Download Ligation of tumour-produced mucins to CD22 dramatically impairs

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.
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Received 17 June 2008/5 September 2008; accepted 17 October 2008
Published as BJ Immediate Publication 17 October 2008, doi:10.1042/BJ20081241
c The Authors Journal compilation c 2009 Biochemical Society