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Biochem. J. (2009) 417, 673–683 (Printed in Great Britain) 673 doi:10.1042/BJ20081241 Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells Munetoyo TODA*†, Risa HISANO*, Hajime YURUGI*, Kaoru AKITA*, Kouji MARUYAMA*, Mizue INOUE*†, Takahiro ADACHI†‡, Takeshi TSUBATA†‡ and Hiroshi NAKADA*†1 *Department of Biotechnology, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan, †CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan, and ‡Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state. INTRODUCTION pathway, resulting in enhanced signal transduction through the BCR. It is generally agreed that CD22 is masked by endogenous cis ligands [8,9]. However, several reports indicated that CD22 is unmasked on subpopulations of resting B-cells or becomes unmasked on a portion of activated B-cells [10,11]. Collins et al. [12] also demonstrated that, although cis ligands mask the binding of sialoside probes, CD22 is still able to interact with glycoprotein ligands expressed on adjacent B- or T-cells. Thus it seems likely that CD22 binds preferentially to glycoproteins that are structurally easily accessible and carry a high level of appropriately linked sialic acids. CD22 exhibits high specificity for sialoside ligands containing the sequence Siaα2−6 GalNAc (where Sia, sialic acid, and GalNAc, N-acetylgalactosamine) [13–16]. The Siaα2−6 GalNAc sequence is present in both the N- and O-linked carbohydrate groups of glycoproteins. Mucins seem to be one type of CD22 ligand with high affinity, because they have a large number of O-glycans with α2,6-sialic acids and exhibit high valency due to their tandem repeat structure. Many tumours arising from epithelial tissues produce mucins. Upon malignant transformation, many epithelial cells produce mucins in abnormal amounts and/or with abnormal glycosylation patterns [17]. It would be interesting to determine whether or not the ligation of mucins to CD22 contributes to some immunological suppression and/or defects in the tumour-bearing state. Murine mammary adenocarcinoma cell lines TA3-Ha and TA3St appear to constitute a matched pair for comparing B-cell function in relation to mucins in vivo, because TA3-Ha cells CD22, a member of the Siglec (sialic acid-binding, immunoglobulin-like lectin) family, is a B-cell-specific glycoprotein that associates with the BCR (B-cell receptor). It is generally agreed that CD22 must be in close proximity to the BCR complex for negative regulation of signalling through recruitment of a SH2 (Src homology 2) domain-containing phosphatase [1]. Although the physiological significance of CD22-mediated ligand binding and adhesion remains unknown, many studies have suggested that CD22 plays an important role in regulation of B-cell function. In fact, B-cells obtained from CD22 gene knockout mice exhibit various biological changes [2,3]. Transgenic mice expressing CD22 without ligand-binding activity revealed that CD22 has physiologically relevant ligand-dependent and -independent functions [4]. The effects on BCR-induced calcium responses, CD22 tyrosine phosphorylation and the recruitment of SHP-1 (SH2 domaincontaining protein tyrosine phosphatase-1) to CD22 are included in these ligand-independent functions, this being consistent with the fact that CD22 physically associates with the BCR to participate in regulation of signalling. However, in B-cells obtained from ST6Gal I (β-galactoside α2,6-sialyltransferase I) knockout mice, which fail to generate CD22 ligands, CD22 causes stronger negative regulation of B-cell signalling in the absence of ligand engagement, suggesting that cis ligands have some effect on signal transduction [5]. Anti-CD22 mAbs [monoclonal Abs (antibodies)] or synthetic sialoside ligands reduce the tyrosylphosphorylation of CD22 and SHP-1 recruitment [6,7]. It is considered that these ligands may sequester the phosphatase from the signal transduction Key words: CD22, immunosuppression, marginal zone B-cell, mucin, sialic acid-binding immunoglobulin-like lectin (Siglec), spleen. Abbreviations used: Ab, antibody; BCR, B-cell receptor; BSM, bovine submaxillary mucin; CHO cell, Chinese hamster ovary cell; DAPI, 4 ,6-diamidino2-phenylindole; ERK, extracellular-signal-regulated kinase; HRP, horseradish peroxidase; KLH, keyhole-limpet haemocyanin; mAb, monoclonal Ab; MAdCAM, mucosal addressin cell adhesion molecule; MDC, marginal zone dendritic cell; MMM, marginal metallophilic macrophage; MZ, marginal zone; MZM, MZ macrophage; NP, (4-hydroxy-3-nitrophenyl)acetyl; SH2 domain, Src homology 2 domain; SHP-1, SH2 domain-containing protein tyrosine phosphatase-1; Siglec, sialic acid-binding, immunoglobulin-like lectin; SSA, Sambucus sieboldiana agglutinin; TI-2, thymus-independent type 2; TMB, 3,3 ,5,5 -tetramethylbenzidine; TNP, 2,4,6-trinitrophenyl. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2009 Biochemical Society 674 M. Toda and others produce a mucin named epiglycanin, but TA3-St cells, a subline of them, do not [18,19]. Epiglycanin is a sialylated membraneassociated glycoprotein with a large mucin-like domain, and the molecule comprises 75–85 % carbohydrate by weight, the T and Tn antigens are major constituents of it, and 16 % of its oligosaccharides are sialylated. In the present study, we demonstrate that tumour-produced mucins bound to CD22 on B-cells, followed by inhibition of signal transduction through the B-cell receptor. The binding of mucins to CD22 in vivo had a crucial effect on the maintenance and function of splenic MZ (marginal zone) B-cells in the tumour-bearing state. Solid phase binding assay Purified epiglycanin or BSM was coated on to Universal-bind 96-well plates (Corning). Serially diluted WT CD22 D2-FLAG or R130A CD22 D2-FLAG was added to the wells, followed by incubation at room temperature (20 ◦C) for 1 h, and then washing with 50 mM sodium phosphate buffer (pH 7.2) containing 0.2 M NaCl and 0.05 % Tween-20. Bound FLAG-tagged CD22 was detected with HRP-conjugated anti-FLAG M2 mAb (Sigma). The enzyme reaction was performed with a TMB (3,3 ,5,5 tetramethylbenzidine) peroxidase substrate (Nacalai Tesque, Kyoto, Japan) and the absorbance at 450 nm was determined. MATERIALS AND METHODS Analysis of signal transduction Mice, cells and materials B-cells were isolated from the splenocytes of normal mice using a MACS B-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). K46μmλ-CD22 and splenic B-cells (2 × 106 cells) were incubated at 37 ◦C for 10 min in the presence or absence of epiglycanin, and then stimulated with NP [(4-hydroxy-3nitrophenyl)acetyl]-BSA and anti-mouse IgM F(ab )2 respectively, at 37 ◦C for 2 min. CD22 was immunoprecipitated from the cell lysate with anti-CD22 mAb (Southern Biotechnologies, Birmingham, AL, U.S.A.). CD22, recruited SHP-1 and phosphorylated CD22 were detected by Western blot analysis using anti-CD22 Ab, anti-SHP-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), and anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY, U.S.A.) respectively. For detection of phosphorylated ERK (extracellular-signalregulated kinase)-1/2, B-cells were stimulated as described above except that the incubation time was 5 min. The cell lysate was subjected to SDS/PAGE followed by Western blotting. Phosphorylated and total ERK-1/2 were detected with anti-phosphorylated ERK-1/2 mAb and anti-ERK-1/2 mAb, respectively (Cell Signaling Technology, Beverly, MA, U.S.A.). A/J mice were purchased from SLC (Shizuoka, Japan). The Kyoto Sangyo University Committee on Animal Welfare approved all animal protocols used in this study. Mice were anaesthetized by inhalation of sevoflurane or intraperitoneal injection of sodium thiopental (50 μg/g of body weight), and all precontions were taken to ensure that the animals did not suffer unduly. The two murine mammary adenocarcinoma sublines (TA3-Ha and TA3-St) were provided by Dr J. Hilkens (The Netherlands Cancer Institute). The cells (5 × 105 ) were inoculated intraperitoneally into 8-week-old female A/J mice. CHO (Chinese hamster ovary)-K1 cells were cultured in Opti-MEM (Invitrogen) supplemented with 2 % FCS (fetal calf serum). Murine monoclonal antibody MLS 128 recognizing the Tn-antigen was prepared as described previously [20]. K46μmλ-CD22 was prepared as described previously [21]. BSM (bovine submaxillary mucin) was purchased from Sigma. Epiglycanin was purified from the ascites fluid of TA3Ha-bearing mice, as described previously [22]. Alexa Fluor® 488-labelled epiglycanin and BSM were prepared using an Alexa Fluor® 488 protein labelling kit (Invitrogen). Western and lectin blot analyses of ascites fluid Flow cytometric analysis Ascites fluid from TA3-Ha- or TA3-St-bearing mice was subjected to SDS/PAGE followed by transfer to a Zeta-probe membrane (BioRad Laboratories). Glycoproteins with a cancer-associated carbohydrate antigen and/or α2,6-linked sialic acids were detected by successive incubation of the membrane with biotinylated MLS 128 mAb or SSA (Sambucus sieboldiana agglutinin; Seikagaku Kogyo, Tokyo, Japan) and HRP (horseradish peroxidase)-conjugated streptavidin (Invitrogen). Splenocytes were prepared from TA3-Ha- or TA3-St-bearing mice on days 2, 3 and 8 after inoculation of tumour cells. After removal of erythrocytes, the leucocytes were incubated for 3 h at 4 ◦C with various combinations of antibodies. The following antibodies were used: anti-CD21 Ab-FITC, anti-CD1d Ab-RPE (BD Biosciences, San Diego, CA, U.S.A.), anti-CD23 Ab-PE and anti-CD45R/B220 Ab-PE-Cy5.5 (eBioscience, San Diego, CA, U.S.A.). Antibody binding was analysed with a FACSort (Becton Dickinson) by gating of cells to the forward and side light-scatter properties of lymphocytes. Preparation of FLAG-tagged soluble CD22 cDNA encoding the N-terminal two Ig domains of CD22 (CD22 D2 cDNA) was generated from total RNA prepared from K46μmλ-CD22 cells by RT–PCR (reverse transcription– PCR) using a pair of primers, 5 -TGAATTCAGACACCATGCGCGTCCATTACC-3 and 5 -CCGGATCCGGTATACTTAACATCCAGACGCACAG-3 . CD22 D2 cDNA was ligated to pUC 18 and then introduced into TOP 10F’ (Invitrogen). A CD22 mutant with Arg130 changed to alanine (R130A) was generated using a site-directed mutagenesis kit (Stratagene). CD22 D2 cDNA and the mutated CD22 D2 cDNA were subcloned into pFLAG CMV-14 (Sigma). The resulting constructs were transfected into CHO-K1 cells using Fugene 6 transfection reagents (Roche Applied Science, Mannheim, Germany). Soluble 3 × FLAGtagged wild-type CD22 (WT CD22 D2-FLAG) and the mutated CD22 (R130A CD22 D2-FLAG) were purified from the conditioned medium of stable transfectants by affinity chromatography on anti-FLAG M2-agarose gels (Sigma). c The Authors Journal compilation c 2009 Biochemical Society Antibody production in TA3-Ha- or TA3-St-bearing mice TA3-Ha or TA3-St cells (5 × 105 cells) were intraperitoneally inoculated into mice. TNP (2,4,6-trinitrophenyl)-Ficoll or TNP-KLH (keyhole-limpet haemocyanin) (10 μg; Biosearch Technologies, Navato, CA, U.S.A.) was injected intravenously on days 5 and 7 after tumour cell implantation, and blood was taken on day 8. Anti-TNP Ab in the sera was quantitated by ELISA on TNP-BSA-coated MaxiSorp plates (Nalge Nunc). Serial dilutions of sera were applied on the plates, and then IgM or IgG was measured using HRP-conjugated goat anti-mouse IgM or anti-mouse IgG Ab (Invitrogen) and TMB as the substrate. Immunochemical observation of spleens Frozen sections of spleens were blocked with PBS containing normal goat serum or 5 % BSA in PBS, and then treated with Impairment of marginal zone B-cells by mucin 675 anti-CD16/32 Ab (BD Biosciences) to block Fc receptors. CD22expressing B-cells, CD1d-expressing MZ B-cells and sinus lining cells were detected by successive incubation with biotinylated anti-mouse CD22 mAb (Southern Biotechnologies) and Alexa 594-labelled streptavidin (Invitrogen), anti-mouse CD1d mAb (BD Biosciences) and Alexa Fluor® 594-labelled streptavidin, and biotinylated anti-mouse MAdCAM (mucosal addressin cell adhesion molecule)-1 mAb (Southern Biotechnologies) and Alexa Fluor® 594-labelled anti-rat IgG Ab (Invitrogen) respectively. For the detection of MZMs (MZ macrophages), MMMs (marginal metallophilic macrophages) and MDCs (MZ dendritic cells), the sections were successively incubated with rat anti-SIGN-R1 mAb (ER-TR9; BMA Biomedicals, Augst, Switzerland), Alexa Fluor® 594-labelled anti-rat IgM Ab (Invitrogen), anti-mouse CD169 (Siglec-1) mAb (AbD Serotec, Oxford, U.K.), Alexa Fluor® 594-labelled anti-rat IgG Ab, anti-mouse CD11c mAb (AbD Serotec) and Alexa Fluor® 594-labelled anti-hamster IgG Ab (Invitrogen) respectively. Treatment of normal mice with BSM BSM (100 μg) was intravenously injected into normal mice every other day (beginning on day 0). Splenocytes were prepared from the mice on day 8 and analysed by flow cytometry as described above. To examine the distribution of labelled epiglycanin or BSM incorporated into the spleen, 100 μg of Alexa Fluor® 488-labelled epiglycanin or BSM was injected into a tail vein. After 20 min, the mice were perfused with 4 % paraformaldehyde through the heart and then their spleens were resected. Sections were treated with anti-CD16/32 Ab to block Fc receptors. To visualize the CD22-expressing B-cells, MZ B-cells or sinus lining cells, spleen sections were stained as described above. Statistical analysis ANOVA and Student’s t test were used to determine the significance of differences between sample means. RESULTS Binding of epiglycanin to CD22 Many studies have suggested a potential function for CD22 ligation of endogenous ligands in regulation of B-cell function. However, the physiological significance of CD22-mediated ligand binding and adhesion in vivo has not been elucidated clearly. In the tumour-bearing state, epithelial cancer cells produce mucins and secrete them into tumour tissues and/or the bloodstream. Recently we demonstrated that MUC2 mucin prepared from human colon cancer cells bound to human CD22 and downmodulated B-cell signalling [23]. To investigate further the effect of mucins on B-cells in the tumour-bearing state, we used mucin-producing (TA3-Ha) and non-producing (TA3-St) mouse mammary adenocarcinoma-bearing mice. TA3-Ha cells produce a mucin named epiglycanin, which is known to be shed from the cell surface and can be detected in ascites fluid as well as in the bloodstream of TA3-Ha-bearing mice [24]. To examine the effect of epiglycanin on B-cell function, we purified epiglycanin from the ascites fluid of TA3-Ha-bearing mice by a conventional method [22]. Purified epiglycanin was subjected to SDS/PAGE followed by carbohydrate and protein staining, and analysis by Western blotting. In general, mucins are silver stained so poorly that if there are any contaminating non-mucin proteins, they are stained densely. Figure 1(A) shows that no other protein could be detected Figure 1 Characteristics of epiglycanin and binding of CD22 to mucins (A) Epiglycanin was purified from ascites fluid of TA3-Ha-bearing mice, and the purified epiglycanin (1.0 μg for silver and PAS (periodate–Schiff) staining, and 0.1 μg for immunoblot detection) was subjected to SDS/PAGE followed by silver staining, PAS staining, and immunochemical detection using MLS 128 mAb after Western blotting (WB). Arrowheads indicate the band of epiglycanin. (B) Ascites fluid was obtained from TA3-Ha- and TA3-St-bearing mice on day 8 after tumour implantation, and then subjected to SDS/PAGE followed by transfer to a Zeta-probe membrane. Glycoproteins with a cancer-associated carbohydrate antigen and α2,6-linked sialic acids were detected using MLS 128 mAb and SSA respectively. (C) FLAG-tagged wild-type CD22 was added to plates coated with the purified epiglycanin (closed circles) or BSM (open triangles). The binding activity was determined using HRP-conjugated anti-FLAG mAb and TMB substrates. Results are means + − S.D. of triplicate determinations. (D) FLAG-tagged wild-type CD22 (closed circles) or mutated CD22 (open circles) was added to plates coated with epiglycanin. The binding activity was detected as described above. and that epiglycanin possessed a cancer-associated carbohydrate antigen such as the Tn antigen, this being consistent with the report by Van den Eijnden et al. [25]. To see if other glycoproteins carrying carbohydrate antigens are present in the ascites fluid of TA3-Ha- or TA3-St-bearing mice, the ascites fluid was directly subjected to SDS/PAGE followed by Western blotting. As shown in Figure 1(B), substantially only epiglycanin was detected in the ascites fluid from TA3-Ha-bearing mice but not in that from TA3St-bearing mice. In addition, the binding of SSA to epiglycanin indicated that epiglycanin is highly modified with α2,6-linked sialic acids, which may be preferential binding sites for CD22. To investigate the binding of epiglycanin to CD22, FLAGtagged wild-type CD22 and mutated CD22 were prepared, and then a plate assay was performed using purified epiglycanin. A mutation was introduced at Arg130 , which is known to be essential for the binding to sialic acids [1]. The wild-type CD22 bound to BSM and epiglycanin in a dose-dependent manner (Figure 1C), but the mutated one did not (Figure 1D), indicating that CD22 specifically bound to epiglycanin. c The Authors Journal compilation c 2009 Biochemical Society 676 M. Toda and others Down-modulation of BCR-mediated signal transduction by CD22 ligation To investigate the effect of epiglycanin on BCR-mediated signal transduction, a stable transfectant (K46μmλ-CD22 cells) expressing CD22 and the BCR recognizing the NP-BSA [21] was incubated with various amounts of epiglycanin at 37 ◦C for 10 min, and then stimulated with NP-BSA for 2 min. From the cell lysates, CD22 and SHP-1 were immunoprecipitated and coimmunoprecipitated, respectively, and then subjected to SDS/ PAGE followed by Western blotting (Figure 2A). Ligation of epiglycanin to CD22 reduced the tyrosyl phosphorylation and recruitment of SHP-1. Next, we examined phosphorylation of ERK-1/2 in the lysate of cells stimulated with NP-BSA for 5 min with a specific mAb (Figure 2B). It should be noted that pre-incubation with epiglycanin prevented the phosphorylation of ERK-1/2 in a dose-dependent manner. Similar experiments were performed with murine splenic B-cells and anti-IgM F(ab )2 as a stimulator. Similarly, treatment of B-cells with epiglycanin reduced the tyrosyl phosphorylation of CD22 as well as the phosphorylation of ERK-1/2 (Figures 2C and 2D). These results indicate that ligation of epiglycanin to CD22 reduces BCR-mediated signal transduction. Reduction of splenic MZ B-cells in mucin-producing adenocarcinoma (TA3-Ha) tumour-bearing mice To examine the effect of mucins in the bloodstream on splenocytes in vivo, TA3-Ha or TA3-St cells were injected intraperitoneally into syngeneic strain A mice. Previously, we reported that splenic B-cells were reduced in mice bearing TA3-Ha cells but not in ones bearing TA3-St cells [23]. To determine if specific subpopulations are responsible for the reduction of splenic B-cells, we analysed the splenic B-cells of tumour-bearing mice on day 8 in relation to the expression of B220, CD21 and CD23 (Figures 3A and 3B). Newly formed B-cells (B220+ CD23low CD21low ) were increased in both types of tumour-bearing mice, probably due to the elevated immune reaction. Follicular B-cells (B220+ CD23high CD21med ) were slightly reduced in both TA3-Ha- and TA3-St-bearing mice compared with control mice. Interestingly, MZ B-cells (B220+ CD23low CD21high ) were significantly reduced in TA3-Ha-bearing mice, whereas this subpopulation in TA3-St-bearing mice was similar to that in control mice. It is well known that splenic MZ B-cells highly express CD21 and CD1d. We compared the expression of CD21 and CD1d in splenocytes prepared from TA3-Ha- and TA3-St-bearing and control mice. As expected, the CD21high CD1dhigh population was clearly reduced in TA3-Ha-bearing mice. In contrast, this subpopulation in TA3-St-bearing mice was similar to that in control mice (Figures 3C and 3D). In addition, this subpopulation in TA3-Ha-bearing mice started to decrease with tumour progression (Figure 3E). We also determined the reduction of the splenic MZ B-cells in TA3-Ha-bearing mice immunochemically. To confirm the location of the MZ, MAdCAM-1 was detected immunochemically (Figure 4A). It is well known that MAdCAM-1 is strongly expressed by sinus-lining cells, and localized on the inner side of the MZ, close to the white pulp [26]. As shown in Figure 4(B), CD1d-positive B-cells in the MZ had almost completely disappeared in TA3-Ha-bearing mice but not in TA3St-bearing mice and control mice. When compared with CD22expressing B-cells (Figure 4C), most of the B-cells in the MZ had disappeared and those in the follicles were slightly reduced in TA3-Ha-bearing mice, this being consistent with the results of flow-cytometric analysis in Figure 3. Next, we administered Alexa Fluor® 488-labelled purified epiglycanin intravenously to c The Authors Journal compilation c 2009 Biochemical Society Figure 2 Down-regulation of BCR-mediated signal transduction on treatment with epiglycanin K46μmλ-CD22 cells (A and B) and splenic B-cells prepared from normal mice (C and D) were stimulated with NP-BSA and goat anti-IgM F(ab )2 respectively, in the presence or absence of epiglycanin. Phosphorylated CD22, recruited SHP-1 and phosphorylated ERK-1/2 were detected as described in the Materials and methods section. normal mice and determined its distribution immunochemically. Epiglycanin was detected around the MAdCAM-1-positive area, indicating the incorporation of epiglycanin into the MZ (Figure 5A). Furthermore, a considerable part of it was associated with CD1d-expressing MZ B-cells (Figures 5B and 5C). It is generally known that blood-borne antigens are taken up by MMMs, MZMs and MDCs in the spleen [27]. To determine whether these cells are also responsible for the trapping of mucins in the bloodstream, we stained sections prepared as above with antibodies against CD169 (Siglec-1), SIGN-R1 (ER-TR9) or CD11c to detect these cells. A portion of labelled epiglycanin was also associated with MMMs as well as MZ B-cells (Figure 6A), but hardly with MZMs or MDCs (Figures 6B and 6C). Impairment of marginal zone B-cells by mucin Figure 3 677 Reduction of splenic MZ B-cells in TA3-Ha-bearing mice (A) Splenocytes were prepared from TA3-Ha or TA3-St-bearing mice on day 8 after tumour implantation, and stained with the combination of PE-Cy5.5-conjugated anti-B220, FITC-conjugated anti-CD21 and PE-conjugated anti-CD23 Ab. Representative flow-cytometry data are shown, and the numbers indicate the percentages of MZ, follicular (FO) and newly formed (NF) B-cells within the B220-positive cell gate. (B) Histograms show the percentages of MZ, follicular (FO) and newly formed (NF) B-cells in the spleens of TA3-Ha- or TA3-St-bearing mice and control mice. ∗ Results represent means + − S.D. (n = 6, each) P < 0.01. (C) Splenocytes prepared as described above were stained with the combination of FITC-conjugated anti-CD21 and RPE-conjugated anti-CD1d Abs. Representative flow-cytometry data are shown and the numbers are the percentages of CD21high CD1dhigh phenotype lymphocytes within the indicated gates. (D) Histograms show high high ∗ (E) Splenocytes the percentages of CD21high CD1dhigh cells on day 8 after tumour implantation. Values represent mean percentages (+ − S.D.) of CD21 CD1d cells (n 6, each). P < 0.01.high were prepared from tumour-bearing and control mice on days 2, 3 and 8 after inoculation, and analysed as described above. Values represent mean percentages (+ S.D.) of CD21 CD1dhigh cells − (n 6, each). c The Authors Journal compilation c 2009 Biochemical Society 678 Figure 4 M. Toda and others Reduction of the splenic MZ B-cells Spleen sections obtained from TA3-Ha- or TA3-St-bearing mice on day 8 after tumour implantation were stained by successive incubation with biotinylated anti-MAdCAM-1 mAb and Alexa Fluor® 594-labelled streptavidin (A), biotinylated anti-CD1d mAb and Alexa Fluor® 594-labelled streptavidin (B) or biotinylated anti-CD22 mAb and Alexa Fluor® 594-labelled streptavidin (C). Nuclei were visualized with DAPI (4 ,6-diamidino-2-phenylindole). WP, white pulp. The boundary lines between WPs and MZs were drawn with reference to the DAPI staining and verified by MAdCAM-1 staining. Impaired TI-2 (thymus-independent type 2) response in TA3-Ha tumour-bearing mice It is generally agreed that the TI-2 response is mainly exhibited by MZ B-cells [28,29]. Thus, we next examined the TI-2 response in TA3-Ha- or TA3-St-bearing mice. A TI-2 antigen, TNP-Ficoll, was injected intravenously into TA3-Ha- or TA3-St-bearing mice on days 5 and 7 after inoculation of the tumour cells. The production of anti-TNP-IgM and -IgG in TA3-Ha-bearing mice decreased to approx. 50 % and 60 % of that in control mice respectively (Figure 7A). In contrast, the level of anti-TNP Ab produced by TA3-St-bearing mice was similar to that in control mice. Furthermore, we also examined the TD (thymus-dependent) response using a TD antigen, TNP-KLH. In these tumour-bearing mice and control mice, similar levels of anti-TNP-IgM and -IgG were produced (Figure 7B). Since MZ B-cells are involved in early B-cell responses, particularly those to blood-borne antigens, these results suggested that the MZ B-cell compartment may be impaired functionally as well as phenotypically in TA3-Ha-bearing mice. c The Authors Journal compilation c 2009 Biochemical Society In vivo effect of BSM on normal mice To investigate further whether or not the intravenously injected mucin has a similar effect on MZ B-cells, BSM was administered to normal mice. When Alexa Fluor® 488-labelled BSM was intravenously administered to normal mice, labelled BSM was definitely detected in the splenic MZ, similarly to epiglycanin (Figure 8A). We also stained immunochemically MZMs, MMMs and MDCs of the spleens obtained from labelled BSM-injected mice. A similar staining pattern (Figure 6) was observed in relation to the distribution of labelled BSM (results not shown). On continuous administration of BSM, MZ B-cells were clearly decreased to about 60 % compared with in control mice (Figure 8B), indicating that the mucin is responsible for the impairment of MZ B-cells. DISCUSSION The interaction of CD22 with glycoconjugates containing α2,6linked sialic acids has been widely proposed to regulate B-cell Impairment of marginal zone B-cells by mucin Figure 5 679 Incorporation of intravenously injected mucin into spleens of normal mice Spleens were resected from mice that had been treated with 4 % paraformaldehyde 20 min after intravenous injection of Alexa Fluor® 488-labelled epiglycanin. Sections were prepared, and sinus-lining cells (A), CD1d-expressing cells (B) and CD22-expressing B-cells (C) were visualized as described in Figure 4. function. It has been reported that cell surface IgM and CD45 are major CD22 ligands in cis. However, the physiological relevance of these interactions has remained unclear. Some reports indicated that, even when CD22 is masked by cis ligands, CD22 can interact with trans ligands that carry high levels of appropriately linked sialic acids. From this point of view, mucins produced by epithelial tumour cells seem to be preferential trans ligands for CD22 in the tumour-bearing state, because many mucins contain appropriately linked sialic acids such as a sialyl-Tn antigen, which has been revealed to be a ligand for CD22 [15], exhibit high valency due to their tandem repeat structure, and are easily accessible to CD22 on the cell surface due to their rod-like structure. In the present study, we used mucin-producing (TA3-Ha) and mucin-non-producing (TA3-St) cloned variants of a mouse mammary adenocarcinoma, because it is known that the mucin named epiglycanin is shed from the cell surface and can be detected in the serum of mice bearing TA3-Ha ascites cells [24]. TA3-Ha cells produced epiglycanin, which was the only glycoprotein reactive to an anti-Tn antigen mAb and SSA (Figure 1B). As shown in Figures 1(C) and 1(D), binding of CD22 to epiglycanin and BSM was confirmed by means of a plate assay. It is well known that CD22 binds to α2,6-linked sialic acid-containing glycoproteins including a sialyl-Tn antigen [15,30]. Epiglycanin carried α2,6-linked sialic acids, which was demonstrated by the binding of SSA (Figure 1B), but rarely the sialyl-Tn antigen, as reported previously [25]. Thus it seems likely that CD22 bound to epiglycanin through these α2,6-linked sialic acids and to BSM through the sialyl-Tn antigen, which is a major O-glycan of BSM [31]. The effect of CD22 ligation on B-cell signal transduction has been studied by using antibodies to CD22. Tuscano et al. [32] reported that antibodies to the CD22 ligand-binding site actually enhance BCR signal transduction. This result and other similar studies [33–35] have been interpreted as follows. The binding c The Authors Journal compilation c 2009 Biochemical Society 680 Figure 6 M. Toda and others Association of intravenously injected mucin with macrophages and dendritic cells in splenic MZ Spleen sections prepared as in Figure 5 were stained with anti-CD169 mAb and Alexa Fluor® 594-labelled anti-rat IgG Ab (A), anti-SIGN-R1 mAb and Alexa Fluor® 594-labelled anti-rat IgM Ab (B) or anti-CD11c mAb and Alexa Fluor® 594-labelled anti-hamster IgG Ab (C). Nuclei were visualized with DAPI. of antibodies to CD22 sequesters it away from the BCR on the cell surface, resulting in its antibody ceasing to function as a negative inhibitor. In contrast, ligation of epiglycanin reduced the phosphorylation of CD22 and recruitment of SHP-1, but unexpectedly the resultant phosphorylation of ERK-1/2 was also reduced clearly in a dose-dependent manner. The difference in the effect on BCR-mediated signal transduction between anti-CD22 antibodies and epiglycanin may be caused by the different valencies to CD22. Epiglycanin is a huge glycoprotein with a molecular mass of approx. 500 kDa and has a rod-like structure of ∼ 500 nm in length [18]. Thus mucins, including epiglycanin, possess an extremely large number of binding sites for CD22. When epiglycanin binds to CD22 on the cell surface, many CD22 molecules around the BCR may be cross-linked, probably leading to an inhibitory effect on movement of these molecules on the cell surface. It is generally agreed that, when the BCR is ligated with antigens, the BCR and CD22 are translocated c The Authors Journal compilation c 2009 Biochemical Society to lipid rafts, and phosphorylation is catalysed by Lyn localized in the lipid rafts. Thus it is speculated that translocation of the BCR and CD22 may be restricted by cross-linking of CD22, probably resulting in down-regulation of ERK-1/2 and CD22 phosphorylation. This possibility is now under investigation. It would be of interest to determine if CD22 ligation with epiglycanin has a negative effect on B-cell function in vivo in the tumour-bearing state. It is noteworthy that a subset of splenic MZ B-cells was dramatically reduced in mucin-producing tumour (TA3-Ha)-bearing mice but not in mucin-non-producing tumour (TA3-St)-bearing ones (Figures 3 and 4). It is generally agreed that CD22 is masked by cis ligands [10], and that cis ligands are important modulators of the activity of CD22 as a regulator of B-cell signalling [5,36]. In addition, Collins et al. [12] demonstrated that, despite the presence of cis ligands, CD22 binds to trans ligands on adjacent B- or T-lymphocytes, causing its redistribution to sites of cell contact. However, it seems likely that Impairment of marginal zone B-cells by mucin Figure 8 681 Effect of intravenously injected BSM on MZ B-cells of normal mice (A) Alexa Fluor® 488-labelled BSM was injected intravenously into normal mice. Spleen sections were obtained and CD22-expressing B-cells were visualized as described in Figure 5. (B) Administration of BSM to normal mice. BSM (100 μg) was injected intravenously into normal mice every other day (beginning on day 0). On day 8, splenocytes were prepared and analysed by flow cytometry as described in Figure 3(C). Representative flow-cytometry data are shown and CD21high CD1dhigh phenotype lymphocytes within the indicated gates are compared. Values in the histograms are percentages relative to that in a control experiment taken as 100 % (n = 9, ∗ each) + − S.D. P < 0.05. Figure 7 TI-2 response in TA3-Ha-bearing mice (A) TA3-Ha- or TA3-St-bearing and control mice were immunized with 10 μg of TNP-Ficoll (intravenously) on days 5 and 7 after inoculation (n = 8, each). On day 8, blood was taken from each mouse, and the levels of anti-TNP IgM and IgG were measured by ELISA. The dots indicate the values for individual specimens examined and the bars indicate mean values. ∗ P < 0.01. (B) TA3-Ha- or TA3-St-bearing and control mice were immunized with 10 μg of TNP-KLH (intravenously), and then anti-TNP IgM and IgG were measured as described above (n = 5, each). The dots indicate the values for individual specimens examined and the bars indicate mean values. trans ligands preferentially bind to CD22 on B-cells expressing low levels of cis ligands. Thus, it is natural that epiglycanin affected MZ B-cells more effectively than other B-cells, because MZ B-cells have a high proportion of unmasked CD22 [11]. In addition, constant exposure of the MZ cells to a large amount of blood and a slow blood-flow rate in the MZ may facilitate the interaction between MZ B-cells and mucins in the bloodstream. To confirm the incorporation of circulating mucins into the MZ cells, labelled epiglycanin and BSM were administered to normal mice, and a part of them were taken up into the splenic MZ (Figures 5, 6A and 8A). Immunostaining of splenic tissues obtained from mucin-administered mice demonstrated that labelled epiglycanin was associated with MZ B-cells and MMMs but rarely with MZMs or MDCs. It is hard to consider that MZ B-celland MMM-associated epiglycanin was derived from its fragments presented by these cells themselves. This is because, even when the mice were perfused at a short time after intravenous injection of the labelled mucins, the incorporated mucins were distributed similarly among the MZ cells (results not shown). Furthermore, it seems unlikely that mucin fragments conjugated with Alexa Fluor® 488 could be loaded on MHC molecules after processing. The incorporation of mucins into MMMs is consistent with the report that CD169 could bind to carcinoma mucins [37]. The binding of mucins to MMMs may also have some effect. Even if this is the case, it seems unlikely that this may be critical to the reduction of MZ B-cells. There have been several reports that MZ B-cells were reduced directly by depletion of CD22 [38] or treatment of mice with anti-CD22 ligand-blocking mAbs [39]. Furthermore, Poe et al. [4] generated two lines of mice expressing mutant CD22 molecules that lack ligand-binding activity to see if the MZ B-cells are reduced due to defective interactions between CD22 and endogenous cis and/or trans ligands. These transgenic mice had a reduced B-cell subpopulation in the splenic MZ, similar to CD22 knockout mice, indicating that the generation and/or maintenance of a MZ B-cell subpopulation require some interactions between CD22 and endogenous ligands. Thus epiglycanin produced by tumours may physiologically prevent some interactions between CD22 and endogenous ligands, and down-modulate B-cell signal transduction, probably leading to a reduction of the MZ B-cell subpopulation in the tumourbearing state. It remains unclear how the splenic MZ B-cells were reduced by the mucins at present. It has been reported that CD22-deficient B-cells predominantly undergo apoptosis following IgM ligation [40] and that apoptosis can be achieved through extensive cross-linking of accessory molecules such as CD22 in addition to BCR engagement [41]. In addition, Poe et al. [4] also reported that mutation of CD22 ligand-binding domains substantially influenced the proliferative potential of B-cells after BCR- or CD40-induced signals and altered B-cell survival. Thus, extensive cross-linking of CD22 by epiglycanin may induce apoptosis. Studies on this are currently in progress. It is well known that splenic MZ B-cells play an important role in the uptake of blood-borne antigens and are responsible for c The Authors Journal compilation c 2009 Biochemical Society 682 M. Toda and others anti-bacterial immunity [28,29]. Antigens with multiple identical epitopes are known to be capable of generating a T-independent B-cell response. It is attractive to speculate that tumour-produced mucins, which generally have multiple identical epitopes, may cause splenic MZ B-cells not to produce an antibody against them. In the current study, we suggested that cancer-derived mucins down-modulate B-cell signal transduction and impaired splenic MZ B-cells by preventing the interaction of CD22 with its endogenous ligands. Many reports suggest that lectin–glycan interactions have many important roles in diverse immune systems [13,42–45]. Since mucins having tandem repeats with multiple O-linked glycans are carriers of various glycoepitopes, mucins secreted by carcinoma cells in the bloodstream may have some other effect on the immune system of patients with cancer by preventing the interaction of lectins with its endogenous ligands. ACKNOWLEDGMENTS We thank Dr J. Hilkens (The Netherlands Cancer Institute) for providing the TA3-Ha and -St cells. FUNDING This study was supported by a grant for Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency. REFERENCES 1 Tedder, T. F., Tuscano, J., Sato, S. and Kehrl, J. H. 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