Lymphopoiesis Prebcr-Dependent Events During B Phosphorylation

Heparan Sulfate and Heparin Enhance ERK Phosphorylation and Mediate preBCR-Dependent Events during B Lymphopoiesis This information is current as of September 29, 2021. Craig D. Milne, Steven A. Corfe and Christopher J. Paige J Immunol 2008; 180:2839-2847; ; doi: 10.4049/jimmunol.180.5.2839 http://www.jimmunol.org/content/180/5/2839 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Heparan Sulfate and Heparin Enhance ERK Phosphorylation and Mediate preBCR-Dependent Events during B Lymphopoiesis1

Craig D. Milne,2 Steven A. Corfe, and Christopher J. Paige

As B lineage cells develop, they interact with cells, proteins, and extracellular matrix components of the surrounding microenvironment. In vitro, one critical checkpoint for developing cells occurs as they lose responsiveness to IL-7. These cells require contact with either stromal cells or other B lineage cells to mature. Our results demonstrate that heparan sulfate and heparin are able to promote this transition when added exogenously to the culture system or when heparan sulfate-bearing cell lines are cocultured with primary B cell progenitors. Addition of heparan sulfate or heparin to LPS-stimulated cultures of primary B cell progenitors resulted in more IgM secreted compared with untreated cultures. Heparan sulfate has been Downloaded from reported to be a ligand for the pre-B cell receptor (preBCR). Extending this observation, we found that treatment of ,preBCR؉ cells with heparan sulfate before anti-␮ stimulation leads to increased phosphorylation of ERK1/2. Consequently -preBCR؉ cells proliferate more in the presence of IL-7 and heparan sulfate, whereas preBCR؊ cells are unaffected, sug gesting that in these experiments, heparan sulfate is not directly affecting IL-7 activity. Heparin treatment of cultures induces .many of the same biological effects as treatment with heparan sulfate, including elevated pERK levels in preBCR؉ cells

However, heparin reduces the proliferation of cells expressing only the preBCR (opposed to both the preBCR and BCR) http://www.jimmunol.org/ possibly due to internalization of the preBCR. Heparan sulfates are present on stromal cells and B lineage cells present in hemopoietic tissues and may provide stimulation to preB cells testing the signaling capacity of the preBCR. The Journal of Immunology, 2008, 180: 2839–2847.

he development of B lineage cells depends on the inte- port of this, Ja¨ck and colleagues (10, 11) developed a soluble gration of signals from several biochemical pathways. preBCR-like molecule and found that it could bind to an uniden- Prominent among these are pathways linked to the pre-B tified 90–135 kDa protein in stromal cell lysates. This interaction T 3 cell receptor (preBCR) and the IL-7R, however, contributions requires the unique tail of ␭5, which interacts with heparan sulfate from accessory molecules such as CD19, CD22, CD45, and others chains associated with this protein. by guest on September 29, 2021 can alter the signaling milieu and influence the development of B We have previously described a culture system that allows for lineage cells (1, 2). The central importance of the preBCR has been the maturation of B cell progenitors from lineage uncommitted recognized for many years and is most easily demonstrated in ex- cells through to the IgM-secreting mature B cell stage (12–14). perimental systems that are either deficient in one or more com- Consequently, this culture system supports any required signaling ponents of the preBCR or in systems that artificially mimic pre- by the preBCR or other receptors necessary to mediate this mat- BCR-dependent signaling (3–5). uration. Ig-secreting B lineage cells can be generated in vitro from Despite these observations, the precise mechanism by which the ϩ B220 bone marrow progenitors by culturing cells under a series preBCR influences B cell development remains elusive and a num- of different conditions. Early phases require IL-7 for proliferation, ber of models have been proposed (6–8). At one extreme, it is survival, and maturation of progenitor cells; however, to reach the thought that simple assembly of preBCR components is sufficient Ig-secreting stage in vitro, cells must be transferred to cultures to generate critical signals that promote development. Others pos- containing LPS. This transition phase is not well understood, but tulate that receptor engagement is needed to initiate signal transduction and a number of ligands have been proposed (9). In sup- has classically required a coculture period between stromal cells and B lineage progenitors (15). This coculture permits B lineage cells to mature to the LPS-responsive stage and, ultimately, secrete Ig. In the absence of this contact-dependent stage, cells do not Ontario Cancer Institute, University Health Network, Department of Immunology, University of Toronto, Toronto, Ontario, Canada respond to LPS and do not secrete Ig. Stromal cells produce many Received for publication August 15, 2007. Accepted for publication December cytokines, growth factors, and adhesion molecules, but it is unclear 17, 2007. which specific molecules are necessary to mediate the maturation The costs of publication of this article were defrayed in part by the payment of page of B lineage cells at this stage. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. One model suggests that an important stimulus for develop- 1 ing B lineage cells is the loss of IL-7 availability. This has been This work was supported by grants from the Terry Fox Foundation, the National ϩ Cancer Institute of Canada, and the Canadian Institute of Health Research. reported to induce maturation of pre-B cells to the sIgM stage; 2 Address correspondence and reprint requests to Dr. Craig Milne, Ontario Cancer however, we have previously shown this not to be true (16). The Institute, 8-105 Princess Margaret Hospital, Toronto, Ontario, Canada M5G 2M9. loss of IL-7 does not change the number of cells reaching the E-mail address: [email protected] sIgMϩ stage; it merely changes the proportion of cells surviving in 3 Abbreviations used in this paper: preBCR, pre-B cell receptor. culture, to favor more sIgMϩ cells at the expense of sIgMϪ pro-B Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 and pre-B cells. Although possibly acting upon similar cells, the www.jimmunol.org 2840 HEPARAN SULFATE AND HEPARIN MEDIATE preBCR-DEPENDENT EVENTS

contact-dependent interactions necessary to mediate the matura- Heparitinase digestion tion of progenitors to the LPS-responsive stage are measured by Ig Cells were treated with heparitinase (from flavobacterium heparinum; secretion, the productive result of LPS-stimulation. These experi- Seikagaku/Associates of Cape Cod catalog no. 100703) at 0.1mU/ml/106 ments do not require IL-7, nor are they hindered by its presence. cells at 37°C for 60 min. Subsequently, cells were washed one time and Previous experiments (15) demonstrated that stromal cells could cultured in the presence of 0.02 mU heparitinase. An additional 0.02mU be replaced at this transition if IL-7-expanded B lineage cells were heparitinase was added every 24 h for the first 3 days of culture. This treatment was sufficient to reduce the epitope detected by the mAb cultured in conditions that promoted cell-cell contact (homotypic clone 10E4. interactions). Progenitors cultured in either condition become responsive to LPS, which subsequently promotes further develop- FACS analysis ment and activation of the cells to secrete IgM. Although much of Cells were washed in FACS buffer (PBS, 3% FCS, and 0.01% sodium the current literature focuses on the interface between stromal cells azide) and resuspended in 96-well, round-bottom assay plates at 104-105 and preB cells, there is less known about potential mediators that cells in 15 ␮l. Labeling was done at 4°C for 15 min in a total volume of 65 ␮l containing optimized dilutions of the following Abs (clone): IgM promote development in the absence of stromal cells, such as in (33.60; made in-house), B220 (RA3-6B2; made in-house), CD2 (RM2-5; our system. For example, we have found that the addition of anti-␮ BD Biosciences), CD19 (MB19-1; eBioscience), CD43 (S7-5; BD Bio- Fab to these cultures prevented maturation (15). We could not sciences), CD117/c-kit (2B8; eBioscience), Ig␬ (187.1; BD Biosciences or detect signaling events initiated within the B cell progenitors ex- polyclonal goat anti-Ig␬; Southern Biotechnology), CD23 (clone B3B4; posed to the anti-␮ Fab and hypothesized that this reagent may BD Biosciences), IgD (10.4; made in-house), CD86 (M5/114; BD Bio- sciences), CD21 (7G6; BD Biosciences), CD22 (Cy34.1; BD Biosciences), block the interaction between the preBCR and an external ligand, BP1 (6C3; BD Biosciences), ␭5 (FS1; made in-house), HSA (M1/69; BD or it may disrupt the formation of oligomeric complexes of the Biosciences), CD5 (53-7; BD Biosciences), Mac1 (BD Biosciences), Downloaded from preBCR. These observations prompted us to design experiments to CD45/LCA (30F11; BD Biosciences), preBCR (SL165; BD Biosciences), test molecules that may mediate cell-cell interactions during B CD44 (KM114; BD Biosciences), syndecan 1 (Seikagaku), CD48 (HM48-1; BD Biosciences), TLR4 (MTS510; eBioscience), GR-1 (RB6- lymphopoiesis. 8C5; BD Biosciences), CD18 (M18/2; made in-house), CD102 (3C4; BD Biosciences), syndecan 4 (KY-8; BD Biosciences), Heparan sulfate (10E4; Materials and Methods Seikagaku), and anti-goat Fc (Jackson ImmunoResearch Laboratiories). ␮ Cell culture Cells were washed twice using 200 l and incubated with secondary re- http://www.jimmunol.org/ agents if needed: streptadivin-perCP or anti-rat IgG (both from BD Bio- B lineage progenitors were harvested from bone marrow (both tibia sciences). In some experiments, propidium iodide was added before anal- and femurs) using a 261⁄2 gauge needle and isolated in MACS buffer ysis to label dead cells. 2ϩ 2ϩ (PBSCa ,Mg , 1 mM EDTA, 0.25% BSA) on ice. B220ϩ cells were iso- FACS analysis was performed using a FACScalibur or FACScan (BD lated from BM directly using the anti-B220 (clone RA3–6B2) Ab coupled Biosciences). Acquisition and analysis were performed using CellQuest to magnetic beads (Miltenyi Biotec) at 5 ␮l/107 total cells. Cells were software version 3.1. positively selected in a VarioMACS magnet with an LS adaptor. Typically, 8–14 ϫ 106 B220ϩ cells were recovered. Cell stimulation and Western blotting Cells were cultured in OptiMEM (Life Technologies) supplemented Cells were rested in OptiMEM medium plus 0.5% FCS in the absence of ␮ with 10% FCS (FCS, lot selected - Life Technologies), 50 M 2-ME, IL-7 for 2–3 h at 37°C. At the indicated time, cells were stimulated with ␮ by guest on September 29, 2021 2.4g/L NaHCO3, and 100 g/ml penicillin-streptomycin or kanamycin. ␮ ␮ Ϫ ϩ 100 g/ml heparin, heparan sulfate, or 5 g/ml anti-CD19. At time 2 B220 cells were cultured at 105/ml in 2 ml of medium containing IL-7 ␮ Ј ␮ min, cells were stimulated with goat anti-mouse F(ab )2 at 25 g/ml. 7 and cultured in a humidified atmosphere at 37°C and 5% CO2 for four days. ϫ ϩ Cells were pelleted at time 0, and lysed at 5 10 /ml in 1% NP40, 150 The resulting population is designated as B220 d4IL-7. IL-7 was obtained mM NaCl, 20 mM Tris-HCl (pH7.4), 10 mM NaF, 1 mM sodium ortho- from a supernatant of the stable-transfected J558 cell line (Dr. Ana Cumano, vanadate, 5 mM sodium pyrophosphate, 1 mM PMSF, 5 ␮g/ml aprotinin, Institute Pasteur, Paris, France) and used at dilution of 1/400, previously cal- and leupeptin (Roche) on ice for 30 min. The detergent insoluble fraction ibrated to be equivalent to 5 ng/ml, or a titration where indicated. ϩ was removed by centrifugation for 10 min at 4°C. To asses mitogen-responsiveness, B220 d4IL-7 cells were washed three A total of 10–15 ␮l of the protein lysates were mixed with 4X NuPage times to remove residual IL-7 and subsequently cultured in 96-well plates sample buffer (Invitrogen/Helixx Technologies) and 0.7M 2-ME. This ␮ ␮ (Costar/Fisher) containing 200 l medium and 15 g/ml LPS (isolated was resolved on a 4–12% Bis-Tris gradient gel (Invitrogen/Helixx from Salmonella typhosa; Sigma-Aldrich catalog no. L6386). S17 stromal Technologies) and transferred to a polyvinylidene difluoride membrane cells were irradiated with 2000 rads and cultured at 1000 cells/well where in 20 mM Tris, 150 mM glycine, and 20% methanol. Membranes were indicated. Alternatively, 5000 cells of indicated cell lines were added to blocked in 5% milk/TBS plus 0.2% Tween 20 (TBST) for Ն1 h. De- wells at the initiation of culture. Cultures were incubated in a humidified tection of phosphorylated ERK (New England Biolabs) was performed, atmosphere at 37°C and 5% CO2 for 7 days without feeding. Subsequently, according to the manufacturer’s instructions. For loading controls, ␮ 50 l of supernatant was sampled for ELISA. membranes were stripped using the Re-Blot Plus recycling kit (Chemi- In some experiments, chondroitin sulfate (from porcine rib cartilage; con International), according to the manufacturer’s instructions. Rep- Sigma-Aldrich catalog no. C-7571), heparin (from porcine intestinal mu- robing for nonphosphorylated ERK (New England Biolabs) was per- cosa; Sigma-Aldrich catalog no. H-3149), or heparan sulfate (from bovine formed, as above. Membranes were washed five times for 5 min each in kidney; Seikagaku/Associates of Cape Cod catalog no. 400700) was in- TBST and probed with a secondary Ab (goat anti-rabbit IgG conjugated cluded in the cultures at the indicated concentrations. to HRP; New England Biolabs) in 5% milk/TBST for 1 h. ECL was performed using ECL substrates (Amersham Pharmacia Biotech) as de- Cell lines scribed by the manufacturer. The following cell lines were used in these studies: S17 (BM stroma), B62, ELISA B62c, B62.1 (IL-7-dependent B lineage lines), 45193C (IL-7-dependent B lineage cell isolated from CD45Ϫ/Ϫ mouse), R5b (IL-7-dependent B lin- EIA plates (Costar/Fisher catalog no. 3590) were coated with 5 ␮g/ml goat eage cell isolated from Rag2Ϫ/Ϫ mouse), CB5 (erythroid cell line), EL4 anti-mouse IgM or IgG (Jackson ImmunoResearch Laboratories) in 0.05M (thymoma), A20 (B lymphoblast), 70Z/3 (pre-B cell line), HC3b (IL-7 Tris-HCl, pH 9.8, 0.15M NaCl for 2–18 h. Plates were washed with dis- dependent B lineage cell isolated from Rag2Ϫ/Ϫ, ␮HC-transgenic), IIB4 tilled water 8 times and patted dry. PBS plus 3% FCS was used to block (proB cell), MC9 (mast cell line), and SCID T (pro-T cell line). plates for 30 min and then washed before the addition of 50 ␮l of culture IL-7-dependent cell lines were generated by limiting dilution of B220ϩ supernatant. Supernatants were added undiluted, at 1/10, and 1/100 serial cells cultured in the presence of IL-7 for 6–8 wk as described previously dilutions. A standard curve was established using purified murine IgM 500 (17). These cells were maintained in culture in OptiMEM medium con- ng/ml and 11, 2-fold serial dilutions. Plates were incubated for Ն4 h and taining 5% FCS and IL-7. Other cell lines were maintained in OptiMEM washed as described. Plate-bound Abs were detected by incubation with medium containing 5% FCS in the absence of IL-7. Adherent cell lines anti-mouse IgM conjugated to HRP for1hatroom temperature. After were passaged by trypsinization for 5 min at 37°C, followed by one wash washing, 50 ␮L of substrate (0.5 mg/mL 2,2,3-ethylbenzthiazoline-6-sul- to remove excess trypsin. fonic acid, 0.05M phosphate citrate buffer, and 0.03% sodium perborate The Journal of Immunology 2841

(Sigma-Aldrich)) was added and the absorbance was read at 405/630 nm on an OptiMax microplate reader (Molecular Devices). Values were sub- tracted for a plate blank and included in the analysis only if they fell within the linear range of the standard curve. Data are presented using GraphPad Prism (GraphPad Software).

Results Contact between primary B cell progenitors and certain cell lines promotes maturation B lineage progenitors can be generated from B220ϩ bone marrow cells cultured in the presence of IL-7 for 4 days (as described ϩ previously in Ref. 16, B220 d4IL-7 cells). These cells remain un- responsive to LPS stimulation unless they undergo a period of coculture with stromal cells or are cultured under conditions that promote contact between the B lineage cells themselves. These assays monitor the emergence of LPS-responsive B cells by measuring the production of IgM found in culture supernatants. Al- though evidence suggests that ␮ is required for the transition to the

LPS-responsive stage, it is unclear what other molecules may func- Downloaded from tion during the contact-dependent events at this stage (15). To help establish the molecular basis for this potent maturation signal, we cocultured a variety of cell lines with primary B cell progenitors ϩ (B220 d4IL-7 cells) to determine which ones were able to promote maturation to the LPS-responsive stage.

We began our screen using the B lineage cell line B62c. In many http://www.jimmunol.org/ ways, this line is similar to the primary B cell progenitors that undergo stromal cell or pre-B-pre-B-mediated maturation (17). ϩ The number of primary B220 d4IL-7 cells used in this culture was substantially lower than the number of B62c cells thus ensuring that nearly all cell-cell interactions occurred between the ϩ B220 d4IL-7 cells and the B62c cells. We found that the primary cells were able to secrete IgM in the presence of B62c, indicating the B62c cells possessed the required attributes to stimulate maturation of the primary B cell progenitors. (Fig. 1A). We failed to by guest on September 29, 2021 detect IgM in cultures of LPS-stimulated B62c in the absence of ϩ B220 d4IL-7 cells. These data indicate that that although B62c cells contributed signals that promoted the maturation of primary B cell progenitors, the cell line itself was unable to respond to LPS stimulation and secrete Ig. Using this assay as a screening tool, we tested a panel of cell lines (Fig. 1B) including IL-7-dependent B lineage cell lines (B62, B62c, B62.1, R5b, 45193C, HC3b, and ␮23), transformed B lineage lines (IIB4, 70Z/3, A20), a T lineage cell line (SCID T), an erythroid line (CB5), a mast cell (MC9), and inert, 10-␮m polystyrene beads. The presence of B62, B62c, B62.1, R5b, 45193C, or 70Z/3 led to significantly increased amounts of IgM detectable in ϩ culture supernatants from B220 d4IL-7 cells compared with when ϩ B220 d4IL-7 cells were cultured in the absence of these cell lines ( p value of Յ0.05). Similar to B62c, none of these cells lines produced detectable levels of IgM when cultured in the absence of ϩ ϩ ϩ FIGURE 1. B220 d4IL-7 cells produce more IgM when cultured in B220 d4IL-7 cells (data not shown), confirming that B220 d4IL-7 ϩ the presence of certain cell lines. A, A total of 200 B220 d4L-7 cells cells are the sole source of IgM detected. In fact, some of the cell were cultured in 96-well plates containing LPS and 1000 irradiated S17 lines are incapable of producing Ig because of inherent mutations cells or 5000 B62c cells where indicated. As a control 5000 B62c were Ϫ/Ϫ Ϫ/Ϫ ␮ ϩ (R5b is RAG2 ; HC3b is RAG2 , IgHC transgenic; 23 is cultured in the absence of B220 d4L-7 cells. The emergence of mitogen Ϫ Ϫ ␮MT / ). In contrast, the inclusion of a number of different cell responsive B cells was monitored 7 days later by ELISA measuring lines (CB5, MC9, A20, and SCID T) or polystyrene beads failed to secreted IgM. Results are expressed as the mean Ϯ SD of five wells. B, ϩ promote B cell maturation as indicated by a lack of IgM in the A total of 200 B220 d4L-7 cells were cocultured with 5000 cells culture supernatants. of the indicated lines. Values of p were calculated using the paired Having established that some cell lines, but not others, have the t test comparing the amount of IgM secreted in the presence or ,p value Ͻ0.005. C ,ءء ,p value Ͻ0.05 ,ء .(ϩ absence of a cell line (none capacity to promote the maturation of B220 d4IL-7 cells, we next ϩ A total of 200 B220 d4L-7 cells were cultured in 96-well plates con- investigated whether this is the result of a soluble factor or re- taining LPS. Five thousand cells of the indicated cell line were cultured quired cell contact-dependent interactions. To distinguish between ␮ ϩ in a transwell containing 0.2- m pores to prevent contact with primary these possibilities, we separated B220 d4IL-7 cells from the cell cells. lines using a transwell system. The transwell membrane has 2842 HEPARAN SULFATE AND HEPARIN MEDIATE preBCR-DEPENDENT EVENTS

0.2-␮m pores, which allow for the exchange of nutrients, gases, peptides, and proteins but not cells. When the cell lines are isolated ϩ from B220 d4IL-7 cells using the transwell, the resulting IgM detectable in culture supernatants is similar to that found when ϩ B220 d4IL-7 cells are cultured in the absence of any cell line (Fig. 1C). Taken together, these data suggest that certain cell lines pro- ϩ vide a contact-dependent interaction to B220 d4IL-7 cells that supports their maturation to the LPS-responsive stage. Supportive cell lines express heparan sulfate ϪϪ Ϫ Having deﬁned two groups of cell lines based on their ability (or ϩ inability) to support contact-mediated maturation of B220 d4IL-7

cells, we next examined the surface protein expression of these ND lines using FACS analysis to identify any similarities or differ- ϩϩϩ ϩϩϩϩ ϩ ϩ ϩ ϩϩϩ ϩ ϩ ences. Table I summarizes these data. We observed that although ϩϩϩ ϩ ϩ ϩ only B lineage cells supported the maturation of B220 d4IL-7 cells, not all B lineage cell lines were capable of doing so. For example, Ϫϩϩϩ ϩ ϩ ϪϩϩϮ ϩ ϩ ND ND A20 is an IgGϩ B cell lymphoma that does not support ND ϩ Downloaded from B220 d4IL-7 cell maturation. Analyzing the phenotypes of the sup- ϩ ϩϪϩϩϩ ϩ ϩ ND ND portive group of cell lines, we observed that they differ in expres- ND ND sion of CD45/B220, HSA, preBCR (using the SL-165 Ab), sIgM, ␬ ND sIg , CD19, CD44, CD2, CD22, syndecan-1, and CD48. In con- ND trast, they all express CD102, CD43, BP-1, and syndecan-4. All

cell lines in the supportive group, with the exception of R5b, also ϩ

express heparan sulfate as detected by the 10E4 mAb. The struc- http://www.jimmunol.org/ ture of heparan sulfate is highly variable and the epitope recognized by the 10E4 clone requires N-sulfated glucosamine residues ND (18). Therefore, it is possible that R5b may express a form of ϩϪ ϪϪ ϮϪ ϪϪϪϪϩ ϩϩ ϩ ϩ ϩϪ ϪϪϪϪϩ heparan sulfate not recognized by 10E4. Notably, this cell line ϩϪ ϪϪ expresses syndecan-4, which is known to be heavily heparan sulfated (19). ND ND ND Recently, heparan sulfate was identiﬁed as a putative ligand for ND the preBCR using a soluble preBCR-like molecule (10). We, therefore, tested whether heparan sulfate or a related molecule, heparin, by guest on September 29, 2021 added exogenously to the culture system could inﬂuence Ϫ ϪϩϩϪϪϪϪϩϩϩϩϩ ϩ B220 d4IL-7 cells stimulated with LPS. In fact, there is a dose- ϩϩ ϩϩϩϪϪϪ ϩϩ

dependent increase in IgM secretion when heparin or heparan sul- ND fate is titrated into the culture (Fig. 2). This is not the case with another extracellular matrix family member, chondroitin sulfate, ND ND ND suggesting that these results are not solely dependent on the presence of charged molecules. Similarly, Chen and colleagues (20)

found that intraperitoneal injections of heparin into mice increased CD19 CD44 CD2 CD22 Syndecan-1 CD48 TLR4 RP105 Gr.1 CD18 Mac-1 CD43 CD102 BP-1 Syndecan-4 Hep. Sulfate

the numbers of plaque-forming cells, suggesting that heparin may ␬ play a role as B lineage cells mature and ultimately secrete Ig. Heparitinase treatment of cultures leads to maturation of B cell Ϯ , Bimodal distribution; ND, FACS not performed. progenitors Heparan sulfates are linear polysaccharide chains consisting of re-

peating disaccharide units of glucuronic acid and N-acetyl glu- a cosamine. Similarly, heparin consists of repeating units of 2-O- sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine. Both molecules exhibit exceptional structural diversity due to substitutions of other saccharide residues and differences in sulfation patterns (18). Therefore, it is possible that commercially prepared ϪϪϪϩϪϪϪϪϩϪ Ϫ Ϫ ϪϪϩϪϪϪϪϩϪϪϪϪϪϪϪϪϪϩϩϪϪ ϩϪϩϪϪϪϪϪϪϪϪϩϪϪϪϪϪϪϩϪϪ ϩϪϩϪϪϪϪϮϩϪ ϩϩϩϪϪϪϩϮϩϪ ϩϩϩϪϮϮϩϩϮϮϮϩϪϪϪ ϪϪϪϩϩϩϪϪϩϩ Ϯ ϩϪϩϪϪϪϪϩϪϩϮϩϪϪϪϪϩϩϩϩϩ ϩϪϩϪϩϪϩϩϪϩϪϩϪϪϪϪ ϩϪϩϪϪϪϩ ϩϩϪϩϩϩϩ ϩϪϩϪϪϪϩϩ ϪϪϪϪϪϪϩϪϩϩϮϪϪϪϪϪ heparan sulfates and heparin may differ in biological outcomes ϩϩϩϩϩϪϮϩϪϩϩϩ ϩϪϩϪϪϪϩ

when compared with native heparan sulfates expressed on the sur- CD45/LCA B220 HSA PreBCR IgM Ig faces of the cells that comprise the hemopoietic microenvironment. To address this issue, we treated supportive cell lines with heparitinase and compared their ability to support the maturation of ϩ

B220 d4IL-7 cells (21, 22). IL-7 ϩ

The effectiveness of the heparitinase treatment can be assessed Summary of FACS analysis of cell lines

by ﬂow cytometry using the 10E4 mAb. Fig. 3A illustrates that 23 B220 MC9 SCID T CB5 IIB4 B62 B62c ␮ B62.1 R5b 45193C 70Z/3 HC3b Cell lines were analyzed for the expression of various markers. non-supportive Supportive cell lines

heparan sulfate can be digested from the surface of B62.1 cells a

using heparitinase, however, expression returns to normal levels Table I. The Journal of Immunology 2843

FIGURE 2. Soluble heparin and heparan sulfate cause an increase in the amount of IgM secreted from LPS-stimulated B lineage cultures. A total of

ϩ Downloaded from 200 B220 d4IL-7 cells were cultured in 96-well plates containing LPS and a titration of heparin, heparan sulfate, or chondroitin sulfate. The emergence of mitogen responsive B cells was monitored 7 days later by ELISA measuring secreted IgM. Results are expressed as the mean Ϯ SE of four independent experiments. http://www.jimmunol.org/ within4hiftheenzyme is washed away. We found that an initial digest using 0.1 mU heparitinase, followed by cultures containing an additional 0.02 mU heparitinase supplemented every 24 h, is sufficient to reduce the 10E4 epitope of heparan sulfate levels for Ն72 h. Longer time points were not assessed. Furthermore, continued exposure to heparitinase had no effect on cell number (data not shown) or viability as assessed by exclusion of propidium iodide (Fig. 3A). To test the role heparan sulfate may play in mediating the con- by guest on September 29, 2021 tact-dependent interactions that support B cell progenitor maturation to the LPS-responsive stage, we compared the amount of IgM ϩ detected from supernatants of B220 d4IL-7 cells cultured with ei- FIGURE 3. Heparitinase treatment of cultures results in more IgM ther supportive cell lines or heparitinase-treated cell lines. It is secreted and more CD2ϩsIgMϪ cells arising. A, B62c cells were stained possible that digestion of heparan sulfates would reduce the sup- for the expression of heparan sulfate using the 10E4 mAb. Some B62c portive capacity of the cell lines and ultimately we would observe were treated with 0.1 mU of heparatinase and stained for the expression Ϯ less IgM. However, this treatment results in a statistically signif- of heparan sulfate or returned to culture containing IL-7 0.02 mU icant, average 2-fold increase in the amount of IgM detected heparitinase. Cells were harvested at the indicated times, stained, and analyzed for the expression of heparan sulfate. Histograms represent ( p value Ͻ0.05; Fig. 3B). In fact, heparitinase treatment of ϩ live-gated cells and are representative of two independent experiments B220 d4IL-7 cells in the absence of the cell lines also resulted in an with two different cell lines. On day 3, cells were also labeled with increased amount of IgM secreted. Considering that these cultures propidium iodide (PI) to examine live/dead cells. Filled histogram rep- are not washed during the 7-day incubation period, heparitinase resents negative staining. Open histograms days 2 and 3 represent he- treatment may result in an increase in free heparan sulfate chains paritinase treated cells. On day 1, cells were treated with heparitinase, present in culture, effectively mimicking the situation in Fig. 2 then allowed to recover at 37°C for 4 h (open histogram, right panel)or where heparan sulfate chains were added exogenously to the cul- examined immediately following treatment (left panel). Data are rep- ture. Alternatively, heparitinase treatment of these cultures may resentative of five experiments with the exception of the 4 h time point ϭ result in the exposure of new carbohydrate epitopes on heparan (n 1). B, Cell lines (B62c, B62.1, or R5b) were treated using 0.1 mU ϩ heparitinase 60 min at 37°C or left untreated. These were subsequently sulfated-proteins that influence B220 d4IL-7 cells, leading to more ϩ used in a coculture system containing 200 B220 BMIL-7 cells and 5000 IgM detected. Ϯ ϩ filler cells, LPS 0.02 mU heparitinase. None represents B220 BMIL-7 The influence of heparin, heparan sulfate, and heparitinase in cells only. The emergence of mitogen responsive B cells was monitored 7 days later by ELISA measuring secreted IgM. Points represent the cultures containing IL-7 mean of 4–5 wells from individual experiments. Bar shown is the mean Experiments presented so far have been performed in the absence average of all experiments. Values of p were calculated using the paired of IL-7 and focused on the transition of progenitor cells to the t test comparing the amount of IgM secreted in the presence or absence LPS-responsive stage and ultimately, Ig-secretion. However, be- of heparitinase. cause the starting population of cells from these experiments ϩ (B220 d4IL-7 cells) is taken from cultures containing IL-7, we next heparan sulfate may augment IL-7-induced proliferation of early B examined whether there were any effects of heparan sulfate or cell progenitors. This may occur by promoting presentation of IL-7 heparin in these cultures. Previous studies have found a link be- to B lineage cells or act as a hybrid molecule with IL-7 to stimulate tween heparan sulfate/heparin and IL-7 and have suggested that pre-pro-B cells (23, 24). 2844 HEPARAN SULFATE AND HEPARIN MEDIATE preBCR-DEPENDENT EVENTS

We have previously shown that CD2ϪsIgMϪ cells require IL-7 for continued growth and survival (16) and that a fraction of the CD2ϪsIgMϪ cells are fated to become CD2ϩsIgMϪ, regardless of the presence or absence of IL-7 in the culture system. To test the effects of heparan sulfate, heparin, or heparitinase, B220ϩ cells were harvested from BM and cultured in the presence of IL-7. Heparinitinase treatment in the presence of IL-7 caused no detectable change in the ratio of CD2ϪsIgMϪ cells to CD2ϩsIgMϪ cells, or in the number of cells surviving after 4 days (Fig. 4). Heparin however, caused a shift in the proportion of cells present. A total of 57% of cells were CD2ϩsIgMϪ after the culture period in the presence of IL-7 and heparin, vs a total of 46% in wells containing IL-7 alone. This appeared to be a result of a loss of CD2ϪsIgMϪ cells in wells containing heparin, suggesting that cells at this stage either may be particularly sensitive to heparin treatment or, alternatively, heparin may cause CD2Ϫ cells to mature to the nonpro- liferating CD2ϩ stage, thus reducing the overall number of cells in the culture over time. Heparan sulfate did not signiﬁcantly alter the

proportion of cells in culture but did result in an elevated number Downloaded from of cells present. These results may support previous ﬁndings that heparan sulfate can augment IL-7-induced proliferation of cells, however, given our results that heparan sulfate can inﬂuence the transition of progenitors to the LPS-responsive stage (in the absence of IL-7), we examined the biochemical outcomes of stimu-

lating cells with heparan sulfate or heparin. http://www.jimmunol.org/

Heparin and heparan sulfate increase signaling via FIGURE 4. Heparan sulfate and heparin influence the number of CD2Ϫ ϩ the preBCR cells in cultures containing IL-7. B220 d4IL-7 cells were cultured in 24-well ␮ The transition of cells from the CD2ϪsIgMϪ to the plates for 2 days in the presence of IL-7 and/or 200 g/ml heparin, heparan ϩ Ϫ sulfate, or 0.02mU heparitinase. Cultures were harvested, counted, and CD2 sIgM stage approximates the transition from large preB analyzed by FACS for the expression of CD2 and sIgM. Results are ex- cells to small preB cells (16) and current models suggest this pressed as the mean Ϯ SE of three independent experiments. results from successful signaling generated by the preBCR on large preB cells. Also, Ja¨ck and colleagues (10, 11) have demonstrated that stromal cell-associated heparan sulfates bind spe- by guest on September 29, 2021 cifically to the ␭5 tail of the preBCR, but not to the mature evated levels of pERK when heparan sulfate or heparin are used to BCR. Given the current data that modifications of heparan sul- pretreat cells in conjunction with anti-␮. These results are consis- fate, either by addition of exogenous molecules or digestion of tent with similar, independently generated cell lines (data not endogenous molecules, has profound affects on the maturation shown). of progenitor cells, we examined biochemical responses of the Heparan sulfate and heparin affect the proliferation of preBCR with varying conditions. ϩ Using the cell line B62c, which was previously shown to ex- preBCR cells press ␮ and ␭5, but not ␬ or ␭, basal levels of pERK representing Given that heparan sulfate and heparin are able to increase levels putative constitutive activity of the preBCR were undetectable by of pERK after anti-␮ stimulation, and given the importance of Western blot. However, when cells were exposed to cross-linking pERK with respect to integrating signals from the IL-7R and the anti-␮ Abs for 2 min, pERK can be detected (Figs. 4 and 5, A). preBCR (29), we examined the ability of cells to proliferate in Heparan sulfate or heparin alone did not induce pERK signaling limiting concentrations of IL-7. Previously, we reported that pre- (data not shown); however, pretreating cells with heparin followed BCRϪ cells have reduced proliferation in ‘low’ picogram concen- by anti-␮ generated increased levels of pERK compared with anti- trations of IL-7 compared with preBCRϩ cells. To test the effects ␮ alone. This suggests that heparin is either recruiting coreceptors of heparan sulfate and heparin on proliferation in IL-7 containing that can reduce the signaling threshold of the preBCR, or that cultures, we measured [3H]thymidine incorporation of R5b, B62c, heparin is modifying the cell surface milieu and allowing the anti- and B62.1 cells under various conditions. R5b cells require high ␮ Ab to be more effective. concentrations of IL-7 to proliferate likely due to the absence of a These findings prompted us to compare the results when cells preBCR (29). Heparin and heparan sulfate do not alter the prolif- are pretreated with an activating, anti-CD19 Ab because CD19 has eration of these cells suggesting that there are no nonspecific in- been reported to establish signaling thresholds of the preBCR (25– teractions that affect proliferation. One model suggests that hepa- 28). Activating the CD19 pathway before anti-␮ stimulation re- ran sulfate may bind IL-7 and increase its effective biological sulted in increased pERK levels, similar to when cells were pre- activity, (23) however these current results demonstrate that, at treated with heparin. least under these specific conditions, heparan sulfate does not yield To determine whether these results depend on the presence of increased proliferation of cells in the presence of IL-7. the preBCR, we used the cell line R5b that was generated from a In contrast to these findings, heparan sulfate increases the ability long-term culture of Rag2Ϫ/Ϫ BM cells, and thus does not express of preBCRϩ B62c cells to proliferate in cultures containing IL-7, the preBCR. Shown in Fig. 5C, R5b cells do not generate pERK in ϳ2-fold (Fig. 6B). Our group has previously reported that prolif- response to heparan sulfate or heparin stimulation. In contrast, eration of preBCRϩ cells is influenced by signals generated from both B62c (preBCRϩ) and B62.1 (preBCRϩ/BCRϩ) generate el- the IL-7R and the preBCR. Based on this model, these data support The Journal of Immunology 2845 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Heparin and heparan sulfate treatment increase signaling through the preBCR. A, B62c cells were rested in OptiMEM medium plus 0.5% FCS in the absence of IL-7 for 2–3 h at 37°C. At time Ϫ30 or Ϫ15 min, cells were stimulated with 100 ␮g/ml heparin or 5 ␮g/ml anti-CD19. Ϫ ␮ Ј At time 2, cells were stimulated with goat anti-mouse F(ab )2 at 25g/ ml. Cells were pelleted at time 0, lysed, and subjected to Western blot analysis using a phospho-specific anti-ERK1/2 Ab or total ERK1/2. B, Band intensities were quantified using ImageJ and are expressed as a ratio of pERK2/ERK2. C, R5b, B62c, or B62.1 cells were treated with heparin or heparan sulfate or left untreated for 15 min before stimulation with goat ␮ Ј anti-mouse F(ab )2 for 2 min. Western blotting was performed as described above. D, Band intensities were quantified as in B. FIGURE 6. Heparan sulfate and heparin treatment influence the proliferation of preBCRϩ cells. Proliferation assays were performed including a titration of IL-7, using R5b cells (A), B62c cells (B), or B62.1 cells (C) and the findings shown in Fig. 5 that heparan sulfate leads to increased 200 ␮g/ml heparin (F), heparan sulfate (E), or IL-7 alone (f). Prolifera- preBCR signaling. tion was assayed by [3H]thymidine incorporation after 4 days in culture. Strikingly, heparin stimulation results in different results. At high concentrations of IL-7, cells proliferate to approximately the same levels whether or not heparin is included in the culture. How- the CD2ϩ stage, resulting in a reduced proliferation of the culture ever, at low concentrations of IL-7, cells cultured in the presence as a whole. of heparin and IL-7 incorporate approximately one tenth as much When the cell line B62.1, which has both the preBCR and the [3H]thymidine as cells cultured with IL-7 alone. This supports the BCR, was cultured in the presence of heparan sulfate and IL-7, data presented in Fig. 4 that the presence of heparin in cultures of more thymidine incorporation was observed when compared with ϩ Ϫ B220 d4IL-7 cells and IL-7 results in more CD2 cells maturing to conditions containing IL-7 alone. This is consistent with the results 2846 HEPARAN SULFATE AND HEPARIN MEDIATE preBCR-DEPENDENT EVENTS

observed with preBCRϩ B62c. However, in contrast to B62c, hep- D-glucosamine with many substitutions by N-sulfate, O-sulfate, or arin resulted in consistently increased thymidine incorporation of N-acetyl groups. Heparitinase cleavage of heparan sulfate reveals B62.1 regardless of IL-7 concentration, suggesting that the pres- a desaturated uronase epitope recognized by the 3G10 mAb and ence of the BCR on B62.1 cells greatly influences the outcome of destroys an N-sulfated glucosamine recognized by the 10E4 Ab. heparin activity on these cells. These results are consistent with This may release small oligosaccharides that are able to stimulate similar, independently generated cell lines (data not shown). B lineage development, similar to conditions where heparan sulfate chains were added exogenously to the culture system. Con- Discussion sidering the widespread distribution of heparan sulfates and their The complex process of B lineage cell development is controlled exceptional structural diversity, it is possible that specific heparan by many factors produced from the surrounding microenviron- sulfate conformers are more or less able to influence B lineage ment. These factors can include cytokines, extracellular matrix development. Whether these conformers are uniquely expressed in molecules, chemokines, adhesion molecules, and cell surface pro- lymphopoietic tissue remains to be determined. teins that act in concert to regulate B lineage cells. In vitro assays Heparin and heparan sulfate appear to influence the preB cell have provided invaluable insights into the biology of many of stage. The results obtained from Ja¨ck and colleagues (10, 11) are these factors and have helped to identify the minimum necessary dependent upon the unique tail of ␭5 binding to heparan sulfate. In components that B lineage cells require during their development. addition, our results reveal that heparin or heparan sulfate poten- These include cytokines IL-11, MGF, and IL-7, however following tiates signaling by the preBCR and may result in the cellular out- these cytokine-dependent phases, B cell progenitors require con- comes observed. In fact, heparin or heparan sulfate in combination tact-dependent interactions to mature to the LPS-responsive stages Downloaded from ␮ (12). Many experimental systems depend on the interactions be- with anti- treatment of cells causes a similar response by pERK ␮ tween stromal cells and B lineage cells, however we have previ- compared with anti-CD19 and anti- . Current models suggest ously recognized that B lineage cells can direct their own matu- CD19 lowers the threshold of activation of the preBCR and BCR, ration if cultured in conditions that promote contact between cells and establishes critical signaling thresholds (25–28). In its ab- (15). One important molecule mediating this transition is the presence, there is an impaired proB to preB cell transition and a severe Ϫ/Ϫ

BCR. Anti-␮ Fab added to cultures prevented the maturation of reduction in mature B cells. CD19 B lineage cells have a http://www.jimmunol.org/ ϩ marked reduction in phospho-ERK and phospho-BTK levels fol- B220 d4IL-7 cells to the LPS-responsive phase, but did not appear to induce signaling. One possibility is that the anti-␮ Fab could lowing stimulation with anti-Ig␤ Abs (27, 31). One possibility is bind to the preBCR and by masking binding sites or steric hin- that B cell-associated heparan sulfate may form a bridge stabiliz- drance, prevent the necessary contact-dependent interactions (30). ing CD19-preBCR interactions, and thus lower the threshold of To screen for other molecules that are important during this activation of the preBCR. cellular transition, we developed an assay whereby cell lines are Previously, co-operation between the preBCR and the IL-7R cultured in excess compared with B cell progenitors, ensuring that resulting in ERK phosphorylation was shown to be critical for most cell-cell contacts occur between cell lines and primary cells, preBCRϩ cells to proliferate in low concentrations of IL-7. Herein, or between the cell lines themselves. None of the cell lines were we demonstrate that when heparan sulfate is added exogenously to by guest on September 29, 2021 capable of producing IgM in these assays. Results demonstrated cultures of preBCRϩ cells and IL-7, more proliferation is ob- that some B lineage cell lines could be as effective as stromal cells served. This suggests a strong link between the ability of heparan ϩ in supporting the maturation of primary B220 d4IL-7 cells to the sulfate to stimulate pERK and the resultant increased proliferation IgM-secreting stage. Although stromal cells are popularly included of these cells. This does not occur with preBCRϪ cells demon- in in vitro assays of B lineage cell development, these results sug- strating that the observed effects are not due to an interaction with gest that with the exception of cytokines, B lineage cells express heparan sulfate and IL-7 directly as has been proposed for other all of the necessary components to direct their maturation. model systems (23). All of the cell lines that could support primary cell maturation Including heparin in these experiments revealed different out- were B lineage cells, and they expressed a number of common comes when compared with heparan sulfate. Like heparan sulfate, proteins. By FACS analysis, BP-1, syndecan-4, and heparan sul- heparin treatment of preBCRϩ cells leads to elevated levels of fate were uniquely expressed by cells that could support matura- pERK. However, unlike heparan sulfate, heparin resulted in a re- tion, but not by other cell lines. BP-1 is a cell surface amino pep- duced proliferation of cells in response to IL-7. Similarly, using tidase that is expressed during the IL-7-responsive stages. ϩ Ϫ B220 BM cells, heparin resulted in a reduced number of CD2 Syndecan-4 is a proteoglycan, decorated with heparan sulfate side cells, whereas heparan sulfate resulted in an increased number of chains, that has been shown to be involved in cell-cell binding and CD2Ϫ cells. One possible explanation for this discrepancy may interactions with the extracellular matrix (19). stem from the observation that heparin treatment of preBCRϩ Previous reports suggest a link between heparin/heparan sulfate and B lineage progenitors. One study found that soluble heparin 70Z/3 cells causes the preBCR to be internalized, but not the BCR can cause the internalization of the preBCR (20). In addition, Ja¨ck on more mature cells (20). Our results demonstrated that heparin ␮ϩ ␭ ϩ and colleagues have identified that stromal cell-associated heparan decreased the ability of B62c cells ( and 5 ) to proliferate, but ␮ϩ ␭ ϩ ␬ϩ sulfate may act as a ligand for the preBCR (10). Another study did not have the same effect on B62.1 cells ( , 5 , and .If found that heparin treatment of Whitlock-Witte cultures prevents heparin caused preBCR structures on B62.1 to be internalized, the formation of lymphocytes (23). However, when the addition of these cells may be protected from the decreased proliferation ob- heparin was delayed, the authors report normal to elevated num- served in B62c by remaining BCR structures that are resistant to bers of B lineage cells. heparin-induced internalization. Our results demonstrate that heparan sulfate-bearing B lineage Ultimately, heparan sulfates are expressed as a normal constit- cells can increase the amount of IgM secreted by LPS-stimulated uent of hemopoietic tissues, both by stromal cells and by devel- ϩ B220 BMIL-7 cells; this is further enhanced by treating cultures oping B lineage cells. Mounting evidence has highlighted the po- with heparitinase. Heparin and heparan sulfate are linear chains tential importance of these molecules during B lymphopoiesis. The consisting of repeating disaccharide subunits of uronic acid and results presented herein substantiate previous findings that heparan The Journal of Immunology 2847 sulfates bind the preBCR by documenting the biological and bio- 14. Ray, R. J., A. Stoddart, J. L. Pennycook, H. O. Huner, C. Furlonger, G. E. Wu, chemical events resulting from manipulating heparan sulfate/hep- and C. J. Paige. 1998. Stromal cell-independent maturation of IL-7-responsive pro-B cells. J. Immunol. 160: 5886–5897. arin as B lineage cells progress. Heparan sulfates may bind the 15. Stoddart, A., H. E. Fleming, and C. J. Paige. 2001. The role of homotypic inter- preBCR and lower the activation threshold needed for signal prop- actions in the differentiation of B cell precursors. Eur. J. Immunol. 31: agation, resulting in enhanced pERK, proliferation, and eventually 1160–1172. 16. Milne, C. D., H. E. Fleming, and C. J. Paige. 2004. IL-7 does not prevent pro- maturation to LPS-responsive stages. 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