DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B Cells and Idiotype Positive Marginal Zone B Cells before and after Immunization with Streptococcus pneumoniae¹

Mature B lymphocytes play an integral role in the adaptive immune response via Ag presentation and Ab secretion. The mature splenic B cell population is divided into the marginal zone (MZ)⁷ and follicular (FO) B cell subsets based on anatomical location, cellular surface molecules, and functional immune responses (reviewed in Ref. 1). MZ B cells respond primarily to T-independent Ags and are proposed to bridge the gap between the rapid Ag nonspecific response and the delayed Ag-specific response. FO B cells respond primarily to T-dependent Ags and are responsible for the generation of long-term memory. However, the exact molecular mechanism by which each subset of B cells function is not fully understood.

MZ B cells are primarily nonrecirculating, located at the outer limit of the white pulp region, and characterized by the expression of IgM^highIgD^lowCD1d⁺CD21^highCD23^low. The MZ B cell repertoire is enriched with B cells expressing germline-encoded BCRs (2, 3, 4), some of which have a low level of self-reactivity. Following activation, MZ B cells increase B7-1 and B7-2 expression, develop into plasmablasts more readily, and are more sensitive to LPS stimulation than their FO counterparts (5, 6). In addition to rapid production of IgM Ab, MZ B cells also possess the ability to capture and shuttle Ag to follicular dendritic cells (7) as well as to efficiently activate naive T cells directly (8), suggesting a potential role for MZ B cells in T cell-dependent Ab responses also. In addition to anatomical location and cellular functions, MZ and FO B cells differentially express a number of cell surface molecules. We have previously shown that CD9, a member of the tetraspanin family, is expressed by the MZ and B1 B cell populations but not by FO B cells (9). Additionally, we identified Fc receptor homologue 3 (FcRH3) as a potentially immunoregulatory molecule expressed by MZ and B1 cells, but not by FO B cells (10). Recently, the scavenger receptor CD36 was identified as a marker predominantly expressed by MZ B cells (11). Taken together, it is clear that MZ B cells fill a specific niche in the splenic environment through unique expression and regulation of specific genes.

The development of DNA microarray technology has allowed for the rapid analysis of genome-wide gene expression profiles. Using this technology, we set out to identify differentially regulated genes between FO and MZ B cells as well as the genes specifically up-regulated or down-regulated following activation. DNA microarray analysis of FACS-sorted resting MZ and FO B cells from MD4 mice revealed 181 genes that are differentially expressed in the resting B cell populations. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. In addition, a comparative DNA microarray analysis of FACS-sorted MZ Id-positive and -negative B cells at 0 and 1 h following i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, revealed genes specifically up-regulated or down-regulated following activation. These results give new insight into the differences between MZ and FO B cells and reveal new candidate genes and pathways to study.

SWR/J and C3H/HeJ samples were kindly provided by T. Waldschmidt (University of Iowa, Iowa City, IA) and were from mice housed at the University of Iowa in specific pathogen-free conditions. MD4 anti-HEL conventional transgenic (Tg) mice were originally obtained from Dr. C. Goodnow (Australian National University, Canberra, Australia) (12). MD4 transgenic mice are on a C57BL/6 (B6) background. M167 Tg mice have been described previously (13). The IL-10/Thy1.1 reporter mice were generously provided by C. Weaver (University of Alabama at Birmingham, Birmingham, AL) as described previously (14). IL-10/Thy1.1 mice were crossed with M167 Tg mice. All mice were bred and housed within the pathogen-free facility at the University of Alabama at Birmingham and used at 6–8 wk of age according to approved animal protocols.

Microarray analysis was performed as described previously (15). Briefly, total RNA was isolated from sort-purified cell populations using an RNeasy Mini kit with on-column Dnase digestion (Qiagen) and, in accordance with expression analysis technical instructions from Affymetrix, cDNA was synthesized. cRNA was synthesized with BioArray high-yield transcript labeling kit (Enzo). Labeled cRNA (∼15 μg) was chemically fragmented for 35 min. at 94°C. Affymetrix MG U74Av2 oligonucleotide GeneChips (Affymetrix) were probed, hybridized, stained, washed, and scanned according to the manufacturer’s protocol at the University of Minnesota Biomedical Genomics Center facility (Minneapolis, MN). Each sort-purified cell population was processed independently as true biological replicates.

FACS analysis was performed as described previously (16). Briefly, total splenocytes were collected, RBCs were lysed with ammonium chloride, and the splenocytes were stained with different combinations of the following Abs: fluorescein (FITC)-, PE-, or allophycocyanin-conjugated anti-mouse CD21, CD23, Thy1.1, CD19 (eBioscience), goat anti-human regulator of G protein signaling (RGS) 10 (RGS10; Santa Cruz Biotechnology), goat anti-mouse D6 β-chemokine receptor, and rabbit anti-human Sharp2/Stra13 (Abcam). All anti-human Abs cross-react with mouse targets. For intracellular FACS analysis, cells were then washed, fixed, and permeabilized using the Cytofix/Cytoperm (BD Biosciences) kit according to manufacturer’s directions. All samples were analyzed using a FACSCalibur flow cytometer or FACSAria cell sorter (BD Biosciences). The data were analyzed using FlowJo software (Tree Star, Inc.).

Western blot analysis was performed as described previously (17). Briefly, following B cell isolation the cells were lysed, total protein was quantitated using a protein quantitation assay (Bio-Rad), and then protein samples (5–20 μg) were resolved by electrophoresis on 10% polyacrylamide gels (Bio-Rad), transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore), probed with goat anti-human RGS10, anti-actin (Santa Cruz Biotechnology), or goat anti-mouse D6 β-chemokine receptor (Abcam), detected with HRP-labeled anti-mouse, goat, and rabbit Abs (Santa Cruz Biotechnology), and developed with the LumiGLO detection kit (Cell Signaling).

Total RNA was isolated from ∼5 × 10⁵ sort-purified MZ B cells using TRIzol reagent (Invitrogen) following the manufacturer’s directions. RT-PCR was performed using the Omniscript RT kit (Qiagen) following the manufacturer’s directions. The following gene-specific primers were used to amplify the cDNA obtained from the RT kit using Fisher Taq and PCR products were resolved using a 1% agarose gel and visualized using ethidium bromide. Primers used were as follows: β-actin, 5′-TACAGCTTCACCACCACAGC-3′ (forward) and 5′-AAGGAAGGCTGGAAAAGAGC-3′ (reverse); D6, 5′-CTTCCAGCTGAACCTTCTGG-3′ (forward) and 5′-CGAGTGCAGAAACAAGGTGA-3′ (reverse); RGS10, 5′-GCCTTAAGAGCACAGCCAAG-3′ (forward) and 5′-CTTTTCCTGCATCTGCTTCC-3′ (reverse); Thy1.1, 5′-ACCAAAACCTTCGCCTGGACTG-3′ (forward) and 5′-TCCTTGGGGTCTTCTACCTTTCTC-3′ (reverse); IL-10, 5′-CATGGGTCTTGGGAAGAGAA-3′ (forward) and 5′-CATTCCCAGAGGAATTGCAT-3′ (reverse); Stra13, 5′-GGATTTGCCCACATGTACC-3′ (forward) and 5′-TCAATGCTTTCACGTGCTTC-3′ (reverse). The annealing temperature for all primers was 60°C.

Expressionist Pro 1.0 (GeneData) was used to generate relative expression values for each transcript using the MAS 5.0 algorithm, default settings, and a scaling factor of 1500 to control for minor cross-chip differences in hybridization intensities. GeneData Expressionist and Microsoft Excel were used for statistical analysis. Hierarchical clustering analysis was performed using Cluster and visualized in TreeView, as described previously (18).

Data with three or more groups were analyzed by a one-way ANOVA and statistical significance was determined by p < 0.05. Data with two groups were analyzed by a two-tailed paired t test and statistical significance was determined by a p < 0.02.

The mature splenic B cell population is divided into MZ and FO B cells based on anatomical location, cellular surface molecule expression, and functional immune responses (reviewed in Ref. 1). DNA microarray analysis was used to determine differences in gene expression profiles between MZ and FO B cell populations. Splenocytes from B6 MD4 Tg mice were sort purified to obtain paired MZ (B220⁺CD21^highCD23^low) and FO (B220⁺CD21^intCD23⁺, where “int” is “intermediate”) B cell samples. Postsort analysis revealed >95% purity of each B cell population (data not shown). MD4 mice carry a heavy and a light chain transgene specific for hen egg lysozyme Ag (12) and were used because >90% of their B cells express the transgenic BCR, thereby potentially reducing the variability due to a polyclonal repertoire. Gene expression was assessed in three replicates of each B cell population the using Affymetrix U74A mouse GeneChip microarray representing ∼11,000 transcripts. Expression levels were quantified using GeneData Expressionist Pro 1.0 software and the data from each array were analyzed to identify the genes that were differentially expressed between the MZ and FO B cell populations. Differential expression was defined as a mean fold change of >2 and p < 0.02 by Student’s t test.

Based on this definition, we identified 181 transcripts differentially expressed between the two populations. Ninety-nine transcripts (∼55% of total) were more highly expressed in MZ B cells relative to FO B cells, whereas 82 transcripts (∼45% of total) were more highly expressed in FO B cells relative to MZ B cells. To better visualize the data, each expression value was divided by the mean expression of all six samples of that transcript and converted into log₂ space. The data were then analyzed by unsupervised hierarchical clustering as described previously (18). The data showed tight clustering of the three replicates of each cell type with a coefficient of correlation between any two replicate samples >0.98. The 181 gene transcripts identified were grouped into the following broad functional classifications: motility/adhesion (Fig. 1,A), immune response (Fig. 1,B), apoptosis (Fig. 1,C), proliferation (Fig. 1,D), transcription factors (Fig. 2,A), signal transduction (Fig. 2,B), metabolism (data not shown), or miscellaneous (data not shown). All 181 genes are listed in Table I.

To determine whether any strain-specific differences exist between MZ and FO B cell gene expression profiles, we expanded our gene expression analysis to include two additional mouse strains, C3H/HeJ (C3H) and SWR/J (SWR). C3H mice have an enlarged MZ B cell population relative to B6 mice while SWR mice have a smaller MZ B cell population relative to B6 mice (data not shown). The 181 transcripts found to be significantly different between FO and MZ B cells were analyzed for their expression levels in C3H and SWR mice, respectively. Although the absolute signal intensities varied across strains (Table I), the fold changes between MZ and FO B cell gene expression were comparable (Fig. 3,A). We identified 29 genes (∼16% of total) that appeared to have different expression profiles between FO and MZ B cells in the C3H and SWR strains relative to the B6 strain (Fig. 3,B and Table II). These strain-specific differences might reflect changes in genes regulating MZ B cell size, strain-specific functional differences, or polymorphisms that influence probe hybridization but have no functional consequences.

MZ B cells provide a rapid response to blood-borne bacterial particulates, in part because of their localization in the spleen. For example, blood-borne Ags accumulate within the splenic MZ as early as 30 min following i.v. immunization (8), giving an opportunity for MZ B cells to sample blood and respond rapidly to an Ag. A number of factors have been shown to play a role in MZ B cell localization within the splenic microenvironment including sphingosine-1-phosphate receptor type 1 (S1P₁) (19) and the presence of MZ macrophages (20) and integrins (21). In addition, in vivo injection of pertussis toxin disrupts MZ localization, suggesting the involvement of G protein-coupled receptor(s) (22). The current microarray data identified a number of molecules that are potentially involved in the migration, localization, and/or retention of MZ B cells in the splenic MZ. Two proteins more highly expressed in MZ B cells relative to FO B cells were the D6 β-chemokine receptor and the RGS10 regulator of G protein signaling protein. To confirm that these two proteins are indeed more highly expressed in MZ B cells, resting splenic MZ and FO B cells were sort purified and analyzed for the level of D6 and RGS10 mRNA (Fig. 4,A) and protein (Fig. 4, B–D) by RT-PCR, Western blotting, and FACS, respectively. Thus, resting MZ B cells express D6 and RGS10 at higher levels than FO B cells with the potential to be involved in MZ B cell localization.

In addition to the differential phenotypes of resting FO and MZ B cells, MZ B cells respond very differently to Ag than FO B cells. Following activation with Ag, MZ B cells increase B7-1 and B7-2 expression, develop into plasmablasts more readily, and are more sensitive to LPS stimulation than their FO counterparts (5, 6). Along with the rapid production of IgM Ab, MZ B cells also possess the ability to efficiently activate naive T cells (8). However, the genes that are rapidly up-regulated and down-regulated in MZ B cells following activation with Ag have not been fully characterized. To determine the gene expression profile of Ag (Id)-positive MZ B cells before and after activation, M167 Tg mice were immunized i.v. with heat-killed S. pneumoniae, R36A, and Id⁺ and Id⁻ MZ B cells were sort purified at 0 and 1 h following immunization. The samples were analyzed via DNA microarray analysis as described for the resting MZ vs FO B cell microarray above. The gene transcripts identified to significantly increase or decrease were grouped into the following broad functional classifications: chemokines (Fig. 5,A), chemokine receptors (Fig. 5,B), cytokines (Fig. 5,C), cytokine receptors (Fig. 5,B), apoptosis (Fig. 6,A), and immune cell markers (Fig. 6 B).

We focused on the Ag responsive Id⁺ MZ B cells and the genes that were regulated following i.v. immunization with R36A. The Id⁺ MZ B cell gene expression profile exhibited an activated phenotype at 1 h post-immunization, as would be predicted. The Id⁺ MZ B cells rapidly down-regulated proapoptotic genes such as multiple caspase proteins, annexin A4, and programmed cell death proteins while concurrently up-regulating anti-apoptotic genes such as Bcl-like proteins. MZ B cells also up-regulated a number of cytokine genes including IL-10, IL-6, TGF-β, and IL-1β while down-regulating many cytokine receptors. Chemokine ligands such as CXCL10, CXCL2, CCL3, CXCL5, and CCL4 were up-regulated while chemokine receptors were either up-regulated (CCR7) or down-regulated (D6, CCR5, and RDC-1). In addition, we cross-referenced the 99 transcripts that were more highly expressed in the MZ B cells relative to FO B cells with the expression profile in the Id⁺ MZ B cells 1 h after activation to determine whether any significant changes occurred (Table III). Six of the 99 genes (6%) more highly expressed in MZ B cells were up-regulated after activation in the MZ Id⁺ B cells while 17 of the 99 genes (17%) were down-regulated. Taken together, these results suggest that Id⁺ MZ B cells have a unique gene expression profile following i.v. immunization with R36A.

A number of interesting genes were identified by DNA microarray analysis on sort-purified MZ Id⁺ and Id⁻ B cells at 0 and 1 h following i.v. immunization with R36A. Two of these genes that warranted further investigation were IL-10 and Stra13.

IL-10 is an immunoregulatory cytokine that plays a role in negatively regulating inflammatory immune responses, and B cells have been shown to secrete IL-10 (23). To confirm whether Id⁺ MZ B cells are activated to secrete IL-10 in response to R36A, we crossed the M167 heavy chain Ig Tg mouse with an IL-10/Thy1.1 reporter mouse in which all IL-10⁺ cells were Thy1.1⁺ (14), immunized with R36A, and analyzed isolated Id⁺ MZ B cells for the presence of IL-10 and Thy1.1 mRNA (Fig. 7,A) and the Thy1.1 reporter protein (Fig. 7,B). As expected, IL-10 and Thy1.1 mRNA increased only in the Id⁺ MZ B cells following immunization with R36A. The difference in degree of induction between IL-10 and Thy1.1 is most likely due to the copy number of the Thy1.1 transgene, which is estimated to be at least 12 copies (14). Stra13 is a basic helix-loop-helix domain-containing class B2 protein that is thought to be a negative regulator of B cells (24). To confirm that Stra13 is up-regulated following activation of MZ B cells, MZ Id⁺ B cells were isolated before and after immunization with R36A and analyzed for the level of Stra13 mRNA (Fig. 7 A). As expected, Stra13 mRNA increased only in the Id⁺ MZ B cells following immunization with R36A. Thus, the DNA microarray analysis of Id⁺ and Id⁻ MZ B cells at 0 and 1 h following immunization with R36A identified multiple genes of interest that were rapidly up-regulated or down-regulated after activation, including IL-10 and Stra13.

Previous data studying FO and MZ B cells have shown that these B cell subsets differ based on their anatomical location in the spleen, cellular surface molecule expression, and effector functions (reviewed in Ref. 1). We set out to identify new genes and pathways that are differentially expressed between FO and MZ B cells and those that were specifically up-regulated or down-regulated within each subset following activation. DNA microarray allows for a high throughput analysis of genomic expression differences between two sample populations. This approach identified 181 genes differentially expressed between resting MZ and FO B cells. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. In addition, DNA microarray analysis of MZ Id⁺ and Id⁻ B cells before (0 h) and after (1 h) R36A immunization revealed many new genes and pathways specifically regulated in the MZ Id⁺ B cells. These findings further our understanding of MZ and FO B cell biology while at the same time identifying new candidate genes and pathways to study.

The MZ vs FO B cell microarray used cells that were isolated by gating on B220 and then sorted based on surface expression patterns of CD21^highCD23^low for MZ B cells and CD21^lowCD23^high for FO B cells. As expected, our DNA microarray results showed higher mRNA expression of CD21 and lower expression of CD23 in MZ B cells relative to FO B cells. MZ B cells are known to express surface CD9 and CD1d whereas FO B cells express little to no CD9 and CD1d (9, 25). Similarly, our microarray results showed a higher expression of CD9 and CD1d on MZ B cells relative to FO B cells. Furthermore, S1P₁ and S1P₃ were previously shown to be expressed at higher levels on MZ B cells relative to FO B cells, while S1P₄ was expressed higher on FO B cells (19). Our data were again consistent with what has been shown in the literature, showing higher expression of S1P₁ and S1P₃ on MZ B cells and higher expression of S1P₄ on FO B cells. Taken together, it appears that our DNA microarray data agree with what has been shown in the literature with respect to known phenotypic differences between MZ and FO B cells, suggesting that our sorted B cell populations were pure and our DNA microarray method of analysis is valid.

One interesting gene more highly expressed in MZ B cells relative to FO B cells was RGS10. RGS10 has been previously confirmed to be specifically expressed in MZ B cells (mRNA) as well as plasma cells (26). RGS10 attenuates signaling pathways via increased GTPase activity to specific G α subunits (27). Phosphorylation by protein kinase A induces its localization to the nucleus (28). Recently, RGS10^−/− mice were reported that exhibited severe osteopetrosis and impaired osteoclast differentiation resulting from the loss of [Ca²⁺]_i oscillation regulation (29), although no immune characterization was reported. Although RGS10 was more highly expressed in resting MZ B cells relative to FO B cells, RGS10 mRNA was not found to be regulated following activation in MZ Id⁺ B cells. However, because chemokine receptors are G protein coupled, a protein that regulates their signaling capacity might play an important role in localization, maintenance, or migration of MZ B cells. For example, RGS1, RGS3, and RGS4 introduction into B cell lines dramatically alters chemokine-induced cell migration (30, 31, 32). Taken together, MZ B cell-specific expression of RGS10 potentially plays a role in regulating the ability to respond to chemokine signals and might play a role in MZ B cell localization.

An additional gene more highly expressed in MZ B cells was D6. D6 is proposed to be a decoy chemokine receptor that has the ability to bind, internalize, and degrade chemokine ligands through a β-arrestin-dependent mechanism, a function termed chemokine scavenging (33, 34, 35, 36). Interestingly, our results show that D6 is more highly expressed in resting MZ B cells relative to FO B cells and that D6 is rapidly down-regulated (10-fold) following activation. Given its proposed property of a chemokine sink and the fact that D6 has not been shown to signal intracellularly, the potential exists that D6 expression on the surface of MZ B cells is involved in keeping them properly localized within the splenic microenvironment. Rapid down-regulation of D6 after activation potentially enhances the migration of MZ B cells to the T:B cell border. D6 expression has been reported in B cells previously (37), although the differential expression in B cell subsets was not investigated. Thus, D6 appears to be an interesting candidate gene potentially involved in MZ B cell localization, maintenance, and/or migration.

Our second DNA microarray experiment was aimed at identifying genes that were specifically up-regulated or down-regulated following activation. Using the M167 Tg mouse, we sort purified MZ Id⁺ and Id⁻ B cells at 0 and 1 h after i.v. immunization with R36A. S1P₁ transcripts were rapidly down-regulated (7.0-fold) following activation only in the Id⁺ MZ B cells, which is in agreement with the findings of Cyster and colleagues (19). S1P₁ has been shown to play a role in the migration of MZ B cells to the T:B border following activation. In addition, the MZ B cell-specific marker, CD9, was increased 2.0-fold following activation, consistent with our previous findings (9). Of additional interest, only the MZ Id⁺ and not the MZ Id⁻ B cells rapidly increased antiapoptotic genes and decreased proapoptotic genes, a phenotype consistent with cellular activation. This shows a remarkable degree of Ag specificity in that virtually no concurrent increases were detected in the MZ Id⁺ and Id⁻ B cell populations at 1 h. Our microarray results appear to agree with a number of well studied genes already published in the literature with respect to MZ B cells, indicating that both our cell sort and microarray analyses are accurate. Consequently, further analysis and weight can be given to the other genes found to be regulated following immunization.

One interesting gene identified by microarray analysis to be specifically increased only in the Id⁺ MZ B cells was IL-10. Interestingly, MZ B cells have been suggested as playing an immunoregulatory role through secretion of IL-10 (38). IL-10 is an immunoregulatory cytokine that plays an important role in negatively regulating inflammatory immune responses. A variety of cells are capable of producing IL-10, including Th2, regulatory T (Treg), B-1, and MZ B cells (39). The effects of IL-10 are mainly immunosuppressive but also depend on which cell type is being affected by IL-10. In experimental autoimmune encephalitis, an experimental model of multiple sclerosis, one study suggested that B cells regulate regulatory T cells via B7 and IL-10 to suppress autoimmune inflammation (40). Besides the immunosuppressive role of IL-10, it has been suggested to play a role in B cell Ab production. The addition of IL-10 to human B cell cultures is reported to increase class switch recombination and the production of IgA and IgG (41, 42, 43). However, the specific B cell subset, its location in the spleen before and after stimulation, and the signals required to produce IL-10 are not fully understood.

Stra13 was another interesting gene that was rapidly up-regulated following activation. Stra13 is a basic helix-loop-helix domain-containing class B2 protein that is thought to be a negative regulator of B cells (24). Stra13^−/− mice develop autoimmune disease characterized by the accumulation of spontaneously activated T and B cells, circulating autoantibodies, infiltration of T and B cells into several organs, and immune complex deposition in glomeruli (44). Stra13 transgenic mice show impaired development of T and B cells, with the expansion of progenitor B and T cells most strongly affected (45). Of interest, Stra13 is developmentally regulated in B cells and decreases after activation in germinal center B cells (45). Our results in Id⁺ MZ B cells show that Stra13 increases after activation, although the functional relevance of this regulation is currently unknown.

The goal of this study was twofold: to identify genes that were differentially expressed between resting FO and MZ B cells and to identify genes that were specifically regulated in MZ Id⁺ B cells following activation. The results generated give a genome-wide look at the genes differentially expressed in FO and MZ B cells that potentially account for their differences in localization and function. Furthermore, the second microarray gave a comparative snapshot at 1 h of the gene expression profiles between Ag-specific vs nonspecific MZ B cells. One major problem with DNA microarray analysis is that many of the genes reported have not been studied, making conclusions difficult. However, a multitude of data is presented here with respect to FO and MZ B cell biology that will facilitate identification of new genes and pathways to explore.

We gratefully acknowledge Dr. Casey Weaver (University of Alabama at Birmingham) for generously sharing the Thy1.1/IL-10 reporter mice.

The authors have no financial conflict of interest.