Polysialic Acid, a Glycan with Highly Restricted Expression, Is
Found on Human and Murine Leukocytes and Modulates
Penelope M. Drake,* Jay K. Nathan,* Christina M. Stock,* Pamela V. Chang,*
Marcus O. Muench,§Daisuke Nakata,¶J. Rachel Reader,?Phung Gip,†Kevin P. K. Golden,*
Birgit Weinhold,#Rita Gerardy-Schahn,#Frederic A. Troy II,¶and Carolyn R. Bertozzi2*†‡
Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell
adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and
potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hema-
topoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor
models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia
glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to
multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV?/?mice, which
lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and de-
creased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and
regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation. The Journal of
Immunology, 2008, 181: 6850–6858.
monosaccharide units, or degree of polymerization (DP), is large,
with reported values of 50–150 ranging up to ?370 residues (1, 2).
This contrasts with the typical N-linked glycan containing 10–12
olysialic acid (polySia)3is an unusual glycan by almost
any definition. Structurally, it comprises repeating sialic
acid monomers with ?2,8 linkages. Its size in terms of
monomers, and is similar to glycosaminoglycans (GAGs), which
average 80–100 residues (3). The synthesis of polySia is also un-
usual: an entire chain is produced by a single enzyme acting on
classical N-linked and, less commonly, O-linked core structures.
Two polysialyltransferases, ST8Sia IV (PST) and ST8Sia II
(STX), with distinct expression patterns are involved in synthesis
of polySia. In contrast, the synthesis of most glycans requires the
coordinated action of many enzymes. Therefore, while glycan
structure is difficult to track genetically, polySia can be localized
by the expression patterns of ST8Sia IV and ST8Sia II. Further-
more, while many glycan structures can modify any number of
protein cores, the polysialyltransferases appear to be highly selec-
tive in their scaffold choices. Aside from autopolysialylation of the
ST8Sia IV and ST8Sia II enzymes, only four other protein carriers
have been identified: the neural cell adhesion molecule (NCAM,
also termed CD56), the ?-subunit of the voltage-gated sodium
channel, CD36, and neuropilin (4–7). Of these, NCAM is by far
the most commonly used scaffold.
Polysialylated NCAM is prominent in the developing nervous
system, where it has been most extensively studied. A large body
of work has shown that polySia affects neuronal functions as var-
ied as migration (8, 9), cytokine response (9, 10), and cell contact-
dependent differentiation (11). Provocatively, these same functions
are vital components of immune function. Leukocytes migrate
throughout the body, guided by specialized cytokines, termed che-
mokines, to effect both homeostatic and inflammatory functions
that are often dictated through cytokine and cell contact-dependent
Intriguingly, expression of both ST8Sia IV and ST8Sia II has
been documented in the immune system, suggesting that polySia is
abundant therein. In the adult human, primary and secondary lym-
phoid organs including the placenta, spleen, thymus, intestine, and
peripheral blood express ST8Sia IV, while ST8Sia II is produced
in the thymus (12). Our recent work in the mouse suggests that
*Department of Chemistry and†Department of Molecular and Cell Biology, Univer-
sity of California, Berkeley, CA 94720;‡Howard Hughes Medical Institute, San Fran-
cisco, CA 94143;§Blood Systems Research Institute, San Francisco, CA 94118 and
Department of Laboratory Medicine, University of California, San Francisco, CA
94121;¶Department of Biochemistry and Molecular Medicine, School of Medicine
and the?Comparative Pathology Laboratory, School of Veterinary Medicine, Univer-
sity of California, Davis, CA 95616; and#Abteilung Zellula ¨re Chemie, Zentrum Bio-
chemie, Medizinische Hochschule Hannover, Germany
Received for publication June 12, 2008. Accepted for publication August 21, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1The research was made possible by a grant from the California Institute for Regen-
erative Medicine (CIRM, Grant RS1-00365). The contents of this publication are
solely the responsibility of the authors and do not necessarily represent the official
views of CIRM or any other agency of the State of California.
P.M.D. conceived and helped to perform and analyze all experiments. J.K.N. con-
ducted immunoblotting and in vivo immunoassays. C.M.S. and K.P.K.G. executed in
vivo immunoassays. P.V.C. performed 5?FU assays and flow cytometric analyses of
peripheral myeloid cells. M.O.M. designed and executed in vitro colony-forming
assays. D.N. and F.A.T. performed the DP analysis. J.R.R. provided pathological
evaluation of ear sections. P.G. prepared and analyzed human NK cells. B.W. and
R.G.-S. provided transgenic mice, mAb 735, and endoneuraminidase N. P.M.D. and
C.R.B. wrote the manuscript.
2Address correspondence and reprint requests to Dr. Carolyn R. Bertozzi, Depart-
ment of Chemistry, B84 Hildebrand Hall No. 1460, University of California, Berke-
ley, CA 94720. E-mail address: email@example.com
3Abbreviations used in this paper: polySia, polysialic acid; CHS, contact hypersen-
sitivity; DL1, Delta-like-1; DNFB, 2,4-dinitrofluorobenzene; DP, degree of polymer-
ization; Endo N, endoneuraminidase N; 5-FU, 5-fluorouracil; GAG, glycosaminogly-
can; NANA, N-acetyl neuraminic acid; NCAM, neural cell adhesion molecule; rh,
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
polySia plays an important role in progenitor trafficking to the
thymus (P. M. Drake, manuscript submitted). Furthermore,
NCAM (CD56) is expressed on two subsets of mature human
lymphocytes: NK cells and NKT cells. Although NCAM has
long been used as a marker for these cell populations (13), its
functional role remains undefined. We postulated that in the
immune system NCAM functions as a scaffold for presentation
of polySia, and that the glycan itself confers function to this
glycoprotein. Moreover, the functional significance of these ob-
servations is unknown.
The documented role of polySia in modulating cell adhesion,
migration, and cytokine response in the nervous system moti-
vated us to investigate the possibility of analogous functions in
the immune system. Herein, we describe polySia expression on
NCAM in human NK cells, as well as mouse hematopoietic
progenitors and myeloid cells. In support of a role for polySia
in the immune system, we demonstrate that human NK cells
regulate the expression and length of polySia with activation
state, and that ST8Sia IV?/?mice have aberrant contact hy-
persensitivity responses and enhanced tumor growth as com-
pared with wild-type mice.
Materials and Methods
Wild-type C57BL/6 and congenic GFP?mice were purchased from The
Jackson Laboratory. Mice were housed in specific pathogen-free condi-
tions. Experiments were approved by the University of California at Ber-
keley’s Animal Care and Use Committee. The generation of ST8Sia IV?/?
and ST8Sia II?/?mice has been described (14, 15).
Human NK cell analysis
Human leukocytes were obtained as buffy coats from the American Red
Cross, Oakland, CA. PBMCs were prepared as described (16). NK cells
were isolated using a magnetic bead-based method (Dynal NK cell nega-
tive isolation kit, Invitrogen). Cells were either lysed directly or cultured in
RPMI 1640 with 10% FBS. Endoneuraminidase N (Endo N, 1/1000 to
1/2000 dilution) and/or 6000 U/ml recombinant IL-2 (National Cancer In-
stitute Preclinical Repository) were added as indicated.
Human bone marrow analysis
Human fetal bone marrow was obtained from elective pregnancy termina-
tions at the University of California San Francisco with approval of the
Committee for Human Research. Bone marrow was harvested from long
bones as previously described (17). Light-density cells were isolated by
centrifugation on a layer of 1.077 g/ml NycoPrep (Axis-Shield). Both total
and light-density cell fractions were studied.
Assessment of polySia polymerization
Human NK cell cultures were pelleted, flash frozen, and stored at ?80°C
until analysis. Pellets from five individual donors were combined, and the
degree of polymerization was determined as described (2).
Abs used for flow cytometry are as follows. From eBioscience: fluorescein-
conjugated anti-CD3? (145-2C11), anti-CD4 (GK1.5), anti-CD11b (M1/
70), anti-CD25 (PC61.5), anti-TCR? (H57–597), anti-Gr-1 (RB6–8C5),
and anti-TER119; PE-Cy5-conjugated Sca-1 (D7), and isotype control rat
IgG2a; and allophycocyanin-conjugated secondary anti-mouse IgG. From
BD Biosciences: purified anti-polySia (12F8), anti-NCAM (NCAM-13),
TCR?? (GL3), anti-NK1.1 (PK136), anti-B220 (RA3-6B2), and isotype
controls mouse IgG2a (G155-178), rat IgG1 (R3-34), rat IgG2a (R35-95),
and rat IgG2b (A95-1); PE-conjugated anti-CD8a (53-6.7), anti-CD3
CD25 (PC61), and anti-CD117 (2B8) and isotype controls rat IgG1
(R3-34) and rat IgG2a (R35-95); PE-Cy5-conjugated anti-CD3 (17A2),
anti-CD44 (IM7). From Jackson ImmunoResearch Laboratories: allo-
phycocyanin-conjugated secondary anti-rat IgM. From Invitrogen: fluores-
cein-conjugated anti-CD3 (S4.1), anti-CD15 (V1MC6), anti-CD34 (581),
and isotype control mouse IgM; PE-conjugated anti-CD7 (CD7-6B7), anti-
CD34 (581), anti-CD45 (HI30), anti-CD105 (SN6), and isotype controls
mouse IgG1, mouse IgG2a, and mouse IgG2b. From Beckman Coulter:
PE-conjugated anti-CD2 (SFCI3Pt2H9-T11). From Exalpha Biologicals:
fluorescein-conjugated anti-CD56 (C5.9). The production of mAb 735 has
been described (18).
Flow cytometry and sorting
Cells were isolated and immediately incubated for 10 min with mouse
BD Fc block (anti-Fc?III/IIR; BD Biosciences), followed by the addi-
tion of Abs for staining. After 20 min cells were washed twice in PBS
and analyzed on a FACSCalibur (BD Biosciences) using CellQuest (BD
Biosciences) software. Hematopoietic stem cells were defined as Lin?
(including TER119, CD3, CD4, CD8, B220, NK1.1, Gr-1, TCR?,
TCR??, CD11b), c-Kit?, Sca-1?. Human cells were analyzed on an
LSR II flow cytometer (BD Biosciences). Metaanalyses were performed
using FlowJo software (Tree Star). Differences between controls, which
were stained with an irrelevant Ab of the same isotype, and the experi-
mentals, which expressed polySia, were calculated using the population
comparison function of FlowJo software. For sorted cells, freshly iso-
lated mouse bone marrow was centrifuged over a layer of Ficoll-Paque
Plus (Amersham Biosciences), and then light-density cells were labeled
with anti-c-Kit (PE) and purified anti-polySia (12F8) followed by anti-
rat IgM (allophycocyanin). Desired cell subsets were sorted using a
Dako-Cytomation MoFlo high-speed sorter (Dako) or a FACSAria (BD
Colony-forming cell assays
Erythroid progenitor cultures were initiated in septuplicate with 2.5 ?
102to 2.0 ? 103cells/dish. Colony-forming cell assays were performed
as previously described (19) in serum-deprived medium with substitu-
tion of 10% FBS for low-density lipoprotein (HyClone Laboratories).
Growth was supported by 100 ng/ml recombinant rat stem cell factor
(Amgen) and 10 U/ml recombinant human (rh) erythropoietin (Amgen).
After 7 days, cultures were scored for burst-forming units erythroid
identified as distinct clusters of erythroid cells with or without myeloid
cells and myeloid colony-forming cells having a dispersed cell mor-
phology. Myeloid progenitors were assayed as previously described
(20). Sorted cells were cultured, in triplicate, at 1.0 ? 102to 2.0 ? 103
cells/dish in the described medium containing 100 ng/ml recombinant
rat stem cell factor, 20 ng/ml rhIL-6, 20 ng/ml recombinant murine
IL-3, 20 ng/ml rhM-CSF (R&D Systems), and 20 ng/ml rhG-CSF (Am-
gen). After 7 days, cultures were scored visually for colonies (50?
cells) and clusters (10–49 cells). The erythroid and myeloid assays
were repeated twice with similar results.
Lymphoid progenitor assay
OP9 cells transfected with Delta-like-1 (DL1) and GFP, or with GFP alone,
were kindly provided by Dr. Juan Carlos Zu ´n ˜iga-Pflu ¨cker (Sunnybrook &
Women’s Research Institute). OP9-GFP and OP9-DL1 cells were cultured
and passaged as described (21). Sorted bone marrow subsets were plated
onto a semiconfluent layer of either OP9-GFP or OP9-DL1 cells in com-
plete DMEM-10 with 1 ng/ml IL-7 (R&D Systems) and 5 ng/ml Flt-3
ligand (R&D Systems). Cocultures were maintained, passaged as described
(21), and harvested at 18 days. Cells were counted with a hemocytometer
and analyzed by flow cytometry for expression of lineage markers. The
experiment was repeated three times with similar results.
5-Fluorouracil (5-FU) recovery
Wild-type mice (6–8 wk) received a single i.p. dose of either 5-FU (150
mg/kg) or vehicle. Following drug administration, bone marrow was har-
vested from two to three mice at each time point (days 1, 2, 7, 10, and 12)
for flow cytometric analyses. The experiment was repeated three times with
Wild-type mice (6–8 wk) were injected i.p. once a day for 5 days with 100
?l of either vehicle (PBS ? 2% BSA) or vehicle containing 2 ?g G-CSF.
Mice, two to three per group, were sacrificed after 5 days. Bone marrow
was harvested for analysis by flow cytometry. The experiment was re-
peated three times with similar results.
Freshly isolated mouse bone marrow and brain samples were disrupted
on ice in 20 mM Tris-Cl (pH 8.0), 140 mM NaCl, 10% glycerol, 1%
Nonidet P-40, 2 mM EDTA, 10 mM NaF, and proteinase inhibitor cock-
tail (Calbiochem). Lysates were separated on a 3–8% Tris-acetate gel
6851 The Journal of Immunology
(Bio-Rad), transferred to nitrocellulose (Bio-Rad), and nonspecific re-
activity was blocked by incubating the blots for 2 h in 5% milk in PBS
with 0.05% Tween 20 (PBST). Blots were then incubated overnight at
4°C in PBST with primary Ab at 1/5000 (anti-polySia, mAb 735) or
1/500 (anti-NCAM, mAb NCAM-13). After washing three times for 5
min in PBST, blots were incubated for 90 min at room temperature in
PBST with HRP-conjugated secondary Abs (Jackson ImmunoResearch
Laboratories) at 1/5000. Blots were washed three times for 5 min in
PBST and signal was detected using SuperSignal West Pico chemilu-
minescence substrate (Pierce).
Bone marrow lysates were prepared as described above from 10 wild-type
and 10 ST8Sia IV?/?mice, and precleared by a 30-min incubation at 4°C
with protein G-Sepharose beads (Invitrogen). Ab-conjugated beads were
prepared by incubating 5 ?g mAb 735 with 50 ?l protein G-Sepharose
beads at 4°C. mAb 735-conjugated beads were added to lysates and tum-
bled for 1 h at 4°C. Following incubation, beads were washed extensively
in ice-cold wash buffer [0.1% Triton X-100, 50 mM Tris-Cl (pH 7.4), 300
mM NaCl, 5 mM EDTA, 0.02% NaN3(w/v)] and then in ice-cold PBS.
PolySia was removed from captured proteins by direct treatment with 2 ?l
of Endo N (3 h at 37°C). Then, the sample was boiled in sample buffer (XT
sample buffer, XT reducing agent; Bio-Rad) and run on a 3–8% Tris-
acetate gel (Bio-Rad). Bands were visualized with a mass spectrometry-
compatible silver stain (Silver Quest; Invitrogen).
Protein digestion and identification by mass spectrometry
The preparation and analysis of samples were performed at the Taplin
Biological Mass Spectrometry Facility, Harvard Medical School. Silver-
stained bands were excised and digested with trypsin, and analyzed by
ESI-LC-MS/MS on an LTQ linear ion-trap mass spectrometer (Ther-
moFisher Scientific). Peptide sequences (and hence protein identity) were
determined by matching protein databases with the acquired fragmentation
pattern by the software program Sequest (ThermoFisher Scientific) (22).
Spectral matches were manually examined and multiple identified peptides
per protein were required.
In vivo progenitor assay
ST8Sia IV?/?mice were crossed with congenic wild-type mice expressing
GFP (stock 004353, The Jackson Laboratory). F1progeny were back-
crossed with ST8Sia IV?/?animals to yield ST8Sia IV?/?;GFP?/?mice,
which were used as bone marrow donors for these studies. Genotype was
confirmed by PCR as previously described (14). Sorted cell populations
were mixed with 1 ? 106whole wild-type bone marrow cells for injection.
Wild-type recipient mice (three per donor subset) were irradiated with one
dose of 900 rad and injected i.v. with prepared cells. After 21 days, recip-
ients were sacrificed and their lymphoid organs analyzed for GFP?cells.
The experiment was repeated twice with similar results.
Contact hypersensitivity (CHS)
2,4-Dinitrofluorobenzene (DNFB) was used to induce CHS in wild-type or
ST8Sia IV?/?mice (3–10 mice per group). Animals were sensitized by
painting bare abdominal skin on 2 consecutive days with 20 ?l of 0.5%
DNFB in acetone/olive oil (4/1). Mice were challenged 7 days later with an
application of 20 ?l of 0.5% DNFB on the right ear, and vehicle alone on
the left ear. The inflammatory response was assessed by measuring ear
thickness with digital calipers at 24, 48, and 72 h. In some cases, animals
were euthanized and their ears removed for histological evaluation. The
experiment was repeated four times with similar results.
Mouse RMA and RMA-S (which have reduced MHC class I expression)
NKT cell tumor cell lines were obtained from the American Type Culture
Collection. Each experimental group (wild-type and ST8Sia IV?/?) con-
tained 5–10 mice (4–12 wk) that were age-matched within 2 wk. RMA or
RMA-S cells (104-105) were injected s.c. into the flank. Palpable tumors
were measured daily with digital calipers. Mice were euthanized after loss
of ?20% of original body weight, when a tumor reached ?1.5 cm or
became ulcerated. Animals surviving 60 days were considered tumor-free.
The experiment was repeated twice with similar results.
NCAM expression with activation. Flow cytometric analyses of total
PBMCs after 24 h in culture with (right) or without (left) the polySia-specific
neuraminidase Endo N (A) and of total PBMCs after 48 h culture in the pres-
ence (heavy line, no fill) or absence (light line, gray fill) of IL-2 (B).
NCAM/CD56?human NK cells modulate polySia and
tion of short to medium-length polySia chains. Purified primary NK cells
from five individual donors were cultured separately for 48 h with or with-
out IL-2, combined and analyzed for DP. Fractions of increasing DP were
collected (x-axis) and analyzed for total sialic acid (as NANA) content
Human NK cell activation results in an increased produc-
row and peripheral myeloid cells. Flow cytometric analyses of total wild-
type (WT) bone marrow comparing polySia and c-Kit expression (A). We
studied four populations: PSAneg/Kithigh(1), PSAlow/Kithigh(2), PSAhigh/
Kithigh(3), and PSAlow/Kitlow(4). These subsets were present in ST8Sia
II?/?mice, but absent from ST8Sia IV?/?animals. In the periphery (B),
wild-type Gr-1?myeloid cells expressed polySia, while the ST8Sia IV?/?
cells displayed fluorescence similar to that of the isotype control. In con-
trast to the human, mouse NK cells did not express polySia (C). DX5?
splenocytes were labeled with isotype control (light line, gray fill) or anti-
polySia (heavy line, no fill) Abs and analyzed by flow cytometry.
ST8Sia IV produces polySia on subsets of mouse bone mar-
6852 POLYSIALIC ACID: MYELOID LINEAGE AND IMMUNE FUNCTION
Hematology and histology
The Univeristy of California Davis Comparative Pathology Laboratory
performed complete blood counts, which were validated by visual exam-
ination of blood smears. Histological evaluation of CHS ear specimens was
Unless otherwise stated, numbers represent the means ? 1 SD.
Statistical significance in tumor assays was calculated using a two-tailed
Mann-Whitney U test. Statistical significance of the remaining data was
calculated using an unpaired two-tailed Student’s t test. p ? 0.05 was
Human NK cells modulate NCAM protein expression and degree
of polysialylation with activation state
Flow cytometric analyses showed that human PBMCs expressed
the NCAM protein scaffold and its polysialic acid modifications,
which were sensitive to the polySia-specific neuraminidase Endo
N (Fig. 1A). Upon activation with IL-2, the cell-surface levels of
both the underlying protein and the attached glycan increased (Fig.
1B; n ? 10). To address whether the observed increase in polySia
expression reflected differential proliferation of CD56dimand
CD56brightNK cell populations during the assay, sorted cells with
these phenotypic characteristics were analyzed in parallel; similar
responses were observed (data not shown). These data suggest that
polySia levels are regulated by NK cell activation.
As the DP of polySia on human primary cells has not been
explored, purified NK cells were cultured with or without IL-2
for 48 h. Samples from five individual donors were combined
for analysis. Intact polySia chains on glycan cores containing
lactosamine were released by endo-?-galactosidase treatment.
This method may have neglected to free polySia that was at-
tached to NCAM through alternate core structures; however, the
relative amounts of total sialic acid released from resting and
activated NK cells were in accord with our flow cytometry (Fig.
1) and immunoblotting data (not shown). The liberated polySia
was analyzed by HPLC as previously described to determine
chain length (2). Total sialic acid content [as N-acetyl neura-
minic acid (NANA)] of HPLC fractions was monitored to track
the relative abundance of each DP population. In both resting
and activated NK cells, a large fraction of total NANA was
associated with small non-polySia glycans (DP 1–10), most
likely reflecting the capping groups of typical N- and O-linked
glycans. Plotting percentage of total NANA vs DP showed that
activation of NK cells increased the abundance of NANA as-
sociated with polySia (Fig. 2). Additionally, chain length,
which was extremely heterogeneous on resting cells, was com-
pressed into the middle range (DP 11–140) upon activation.
throid and myeloid progenitors. PolySia (PSA)/c-Kit-defined subsets were
sorted from wild-type bone marrow and plated in culture using conditions
that promote erythroid (A) or myeloid (B) development. A, Cells were
maintained for 7 days in methyl-cellulose with erythropoietin, stem cell
factor, and IL-3 and then analyzed microscopically for burst-forming units
erythroid (BFU-E) and CFU granulocyte-macrophage (CFU-GM). BFU-E
and CFU-GM counts were combined to determine the total number of
colonies formed. B, Bone marrow cells were maintained in culture for 7
days and then scored microscopically for colonies (?50 cells) and clusters
(10–49 cells). The results of two separate experiments are shown.
The PSAneg/Kithighand PSAlow/Kithighsubsets contain ery-
phoid progenitors. PolySia (PSA)/c-Kit-defined subsets were sorted from
wild-type bone marrow and placed in coculture with OP9 stromal cells
expressing either GFP and DL1 or GFP alone. OP9-DL1 promotes the
differentiation of lymphoid progenitors into T cells and NK cells, while
OP9-GFP supports the development of B cells and NK cells. Differentia-
tion was monitored by flow cytometry; results after 18 days in culture are
shown. B cells were identified by B220 expression, and NK cells by cell
surface DX5. T cell development was monitored by appearance of double-
negative stage 2 and 3 cells, identified by expression of CD44 and CD25.
Representative data from one experiment are shown. The experiment was
repeated three times with similar results.
The PSAneg/Kithighand PSAlow/Kithighsubsets contain lym-
Table I. Expression of Ags analyzeda
55 ? 11
7.4 ? 1.7
0.02 ? 0.17
0.26 ? 0.25
65 ? 6
95 ? 2
66 ? 2
21 ? 3
9 ? 0.3
61 ? 0.55
96 ? 0.2
99 ? 0.3
9.8 ? 0.9
31.4 ? 0.9
73 ? 5.2
38.6 ? 0.1
2 ? 1.7
27 ? 1.6
9 ? 0.01
59 ? 0.1
an ? 3. The expression of the following antigens was also analyzed (data not shown): CD150, CD48, CD244.2, HSA, FLT3,
IL-7R, Thy1, CD27, TER119, B220, CD3, CD4, CD8, TCR?, TCR??, NK1.1, CD11b, CD11c, CD25, and CD36.
6853The Journal of Immunology
ST8Sia IV catalyzes polySia expression by populations of mouse
bone marrow cells during myeloid differentiation
To analyze the potential immunological properties of polySia,
we used a mouse model. First, we characterized the expression
patterns of polySia on immune subsets in wild-type animals. In
contrast to their human counterparts, polySia was not detectable
on mouse NK cells (Fig. 3C). This finding was consistent with
RT-PCR analyses on sorted NK cells that revealed an absence
of NCAM and ST8Sia IV (data not shown). Interestingly, robust
polySia expression was detected on wild-type mouse bone mar-
row subsets (Fig. 3A). This expression was conserved in ST8Sia
II?/?mice but absent in ST8Sia IV?/?mice, indicating that the
latter enzyme was responsible for polySia on these cells.
Bone marrow polySia expression correlated with receptor ty-
rosine kinase c-Kit expression, suggesting that the polySia?cells
were hematopoietic progenitors. We defined four subsets accord-
ing to their relative levels of polySia (PSA) and c-Kit (Kit)
(PSAneg/Kithigh, PSAlow/Kithigh, PSAhigh/Kithigh, PSAlow/Kitlow;
Fig. 3A). The populations comprised 12%, 6%, 12%, and 42%,
respectively, of total c-Kit?bone marrow cells. Extensive pheno-
typic analyses by flow cytometry suggested that these subsets com-
prised cells that were differentiating along a myeloid pathway (Ta-
ble I). The PSAneg/Kithighpopulation included hematopoietic stem
cells (defined as Lin?, c-Kit?, Sca-1?) and most appeared to be
progenitors as they were Lin?and expressed CD34. The second
population (PSAlow/Kithigh) comprised more committed progeni-
tors with near uniform expression of CD34, and to a lesser extent
CD11b and Gr-1. As differentiation proceeded, evidenced by a
reduction in CD34 expression and an increase in CD11b and Gr-1
expression, PSA levels dramatically increased in parallel (PSAhigh/
Kithigh). The fully differentiated progeny (CD34?/?, CD11b?, Gr-
1high) had polySia levels that were comparable to the progenitors
(PSAlow/Kitlow). Consistent with the latter population containing
mature myeloid cells, we found low levels of polySia expression
on peripheral wild-type, but not ST8Sia IV?/?, Gr-1?splenocytes
(Fig. 3B). Thus, myeloid differentiation is characterized by a wave
of high levels of polySia expression. In contrast to the mouse,
human fetal bone marrow did not contain these polySia?myeloid
populations, and human peripheral myeloid cells did not express
polySia (data not shown). The polySia?subset in human fetal
bone marrow consisted of NK cells, as determined by the pheno-
type: CD56?, CD7?, CD33?, and CD34?(data not shown). Note
that the observed differences between adult mouse bone marrow
and fetal human bone marrow may be attributable to variations in
polySia expression during development.
In vitro progenitor studies confirm the phenotypic analyses of
the polySia expressing subsets
To confirm the phenotypic analyses, the bone marrow populations
defined by polySia and c-Kit were sorted by flow cytometry and
tested in vitro and in vivo for their ability to give rise to various
immune lineages. For these experiments, the PSAneg/Kithighsubset
served as a positive control, as hematopoietic stem cells were con-
tained in this population (data not shown). In colony-forming as-
says testing erythroid and myeloid potential, both the positive con-
trol and the progenitor subset, PSAlow/Kithigh, gave rise to these
lineages, forming both erythroid blasts and colonies, as well as
myeloid colonies and clusters (Figs. 4). Of the more differentiated
populations, the immature myeloid cells, PSAhigh/Kithigh, showed
an intermediate ability to form myeloid clusters and did not pro-
duce erythroid populations. The fully differentiated PSAlow/Kitlow
cells did not produce colonies in either assay.
To test lymphoid progenitor potential, sorted bone marrow sub-
sets were cocultured with OP9 cells transfected with a GFP vector
control, or with GFP and the Notch ligand, DL1. In this assay,
multipotent progenitors develop into B cells when cultured on
OP9-GFP feeders, while DL1 induces T cell development. NK
cells develop at lower efficiencies under both conditions. In accord
with the phenotypic data and colony-forming assays, the positive
control, PSAneg/Kithigh, and the progenitor population, PSAlow/
Kithigh, produced all the lymphoid lineages, the latter with a 10-
fold reduction in efficiency as compared with the former (Fig. 5).
temporally linked. Wild-type mice were injected with a single dose of 5-FU
to deplete cycling cells, and then sacrificed at various intervals over a
12-day period. The loss and restoration of the PSA/c-Kit-defined subsets
was monitored by flow cytometry.
Development of the polySia (PSA)/c-Kit-defined subsets is
PSAhigh/Kithigh, and PSAlow/Kitlowsubsets. Wild-type mice were injected
every 24 h with either vehicle alone or vehicle containing 2 ?g of G-CSF.
Animals were sacrificed after 5 days and polySia/c-Kit-defined bone mar-
row subsets were monitored by flow cytometry. The change in percentage
of total bone marrow was calculated for each population. The results of two
separate experiments were combined and plotted in this graph. ?, p ? 0.007.
G-CSF increases the production of the PSAlow/Kithigh,
Table II. Number (?103) of recovered GFP?cells expressing the designated cell-surface Ag/1000
TER119Gr-1 Gr-1CD14 DX5B220
59 ? 47
33 ? 5.6
1.2 ? 0.8
1.6 ? 0.6
117 ? 88
50 ? 34
2.1 ? 0.3
2.1 ? 0.7
320 ? 58
273 ? 47
10.7 ? 10.7
0.5 ? 0.9
172 ? 108
162 ? 45
6.6 ? 7.7
0.08 ? 0.13
29 ? 18
26 ? 11
1.4 ? 1.8
249 ? 194
206 ? 47
9 ? 15
1.9 ? 1.1
6854POLYSIALIC ACID: MYELOID LINEAGE AND IMMUNE FUNCTION
The immature myeloid subset, PSAhigh/Kithigh, did not produce
NK or T cells and generated almost 1000-fold fewer B cells than
the positive control population. The mature myeloid population,
PSAlow/Kitlow, did not proliferate.
In vivo progenitor studies confirm the phenotypic and in vitro
analyses of the polySia expressing subsets
For in vivo experiments, GFP?congenic wild-type mice were
used as donors so that engrafted lineages could be identified using
this fluorescent reporter. Flow-sorted bone marrow subsets
(?28,000 cells/mouse) were injected with a survival dose (1 ? 106
cells/mouse) of wild-type bone marrow into irradiated wild-type
recipient animals. Three weeks later mice were sacrificed and or-
gans analyzed for GFP?populations (Table II). Both the positive
control, PSAneg/Kithigh, and the progenitor population, PSAlow/
Kithigh, gave rise to erythroid (TER119?), myeloid (Gr-1?and
CD14?), and lymphoid (DX5?and B220?) cells in the bone mar-
row and spleen. No statistically significant differences were noted
in the numbers of cells recovered from these two donor popula-
tions. In contrast, the numbers of GFP?cells isolated from the
immature myeloid subset, PSAhigh/Kithigh, and the mature myeloid
population, PSAlow/Kitlow, were reduced by an average of ?40-
fold and ?500-fold, respectively, as compared with the progenitor
Next, we asked whether development of the four bone marrow
subsets was temporally linked. Mice were treated with one bolus of
5-FU to deplete cycling cells, and the disappearance and reappear-
ance of the polySia/c-Kit-defined populations was followed over
12 days. As expected, the depletion and recovery of the progeni-
tors preceded by 1 or 2 days that of the mature subsets (Fig. 6).
Finally, we stimulated myeloid development and characterized
the response of the bone marrow subsets. Briefly, wild-type mice
were injected every 24 h with G-CSF for 5 days to induce expan-
sion of the myeloid compartment, and bone marrow was analyzed
by flow cytometry. In accord with the preceding phenotypic in
vitro and in vivo data, both the progenitor population (PSAlow/
Kithigh) and the immature and mature myeloid subsets (PSAhigh/
Kithighand PSAlow/Kitlow) significantly expanded in response to
G-CSF treatment as compared with vehicle-treated control mice
(p ? 0.01; Fig. 7). Collectively, the data in Table II and Figs. 3–7
reveal that polySia expression is expressed and modulated during
myeloid development in the mouse.
Given the expression of polySia on hematopoietic progenitors
and myeloid cells, we asked whether there were obvious changes
in the distribution of immune subsets in ST8Sia IV?/?as com-
pared with wild-type mice. Hematological analyses revealed min-
imal differences: a statistically significant, slight increase in the
percentage of lymphocytes in peripheral blood, and a correspond-
ing although not significant decrease in the percentage of circulat-
ing monocytes and neutrophils (data not shown). No changes in the
numbers or phenotype of peripheral myeloid lineages were noted
(data not shown).
NCAM is the scaffold for myeloid expression of polySia
As this study was the first description of polySia on myeloid
cells, the underlying protein scaffold was unknown. Candidate
proteins that are expressed by other cell types that carry polySia
modifications include NCAM and CD36. To identify the scaf-
fold on the myeloid subsets that were the subject of this inves-
tigation, we immunoprecipitated wild-type and ST8Sia IV?/?
marrow cells is the scaffold for polySia. Polysialylated
proteins were immunoprecipitated from lysates of wild-
type (WT) or ST8Sia IV?/?(IV?/?) bone marrow
(BM) cells and Endo N treated (A). Recovered proteins
were silver stained and two bands were visible in wild-
type, but not in ST8Sia IV?/?, samples. The bands were
isolated and both were identified as NCAM, on the basis
of two peptides each, by mass spectrometry. Flow cy-
tometry using anti-NCAM and anti-c-Kit on wild-type
mouse bone marrow cells showed NCAM was ex-
pressed on populations that were analogous to those ob-
served by staining with anti-polySia and anti-c-Kit (B).
Immunoblotting with anti-polySia and anti-NCAM
mAbs showed a polySia signal in wild-type (WT) and
ST8Sia II?/?bone marrow (BM), but not ST8Sia IV?/?
bone marrow. In contrast, an NCAM signal was ob-
served in association with all of the samples (C). Adult
wild-type brain lysate was used as a positive control for
both polySia and NCAM signals, and Endo N-treated
(?EN) wild-type bone marrow was used as a negative
control for polySia expression. Three major isoforms of
NCAM were visible in brain lysates, while two (of
?140 and ?120 kDa) were present in bone marrow
samples. Note that the polysialyated bands migrated
with a lower mobility than those recognized by anti-
NCAM, as only a fraction of this molecule carried these
NCAM on the surface of mouse bone
6855 The Journal of Immunology
bone marrow lysates with an anti-polySia Ab, treated the pre-
cipitatate with Endo N to remove polySia, and separated the
deglycosylated proteins by SDS-PAGE. Following electro-
phoresis, two silver stained bands of ?120 and 140 kDa were
observed in the wild-type, but not in the ST8Sia IV?/?, samples
(Fig. 8A). The bands were excised and analyzed by electrospray
mass spectrometry (LTQ linear ion trap), which identified both
bands, on the basis of two peptides each, as NCAM (data not
shown). In accord with this finding, both flow cytometry and
immunoblotting confirmed the presence of NCAM on the rele-
vant cells in mouse bone marrow (Fig. 8, B and C).
ST8Sia IV?/?mice exhibit exaggerated contact hypersensitivity
and an inability to control growth of engrafted tumors
Next we tested immune responses in ST8Sia IV?/?mice. First,
we used a CHS assay. Wild-type and ST8Sia IV?/?mice were
sensitized with the hapten DNFB; a week later, they were chal-
lenged with an application of DNFB to one ear, and vehicle
alone to the other ear. Wild-type mice responded as expected,
with peak swelling observed around 24 h (23). Interestingly, the
ST8Sia IV?/?response equaled or exceeded the wild-type re-
sponse at 24 h, and inflammation continued to increase through
48 and 72 h. Fig. 9A shows the results of a representative ex-
periment in which ST8Sia IV?/?ear thickness was statistically
increased at all time points as compared with the response of
We also assessed the CHS response at 72 h on a histological
level and noted significantly more inflammation and edema after
DNFB treatment of ST8Sia IV?/?mice as compared with con-
trols, whose lesions were much less severe (Fig. 9B). Injury to the
epithelium of ST8Sia IV?/?ears was also more pronounced, with
diffuse epidermal hyperplasia, intercellular edema, and ulceration.
The ST8Sia IV?/?vehicle-treated ears also demonstrated minimal
to mild edema whereas the wild-type controls had no significant
We used immunosensitive and immunoresistant cell lines to
test the response of ST8Sia IV?/?mice to a tumor challenge.
RMA-S cells, which have reduced MHC class I expression, are
sensitive to NK killing, while the parental RMA cells form
tumors in wild-type animals (24). In initial experiments,
RMA-S cells formed tumor masses in NK cell-depleted wild-
type animals (25), but not wild-type or ST8Sia IV?/?mice.
This finding suggested that the NK cell compartment of ST8Sia
IV?/?mice was intact (data not shown). In contrast, injection
of RMA cells into wild-type, ST8Sia IV?/?, or the immunode-
ficient TCR??/?mice led to uncontrolled tumor growth in all
cohorts, requiring euthanasia of the animals when their tumors
exceeded acceptable size limitations (Fig. 10). Importantly, tu-
mor growth in ST8Sia IV?/?mice was significantly faster than
in wild-type mice, and was comparable to the rate observed in
TCR??/?mice (p ? 0.02).
ST8Sia IV?/?mice. Wild-type and ST8Sia IV?/?mice were sensitized
to DNFB. One week later the left ear was treated with DNFB and the
right ear with vehicle alone as a negative control. We assessed the
inflammatory response by measuring ear thickness with digital calipers
at 24, 48, and 72 h (A). Each ear measurement was taken three times.
The data represent the average ear thickness ? SD for the DNFB-
treated ears of all mice (n ? 3 wild-type (WT), n ? 3 ST8Sia IV?/?).
At each time point, ST8Sia IV?/?mice had significantly more ear
swelling than did wild-type animals (?, p ? 0.01; ??, p ? 0.001).
Histological analyses showed excessive tissue damage observed in
ST8Sia IV?/?mice as compared with wild-type mice (B). Vehicle- (i
and ii) and DNFB-treated (iii and iv) ears were removed, fixed, sec-
tioned, and stained with H&E for histological analysis. Asterisks denote
areas of edema, which appeared as empty spaces. Bar ? 60 ?m.
The contact hypersensitivity response is excessive in
IV?/?mice than in wild-type animals. Wild-type,
ST8Sia IV?/?, and TCR??/?mice (negative controls
for immunocompetence) were injected subcutaneously
with 104-105RMA cells. When tumors became ulcer-
ated, or grew larger than 1.5 cm, or mice lost ?20% of
their original body weight, animals were sacrificed. An-
imals surviving more than 60 days were considered tu-
mor-free; all deaths are plotted on the graph. There was
a statistically significant difference in survival rates be-
tween wild-type and ST8Sia IV?/?animals (p ? 0.02,
two-tailed Mann-Whitney U test).
RMA tumors grow faster in ST8Sia
6856POLYSIALIC ACID: MYELOID LINEAGE AND IMMUNE FUNCTION
PolySia expression and function have been extensively studied in
the nervous system, but the distribution and role of polySia in the
immune compartment is still undefined. Herein we demonstrated
the presence of polySia on human NK cells and its absence on
other immune subsets, a finding that is in accord with two earlier
reports (26, 27). NK cells up-regulated the expression of polySia
and its scaffold NCAM upon activation by IL-2 treatment. This
coregulation suggested that the heightened polySia signals were
due to an up-regulation of both scaffold and glycan, rather than
solely attributable to expanded polySia chain length. In support of
this conclusion, a comparison of DP on resting and activated cells
showed that upon activation total polySia was increased and the
DP became more homogenous, tending toward small- to medium-
length chains. To our knowledge, this is the first elucidation of
polySia DP on primary human cells. The DP values we observed
were slightly larger than analogous structures that were isolated
from cell lines and chick brain (1, 28). Although this distinction
may be due to real differences among samples, it more likely re-
flects the fact that our method protects samples from acid-cata-
lyzed internal hydrolysis. Importantly, this result shows that the
size of polySia chains on human immune cells is comparable to
that of GAGs, polysaccharides that play critical structural and
functional roles in the extracellular matrix compartment. The re-
peating negative charge present on polySia is also reminiscent of
highly anionic GAGs, and this characteristic is likely critical to its
Mouse NK cells did not express NCAM or polySia; however,
both were coexpressed in mouse bone marrow. Immunoprecipita-
tion of bone marrow lysates with anti-polySia Abs and identifica-
tion of the resulting bands by mass spectrometry demonstrated that
NCAM was the underlying scaffold. Analyses of ST8Sia II?/?and
ST8Sia IV?/?mice indicated that the latter enzyme produced the
Flow cytometry revealed four bone marrow subsets based on
expression of polySia and c-Kit. These populations (PSAneg/
Kithigh, PSAlow/Kithigh, PSAhigh/Kithigh, PSAlow/Kitlow) bore cell
surface Ags that were characteristic of myeloid differentiation. The
PSAneg/Kithighsubset contained hematopoeitic progenitors, the
Kithigh, PSAlow/Kitlowgroups, immature and mature myeloid cells,
respectively. These findings were confirmed by in vitro and in vivo
functional studies. 5-FU experiments demonstrated that the devel-
opmental kinetics of the four populations were temporally linked,
and thus these populations shared a common lineage. Finally, the
progenitor population, PSAlow/Kithigh, and the immature and ma-
ture myeloid subsets, PSAhigh/Kithighand PSAlow/Kitlow, respec-
tively, all expanded in response to treatment with G-CSF, further
evidence that these populations are myeloid.
We observed a dramatic different in CHS response in ST8Sia
IV?/?, which sustained augmented inflammation, as compared
with wild-type. This striking observation elicits some interesting
hypotheses. Considering possible roles for polySia in the immune
system, we noted that both NK and myeloid cells are cytotoxic
populations, carrying small positively charged antimicrobial pep-
tides (AMPs) (29–31). Negatively charged GAGs such as heparan
sulfate can bind and neutralize AMPs (32, 33), and thus it is pos-
sible that polySia with its structural similarities to GAGs has par-
allel activities. Expression of polySia on cytotoxic cells may lo-
calize AMPs released from leukocytes to prevent damage of
surrounding tissues. The excessive CHS inflammatory response
noted in ST8Sia IV?/?mice is consistent with this notion, as gran-
ulocytes that were initially drawn to the site might have injured
tissues by uncontrolled AMP leakage. In turn, damaged tissue
would release proinflammatory signals, upregulating the immune
response (34). This cycle is consistent with the protracted inflam-
matory reaction, the increased tissue damage, and the elevated leu-
kocytic infiltrate noted in ST8Sia IV?/?DNFB-treated ears rela-
tive to wild-type.
Additionally, GAGs, which bind cytokines and chemokines,
modulate their availability and local concentration by either pre-
senting or sequestering these molecules. It is possible that polySia
plays a similar role. Studies in the nervous system have already
demonstrated that neuronal responses to the cytokines brain-de-
rived neurotrophic factor (BDNF) and platelet-derived growth fac-
tor (PDGF) are affected by polySia expression, although the mech-
anism has not been elucidated (9, 10). As the development and
function of the immune system depend heavily on the influence of
cytokines, polySia could have a great impact. However, its effects
would likely be complex since cytokines that regulate signaling
networks that, in turn, direct fate decisions, trafficking, and acti-
vation might be involved. The decreased immune response to ec-
topic tumors noted in ST8Sia IV?/?mice raises the intriguing
possibility that these processes are influenced by polySia.
To our knowledge, this is the first demonstration of either poly-
Sia or NCAM expression on myeloid cells. The discovery of such
expression in the mouse provides an opportunity to manipulate and
study the immunological role of these molecules in a biological
context, including in vivo models. The findings could have impor-
tant implications for human health, as polySia and NCAM are not
only expressed on human NK cells but also decorate the surfaces
of a variety of tumors and bacteria. For instance, polysialylated
NCAM, which is found on a number of cancers including gliomas,
small-cell lung carcinomas, and Wilms’ tumors, is positively as-
sociated with metastasis and disease progression (35–37). We pos-
tulate that tumors co-opt the immune system’s strategy of using
polySia to modulate responses to chemokines and growth factors,
thus gaining a competitive advantage. Regarding bacteria, polySia
expression is correlated with the virulence of pathogenic strains of
Escherichia coli K1 and group B Neisseria meningitidis (38, 39).
It is possible that bacterial polySia binds and neutralizes AMPs,
sequestering them at a safe distance from the cell wall. Similarly,
a subset of chemokines also has antibiotic activity (40), and poly-
Sia, which has been shown to modulate chemokine functions (9,
10), may protect bacteria from this threat. Collectively, our data
point to a role for polySia in the complex processes involved in
immunological development and host defense.
The authors have no financial conflicts of interest.
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