Hyper-Ag-specific Ab Production in NC/Nga Mice is not associated with Deletion Polymorphism in the Promotor Region of the FcγRIIB Gene

Problem statement: NC/Nga (NC) mice produced high levels of ovalbumin (OVA)specific IgG, IgG1 and IgG2a. We previously found d eletion polymorphisms in the promoter region of fcgr2b in NC mice. To determine whether this mutati on causes a hyper-humoral immune response, we generated congenic BALB/c mice carrying the NC-type fcgr2b allele (NC fcgr2b) and analyzed humoral immune response and Fc γRIIB on germinal center (GC) B cells. Approach: BALB/c, NC and BALB/cNC fcgr2b congenic mice were immunized with OVA 2 times at a 2-week-interval. Levels of OVA-specific IgG, IgG1 and IgG2a in serum were determined by ELISA. Four-color (antiB220, anti-IgD, 2.4G2 and PNA) flow cytometry analy sis was performed on splenocytes obtained from OVA-immunized mice and levels of Fc γRIIB in GC (IgD-PNA) and non-GC (IgD+PNA ) B cells were analyzed. Results: Although perturbed up-regulation of Fc γRIIB on GC B was observed in congenic mice, levels of OVA-specific Abs were c omparable to those in BALB/c mice. Conclusion: NC fcgr2b affects the level of Fc γRIIB in GC B cells but that the reduced Fc γRIIB expression is not related to enhanced Ag-specific A b responses in NC mice.


INTRODUCTION
FcγRIIB, a low-affinity FcR for IgG, acts as a negative feedback regulator by inhibiting B Cell Receptor (BCR)-mediated activation signal through an immunotyrosine-based inhibition motif when these two receptors are co-cross-linked by Ags and IgGcontaining immune complex [1][2][3] . NC/Nga (NC) mice have been shown to develop human atopic-like skin lesions with elevated serum IgE level when kept in conventional conditions [4,5] . In the course of investigating these mechanisms, we found that NC mice produced a higher level of Ag-specifc IgG2a than did BALB/c mice and that NC mice have three deletion sites in regulatory regions of the FcγRIIB gene, two in the promoter region and one in the third intron [6] . We investigated the role of the NC-type fcgr2b allele (NC fcgr2b) in humoral immune response in (BALB/c x NC) x BALB/c or (BALB/c x NC) x NC backcross mice. Results of analysis showed that hyper Ag-specific IgG2a is not controlled by NC fcgr2b [6] . However, the role of NC fcgr2b in Ab response is not fully understood. Establishment of congenic mice for NC fcgr2b made it feasible to examine the in vivo effect of fcgr2b allele polymorphism. In this study, we examined the effect of NC fcgr2b on Ag-specific Ab response in a congenic mouse strain.

MATERIALS AND METHODS
Mice: Specific pathogen-free female NC and BALB/c mice were purchased from SLC (Hamamatsu, Japan). Congenic BALB/c mice carrying NC fcgr2b were generated by backcrossing ten times (BALB/c × NC)F1 mice × BALB/c mice. Genotyping for NC fcgr2b was done using anti-Ly 17.2 mAb. Anti-Ly 17.2 mAb reacts with BALB/c-type FcγRIIB but not with that of NC. Backcross mice showing reduced levels of Ly 17.2 staining were judged as being positive for NC fcgr2b.
Statistical analysis: Statistical analysis was done by one-way ANOVA followed by Sheffe's multiple comparison test. Values in the text are means ± SD. Differences were considered significant at p<0.05.

RESULTS AND DISCUSSION
NC mice produced significantly higher levels of Ag-specific IgG, IgG1 and IgG2a Ab than did BLAB/c mice when immunized with OVA. Quantitative analysis using serially diluted serum revealed that the amounts of Ag-specific IgG2a Ab in NC/Nga mice were -100times greater than those in BALB/c mice (data not shown). In congenic mice carrying NC fcgr2b, levels of OVA-specific Ab response were comparable to those in BALB/c mice (Fig. 1). These results indicate that NC fcgr2b does not cause hyper IgG production and that genetic factors other than NC fcgr2b contribute to the hyper Ag-specific Ab response in NC mice.
The Germinal Center (GC) is a microenvironment formed in the follicles of secondary lymphoid organs by highly proliferative B cells responding to T celldependent Ags. It has been shown that FcγRIIB on GC B cell regulates Ab-Forming Cells (AFC) [7,8] . BCR and FcγRIIB are cross-linked when GC B cells interact with Ag, resulting in inhibition of AFC differentiation [9] . We evaluated the expression of FcγRIIB in GC B cells because down-regulation of FcγRIIB in the GC has been reported in NZB mice, which have deletion mutation of fcg2b similar to that in NC mice [10][11][12] . Figure 2 shows FcγRIIB expression levels in GC (PNA high IgD -) and non-GC (PNA low IgD + ) B cells in BALB/c and congenic mice at the time of primary immune response by OVA immunization. Expression of FcγRIIB was upregulated in GC B cells compared to that in non-GC B cells in BALB/c mice, whereas expression of FcγRIIB in congenic mouse GC B cells was two-fold lower than that in BALB/c mice. Therefore, NC fcgr2b affects the level of FcγRIIB expression in GC B cells but does not affect Ag-specific Ab levels. Previous studies showed that expression of FcγRIIB in GC B cells is down-regulated in NZB mice [11,12] . However, a recent study by Rahman showed that expression of FcγRIIB in GC B cells is unchanged in autoimmuneprone mice by using multiple GC B cell maker Ags [13] . Our results showing increased expression of FcγRIIB in GC B cells are consistent with Rahman's report. The data are representative of two independent experiments of pooled samples from two to three mice [6] Xiu examined the effect of NZB-type deletion polymorphism on transcriptional regulation of the fcgr2b gene and showed defective transcription activity in an NZB-derived segment due to absence of transcription by AP4, which binds to the polymorphic 13-nucleotide deletion site [12] . Since NC fcgr2b has the same mutation, this mutation might affect induction of FcγRIIB level. The finding that NC fcgr2b does not induce hyper Ab production is unexpected because NZB-type fcgr2b congenic B6 mice show a stronger anti-KLH Ab response than do B6 mice [12] . Polymorphisms in putative regulatory regions of the FcγRIIB gene in NZB and NC mice are identical. There are several possible reasons for the different results. First, genetic background might influence the effect of NC fcgr2b on Ab response since BALB/c mice are known to be a mouse strain that induce Th2 response and show strong Ab response compare to B6 mice [14,15] . FcγRIIB-deficient mice develop autoantibodies and glomerulonephritis with a pathology resembling that of human lupus on B6 background. The same mutation on the BALB/c background does not lead to spontaneous disease, suggesting that the effect of FcγRIIB on susceptibility to the disease is different in BALB/c and B6 strains [16] . Several candidate genes for regulating autoantibody production proximal to fcgr2b have been reported [17][18][19] . Thereby, contribution of these genes on Ag-specific Ab production is not excluded at present stage.

CONCLUSION
The present study showed that NC fcgr2b affects the level of FcγRIIB in GC B cells but that the reduced FcγRIIB expression is not related to enhanced Agspecific Ab responses in NC mice. In addition, the results showed the contribution of the deletion mutation in the promoter region of fcgr2b to the humoral immune response might be limited.