Egr1^+/- mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a pathogen-free barrier facility at Hanyang University (Seoul, South Korea). Egr1^+/- mice were incross to generate Egr1^-/- mice. When Egr1^-/- mice and their wild-type (WT) littermates were 6~12-weeks old, they were injected i.p. with a mixture of 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin (NP-KLH; 100 µg/mouse; Biosearch Technologies) and alum (Thermo Scientific). Our study was approved by the Institutional Animal Care and Use Committee (HY-IACUC-13-053).

Erythrocyte-depleted spleen cell fractions from WT and Egr1^-/- mice were stimulated with 20 ng/ml PMA and 1 µM ionomycin (Sigma-Aldrich) for either 1 or 3 h. Total RNA was isolated using Trizol reagent (Life Technologies). Semi-quantitative and quantitative RT-PCR assays were conducted as described previously (13). The mRNA expression levels of each gene were normalized to that for the gene encoding β2-microglobulin (B2m). Differences between samples were normalized using the cycle threshold method (ΔC_t). We used specific oligonucleotide primers targeting Egr1 (5'-AAC CGG CCC AGC AAG ACA CC-3' and 5'-TGG CAA ACT TCC TCC CAC AAA T-3'), Egr2 (5'-CTT CAG CCG AAG TGA CCA CC-3' and 5'-GCT CTT CCG TTC CTT CTG CC-3'), Egr3 (5'-CAA CGA CAT GGG CTC CAT TC-3' and 5'-GGG CAG GCT GCC GAA TCC CG-3'), and B2m (5'-CAG TGT GAG CCA GGA TAT AG-3' and 5'-TGA CCG GCT TGT ATG CTA TC-3').

Splenic single-cell suspensions from WT and Egr1^-/- mice were stained and analyzed using a FACS_{Canto II} (BD Biosciences) as described previously (14). The following mAbs and reagents were purchased from BD Biosciences or eBioscience: anti-GL7-FITC, anti-CD4-PE, anti-B220-PerCP, anti-CD11C-allophycocyanin, anti-CD138-allophycocyanin, 7-AAD, and Annexin V-FITC.

Naive GL7^-CD138^-CD11c^-B220⁺ B cells from the spleens of WT and Egr1^-/- mice were sorted using a FACS_{Aria III} (BD Biosciences) and stained with 3 µM CFSE (Molecular Probes). Cells at 1×10⁶/ml were cultured in the presence of 20 µg/ml LPS (Sigma-Aldrich) and 10 ng/ml IL-4 (Peprotech), or 5 µg/ml goat anti-mouse IgM (Jackson Immune Research) and 10 ng/ml IL-4. After culturing for 4 days, cells were subjected to flow cytometry.

Sera were collected from pre- and post-immunized mice and assayed by ELISA to determine levels of NP-specific Abs. In brief, sera were diluted 1:100,000 in PBS (WelGene) and dispensed into 96-well immunosorbent plates (Nunc) pre-coated with 10 µg/ml NP₈ conjugated to BSA (Biosearch Technologies). A serum sample containing the highest titer of anti-NP₈ IgG was serially diluted and used as a standard. Plates were incubated with an anti-mouse IgG conjugated to biotin (Sigma-Aldrich), followed by streptavidin conjugated to HRP (BD Biosciences).

We examined whether depletion of Egr1 induces compensatory expression of other Egr genes. Egr1 mRNA were detected in unstimulated WT B cells, and were more abundant 1 h later in response to stimulation with PMA and ionomycin. We failed to detect Egr1 mRNA in Egr1^-/- B cells, regardless of whether cells were stimulated (Fig. 1). We also found that Egr2 and Egr3 mRNA were expressed at similar levels in WT and Egr1^-/- B cells. Our findings show that deficient Egr1 expression did not induce compensatory expression of Egr2 and Egr3. Thus, our Egr1^-/- knockout model appears to be appropriate for investigating the role(s) of Egr-1.

Given that a deficiency in Egr-1 affects normal development of B-lineage cells (78), we assessed whether Egr1^-/- mice have an abnormal composition of spleen cells. We observed similar proportions of B220⁺ and CD4⁺ cells among WT and Egr1^-/- mice (Fig. 2). The proportions of B220⁺GL7^- naive B cells and B220⁺GL7⁺ germinal center B cells within the whole B cell population were similar between WT and Egr1^-/- mice. Therefore, Egr-1 activity does not appear to be necessary for the development of naive B cells, CD4⁺ T cells, or germinal center B cells.

We sought to determine whether a deficiency in Egr-1 affects the proliferation and apoptosis of Egr1^-/- B cells, and whether naive Egr1^-/- B cells can effectively differentiate into plasma cells. Naive B cells sorted from WT and Egr1^-/- mice were labeled with CFSE and cultured with LPS plus IL-4 or anti-IgM Ab plus IL-4, and then subjected to flow cytometry. The vast majority of cells proliferated, as determined by the decrease in CFSE intensity (CFSE^dim cells), when treated with both types of stimulants (Fig. 3). The proportions of CFSE^dim cells were not significantly different between WT and Egr1^-/- mice, regardless of the stimulants used (88.7±1.2 for LPS/IL-4-treated WT versus 89.5±2.1 for LPS/IL-4-treated KO, p=0.67; 88.2±1.4 for anti-IgM/IL-4-treated WT versus 82.9±4.5 for anti-IgM/IL-4-treated KO, p=0.46). In addition, the proportions of early apoptotic (Annexin V⁺7-AAD^-) and late apoptotic (Annexin V⁺7-AAD⁺) cells were equivalent between WT and Egr1^-/- mice. However, the proportion of CD138⁺CFSE^dim cells among Egr1^-/- cells was approximately 50% of that seen for WT cells. These results demonstrate that a deficiency in Egr-1 impaired the differentiation of B cells into plasma cells, while proliferation and apoptosis of these cells were unaffected. Thus, Egr-1 appears to play an important role in plasma cell differentiation, and its depletion cannot be adequately compensated for by other Egr family members. Results from previous studies have shown that Egr-1 activity is associated with the proliferation of mature B lymphoma cell lines (29). Therefore, it is likely that, rather than not participating in proliferation, the functions of Egr-1 are redundant and compensated for by other Egr family members, as suggested previously (8).

To validate the in vitro effects of Egr-1 deficiency on plasma cell development, we immunized Egr1^-/- mice and their WT littermates with NP-KLH and alum. We then determined serum titers of Abs with high affinity for NP at 14 and 21 days post-immunization (dpi). The anti-NP₈ IgG was detected at both dpi; its titer was significantly lower in Egr1^-/- mice at 21, but not 14, dpi than that in WT mice (Fig. 4). Our findings reveal that Egr-1 is involved in the production of Abs, possibly by promoting the development of plasma cells through a TD pathway.

Results from several studies have indicated that Egr-1 is possibly associated with plasma cell development. When chronic lymphocytic leukemia B cells were stimulated with phorbol esters, Egr-1 was upregulated, giving rise to cells with a plasmacytic phenotype (15). Egr-1 could bind to the GC-box element of human PRDM1, which encodes Blimp-1, a master regulator of plasma cell differentiation (16). In the light of this result, we speculate that the effect of Egr-1 in the differentiation of B cells into plasma cells may be mediated by the induction of Blimp-1 expression. Transgenic mice lacking all Egr family members contain lower numbers of Ab-secreting cells than their WT counterparts (8). However, these studies have provided relatively little information regarding the role of Egr-1 during plasma cell differentiation.

In summary, we found that the proliferation and death of B cells was unaffected by the absence of Egr-1, indicating that Egr-1 is not required for these processes. We also showed that Egr1^-/- B cells could not efficiently differentiate into plasma cells in vitro and in vivo, which was a strong indicator of the crucial and non-redundant role of Egr-1 in the commitment of B cells to a plasma cell fate.