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. Author manuscript; available in PMC: 2014 Mar 26.
Published in final edited form as: J Immunol. 2008 May 15;180(10):6663–6674. doi: 10.4049/jimmunol.180.10.6663

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 pneumoniae 1

Nicholas W Kin *,#, Dianna M Crawford †,3,#, Jiabin Liu †,4, Timothy W Behrens †,5, John F Kearney *,6
PMCID: PMC3966313  NIHMSID: NIHMS556235  PMID: 18453586

Abstract

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. 99 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, idiotype positive and negative MZ B cells were sort-purified before (0 hour) or after (1 hour) 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 hour following immunization in the idiotype 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 antigen-specific MZ B cells following interaction with antigen.

Keywords: MZ B cell, FO B cell, microarray, cytokine, idiotype

Introduction

Mature B lymphocytes play an integral role in the adaptive immune response via antigen presentation and antibody secretion. The mature splenic B cell population is divided into the marginal zone (MZ) and follicular (FO) B cell subsets based on anatomical location, cellular surface molecules, and functional immune responses [reviewed in (1)]. MZ B cells respond primarily to T-independent antigens and are proposed to bridge the gap between the rapid antigen non-specific response and the delayed antigen-specific response. FO B cells respond primarily to T-dependent antigens 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 non-recirculating, located at the outer limit of the white pulp region, and characterized by the expression of IgMhiIgDloCD1d+CD21hiCD23lo. The MZ B cell repertoire is enriched with B cells expressing germline-encoded B cell receptors (2-4), 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 antibody, MZ B cells also possess the ability to capture and shuttle antigen to follicular dendritic cells (7) as well as efficiently activate naive T cells directly (8), suggesting a potential role for MZ B cells in T cell-dependent antibody responses as well. 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 MZ and B1 B cell populations but not by FO B cells (9). Additionally, we identified Fc Receptor Homolog 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. 99 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 idiotype positive and negative B cells at 0 and 1 hr 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.

Materials and Methods

Animals

SWR/J and C3H/HeJ samples were kindly provided by T. Waldschmidt (University of Iowa, Iowa City, Iowa) and were from mice housed at the University of Iowa in specific pathogen-free conditions. MD4 anti-HEL conventional transgenic mice were originally obtained from Dr. C. Goodnow (Australian National University, Canberra, Austrailia) (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 Casey Weaver (University of Alabama at Birmingham, Birmingham, Alabama), 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 to 8 weeks of age according to approved animal protocols.

DNA Microarray Analysis

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 Inc., Valencia, CA), and, in accordance with the Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA), cDNA was synthesized. CRNA was synthesized with BioArray High-Yield Transcript Labeling kit (Enzo, New York, NY). Labeled cRNA (~15 μg) was chemically fragmented for 35 min. at 94°C. Affymetrix MG U74Av2 oligonucleotide GeneChips (Affymetrix, Santa Clara, CA) were probed, hybridized, stained, washed, and scanned according to the manufacturer’s protocol at the University of Minnesota Biomedical Genomics Center facility. Each sort-purified cell population was processed independently as true biological replicates.

Flow Cytometry and Cell Sorting

FACS analysis was performed as described previously (16). Briefly, total splenocytes were collected, red blood cells lysed with ammonium chloride, and stained with different combinations of the following antibodies: fluorescein (FITC), phycoerythrin (PE), or allophycocyanin (APC) conjugated anti-mouse CD21, CD23, Thy1.1, CD19 (eBiosciences, San Diego, CA), goat anti-human RGS10 (Santa Cruz Biotechnology, Inc.), goat anti-mouse D6 beta chemokine receptor, and rabbit anti-human Sharp2/Stra13 (abcam Cambridge, MA). All anti-human antibodies cross react with mouse targets. For intracellular FACS analysis, cells were then washed, fixed, and permeablized 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, San Jose, CA). The data were analyzed using FLOWJO software (Tree Star, Inc.).

Western Blot Analysis

Western blot analysis was performed as described previously (17). Briefly, following B cell isolation, cells were lysed, total protein quantitated using a protein quantitation assay (BioRad, Inc.), and protein samples (5-20 μg) were resolved by electrophoresis on 10% polyacrylamide gels (BioRad, Inc.), transferred to Immobilon-P PVDF membranes (Millipore), probed with either goat anti-human RGS10, anti-actin (Santa Cruz Biotechnology, Inc.), goat anti-mouse D6 beta chemokine receptor (abcam Cambridge, MA), and detected with horseradish peroxidase (HRP)-labeled anti-mouse, goat, and rabbit antibodies (Santa Cruz Biotechnology, Inc.), and developed with the LumiGlo Detection Kit (Cell Signaling).

RNA isolation and PCR

Total RNA was isolated from approximately 5 × 105 sort-purified MZ B cells using TRIZOL Reagent (Invitrogen, Carlsbad, CA) following manufacturer’s directions. RT-PCR was performed using Omniscript RT Kit (Qiagen, Valencia, CA) following manufacturers 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: β-actin - 5′-TACAGCTTCACCACCACAGC-3′ and 5′-AAGGAAGGCTGGAAAAGAGC-3′; D6 - 5′-CTTCCAGCTGAACCTTCTGG-3′ and 5′-CGAGTGCAGAAACAAGGTGA-3′; RGS10 - 5′-GCCTTAAGAGCACAGCCAAG-3′ and 5′-CTTTTCCTGCATCTGCTTCC-3′; Thy1.1 - 5′-ACCAAAACCTTCGCCTGGACTG-3′ and 5′-TCCTTGGGGTCTTCTACCTTTCTC-3′; IL-10 - F-CATGGGTCTTGGGAAGAGAA, R-CATTCCCAGAGGAATTGCAT; Stra13 - 5′-GGATTTGCCCACATGTACC-3′ and 5′-TCAATGCTTTCACGTGCTTC-3′ (60°C annealing temperature for all primers).

Data Processing

GeneData Expressionist Pro 1.0 (GeneData Inc., Waltham, MA) 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 (Microsoft Corp., Seattle, WA) were used for statistical analysis. Hierarchical clustering analysis was performed using CLUSTER and visualized in TREEVIEW, as described previously (18).

Statistics

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

Results

DNA Microarray Analysis of resting MZ and FO B cells

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 (1)]. DNA microarray analysis was employed to determine differences in gene expression profiles between MZ and FO B cell populations. Splenocytes from B6 MD4 transgenic mice were sort-purified to obtain paired MZ (B220+, CD21hi, CD23low) and FO (B220+, CD21int, CD23pos) B cell samples. Post-sort analysis revealed greater than 95% purity of each B cell population (data not shown). MD4 mice carry a heavy and light chain transgene specific for hen egg lysozyme antigen (12) and were used because greater than 90% of their B cells express the transgenic B cell receptor, thereby potentially reducing the variability due to a polyclonal repertoire. Gene expression was assessed in three replicates of each B cell population using Affymetrix U74A mouse GeneChip microarray, representing approximately 11,000 transcripts. Expression levels were quantified using GeneData Expressionist Pro 1.0 software and the data from each array was 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 > 2 and p < 0.02 by Student’s T test.

Based on this definition, we identified 181 transcripts differentially expressed between the two populations. 99 transcripts (approximately 55% of total) were more highly expressed in MZ B cells relative to FO B cells while 82 transcripts (approximately 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 was 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 greater than 0.98. The 181 gene transcripts identified were grouped into the following broad functional classifications: Figure 1 (A) motility/adhesion, (B) immune response, (C) apoptosis, (D) proliferation, Figure 2 (A) transcription factors, (B) signal transduction, metabolism (data not shown), or miscellaneous (data not shown). All 181 genes are listed in Table 1.

Figure 1. Expression profile of differentially expressed genes between FO and MZ B cells.

Figure 1

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DNA microarray analysis identified 181 genes that were significantly different in sort-purified follicular (FO) vs. marginal zone (MZ) B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change > 2 and a p value < 0.02 by T-test. The differentially expressed genes were grouped into various functional categories (A) Motility/Adhesion, (B) Immune Response, (C) Apoptosis, and (D) Proliferation. 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 is unsupervised.

Figure 2. Expression profile of differentially expressed genes between FO and MZ B cells.

Figure 2

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DNA microarray analysis identified 181 genes that were significantly different in sort-purified follicular (FO) vs. marginal zone (MZ) B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change > 2 and a p value < 0.02 by T-test. The differentially expressed genes were grouped into various functional categories (A) Transcription Factors and (B) Signal Transduction. 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 is unsupervised.

Table 1.

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

Relative Expression Relative Expression Relative Expression
Affy ID Gene Symbol Gene Title B6 FO B6 MZ Fold Difference (MZ/FO) SWR FO SWR MZ Fold Difference(MZ/FO) C3H FO C3H MZ Fold 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 P1a2g7 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 gamma-3 heavy chain precursor 310 2436 7.9 829 1541 1.9 1281 2699 2.1
100912 at Dph5 DPH5 homolog (S. cerevisiae) 1229 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, non-receptor 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 Bh1hb2 basic helix-loop-helix domain containing 272 1490 5.5 5828 8002 1.4 5729 6426 1.1
160629 at Rgs10 regulator of G-protein signalling 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 (Drosophila) 7962 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 72 86 4.0
102721 at IgG3 Ig gamma-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 Mpeg1 macrophage expressed gene 1 2402 8938 3.7 491 1982 4.0 1743 5743 3.3
160487 at My14 myosin, light polypeptide 4 942 3407 3.6 2627 6593 2.5 2335 6783 2.9
102223 at Pp1 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 Rgs10 regulator of G-protein signalling 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-coniugating enzyme E2R 2 9117 28949 3.2 5367 13029 2.4 10403 17285 1.7
102914 s at Bc12a1 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 G1p2 interferon, alpha-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 Tnf receptor-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 S1P1 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 alpha 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 S1P3 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
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 P1ac8 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 S1c39a6 solute carrier family 39 (metal ion transporter) 862 2007 2.3 1337 2067 1.5 971 1825 1.9
95358 at Pip5k2a phosphatidylinositol-4-phc sphate 5-kinase 4119 9541 2.3 2763 5125 1.9 3479 6084 1.7
93084 at S1c25a4 solute carrier family 25 (adenine 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
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Identification of strain-specific differences in gene expression between resting FO and MZ B cells

To determine if 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. While the absolute signal intensities varied across strains (Table 1), the fold changes between MZ and FO B cell gene expression were comparable (Fig. 3A). We identified 29 genes (approximately 16% of total) that appeared to have a different expression profile between FO and MZ B cells in the C3H and SWR strains relative to the B6 strain (Fig. 3B and Table 2). 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.

Figure 3

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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 > 2 and a p value < 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 is unsupervised.

Table 2.

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

Relative Expression Relative Expression Relative Expression
Affy_ID Gene Symbol Gene Title B6FO B6MZ Fold Difference (MZ/FO) SWR FO SWR MZ Fold Difference (MZ/FO) C3H FO C3H MZ Fold 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), beta 1911 4882 2.6 12638 12321 0.97 20455 13386 0.65
100516_at Chka choline kinase alpha 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
9443l_at St6gal1 beta galactoside alpha 2,6 sialyltransferase 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, beta 2 5831 1961 3.0 8025 9318 0.86 3784 5394 0.70
98083_at Klf6 Kruppel like factor 6 4002 1522 2.6 14189 19034 0.75 18319 14977 1.22
99622_at Klf4 Kruppel-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, alpha 15 689 315 2.2 51 136 0.37 84 136 0.61
98335_at Recc1 replication factor C 1 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
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D6 Beta Chemokine Receptor and RGS10 Are More Highly Expressed in MZ than FO B cells

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 antigens accumulate within the splenic marginal zone 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 antigen. A number of factors have been shown to play a role in MZ B cell localization within the splenic microenvironment including S1P1 (19), the presence of marginal zone macrophages (20), and integrins (21). In addition, in vivo injection of pertussis toxin disrupts MZ localization, suggesting 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 marginal zone. Two proteins more highly expressed in MZ B cells relative to FO B cells were the D6 beta chemokine receptor (D6) and the regulator of G-protein signaling (RGS10) 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. 4A) and protein (Fig. 4B-D) by RT-PCR, Western blot, 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.

Figure 4

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

Gene Expression Profile of MZ Id+ B cells before and after stimulation

In addition to the differential phenotype of resting FO and MZ B cells, MZ B cells respond very differently to antigen than FO B cells. Following activation with antigen, 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 antibody, 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 antigen have not been fully characterized. To determine the gene expression profile of antigen (idiotype) positive MZ B cells before and after activation, M167 Tg mice were immunized i.v. with heat-killed Streptococcus pneumoniae, R36A, and Id+ and Id− MZ B cells were sort-purified at 0 and 1 hour 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: Figure 5 (A) chemokines, (B) chemokine receptors, (C) cytokines, (D) cytokine receptors, Figure 6 (A) apoptosis (B) immune cell markers.

Figure 5. Regulated genes in Idiotype Positive MZ B cells after Activation.

Figure 5

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MZ Id+ (Ag+) and Id− (Ag−) B cells were isolated from M167 Tg mice at 0 and 1 hour 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 hr after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories (A) Chemokines, (B) Chemokine Receptors, (C) Cytokines, and (D) Cytokine Receptors. 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 is unsupervised.

Figure 6. Regulated genes in Idiotype Positive MZ B cells after Activation.

Figure 6

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MZ Id+ (Ag+) and Id− (Ag−) B cells were isolated from M167 Tg mice at 0 and 1 hour 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 hr after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories (A) Apoptosis and (B) Immune Cell Markers. 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 is unsupervised.

We focused on the antigen 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 hr post immunization, as would be predicted. The Id+ MZ B cells rapidly down regulated pro-apoptotic 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 hour after activation to determine if any significant changes occurred (Table 3). 6 of 99 genes (6 %) more highly expressed in MZ B cells were up regulated after activation in the MZ Id+ B cells while 17 of 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 3.

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

Relative Expression Relative Expression
Affy_ID Gene Symbol Gene Title B6 FO B6 MZ Fold Difference (MZ/FO) MZ Id+ 0hr MZ Id+ 1hr Fold Difference (1hr/0hr)
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 ol 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 specilicity phosphatase 16 787 4075 5.2 312 9 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 sullate (glucosamine) 3-O-sullotranslerase 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
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Id+ MZ B cells upregulate IL-10 and Stra13 in response to R36A immunization

A number of interesting genes were identified by DNA microarray analysis on sort-purified MZ Id+ and Id− B cells at 0 and 1 hour 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 immunoglobulin tg mouse with an IL-10/Thy1.1 reporter mouse in which all IL-10+ cells are Thy1.1+ (14), immunized with R36A, and analyzed isolated Id+ MZ B cells for the presence of IL-10 and Thy1.1 mRNA (Fig. 7A) and the Thy1.1 reporter protein (Fig. 7B). 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. 7A). 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 hours 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.

Figure 7

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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 hours after immunization and total RNA was isolated. (A) RT-PCR was performed using gene-specific primers for IL-10, Thy1.1, Stra13, and actin. The expression level of (B) Thy1.1 was determined via FACS analysis on gated MZ Id+ B cells at 24 hours following R36A immunization. MZ Id+ B cells were approximately 5% (PBS) and 20% (R36A) positive for Thy1.1 respectively.

Discussion

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 (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. 99 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 (0hr) and after (1hr) 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 CD21hiCD23lo for MZ B cells and CD21loCD23hi 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, while 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 being expressed higher on FO B cells (19). Our data was 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 agrees 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-alpha subunits (27). Phosphorylation by PKA induces its localization to the nucleus (28). Recently, RGS10−/− mice were reported and exhibited severe osteopetrosis and impaired osteoclast differentiation resulting from the loss of [Ca2+]i oscillation regulation (29), though no immune characterization was reported. While 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, since 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 (30-32). 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 (33-36). Interestingly, our results show that D6 is more highly expressed in resting MZ B cells relative to FO B cells and D6 is rapidly downregulated (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), though 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 hr post i.v. immunizaton 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 Cyster et. al. (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 MZ Id− B cells rapidly increased anti-apoptotic genes and decreased pro-apoptotic genes, a phenotype consistent with cellular activation. This shows a remarkable degree of antigen 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 to play 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, Treg, B-1 and MZ B cells (39). The effects of IL-10 are mainly immunosuppressive, but also depend on what cell type is being affected by IL-10. In EAE, an experimental model of MS, one study suggested that B cells regulate Treg 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 antibody production. Addition of IL-10 to human B cell cultures is reported to increase class switch recombination and production of IgA and IgG (41-43). 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 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 two fold; 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 one hour of the gene expression profiles between antigen-specific versus non-specific 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 present here with respect to FO and MZ B cell biology, which will facilitate identification of new genes and pathways to explore.

Acknowledgements

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

Footnotes

1

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

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