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International Immunology, Vol. 11, No. 6, 871-879, June 1999
© 1999 Japanese Society for Immunology

CpG oligodeoxynucleotides rescue BKS-2 immature B cell lymphoma from anti-IgM-mediated growth inhibition by up-regulation of egr-1

Seong-Su Han1,2,3, Seung-Tae Chung1,3, D. A. Robertson1,2, Ralph L. Chelvarajan1,2 and Subbarao Bondada1,2

1 Department of Microbiology and Immunology, and
2 Sanders-Brown Research Center on Aging, University of Kentucky, Lexington, KY 40536, USA

Correspondence to: S. Bondada, 329 Sanders-Brown Building, University of Kentucky, Lexington, KY 40536, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Cross-linking of the IgM antigen receptor on an immature B cell lymphoma (BKS-2) induces growth arrest and apoptosis. This is accompanied by down-regulation of the immediate early genes, egr-1 and c-myc, and a reduction in NF-{kappa}B activity. Anti-IgM-induced growth arrest and apoptosis of this murine B cell lymphoma were prevented by oligodeoxynucleotides (ODN) containing the CpG motif, which are also known to be stimulatory for mature and immature B cells. The CpG but not non-CpG ODN rescued BKS-2 cells from anti-IgM-mediated growth inhibition by up-regulation of egr-1 and c-myc expression as well as by restoring NF-{kappa}B activity. Interestingly, changes in egr-1 expression occurred more rapidly than in c-myc expression. Also the c-myc levels remained high up to 6 h after addition of the anti-IgM, which was also the time until which the addition of CpG could be delayed without affecting its ability to provide complete protection. This CpG-induced rescue of B lymphoma cells was blocked by antisense egr-1 ODN, suggesting that the expression of egr-1 is important for the effects of CpG ODN on the growth and survival of BKS-2 cells.

Keywords: B cell lymphoma, BKS-2, CpG oligonucleotide, egr-1


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
As a part of the immune defense mechanism, vertebrates have developed a remarkable system to distinguish their own DNA from prokaryotic DNA. Bacterial DNA contains unmethylated CpG dinucleotides at the expected frequency (once every 16 bases) but in vertebrates the CpG dinucleotides occur at approximately one-fifth the expected frequency and 60–80% of these dinucleotides are highly methylated (1). Bacterial DNA as well as oligodeoxynucleotides (ODN) containing the CpG dinucleotide have mitogenic effects for B lymphocytes, macrophages and NK cells in mice. CpG ODN have been reported to induce secretion of IL-6, IL-12, IFN-{gamma} and tumor necrosis factor (TNF)-{alpha} from murine splenocytes (2–5), elevation of NK cell activity as well as differentiation of mature B cells (2–10). A CpG dinucleotide flanked by two 5'-purines (especially GA) and two 3'-pyrimidines (especially TT) called the CpG motif was found to be optimal for B cell activation (2,3,7,11).

The engagement of B cell antigen receptor (BCR) on immature B cells results in anergy or clonal deletion, while mature B cells proliferate and differentiate into antibody-producing cells and memory cells in response to BCR cross-linking (12–14). It is well established that signaling through the BCR induces a variety of biochemical changes, such as activation of protein tyrosine kinases and protein kinase C (12), elevation of intracellular Ca2+ (13,15), induction of the immediate early genes, egr-1, c-fos and c-myc (16–18), up-regulation of NF-{kappa}B (17,18) and induction of cyclin E and cdk2 activities in mature B cells (19). egr-1, an immediate early gene, encodes a nuclear protein that can bind to the regulatory element GCG(G/T)GGGCG in a zinc-dependent manner (20, 21). egr-1 mRNA is induced within 30 min after cross-linking of BCR in mature splenic lymphocytes and the Egr-1 protein has been shown to be a transcriptional activator in B cells, as well as in other cell types (20,22,23). Studies with egr-1 antisense ODN have demonstrated that the induction of egr-1 is necessary for antigen receptor-mediated signaling of B and T lymphocytes (24,25). However, in contrast to mature B cells, anti-IgM stimulation failed to induce egr-1 expression in some immature B cell lines (21).

Normal immature B cells and B lymphoma cell lines such as WEHI-231 and BKS-2, which have an immature phenotype, are growth inhibitors in response to BCR cross-linking (14,15,17,18). This is thought to be a model for B cell tolerance induction. Lymphoma models have been utilized extensively to understand biochemical signals involved in BCR-induced growth arrest and apoptosis. In this context, WEHI-231 and BKS-2 B lymphoma cells have been shown to undergo apoptosis in response to BCR cross-linking (14,15,17,18). Studies with WEHI-231 cells have demonstrated that down-regulation of c-myc expression and inhibition of NF-{kappa}B activity may be essential for the BCR-induced apoptosis response (17,18,26,27). Inhibition of NF-{kappa}B by the protease inhibitors TPCK or PDTC induces apoptosis of WEHI-231 B lymphoma cells (26). Also, the unresponsiveness of CD5-expressing peritoneal B cells to BCR cross-linking is related to an inability to induce NF-{kappa}B activity (28). CD40-mediated signaling rescues anti-IgM-treated WEHI-231 cells from apoptosis by restoring NF-{kappa}B binding activity (29). Similarly, WEHI-231 cells stimulated by CpG ODN are protected from anti-IgM-induced apoptosis, which is accompanied by restoration of c-myc expression (30,31), but the importance of NF-{kappa}B for this protection has not yet been investigated.

The role of the egr-1 gene expression in lymphoma growth regulation by anti-IgM and the CpG ODN was unclear since most WEHI-231 cell lines do not express the egr-1 gene constitutively. We have been studying another B cell lymphoma, BKS-2, which also has an immature phenotype and undergoes growth inhibition in response to BCR cross-linking (14,15,32–35). Unlike WEHI-231, BKS-2 has been maintained by in vivo serial passage and expresses egr-1 constitutively (25,32). Recently, we have demonstrated that egr-1 plays a critical role in survival of BKS-2 immature B cell lymphoma following anti-IgM treatment (25). In the present work, we show that CpG ODN can rescue BKS-2 cells from anti-IgM-induced apoptosis by increasing expression of egr-1 and c-myc genes and an increase in NF-{kappa}B activity.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell line
Isolation and characterization of BKS-2 immature B cell lymphoma has been described elsewhere (32,33). This lymphoma originated in a CBA/N mouse transferred with spleen cells from aged CBA/Ca mice. The characteristics of BKS-2 cells are similar to immature B cells in that they expressed high levels of IgM but have very little or no IgD. BKS-2 cells are monoclonal in origin and are grown in CBA/N mice or SCID mice as splenic tumor by serial i.v. injections. These cells attain maximal growth (4–6x108 ) in ~7–10 days and are collected for experimental use at this stage. BKS-2 cells from CBA/N or SCID hosts behave similarly (34,35) and cells from the former strain were used in most experiments. The depletion of T cells was performed as described previously (34).

Reagents
The characteristics of the monoclonal rat anti-mouse µ chain antibody, AK11, have been described previously (14). This hybridoma cell line obtained from Dr R. Noelle (Dartmouth Medical School, Hanover, NH) was grown in vitro and the culture supernatant was affinity purified by passing through a column containing mouse IgM protein (HPCM 2, a hybridoma from BALB/c origin) coupled to agarose beads (BioRad, Hercules, CA) (14). In experiments aimed at blocking egr-1 gene expression, we used a phosphorothioate-capped antisense egr-1 oligonucleotide (5'-GsCsGGGGTGCAGGGGCACAsCsT-3') and a control phosphorothioate-capped nonsense egr-1 oligonucleotide (5'-CsGsCCGCACCACCGCGAGTsCsA-3'). The phosphorothioate oligonucleotides 1d, containing the CpG dinucleotide, and 1a, not containing the CpG dinucleotide (for control), were as described by Krieg et al. (7). Their sequences are: 1a: GCT AGA TGT TAG CGT; 1d: GCA TGA CGT TGA GC. All ODN were purchased from the Regional DNA Synthesis Laboratory (Calgary, Alberta, Canada). The ODN were column purified and were found to be free of endotoxin by the limulus ameobocyte assay.

Proliferation assay
BKS-2 cells (3x104/well) were cultured in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA) in 0.2 ml of 1:1 mixture of Isocove's and Ham's F 12 (IF-12) medium containing supplements and 5% FCS as described previously (34). Preliminary studies showed that 48 h is an optimal time to observe maximal inhibition by anti-µ and the stimulatory effects of the CpG ODN. Hence, the cultures were incubated at 37°C with 5% CO2 for various time points or 48 h and were pulsed with 1 µCi of [3H]thymidine (sp. act. 2 Ci/mmol; New England Nuclear, Boston, MA) during the last 4 h culture period. The cultures were harvested and [3H]thymidine incorporation was determined by a Matrix-96 ß-counter (Packard, Downers Grove, IL).

Apoptosis measurement
Apoptosis was monitored by DNA fragmentation assay using the Puregene DNA isolation kit (Gentra System, Minneapolis, MN) according to the manufacturer's protocol. Briefly, 5x106 BKS-2 cells were cultured for 48 h, washed and harvested. Pellets were resuspended in 600 µl of cell lysis solution containing 3 µl of RNase (4 mg/ml) and incubated at 37°C for 1 h. Samples were treated with 200 µl of protein precipitation solution and centrifuged at 15,000 g for 3 min. To the supernatant, 600 µl of isopropanol was added to precipitate the DNA and centrifuged at 15,000 g for 10 min. The pellet was washed with 70% ethanol and dissolved in DNA hydration buffer. The sample was loaded onto a 1.8% agarose gel containing ethidium bromide. Electrophoresis was carried out in 1xTAE buffer (40 mM Tris–acetate and 1 mM EDTA).

Preparation of nuclear extracts
Nuclear extracts were obtained from 10x106 BKS-2 cells using a modification of the method described by Dent and Latchman (36). Cells were lysed in 400 µl of Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT and 0.2 mM PMSF) by incubating at 4 °C for 10 min. After a 6 min centrifugation, the pellet was resuspended in 100 µl of ice-cold buffer C [20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 20% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM DTT and 0.2 mM PMSF], incubated at 4°C for 20 min, centrifuged for 6 min, and the resulting pellet was resuspended in 100 µl of ice-cold buffer C, aliquoted and stored at –70°C. The protein content of the final extracts was estimated using the BCA protein estimation kit according to the manufacturer's protocol (BioRad).

Electrophoretic mobility shift assay (EMSA)
EMSA was performed using a DNA–protein binding detection kit for NF-{kappa}B binding according to the manufacturer's protocol (Gibco/BRL, Gaithersburg, MD). Briefly, the NF-{kappa}B oligonucleotide probe was labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase and purified on a Nick column (Pharmacia Biotech, Uppsala, Sweden). The binding reaction was carried out in a total volume of 25 µl containing 5 µl of incubation buffer [10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 4% (v/v) glycerol and 0.1 mg/ml sonicated salmon sperm DNA] with 10 µg of nuclear extracts and 100,000 c.p.m. of the labeled probe. A 160-fold excess of the cold competitor was added where necessary. After 20 min of incubation at room temperature, 2 µl of 0.1% bromophenol blue was added and samples were electrophoresed through a 6% non-denaturing polyacrylamide gel at 150 V in a cold room. Finally, the gels were dried and exposed to X-ray film.

Northern analysis
Total RNA was isolated with TRI reagent (Sigma, St Louis, MO) according to the manufacturer's protocol and Northern analysis was performed as described previously (25). The probe for egr-1 was prepared with a 3.1 kb cDNA fragment from pCMV-EGR-1 (37), and the probe for c-myc was prepared with a 4.8 kb cDNA fragment generated by XbaI and BamHI double-digestion of the plasmid, pSV-c-myc 1 (purchased from ATCC, Rockville, MD).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Stimulation of BKS-2 cells with CpG ODN induces an increase in proliferation and a dramatic up-regulation of egr-1 and c-myc expression
In order to examine whether CpG ODN could increase BKS-2 cell proliferation similar to its growth stimulatory effects on normal B lymphocytes (4,6,7), we cultured BKS-2 cells with various concentrations of 1d CpG or 1a non-CpG ODN. As shown in Fig. 1Go(A), 1d CpG ODN increased proliferation of BKS-2 cells up to 170% at 10 µM, whereas the control non-CpG ODN (1a) did not affect BKS-2 proliferation. In contrast to the substantial increase in proliferation seen when normal B lymphocytes were treated with CpG ODN (4,6,7,25), BKS-2 cell proliferation was enhanced only slightly, presumably because BKS-2 cells were already in cycle. When we treated BKS-2 cells with the bacterial lipopolysaccharide (LPS) similar results were obtained (data not shown).




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  		Fig. 1. The effect of CpG ODN on BKS-2 cell growth and immediate early gene expression. (A) BKS-2 cells (3x104/well) were cultured with the various concentrations of 1d CpG ODN or 1a non-CpG ODN for 48 h. Cells were pulsed with [3H]thymidine during the last 4 h of culture. Data represent the mean ± SE of triplicate determinations. Results are presented as percent of control, which was medium only, and the response in the control was 57059 ± 2190 c.p.m./culture. The first point indicating 0.0001 µM is really with no ODN but was given this value since the log scale does not allow a zero value. (B) CpG ODN up-regulates egr-1 and c-myc mRNA level in BKS-2 cells. BKS-2 cells were cultured with 1.0 µM of 1d CpG ODN or 1a non-CpG ODN as a control for the indicated times. Egr-1 and c-myc mRNA levels were assessed by Northern analysis (15 µg of total RNA/lane) as described under Methods. GAPDH was used as a control for equal RNA loading. Zero time point indicates unstimulated cells. This experiment was repeated at least 3 times with similar results.

 
To determine whether this increase in BKS-2 cell growth induced by CpG ODN was due to any changes in egr-1 and c-myc mRNA levels, Northern analysis was performed with total RNA isolated from BKS-2 cells cultured with 1.0 µM of 1d CpG or 1a non-CpG ODN for various periods of time. The mRNA level of egr-1 was up-regulated dramatically as early as 30 min following stimulation with the 1d CpG ODN but not with 1a, the control non-CpG ODN (lane 1 versus lanes labeled 0.5 h with 1a or 1d, Fig. 1BGo). Subsequently, the mRNA level of egr-1 began to decrease so that by 24 h egr-1 mRNA level was barely detectable following stimulation with CpG ODN (Fig. 1BGo). In contrast, throughout this time period, the egr-1 levels remained almost unchanged in BKS-2 cells treated with the control ODN.

BKS-2 cells were found to express high levels of c-myc constitutively, which was also increased dramatically as early as 30 min after treatment with CpG ODN (Fig. 1BGo). This increase in c-myc peaked at 3 h, although the levels at 24 h were higher than those seen in unstimulated BKS-2 cells (Fig. 1BGo). Interestingly, c-myc mRNA was up-regulated and peaked at 3 h just after the peak of egr-1 mRNA accumulation. However, 1a non-CpG ODN did not induce any alterations in the c-myc mRNA levels (Fig. 1BGo). These results indicated that the CpG ODN-mediated enhancement of BKS-2 cell proliferation may be due to the increased expression of egr-1 and c-myc genes.

CpG ODN rescues BKS-2 cells from anti-IgM-mediated growth arrest in a dose- and time-dependent manner via up-regulation of egr-1 and c-myc expression
Previously we showed that anti-IgM induced growth inhibition in BKS-2 cells (14,32–35), which was accompanied by a decrease in egr-1 expression (25). We tested if the 1d CpG ODN that enhanced egr-1 expression can overcome the growth inhibitory effects of anti-IgM. BKS-2 cells were cultured for 48 h with various concentrations of 1d CpG or 1a non-CpG ODN along with 5 µg/ml of anti-IgM. BKS-2 cells were rescued from anti-IgM-mediated growth arrest by treatment with very low concentrations (0.05 µM) of 1d CpG ODN with 100% rescue at 0.2 µM of 1d CpG (Fig. 2AGo). At higher concentrations, the response was more than control, as expected from the stimulatory effect of 1d CpG ODN alone on BKS-2 cells (Fig. 1AGo). The control non-CpG ODN (1a) did not significantly alter the anti-IgM induced growth inhibition.





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  		Fig. 2. Effect of CpG ODN on anti-IgM mediated growth arrest of BKS-2 cells and immediate early gene expression. (A) BKS-2 cells (3x104/well) were cultured with various concentrations of 1d CpG or 1a non-CpG ODN as indicated with 5.0 µg/ml of anti-IgM for 48 h. Cells were pulsed with [3H]thymidine during the last 4 h culture. Data represent the mean ± SE of triplicate determinations. Results are presented as percent control, which was BKS-2 cells cultured in medium only. The responses of BKS-2 cells in control cultures were: medium only 59,582 ± 1618; anti-IgM only 4548 ± 191. The first point indicating 0.00001 µM is with no ODN but was given this value since the log scale does not allow a zero value. (B) Delayed addition of CpG ODN rescues BKS-2 cells from anti-IgM-mediated growth arrest. BKS-2 cells (3x104/well) were cultured with 1.0 µM of 1d CpG ODN or 1a non-CpG ODN for various time periods as indicated after the addition of 5 µg/ml of anti-IgM for 48 h. Cells were pulsed with [3H]thymidine during the last 4 h of culture. Data represent the mean ± SE of triplicate determination. (C) CpG ODN rescues BKS-2 cells from anti-IgM-mediated growth arrest by up-regulation of egr-1 and c-myc mRNA level. BKS-2 cells were cultured with 5.0 µg/ml of anti-IgM plus 1.0 µM of 1d CpG ODN or 1a non-CpG ODN as a control for various periods of time as indicated. egr-1 and c-myc mRNA levels were assessed by Northern analysis (15 µg of total RNA/lane) as described under Methods. GAPDH was used as a control for equal RNA loading. Zero time point indicates unstimulated cells. This experiment was repeated two more times with similar results.

 
To see if the CpG ODN could reverse the inhibitory pathway that was already initiated by BCR cross-linking, the 1d CpG or 1a non-CpG ODN were added at various times after the addition of anti-IgM to the BKS-2 cell cultures. As shown in Fig. 2Go(B), anti-IgM reduced the BKS-2 cell growth to 15% of untreated cells while the 1d CpG ODN restored BKS-2 cell proliferation to 120% even if it was added up to 3 h after anti-IgM treatment. At later time points the rescue effect decreased steadily and remained as high as 80% of control even when the CpG ODN were added 12 h after exposure to anti-IgM. This result revealed that 1d CpG ODN rescued BKS-2 cells from anti-IgM-mediated growth arrest in a time-dependent manner (Fig. 2BGo). 1a non-CpG ODN did not rescue BKS-2 cell growth response at any time point tested (Fig. 2A and BGo).

As BKS-2 cells exhibited an up-regulation of egr-1 and c-myc mRNA levels upon treatment with 1d CpG ODN and a down regulation of egr-1 with anti-IgM alone (Fig. 1BGo) (25), we examined whether the rescue effect of 1d CpG ODN was correlated with restoration of egr-1 levels. In agreement with our previous results, anti-IgM inhibited the expression of egr-1 (25) (left-most lanes versus the right-most lane), which was not restored by the addition of the control 1a non-CpG ODN. Interestingly, the 1d CpG ODN restored the expression of egr-1 that was higher than in the control cultures in accordance with the results shown in Fig. 2Go(C). However, the egr-1 levels began to fall by 1 h and became undetectable by 3 h. Like egr-1, the levels of c-myc also were elevated as early as 0.5 h but remained above the control levels until 6 h post-stimulation. By 24 h of culture the c-myc mRNA levels were less than the control (last two lanes versus lane 1 in Fig. 2CGo). The changes in c-myc were the same whether or not the control non-CpG was added to the anti-IgM treated BKS-2 cells. Equal loading of RNA in all lanes was shown by probing for the GAPDH gene. The c-myc mRNA levels remained high up to 6 h after stimulation with anti-IgM and CpG ODN by which time egr-1 mRNA was already reduced to near background levels. Interestingly, 6 h is also the time period up to which the addition of 1d CpG ODN could be delayed to obtain a complete rescue of BKS-2 cells from anti-IgM-induced growth inhibition (Fig. 2B and CGo). These facts implicate strongly that egr-1 and c-myc mRNA levels are correlated with survival of BKS-2 cells and play a key role in the ability of 1d CpG ODN to induce BKS-2 cell proliferation.

CpG ODN rescues BKS-2 cells from anti-IgM-mediated apoptosis
Previously we showed that anti-IgM-induced growth inhibition was due mainly to increased apoptosis of BKS-2 cells (25,33). Therefore, it was important to see if the 1d CpG ODN were rescuing BKS-2 cells from such apoptotic death or if they were simply enhancing the growth of a small, resistant population. As shown in Fig. 3Go, anti-IgM induced a significant fragmentation of BKS-2 DNA which was completely blocked by the presence of 1d CpG ODN but not the control 1a non-CpG ODN.



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  		Fig. 3. CpG ODN rescues BKS-2 cells from anti-IgM mediated apoptosis. 5x106 BKS-2 cells were cultured for 48 h with following treatments: lane 2, untreated; lane 3, 1 µM of 1d CpG; lane 4, 1 µM of 1a non-CpG; lane 5, 5 µg/ml of anti-IgM; lane 6, 5 µg/ml of anti-IgM + 1 µM of 1d CpG; lane 7, 5 µg/ml of anti-IgM + 1 µM of 1a non-CpG. Cells were harvested and DNA was extracted and analyzed by agarose gel electrophoresis. Lane 1 shows the 1 kb ladder.

 
CpG ODN induced NF-{kappa}B binding activity from anti-IgM-mediated repression
Studies with WEHI-231 B lymphoma cells showed that the c-myc gene has two NF-{kappa}B binding sites in its promoter and that NF-{kappa}B might be important for c-myc expression (18,27). Since the mRNA level of c-myc was up-regulated by 1d CpG ODN in our present results, we examined whether 1d CpG ODN could induce NF-{kappa}B binding activity using the EMSA technique.

Consistent with the data using WEHI-231 cells, anti-IgM treatment of BKS-2 cells decreased NF-{kappa}B binding activity (Fig. 4Go, cf. lanes 3 and 2). The addition of 1d CpG ODN alone increased NF-{kappa}B binding activity in BKS-2 cells but treatment with 1a non-CpG ODN did not have any effect on NF-{kappa}B binding activity (Fig. 4Go, lanes 4 and 5). Interestingly, the repression of NF-{kappa}B binding activity induced by anti-IgM treatment to BKS-2 was reversed dramatically by the addition of 1d CpG ODN with anti-IgM simultaneously (Fig. 4Go, lanes 3 and 6). The control non-CpG ODN did not restore NF-{kappa}B activity in anti-IgM treated BKS-2 cells (Fig. 4Go, lanes 3 and 7). This result is consistent with the possibility that 1d CpG ODN activates NF-{kappa}B, which can bind to the c-myc promoter regulatory region and stimulate its expression. In addition to NF-{kappa}B, BKS-2 cells also have a faint faster migrating band, which is likely to be due to a small amount of the p50/p50 homodimer (Fig. 4Go). Anti-IgM repressed this presumed p50/p50 homodimer like NF-{kappa}B, which was restored to basal levels by the 1d CpG ODN (Fig. 4Go, lanes 3 and 6). However, the treatment with 1d CpG ODN alone did not affect the basal level of p50 homodimer which was seen in higher exposures of these gels in unstimulated as well as anti-IgM-treated cells (Fig. 4Go, lanes 2 and 4). These results suggested that NF-{kappa}B (p65/p50), but not the p50/p50 homodimer, was the major transcription factor important for the survival of BKS-2 cells. The definition of the exact role of p50/p50 in B lymphoma growth requires additional studies with more sensitive techniques.



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  		Fig. 4. CpG ODN induced NF-{kappa}B binding activity in BKS-2 cells treated with anti-IgM. Nuclear extracts were prepared from 10x106 BKS-2 cells treated with following reagents for 1 h: lane 1, probe only; lane 2 , no treatment; lane 3, 5 µg/ml of anti-IgM; lane 4, 1 µM of 1d CpG; lane 5, 1 µM of 1a non-CpG; lane 6, 5 µg/ml of anti-IgM + 1 µM of 1d CpG lane 7, 5 µg/ml of anti-IgM + 1 µM of 1a non-CpG; lane 8, 5 µM of 1d CpG/160-fold excess of competitor. The cell extracts from these treatments were used in an EMSA as described in Methods.

 
CpG ODN protects antisense egr-1 ODN-induced growth inhibition and apoptosis of BKS-2 cells
To verify the importance of egr-1 expression for the growth-promoting effects of CpG ODN on BKS-2 cells, we utilized antisense egr-1 ODN to specifically suppress egr-1 mRNA. Results shown in Fig. 5Go(A) demonstrated that antisense, but not nonsense, egr-1 ODN inhibits BKS-2 cell growth in a dose-dependent manner, which is in agreement with our previous results (25). Figure 5Go(B) shows the effect of CpG ODN on antisense egr-1 ODN-induced growth inhibition of BKS-2 cells. As shown in Fig. 5Go(B), BKS-2 cell proliferation increased slightly (120% of control) upon the addition of 1d CpG ODN alone but 1a non-CpG ODN alone did not have any effect. When nonsense egr-1 ODN was added to BKS-2 cells with 1d CpG or 1a non-CpG ODN simultaneously, the growth responses were not altered (Fig. 5BGo) because nonsense egr-1 ODN alone did not have any effect on BKS-2 cells (Fig. 5AGo). However, the treatment of BKS-2 cells with the 1d CpG ODN, but not the control 1a non-CpG ODN, partially overcame the growth arrest of BKS-2 cells induced by the antisense egr-1 ODN (Fig. 5BGo).




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  		Fig. 5. CpG ODN rescues BKS-2 cells from antisense egr-1 ODN induced growth arrest. (A) BKS-2 cells (3x104 /well) were cultured with various concentrations of antisense or nonsense egr-1 ODN as indicated for 48 h. Cells were pulsed with [3H ]thymidine during the last 4 h culture. Data represent the mean ± SE of triplicate determinations. The first point indicating 0.01 µM was without ODN but was given this value since the log scale does not allow a zero value. This experiment was repeated at least 3 times with similar results. Proliferation response of untreated BKS-2 cells was 40,339 ± 915 c.p.m. (B) CpG ODN antagonizes the inhibitory effect of antisense egr-1 on BKS-2 cell proliferation. BKS-2 cells (3x104/well) were cultured with various concentrations of antisense (AS) or nonsense (NS) egr-1 ODN with 1.0 µM of 1d CpG ODN or 1a non-CpG ODN for 48 h. Cells were pulsed with [3H]thymidine during the last 4 h of culture. Data represent the mean ± SE of triplicate determinations. Results are shown as percent control response with untreated BKS-2 cells as 100%. The thymidine incorporation in the untreated group was 45088 ± 1971 c.p.m.. The first point indicating 0.01 µM is with no ODN but was given this value since the log scale does not allow a zero value. Results from one out of three similar experiments are shown.

 
In order to examine whether the CpG ODN antagonized the inhibitory effect of antisense egr-1 ODN by preventing apoptosis, we performed a DNA fragmentation assay (Fig. 6Go). Antisense egr-1 ODN alone induced apoptosis of BKS-2 cells (Fig. 6Go, lane 3) which was partially inhibited by 1d CpG ODN (Fig. 6Go, lane 5) but not by 1a non-CpG ODN (Fig. 6Go, lane 7). The nonsense egr-1 ODN did not induce apoptosis either alone (Fig. 6Go, lane 4) or when added with 1d CpG or 1a non-CpG ODN (Fig. 6Go, lanes 6 and 8). This result was consistent with the [3H]thymidine incorporation assay in Fig. 5Go(B), which showed that the 1d CpG ODN reversed antisense egr-1 ODN-induced growth inhibition of BKS-2 cells (up to 60%). These results provided further evidence for a key role of egr-1 gene in the survival of BKS-2 cells and the growth-promoting effects of CpG ODN.



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  		Fig. 6. Antisense egr-1-induced apoptosis of BKS-2 cells is partially overcome by CpG ODN. BKS-2 cells (5x106) were cultured for 48 h with following treatments: lane 2, untreated; lane 3, 25 µM of AS egr-1; lane 4, 25 µM of nonsense egr-1; lane 5, 1 µM of 1d CpG + 25 µM of AS egr-1; lane 6, 1 µM of 1d CpG + 25 µM of NS egr-1; lane 7, 1 µM of 1a non-CpG + 25 µM of AS egr-1; lane 8, 1 µM of 1a non-CpG + 25 µM of NS egr-1. Cells were harvested, and DNA was extracted and analyzed by agarose gel electrophoresis. Lane 1 shows the 1 kb ladder.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Recent studies demonstrated that bacterial DNA as well as unmethylated CpG ODN can activate a variety of hematopoietic cells like B lymphocytes, NK cells, macrophages, etc. However, so far the molecular basis of the action of bacterial DNA containing CpG dinucleotides or synthetic CpG ODN on leukocytes has not been investigated in detail. In this report, BKS-2, an immature B cell lymphoma cell line, is used as a model to study the role of the immediate early genes, egr-1 and c-myc, in CpG ODN-mediated B cell proliferation. CpG ODN enhance the proliferation of the BKS-2 cells, which is accompanied by a strong increase in the mRNA levels of egr-1 and c-myc (Fig. 1Go). Our data reveal that the mRNA levels of egr-1 and c-myc, and the DNA binding activity of NF-{kappa}B are down-regulated when BKS-2 cells undergo growth arrest and apoptosis upon anti-IgM engagement (Figs 2C and 4GoGo). Addition of CpG ODN to anti-IgM-treated BKS-2 cells not only rescues them from apoptosis, but also restores the expression of both c-myc and egr-1 mRNA as well as the activity of NF-{kappa}B.

BKS-2 cells treated with antisense egr-1 ODN undergo growth arrest and apoptosis (Figs 5 and 6GoGo), which are partially overcome by up-regulation of egr-1 message by 1d CpG (Figs 1 and 2GoGo), suggesting that egr-1 is one of the key genes regulated by 1d CpG ODN. The importance of egr-1 gene expression for CpG protection is consistent with the previous data showing an essential role of this gene product in B cell growth responses. Wild-type BKS-2 cells that undergo apoptosis with anti-IgM down-regulate egr-1 while mutant BKS-2 cells that are resistant to anti-IgM induced apoptosis up-regulate egr-1 upon BCR cross-linking (25). In WEHI-231, another lymphoma cell line, LPS-mediated protection from anti-IgM induced apoptosis is also accompanied by elevated egr-1 expression (20,21). Our results are in agreement with those of Yi et al. (31) on the ability of CpG ODN to protect WEHI-231 cells from anti-IgM-induced apoptosis and in restoring c-myc expression, but provide new information concerning the causative role of egr-1 expression in mediating the growth effects of CpG ODN.

The egr-1 gene is widely expressed and has initially been found to be important for the growth, as well as apoptosis responses, of a variety of cells (20,21,25). This immediate early gene product has been implicated to be important for ionizing radiation-induced apoptosis of prostate cancer cell lines and its expression negatively correlates with transformation in several breast cancer cell lines (38,39). This may relate to the negative regulatory function of this protein, i.e. its ability to compete with Sp-1 for similar binding sites in key promoters and/or its association with Nab1 and Nab2 proteins that act as transcriptional repressors (40,41). In contrast, metabolic activity of mature B cells and growth of B cell lymphomas have been shown to be suppressed by inhibition of egr-1 gene with antisense oligonucleotides (21,25). In this regard, Egr-1 has been shown to regulate the expression of several genes like CD44, c-myc, NF-{kappa}B1, thymidylate kinase, cyclin D1 and platelet-derived growth factor that are important for cell survival and proliferation (20–25). Our studies further support the notion that the egr-1 gene product plays a positive role during the B cell growth response.

Although several genes are induced by CpG, the temporal expression of egr-1 and c-myc in cells treated with anti-IgM, CpG ODN or both, suggests that the induction of c-myc may be dependent upon egr-1 expression. It is well established that apoptosis of immature B cells is coupled with the down-regulation of c-myc expression (17,18,27,29–31). The apoptosis of WEHI-231 cells mediated by anti-IgM engagement is induced by down-regulation of c-myc expression below basal levels seen in unstimulated cells following a big transient increase at early time points (27). On the other hand, mutants of WEHI-231 cells that are refractory to anti-IgM-mediated apoptosis have a 2-fold higher uninduced level of c-myc than that of wild-type WEHI-231 cells and do not decrease below basal levels after anti-IgM engagement (42). Furthermore, the expression of c-myc and cdk2/cyclin E component for G1/S transition in cell cycle is stimulated in mature B cells activated with anti-IgM treatment, but not in immature B cells that undergo apoptosis with anti-IgM (19). The gene for the protein phosphatase, Cdc25a, has twoc-myc binding sites in its intron and the binding of c-myc to these sites induces Cdc25a expression, which in turn regulates G1/S transition by dephosphorylating the cdk2/cyclin E complex (43,44). These previous reports strongly implicate that c-myc may determine the fate of the B cell in terms of death or survival by regulating cell cycle entry and progression.

Expression of c-myc gene may be enhanced either through a direct effect of egr-1 protein binding to specific sites in the c-myc promoter or indirectly by up-regulation of NF-{kappa}B expression. The c-myc gene has two NF-{kappa}B binding sites in its promoter and is regulated by binding of NF-{kappa}B to its binding sites in the c-myc promoter (18,27,45). The result that 1d CpG ODN rescues BKS-2 cells from anti-IgM mediated apoptosis by induction of NF-{kappa}B binding activity (Fig. 4Go) suggests that CpG ODN may regulate c-myc expression via induction of NF-{kappa}B binding activity. Accordingly, Cogswell et al. reported that the NF-{kappa}B1 gene, which encodes a 105 kDa protein, the precursor of the p50 component of NF-{kappa}B, contains an Egr-1 binding site in its promoter and the binding of Egr-1 protein at this site activates the expression of NF-{kappa}B1 in T lymphocytes (46). Alternatively, TNF-{alpha}, a gene known to have Egr-1 binding sites, may be stimulated by CpG and in turn could up-regulate NF-{kappa}B and protect BKS-2 cells (21,47,48). This predicts that the effects of CpG ODN should be blocked by antibodies to TNF-{alpha} and/or its receptor.

In summary, the results reported in this paper present evidence for a molecular basis for the action of CpG ODN. CpG ODN stimulates proliferation and rescues BKS-2 immature B cell lymphoma from anti-IgM-mediated growth arrest and apoptosis by up-regulation of the immediate early genes, egr-1 and c-myc, and by NF-{kappa}B activation.


   Acknowledgments
 
Our thanks are due to Drs Charlie Snow and C. Venkataraman for a critical review of the manuscript. S.-T. C. was supported in part by a grant from Korea Food and Drug Administration. This work was supported in part by the NIH grants AI21490 and AG05731 to S. B.


   Abbreviations
 
BCRB cell receptor
EMSAelectrophoretic mobility shift assay
LPSlipopolysaccharide
ODNoligodeoxynucleotide
TNFtumor necrosis factor

   Notes
 
3 Present address: Division of Immunotoxicology, Korea Food and Drug Administration, Seoul 122-020, Korea Back

Transmitting editor: A. Singer Back

Received 6 October 1998, accepted 25 January 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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