Formation of Dendritic Processes Lymphocytes and Can Transmit A

Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes

This information is current as Yoshio Yamashita, Kenji Oritani, Erina K. Miyoshi, of September 24, 2021. Randolph Wall, Merton Bernfield and Paul W. Kincade J Immunol 1999; 162:5940-5948; ; http://www.jimmunol.org/content/162/10/5940 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes1

Yoshio Yamashita,* Kenji Oritani,* Erina K. Miyoshi,† Randolph Wall,† Merton Bernﬁeld,‡ and Paul W. Kincade2*

Our previous studies indicated that stromal cell-derived syndecan-4 might mediate some form of communication with pre-B cells in bone marrow. We now report additional aspects of this recognition and show that syndecan-4 is also present on pre-B cells. Indeed, the molecule is acquired at an early stage of differentiation and retained until mature B cells undergo Ig isotype switching. mAbs developed to two portions of the syndecan-4 protein core were used to probe possible functions on B lineage lymphocytes. Syndecan-4 ligation had no obvious influence on B lymphocyte formation or activation, but this treatment caused a dramatic morphological change in appropriately stimulated leukocytes. Extended filopodia appeared on transfected Ba/F3 or FDCP-1 cells, as well as activated B cell blasts that were placed on syndecan-4 Ab-coated surfaces. The dendritic processes contained polymer- Downloaded from ized actin as well as pp52(LSP1), a prominent F-actin binding protein in lymphocytes. The cytoplasmic domain of syndecan-4 was not required for this response. Shape changes of this type could facilitate interactions between B lymphocytes and other components of the immune system. Not only is syndecan-4 a useful marker for discriminating normal B lineage lymphocyte subsets, but our results suggest new ways for the syndecans to participate in immune responses. The Journal of Immunology, 1999, 162: 5940–5948. http://www.jimmunol.org/ ell-surface heparan sulfate proteoglycans (HSPGs)3 rep- regulated with respect to developmental expression and cell-type resent an abundant and widely expressed group of mac- specificity as well as the extent and nature of glycosylation. An C romolecules. Of major interest is the ability of HSPGs to early finding was that syndecan-1 is present on pre-B cells, lost interact with heparin-binding growth factors and chemokines. The immediately before B cell maturation, and then reexpressed upon productive binding of some factors to transducing receptors can be differentiation to the plasma cell stage (6). Northern blots of lym- critically dependent on HSPGs. The best characterized cell-surface phoid tissues and transformed cell lines suggest that lymphocytes HSPGs include syndecans, glypicans, betaglycan, and certain may express additional syndecans, including syndecan-4 (7). We CD44 family proteins (1). Representatives of these categories are have now explored this issue in greater detail with syndecan-4- known to be expressed in bone marrow, and interactions with var- specific mAbs and nontransformed cells. Our results revealed that by guest on September 24, 2021 ious HSPG ligands may have important consequences for the pro- syndecan-4 coincides with developmental progression of B lineage duction of hemapoietic cells (2–4). Syndecan-4 was one of seven precursors. Ectopic expression and ligation of this glycoprotein stromal cell products that we identified with a new two-step clon- caused dramatic morphological changes that may facilitate inter- ing strategy (5). A soluble fusion protein containing some extracellular communication. HSPGs of this kind may contribute to cellular domains of syndecan-4 bound specifically to pre-B cells, formation as well as maintenance of the humoral immune system. suggesting it might be important for lymphocyte interactions with stromal cells. We extended these findings by using fusion proteins Materials and Methods containing the full length extracellular domain of syndecan-4 and Experimental animals now report recognition requirements by counter-receptors on BALB/c and CB17 scid/scid mice were obtained from the Oklahoma Med- pre-B cells. ical Research Foundation Laboratory Animal Resources Center. All exper- A second aim of this study was to investigate the distribution of iments reported here were done with female mice at 4–8 wk of age. Wistar syndecan-4 in lymphohematopoietic tissues. Syndecans are highly rats were purchased from Harlan (Indianapolis, IN). Cell lines B lineage cell lines (BCB10, BC7.12, 1A9, 70Z/3, WEHI231, CH12, *Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, BCL1, and SP2/0), T lineage cell lines (2B4 and BW5147), fibroblast cell † Oklahoma City, OK 73104; Department of Microbiology and Immunology, Univer- lines (NIH 3T3 and L929), stromal cell lines (BMS2 and ST2), macrophge sity of California, Los Angeles School of Medicine, Los Angeles, CA 90095; and cell lines (J774A.1 and P388D1), a human renal carcinoma cell line trans- ‡Joint Program in Neonatology, Harvard Medical School, Boston, MA 02115 fected with large T Ag (293T), and the Jurkat human T lymphoma were Received for publication October 7, 1998. Accepted for publication February maintained as previously described (5). The Ba/F3 and FDC-P1 immature 19, 1999. hematopoietic cell lines were grown in medium conditioned by IL-3-trans- The costs of publication of this article were defrayed in part by the payment of page fected Chinese hamster ovary cells. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Transfection 1 These studies were supported by Grants AI 20069, AI 33085, CA 12800, GM 40185 Native murine syndecan-4 cDNA was inserted into the pEF-BOS vector (a and HD 06763, CA 28735, and HL 56398 from the National Institutes of Health. kind gift from Dr. S. Nagata, Osaka Bioscience Institute, Osaka, Japan), 2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Oklahoma and 2 ␮g of plasmid was then transfected into 293T cells using a calcium Medical Research Foundation, Immunobiology and Cancer Program, 825 N.E. 13th phosphate method. After 2 days of culture, 293T cells were detached with Street, Oklahoma City, OK 73104. E-mail address: [email protected] PBS containing 0.2 mM EDTA and stained with the KY series of mAbs. 3 Abbreviations used in this paper: HSPG, heparan sulfate proteoglycans; HS, heparan Truncated forms were also used that lacked the cytoplasmic domain of sulfate; int, intermediate. syndecan-4 and had the transmembrane domain from tissue factor with

either the full extracellular or amino terminal portions of syndecan-4 as crotiter plates, and rat mAbs were added. HRP-labeled goat anti-rat IgG previously described (5). For stable transfection, the pRc/RSV vector con- (HϩL) (Zymed) and peroxidase substrate (ABTS kit from Zymed) were taining the neomycin resistance gene was purchased from Invitrogen then used for their detection. Results are presented as mean OD Ϯ SD from (Carlsbad, CA). Both plasmid DNAs were added to suspensions of either triplicate wells. FDC-P1 or Ba/F3 cells in a cuvette (gene pulser cuvette; Bio-Rad Labo- ␮ ratories, Richmond, CA) and a 0.35 kV, 960 F pulse was applied with a Colony-forming cell assays gene pulser (Bio-Rad). Transfected cells were cultured for 24 h and then selected in the presence of 1 mg/ml G418 (Sigma, St. Louis, MO). G418- Murine bone marrow cell populations were suspended in 1 ml of assay resistant clones were selected for intensity of syndecan-4 expression by medium as previously described (10). The semisolid agar cloning assay for flow cytometry. B lymphocyte precursors was done with 2 ng recombinant mouse IL-7 (Upstate Biotechnology, Lake Placid, NY). The granulocyte-macrophage Expression of soluble recombinant Ig fusion proteins progenitor assay (CFU-c) was done with 25 ␮l of 10-fold concentrated L cell-conditioned medium as a source of CSF. All cloning assays were per- A pEF-BOS-derived IgG1 expression plasmid was constructed essentially formed in 35-mm Petri dishes and were incubated at 37°C, 5% CO . Col- as described (5). These constructs were introduced into DH5␣, expanded, 2 onies were scored on day 6. To separate syndecan-4 positive and negative and purified for transfection. Purified plasmids were transfected into 293T populations from bone marrow, cells were stained with biotinylated KY/ cells with a calcium phosphate method. After 4 days of culture, superna- 8.2 mAb followed by PE-streptavidin. After two washes, cells were sorted tants were collected and used for immunofluorescence staining or ELISA. using the FACStarPlus cell sorter (Becton Dickinson). Preparation of mAbs Spreading assay and confocal microscopy Wistar rats were immunized six times with BCB10, a pre-B cell line found by RT-PCR to strongly express syndecan-4. Popliteal lymph nodes were mAbs were bound to glass Lab-Tek chamber slides (Nalge Nunc Interna- removed and fused with SP2/0 myeloma cells (American Type Culture tional, Naperville, IL) by incubation at 4°C overnight at concentrations Downloaded from Collection, Manassas, VA). Over 2000 hybridoma supernatants were ranging from 10 ␮g/ml to 100 ␮g/ml. The wells were washed twice with screened by ELISA for reactivity to a syndecan-4 Ig fusion protein. The HBSS (Life Technologies) and then blocked with 1% BSA or 10% FBS. resulting Abs (designated the KY series) were IgG2a,␬. Abs were purified Cells were plated on mAb-coated wells in culture medium and incubated from the ascites fluid of CB17 scid/scid mice that had been transplanted for4hat37°C. Photomicrographs were made after examination by phase with these hybidomas. contrast microscopy. Viable cells were stained with a monoclonal anti- syndecan-4 Ab different from that used for coating slides. The biotin-la- Immunofluorescence staining beled primary reagent was revealed by staining with cychrome-labeled http://www.jimmunol.org/ The fluorescent Abs used in these experiments were as follows. The pri- streptavidin, and the cells were washed and fixed with 3.7% formaldehyde mary reagents FITC-conjugated anti-mouse CD3, CD19, CD24 (M1/69), plus 0.1% Triton X-100. Some of the fixed chamber slides were stained BP-1, Thy1.2, Ly-6G (GR-1), PE-conjugated anti-mouse TER119, CD5, with PE-labeled phalloidin (Molecular Probes, Eugene, OR). Additional CD43, and APC-conjugated CD45R (B220) were obtained from Phar- slides were stained with a purified polyclonal goat Ab to pp52, followed by Mingen (San Diego, CA). PE-conjugated anti-mouse IgA was obtained FITC-labeled anti-goat IgG. After removal of chambers, slides were from Southern Biotechnology Associates (Birmingham, AL). FITC-conju- mounted with the Prolong anti-fade kit (Molecular Probes) and examined gated anti-Mac-1/CD11b was obtained from Boehringer Mannheim Bio- with a TCS-NT laser scanning confocal microscope (Leica, Heidelberg, chemicals (Indianapolis, IL). The new rat Ab against mouse IL-7R␣ (SB/ Germany). 199; IgG2b,␬) was established in our laboratory (Y. Yamashita et al., manuscript in preparation). The secondary reagent PE-streptavidin (Vector, Results by guest on September 24, 2021 Berlingame, CA) was used in dual staining experiments, and RED613- streptavidin (Life Technologies, Gaithersburg, MD) was added for three- The complete extracellular domain of murine syndecan-4 or four-color staining. FITC-conjugated anti-mouse IgM and FITC-avidin selectively recognizes B lymphocyte lineage cells, and heparan were obtained from Zymed (San Francisco, CA) sulfation is required To minimize nonspecific binding, cells were preincubated with the anti- Fc receptor mAb, 2.4G2 (American Type Culture Collection), and 10% We previously demonstrated that an N-terminal fragment of syn- normal rat serum for 20 min on ice and then washed. Cells were then decan-4, when expressed as a fusion protein with human IgG1, incubated with the appropriate combinations of primary Abs in staining binds to a pre-B cell line (5). The full-length, native sequence of medium (PBS with 2% FCS, 0.1% NaN3) on ice for 20 min, washed twice syndecan-4 has now been obtained and, with the exception of a with staining medium, then incubated an additional 20 min with PE- streptavidin (Vector) for dual staining and with RED613-streptavidin (Life 2-nt insertion in the noncoding region, was identical to one re- Technologies) for three- or four-color staining experiments, and finally cently published (11). It has a high degree of homology with other washed twice with staining medium. Flow cytometry analysis was con- members of the syndecan family and is very similar to the synde- ducted using a FACSCalibur (Becton Dickinson, San Jose, CA). can-4 characterized in other species (data not shown). Because the For detection of fusion protein binding, cells were stained with culture original fusion protein contained only the first 71 residues, we supernatants from 293T cells transfected with the F-syndecan-4-Ig/ pEFBOS plasmid. FITC-conjugated goat anti-human IgG (Southern Bio- prepared a second fusion protein containing 122 aa to assess biotechnology Associates) was used as a secondary Ab. Supernatants contain- logical activity of the native syndecan-4 extracellular domain. We ing soluble CD44-Ig were used for negative controls. Staining efficiency also conducted experiments to further define the nature of the syn- was improved by inclusion of MnCl2 (5 mM) in the staining buffer (5) and decan-4 interaction with pre-B cells. by performing incubations at room temperature. Digestion with heparatinase (Seikagaku, Ijamsville, MD) was performed as described by Stanley The full-length ectodomain recognized the BC7.12 pre-B cell et al. (8) except that the reactions were done in PBS containing 0.1% BSA. line (Fig. 1) in a manner indistinguishable from the truncated ver- sion, demonstrating that the recognition portion of syndecan-4 is ELISA located in the amino terminus and functional irrespective of Capturing mAbs were coated onto 96-well microtiter plates, and fusion whether the membrane proximal domain is present. In addition to proteins were added. HRP-labeled goat anti-human IgG Ab (Southern Bio- bone marrow pre-B cells, syndecan-4-Ig bound to a small number technology Associates) was then used for detection of fusion proteins. of B cells in spleen (not shown). Binding of the full-length domain Color was developed with a peroxidase substrate, and OD at 405 nm were measured on a microplate reader. The purified syndecan-1 ectodomain was was influenced by divalent cations and dependent on HSPG mod- 2ϩ prepared as described elsewhere (9). GST-fusion proteins of the syndecan- ifications (Fig. 1). The presence of 5 mM Mn increased binding 2–4 ectodomains were prepared by inserting the cDNA corresponding to efficiency similar to the 71-aa fragment (5). However, treatment the extracellular domain of each syndecan into the pGEX-2T expression with 5 mM EDTA, an amount sufficient to totally block binding of vector (Pharmacia, Piscataway, NJ), in frame with the sequence encoding GST, expressing the vectors in Escherichia coli and purifying the fusion the stromal interaction molecule (SIM) fusion protein (5), only proteins by absorption and elution from glutathione Sepharose 4B (Phar- slightly interfered with syndecan-4-Ig. Although the target of syn- macia). The ectodomain and fusion proteins were coated onto 96-well mi- decan-4-Ig on pre-B cells is not known, pretreatment of BC7.12 5942 MURINE SYNDECAN-4

Table I. Recognition by KY mAbs of truncated and full-length syndecan-4

Fusion Proteins

mAbs N-Syndecan-4-Iga F-Syndecan-4-Iga CD44-Ig

None 0.038 Ϯ 0.01b 0.015 Ϯ 0.005 0.037 Ϯ 0.029 KY/8.2 1.173 Ϯ 0.024 1.184 Ϯ 0.078 0.017 Ϯ 0.017 KY/103 0.299 Ϯ 0.021 1.073 Ϯ 0.015 0.037 Ϯ 0.013 YP/28c 0.153 Ϯ 0.031 0.147 Ϯ 0.018 1.249 Ϯ 0.025

a N-Syndecan-4-Ig contains the N-terminal portion of syndecan-4 (71 aa), and F-Syndecan-4-Ig is composed of all 122 aa of the syndecan-4 extracellular domain. b The results represent mean OD Ϯ SD from triplicate determinations by ELISA. c YP/28 is an Ab to murine CD44 established in our laboratory.

Syndecan-4 is preferentially expressed on B lineage cells in bone marrow The new mAbs were used to investigate the distribution of syndecan-4 on hematopoietic cells by multiparameter flow cytometry Downloaded from (Fig. 2). Substantial numbers of nucleated cells in the bone marrow FIGURE 1. Heparin and heparitinase treatment block recognition of were syndecan-4 positive. Approximately 85% of the syndecan-4ϩ pre-B cells by syndecan-4, but divalent cations are not required. Flow cells expressed the B lymphocyte lineage marker CD19, although cytometry results are shown for binding of a soluble syndecan-4-Ig fusion there was not complete concordance between these two markers. protein (shaded histograms) to the BC7.12 pre-B cell line. Staining was Few, if any, syndecan-4-bearing cells were myeloid (expressed performed in Tris-buffered saline alone or with EDTA (5 mM), heparin (5 ␮ ␮ either GR1/Ly-6G or Mac-1/CD11b), and only a small fraction g/ml), chondroitin sulfate A (5 g/ml), or heparitinase (0.1 U/ml). Back- ϩ http://www.jimmunol.org/ ground levels of fluorescence obtained with a control CD44-Ig fusion pro- were in the erythroid lineage (TER119 ). Restriction of synde- tein are shown as open histograms. can-4 expression to B cell precursors suggested that the molecule might be acquired as a consequence of lymphocyte maturation. Therefore, additional Abs were used to resolve subsets of lympho- cells with heparatinase nearly abolished the recognition reaction. cyte precursors (12). Binding was also inhibited by the presence of soluble heparin but Presumptive early precursors (fraction A, CD45R/B220ϩ not chondroitin sulfate A, pointing to the involvement of HSPGs. CD43ϩ CD24Ϫ) lacked syndecan-4 (Fig. 3), but the marker was Finally, the fusion protein was inactive when prepared in 293T progressively acquired by more mature cells. While only half of cells treated with sodium chlorate, an inhibitor of sulfation (data fraction C cells (distinguished by BP-1 expression) were positive, by guest on September 24, 2021 not shown). These observations are consistent with the heparan this increased to 70% of pre-B cells (fraction D that lacks CD43; sulfate (HS) chains of syndecan-4 interacting with some HSPG on Fig. 4A) and nearly all sIgMϩ B cells (fractions E and F, distin- pre-B cells. guished by sIgM densities) (Fig. 4, B and C). IL-7 is essential for B lymphocyte formation in mice, and early Establishment of Abs to the protein core of syndecan-4 lymphocyte precursors can be resolved on the basis of IL-7R ex- From a panel of mAbs generated against syndecan-4, two clones pression (13). Flow cytometry revealed that while virtually all syn- (KY/8.2 and KY/103) that appeared to recognize distinct epitopes decan-4-bearing lymphocytes also expressed the IL-7R, substantial were further characterized (Table I). The KY/8.2 mAb had equal numbers of IL-7Rϩ cells lacked syndecan-4 (Fig. 3). This might reactivity with both the truncated (71 aa) and full-length (122 aa) suggest a sequence through which IL-7-responsive lymphocyte ectodomain of syndecan-4 fusion proteins, whereas KY/103 de- precursors acquire this glycoprotein. A subset of IL-7Rϩ cells is tected only the latter. These observations suggest that KY8.2 rec- capable of responding to IL-7 in semisolid agar, and we used cell ognizes an epitope in the N-terminal portion while KY/103 targets sorting to assess their expression of syndecan-4 (Table III). La- residues in the membrane proximal region. Both Abs specifically beling of lymphocytes with the syndecan-4-specific Abs did not recognized syndecan-4-GST fusion proteins prepared in bacteria influence their responsiveness to IL-7 (data not shown), and cells and did not bind to representatives of the other three members of capable of IL-7-dependent clonal proliferation were present in the syndecan family (Table II). These reagents also stained syn- both syndecan-4ϩ and syndecan-4Ϫ populations. In contrast, CSF- decan-4-transfected 293T cells (data not shown). We conclude that responsive myeloid progenitors appeared to lack this marker and the Abs are specific to syndecan-4 and that posttranslational mod- were enriched among the syndecan-4Ϫ cells (Table III). Synde- ification of the protein core is not required for recognition. can-4 was not expressed on dendritic cells that were expanded by

Table II. Speciﬁcity of mAbs for syndecan-4

Murine Syndecan Family

mAbs Syndecan-1 (ectodomain) Syndecan-2-GST Syndecan-3-GST Syndecan-4-GST

None 0.048 Ϯ 0.001 0.047 Ϯ 0.003 0.044 Ϯ 0.002 0.044 Ϯ 0.001 KY/8.2 0.062 Ϯ 0.002 0.071 Ϯ 0.003 0.072 Ϯ 0.002 1.532 Ϯ 0.055 KY/103 0.059 Ϯ 0.003 0.061 Ϯ 0.001 0.060 Ϯ 0.001 1.360 Ϯ 0.062 Control Aba 0.059 Ϯ 0.003 0.063 Ϯ 0.001 0.059 Ϯ 0.001 0.064 Ϯ 0.006

a Control Ab is an isotype-matched control for the KY mAbs. The Journal of Immunology 5943

FIGURE 2. Syndecan-4 is preferentially expressed by B lymphocyte lineage cells in bone marrow. Two-color staining was performed with mAb FIGURE 4. Syndecan-4 is uniformly present on newly formed B lym- directed to syndecan-4 and markers of B-lineage cells (CD19 and surface phocytes. Flow cytometry gating parameters that were used to resolve small pre-B cells (fraction D, CD45Rlow CD43Ϫ sIgMϪ, shown in A),

IgM), T cells and stem cells (Thy1.2), myeloid cells (Ly-6G and CD11b), Downloaded from low Ϫ ϩ or erythroid cells (TER119). Quadrants are indicated to show levels of newly formed B cells (fraction E, CD45R CD43 sIgM , B), and more high ϩ background staining observed with appropriate control Abs. The percent- mature B cells (fraction F, CD45R sIgM , C) are shown in the left ages of cells in each quadrant are indicated in the upper right quadrant. panel. The right columns represent expression of syndecan-4 on these three lymphocyte subsets. Thick lines depict background staining obtained with isotype-matched control Abs. culture of bone marrow cells for 1 wk with 20 ng/ml of GM-CSF or on myeloid cells grown in long-term bone marrow cultures (data http://www.jimmunol.org/ not shown). less than syndecan-1 on these cell types. Surface display of syn- Screening of established cell lines revealed that syndecan-4 was decans 1 and 4 was also demonstrated by immunofluorescent stain- present on IL-7-responsive lymphocyte clones (Table IV). Matur- ing of viable primary stromal cells in Whitlock-Witte cultures (not ing lymphocyte precursors expressed more syndecan-1 than syn- shown). We conclude that syndecan-4 is predominantly associated decan-4, but the former is known to be lost at the B cell stage and with stromal cells and B lymphocyte lineage precursors in bone reacquired by Ab-secreting plasma cells (6) (Table IV). While B marrow. Acquisition of the syndecan-4 protein core on lympho- lymphomas expressed syndecan-4, very little was present on my- cytes reflects their degree of maturation, but does not precisely eloma cells. As expected from our previous study and work by correspond to their ability to respond to IL-7. others (3, 5), syndecan-4 was present on several stromal cell and by guest on September 24, 2021 fibroblast lines. While the level of expression was always above Syndecan-4 is present on most, but not all, mature B background and more remarkable with biotin-labeled Abs, it was cell subsets In contrast to syndecan-1 (6), syndecan-4 is expressed by most splenic B cells as well as an extremely small population of T cells (Fig. 5). While over 90% of the CD19ϩ or IgMϩ B cells in spleen or lymph nodes displayed syndecan-4, a distinct subset of B cells was always observed to lack this protein. The small subset of splenic B cells that display CD5 were not distinguished by absence of syndecan-4 (data not shown). A more detailed analysis was made of CD5ϩ and CD5Ϫ B cells in the peritoneal cavity (Fig. 6). While CD5ϩ B cells tended to have a lower average density of syndecan-4, there was heterogeneity among both subsets. Addi- tional subsets of mature B cells were resolved by four-color flow

Table III. Syndecan-4 is expressed on lymphoid, but not myeloid, progenitors

No. of Colonies

FIGURE 3. Syndecan-4 is present in marrow on subsets of pro-B cells Syndecan-4ϩ Syndecan-4Ϫ that also express IL-7R. Four-color ﬂow cytometry was used to resolve subsets of B lymphocyte lineage cells in bone marrow cell suspensions. CFU-IL7 Expt. 1 46 Ϯ 449Ϯ 12 The top left panel depicts gating parameters that were used to evaluate ϩ ϩ Expt. 2 83 Ϯ 24 81 Ϯ 8 CD45R CD43 precursors. This population was then analyzed further CFU-c with respect to syndecan-4 and three other markers (second row of panels). Expt. 1 1 Ϯ 245Ϯ 11 While there was little syndecan-4 on very early precursors (fraction A, Expt. 2 3 Ϯ 567Ϯ 12 CD24Ϫ), ϳ50% of B cells had acquired syndecan-4 by fraction C (BP-1ϩ). Note that virtually all syndecan-4ϩ lymphocytes also express the IL-7R␣- Syndecan-4-positive and -negative cells were isolated by ﬂow cytometry and cell sorting as described in Materials and Methods. The data are presented as mean num- chain. Quadrants were set to indicate backgrounds obtained with irrelevant bers of colonies per 105 cells cultured in semisolid agar with IL-7 (CFU-IL7) or L control mAbs. cell-conditioned medium (CFU-c). 5944 MURINE SYNDECAN-4

Table IV. Cell surface expression of syndecan-1 and -4 by mouse cell lines

MFIa

Cell Line Characteristics Syndecan-1 Syndecan-4

BCB10 Pre-B 0.5 85.6 BC7.12 Pre-B 0.2 81.1 1A9 Pre-B 20.1 51.5 70Z/3 Pre-B 44.4 135.7 WEHI231 B 0.0 44.8 CH12 B 0.1 51.8 BCL1 B 0.4 42.1 FIGURE 6. Peritoneal B1a cells display low levels of syndecan-4. Peri- SP2/0 Myeloma 139.5 3.6 toneal lavage cells were stained with FITC-IgM, PE-CD5, and biotin-KY/ 2B4 T hybridoma 13.8 0.4 8.2 plus streptavidin-RED613. Levels of syndecan-4 expression on the B1a BW5147 Thymoma 21.9 0.2 ϩ ϩ subset (A, CD5 sIgM cells) are compared with those present on B1b J774A.1 Macrophage 0.3 5.0 Ϫ ϩ P388D1 Macrophage 5.9 19.6 plus B2 subsets (B, CD5 sIgM cells). Each histogram also contains the BMS2 Bone marrow stromal 36.8 3.9 isotype control for comparison (hatched lines). ST2 Bone marrow stromal 32.1 2.1 NIH3T3 Fibroblast 68.5 37.3 L Fibroblast 41.6 2.6 creases expression, but isotype-switched memory B cells may Downloaded from P815 Mastocytoma 0.0 0.5 markedly down-regulate syndecan-4. WEHI-3 Myelomonocytic 0.2 3.7 Ba/F3 Immature hematopoietic 0.0 0.2 Syndecan-4 mediated changes in cell shape FDCP1 Multipotential stem 0.3 0.0 Syndecan-4 functions were tested by ectopic expression in two a Results are represented as the mean ﬂuorescence intensity (MFI) of cells stained with biotinylated 281.2 (anti-syndecan-1) or KY/8.2 (anti-syndecan-4) followed by early hemapoietic cell lines (FDCP-1 and BaF/3) demonstrated not

FITC-avidin minus the MFI of the cells stained with FITC-avidin alone. to bear this molecule (Table IV). Transfected cells expressed high http://www.jimmunol.org/ levels of syndecan-4 protein, with no apparent inﬂuence on growth rates, IL-3 dependence, or self-adhesiveness. The addition of sol- cytometry, and none of them correlated well with syndecan-4Ϫ B uble Abs to syndecan-4 caused modest aggregation and did not cells (see Discussion). abrogate their requirement for growth factors. Thus, syndecan-4 Syndecan-4 is stable during B cell activation but absent from Ig made by these cells did not mediate strong homophilic recognition. isotype-switched lymphocytes A remarkable morphological change occurred when syndecan- 4-transfected cells contacted immobilized syndecan-4 Abs. In Syndecan-4 expression on B cells might be modulated as a con- these experiments, transfected cells were added to slides coated sequence of, or in parallel with, Ag experience. Blast cells were with anti-syndecan mAbs as a mimic of potential syndecan-4 li- by guest on September 24, 2021 generated in culture by stimulating resting splenic B cells with gands. Within4hofattachment, syndecan-4-transfected FDC-P1 LPS, an Ab to CD40 or RP105. Each of these activated lympho- cells developed numerous long, thin ﬁlopodia (Fig. 8, D and G) cyte populations continued to display high densities of syndecan-4 that frequently displayed antenary branching. Nearly all of the (not shown). Furthermore, expression was more homogeneous transfected cells responded in this way, but did not do so in syn- than on corresponding control cells held in medium alone. chronous fashion. No morphological changes were observed when We then investigated syndecan-4 expression on B cells that have control rat IgG was used to coat slides (Fig. 8, E and H). The undergone Ig isotype switching. Peyer’s patches are known to con- formation of dendritic processes was more apparent in wells tain substantial numbers of B cells that display IgA but have lost coated with KY/8.2 (N-terminal epitope) than KY/103 (membrane the ability to synthesize IgM. These cells completely lacked syn- proximal epitope), and in both cases was uniformly and completely decan-4 (Fig. 7A). In contrast, IgM-bearing cells in this mucosal tissue displayed the same heterogeneity in syndecan-4 density noted with splenic B cells (Fig. 7B). Similar to IgAϩ cells, IgGϩ B cells in the spleen also lacked syndecan-4 (data not shown). Collectively, these results indicate that B cells leave the bone marrow bearing syndecan-4. Entry into a proliferative response in-

FIGURE 5. Most mature B lymphocytes express syndecan-4, and the FIGURE 7. B cells that have undergone Ig isotype switching lack syn- marker is preferentially present on B cells in murine spleen. Two-color decan-4. Cells from Peyer’s patches were pooled and stained with FITC- ﬂow cytometry was used to characterize spleen cell suspensions, with gat- anti-IgM, PE-anti-IgA, and biotin-KY/8.2 plus streptavidin-RED613. The ing based on lymphocyte light scatter (not shown). Negative control stain- right panels illustrate the staining pattern for syndecan-4 on the resident ing is represented by quadrants in each panel, along with percentages of IgAϩ (A) and IgMϩ B cells (B). The isotype controls are shown as dark cells in each of the quadrants. histograms. The Journal of Immunology 5945

FIGURE 8. Ligation of syndecan-4 induces filopodia on syndecan-4-transfected cells, and the extracellular domains of syndecan-4 are sufficient for this response. Levels of syndecan-4 expression on FDCP-1 cells determined by flow cytometry are illustrated in the left column of histograms. Photomi- crographs are shown for transfected cells incubated in chamber slides coated with the KY/8.2 Ab to syndecan-4 (middle column) or a control Ab (far right column). Syndecan-4-transfected, but not mock- transfected, cells attached to this substrate, but not to slides coated with control rat IgG. Downloaded from

blocked by addition of soluble anti-syndecan mAbs or soluble syn- Discussion http://www.jimmunol.org/ decan-4-Ig fusion proteins (data not shown). Neither the trans- This study builds on our previous findings that stromal cells pro- membrane or cytoplasmic domains were required inasmuch as duce syndecan-4 and that syndecan-4 from this source can recog- constructs with the extracellular portion of syndecan-4 fused to the nize pre-B cells. We now show that the interaction depends on HS transmembrane domain of tissue factor also mediated these dra- present on both syndecan-4 and pre-B cells. Newly developed matic morphological changes (Fig. 8G). mAbs allowed us to determine that syndecan-4 is expressed from The filopodia were brightly stained by phalloidin (not shown), an early pro-B cell step, through immature and mature B cell suggesting local actin polymerization, and we wondered if other cytoskeletal components might be present. The F-actin binding stages. It is almost, but not entirely, restricted to B lymphocyte protein pp52/LSP1, which contributes to the morphological fea- lineage cells in bone marrow and absent from B cells that have by guest on September 24, 2021 tures in hairy cell leukemia,4 was of particular interest because of undergone Ig isotype switching. We also show that syndecan-4 its involvement in cytoskeletal dynamics and cell motility (14, 15). ligation on appropriately activated B lymphocytes leads to dra- Confocal microscopy revealed that pp52 was distributed through- matic morphological changes and extrusion of prominent filopo- out filopodia of syndecan-4-ligated cells (Fig. 9B). In contrast, dia. Extracellular matrix components, cytokines, chemokines, and syndecan-4 was more concentrated on the cell body (Fig. 9A). other known syndecan ligands could influence B cells and their Therefore, while syndecan-4 was not localized to the focal adhe- precursors via interaction with syndecan-4. sions, ligation through this molecule was associated with cytoskel- Syndecan-4 on stromal cells could mediate bidirectional com- etal reorganization involving actin and pp52. munication with B lymphocyte precursors. We demonstrate here that recognition of pre-B cells occurs when the entire extracellular Filopodia are induced by syndecan-4 ligation of activated domain of syndecan-4 is expressed in a fusion protein and that this normal B cells recognition is inhibited by heparin, but not chondroitin sulfate A Normal splenic B cells stimulated to form lymphoblast aggregates (Fig. 1). Heparitinase (HS lyase I, specific for linkages in HS were subsequently placed on anti-syndecan-4-coated slides. Blasts chains) treatment of pre-B cells destroyed binding. Furthermore, induced by treatment with anti-CD40 developed dendritic pro- the syndecan-4-Ig fusion protein lost activity when prepared in cesses similar to those described above for transfected cells except sodium chlorate-treated cells, a technique that reduces GAG sul- that the processes were less numerous and prominent. Approxi- fation (our unpublished observations). These results would be mately 70–80% of the blasts responded, suggesting that they were compatible with the HS chains of syndecan-4 interacting with an- more heterogeneous than the syndecan-4-transfected cells de- other HSPG on pre-B cells, and an obvious candidate would be scribed above. In contrast, cells stimulated with other activation syndecan-4 itself. signals including LPS or anti-RP105 did not undergo morpho- A previous study demonstrated that the cellular environment can logical changes even though they displayed high densities of be critical for appropriate posttranslational modification and adhe- syndecan-4 (data not shown). Indeed, RP105-activated B cells sive receptor properties of syndecans (16). The early progenitor showed little tendency even to attach. These findings demon- (FDCP-1 and Ba/F3) and human T leukemia (Jurkat) or renal cell strate that normal B cells can undergo dramatic shape changes carcinoma (293T) cells used here may differ from normal B cell on contact with ligands for syndecan-4, but the initial route of precursors with respect to protein glycanation. Indeed, the former activation is important. two lines do not express HS on their surfaces (17). Therefore, it is uncertain whether syndecans, CD44, or other HSPGs on pre-B cells represent ligands for the syndecan-4 made by stromal cells. 4 E. K. Miyoshi, P. L. Stewart, P. W. Kincade, M. B. Lee, R. Wall, and A. A. Thompson. Elevated pp52 (LSP1) expression and altered distribution correlates with HSPGs are bound by a variety of lymphocyte-derived ligands. For the distinctive cell morphology of hairy cell leukemia. Submitted for publication. example, CD45, a lymphocyte molecule not known to bear GAG 5946 MURINE SYNDECAN-4

chains, may recognize HSPGs on stromal cells (18). It is possible that CD45 binds to other HSPGs known to be made by stromal cells (3). The core protein of syndecan-4 has been shown to have a ligand on fibroblasts (19), so the range of possible syndecan-4-mediated interactions may be even greater than those revealed here. Sanderson and colleagues found that syndecan-1 was present on pre-B cells, down-regulated at the mature B cell stage and reexpressed by plasma cells (6). Much broader expression of syndecan-4 was suggested by a more recent study, where Northern blot- ting revealed transcripts in a series of transformed cell lines (7). We used Abs specific for two syndecan-4 extracellular domains to document when this HSPG is acquired and lost by cells of the B lymphocyte lineage. Some fraction B cells (CD45Rϩ CD43ϩ CD24ϩ BP-1Ϫ cells) display syndecan-4, but the marker is not uniformly present on a slightly more mature category of bone marrow precursors (fraction C; CD45Rϩ CD43ϩ CD24ϩ BP-1ϩ cells). Many fraction B cells have undergone at least the first step

(DH to JH) of Ig gene rearrangement and some precursors in this

category proliferate in the presence of IL-7 (20–23). While syn- Downloaded from decan-4 initially appears during an early stage of B lymphocyte development, the onset of expression is not perfectly synchronized with other maturational changes. Expression of this protein may correspond to a previously unrecognized milestone in B lymphocyte lineage differentiation.

Heparitinase treatment of pre-B cells reduces their ability to http://www.jimmunol.org/ bind and respond to IL-7 (24). Therefore, syndecan-4, or another HSPG, acts as a coreceptor for this cytokine, possibly in a manner similar to that proposed for the basic fibroblast growth factor receptor (25). While most syndecan-4ϩ pro-B cells express one component of IL-7R (IL-7R␣), approximately half of the IL-7R␣ϩ cells lack syndecan-4 (Fig. 3). This corresponds to the finding that nearly equal numbers of IL-7-responding precursors were present in fractions sorted on the basis of syndecan-4 expression (Table III). Similar results were obtained by sorting on the basis of syn- by guest on September 24, 2021 decan-1 expression (not shown). It is possible that syndecan-negative precursors acquire this HSPG when placed in culture. Thresholds of responsiveness of B lineage precursors to IL-7 change with successful Ig gene rearrangement (49), potentially a result of HSPG expression. In contrast to lymphocyte precursors, myeloid progenitors in bone marrow did not express syndecan-4. Syndecan-4 may be a unique marker for resolving mature B lymphocyte subsets. Approximately 10% of B cells in spleen and lymph node lack syndecan-4, and these cells do not closely correlate with previously described populations. For example, the well studied “B1a” lymphocytes are not discriminated from “conventional” B2 cells on the basis of syndecan-4 expression (Fig. 6) and are less frequent in spleen than syndecan-4-negative B cells (26). We found syndecan-4-positive and syndecan-4-negative B cells among subsets that were gated for CD21Ϫ CD23Ϫ (newly formed B cells); CD21ϩ CD23Ϫ (marginal zone B cells); or CD21ϩ CD23ϩ (follicular B cells) (27–29). Syndecan-4Ϫ B cells had “mature” properties in that they were IgMlow IgDhigh class IIhigh and CD23ϩ. They also included CD24int as well as CD24high populations. Of particular interest, the distinct subset of IgMϪ IgAϩ B cells in Peyer’s patches and the IgMϪ IgGϩ B cells in spleen uniformly lacked syndecan-4 (Fig. 7 and data not shown). FIGURE 9. Filopodial extensions of syndecan-4 Ab-ligated cells con- Therefore, syndecan-4 might be down-regulated as B cells emerge tain pp52(LSP1), while syndecan-4 is concentrated on the main cell body. from immune responses as Ig isotype-switched memory cells. Fur- Confocal laser scanning micrographs are shown for stable syndecan-4- Ϫ ther study may reveal that many syndecan-4 B cells are long transfected FDCP-1 cells incubated on KY/8.2 mAb-coated chamber slides. Viable cells were surface stained for syndecan-4 with biotin-KY/ lived and have other properties ascribed to memory cells. 103 plus streptavidin-Cychrome. The cells were then fixed, permeabilized, Syndecan-4 is known to be actively shed from endothelial cells and stained with goat anti-mouse pp52 plus anti-goat Ig FITC. Pseudo and this process is modulated by growth factors (30). It will be color images are shown of syndecan-4 alone (green color, A), pp52 alone important to learn if the same is true for B lineage lymphocytes (red color, B), and both merged (C). Bar ϭ 10 ␮m. and stromal cells, because soluble syndecan-4 may condition the The Journal of Immunology 5947 immediate microenvironment. As one example, syndecan-4 has can-4 transmitting a signal for morphological change. The cyto- recently been found to bind proteases and modify their interaction plasmic domain of syndecan-4 can apparently associate with and with anti-proteases (31). Interestingly, stromal cells and pre-B activate protein kinase C (46), but the transmembrane and cyto- cells express several ectoproteases (32). Cell bound and/or soluble plasmic domains of syndecan-4 were not required for the morpho- syndecans could modify their activity. Many other roles have been logical response seen here. One possibility is that the ectodomain proposed for HSPGs, including their ability to serve as receptor of syndecan-4 can interact with other transmembrane-spanning components and docking sites for cytokines and chemokines (33). proteins to transmit signals. Of particular interest is the recent find- It was recently shown that chemokines are necessary for produc- ing that syntenin interacts with the cytoplasmic domains of syn- tion of B lymphocytes as well as for organization of peripheral decans and overexpression of syntenin caused membrane exten- lymphoid tissues (34, 35). We have shown that syndecan-4 density sions (47). Cdc42 is a member of the Rho family of small GTP- changes with B lymphocyte lineage differentiation and is notably binding proteins (48). Over-expression of Cdc42 in macrophages absent from isotype-switched B cells. It will be interesting to see results in filopodia formation and a remarkably similar morphol- if this correlates with changes in ability to capture and respond to ogy to the activated B cells described here (compare Fig. 4a of a newly described, B cell-specific chemokine (36, 37). Ref. 48 to Fig. 6 in this paper). It will be important to learn more There are now exceptions to the conventional view that B lym- about the biochemical basis for filopodia formation in activated B phocytes have a strictly round morphology. Activated B cell blasts lymphocytes. placed on surfaces coated with Abs to several Ags generate ex- The Abs we prepared recognize protein epitopes in two separate tended filopodia (38–40). In these circumstances, resting B cells regions of the extracellular domain of syndecan-4. To date, we were first exposed to anti-Ig Ab in addition to IL-4. We found that have found no biological activities influenced by these Abs other Downloaded from Abs to syndecan-4 similarly trigger filopodia production by trans- than the morphological change in B cells. For example, the Abs fected FDCP-1 and Ba/F3 cells without cytokine stimulation (Fig. had no obvious effect on production of myeloid or lymphoid cells 8). Resting normal B cells had to be previously activated for this in long-term bone marrow cultures, the responsiveness of pre-B response and the pathway of initial B lymphocyte activation was cells to IL-7, the activation of mature B cells by LPS, or by Abs important. B cell blasts prepared by ligation of CD40 but not those directed to CD38, CD40, or RP105 Ags. Syndecan-4 functions

prepared by stimulation via RP105 or LPS made filopodia in re- might overlap with those of other HSPGs or our Abs might not be http://www.jimmunol.org/ sponse to subsequent contact with syndecan-4 Ab. Memory B cells directed to critical portions of the syndecan-4 molecule. Regard- isolated from human tonsils have irregular outlines and could rep- less, this first study of syndecan-4 expression on normal, untrans- resent in vivo homologues of the cells we generated (41). Such formed lymphocytes provides a basis for further investigation of extended dendritic processes could increase surface area and fa- HSPG-dependent biological processes in the immune system. cilitate physical contact between Ag-presenting B cells and other Moreover, the observations suggest means through which synde- cells of the immune system. can-4 might facilitate intercellular communication between B lin- We found that pp52(LSP1) phosphoprotein was present eage lymphocytes and surrounding cells. throughout the filopodia of syndecan-4-ligated cells. Staining with phalloidin revealed colocalization of polymerized actin in these Acknowledgments by guest on September 24, 2021 processes as expected because pp52 binds F-actin and is closely We thank Drs. Ralph Sanderson and Lisa Borghesi for helpful advice and colocalized with F-actin in microfilament-rich cell surface projec- discussions. tions on leukocytes (14, 15). Hairy cell leukemia provides an example where B lineage lymphocytes have an irregular outline (42), and levels of pp52(LSP1) have recently been found to correlate References with the appearance of hairy cell projections.4 Furthermore, ele- 1. Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Biology of the syndecans: a family of transmembrane heparan vated pp52(LSP1) expression has been implicated in cytoskeleton- sulfate proteoglycans. Annu. Rev. Cell Biol. 8:365. mediated morphological and functional defects in neutrophils (43). 2. Siczkowski, M., D. Clarke, and M. Y. Gordon. 1992. Binding of primitive he- Lebakken and Rapraeger observed morphological changes in matopoietic progenitor cells to marrow stromal cells involves heparan sulfate. Blood 80:912. lymphoblastoid cells that were transfected with syndecan-1 and 3. Drzeniek, Z., B. Siebertz, G. Sto¨cker, U. Just, W. Ostertag, H. Greiling, and exposed to surfaces coated with syndecan ligands or an Ab to H. D. Haubeck. 1997. Proteoglycan synthesis in haematopoietic cells: isolation syndecan-1 (44). Although spreading and lamellipodia formation and characterization of heparan sulphate proteoglycans expressed by the bone- marrow stromal cell line MS-5. Biochem. J. 327:473. were more prominent than in our experiments, they noted the ap- 4. Gupta, P., J. B. McCarthy, and C. M. Verfaillie. 1996. Stromal fibroblast heparan pearance of filopodia with time in culture. While the cytoplasmic sulfate is required for cytokine-mediated ex vivo maintenance of human long- term culture-initiating cells. Blood 87:3229. tail of syndecan-1 was not required, the authors concluded from 5. Oritani, K., and P. W. Kincade. 1996. Identification of stromal cell products cytochalasin D treatment that the response is dependent on trans- which interact with pre-B cells. J. Cell. Biol. 134:771. mission of signals via the cytoskeleton. 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