, and NF-ItNR binding sites, data not shown). In another case, treatment with anti-mu resulted in the induction of a DNA binding activity (Fig. 4 predominant band in panel E5). On the other hand, a substantial reduction was observed in the level of OTF2 (compare band 2 in lane 4 with lane 5 in OCTA panel). Densitometric quantitation of the OTF2 retardation bands from several experiments using extracts from two separate stimulations revealed a consistant 50% difference in binding activity. Considering the fact that more cells are stimulated with LPS + anti-mu than with LPS alone (see below), the difference in binding activity on a per cell basis would be even greater. Bearing in mind that OTF2 plays a central role in heavy chain transcription, this reduction may indeed be responsible for the turn-off of heavy chain expression resulting from anti-mu treatment. In order to further quantify the amount of the DNA-binding proteins interacting with the enhancer region, we used antibodies specific for the Octamer binding proteins, i.e. OTF-1, OTF-2 and POU domains. The antibodies were raised by immunizing rabbits with recombinant fusion octamer binding proteins, and

were tested for their specificities by inhibition of the binding capacity of octamer-binding proteins to the octamer motif as shown in a gel retardation assay (Fig. SA). These antibodies were used to quantify the amount of octamer binding proteins existing in B-cell nuclei by applying immunofluorescent staining technique and visualized by using a UV-microscope (Fig. SB) and by confocal microscopic analysis (Fig SC and Table 1). We stimulated resting B-cells with LPS or LPS+anti-mu, harvested cells every day from day 1-4, and stained nuclei with antibodies against octamer binding proteins. We could not observe any significant fluorescent staining when the nuclei were obtained from resting B-cells, day 1 or day 2 activated B-cells (data not shown). Only the nuclei isolated from day 3-4 showed significant immunofluorescence with these antibodies. As shown in Fig. SB, after 3-4 days of activation, 20-30% of nuclei from LPS stimulated B-cells were positive with OTF2 antibodies, while 80-90% of nuclei stimulated with LPS +anti-mu were positively stained. These data are consistent with the level of activation seen from cell cycle analysis (Seyschab et al., 1989; Chen, 1990b). There were no differences in the fluorescence intensities within individual nuclei of either ubiquitous OTF-1 or POU domain

Fig. 5. Quantitation of OTF protein concentration at the single cell level using immunofluorescence. (A) Antibody specificity was assayed by a change in the mobility shift of the octamer binding protein in nuclear extracts from HeLa and BJAB. The DNA probe used contains the H2B-octamer as described in Gerster and Roeder (1988). The binding conditions were essentially identical to those in Fig. 4, except that the reaction mixes were preincubated at 30°C for 30 minutes before the probe was added. Samples contained nuclear extracts isolated from HeLa (lanes 1-4) or BJAB (lanes 5-8) and preimmune serum (designated N, lanes 1,3,5,7) or immune serum (designated 0) against a fusion protein containing the N-terminus of OTF-2 (lanes 2 & 6), or against a fusion protein containing the POU-specific region of the OTF's (lanes 4 & 8). N.S. is a non-specific band appearing with all immune precipitations. (B) Immunofluorescent intensity on LPS or LPS+anti-mu treated single B-cells as judged by immunotyping. Cells stimulated with LPS (L, panels a-d) or LPS +anti-mu (Lu, panels e-h) were incubated with OTF2-specific immune serum (panels a & e), OTFI-specific serum (panels b & f), POU-specific serum (panels c & g), or preimune serum (panels d & h). Magnification is IOOX. (C) Quantitation of flourescent signal of OTF2 staining by confocal image processing. Nuclei from cells treated with LPS (panel A) or LPS+anti-mu (panel B) were stained as described. Magnification is IOOX. Fluorescence of individual nuclei (indicated with triangles) was quantified for LPS (panels A-1 through A-3) or LPS+anti-mu (panels B-l through B-3) treatment using image processing. Nuclei which stained above background were divided into three groups-low level staining intensity (70-105 units) is depicted as green, intermediate staining intensity (105-170 units) is depicted as blue, and high level staining intensity (170-250 units) is depicted as red. The percentage of nuclei in each staining group is indicated in Table 1.

5988 Nucleic Acids Research, Vol. 19, No. 21 (Fig. SB, panels b,f,c,g). However, there was a significant difference in fluorescent intensity when both sets of nuclei were stained with anti-OTF2 antibodies (Fig. SB, panels a & e). In addition, the pattern of staining with OTF2 antibodies in LPS treated cells showed an unusual pattern; the nuclear staining had a granular appearance with the nucleolus unstained (panel a). On the other hand a more diffuse staining pattern was observed in nuclei from LPS +anti-mu treated cells (panel e), and in both sets of nuclei stained with the other antibodies (panels b, c, f, and g). Quantitative immunofluorescence of OTF2 staining was further measured using confocal microscopy combined with computer analysis as shown in Fig. SC and summarized in Table 1. In individual nuclei from the LPS treated sample (panel A) 26.4% of the nuclei stained brightly (red color), whereas in the LPS + anti-mu treated nuclei (panel B), none stained brightly. This represents a drastic reduction of nuclei which stain brightly for OTF2 when LPS treatment is combined with anti-mu. There was also a higher percentage of cells which stained at an intermediate level with LPS vs. LPS+anti-mu (blue color, 3 1.7% vs 7.3 %). At the individual nuclei level, there is a granular, uneven distribution of OTF2 fluorescence in the LPS-treated sample (Figure 5C, panel A). This distribution is reflected by the biphasic or triphasic intensity patterns for individual nuclei in this sample (panels A-I to A-3). In contrast nuclei from LPS + anti-mu treated samples have a more even distribution of OTF2 fluorescence (panel B), with even distributions of fluorescent intensities (panels B-1 to B-3). These data indicate that OTF2 has a very unusual localization in nuclei from LPS-treated cells.

DISCUSSION Resting B-cells can be stimulated in various ways to proliferate and differentiate. The end point of B-cell differentiation is the plasma cell, which vigorously produces immunoglobulins. At the molecular level, the Ig loci are transcriptionally activated and RNA polymerase II activity is abundant at the mu-delta and kappa loci (Yuan and Tucker, 1984; Chen-Bettecken et al., 1987; Chen, 1990a). Plasma cells are short lived cells, destined to die. In vitro these characteristics are reproduced with LPS stimulation of resting splenic B-cells. An alternate pathway of B-cell activation results in proliferation without differentiation into plasma cells, generating memory B-cells (MacLennan and Gray, 1986). Since B cell memory probably requires the persistence of antigen, antigenic activation under certain conditions results in the generation of memory B-cells (Gray and Skarvall, 1988). Activation by LPS + anti-mu treatment may mimic this alternate pathway. An interesting implication of this would be that induction of memory cell formation would be dominant to plasma cell formation, since anti-mu effects override LPS effects. It is important to keep in mind that the inhibitory effects at the transcriptional level are not the primary consequence of antiInitially the switch from membrane form to the secretory form of mu-mRNA is inhibited, and Ig-transcription is not affected (Chen-Bettecken et al., 1985); only later is transcription regulation affected. When we used constructs encoding either the membrane form or the secretory form of the mu-heavy chain for transfection at late stages of culture, the two variants gave results identical with those of the complete gene (Fig. 2). All three constructs are equally downregulated as well as the endogenous mu-RNA in the LPS + anti-mu doubly treated B-cells. These data are consistent with the observation that the mu treatment.

specific inhibition of secretory mu-RNA expression in the doubly treated B-cells occurs early in the culture (day 2), and later on (day 4), all forms of mu-RNA are downregulated. We have not yet successfully transfected resting B-cells, and the analysis of earlier events awaits another approach. LPS activation was found to induce a whole series of different DNA-binding activities in resting B-cells (data not shown). Thus, the lack of heavy chain transcription in resting B-cells correlates with the absence of nuclear proteins which bind to its enhancer and promoter. It is also intriguing to wonder whether other genes, e.g. adhesion molecules, interleukin receptors, or other transcription factors, might also respond to the changes in DNA binding activities between the different differentiation pathways. In our effort to analyze the effect of anti-mu treatment at the transcriptional level, we were able to demonstrate that the transcriptional effect can be mimicked using transfected immunoglobulin heavy chain gene, and that the mu-enhancer itself is also a target of the down modulation effect. Although there were no apparent qualitative changes in the presence of specific nuclear binding proteins, some quantitative changes were observed. One significant change was the 50% higher level of OTF2 binding activity in cells treated with LPS alone as compared with cells treated with LPS +anti-mu (Fig. 4). This difference in OTF2 levels is also reflected in the higher percentage of brightly staining nuclei from cells treated with LPS alone as judged by confocal image processing (Fig. SC and Table 1). Thus, at least one effect of antigenic stimulation for B-cells would be the partial downregulation of Ig transcription factor concentration. An important question is whether this difference in nuclear factor activity is sufficient to account for the 4 - 8 fold difference in IgH enhancer activity (Fig. 3). Recently it has been demonstrated, with factors NF-AT and NF-xB, that small changes in transcription factor concentrations can have dramatic effects on transcriptional activation (Fiering, et al., 1990). The threshold effect in nuclear factor concentration observed results in a bimodal pattern of target gene expression; an individual cell is either on or off, with no intermediate levels of expression. Indeed, we find that individual cells appear to express either high levels or no C,t transcripts by in situ hybridization (see Fig. 1). The downregulation of VH promoter activity (Hogbom, et al., 1987) might also be due to the alteration in OTF2 activity. The combination of promoter and enhancer inhibition could easily account for the 10-20 fold difference in Ig gene expression we observed with the transfected constructs (Fig. 2). The other effect observed is an increase in the level of tE5 binding activity. Evidence for at least two different activities interacting with the /E5 motif have been recently demonstrated (Ruezinsky, et al., 1991). The protein encoded by the ITF-l gene can act as a positive transcription factor, and can function to displace an enhancer repressor which also interacts with this region. In our experiments, the predominant retarded band with the 1tE5 probe (Fig. 4) is found present at hgih levels in the T cell extract (BW5 147) but very low levels in the B cell line extract (J558L); this would be an appropriate cell-type distribution for this putative enhancer repressor. Thus, the induction of this binding activity in LPS+anti-mu treated cells might also be involved in enhancer downregulation. In addition, post-translational modifications, such as phosphorylation (Tanaka and Herr, 1990; Pierani, et al., 1990), affecting transcriptional activation rather than DNA-binding activities might also contribute to the substantial inhibition of heavy chain transcription by anti-mu treatment. Another potential

Nucleic Acids Research, Vol. 19, No. 21 5989 mechanism for changing the effective activity of nuclear transcription factors without substantially changing their total concentration would be to change their compartmentalization within the nucleus. Indeed, we have observed different nuclear staining patterns within the nuclei isolated from the two types of stimulated cells (see Fig. 5B and C). Resting B-cells are poised at a branch point in differentiation. A cell which is specific for a particular antigen must not only terminally differentiate into an Ig secreting plasma cell, but also develop into a memory cell to provide for rapid responses in the future. The differential response of splenic B-cells to LPS with or without crosslinking of the antigen receptor may mimic this situation. Cells receiving a single signal would terminally differentiate toward the plasma cell state, while cells receiving two signals would head towards the memory cell state. Indeed, terminal differentiation under the circumstances may be a form of tolerance induction. Thus, the stimulation by one cell surface signal vs. two decides the choice between differentiation and death vs. memory and long life.

ACKNOWLEDGEMENTS We thank L.Staudt, P.Gruss and those referred to in (4) for plasmids, U.Kaempf, D.Thorpe and H.Y.Mok for technical assistance, and H.P.Stahlberger for graphic work. We thank R.Lauster, S.Bauer and H.-J.Thiesen for critical reading and help with this manuscript, and we thank Nicole Schoepflin for its preparation. The first author would also like to acknowledge the help of W.Schaffner with primary B-cell transfections. The Basel Institute for Immunology was founded and is supported by F.Hoffmann-La Roche & Co., Ltd., Basel, Switzerland. This work was also supported by Public Health Service grants A127397, CA42567, and RR05869 from the National Institutes of Health (R.G.R.) and with general support from the Pew Trusts to the Rockefeller University.

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