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The Journal of Immunology, 2005, 175: 1440-1449.
Copyright © 2005 by The American Association of Immunologists

Failed Up-Regulation 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

Ziaur S. M. Rahman and Tim Manser1

Department of Microbiology and Immunology and Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fc{gamma}RIIB, a low-affinity FcR for IgG, inhibits BCR-mediated activation when these two receptors are cocross-linked by Ags and IgG-containing immune complexes. Although a role for Fc{gamma}RIIB in the germinal center (GC) reaction has been proposed, conflicting results have been published regarding the levels of Fc{gamma}RIIB expressed on GC B cells in normal and autoimmune-prone mice and humans. In the present study, we investigate this issue in detail in mice by using multiple GC B cell markers, two different antigenic systems, primary and secondary GC responses, and by excluding the influence of splenic influx of immature B cells and passive acquisition of Fc{gamma}RIIB from follicular dendritic cells. Our results are in concordance with previous data indicating that Fc{gamma}RIIB expression is up-regulated on GC B cells in normal mice. In contrast, we observe comparable levels of Fc{gamma}RIIB on GC and non-GC B cells in New Zealand White, New Zealand Black, and B6.Sle1 autoimmune-prone strains. Therefore, we suggest that these strains exhibit failed up-regulation of Fc{gamma}RIIB on GC B cells, rather than down-regulation, as previously suggested. Also, in contrast to previous indications, this perturbed regulation is not uniquely associated with deletion polymorphisms in the promoter region of the Fc{gamma}RIIB gene but does appear to be independent of genetic background. Finally, we present evidence indicating that Fc{gamma}RIII, a low-affinity activating IgG FcR, is expressed on the GC B cells of normal but not autoimmune-prone mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Three types of FcRs for IgG are widely expressed on hemopoietic cell lineages. These Fc{gamma}Rs play important roles in maintaining homeostasis of the Ab response and link IgG Abs with effector responses (1, 2, 3). They have either high (Fc{gamma}RI) or low (Fc{gamma}RII and III) affinity for IgGs, and the low-affinity Fc{gamma}Rs can only bind IgG in the form of immune complexes (ICs) 2 (4). Fc{gamma}RI and Fc{gamma}III are activating receptors, which mediate signaling through a common {gamma}-chain. In contrast, Fc{gamma}RIIB is an inhibitory receptor that contains an ITIM in its cytoplasmic tail (5, 6). Fc{gamma}RIIB1 and Fc{gamma}RIIB2 are two isoforms of Fc{gamma}RIIB, resulting from the alternative splicing of RNA encoded by a single gene (4, 5, 7, 8). Murine B cells appear to lack expression of Fc{gamma}RIIB2 and activating Fc{gamma}Rs but express Fc{gamma}RIIB1 (5, 9).

Upon IC-mediated coaggregation with the BCR, Fc{gamma}RIIB1 recruits SHIP to its ITIM motif. SHIP then hydrolyzes phosphatidylinositol 3,4,5-triphosphate to phosphatidylinositol 3,4-triphosphate (10, 11, 12, 13), resulting in the dissociation of pleckstrin homology-domain containing proteins such as Bruton’s tyrosine kinase and phospholipase C{gamma} from the inner leaflet of the plasma membrane (10, 11). This precludes the intracellular calcium mobilization necessary to obtain a sustained B cell activation response (11, 13). Extensive cross-linking of Fc{gamma}RIIB alone has also been reported to induce B cell apoptosis via an ITIM/SHIP-independent pathway (14, 15, 16).

Germinal centers (GCs) are microenvironments formed in the follicles of secondary lymphoid organs by highly proliferative B cells responding to T cell-dependent Ags. Somatic hypermutation alters the variable regions of BCRs expressed by GC B cells (17, 18, 19). Thus, a diverse repertoire of B cells is generated in GCs. Follicular dendritic cells (FDCs) are the major stromal cell type in GCs and can trap Ags on their surface in the form of ICs via complement receptors (20, 21, 22) and FcRs (23, 24, 25). Several models for the GC response have suggested that GC B cells with high affinity and specificity for Ags in ICs on FDCs are selected, at the expense of their low-affinity and autoreactive counterparts, into the memory B cell compartment (19, 26, 27).

Such models have primarily focused on the role of the BCR, but a few have also considered an influence of Fc{gamma}RIIB in this specificity maturation process. Tarlinton and Smith (28) have suggested that Fc{gamma}RIIB on GC B cells regulates Ab-forming cells (AFCs) vs memory B cell development. According to their model, early during the response GC B cells predominantly differentiate into AFCs. Late in the response, when high titers of IgG specific for the driving Ag have been produced, ICs become the major form of this Ag in the GC. As a result, the BCR and Fc{gamma}RIIB are cocross-linked when GC B cells interact with Ags, resulting in inhibition of the AFC differentiation pathway and promotion of memory B cell production.

Ravetch and Lanier (29) have proposed that the net outcome of B cell selection in the GC response depends on the relative strength of opposing signals from the BCR and Fc{gamma}RIIB. In their model, engagement of ICs by Fc{gamma}RIIB on GC B cells with low affinity for Ags, or B cells with autoreactivity, predominates over BCR engagement of Ags in these ICs, resulting in cell death. Conversely, coengagement of the BCR and Fc{gamma}RIIB by ICs on high-affinity GC B cells culminates in signaling that inhibits proliferation and apoptosis, leading to survival and progression to memory B cells (14).

These models have not considered how modulation of Fc{gamma}RIIB surface levels might influence positive and negative selection of GC B cells. Conflicting results have been published regarding the levels of Fc{gamma}RIIB expression on GC B cells in different strains of mice and in humans. Three types (New Zealand Black (NZB), New Zealand White (NZW), and C57BL/6) of murine Fc{gamma}RIIB alleles have been described previously (30). It was reported that Fc{gamma}RIIB expression is up-regulated on activated (31, 32) and GC B cells (30, 33) in mice carrying the C57BL/6 allele and down-regulated on GC B cells in mice with the NZB allele and to a lesser extent on GC B cells in mice containing the NZW allele (30, 34). This reduced expression of Fc{gamma}RIIB associated with the NZB and NZW alleles was suggested to have the potential to accelerate autoimmune disease (30, 34).

Down-regulation of Fc{gamma}RIIB on GC B cells was argued to be due to small, polymorphic deletions in the promoter regions of the Fc{gamma}RIIB gene in NZB mice (30, 33, 34). These promoter deletions overlap putative transcription factor (AP-4 and S box) binding sites and chimeric Fc{gamma}RIIB reporter constructs containing these deletions drive lower levels of transcription in B cell lines than constructs lacking these deletions (33). High titers of antinuclear Abs develop in Fc{gamma}RIIB-deficient C57BL/6 (B6.RIIB–/–) mice (35), raising the possibility that down-regulation of Fc{gamma}RIIB in autoimmune strains may be functionally equivalent to an Fc{gamma}RIIB deficiency.

However, data from our laboratory suggested that levels of Fc{gamma}RIIB were down-regulated on primary GC B cells in nonautoimmune C57BL/6 mice (36). Adding to the confusion, data inconsistent with many of the results from mice have been obtained from the analysis of Fc{gamma}RIIB expression on human B cells. GC B cells in normal human tonsil and other reactive lymphoid tissues have been reported to express low or undetectable levels of Fc{gamma}RIIB (37). In addition, elevated levels of Fc{gamma}RIIB were found on EBV-transformed B cells and peripheral blood B cells from human donors with the 2B.4 haplotype of Fc{gamma}RIIB promoter region polymorphisms (38). The Fc{gamma}RIIB 2B.4 haplotype is associated with systemic lupus erythematosus (39). Collectively, previous data on whether and in which direction Fc{gamma}RIIB levels on GC B cells are modulated in normal and autoimmune-prone situations have failed to reach consensus. In the studies reported here, we have attempted to resolve this controversy in the case of the murine GC response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6, BALB/c, NZB, NZW, and 129/Sv mice were purchased from The Jackson Laboratory. B6.Sle1 mice have been previously described (40, 41, 42) and were kindly provided by Drs. C. Mohan and E. Wakeland (University of Texas Southwestern Medical Center, Dallas, TX). B6.RIIB–/– mice (35, 43) were purchased from The Jackson Laboratory or kindly supplied by Dr. J. Ravetch (Rockefeller University, New York, NY). Animals were maintained in a pathogen-free barrier facility. All mice used in these studies were 8–13 wk old.

Mixed bone marrow (BM) chimeras

Mixed BM chimeras were generated through adoptive transfer of a 1:1 mixture of BM from C57BL/6 and µMT (C57BL/6-Igh-6tm/Cgn) mice into lethally irradiated (1000-rad whole body irradiation) C57BL/6 control and B6.RIIB–/– mice as described previously (44). All chimeras were allowed to reconstitute for at least 40 days before immunizations.

Immunizations

C57BL/6 and B6.Sle1 mice were immunized i.p. with SRBCs (Lampire Biological Laboratories) as described for primary responses (36, 45), and half this number of SRBCs was used for induction of secondary immune responses. Chimeric mice were immunized with 50 µg of (4-hydroxy-3-nitrophenyl) acetyl chicken {gamma}-globulin (NP16-CGG) in alum for primary responses and with 10 µg of NP16-CGG in PBS for secondary responses, as described previously (46). Secondary responses were induced by i.p. boosting 4–5 wk after primary immunization with 10 µg of NP16-CGG in PBS.

Abs and other reagents

Abs and other reagents used for flow cytometry and immunohistology included the following: FITC-GL7; PE, FITC and PE-Texas red-anti-B220 (clone RA3-6B2); PE-anti-Fc{gamma}RIIB/Fc{gamma}RIII (clone 2.4G2); PE-anti-IgD (clone 11-26); rat IgG Ab to mouse FDCs (FDC-M1); streptavidin (SA)-CyChrome; PE-anti-NK1.1 and FITC-anti-2B4 (BD Pharmingen); biotin-(Fab')2 mouse anti-rat IgG (Jackson ImmunoResearch Laboratories); biotin-anti-IgD (clone 11-26; Southern Biotechnology Associates); SA-PE; avidin-Alexa Fluor 488 (Molecular Probes); and FITC-peanut lectin (agglutinin) (PNA) (Vector Laboratories). The K9.361 hybridoma was a kind gift from Dr. U. Hammerling (Sloan-Kettering Memorial Hospital, New York, NY), and the mAb it produces was biotinylated in our laboratory.

Immunohistology

Spleen cryostat sections (5–6 µm) were prepared as described previously (46). Immunohistology was performed using immunofluorescent staining, and the stained sections were analyzed using a fluorescence microscopy (Leitz Diaplan), and images were captured as described previously (44).

Flow cytometry

Four-color flow cytometric analysis was done on cell suspensions prepared from spleens of immunized mice and stained with multiple combinations of the Abs and reagents listed above. Biotinylated Abs were detected with SA-CyChrome. Stained cells were processed using a Coulter Epics XL/MCL analyzer. These data were analyzed using FlowJo software (Tree Star).

Cell sorting, RNA extraction, and real-time RT-PCR

B220+PNA–IgD+ and B220+PNA+IgDlow/neg mantle zone (non-GC) and GC B cells, respectively, from SRBC-immunized C57BL/6 and B6.Sle1 mice were purified using a MoFlo fluorescent activated high-speed sorter (DakoCytomation). NK1.1+2B4+ NK cells were also sorted from naive C57BL/6 mice and used as a positive control in some experiments. RNA was extracted from the sorted cells using the RNeasy mini kit (Qiagen), following the manufacturer’s instructions. RNA was reverse transcribed with TaqMan reverse transcription reagents (Applied Biosystems). Real-time RT-PCR and raw relative quantification (RQ) values for Fc{gamma}RIIB and RIII gene expression, using GAPDH as an endogenous control, were collected using the ABI Prism 7000 sequence detection system (Abbott Diagnostics), using TaqMan gene expression assays and universal master mix with AmpErase uracil N-glycosylase (Applied Biosystems).

PCR, cloning, and sequencing

Genomic DNA was extracted from mouse tail tissue. PCR amplification of the putative regulatory regions of the Fc{gamma}RIIB gene was achieved with the following pairs of primers: 1) 5'-GCTGCAGAATCTGAGAAAC-3' (5' primer) and 5'-AGTACCCAGAGAACAGAC-3' (3' primer) for the promoter and 5' transcribed region; 2) 5'-GTGTGCGTTCTCACTTGCTGC-3' (5') (30) and 5'-CTCCTGAGATCTACCCTGCT-3' (3'); and 3) 5'-CGTGTCAAAACCAAAAGTGT-3' (5') and 5'-ACATGGGACAGGAATGCTA-3' (3') for regions 3 and 4, respectively, located in the third intron. PCR product purification, cloning, and sequencing analyses were done as described previously (47).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Elevated levels of Fc{gamma}RIIB on GC B cells in C57BL/6 mice

We first investigated whether past discrepant results regarding levels of Fc{gamma}RIIB expression on murine GC B cells might have resulted from the use of different antigenic systems, immunization protocols, or cell surface phenotypic qualities (GL7 vs PNA binding, IgDlow vs IgDneg) to define GC B cells in flow cytometric studies. We also considered the potential confounding influence of influx of B220lowGL7+/PNA+ immature B cells into the spleen because this often takes place in mice immunized with adjuvant (48, 49).

No single-cell surface marker unambiguously distinguishes GC B cells from other B cell subsets, and B220+PNAhigh cells do contain GL7– and IgD+ cells (36, 45). Therefore, to establish a more rigorous operative definition of GC B cell phenotype for flow cytometry, we first examined primary splenic GC responses in SRBC-immunized B6 mice using either GL7 or PNA, in combination with anti-B220 and anti-IgD (Fig. 1A). Based on B220 and GL7/PNA staining, we divided B cells into B220high/GL7high or PNA+, or B220high/GL7– or PNA– mature B cells and B220low/GL7high or PNA+, or B220low/GL7– or PNA– immature B cell subpopulations (rectangular gates, Fig. 1A, upper and lower left panels). We then defined B220high/PNA– or GL7–/IgD+, and B220high/PNA+ or GL7high/IgDlow and B220high/PNA+ or GL7high/IgDneg cells as non-GC and two subsets of GC B cells, respectively (rectangular gates in Fig. 1A, upper and lower right panels).



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  		FIGURE 1. Flow cytometric definition of GC B cell phenotype and levels of Fc{gamma}RIIB expression on these cells. A, Splenocytes from SRBC-immunized C57BL/6 mice on day 9 of the immune response were stained with either GL7 or PNA, in combination with anti-B220 and anti-IgD. B220highGL7+/PNA+ or B220highGL7–/PNA– mature and B220lowGL7+/PNA+ or B220lowGL7–/PNA– immature populations are shown in upper and low rectangular gates, respectively. Upper and lower right panels, Non-GC (B220highGL7–/PNA–IgD+) and GC (B220highGL7+/PNA+IgDlow/neg) B cells are shown in rectangular gates. B, GC vs non-GC B cell populations from SRBC-immunized C57BL/6 and B6.RIIB–/– mice are shown in the three rectangular gates defined in A. The levels of staining with 2.4G2 (left two panels) and K9.361 (right two panels) on non-GC (red histogram) and GC B cells (blue and green histograms for B220highGL7+/PNA+IgDlow and B220highGL7+/PNA+IgDnegative, respectively) are shown. These data represent two independent experiments, each including pooled samples from two to three mice of each strain.

 
Using these definitions of B cell phenotype, we performed four-color flow cytometric analyses on splenocytes obtained from SRBC-immunized B6 and B6.RIIB–/– mice on day 9 of the immune response to measure Fc{gamma}RIIB levels on B cell subpopulations. The mAb 2.4G2 that recognizes the extracellular domains of both Fc{gamma}RII and III and the mAb K9.361, which is specific for Fc{gamma}RIIB (Ly17.2 allotype) in B6 mice, were used. As shown in Fig. 1B, compared with non-GC B cells (red lines in histograms), the levels of staining with K9.361 were elevated on both subsets of GC B cells (blue and green lines in histograms for B220high/PNA+ or GL7high/IgDlow and B220high/PNA+or GL7high/IgDneg cells, respectively) in B6 mice using both PNA and GL7, whereas background levels of staining were observed in B6.RIIB–/– mice. Similar results were obtained using 2.4G2 on the two GC B cell subpopulations in B6 mice. However, the levels of 2.4G2 but not K9.361 staining were somewhat higher on GL7+ GC as compared with non-GC B cells in B6.RIIB–/– mice, possibly indicating low-level expression of Fc{gamma}RIII. Among B220low cells, both GL7high and PNA+ subpopulations displayed increased levels of 2.4G2 staining in B6 mice, as compared with B220high/GL7– or PNA– non-GC B cells (data not shown).

Most previous studies have evaluated Fc{gamma}RIIB expression on GC B cells during secondary immune responses (30, 33, 34), while our laboratory’s past studies were restricted to the primary immune response (36). Therefore, we next examined Fc{gamma}RIIB expression on various B cell subpopulations (defined as described above) in the spleens of B6 mice on day 4 after secondary SRBC immunization. These experiments yielded results analogous to those obtained from the analysis of the primary anti-SRBC GC response (Fig. 2 and data not shown). We also evaluated Fc{gamma}RIIB expression in both primary and secondary responses of B6 mice immunized with NP16-CGG (which induces less robust GC responses than SRBCs) as described above and obtained results concordant with those found using SRBCs (data not shown).



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  		FIGURE 2. Levels of Fc{gamma}RIIB expression on GC B cells in the secondary immune response. Four-color (anti-B220, anti-IgD, 2.4G2 or K9.361, and GL7 or PNA) flow cytometry analysis was performed on splenocytes obtained from SRBC-immunized C57BL/6 mice on day 4 of the immune response. As described in the legend to Fig. 1, non-GC and two subsets of GC B cells are shown in rectangular gates for both GL7 and PNA. The staining intensity of 2.4G2 and K9.361 for non-GC (red) and GC (blue and green) B cells, as described in Fig. 1, is shown. These data are representative of three independent experiments of pooled samples from two to three mice.

 
Increased levels of Fc{gamma}RIIB on GC B cells are not the result of passive acquisition from FDCs, and Fc{gamma}RIIB expression on stromal elements, including FDCs, has no apparent influence on the regulation of Fc{gamma}RIIB on GC B cells

During the GC reaction, FDCs express very high levels of Fc{gamma}RIIB (36, 44). Therefore, we tested whether the increased levels of Fc{gamma}RIIB on GC B cells resulted from passive acquisition from FDCs before or during the experimental manipulations necessary for flow cytrometric analysis. These same studies allowed an evaluation of whether Fc{gamma}RIIB on radioresistant stromal elements, including FDCs, had any influence on the regulation of Fc{gamma}RIIB on GC B cells. We generated the following mixed BM chimeras: B6 + B6.µMT (B cell deficient) -> B6.RIIB–/–, in which B cells are derived from the pure B6 background, and lymphocytes and myeloid cells are Fc{gamma}RIIB sufficient, whereas stromal elements, including FDCs, are Fc{gamma}RIIB deficient, and the control chimeras B6 + B6.µMT -> B6, in which lymphocytes, FDCs, and myeloid cells all are Fc{gamma}RIIB sufficient.

These chimeric mice were immunized with NP16-CGG and evaluated for Fc{gamma}RIIB expression on splenic GC B cells (defined as described above, except sIgDlow and sIgD– cells were analyzed together) on days 12 and 4 of the primary and secondary GC responses, respectively. The levels of 2.4G2 and K9.361 staining (Fig. 3, A and B) were similarly higher on GC B cells (blue lines in histograms) as compared with non-GC B cells (red lines in histograms) in both primary (Fig. 3A) and secondary (Fig. 3B) GC responses in the presence (B6 + B6.µMT -> B6) or absence (B6 + B6.µMT -> B6.RIIB–/–) of Fc{gamma}RIIB on FDCs and stromal elements.



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  		FIGURE 3. Fc{gamma}RIIB expression on GCs B cells in BM chimeric mice. Four-color flow cytometry analysis, conducted and illustrated as described in Figs. 1 and 2 with the exception that IgDlow and IgD– GC B cells were grouped, was performed on splenocytes obtained on day 12 of the primary (A) and day 4 of the secondary (B) responses of NP16-CGG-immunized B6 + B6.µMT -> B6 and B6 + B6.µMT -> B6.RIIB–/– chimeric mice. The data presented were obtained from the pooled cells of two to three mice of each group and are representative of those obtained in five independent experiments.

 
Perturbed up-regulation of Fc{gamma}RIIB on GC B cells in autoimmune-prone mice

We next examined whether Fc{gamma}RIIB expression levels were up-regulated on the GC B cells of the NZB and NZW mouse strains that contain the two other previously described Fc{gamma}RIIB alleles, as well as in B6.Sle1 mice, using the protocol described above. B6.Sle1 (also called Sle1) is a congenic strain that carries the 37- cM long Sle1 susceptibility interval of NZW origin from the NZM2410 autoimmune strain, a recombinant inbred line derived from NZW and NZB (40, 41, 42). The NZM2410/Sle1 interval in B6.Sle1 mice has been reported to contain the Fc{gamma}RIIB gene, allowing a potential assessment of whether B6 background genes might epistatically influence expression of a non-B6 Fc{gamma}RIIB allele on GC B cells. Given the mixed origin of the B6.Sle1 strain, we first performed preliminary experiments with K9.361 and found that this mAb did not recognize the B6.Sle1 allotypic form of Fc{gamma}RIIB (data not shown), supporting the conclusion that Fc{gamma}RIIB in these mice is of the Ly17.1 allotype derived from NZM2410 mice (41, 42, 50, 51). Consequently, only the 2.4G2 mAb could be used in these studies to examine levels of Fc{gamma}RIIB on GC B cells.

As illustrated in Fig. 4, in contrast to splenic GC B cells in B6 mice (top row), we found little or no elevation of 2.4G2 staining levels on splenic GC B cells (blue and green lines in histograms for B220highPNA+ or GL7highIgDlow and B220highPNA+ or GL7highIgDneg cells, respectively) as compared with non-GC B cells (red lines in histograms) on day 9 of the primary anti-SRBC response of NZB, NZW, or B6.Sle1 mice. Analogous results were obtained on day 4 of the secondary anti-SRBC response (data not shown). Moreover, little or no difference in 2.4G2 staining levels were observed among either GC or non-GC B cells in these strains. These data indicate a defect in up-regulation of Fc{gamma}RIIB expression on GC B cells in these strains of mice.



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  		FIGURE 4. Fc{gamma}RIIB expression on GC B cells in autoimmune prone mouse strains. Splenocytes obtained from SRBC-immunized C57BL/6, NZW, NZB, and B6.Sle1 mice on day 9 of the immune response were stained, analyzed, and the data illustrated as detailed in the legend to Fig. 1. These data are representative of two individual experiments in which pooled samples from two to three mice of each strain were analyzed.

 
Also in contrast to our findings in B6 mice, B220low/GL7high or PNA+ spleen cells revealed heterogeneous but generally lower levels of 2.4G2 staining than B220high/GL7– or PNA– non-GC B cells (data not shown) in NZB, NZW, and B6.Sle1 mice. This suggests that altered regulation of Fc{gamma}RIIB expression extends to the immature B cell compartment of these strains, but more rigorous studies will be required to define the lineage identities of the cells in this subpopulation.

Fc{gamma}RIIB expression in B6.Sle1 GCs

We next investigated whether the altered expression of Fc{gamma}RIIB we observed by flow cytometry on B6.Sle1 GC B cells was evident on GC B cells and FDCs defined histologically. Parallel spleen sections obtained on day 9 of the anti-SRBC response were stained with 2.4G2 and anti-IgD (Fig. 5, left panels) and FDC-M1 and GL7 (Fig. 5, right panels) to evaluate the expression of Fc{gamma}RIIB on GC B cells and FDCs, respectively. Although 2.4G2 staining in B6 and B6.Sle1 GC FDC-rich regions was comparable, this staining was undetectable in B6.Sle1 GCs in areas deficient in FDCs. In the FDC-poor regions of B6.RIIB–/– GCs, the intensity of 2.4G2 staining was much lower than in analogous regions of B6 GCs but higher than in these regions in B6.Sle1 GCs, indicating the possibility of low-level expression of Fc{gamma}RIII on B6.RIIB–/– but not B6.Sle1 GC B cells. This was consistent with our flow cytometry data in which GL7+ GC B cells revealed a higher level of 2.4G2 staining as compared with non-GC B cells in B6.RIIB–/– mice (Fig. 1B).



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  		FIGURE 5. Fc{gamma}RIIB expression on B cells and FDCs in B6.Sle1 GCs. Adjacent spleen sections, obtained from SRBC-immunized B6, B6.RIIB–/–, and B6.Sle1 mice on day 9 of the primary immune response, were stained with 2.4G2 (red) and anti-IgD (green) and FDC-M1 (red) and GL7 (green). Two representative GCs from each strain of mice are illustrated. These data are representative of those obtained from three mice from each strain. Original magnification of images, x250.

 
Both Fc{gamma}RIIB and Fc{gamma}RIII RNA levels are up-regulated in C57BL/6 and down-regulated in B6.Sle1 GC B cells

Our analysis of B6 and B6.RIIB–/– mice indicated that the majority of 2.4G2 staining on GC B cells was due to expression of Fc{gamma}RIIB. Nonetheless, because this mAb recognizes the extracellular domains of both Fc{gamma}RIIB and Fc{gamma}RIII, a fraction of the 2.4G2 staining differences observed in the autoimmune strains could have resulted from altered expression of Fc{gamma}RIII. Therefore, we went on to measure levels of RNA encoding these proteins in sorted B220+PNA+IgDlow/neg (GC) and B220+PNA–IgD+ (non-GC) B cells obtained on day 9 of the SRBC response via real-time RT-PCR. In this analysis, B220high and low B cells were not distinguished because the small percentage of B220low PNA+ immature B cells we observed in our studies (≤5% of total B220+/PNA+ cells) were unlikely to overtly influence the results. Fc{gamma}RIIB mRNA levels were found to be 2-fold higher in GC compared with non-GC B cells in B6 mice but were at least 5-fold lower in B6.Sle1 GC B cells (Fig. 6A). We also observed a substantial level of Fc{gamma}RIII RNA expression in B6 GC B cells as compared with undetectable levels in non GC B cells and nearly undetectable levels in B6.Sle1 GC and non-GC B cells (Fig. 6B).



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  		FIGURE 6. Levels of Fc{gamma}RIIB and Fc{gamma}RIII RNA in GC B cells. RNA was extracted from FACS-purified non-GC (B220+PNA–IgD+) and GC (B220+PNA+IgDlow/neg) B cells obtained on day 9 of the SRBC immune response of B6 and B6.Sle1 mice. Raw RQ values of Fc{gamma}RIIB (A) and Fc{gamma}RIII (B) expression in non-GC (black bars) and GC (green bars) B cells in B6 and B6.Sle1 mice were obtained via real-time RT-PCR by setting the RQ value for non-GC B cells as 1. RNA from purified NK cells (black bars) in the lower panel served as a positive control for Fc{gamma}RIII gene expression. Real-time RT-PCR amplification was done in triplicate, from which the error bars were generated. These data are representative of pooled cells of three mice of each genotype obtained from a single experiment.

 
Promoter region deletion polymorphisms are not correlated with altered expression of Fc{gamma}RIIB on GC B cells

We next sequenced the promoter and 5' transcribed region, and various other putative regulatory regions of the Fc{gamma}RIIB gene isolated from the genomic DNA of B6, NZB, 129/Sv, NZW, and B6.Sle1 mouse strains. As shown in Fig. 7A, the sequence of the Fc{gamma}RIIB promoter region in NZW mice was found to be identical to that in B6.Sle1 mice, supporting the conclusion that the Fc{gamma}RIIB allele in B6.Sle1 mice is derived from the NZW genomic interval of NZM2410 mice. Consistent with previous data, we found 13- and 3-nt deletions in the promoter region, and a 16-nt insertion in the first intron of the NZB and 129/Sv Fc{gamma}RIIB genes (30). However, these polymorphisms were absent in the Fc{gamma}RIIB alleles of NZW and B6.Sle1 mice (Fig. 7A). Our sequencing results for putative regulatory regions 3 and 4 in the third intron were also in concordance with previous data in that the NZB (NZB and 129/Sv mice) and NZW alleles (NZW and B6.Sle1 mice) shared a 4-nt deletion at position 4133–4136 in region 3, and in region 4, a 24-nt insertion was observed only in the NZW allele. These sequencing data are summarized in Table I.



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  		FIGURE 7. Polymorphisms in putative regulatory regions of the Fc{gamma}RIIB gene. Nucleotide sequences of the promoter and 5' transcribed region (A) and regions 3 (B) and 4 (C) in the third intron of the Fc{gamma}RIIB gene in several nonautoimmune and autoimmune-prone strains of mice (C57BL/6, 129/Sv, NZB, NZW, and B6.Sle1) are shown as compared with the previously published sequence from BALB/C in this region. Numbers indicate the nucleotide position relative to the major transcription initiation site (+1). A dash indicates sequence identity, asterisks indicate deletions, and nucleotide differences are shown explicitly. Exon sequences are shown in underlined italic font. The location of a small insertion in the first intron (A) of 129/Sv and NZB mice is shown using arrows.

 

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  		Table I. Polymorphisms in the putative regulatory regions of the Fc{gamma}RIIB gene

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Defining the expression levels of Fc{gamma}RIIB on GC B cells is a crucial first step in evaluating models that propose a central role for this inhibitory FcR in the clonal selection processes that take place during the GC response (14, 28, 29). Previously, several groups, including our own, have conducted such investigations, and inconsistent results were obtained (30, 33, 34, 36). Therefore, we felt compelled to comprehensively revisit the issue of whether and to what extent Fc{gamma}RIIB expression was altered on mouse GC B cells. Our analysis included the use of two different antigenic systems, multiple GC B cell markers, both primary and secondary immune responses, and also addressed the possible influences of B220lowGL7+or PNA+ immature BM cells, and passive acquisition of Fc{gamma}RIIB from FDCs. In total, our new data are in agreement with previous suggestions that levels of expression of Fc{gamma}RIIB are up-regulated on GC B cells in normal mice (30, 33).

In contrast, most previous studies have reported reduced levels of Fc{gamma}RIIB on GC, as compared with non-GC B cells in autoimmune-prone mice (30, 34). In NZB mice, this down-regulation was argued to result from small, polymorphic deletions in the promoter region of the Fc{gamma}RIIB gene that overlap putative transcription factor binding sites (30, 33, 34). In support of this idea, reduced levels of Fc{gamma}RIIB on GC B cells did not appear to be influenced by genes in the NZB background because GC B cells in congenic B6 mice containing a 4-cM interval from NZB, including the Fc{gamma}RIIB gene, displayed this same down-regulation (33). However, past studies also found 4- to 6-fold lower Fc{gamma}RIIB levels on GC B cells in NZW mice, despite the fact that the promoter region deletion polymorphisms were reported to be absent in NZW-type Fc{gamma}RIIB alleles (30, 34). This observation casts doubt on the idea that these promoter deletions were solely responsible for reduced Fc{gamma}RIIB expression on the GC B cells of autoimmune-prone mice.

In the present studies, we found comparable levels of Fc{gamma}RIIB on NZB, NZW, and B6.Sle1 non-GC and GC B cells and non-GC B cells in B6 mice. Therefore, we interpret the differences in Fc{gamma}RIIB expression on GC B cells in NZB, NZW, and B6.Sle1 mice as compared with B6 mice as lack of up-regulation, rather than down-regulation. Nonetheless, real-time RT-PCR analysis of Fc{gamma}RIIB RNA levels in sorted populations of GC B cells demonstrated a substantial reduction in the levels of this RNA in the GC B cells of B6.Sle1 mice. This could be explained by diminution of the transcriptional activity of the Fc{gamma}RIIB gene or destabilization of Fc{gamma}RIIB RNA. Whatever the mechanism, our Fc{gamma}RIIB gene cloning and sequencing studies show that alteration in Fc{gamma}RIIB expression on GC B cells is not uniquely associated with the deletion polymorphisms in the promoter region of the NZB-type Fc{gamma}RIIB allele.

The differences in the results we report here and previously published findings, including our own, do not appear to have arisen from the use of distinct Ag systems or the study of primary vs secondary GC responses. Variability in levels of passive Fc{gamma}RIIB acquisition by GC B cells from FDCs before or during preparation of spleens for flow cytometry also seems unlikely, as our BM chimera studies did not provide support for this caveat. Moreover, we found that FDCs in B6.Sle1 GCs appear to express levels of Fc{gamma}RIIB comparable to those in B6 GCs. It remains possible that the flow cytometric parameters used to define GC B cells in past studies were inaccurate. In this regard, our analyses of the B220low/GL7high or PNA+ subpopulations in the spleens of NZB-, NZW-, and B6.Sle1-immunized mice revealed that these subpopulations appeared to express levels of Fc{gamma}RIIB lower than on either B220high/GL7– or PNA– non-GC B cells or B220high/GL7high or PNA+ GC B cells in these mice.

At present, we can only speculate on the mechanism(s) that results in the failure of Fc{gamma}RIIB levels to be up-regulated on the GC B cells of mice bearing the NZB and NZW Fc{gamma}RIIB alleles. Our sequencing studies confirmed previous reports (30) that both these alleles share polymorphisms in the promoter and other putative regulatory regions that are not present in the B6 allele. For example, a deletion polymorphism is located in an alternating purine/pyrimidine-rich region in the third intron of the NZB and NZW alleles. This region contains putative NF-{kappa}B-like binding sites and elements associated with gene silencer activity (52, 53). Future studies are required to determine whether this region plays a role in regulation of Fc{gamma}RIIB gene expression patterns and whether the deletion in this area alters this regulation.

Our analysis of B6.Sle1 GC B cells suggests that if any of the polymorphisms that distinguish the New Zealand alleles from the B6 allele play a role in the perturbed expression of Fc{gamma}RIIB on GC B cells in autoimmune-prone strains of mice, they do so in a manner independent of epistatic interactions with background genes in the New Zealand strains. Nevertheless, Wakeland and colleagues (42) have shown that autoantibody production in B6.Sle1 mice is regulated by a nonoverlapping cluster of functionally related but independently acting genes in the Sle1 interval (named Sle1a, Sle1b, Sle1c, and Sle1d). Subsequently, this group has identified complement receptor 2 as a potential candidate gene for Sle1c locus (54) and the SLAM/CD2 gene cluster as a candidate for the Sle1b locus (55).

During the course of these fine-mapping genetic studies, the Fc{gamma}RIIB gene was segregated from the Sle1b interval, and a new B6.NZM2410FcR congenic strain was created (55). Interestingly, this congenic strain was shown to be negative for spontaneous antinuclear Ab production. These data are consistent with our studies showing the B6.Sle1 Fc{gamma}RIIB allele is of the NZW type and exclude the possibility of this form of the Fc{gamma}RIIB gene being predominantly responsible for loss of B cell tolerance in B6.Sle1 mice. Future studies of Fc{gamma}RIIB expression on the GC B cells in individual Sle1a, Sle1b, Sle1c, Sle1d, and Fc{gamma}RIIB subcongenic strains will clearly be helpful in evaluating the relative role of polymorphisms in the Fc{gamma}RIIB gene and other genes that act epistatically in producing the altered expression of Fc{gamma}RIIB on GC B cells characteristic of autoimmune-prone mice.

Such considerations raise the more pressing question of whether failed up-regulation of Fc{gamma}RIIB expression on GC B cells would have any functional impact on the positive or negative clonal selection processes that operate during the GC response. Ravetch and colleagues (35, 43) showed that a complete Fc{gamma}RIIB deficiency on the B6 background led to spontaneous antinuclear Ab production and, ultimately, fulminant autoimmune disease. However, subsequent studies from this group revealed that this genetic configuration perturbed the regulation of receptor editing during the primary development of autoreactive B cells and IgG autoantibody production by Ab secretory cells (56). Moreover, the levels of Fc{gamma}RIIB expressed on non-GC B cells in normal mice and, by extension of the results we report in the present study, on the GC B cells of autoimmune prone mice are presumably sufficient to allow IC-mediated coligation with the BCR and inhibition of BCR-mediated activation pathways. Nonetheless, the elevated levels of Fc{gamma}RIIB expressed on the GC B cells of normal mice may result in more facile homoaggregation of this receptor, a process that can induce apoptosis (14, 16). As ICs progressively become the predominant form of Ags during the GC response, the increased probability of Fc{gamma}RIIB homoaggregation on GC B cells may allow the subpopulations of these cells expressing low-affinity or autoreactive BCRs to be more efficiently deleted. We are currently conducting experiments to test this idea.

Finally, during the course of our studies, we observed significant levels of 2.4G2 staining on GL7+ GC B cells in B6.RIIB–/– mice, suggesting that Fc{gamma}RIII might be expressed on GC B cells in normal mice. This idea was supported by RT-PCR analyses, showing that amounts of Fc{gamma}RIII RNA were substantially higher in B6 GC B cells as compared with non-GC B cells. Interestingly, this elevation was not observed in B6.Sle1 GC B cells, indicating a perturbed up-regulation phenomenon analogous to that observed for Fc{gamma}RIIB. Given the tight linkage of the Fc{gamma}RII and Fc{gamma}RIII genes (57, 58, 59), it is tempting to speculate that a single chromosomal regulatory mechanism is responsible for the changes in the levels of expression of these two FcRs we have observed. If future studies, including the analysis of Fc{gamma}RIII-deficient mice as controls, demonstrate that Fc{gamma}RIII is expressed on GC B cells as a functional receptor capable of inducing activation signals, extensive alterations in models that propose a central role for ICs in regulating the outcome of the GC reaction in normal and autoimmune situations will be warranted.


   Acknowledgments
 
We thank Scot Fenn for technical assistance and all members of the Manser laboratory for their indirect contributions. We also thank Drs. Chandra Mohan and Edward Wakeland (University of Texas Southwestern Medical Center) for providing B6.Sle1 mice and Dr. Jeffrey Ravetch (Rockefeller University) for supplying B6.RIIB–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Tim Manser, Department of Microbiology and Immunology and The Kimmel Cancer Center, Jefferson Medical College, Bluemle Life Sciences Building 708, 233 South 10th Street, Philadelphia, PA 19107-5541. E-mail address: manser{at}mail.jci.tju.edu Back

2 Abbreviations used in this paper: IC, immune complex; GC, germinal center; FDC, follicular dendritic cell; AFC, Ab-forming cell; NZB, New Zealand Black; NZW, New Zealand White; BM, bone marrow; NP16-CGG, (4-hydroxy-3-nitrophenyl) acetyl chicken {gamma}-globulin; SA, streptavidin; PNA, peanut lectin (agglutinin); RQ, relative quantification. Back

Received for publication April 15, 2005. Accepted for publication May 20, 2005.


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