Marginal zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and follicular (FO) B cells were sort purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two 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. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, Id-positive and -negative MZ B cells were sort purified before (0 h) or after (1 h) i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up-regulated or down-regulated at 1 h following immunization in the Id-positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs FO B cells and the specific regulation of gene expression in Ag-specific MZ B cells following interaction with Ag.

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 IgMhighIgDlowCD1d+CD21highCD23low. 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+CD21highCD23low) and FO (B220+CD21intCD23+, 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.

FIGURE 1.

Expression profile of differentially expressed genes between FO and MZ B cells. DNA microarray analysis identified 181 genes that were significantly different in sort-purified FO vs MZ B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change of >2 and p < 0.02 by t test. The differentially expressed genes were grouped into various functional categories as follows: motility/adhesion (A), immune response (B), apoptosis (C), and proliferation (D). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sort-purified FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

FIGURE 1.

Expression profile of differentially expressed genes between FO and MZ B cells. DNA microarray analysis identified 181 genes that were significantly different in sort-purified FO vs MZ B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change of >2 and p < 0.02 by t test. The differentially expressed genes were grouped into various functional categories as follows: motility/adhesion (A), immune response (B), apoptosis (C), and proliferation (D). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sort-purified FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

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FIGURE 2.

Expression profile of differentially expressed genes between FO and MZ B cells. DNA microarray analysis identified 181 genes that were significantly different in sort-purified FO vs MZ B cells from MD4 Tg mice (B6 background). The identified transcripts have a fold change of >2 and p < 0.02 by t test. The differentially expressed genes were grouped into various functional categories as follows: transcription factors (A) and signal transduction (B). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

FIGURE 2.

Expression profile of differentially expressed genes between FO and MZ B cells. DNA microarray analysis identified 181 genes that were significantly different in sort-purified FO vs MZ B cells from MD4 Tg mice (B6 background). The identified transcripts have a fold change of >2 and p < 0.02 by t test. The differentially expressed genes were grouped into various functional categories as follows: transcription factors (A) and signal transduction (B). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

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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.

FIGURE 3.

Identification of strain-specific differences in gene expression profiles between FO and MZ B cells. Gene expression profile of splenic FO and MZ B cells from B6, SWR, and C3H mice. The profile includes 181 gene transcripts with a fold change of >2 and p < 0.02 by t test. A, Hierarchical analysis of 152 genes with consistent regulation across the three mouse strains. B, Hierarchical analysis of 29 genes with strain-specific differences in MZ vs FO gene expression profiles. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that strain. Each column represents one sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of genes was unsupervised.

FIGURE 3.

Identification of strain-specific differences in gene expression profiles between FO and MZ B cells. Gene expression profile of splenic FO and MZ B cells from B6, SWR, and C3H mice. The profile includes 181 gene transcripts with a fold change of >2 and p < 0.02 by t test. A, Hierarchical analysis of 152 genes with consistent regulation across the three mouse strains. B, Hierarchical analysis of 29 genes with strain-specific differences in MZ vs FO gene expression profiles. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that strain. Each column represents one sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of genes was unsupervised.

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Table II.

Genes differentially expressed between FO and MZ B cells from B6, SWR, and C3H strains of mice

Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
93195_at Mfhas1 Malignant fibrous histiocytoma-amplified sequence 1143 5462 4.8 2857 2424 0.85 3446 3592 1.04 
160069_at Gmnn Geminin 422 1174 2.8 376 323 0.86 229 445 1.94 
93833_s_at Hist1h2bc Histone 1, H2bc 773 2093 2.7 617 697 1.13 1189 536 0.45 
161788_f_at S1P1 Sphingolipid G protein-coupled receptor 1 565 1476 2.6 1332 747 0.56 936 562 0.60 
94995_at  RIKEN cDNA A030007L17 gene 848 2186 2.6 926 734 0.79 1184 817 0.69 
92925_at Cebpb CCAAT/enhancer binding protein (C/EBP), β 1911 4882 2.6 12638 12321 0.97 20455 13386 0.65 
100516_at Chka Choline kinase α 874 2195 2.5 1728 1646 0.95 880 728 0.83 
160841_at Dbp D site albumin promoter binding protein 239 540 2.3 226 129 0.57 293 336 1.15 
102104_f_at  Est 2147 4844 2.3 1411 2614 1.85 1284 1133 0.88 
99024_at Mxd4 Max dimerization protein 4 5977 12958 2.2 6053 6769 1.12 6873 6672 0.97 
95387_f_at Sema4b Semaphorin 4B 8465 17775 2.1 4483 3837 0.86 4046 4411 1.09 
103460_at Ddit4 DNA damage-inducible transcript 4 3274 6711 2.0 1631 1280 0.78 1275 3062 2.40 
100573_f_at Gpi1 Glucose phosphate isomerase 1 1410 2889 2.0 3126 1986 0.64 1911 2109 1.10 
98868_at Bcl2 B cell leukemia/lymphoma 2 1203 2437 2.0 1411 1261 0.89 1030 833 0.81 
94431_at St6gal1 β-Galactoside α-2,6-ialyltransferase 1 1562 331 4.7 922 410 2.25 850 872 0.98 
103504_at Ssbp2 Single-stranded DNA binding protein 2 1538 335 4.6 85 224 0.38 496 182 2.72 
98918_at Txndc5 Thioredoxin domain containing 5 5318 1447 3.7 745 893 0.83 3928 1955 2.01 
104523_at Lrrc8c Leucine rich repeat containing 8 family, member C 1533 483 3.2 905 1098 0.82 970 693 1.40 
97890_at Sgk Serum/glucocorticoid regulated kinase 1107 351 3.2 1857 1923 0.97 1674 717 2.33 
93193_at Adrb2 Adrenergic receptor, β2 5831 1961 3.0 8025 9318 0.86 3784 5394 0.70 
98083_at Klf6 Krüppel-like factor 6 4002 1522 2.6 14189 19034 0.75 18319 14977 1.22 
99622_at Klf4 Krüppel-like factor 4 697 278 2.5 10472 25698 0.41 18855 23655 0.80 
102892_at Kcnab2 Potassium voltage-gated channel 2707 1133 2.4 2525 2350 1.07 1751 1970 0.89 
100554_at Pdlim1 PDZ and LIM domain 1 (elfin) 2352 1009 2.3 443 336 1.32 147 243 0.61 
97203_at Marcksl1 MARCKS-like 1 2027 900 2.3 5719 7047 0.81 5684 6025 0.94 
94753_at Gna15 Guanine nucleotide binding protein, α15 689 315 2.2 51 136 0.37 84 136 0.61 
98335_at Rece1 Replication factor C1 2339 1104 2.1 1421 1647 0.86 1758 1339 1.31 
101502_at Tgif TG interacting factor 2690 1292 2.1 27576 36312 0.76 23520 24132 0.97 
100576_at Pafah1b3 Platelet-activating factor acetylhydrolase 3106 1545 2.0 1069 1280 0.84 1627 988 1.65 
Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
93195_at Mfhas1 Malignant fibrous histiocytoma-amplified sequence 1143 5462 4.8 2857 2424 0.85 3446 3592 1.04 
160069_at Gmnn Geminin 422 1174 2.8 376 323 0.86 229 445 1.94 
93833_s_at Hist1h2bc Histone 1, H2bc 773 2093 2.7 617 697 1.13 1189 536 0.45 
161788_f_at S1P1 Sphingolipid G protein-coupled receptor 1 565 1476 2.6 1332 747 0.56 936 562 0.60 
94995_at  RIKEN cDNA A030007L17 gene 848 2186 2.6 926 734 0.79 1184 817 0.69 
92925_at Cebpb CCAAT/enhancer binding protein (C/EBP), β 1911 4882 2.6 12638 12321 0.97 20455 13386 0.65 
100516_at Chka Choline kinase α 874 2195 2.5 1728 1646 0.95 880 728 0.83 
160841_at Dbp D site albumin promoter binding protein 239 540 2.3 226 129 0.57 293 336 1.15 
102104_f_at  Est 2147 4844 2.3 1411 2614 1.85 1284 1133 0.88 
99024_at Mxd4 Max dimerization protein 4 5977 12958 2.2 6053 6769 1.12 6873 6672 0.97 
95387_f_at Sema4b Semaphorin 4B 8465 17775 2.1 4483 3837 0.86 4046 4411 1.09 
103460_at Ddit4 DNA damage-inducible transcript 4 3274 6711 2.0 1631 1280 0.78 1275 3062 2.40 
100573_f_at Gpi1 Glucose phosphate isomerase 1 1410 2889 2.0 3126 1986 0.64 1911 2109 1.10 
98868_at Bcl2 B cell leukemia/lymphoma 2 1203 2437 2.0 1411 1261 0.89 1030 833 0.81 
94431_at St6gal1 β-Galactoside α-2,6-ialyltransferase 1 1562 331 4.7 922 410 2.25 850 872 0.98 
103504_at Ssbp2 Single-stranded DNA binding protein 2 1538 335 4.6 85 224 0.38 496 182 2.72 
98918_at Txndc5 Thioredoxin domain containing 5 5318 1447 3.7 745 893 0.83 3928 1955 2.01 
104523_at Lrrc8c Leucine rich repeat containing 8 family, member C 1533 483 3.2 905 1098 0.82 970 693 1.40 
97890_at Sgk Serum/glucocorticoid regulated kinase 1107 351 3.2 1857 1923 0.97 1674 717 2.33 
93193_at Adrb2 Adrenergic receptor, β2 5831 1961 3.0 8025 9318 0.86 3784 5394 0.70 
98083_at Klf6 Krüppel-like factor 6 4002 1522 2.6 14189 19034 0.75 18319 14977 1.22 
99622_at Klf4 Krüppel-like factor 4 697 278 2.5 10472 25698 0.41 18855 23655 0.80 
102892_at Kcnab2 Potassium voltage-gated channel 2707 1133 2.4 2525 2350 1.07 1751 1970 0.89 
100554_at Pdlim1 PDZ and LIM domain 1 (elfin) 2352 1009 2.3 443 336 1.32 147 243 0.61 
97203_at Marcksl1 MARCKS-like 1 2027 900 2.3 5719 7047 0.81 5684 6025 0.94 
94753_at Gna15 Guanine nucleotide binding protein, α15 689 315 2.2 51 136 0.37 84 136 0.61 
98335_at Rece1 Replication factor C1 2339 1104 2.1 1421 1647 0.86 1758 1339 1.31 
101502_at Tgif TG interacting factor 2690 1292 2.1 27576 36312 0.76 23520 24132 0.97 
100576_at Pafah1b3 Platelet-activating factor acetylhydrolase 3106 1545 2.0 1069 1280 0.84 1627 988 1.65 

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.

FIGURE 4.

MZ B cells express higher levels of D6 and RGS10 relative to FO B cells. Resting splenic MZ and FO B cells were sort purified and total RNA and protein were isolated. Resting MZ B cells express higher mRNA (A) and protein (B) levels of D6 and RGS10 as determined by RT-PCR and Western blotting, respectively. Total splenocytes were isolated and analyzed via FLOW cytometry. The expression levels of D6 isotype (0.3%), MZ B cell (88.8%), and FO B cell (11.8%) (C) and RGS10 isotype (0.2%), MZ B cell (82.1%), and FO B cell (1.1%) (D) are displayed as histogram plots.

FIGURE 4.

MZ B cells express higher levels of D6 and RGS10 relative to FO B cells. Resting splenic MZ and FO B cells were sort purified and total RNA and protein were isolated. Resting MZ B cells express higher mRNA (A) and protein (B) levels of D6 and RGS10 as determined by RT-PCR and Western blotting, respectively. Total splenocytes were isolated and analyzed via FLOW cytometry. The expression levels of D6 isotype (0.3%), MZ B cell (88.8%), and FO B cell (11.8%) (C) and RGS10 isotype (0.2%), MZ B cell (82.1%), and FO B cell (1.1%) (D) are displayed as histogram plots.

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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).

FIGURE 5.

Regulated genes in Id+ MZ B cells after activation. MZ Id+ (Ag+) and Id (Ag) B cells were isolated from M167 Tg mice at 0 and 1 h after i.v. immunization with heat killed S. pneumoniae R36A. DNA microarray analysis identified genes that were significantly up-regulated and down-regulated in the Id+ MZ B cells 1 h after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories as follows: chemokines (A), chemokine receptors (B), cytokines (C), and cytokine receptors (D). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

FIGURE 5.

Regulated genes in Id+ MZ B cells after activation. MZ Id+ (Ag+) and Id (Ag) B cells were isolated from M167 Tg mice at 0 and 1 h after i.v. immunization with heat killed S. pneumoniae R36A. DNA microarray analysis identified genes that were significantly up-regulated and down-regulated in the Id+ MZ B cells 1 h after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories as follows: chemokines (A), chemokine receptors (B), cytokines (C), and cytokine receptors (D). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

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FIGURE 6.

Regulated genes in Id+ MZ B cells after activation. MZ Id+ (Ag+) and Id (Ag) B cells were isolated from M167 Tg mice at 0 and 1 h after i.v. immunization with heat killed S. pneumoniae R36A. DNA microarray analysis identified genes that were significantly up-regulated and down-regulated in the Id+ MZ B cells 1 h after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories as follows: apoptosis (A) and immune cell markers (B). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

FIGURE 6.

Regulated genes in Id+ MZ B cells after activation. MZ Id+ (Ag+) and Id (Ag) B cells were isolated from M167 Tg mice at 0 and 1 h after i.v. immunization with heat killed S. pneumoniae R36A. DNA microarray analysis identified genes that were significantly up-regulated and down-regulated in the Id+ MZ B cells 1 h after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories as follows: apoptosis (A) and immune cell markers (B). Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes was unsupervised.

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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.

Table III.

Genes more highly expressed in MZ B cells relative to FO B cells and specifically regulated in MZ Id+ B cells following activation

Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)MZ Id+ (0 h)MZ Id+ (1 h)Fold Difference (1 h/0 h)
95462_at Bzw2 Basic leucine zipper and W2 domains 2 1482 8442 5.7 3855 13947 3.6 
92217_s_at Gp49 Glycoprotein 49 A/B 161 2762 17.2 2530 13563 5.4 
100325_at Gp49 Glycoprotein 49 A/B 394 5124 13.0 2530 13563 5.4 
93013_at Id2 Inhibitor of DNA binding 2 2417 6577 2.7 9220 46793 5.1 
95661_at CD9 CD9 antigen 480 2677 5.6 8265 16347 2.0 
104701_at Bhlhb2 Basic helix-loop-helix domain containing 272 1490 5.5 6307 22934 3.6 
102914_s_at Bcl2a1 B cell leukemia/lymphoma 2 related protein A1 3284 10193 3.1 30696 152568 5.0 
161788_f_at S1P1 Sphingolipid G-protein-coupled receptor 1 565 1476 2.6 129 18 0.14 
97740_at Dusp16 Dual specificity phosphatase 16 787 4075 5.2 312 0.03 
93101_s_at Nedd4 Neural precursor cell expressed 660 3176 4.8 3668 565 0.15 
99071_at Mpeg1 Macrophage expressed gene 1 2402 8938 3.7 55 13 0.24 
95758_at Scd2 Stearoyl-Coenzyme A desaturase 2 4483 12233 2.7 6858 821 0.12 
160711_at Decr1 2,4-dienoyl CoA reductase 1, mitochondrial 187 553 3.0 670 364 0.54 
160069_at Gmnn Geminin 422 1174 2.8 905 216 0.24 
102410_at Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 575 1392 2.4 2749 254 0.09 
97949_at Fgl2 Fibrinogen-like protein 2 314 752 2.4 570 242 0.42 
98417_at Mx1 myxovirus (influenza virus) resistance 1 260 608 2.3 428 233 0.54 
160841_at Dbp D site albumin promoter binding protein 239 540 2.3 516 121 0.23 
95387_f_at Sema4b Semaphorin 4B 8465 17775 2.1 1495 271 0.18 
98026_g_at Evi2a Ecotropic viral integration site 2a 3528 7178 2.0 618 271 0.44 
93430_at Cmkor1 Chemokine orphan receptor 1 78 4369 56.0 8291 1092 0.13 
160495_at Ahr Aryl-hydrocarbon receptor 31 249 8.0 735 404 0.55 
98309_at Ccbp2 Chemokine binding protein 2 481 3549 7.4 3011 310 0.10 
103422_at CD1d CD1d antigen 1999 12449 6.2 22540 4484 0.20 
Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)MZ Id+ (0 h)MZ Id+ (1 h)Fold Difference (1 h/0 h)
95462_at Bzw2 Basic leucine zipper and W2 domains 2 1482 8442 5.7 3855 13947 3.6 
92217_s_at Gp49 Glycoprotein 49 A/B 161 2762 17.2 2530 13563 5.4 
100325_at Gp49 Glycoprotein 49 A/B 394 5124 13.0 2530 13563 5.4 
93013_at Id2 Inhibitor of DNA binding 2 2417 6577 2.7 9220 46793 5.1 
95661_at CD9 CD9 antigen 480 2677 5.6 8265 16347 2.0 
104701_at Bhlhb2 Basic helix-loop-helix domain containing 272 1490 5.5 6307 22934 3.6 
102914_s_at Bcl2a1 B cell leukemia/lymphoma 2 related protein A1 3284 10193 3.1 30696 152568 5.0 
161788_f_at S1P1 Sphingolipid G-protein-coupled receptor 1 565 1476 2.6 129 18 0.14 
97740_at Dusp16 Dual specificity phosphatase 16 787 4075 5.2 312 0.03 
93101_s_at Nedd4 Neural precursor cell expressed 660 3176 4.8 3668 565 0.15 
99071_at Mpeg1 Macrophage expressed gene 1 2402 8938 3.7 55 13 0.24 
95758_at Scd2 Stearoyl-Coenzyme A desaturase 2 4483 12233 2.7 6858 821 0.12 
160711_at Decr1 2,4-dienoyl CoA reductase 1, mitochondrial 187 553 3.0 670 364 0.54 
160069_at Gmnn Geminin 422 1174 2.8 905 216 0.24 
102410_at Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 575 1392 2.4 2749 254 0.09 
97949_at Fgl2 Fibrinogen-like protein 2 314 752 2.4 570 242 0.42 
98417_at Mx1 myxovirus (influenza virus) resistance 1 260 608 2.3 428 233 0.54 
160841_at Dbp D site albumin promoter binding protein 239 540 2.3 516 121 0.23 
95387_f_at Sema4b Semaphorin 4B 8465 17775 2.1 1495 271 0.18 
98026_g_at Evi2a Ecotropic viral integration site 2a 3528 7178 2.0 618 271 0.44 
93430_at Cmkor1 Chemokine orphan receptor 1 78 4369 56.0 8291 1092 0.13 
160495_at Ahr Aryl-hydrocarbon receptor 31 249 8.0 735 404 0.55 
98309_at Ccbp2 Chemokine binding protein 2 481 3549 7.4 3011 310 0.10 
103422_at CD1d CD1d antigen 1999 12449 6.2 22540 4484 0.20 

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.

FIGURE 7.

IL-10 and Stra13 are increased following R36A immunization. M167 Tg mice were crossed with an IL-10/Thy1.1 reporter mouse and immunized i.v. with R36A. MZ Id+ B cells were sort purified at 0, 1, and 4 h after immunization and total RNA was isolated. A, RT-PCR was performed using gene-specific primers for IL-10, Thy1.1, Stra13, and actin. B, The expression level of Thy1.1 was determined via FACS analysis on gated MZ Id+ B cells at 24 h following R36A immunization. MZ Id+ B cells were ∼5% (PBS) and 20% (R36A) positive for Thy1.1, respectively.

FIGURE 7.

IL-10 and Stra13 are increased following R36A immunization. M167 Tg mice were crossed with an IL-10/Thy1.1 reporter mouse and immunized i.v. with R36A. MZ Id+ B cells were sort purified at 0, 1, and 4 h after immunization and total RNA was isolated. A, RT-PCR was performed using gene-specific primers for IL-10, Thy1.1, Stra13, and actin. B, The expression level of Thy1.1 was determined via FACS analysis on gated MZ Id+ B cells at 24 h following R36A immunization. MZ Id+ B cells were ∼5% (PBS) and 20% (R36A) positive for Thy1.1, respectively.

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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 CD21highCD23low for MZ B cells and CD21lowCD23high 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.

Table I.

Genes differentially expressed between FO and MZ B cells in B6, SWR, and C3H mouse strains

Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
93430_at Cmkor1 Chemokine orphan receptor 1 78 4369 56.0 12 2081 178.5 107 3147 29.3 
97967_at Plxnd1 Plexin D1 70 3800 54.4 70 107 1.5 44 50 1.1 
102910_at Abcb1a ATP-binding cassette, sub-family B (MDR/TAP) 48 953 19.8 28 716 25.3 52 596 11.6 
92217_s_at Gp49 Glycoprotein 49 A/B 161 2762 17.2 559 2146 3.8 582 2470 4.2 
101587_at Ephx1 Epoxide hydrolase 1, microsomal 235 3410 14.5 3682 4366 1.2 4074 4099 1.0 
100325_at Gp49 Glycoprotein 49 A/B 394 5124 13.0 716 4134 5.8 1009 4883 4.8 
96865_at Marcks Myristoylated alanine rich protein kinase C substrate 1183 14871 12.6 952 3840 4.0 1085 5057 4.7 
97105_at C230027N18Rik RIKEN cDNA C230027N18 gene 269 3142 11.7 564 2822 5.0 417 2519 6.0 
101923_at Pla2g7 Phospholipase A2, group VII 284 2408 8.5 24 669 28.2 89 1230 13.9 
160495_at Ahr Aryl-hydrocarbon receptor 31 249 8.0 461 1477 3.2 737 2594 3.5 
93411_at Sema7a Semaphorin 7A 788 6214 7.9 1038 4241 4.1 826 2926 3.5 
102722_g_at IgG3 Ig γ-3 heavy chain precursor 310 2436 7.9 829 1541 1.9 1281 2699 2.1 
100912_at Dph5 DPH5 homolog (S. cerevisiae1229 9553 7.8 687 3390 4.9 954 4768 5.0 
98309_at Ccbp2 Chemokine binding protein 2 481 3549 7.4 85 895 10.5 343 526 1.5 
97487_at Serpine2 Serine (or cysteine) peptidase inhibitor 219 1377 6.3 148 747 5.1 203 1708 8.4 
103422_at CD1d CD1d antigen 1999 12449 6.2 1339 8025 6.0 2393 5418 2.3 
161058_f_at R74862 Expressed sequence R74862 52 313 6.1 17 131 7.8 79 184 2.3 
92356_at Ptpn22 Protein tyrosine phosphatase, nonreceptor type 22 1775 10160 5.7 4668 13103 2.8 7670 20357 2.7 
95462_at Bzw2 Basic leucine zipper and W2 domains 2 1482 8442 5.7 1678 4786 2.9 3611 17670 4.9 
95661_at CD9 CD9 antigen 480 2677 5.6 127 2212 17.4 550 2737 5.0 
104701_at Bhlhd2 Basic helix-loop-helix domain containing 272 1490 5.5 5828 8002 1.4 5729 6426 1.1 
160629_at Res10 Regulator of G-protein signaling 10 450 2368 5.3 135 1234 9.2 419 838 2.0 
97740_at Dusp16 Dual specificity phosphatase 16 787 4075 5.2 1214 3889 3.2 1173 4992 4.3 
102924_at Dtx1 Deltex 1 homolog (Drosophila7962 39206 4.9 9975 29520 3.0 7015 31023 4.4 
93101_s_at Nedd4 Neural precursor cell expressed 660 3176 4.8 480 1244 2.6 645 1626 2.5 
93195_at Mfhas1 Malignant fibrous histiocytoma amplified sequence 1143 5462 4.8 2857 2424 0.8 3446 3592 1.0 
101584_at Rsu1 Ras suppressor protein 1 2028 9591 4.7 2007 4411 2.2 1809 7286 4.0 
102721_at IgG3 Ig γ-3 heavy chain precursor 812 3415 4.2 1344 1948 1.4 1766 2882 1.6 
98433_at Bid BH3 interacting domain death agonist 1622 6473 4.0 689 1926 2.8 495 1277 2.6 
101516_at CD59a CD59a antigen 527 2091 4.0 410 1617 3.9 533 1817 3.4 
102644_at Mdfic MyoD family inhibitor domain containing 784 3083 3.9 533 1751 3.3 1426 2847 2.0 
99071_at Mpeg 1 Macrophage expressed gene 1 2402 8938 3.7 491 1982 4.0 1743 5743 3.3 
160487_at Myl4 Myosin, light polypeptide 4 942 3407 3.6 2627 6593 2.5 2335 6783 2.9 
102223_at Ppl Periplakin 1294 4673 3.6 1482 2177 1.5 1404 2906 2.1 
96283_at Itm2C integral membrane protein 2C 1458 5254 3.6 881 1943 2.2 1073 3249 3.0 
161765_f_at Res10 Regulator of G-protein signaling 10 535 1792 3.4 365 963 2.6 290 733 2.5 
94958_at 1110013L07Rik RIKEN cDNA 1110013LC7 gene 486 1575 3.2 220 548 2.5 129 336 2.6 
101897_g_at CD1d CD1d antigen 5039 16158 3.2 2039 6961 3.4 3129 7065 2.3 
102289_r_at CD21 Complement receptor 2 931 2964 3.2 2116 4053 1.9 1348 3034 2.3 
97460_at Ube2r2 Ubiquitin-conjugating enzyme E2R 2 9117 28949 3.2 5367 13029 2.4 10403 17285 1.7 
102914_s_at Bcl2a1 B cell leukemia/lymphoma 2 related protein A1 3284 10193 3.1 15571 28095 1.8 22037 32334 1.5 
95084_f_at Grhpr Glyoxylate reductase/hydroxypyruvate reductase 2362 7175 3.0 1612 3071 1.9 1565 2941 1.9 
160711_at Decr1 2,4-Dienoyl CoA reductase 1, mitochondrial 187 553 3.0 267 270 1.0 201 437 2.2 
100397_at DAP12 TYRO protein tyrosine kinase binding protein 4658 13714 2.9 751 2767 3.7 2109 3985 1.9 
96735_at Stard10 START domain containing 10 2538 7449 2.9 2026 2932 1.4 1349 1734 1.3 
92587_at Fdx1 Ferredoxin 1 1631 4714 2.9 1510 2957 2.0 2305 3744 1.6 
104298_at 2310044G17Rik RIKEN cDNA 2310044G17 gene 1290 3689 2.9 1797 2292 1.3 1320 4032 3.1 
104299_at Zdhhc14 Zinc finger, DHHC domain containing 14 1176 3359 2.9 328 651 2.0 794 1865 2.4 
160941_at Pde8a Phosphodiesterase 8A 383 1085 2.8 690 1016 1.5 414 1013 2.4 
98822_at Gip2 Interferon, α-inducible protein 1381 3885 2.8 1335 2609 2.0 1244 3258 2.6 
98033_at 1100001H23Rik RIKEN cDNA 1100001H23 gene 4159 11695 2.8 4474 7701 1.7 6414 9391 1.5 
94186_at Traf1 TNFR-associated factor 1 1612 4530 2.8 1740 4258 2.4 1538 4368 2.8 
160069_at Gmnn Geminin 422 1174 2.8 376 323 0.9 229 445 1.9 
95758_at Scd2 Stearoyl-coenzyme A desaturase 2 4483 12233 2.7 1094 2501 2.3 703 1147 1.6 
100880_at 9830147J24Rik RIKEN cDNA 9830147J24 gene 1271 3463 2.7 733 951 1.3 676 1657 2.5 
92850_at Rrbp1 Ribosome binding protein 1 2821 7681 2.7 3262 6051 1.9 2147 5191 2.4 
93013_at Id2 Inhibitor of DNA binding 2 2417 6577 2.7 2786 11282 4.0 6513 15250 2.3 
93261_at lgmn Legumain 2555 6932 2.7 1582 3143 2.0 1838 2832 1.5 
93833_s_at Hist1h2bc Histone 1, H2bc 773 2093 2.7 617 697 1.1 1189 536 0.5 
96688_at Tmem77 Transmembrane protein 77 728 1935 2.7 320 860 2.7 572 1211 2.1 
160762_at Abr Active BCR-related gene 706 1845 2.6 736 2020 2.7 620 1084 1.7 
161788_f_at SlP1 Sphingolipid G protein-coupled receptor 1 565 1476 2.6 1332 747 0.6 936 562 0.6 
93483_at Hck Hemopoietic cell kinase 5919 15436 2.6 2869 9495 3.3 2498 6070 2.4 
94995_at A030007L17Rik RIKEN cDNA A030007L17 gene 848 2186 2.6 926 734 0.8 1184 817 0.7 
92925_at Cebpb CCAAT/enhancer binding protein (C/EBP), beta 1911 4882 2.6 12638 12321 1.0 20455 13386 0.7 
100516_at Chka Choline kinase α 874 2195 2.5 1728 1646 1.0 880 728 0.8 
104712_at Myc Myelocytomatosis oncogene 906 2242 2.5 4293 11096 2.6 4967 12611 2.5 
92352_at SlP3 Sphingolipid G protein-coupled receptor 3 1522 3765 2.5 1223 1791 1.5 1370 2343 1.7 
98931_at Gns Glucosamine (N-acetyl)-6-sulfatase 2595 6366 2.5 3560 4612 1.3 2685 4575 1.7 
102410_at Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 575 1392 2.4 510 2503 4.9 9768 14854 1.5 
          (Table continues 
Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
93430_at Cmkor1 Chemokine orphan receptor 1 78 4369 56.0 12 2081 178.5 107 3147 29.3 
97967_at Plxnd1 Plexin D1 70 3800 54.4 70 107 1.5 44 50 1.1 
102910_at Abcb1a ATP-binding cassette, sub-family B (MDR/TAP) 48 953 19.8 28 716 25.3 52 596 11.6 
92217_s_at Gp49 Glycoprotein 49 A/B 161 2762 17.2 559 2146 3.8 582 2470 4.2 
101587_at Ephx1 Epoxide hydrolase 1, microsomal 235 3410 14.5 3682 4366 1.2 4074 4099 1.0 
100325_at Gp49 Glycoprotein 49 A/B 394 5124 13.0 716 4134 5.8 1009 4883 4.8 
96865_at Marcks Myristoylated alanine rich protein kinase C substrate 1183 14871 12.6 952 3840 4.0 1085 5057 4.7 
97105_at C230027N18Rik RIKEN cDNA C230027N18 gene 269 3142 11.7 564 2822 5.0 417 2519 6.0 
101923_at Pla2g7 Phospholipase A2, group VII 284 2408 8.5 24 669 28.2 89 1230 13.9 
160495_at Ahr Aryl-hydrocarbon receptor 31 249 8.0 461 1477 3.2 737 2594 3.5 
93411_at Sema7a Semaphorin 7A 788 6214 7.9 1038 4241 4.1 826 2926 3.5 
102722_g_at IgG3 Ig γ-3 heavy chain precursor 310 2436 7.9 829 1541 1.9 1281 2699 2.1 
100912_at Dph5 DPH5 homolog (S. cerevisiae1229 9553 7.8 687 3390 4.9 954 4768 5.0 
98309_at Ccbp2 Chemokine binding protein 2 481 3549 7.4 85 895 10.5 343 526 1.5 
97487_at Serpine2 Serine (or cysteine) peptidase inhibitor 219 1377 6.3 148 747 5.1 203 1708 8.4 
103422_at CD1d CD1d antigen 1999 12449 6.2 1339 8025 6.0 2393 5418 2.3 
161058_f_at R74862 Expressed sequence R74862 52 313 6.1 17 131 7.8 79 184 2.3 
92356_at Ptpn22 Protein tyrosine phosphatase, nonreceptor type 22 1775 10160 5.7 4668 13103 2.8 7670 20357 2.7 
95462_at Bzw2 Basic leucine zipper and W2 domains 2 1482 8442 5.7 1678 4786 2.9 3611 17670 4.9 
95661_at CD9 CD9 antigen 480 2677 5.6 127 2212 17.4 550 2737 5.0 
104701_at Bhlhd2 Basic helix-loop-helix domain containing 272 1490 5.5 5828 8002 1.4 5729 6426 1.1 
160629_at Res10 Regulator of G-protein signaling 10 450 2368 5.3 135 1234 9.2 419 838 2.0 
97740_at Dusp16 Dual specificity phosphatase 16 787 4075 5.2 1214 3889 3.2 1173 4992 4.3 
102924_at Dtx1 Deltex 1 homolog (Drosophila7962 39206 4.9 9975 29520 3.0 7015 31023 4.4 
93101_s_at Nedd4 Neural precursor cell expressed 660 3176 4.8 480 1244 2.6 645 1626 2.5 
93195_at Mfhas1 Malignant fibrous histiocytoma amplified sequence 1143 5462 4.8 2857 2424 0.8 3446 3592 1.0 
101584_at Rsu1 Ras suppressor protein 1 2028 9591 4.7 2007 4411 2.2 1809 7286 4.0 
102721_at IgG3 Ig γ-3 heavy chain precursor 812 3415 4.2 1344 1948 1.4 1766 2882 1.6 
98433_at Bid BH3 interacting domain death agonist 1622 6473 4.0 689 1926 2.8 495 1277 2.6 
101516_at CD59a CD59a antigen 527 2091 4.0 410 1617 3.9 533 1817 3.4 
102644_at Mdfic MyoD family inhibitor domain containing 784 3083 3.9 533 1751 3.3 1426 2847 2.0 
99071_at Mpeg 1 Macrophage expressed gene 1 2402 8938 3.7 491 1982 4.0 1743 5743 3.3 
160487_at Myl4 Myosin, light polypeptide 4 942 3407 3.6 2627 6593 2.5 2335 6783 2.9 
102223_at Ppl Periplakin 1294 4673 3.6 1482 2177 1.5 1404 2906 2.1 
96283_at Itm2C integral membrane protein 2C 1458 5254 3.6 881 1943 2.2 1073 3249 3.0 
161765_f_at Res10 Regulator of G-protein signaling 10 535 1792 3.4 365 963 2.6 290 733 2.5 
94958_at 1110013L07Rik RIKEN cDNA 1110013LC7 gene 486 1575 3.2 220 548 2.5 129 336 2.6 
101897_g_at CD1d CD1d antigen 5039 16158 3.2 2039 6961 3.4 3129 7065 2.3 
102289_r_at CD21 Complement receptor 2 931 2964 3.2 2116 4053 1.9 1348 3034 2.3 
97460_at Ube2r2 Ubiquitin-conjugating enzyme E2R 2 9117 28949 3.2 5367 13029 2.4 10403 17285 1.7 
102914_s_at Bcl2a1 B cell leukemia/lymphoma 2 related protein A1 3284 10193 3.1 15571 28095 1.8 22037 32334 1.5 
95084_f_at Grhpr Glyoxylate reductase/hydroxypyruvate reductase 2362 7175 3.0 1612 3071 1.9 1565 2941 1.9 
160711_at Decr1 2,4-Dienoyl CoA reductase 1, mitochondrial 187 553 3.0 267 270 1.0 201 437 2.2 
100397_at DAP12 TYRO protein tyrosine kinase binding protein 4658 13714 2.9 751 2767 3.7 2109 3985 1.9 
96735_at Stard10 START domain containing 10 2538 7449 2.9 2026 2932 1.4 1349 1734 1.3 
92587_at Fdx1 Ferredoxin 1 1631 4714 2.9 1510 2957 2.0 2305 3744 1.6 
104298_at 2310044G17Rik RIKEN cDNA 2310044G17 gene 1290 3689 2.9 1797 2292 1.3 1320 4032 3.1 
104299_at Zdhhc14 Zinc finger, DHHC domain containing 14 1176 3359 2.9 328 651 2.0 794 1865 2.4 
160941_at Pde8a Phosphodiesterase 8A 383 1085 2.8 690 1016 1.5 414 1013 2.4 
98822_at Gip2 Interferon, α-inducible protein 1381 3885 2.8 1335 2609 2.0 1244 3258 2.6 
98033_at 1100001H23Rik RIKEN cDNA 1100001H23 gene 4159 11695 2.8 4474 7701 1.7 6414 9391 1.5 
94186_at Traf1 TNFR-associated factor 1 1612 4530 2.8 1740 4258 2.4 1538 4368 2.8 
160069_at Gmnn Geminin 422 1174 2.8 376 323 0.9 229 445 1.9 
95758_at Scd2 Stearoyl-coenzyme A desaturase 2 4483 12233 2.7 1094 2501 2.3 703 1147 1.6 
100880_at 9830147J24Rik RIKEN cDNA 9830147J24 gene 1271 3463 2.7 733 951 1.3 676 1657 2.5 
92850_at Rrbp1 Ribosome binding protein 1 2821 7681 2.7 3262 6051 1.9 2147 5191 2.4 
93013_at Id2 Inhibitor of DNA binding 2 2417 6577 2.7 2786 11282 4.0 6513 15250 2.3 
93261_at lgmn Legumain 2555 6932 2.7 1582 3143 2.0 1838 2832 1.5 
93833_s_at Hist1h2bc Histone 1, H2bc 773 2093 2.7 617 697 1.1 1189 536 0.5 
96688_at Tmem77 Transmembrane protein 77 728 1935 2.7 320 860 2.7 572 1211 2.1 
160762_at Abr Active BCR-related gene 706 1845 2.6 736 2020 2.7 620 1084 1.7 
161788_f_at SlP1 Sphingolipid G protein-coupled receptor 1 565 1476 2.6 1332 747 0.6 936 562 0.6 
93483_at Hck Hemopoietic cell kinase 5919 15436 2.6 2869 9495 3.3 2498 6070 2.4 
94995_at A030007L17Rik RIKEN cDNA A030007L17 gene 848 2186 2.6 926 734 0.8 1184 817 0.7 
92925_at Cebpb CCAAT/enhancer binding protein (C/EBP), beta 1911 4882 2.6 12638 12321 1.0 20455 13386 0.7 
100516_at Chka Choline kinase α 874 2195 2.5 1728 1646 1.0 880 728 0.8 
104712_at Myc Myelocytomatosis oncogene 906 2242 2.5 4293 11096 2.6 4967 12611 2.5 
92352_at SlP3 Sphingolipid G protein-coupled receptor 3 1522 3765 2.5 1223 1791 1.5 1370 2343 1.7 
98931_at Gns Glucosamine (N-acetyl)-6-sulfatase 2595 6366 2.5 3560 4612 1.3 2685 4575 1.7 
102410_at Hs3st1 Heparan sulfate (glucosamine) 3-O-sulfotransferase 575 1392 2.4 510 2503 4.9 9768 14854 1.5 
          (Table continues 
Table 1A.

(Continued)

Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
97949_at Fg12 Fibrinogen-like protein 2 314 752 2.4 126 541 4.3 234 1144 4.9 
101495_at CD81 CD81 antigen 8639 20584 2.4 5250 7579 1.4 5062 8355 1.7 
98092_at Plac8 Placenta-specific 8 56686 134293 2.4 37938 75591 2.0 29999 77352 2.6 
98417_at Mx1 Myxovirus (influenza virus) resistance 1 260 608 2.3 190 309 1.6 104 206 2.0 
103459_at Slc39a6 Solute carrier family 39 (metal ion transporter) 862 2007 2.3 1337 2067 1.5 971 1825 1.9 
95358_at Pip5k2a Phosphatidylinositol-4-phosphate 5-kinase 4119 9541 2.3 2763 5125 1.9 3479 6084 1.7 
93084_at Slc25a4 Solute carrier family 25 (ademine translocator) 4789 11081 2.3 3596 5997 1.7 6120 7659 1.3 
102217_at Gprk5 G protein-coupled receptor kinase 5 772 1784 2.3 450 1351 3.0 601 668 1.1 
Affymetrix IdentifierGene SymbolGene TitleRelative ExpressionRelative ExpressionRelative Expression
B6 FOB6 MZFold Difference (MZ/FO)SWR FOSWR MZFold Difference (MZ/FO)C3H FOC3H MZFold Difference (MZ/FO)
97949_at Fg12 Fibrinogen-like protein 2 314 752 2.4 126 541 4.3 234 1144 4.9 
101495_at CD81 CD81 antigen 8639 20584 2.4 5250 7579 1.4 5062 8355 1.7 
98092_at Plac8 Placenta-specific 8 56686 134293 2.4 37938 75591 2.0 29999 77352 2.6 
98417_at Mx1 Myxovirus (influenza virus) resistance 1 260 608 2.3 190 309 1.6 104 206 2.0 
103459_at Slc39a6 Solute carrier family 39 (metal ion transporter) 862 2007 2.3 1337 2067 1.5 971 1825 1.9 
95358_at Pip5k2a Phosphatidylinositol-4-phosphate 5-kinase 4119 9541 2.3 2763 5125 1.9 3479 6084 1.7 
93084_at Slc25a4 Solute carrier family 25 (ademine translocator) 4789 11081 2.3 3596 5997 1.7 6120 7659 1.3 
102217_at Gprk5 G protein-coupled receptor kinase 5 772 1784 2.3 450 1351 3.0 601 668 1.1 

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

This work was supported by research funds from the National Institutes of Health Grant AI14782. N.W.K. is a recipient of a Training Grant Postdoctoral Fellowship Award from National Institutes of Health Grant T32 AI7051.

7

Abbreviations used in this paper: MZ, marginal zone; B6, C57BL/6; FO, follicular; S1P1, 3, or 4, sphingosine-1-phosphate receptor type 1, 3, or 4; RGS, regulator of G protein; Tg, transgenic.

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