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The Journal of Immunology

Evidence That Marginal Zone B Cells Possess an Enhanced Secretory Apparatus and Exhibit Superior Secretory Activity1 Kathryn E. Gunn2 and Joseph W. Brewer3 Marginal zone B (MZB) cells are the first splenic B cells to initiate Ab secretion against polysaccharide-encapsulated Ags in vivo. This swift MZB cell response can be reproduced in vitro as LPS treatment induces Ab secretion in as little as 12 h. Conversely, in vitro LPS treatment of splenic follicular B (FOB) cells results in Ab secretion after 2–3 days. The basis for these distinct response kinetics is not understood. We performed ex vivo analysis of resting and LPS-stimulated murine MZB and FOB cells and found that MZB cells express higher levels of the LPS TLR complex RP105/MD-1 and respond to much lower concentrations of LPS than do FOB cells. Furthermore, increasing doses of LPS do not accelerate the kinetics by which FOB cells transition into Ab secretion. Ultrastructural analysis of resting cells demonstrated that rough endoplasmic reticulum is more abundant in MZB cells than in FOB cells. Additionally, RT-PCR and immunoblot analyses revealed that numerous endoplasmic reticulum resident chaperones and folding enzymes are expressed at greater levels in resting MZB cells than in resting FOB cells. Although both LPS-stimulated MZB and FOB cells increase expression of these factors, MZB cells exhibit a more rapid increase that correlates with accelerated kinetics of Ab secretion and higher per cell output of secreted IgM. These data indicate that MZB cells are equipped for exquisite sensitivity to bacterial components like LPS and poised for rapid, robust Ab production, making MZB cells ideally suited as frontline defenders in humoral immunity. The Journal of Immunology, 2006, 177: 3791–3798.

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ost blood flowing through the spleen exits circulation via the marginal sinus, bringing blood-borne pathogens and microbial products into close proximity with marginal zone B (MZB)4 cells, a population constituting only 5–10% of total splenic B cells (1, 2). The juxtaposition of MZB cells (IgMhighIgDlowCD21highCD23low) (3– 6) to the bloodstream allows this subset to initiate rapid T-independent IgM responses to blood-borne Ags (7). In contrast, follicular B (FOB) cells (IgMlowIgDhighCD21intCD23high) (3– 6) reside in the splenic follicle bordering the periarteriolar T cell zone, a position that facilitates interaction with T cells and participation in T-dependent immune responses (2). The MZB cell IgM response can be observed in vitro using the bacterial component LPS as a stimulant. LPStreated MZB cells respond rapidly, exhibiting robust Ab secretion, proliferation, and up-regulation of costimulatory molecules (2). While LPS treatment of FOB cells induces these same events, they occur neither as quickly nor as strongly as in MZB cells (2). The basis for these remarkable differences between MZB and FOB cells is poorly understood.

Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153 Received for publication March 20, 2006. Accepted for publication June 29, 2006. 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 National Institutes of Health Grants NIH-T32 AI007508 (to K.E.G.) and GM61970 (to J.W.B.). 2 Current address: Laboratory of Protein Dynamics and Signaling, National Cancer Institute at Frederick, 1050 Boyles Street, Building 560, Room 22-103, Frederick, MD 21702 3 Address correspondence and reprint requests to Dr. Joseph W. Brewer, Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153. E-mail address: [email protected] 4 Abbreviations used in this paper: MZB, marginal zone B; FOB, follicular B; ER, endoplasmic reticulum; UPR, unfolded protein response; TRAP␣, translocon-associated protein ␣; RER, rough ER.

Copyright © 2006 by The American Association of Immunologists, Inc.

A prevailing theory is that MZB cells are poised to respond, perhaps as a result of prior Ag encounter (8). This is plausible given that MZB cells exhibit a “preactivated” phenotype. Specifically, freshly isolated MZB cells express markers consistent with previous Ag exposure such as increased abundance of transcripts encoding Blimp-1 (7), a master regulator of plasma cell differentiation, higher cell surface levels of CD44, B7.1, and B7.2, and decreased surface levels of L-selectin and IgD (2), as compared with FOB cells. Importantly, MZB cells exhibit high levels of cell surface RP105, a member of the TLR family of innate immune receptors (9). The TLR complexes TLR4/MD-2 and RP105/MD-1 are essential for the B cell LPS response because B cells deficient for TLR4 (10, 11), MD-2 (12), RP105 (13), or MD-1 (14) are hyporesponsive to LPS. The levels of TLRs expressed on cells can impact the magnitude of the response to ligand (15–17). In keeping with this, MZB cells are triggered to proliferate by lower concentrations of LPS than FOB cells (3). Therefore, it follows that high TLR levels might also facilitate rapid Ab secretion when MZB cells encounter stimuli like LPS. A hallmark of Ab-secreting B cells is a highly developed secretory pathway, most notably an elaborate endoplasmic reticulum (ER) network where Ig chains are synthesized and assembled into functional Abs (18, 19). Expansion of the ER during the differentiation process is accompanied by increased expression of a large number of genes encoding proteins that function throughout the secretory pathway, including components of the ER translational apparatus, ER resident molecular chaperones, ER resident oxidoreductases that facilitate oxidative protein folding, and proteins involved in vesicular transport (19 –22). Taken together, these factors provide an environment appropriately equipped for high-rate Ab biosynthesis and secretion (23). This entire process of ER biogenesis was linked recently to the transcription factor XBP-1, a basic leucine zipper protein required for the development of Absecreting B cells (24, 25). XBP-1 has the capacity to orchestrate ER expansion (22, 26) and, functioning downstream of Blimp-1, is essential for optimal expression of many secretory pathway components that are up-regulated as B cells transition into Ab secretion 0022-1767/06/$02.00

3792 (22). The active form of XBP-1, termed XBP-1(S), is generated when the unfolded protein response (UPR), a complex signaling system that emanates from the ER (23, 27), alters the reading frame of XBP-1 transcripts via a novel mRNA splicing mechanism (28 –30). ER abundance, expression of ER components, and induction of XBP-1(S), all of which are linked to secretory capacity, have not been assessed in MZB vs FOB cells. To further delineate the “preactivated” phenotype of MZB cells, we evaluated freshly isolated and LPS-stimulated MZB and FOB cells for TLR expression, ER abundance, and expression of genes that regulate the secretory pathway. In the present study, we report that MZB cells have distinctive molecular and ultrastructural profiles that fit well with their ability to rapidly initiate Ab production. In addition, we show that MZB cells are more proficient Ab producers than FOB cells. These data provide further insight into the means by which MZB cells are poised to swiftly and efficiently respond to blood-borne Ags, enabling timely Ab-mediated control of infections.

MZB CELLS AND RAPID IgM SECRETION chain, XBP-1, BiP, and GAPDH as described previously (32). Additional gene specific primers include: ERdj4, 5⬘-AGTCTGCCTCAGAGCGA CAA-3⬘ and 5⬘-CCTCTTTGTCCTTTGCCATTGGTA-3⬘; MD-1, 5⬘-AT TCTGACTTCTCCGAGC-3⬘ and 5⬘-CTCCCTGTGGAACATCAA-3⬘; RP105, 5⬘-AGGTGTCAATGAAATTCC-3⬘ and 5⬘-CCTGCAATGTC ATTTCC-3⬘; ␤-actin, 5⬘-CCTAAGGCCAACCGTGAAAAG-3⬘ and 5⬘TCTTCATGGTGCTAGGAGCCA-3⬘; GRP94, 5⬘-TTGAAAAAACTGT GTGGGATTGGG-3⬘ and 5⬘-TGTCGTTATACTTCTCATCAGCAA-3⬘; ERp44, 5⬘-CCAGATGTTGCATCCAATTT-3⬘ and 5⬘-GCACAGTCAT CATGCAAAAT-3⬘; and ERO1L␤, 5⬘-CTGTGATGTCGAGACCATC GATAAGTTTA-3⬘ and 5⬘-GGTTGGCTTCCTCAGAATACTTGTA-3⬘. PCR products were resolved on 1.5% agarose gels and visualized by Vistra Green stain (Amersham Biosciences) and fluorescence imaging on a Typhoon fluorimager (Amersham Biosciences) (32). Semiquantitative analysis of gene expression was performed using ImageQuant software. Quantitation by volume integration was used to evaluate the relative density of bands. MZB and FOB cell data were normalized using the ␤-actin signal. The FOB cell value was assigned a value of 1, and MZB cell expression level was expressed as the fold higher expression by MZB cells over that of FOB cells.

DNA-PAGE

Materials and Methods Animals C57BL/6 female mice were purchased (The Jackson Laboratory) and used for phenotypic and functional studies at 12–25 wk of age according to a protocol approved by the Loyola University Medical Center Institutional Animal Care and Use Committee.

Flow cytometry analyses Anti-CD23-PE (B3B4) and anti-CD21-FITC (7G6) were purchased from BD Pharmingen. Anti-MD-1-BIOT (MD14) and streptavidin-allophycocyanin were purchased from eBioscience. Anti-CD180-BIOT (RP/14) was purchased from BioLegend. Data from stained cell samples were acquired using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software (BD Biosciences).

MZB and FOB cell isolation Spleens were harvested from C57BL/6 female mice, and B cells were isolated by RBC depletion and complement-mediated T cell depletion, followed by Lympholyte M gradient. Following RBC lysis, splenocytes were incubated 30 min at 4°C with culture supernatant from the anti-Thy1.2secreting 30-H12 hybridoma obtained from Dr. D. Quinn (University of Ulster, Coleraine, Ireland). Cell suspensions were then incubated with Low-Tox M rabbit complement (Cedarlane Laboratories) for 30 min at 37°C. Cells were washed in HBSS/2% FCS, and viable B cells were isolated via centrifugation for 20 min over Lympholyte M (Cedarlane Laboratories). Viable cells were isolated from the medium/Lympholyte M interface, washed, and resuspended in medium supplemented with 20% FCS. This procedure yielded ⱖ95% pure B cells. CD21intCD23high FOB B cells and CD21highCD23low MZB B cells were sorted to ⱖ99% purity using a FACStarPlus (BD Biosciences), a MoFlo (DakoCytomation), or a FACSAria (BD Biosciences).

Cell culture Sorted cells were plated in RPMI 1640 supplemented as previously described (31) at a density of 5 ⫻ 105 cells/ml in flat-bottom wells and cultured with 20 ␮g/ml LPS (Escherichia coli 055:B5; Sigma-Aldrich). Cells were then isolated, washed, and used for various analytical assays.

ELISPOT and ELISA ELISPOT assays were performed as previously described (32) using 2-fold serial dilutions of B cells. For ELISA, B cells were washed twice in medium and then cultured at 5 ⫻ 105 cells/ml for various intervals. Culture supernatants were assessed for IgM content by ELISA as described previously (31).

Semiquantitative RT-PCR analysis of gene expression Total RNA was isolated from MZB and FOB cells using the Qiagen RNeasy RT-PCR kit (Qiagen). cDNA was prepared using oligo(dT) primers and the Superscript First Strand Synthesis System for RT-PCR (Invitrogen Life Technologies). Four-fold serial dilutions of cDNA were subjected to PCR amplification using gene-specific primers for ␮m, ␮s, ␬, J

DNA-PAGE was used to assess UPR-mediated splicing of XBP-1 mRNA. The primers used to PCR-amplify XBP-1 from cDNA flank the 26-nt intron that is excised upon UPR activation and yield products of 237 and 211 nt from unspliced and spliced XBP-1 mRNA, respectively. These products were resolved on 5% polyacrylamide gels (1.5 mm in thickness) containing 0.5⫻ Tris/borate/EDTA (TBE) buffer (89 mM Tris borate and 2 mM EDTA (pH 8.3)) and 13.3% glycerol using 0.5⫻ TBE running buffer. DNA products were visualized by Vistra Green stain and fluorescence imaging on a Typhoon fluorimager. ImageQuant software was used for data analyses.

SDS-PAGE and immunoblotting Cells were lysed on ice in lysis buffer (0.5% Nonidet P-40, 0.5% deoxycholate, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 ␮g/ml leupeptin, 40 ␮g/ml aprotinin, and 100 ␮g/ml PMSF). Lysates from an equal number of cells were resolved by standard reducing SDS-PAGE. Culture supernatants were resolved on nonreducing 2.5% polyacrylamide/0.5% agarose/SDS gels as described previously (32, 33). Chemiluminescent immunoblotting of Ig ␮ and ␬ chains was performed as described previously (31). Polyclonal rabbit antisera against GRP94, calnexin, ERdj3, and BiP/GRP78 were generously provided by Dr. L. Hendershot (St. Jude Children’s Research Hospital, Memphis, TN). Rabbit anti-translocon-associated protein ␣ (TRAP␣) was a gift from Dr. C. Nicchitta (Duke University, Durham, NC). Rabbit anti-ERp72 (StressGen Biotechnologies), rabbit anticalreticulin (StressGen Biotechnologies), rabbit anti-XBP-1 (Santa Cruz Biotechnology), and mouse anti-␤ actin mAb (clone AC-15) (SigmaAldrich) were purchased. Donkey anti-mouse IgG HRP and donkey antirabbit IgG HRP were purchased (Jackson ImmunoResearch Laboratories).

Electron microscopy Sorted MZB and FOB cells were pelleted by centrifugation, and thin sections were prepared for transmission electron microscopy (model H-600; Hitachi) as described previously (26). Images were captured on 35-mm film and processed using Adobe Photoshop software.

Stereological measurements of rough ER (RER) and cell size Two independent stereological sets containing electron micrographs of freshly isolated MZB and FOB cells were analyzed. Magnification of ⫻15,000 was used for RER and mitochondria, ⫻3,000 for cytoplasm to nucleus ratio, and ⫻40 for cell size. RER abundance was determined by measuring the volume fraction (Vv) with a grid containing test points in the linear array (1.25 cm spacing). Vv is equal to the number of test points falling on RER divided by the number of test points falling on the containing space. The ratio of cytoplasm to nucleus was determined by measuring the number of test points falling in the cytoplasm divided by the number of test points falling in the nucleus. Average cell size was determined from light microscopy analysis of thick sections using the Scion image program. Average pixels per cell were determined and converted to micrometers (3.086 pixels/1 ␮m). The mean cell area (␮m2) was determined in three fields, and SEM was calculated.

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Results TLR expression and LPS response of MZB and FOB cells The mechanistic basis for the distinct kinetics by which MZB and FOB cells initiate Ab secretion in response to LPS stimulation has not been defined. We reasoned that MZB cells possess a greater capacity for LPS signal transduction than do FOB cells. LPS signaling in B cells depends partly upon the TLR complex RP105/ MD-1. Therefore, we first assessed expression of RP105/MD-1 in MZB and FOB cells. Semiquantitative RT-PCR analyses revealed that MZB cells express ⬃4-fold greater levels of transcripts encoding RP105 and MD-1 than do FOB cells (Fig. 1A). These data correlated with flow cytometric analyses demonstrating higher levels of cell surface RP105 and MD-1, 2.3- and 2.8-fold, respectively, on MZB vs FOB cells (Fig. 1B). The higher quantity of RP105/MD-1 on MZB cells is in agreement with published data demonstrating that these B cells exhibit enhanced sensitivity to low concentrations of LPS as measured by proliferation (2). Building upon this information, we cultured MZB and FOB cells in vitro in the presence of various LPS concentrations and assessed Ab secretion over 4 days. MZB cells secreted IgM in response to LPS in a dose-dependent fashion, responding to as little as 0.2 ␮g/ml LPS, whereas FOB cells did not respond to doses ⬍ 2.0 ␮g/ml (Fig. 1C). Moreover, the kinetics of FOB cell differentiation were not accelerated by increasing the LPS concentration (Fig. 1C). These data indicate that MZB cells are specialized in detecting and differentiating into Ab-secreting cells in response to the TLR ligand LPS. Morphologic and ultrastructural analysis of MZB and FOB cells As resting B cells transition into Ab secretion, they increase secretory capacity by expanding the exocytic pathway, most notably the RER, the site of Ig translation and assembly. Given the preactivated phenotype of MZB cells and their ability to rapidly initiate Ab secretion, we hypothesized that the secretory network might be more developed in MZB cells than FOB cells. Ultrastructural analysis of resting MZB and FOB cells by transmission electron microscopy revealed that in resting MZB cells the percentage of cytoplasm containing RER is ⬃2-fold greater than in resting FOB cells (Fig. 2, A and B). Consistent with increased abundance of RER, we found the level of TRAP␣, a component of ER translocons, to be higher in MZB cells than in FOB cells (Fig. 2C). In addition to RER expansion, Ab-secreting B cells undergo other morphologic changes, including increased cell size and ratio of cytoplasmic to nuclear area (C:N). In agreement with previous histological analyses (2), we found that resting MZB cells are slightly larger (⬃1.6-fold) than resting FOB cells (Fig. 2D), while both resting MZB and FOB cells display C:N ratios of ⬃0.8 (Fig. 2E). These data indicate that resting MZB cells have a modestly expanded RER but have not yet achieved other morphologic alterations characteristic of fully developed Ab-secreting cells. Expression of Blimp-1 and XBP-1 in resting MZB and FOB cells Ab secretion depends on the transcription factors Blimp-1 (34) and XBP-1(S) (24, 25). Blimp-1 represses transcription of Pax5 (35), a repressor of XBP-1 (36), and is required for up-regulated expression of XBP-1 mRNA in LPS-stimulated splenic B cells (22). A novel mRNA splicing mechanism initiated by the UPR pathway modifies XBP-1 transcripts to yield XBP-1(S) protein (28 –30), a bZIP factor that has been implicated in the biogenesis of RER (22, 26). Blimp-1 has been shown previously to be expressed at higher levels in MZB cells than in FOB cells (7). Therefore, we reasoned that MZB cells might exhibit elevated expression of XBP-1. As

FIGURE 1. LPS responsiveness of MZB and FOB cells. A, Freshly isolated MZB and FOB cells were assessed for mRNA expression of RP105, MD-1, and ␤-actin. In semiquantitative RT-PCR assays of total RNA, serial 4-fold dilutions of reverse-transcribed cDNA were used for PCR amplification of the indicated targets. Negative controls (NTC, no template; ⫺RT, no reverse transcriptase) for PCR and cDNA synthesis, respectively, are shown. Data representative of three independent experiments are depicted. B, Total splenic B cells were stained with anti-CD21-FITC, antiCD23-PE, and anti-RP105-BIOT (left panel) or anti-MD-1-BIOT (right panel) Abs. Streptavidin-allophycocyanin was used as a secondary staining reagent. Filled histogram, CD21intCD23high cells (FOB cells); open histogram, CD21highCD23neg cells (MZB cells). Data representative of three independent experiments are shown. C, MZB (top panel) and FOB (bottom panel) cells were cultured with the indicated doses of LPS for 3 days. The amount of IgM in culture supernatants on each day was assessed by IgM-specific ELISA (BLD, below limit of detection). While the amount of IgM that accumulated in culture supernatants varied among experiments, MZB cells consistently secreted IgM in response to LPS at concentrations as low as 0.2 ␮g/ml, and FOB cells did not exhibit augmented IgM secretion to increasing doses of LPS. Data from one of three such experiments are shown.

expected (7), we found Blimp-1 mRNA to be more abundant in resting MZB cells than in FOB cells (Fig. 3A). However, this was not the case for XBP-1 transcripts (Fig. 3A). In resting MZB and FOB cells, both the unprocessed and processed forms of XBP-1 message were detectable and expressed similarly between the two subpopulations (Fig. 3B). Neither MZB nor FOB cells expressed

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FIGURE 2. Morphologic and ultrastructural analysis of MZB and FOB cells. Thin sections were prepared from freshly isolated MZB and FOB cells and examined by transmission electron microscopy. A, Representative micrographs of MZB and FOB cells at ⫻15,000 magnification. Arrows indicate RER. B, Stereological analysis of RER abundance was performed on electron micrographs, and the mean ⫾ SEM of the RER to cytoplasm ratio are plotted (n ⫽ 26). Data representative of two independent experiments are shown. C, Immunoblot analysis of TRAP␣, a component of ER translocons, and ␤-actin in freshly isolated MZB and FOB cells. The data depicted were derived from the same immunoblot; intervening lanes have been removed. D, Average cell size of MZB and FOB cells was determined from light microscopy analysis of thick sections using the Scion image program. The mean cell area (␮m2) was determined in three fields of ⬎70 cells each and is plotted ⫾ SEM. Data representative of two independent experiments are shown. E, The amount of cytoplasm relative to the nucleus was determined by stereological analysis of electron micrographs and plotted as the mean ⫾ SEM (n ⫽ 28). Data representative of two independent experiments are shown.

detectable levels of XBP-1(S) protein before LPS stimulation (Fig. 3C). These data indicate that the preactivated status of resting MZB cells does not include augmented UPR activity and further suggest that the enhanced RER in MZB cells is not mediated by XBP-1(S). Gene expression in MZB and FOB cells The elevated abundance of RER in resting MZB cells prompted us to evaluate expression of Ig chains and components of the ER protein folding machinery in MZB and FOB cells. Consistent with the absence of Ab secretion, semiquantitative RT-PCR analyses revealed that resting MZB and FOB cells express extremely low levels of transcripts encoding secretory ␮ H chain (us), ␬ L chain (␬), and J chain (J chain) (Fig. 4A). As expected, LPS-stimulated MZB cells rapidly increased expression of these transcripts and

MZB CELLS AND RAPID IgM SECRETION

FIGURE 3. Analysis of Blimp-1 and XBP-1 expression in MZB and FOB cells. A, Freshly isolated MZB and FOB cells were assessed for mRNA expression of Blimp-1, XBP-1, and ␤-actin. In semiquantitative RT-PCR assays of total RNA, serial 4-fold dilutions of reverse-transcribed cDNA were used for PCR amplification of the indicated targets. Negative controls (NTC, no template; ⫺RT, no reverse transcriptase) for PCR and cDNA synthesis, respectively, are shown. Data representative of three independent experiments are depicted. B, Freshly isolated MZB and FOB cells were assessed for UPR-mediated processing of XBP-1 mRNA. Samples of XBP-1 RT-PCR as in A were resolved by PAGE to separate products corresponding to unprocessed and UPR-processed XBP-1 mRNA. The percentage of total XBP-1 mRNA scoring as unprocessed and UPR-processed are plotted as the mean ⫾ SEM (n ⫽ 3). C, Immunoblot analysis of XBP-1(S) and ␤-actin in freshly isolated MZB and FOB cells. FOB cells cultured with LPS for 3 days served as a positive control for XBP-1(S) detection. The data depicted were derived from the same immunoblot; intervening lanes have been removed. Data representative of two independent experiments are shown.

their protein products (Fig. 4), while FOB cells required longer intervals of stimulation to reach similar levels of expression (Fig. 4). A large cohort of ER resident proteins plays critical roles in the synthesis and maturation of nascent polypetides in the exocytic pathway. These factors include resident ER proteins such as GRP94 and calnexin, the molecular chaperone BiP/GRP78, and enzymes such as ERO1-L␤, ERp44, and ERp72 that regulate oxidative protein folding and thiol-mediated retention of IgM assembly intermediates (18). Interestingly, we found that transcripts encoding many ER resident proteins and folding enzymes were elevated (2- to 3-fold) in resting MZB cells compared with resting FOB cells (Fig. 5A). The abundance of all transcripts assessed

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FIGURE 4. Analysis of Ig expression in MZB and FOB cells. A, MZB and FOB cells were treated with LPS for the indicated intervals and then assessed for mRNA expression of ␮m, ␮s, ␬, J chain, and ␤-actin by RTPCR analysis. Negative controls (NTC, no template; ⫺RT, no reverse transcriptase) for PCR and cDNA synthesis, respectively, are shown. Data representative of four independent experiments are shown. B, MZB and FOB cells were cultured with LPS for the indicated intervals and then assessed for protein expression of ␮m, ␮s, ␬, and ␤-actin by immunoblotting. Data representative of two independent experiments are shown.

increased in both populations after 1 day of LPS stimulation, and all were expressed more strongly (between 4- and 7-fold) in MZB vs FOB cells (Fig. 5A). Moreover, immunoblot analyses revealed that some of these ER proteins such as BiP/GRP78, ERdj3, GRP94, and ERp72 were detectable in resting MZB cells and increased within 1 day of LPS stimulation (Fig. 5B). In contrast, these same factors were more weakly expressed in resting FOB cells and then increased during LPS stimulation to levels comparable to those displayed by MZB cells (Fig. 5B). Interestingly, increased levels of many ER chaperones and folding assistants were apparent before the appearance of readily detectable XBP1(S) protein in both LPS-stimulated MZB and FOB cells (Fig. 5B). These results provide evidence that the protein folding machinery of the ER is enhanced in resting MZB cells and rapidly undergoes further development upon LPS activation.

FIGURE 5. Analysis of ER protein folding machinery expression in MZB and FOB cells. A, MZB and FOB cells were cultured with LPS for the indicated intervals and assessed for mRNA expression of BiP, ERdj4, GRP94, ERp44, and ERO1-L␤ using semiquantitative RT-PCR analysis. The data are plotted as the ratio of the MZB cell expression level for each transcript to that of FOB cells. Data representative of two independent experiments that both revealed enhanced levels of these transcripts in MZB vs FOB cells are shown. B, MZB and FOB cells were cultured with LPS for the indicated intervals and assessed for protein expression of XBP-1(S), BiP, ERdj3, GRP94, ERp72, calreticulin, calnexin, and ␤-actin by immunoblotting. Analysis of ␬ L chains served as a positive control for differentiation. Data representative of two independent experiments are shown.

Secretory output of MZB and FOB cells While it is well established that MZB cells initiate Ab production more rapidly than FOB cells upon LPS stimulation (2, 3), the rate at which these two types of responding B cells secrete Ig has not been evaluated. We reasoned that resting MZB cells, possessing enhanced RER and elevated levels of ER resident folding assistants (Figs. 2, A and B, 5), might have the capacity for increased Ab output, as well as accelerated kinetics of secretion as compared with FOB cells. Using a combination of ELISA and ELISPOT assays, we determined the average per cell IgM secretion rate for MZB and FOB cells at various intervals of LPS stimulation. Indeed, on each day assessed, MZB cells secreted ⱖ3-fold more IgM per hour as did FOB cells on a per cell basis (Fig. 6A). Immunoblot

analysis of culture supernatants revealed that LPS-stimulated MZB and FOB cells both secreted fully assembled polymeric IgM that resolved as IgM pentamers and hexamers (Fig. 6B) (37). Interestingly, MZB cells secreted a greater proportion of hexameric IgM than did FOB cells, a characteristic that is also exhibited by peritoneal B cells (38). Neither MZB nor FOB cells secreted readily detectable amounts of IgM assembly intermediates (Fig. 6B), demonstrating that the increased IgM output by MZB cells does not compromise the quality control mechanisms in the ER that guard against improper secretion of incompletely assembled IgM (39, 40). Therefore, in addition to their rapid transition into secretion,

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FIGURE 6. Analysis of secretory IgM output by MZB and FOB cells. A, MZB and FOB cells were cultured in the presence of LPS for the indicated intervals. On each day, IgM secretion was assessed by ELISPOT assay and secretion cultures followed by ELISA. Data were combined to yield the average per cell IgM secretory rate (BLD, below limit of detection). Data representative of three independent experiments are shown. On day 3 of LPS stimulation, the IgM secretory rate was 4.7 ⫾ 1.3-fold higher for MZB vs FOB cells (n ⫽ 3). B, MZB and FOB cells were cultured with LPS for the indicated intervals, and IgM content in cell culture supernatants was determined by ELISA. Equal amounts of IgM were resolved by agarose/SDS-PAGE under nonreducing conditions and assessed for IgM polymeric structure by ␮ H chain-specific immunoblotting. IgM pentamers, (␮2L2)5, and hexamers, (␮2L2)6, were detected. Data representative of three independent experiments are shown.

LPS-activated MZB cells are more proficient in Ab production than are FOB cells.

Discussion The rapid and robust activity exhibited by MZB cells early during immune responses has previously been interpreted to reflect the preactivated status of this B cell subpopulation. In the present study, we have further delineated the phenotype of resting MZB cells by providing, to our knowledge, the first comparative analysis of cellular ultrastructure in MZB vs FOB cells. Our study also represents the first extensive examination of key cellular proteins involved in plasma cell differentiation and Ig biosynthesis during the LPS-induced transition of MZB and FOB cells into Ab-secreting cells. We have shown that, in comparison to their FOB coun-

MZB CELLS AND RAPID IgM SECRETION terparts, MZB cells express higher levels of the LPS TLR complex RP105/MD-1, exhibit a more highly developed RER, and express elevated levels of various components of the ER protein folding machinery. These MZB cell characteristics fit well with their ability to swiftly initiate Ab secretion upon stimulation, and as we have demonstrated (Fig. 6A), correlate with enhanced Ab output. Our data demonstrate that MZB cells exhibit greater expression of RP105 and MD-1, both at the transcript level and on the cell surface (Fig. 1, A and B). Similar findings regarding cell surface expression of RP105 were reported recently by Nagai et al. (9). These investigators proposed that high levels of RP105 underlie the importance of this receptor for effective Ab responses to Tindependent Ags. In support of this model, we found that MZB cells secrete Ab in response to LPS concentrations 10-fold lower than the doses required to trigger FOB cells (Fig. 1C). Given that elevated expression of TLRs corresponds with enhanced capacity to respond to ligand (15–17), our data and the work of Nagai et al. (9) suggest that heightened RP105/MD-1 expression facilitates rapid LPS-induced MZB cell proliferation and IgM secretion. However, it is also possible that there are inherent differences in the signaling machinery found in MZB and FOB cells. It would be of interest to compare the levels and activities of signaling molecules downstream of TLR4/MD-2, RP105/MD-1, and the BCR in MZB and FOB cells, although such biochemical studies would likely be technically challenging due to the limited number of MZB cells per mouse. Profiling the signaling capacity of these B cell subsets might also yield insight into the molecular mechanisms that underlie the enhanced sensitivity of MZB cells to bacterial B cell superantigens (41, 42). The morphologic signature of an Ab-secreting B cell is an elaborate network of RER, the platform for Ig production. To date, most studies of the exocytic pathway in Ab-secreting B cells have focused on RER expansion after LPS stimulation (19, 43), with little attention given to the status of the RER in resting cells. Previous immunofluorescence, immunohistochemical and flow cytometric analyses by the Kearney laboratory indicated that freshly isolated MZB cells are larger than FOB cells (2, 3). Extending these studies, we have quantified the difference in cell size and demonstrated that resting MZB cells contain more RER than resting FOB cells (Fig. 2, A and B). In agreement with these data, we found that resting MZB cells express higher levels of various ER resident proteins, including translocon subunits (Fig. 2C), molecular chaperones and their cofactors, and folding enzymes (Fig. 5, A and B). Therefore, we propose that the increased abundance of RER and protein biosynthetic machinery allows resting MZB cells to more rapidly initiate synthesis, assembly, and secretion of Ig molecules. The notion that MZB cells are, at least, partially “prepared” for Ig production is reminiscent of the proposal by van Anken et al. (21) that, upon activation, B cells first initiate a “preparative” phase that readies the cell for optimal Ab production. Using a proteomics approach, these investigators found that LPS-stimulated I.29␮⫹ B lymphoma cells undergo changes in metabolic capacity and secretory machinery that precede peak Ig synthesis and secretion (21). Data from studies of splenic B cells (25) and B cell lines (21, 31) suggest that, once the differentiation process is fully engaged, Ig synthesis leads to maximal levels of the XBP-1(S) transcription factor, which orchestrates many events, including optimal translation of IgH chains (44), expansion of the entire secretory apparatus (22, 26), and proliferation of mitochondria (22). This model is largely consistent with our data regarding the kinetics for induction of Ig synthesis, appearance of XBP-1(S) protein, and Ig secretion exhibited by LPS-stimulated MZB and FOB cells. Given the differences we observed in the abundance of RER and

The Journal of Immunology secretory pathway machinery in resting MZB vs FOB cells, we propose that a “preparative” phase is already in progress in resting MZB cells, whereas the XBP-1(S)-mediated events are triggered after stimulation. By contrast, the entire differentiation program must be launched and executed upon activation of FOB cells. The observation of distinct phenotypes for resting naive MZB and FOB cells prompts several questions. First, what transcription factor(s) are responsible for the enhanced secretory apparatus of resting MZB cells? As we (Fig. 3A) and others (7) have shown, MZB cells express higher levels of Blimp-1 transcripts. However, we found no evidence for enhanced expression of XBP-1 in MZB cells (Fig. 3A). Moreover, we were unable to detect XBP-1(S) protein in resting MZB cells (Fig. 3C), suggesting that the increased amount of RER and elevated expression of ER proteins in these cells occurs by a XBP-1-independent mechanism. While Blimp-1 and XBP-1 are both required for a large program of inducible gene expression in LPS-stimulated B cells, a smaller cohort of genes are up-regulated downstream of Blimp-1 in a XBP1-independent fashion in such cells (22). Therefore, we reason that Blimp-1 itself might mediate some of the traits of resting MZB cells that we have shown here. A thorough analysis of MZB cell development and phenotype in prdm1flox/floxCD19Cre/⫹ mice in which Blimp-1, encoded by prdm1, is specifically deleted in B lymphocytes is warranted (34). In addition, the transcription factor ATF6␣ might also be involved in shaping the profile of resting MZB cells because this UPR-regulated bZIP protein has the capacity to enhance expression of many ER chaperones and folding enzymes (45). Second, is the MZB phenotype rooted in a unique developmental program or does it stem from occasional Ag stimulation facilitated by the juxtaposition of MZB cells to the bloodstream? It is generally accepted that MZB cells exhibit traits consistent with previous Ag encounter (2, 8), but this has not been demonstrated directly. In this regard, MZB cells develop in germfree rats in the absence of exogenous Ag and are phenotypically similar to those found in conventionally raised rats (46), indicating that microbial Ags are not essential for the generation of this B cell subset. This does not rule out a possible role for self-Ags or Ags derived from the microbiota of normal mice in modulating the phenotype of MZB cells. In support of this idea, a recent study of splenic B cells demonstrated that cross-linking the BCR up-regulates transcript levels for XBP-1 and a number of ER chaperones and induces the generation of small amounts of cytoplasmic membrane-bound tubular structures, presumably ER (47). Therefore, it is possible that signals received through the BCR either by developing MZB cells or mature MZB cells in situ might elicit enhancement of their secretory machinery. In summary, our data provide clear evidence that the preactivated phenotype of MZB cells includes partial preparedness for Ab production. Much remains to be defined concerning the mechanisms that coordinate the impressive protein biosynthetic and secretory capacity of MZB cells, yielding soluble Ab within hours of Ag encounter and sustaining high-rate Ab output for 3– 4 days. In light of recent data suggesting a link between copious Ig synthesis, impaired proteasomal activity, and the apoptosis of LPS-stimulated B cells (48), it will be particularly interesting to ask whether a connection exists between heightened secretory activity and the short lifespan typical of Ab-secreting MZB cells.

Acknowledgments We thank Patricia Simms (Loyola University Medical Center Flow Cytometry Facility), James Marvin and Ryan Duggan (University of Chicago Flow Cytometry Facility), Dr. John McNulty and Linda Fox (Loyola University Imaging Facility), and LeeTerry Moore for expert technical assistance.

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Disclosures The authors have no financial conflict of interest.

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