Hyper-Ag-specific Ab production in NC/Nga mice is not associated with deletion polymorphism in the promotor region of the Fc[gamma]RIIB gene.
Abstract: Problem statement: NC/Nga (NC) mice produced high levels of ovalbumin (OVA)specific IgG, IgG1 and IgG2a. We previously found deletion polymorphisms in the promoter region of fcgr2b in NC mice. To determine whether this mutation 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[gamma]RIIB on germinal center (GC) B cells. Approach: BALB/c, NC and BALB/c- NC 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 analysis was performed on splenocytes obtained from OVA-immunized mice and levels of Fc[gamma]RIIB in GC (IgD-[PNA.sup.high]) and non-GC (IgD+[PNA.sup.low]) B cells were analyzed. Results: Although perturbed up-regulation of Fc[gamma]RIIB on GC B was observed in congenic mice, levels of OVA-specific Abs were comparable to those in BALB/c mice. Conclusion: NC fcgr2b affects the level of Fc[gamma]RIIB in GC B cells but that the reduced Fc[gamma]RIIB expression is not related to enhanced Ag-specific Ab responses in NC mice.

Key words: NC/Nga mice, Fc[gamma]RIIB, antibody, congenic mice
Article Type: Report
Subject: Albumin (Physiological aspects)
Albumin (Research)
Immune response (Physiological aspects)
Immune response (Genetic aspects)
Immune response (Research)
Immunoglobulins (Physiological aspects)
Immunoglobulins (Research)
Authors: Sakai, Tohru
Nakamoto, Mariko
Suto, Emi
Pub Date: 07/01/2009
Publication: Name: American Journal of Immunology Publisher: Science Publications Audience: Professional Format: Magazine/Journal Subject: Biological sciences Copyright: COPYRIGHT 2009 Science Publications ISSN: 1553-619X
Issue: Date: July, 2009 Source Volume: 5 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: SIC Code: 2836 Biological products exc. diagnostic
Geographic: Geographic Scope: Japan Geographic Code: 9JAPA Japan
Accession Number: 208134191

Fc[gamma]RIIB, 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 IgG-containing immune complex [1-3]. 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 Fc[gamma]RIIB 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.


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 x NC)F1 mice x 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 Fc[gamma]RIIB but not with that of NC. Backcross mice showing reduced levels of Ly 17.2 staining were judged as being positive for NC fcgr2b.

Immunization and flow cytometric analysis: Mice were intraperitoneally immunized with 10 [micro]g of Ovalubumin (OVA) (Sigma Chemical Co., MO, USA) absorbed in 1 mg of Aluminium Hydroxide Gel Adjuvant (HCI Biosector, Denmark) 2 times at a 2week interval. Serum OVA-specific Ab levels were measured by the standard method. Spleen cells from OVA-immunized mice were stained FITC-anti-IgD mAb, PE-anti-Fc[gamma]RIIB/FcgRII mAb (clone 2.4G2), APC-anti-B220 mAb and biotin-peanut lectin (aggutinin) (PNA), followed by PerCP-streptavidin. Stained cells were analyzed by FACSCalibur and CellQuest software (BD Biosciences, Mountain View, CA).

Statistical analysis: Statistical analysis was done by one-way ANOVA followed by Sheffe's multiple comparison test. Values in the text are means [+ or -] SD. Differences were considered significant at p<0.05.


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 ~100-times 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 cell-dependent Ags. It has been shown that Fc[gamma]RIIB on GC B cell regulates Ab-Forming Cells (AFC) [7,8]. BCR and Fc[gamma]RIIB are cross-linked when GC B cells interact with Ag, resulting in inhibition of AFC differentiation [9]. We evaluated the expression of Fc[gamma]RIIB in GC B cells because down-regulation of Fc[gamma]RIIB in the GC has been reported in NZB mice, which have deletion mutation of fcg2b similar to that in NC mice [10-12]. Figure 2 shows Fc[gamma]RIIB expression levels in GC ([PNA.sup.high] IgD-) and non-GC ([PNA.sup.low] [IgD.sup.+]) B cells in BALB/c and congenic mice at the time of primary immune response by OVA immunization. Expression of Fc[gamma]RIIB was upregulated in GC B cells compared to that in non-GC B cells in BALB/c mice, whereas expression of Fc[gamma]RIIB in congenic mouse GC B cells was two-fold lower than that in BALB/c mice. Therefore, NC fcgr2b affects the level of Fc[gamma]RIIB expression in GC B cells but does not affect Ag-specific Ab levels. Previous studies showed that expression of Fc[gamma]RIIB in GC B cells is down-regulated in NZB mice [11,12]. However, a recent study by Rahman showed that expression of Fc[gamma]RIIB in GC B cells is unchanged in autoimmuneprone mice by using multiple GC B cell maker Ags [13]. Our results showing increased expression of Fc[gamma]RIIB in GC B cells are consistent with Rahman's report. 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 Fc[gamma]RIIB 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 Fc[gamma]RIIB 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]. Fc[gamma]RIIB-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 Fc[gamma]RIIB 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 [17-19]. Thereby, contribution of these genes on Ag-specific Ab production is not excluded at present stage.




The present study showed that NC fcgr2b affects the level of Fc[gamma]RIIB in GC B cells but that the reduced Fc[gamma]RIIB expression is not related to enhanced Ag-specific 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.


[1.] Amigorena, S., C. Bonnerot, J.R. Drake, D. Choquet and W. Hunziker et al., 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science, 256: 1808-1812. http://cat.inist.fr/?aModele=afficheN&cpsidt=5433478

[2.] Bolland, S. and J.V. Revetch, 1999. Inhibitory pathways triggered by ITIM-containg receptors. Adv. Immunol., 72: 149-177. http://www.ncbi.nlm.nih.gov/pubmed/10361574

[3.] Takai, T., 2002. Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol., 2: 580-592. http://www.ncbi.nlm.nih.gov/pubmed/12154377

[4.] Matsuda, H., N. Watanabe, G.P. Geba, J. Sperl and M. Tsudzuki et al., 1997. Development of atopic dermatitis-like skin lesion with hyperproduction in NC/Nga mice. Int. Immunol., 9: 461-466. http://www.ncbi.nlm.nih.gov/pubmed/9088984

[5.] Matsumoto, M., C. Ra, K. Kawamoto, H. Sato and A. Itakura et al., 1999. IgE hyperproduction through enhanced tyrosine phosphorylation of Janus kinase 3 in NC/Nga mice, a model for human atopic dermatitis. J. Immunol., 162: 1056-1063. http://cat.inist.fr/?aModele=afficheN&cpsidt=9913321

[6.] Sakai, T., M. Kogiso, K. Mitsuya, T. Komatsu and S. Yamamoto, 2006. Deletion polymorphisms in the promotor region of Fcg receptor IIB is not associated with antigen-specific IgG2a and IgG2b antibody responses in NC/Nga Mice. Microbiol. Immunol., 50: 475-480. http://cat.inist.fr/?aModele=afficheN&cpsidt=17952925

[7.] Berek, C., A. Berger and M. Apel, 1991. Maturation of the immune response in germinal centers. Cell, 90: 293-301. http://www.ncbi.nlm.nih.gov/pubmed/1760840

[8.] Jacob, J., G. Kelsoe, K. Rajewsky and U. Weiss, 1991. Intraclonal generation of antibody mutants in germinal centers. Nature, 354: 389-392. http://cat.inist.fr/?aModele=afficheN&cpsidt=5174446

[9.] Tarlinton, D.M. and K.G. Smith, 2000. Dissecting affinity maturation: A model explaining selection of antibody-forming cells and memory B cells in the germinal center. Immunol. Today, 21: 436-441. http://www.ncbi.nlm.nih.gov/pubmed/11738998

[10.] Luan, J.J., R.C. Monteiro, C. Sautes, G. Fluteau and L. Eloy et al., 1996. Defective Fc[gamma]RII gene expression in macrophages of NOD mice. J. Immunol., 157: 4707-4716. http://www.jimmunol.org/cgi/content/abstract/157/10/4707

[11.] Jiang, Y., S. Hirose, M. Abe, R. Sanokawa-Akakura and M. Ohtsuji et al., 2000. Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility. Immunogenetics, 51: 429-435. http://www.ncbi.nlm.nih.gov/pubmed/10866109

[12.] Xiu, Y., K. Nakamura, M. Abe, N. Li and X.S. Wen et al., 2002. Transcriptional regulation of Fcgr2b by polymorphic promoter region and its contribution to humoral immune responses. J. Immunol., 169: 4340-4346. http://www.ncbi.nlm.nih.gov/pubmed/12370366

[13.] Rahman, Z.S.M. and T. Manser, 2005. Failed upregulation of the inhibitory IgG Fc receptor Fc[gamma]RIIB on germinal center B cells in autoimmune-prone mice is not associated with deletion polymorphisms in the promoter region of the Fc[gamma]RIIB gene. J. Immunol., 175: 1440-1449. http://www.ncbi.nlm.nih.gov/pubmed/16034080

[14.] Reiner, S.L. and R.M. Locksley, 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol., 12: 151-177. http://www.ncbi.nlm.nih.gov/pubmed/7612219

[15.] Sher, A. and R.L. Coffman, 1992. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Annu. Rev. Immunol., 10: 385-409. http://www.ncbi.nlm.nih.gov/pubmed/1590992

[16.] Terasenko, T., H.K. Kole and S. Bolland, 2008. A lupus-suppressor BALB/c locus restricts IgG2 autoantibodies without altering intrinsic B cell-tolerance mechanisms. J. Immunol., 180: 3807-3814. http://www.ncbi.nlm.nih.gov/pubmed/18322187

[17.] Boackle, S.A., V.M. Holers, X. Chen, G. Szakonyi, D.R. Karp, E.K. Wakeland and L. Morel, 2001. Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein. Immunity, 15: 775-785. http://cat.inist.fr/?aModele=afficheN&cpsidt=13380515

[18.] Morel, L., K.R. Blenman, B.P. Croker and E.K. Wakeland, 2001. The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related gene. Proc. Natl. Acad. Sci. USA., 98: 1787-1792. http://adsabs.harvard.edu/abs/2001PNAS...98.1787M

[19.] Rozzo, S., J.J.D. Allard, D. Choubey, T.J. Vyse, S. Izui, G. Peltz and B.L. Kotzin, 2001. Evidence for an interferon-inducible gene, ifi202, in the susceptibility to systemic lupus erythematosus. Immunity, 15: 435-443. http://www.ncbi.nlm.nih.gov/pubmed/11567633

Corresponding Author: Tohru Sakai, Department of Public Health and Applied Nutrition, Institution of Health Bioscience, University of Tokushima Graduate School, Tokushima, Tokushima 770-8503, Japan Tel: +81-88-633-7096 Fax: +81-88-633-9427

Tohru Sakai, Mariko Nakamoto and Emi Suto Department of Public Health and Applied Nutrition, Institution of Health Bioscience, University of Tokushima Graduate School, Tokushima, Tokushima 770-8503, Japan
Gale Copyright: Copyright 2009 Gale, Cengage Learning. All rights reserved.