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Prepublished online as a Blood First Edition Paper on April 24, 2003; DOI 10.1182/blood-2003-03-0836.
Blood, 1 September 2003, Vol. 102, No. 5, pp. 1678-1685
Impaired selectin-ligand biosynthesis and reduced inflammatory responses in
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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-1,4-galactosyltransferase (4GalT) plays a key role in these
processes. Recently isolated 6 4GalT genes are candidates, but
their individual roles, including those in selectin-ligand biosynthesis,
remain to be elucidated. More than 80% of the core 2 O-glycans on the
leukocyte membrane glycoproteins of 4GalT-I–deficient mice lacked
galactose residues in -1,4 linkage, and soluble P-selectin binding to
neutrophils and monocytes of these mice was significantly reduced, indicating
an impairment of selectin-ligand biosynthesis. 4GalT-I–deficient
mice exhibited blood leukocytosis but normal lymphocyte homing to peripheral
lymph nodes. Acute and chronic inflammatory responses, including the contact
hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses, were
suppressed, and neutrophil infiltration into inflammatory sites was largely
reduced in these mice. Our results demonstrate that 4GalT-I is a major
galactosyltransferase responsible for selectin-ligand biosynthesis and that
inflammatory responses of 4GalT-I–deficient mice are impaired
because of the defect in selectin-ligand biosynthesis. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Selectin ligands such as sLex and sulfated sLex are
mainly expressed at the terminus of N-acetyl lactosamine repeats on
serine/threonine (O)–Linked oligosaccharides
(O-glycans).7-9
In the biosynthesis of O-glycans,
Gal1
3GalNAc
1Ser/Thr (core 1) is formed by the action
of recently cloned core 1
-1,3-galactosyltransferase10
(core 1 3GalT). Core 1 is then converted to
GlcNAc16(Gal13)GalNAc1Ser/Thr (core 2) by
core 2 -1,6-N-acetylglucosaminyltransferase (core 2 6GnT)
(Figure 1). Studies of core 2
6GnT-deficient mice have revealed that core 2 O-glycans are
essential for leukocyte emigration during inflammation but not for lymphocyte
trafficking under normal
circumstances.11
These results suggest that most of the E- and P-selectin ligands are expressed
on core 2 O-glycans. Recently, 6-sulfo sLex on extended
core 1 O-glycans has been identified as one of the L-selectin
ligands.12
-1,4-Galactosyltransferase (4GalT) and
-1,3-N-acetylglucosaminyltransferase (3GnT) act
alternatively to extend N-acetyl lactosamine repeats on core 2
O-glycans, and 4GalT is also required to synthesize the
sLex epitope in collaboration with -2,3-sialyltransferase
(ST) and -1,3-fucosyltransferase (FucT)
(Figure 1). Among these
glycosyltransferases, Fuc-TVII has been shown to be mainly responsible for the
fucosylation of
sLex.13,14
However, the contribution of the 4GalT genes in the
biosynthesis of selectin ligands remains to be elucidated.
|
4GalT is involved in the biosynthesis of biologically important
galactose-containing oligosaccharides, such as stage-specific embryonic
antigen-1 (SSEA-1 = Lex), sLex, and 6-sulfo
sLex. SSEA-1, which is expressed specifically on preimplantation
embryos, has been suggested to play a role in morula compaction and blastocyst
formation.15
However, 4GalT-I–deficient embryos form normal compacted morulae
and blastocysts. Polysialic acid (PSA) and human natural killer-1 (HNK-1)
carbohydrate are expressed on the outer chains of the
Gal14GlcNAc-R backbone. These carbohydrates, which are expressed
on neuronal cell adhesion proteins, are thought to be involved in neuronal
cell
recognition.16,17
In support of this idea, we and another group showed that mice deficient in
the biosynthesis of HNK-1 and PSA exhibit impaired synaptic plasticity,
respectively.18,19
To elucidate the biologic relevance of these galactose residues and their
outer chains, we and another group previously generated
4GalT-I–deficient
mice.20,21
These mice exhibit growth retardation and semi-lethality before weaning
because of the augmented proliferation and abnormal differentiation of small
intestinal epithelial
cells.20 In
contrast, PSA and HNK-1 are expressed normally, and no neuronal defects are
detected in 4GalT-I–deficient
mice,22 suggesting
that a 4GalT gene(s) other than 4GalT-I is
responsible for the biosynthesis of PSA and HNK-1. Although approximately half
the 4GalT-I–deficient mice die before the weaning period, the rest
can be used for further study. In the present study, selectin-ligand
biosynthesis and the effect of selectin-ligand deficiency were examined in
4GalT-I–deficient mice.
Seven 4GalT genes (4GalT-I to -VII)
have been isolated thus
far,23 and their
individual roles, including those in selectin-ligand biosynthesis, remain to
be elucidated. Because 4GalT-VII acts on xylose, 4GalT-I to
–VI can catalyze the Gal14GlcNAc linkage and thus are good
candidates for the synthesis of selectin ligands such as sLex and
6-sulfo
sLex.23
Here, to evaluate the contribution of 4GalT-I in selectin-ligand
biosynthesis, the carbohydrate structures of leukocytes from
4GalT-I–deficient mice and the inflammatory responses of these
mice were examined. More than 80% of the core 2 O-glycans on the
leukocyte membrane glycoproteins of 4GalT-I–deficient mice lacked
galactose residues in -1,4 linkage, and the binding of soluble
P-selectin to the neutrophils and monocytes of 4GalT-I–deficient
mice was reduced. These results indicate that selectin-ligand biosynthesis was
severely impaired in 4GalT-I–deficient mice. Furthermore,
4GalT-I–deficient mice exhibited peripheral blood leukocytosis and
reduced acute and chronic inflammatory responses, including contact
hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses,
indicating a selectin-ligand deficiency. Our results clearly demonstrate that
among the 4GalT gene family,
4GalT-I is a major galactosyltransferase responsible for
selectin-ligand biosynthesis.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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4GalT-I–deficient mice
The generation of 4GalT-I–deficient mice was described
previously.20
4GalT-I–/– mice on
a mixed background between 129/Sv and C57BL/6 were used for the experiments,
with 4GalT-I+/–
littermates as controls. Carbohydrate structures of the serum glycoproteins
between 4GalT-I+/+ and
4GalT-I+/– mice were
indistinguishable,24
and no overt phenotypes were observed in the
4GalT-I+/– mice. The
mice were kept under specific pathogen-free conditions in an environmentally
controlled clean room at the Laboratory Animal Research Center, Institute of
Medical Science, University of Tokyo and at the Institute for Experimental
Animals, Kanazawa University. Experiments were conducted according to
institutional ethical guidelines for animal experiments and safety guidelines
for gene manipulation experiments.
Preparation of O-glycans from splenocytes
Splenocytes were prepared by grinding the spleen with the plunger of a disposable syringe, passing the ground spleen through nylon mesh, and suspending the cells in minimum essential medium (MEM). They were then treated twice with hemolysis buffer (17 mM Tris-HCl containing 140 mM NH4Cl, pH 7.2) to remove red blood cells and were passed through nylon mesh to remove debris. The cells were washed twice with phosphate-buffered saline (PBS), homogenized in 10 mM Tris-HCl containing 1 mM EDTA (ethylenediaminetetraacetic acid), pH 7.4 (TE buffer), and spun at 150g for 5 minutes. The supernatant was spun at 25 000g for 30 minutes, and the precipitate was washed twice with TE buffer and once with 10 mM ammonium acetate. The precipitated membrane fraction was lyophilized and delipidated as previously described.25 Delipidated ghosts were suspended in 1 mL 0.05 M NaOH containing 1 M sodium borohydride and were incubated at 45°C for 16 hours. Released glycans were purified according to the method described previously.26
Analysis of O-glycans by HPAEC-PAD
High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed using a Bio-LC system (Dinex, Sunnyvale, CA) equipped with a CarboPac PA-1 column (4 x 250 mm) and a pulsed amperometric detector. O-glycans were eluted with a linearly increasing concentration of 0 to 400 mM sodium acetate in 100 mM NaOH from 0 to 30 minutes. O-glycans were also desialylated by mild acid hydrolysis with 0.01 M HCl at 100°C for 20 minutes and analyzed by HPAEC-PAD using an isocratic elution with 22 mM NaOH.
Flow cytometric analysis
Splenocytes were prepared as described above except that they were treated with the hemolysis buffer only once. Splenocytes (2 x 106 cells) were blocked with antimouse CD16/CD32 for 15 minutes and were incubated with the first antibody or human–P-selectin immunoglobulin M (IgM) for 45 minutes. When antirat IgG2b was used as the second antibody, mouse serum was used for blocking instead of antimouse CD16/CD32. The splenocytes were washed, incubated with R-phycoerythrin (PE)– or fluorescein isothiocyanate (FITC)–conjugated second antibodies for 45 minutes, washed again, and incubated with 7-amino actinomycin D (Sigma, St Louis, MO). Incubations with antibodies were carried out on ice. Data were analyzed on a FACScan flow cytometer using Lysis II software (Becton Dickinson, Mountain View, CA).
A human P-selectin–IgM expression plasmid, human P-selectin–IgM/pcDNA1.1, which bears part of the human P-selectin cDNA containing the NH2-terminal lectin domain, the epidermal growth factor (EGF)–like domain, and the first 2 complement-binding protein-like domains (provided by Ono Pharmaceutical, Osaka, Japan), and the µ heavy chain of human IgM (provided by Dr A. Traunecker, Basel Institute for Immunology, Switzerland) was transfected into human embryonic kidney-derived 293T cells (provided by Dr Y. Kanakura, Osaka University Graduate School of Medicine, Japan). The pAdVAntage vector (Promega, Madison, WI) was cotransfected with human P-selectin–IgM/pcDNA1.1 to enhance protein expression levels. Four days after transfection, the culture medium containing the P-selectin–IgM was collected and stored at –80°C until use.
Antimouse CD16/CD32 (2.4G2), antimouse Gr-1 (RB6-8C5), and antimouse PSGL-1 (2PH1), were purchased from PharMingen (San Diego, CA). PE-antimouse F4/80 and FITC-antirat IgG were purchased from Caltag (Burlingame, CA), and FITC-antihuman IgM was purchased from American Qualex (San Clemente, CA). PE-antirat IgG was kindly provided by Dr T. Yoshimoto (Institute for Medical Sciences, University of Tokyo, Japan).
Colony-forming activity of bone marrow cells
Nonadherent mononuclear cells (1 x 104) were prepared from
the bone marrow of
4GalT-I–/–
(n = 5) and 4GalT-I+/–
(n = 5) mice (2-3 months old) and plated into 0.45% methylcellulose in the
presence of 50 ng/mL granulocyte–colony-stimulating factor (G-CSF)
(provided by Chugai Pharmaceutical, Tokyo, Japan), 2 U/mL EPO (provided by
Kirin Brewery, Tokyo, Japan), or 20 ng/mL interleukin-3 (IL-3; Amgen, Thousand
Oaks, CA). Colonies were counted after 2 weeks.
Acute inflammation
Zymosan-induced inflammation was carried out as described
previously.27
4GalT-I–/– (n =
10) and 4GalT-I+/– (n =
8) mice were anesthetized with ketamine and xylazine, and 20 µL 2% zymosan
A (Sigma) and saline were injected subcutaneously into the right and left
earlobes, respectively, using a microsyringe (Hamilton, Reno, NV) with a
30-gauge needle. At 8 hours after the injection, when the swelling reached
peak levels, the mice were killed, and a disk of each earlobe was removed
using a 6-mm biopsy punch and weighed. Ear swelling was calculated as follows:
[Increase of ear swelling (%)] = ([weight of challenged earlobe] –
[weight of vehicle-treated earlobe]) x 100/[weight of vehicle-treated
earlobe].
CHS response
4GalT-I–/– (n =
11) and 4GalT-I+/– (n =
11) mice were sensitized by the epicutaneous application of 100 µL 7%
2,4,6-trinitrochlorobenzene (TNCB) (Tokyo Chemical, Tokyo, Japan) in
acetone/olive oil (3:1, vol/vol) onto shaved abdomens. On day 5 after
sensitization, 20 µL 1% TNCB solution and the solvent alone were applied
epidermally onto the right and left earlobe, respectively. Mice were killed,
and earlobe biopsies were taken as described above 24 hours after the second
challenge, when the swelling reached peak levels. Each ear disk was weighed,
and the ear swelling was calculated as described above.
DTH response
4GalT-I–/– (n =
8) and 4GalT-I+/– (n =
8) mice were sensitized by the subcutaneous injection of 200 µL 1.25 mg/mL
methylated bovine serum albumin (mBSA) (Sigma) in PBS/complete Freund adjuvant
(1:1, vol/vol) into the tail roots. On day 7 after the sensitization, 20 µL
10 mg/mL mBSA in PBS and PBS alone were applied epidermally onto the right and
left footpad, respectively. Mice were killed, and footpad thickness was
measured 24 hours after the second challenge, when the swelling reached peak
levels. Footpad thickness was calculated as follows: [Increase of footpad
thickness (%)] = ([thickness of challenged footpad] – [thickness of
vehicle-treated footpad]) x 100/[thickness of vehicle-treated
footpad].
Histology
Tissues were fixed in 10% neutral formalin, dehydrated, and embedded in paraffin wax according to standard procedures. Footpads were defatted with methanol and chloroform (1:1) and were decalcified with 5% formic acid before they were embedded in paraffin wax. Sections of 6 µm were made and stained with Weigert hematoxylin-eosin.
Myeloperoxidase assay
Ear disks (obtained with a 6-mm punch) were homogenized in 400 µL PBS and sonicated for 30 seconds. The homogenates were frozen and thawed 3 times and again were sonicated for 30 seconds. After the debris was removed by centrifugation, myeloperoxidase (MPO) activity in the supernatants was measured as described.28 The MPO assay was carried out in 400 µL 50 mM potassium-phosphate buffer (pH 6.0) containing 0.4 mg/mL O-phenylenediamine (Sigma) and 0.05% H2O2 for 20 minutes. The reaction was stopped by the addition of 100 µL0.4 M H2SO4, and OD 490 nm was measured. MPO activity was calculated using commercial peroxidase (Sigma) as a standard.
Statistical analysis
Statistical evaluation was carried out by means of the Student t
test or the Welch t test according to the Levene test for equality of
variance between 4GalT-I–deficient and control mice. A 2-sided
level of P < .05 was accepted as statistically significant. Data
are presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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4GalT-I–deficient
leukocytes
Sialyl Lex is most abundantly expressed at the terminus of
N-acetyl lactosamine disaccharide repeats on core 2
O-glycans.3
As shown in Figure 1, a
4GalT gene must be involved in the biosynthesis of the
N-acetyl lactosamine disaccharide repeats and terminal
sLex epitope on the core 2 branch. To clarify the contribution of
the 4GalT-I gene in the core 2 O-glycan biosynthesis
on leukocytes, the O-glycan structures of splenocyte membrane
glycoproteins prepared from
4GalT-I+/– and
4GalT-I–/– mice
were analyzed by HPAEC-PAD.
Eight major glycan peaks were detected in samples from the
4GalT-I+/– mice, and 6
major peaks were seen in samples from the
4GalT-I–/– mice
(Figure 2A-B). Considering
their elution positions compared with those of authentic O-glycan
standards from fetuin, sheep erythrocytes, erythrocytes from
4GalT-I–/–
mice,25,26
and their previous structural
analysis,26 we
deduced the structures giving rise to each peak to be those shown in
Figure 2C. Thus, the glycans
with 1,4-galactosylated core 2 branch structures corresponding to peaks
4, 6, and 8 in the
4GalT-I+/– sample were
only faintly detected or not detected in the
4GalT-I–/– sample,
and a glycan with an abnormal core 2 branch that lacked galactose residues in
-1,4 linkage (peak 2) was abundantly detected in the
4GalT-I–/– sample.
In contrast, core 1 O-glycans were equally synthesized in both
mice.
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To further confirm the impaired 1,4-galactosylation of core 2
branches in the
4GalT-I–/– sample,
the O-glycans of splenocyte membrane glycoproteins were mildly
hydrolyzed to remove sialic acids and were analyzed by HPAEC-PAD. The
resultant asialo O-glycans from the
4GalT-I+/– sample gave 2
peaks corresponding to a core 1 disaccharide, Gal13GalNAc-ol
(70%), and a core 2 tetrasaccharide,
Gal13(Gal14GlcNAc16)GalNAc-ol (30%)
(Figure 3A). In contrast, the
similarly treated sample from
4GalT-I–/– mice
was composed of the core 1 disaccharide (72%), a core 2 trisaccharide,
Gal13(GlcNAc16)GalNAc-ol (24%), and the core 2
tetrasaccharide (4.0%) (Figure
3B). The peak area ratio of the core 1 disaccharide was nearly the
same for both mice. However, the core 2 tetrasaccharide was dramatically
reduced for the
4GalT-I–/– mice.
The ratio of core 2 trisaccharide (1,4-ungalactosylated) to the
tetrasaccharide (1,4-galactosylated) revealed that most (86%) core 2
branch lacked galactose residues in -1,4 linkage in the leukocytes of
4GalT-I–/– mice,
as had also been observed in their
erythrocytes.25
These results clearly indicate that 4GalT-I plays a central role in the
formation of the galactosylated core 2 branch, which is a prerequisite for
further elongation of the polylactosamine chains.
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Selectin-ligand formation on 4GalT-I–deficient
neutrophils and monocytes
Selectin-ligand formation on the surface of neutrophils and monocytes from
4GalT-I–/– mice
was analyzed by flow cytometry using a chimeric human P-selectin IgM molecule.
The binding of P-selectin to splenic neutrophils (Gr-1–positive) and
splenic monocytes (F4/80-positive) was markedly reduced in
4GalT-I–/– mice
compared with 4GalT-I+/–
mice (Figure 4). Because PSGL-1
is the predominant ligand for P-selectin on
neutrophils,29 the
expression of PSGL-1 on neutrophils and monocytes was compared between
4GalT-I–/– and
4GalT-I+/– mice and was
found to be the same (Figure
4C). These data, together with the impaired core 2
O-glycan biosynthesis, indicate that the biosynthesis of the
selectinligand oligosaccharide is severely reduced, though not completely
abrogated, in 4GalT-I–deficient neutrophils and monocytes.
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Leukocytosis and neutrophilia of 4GalT-I–deficient
mice
Leukocytosis and neutrophilia are commonly observed in selectin-deficient
mice and selectin ligand-deficient
mice.11,13,14,30
Here, the total white blood cell count in the peripheral blood was elevated
2.2-fold in the
4GalT-I–/– mice
compared with the
4GalT-I+/– mice
(Figure 5A,
Table 1). Neutrophil and
lymphocyte counts were also increased 2.3-and 2.5-fold, respectively
(Figure 5B,
Table 1). Therefore,
4GalT-I–deficient mice also showed leukocytosis and
neutrophilia.
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To clarify whether leukocytosis was caused by abnormal hematopoiesis in the
bone marrow, the colony-forming activity of bone marrow cells was measured.
Colony-forming units (CFUs) in the presence of G-CSF or IL-3 were comparable
between 4GalT-I–/–
and 4GalT-I+/– mice,
suggesting that the hematopoietic differentiation of
4GalT-I–deficient bone marrow cells into the leukocyte lineage was
normal (Figure 5C). In
contrast, CFUs in the presence of EPO were significantly increased in the
4GalT-I–/– mice
(Figure 5C). Red blood cell
counts, hematocrit values, and hemoglobin concentrations in the peripheral
blood were slightly but significantly reduced in the
4GalT-I–/– mice
(Figure 5A). The high response
to EPO might have been attributed to the mild anemia observed in the
4GalT-I–/–
mice.
In addition to peripheral blood leukocytosis, splenomegaly and
extramedullary hematopoiesis in the spleen and liver were often observed in
the 4GalT-I–deficient mice
(Figure 5D and data not shown).
Microscopic observation of spleen and liver sections showed extramedullary
hematopoiesis, including the presence of erythroid precursors, granulocytes,
and megakaryocytes, in the
4GalT-I–/– mice
(data not shown).
Normal lymphocyte homing to peripheral lymph nodes
Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by
L-selectin on lymphocytes and L-selectin ligands, such as 6-sulfo
sLex on the HEVs of PLNs.
4GalT-I–/– mice
had cervical, axillary, and mesenteric LNs that were comparable in weight to
those of 4GalT-I+/– mice
(Figure 5D). In addition, an in
vivo lymphocyte homing assay using 5-chloromethylfluorescein diacetate
(CMFDA)–labeled wild-type
lymphocytes13
demonstrated that lymphocyte trafficking to PLNs was normal in
4GalT-I–/– mice
(data not shown). These results suggest that lymphocyte homing to PLNs was not
affected in the 4GalT-I–deficient mice.
Acute inflammation of 4GalT-I–deficient mice
Cell adhesion through selectins and their ligands plays an important role
in acute and chronic inflammation. Zymosan-induced dermatitis is a model in
which acute inflammation is mediated by E- and
P-selectins.27
Zymosan-induced ear swelling was significantly, but not completely, suppressed
in 4GalT-I–/– mice
compared with 4GalT-I+/–
mice (Figure 6A). Neutrophil
trafficking into the inflamed earlobe as measured by MPO activity was not
induced by the zymosan treatment in
4GalT-I–/– mice,
whereas in 4GalT-I+/–
mice it was induced 2-fold (Figure
6B). Therefore, acute inflammation induced by zymosan was
significantly suppressed in the 4GalT-I–deficient mice, probably
because of a defect in neutrophil trafficking.
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CHS and DTH responses of 4GalT-I–deficient mice
CHS is a typical chronic cutaneous inflammation in which selectins are
known to be
involved.31 TNCB
was applied epidermally onto the earlobe 5 days after sensitization by the
epicutaneous application of TNCB onto the abdomen. The increase in ear
swelling 24 hours after the second challenge was significantly, but not
completely, suppressed in
4GalT-I–/– mice
compared with 4GalT-I+/–
mice (Figure 7A). In addition,
neutrophil trafficking as measured by MPO activity after the sensitization was
also reduced in the
4GalT-I–/– mice
(Figure 7B).
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Although CHS was once thought to be a prototypic DTH response, recent
reports indicate that CHS and DTH are mediated by major histocompatibility
complex (MHC) class 1-restricted CD8+ T cells and MHC class
2-restricted CD4+ T cells,
respectively.32 The
mBSA-induced DTH responses were compared between the
4GalT-I–deficient and control mice. An increase in footpad
thickness induced by the epicutaneous application of mBSA after sensitization
was significantly, but not completely, suppressed in
4GalT-I–/– mice
compared with 4GalT-I+/–
mice (Figure 8A). Leukocyte
infiltration into the dermis of the footpad was induced by the mBSA treatment
in 4GalT-I+/– mice, but
it was suppressed in
4GalT-I–/– mice
(Figure 8B). Therefore, chronic
inflammation such as CHS and DTH was also suppressed in the
4GalT-I–deficient mice.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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4GalT-I–deficient mice
4GalT-I was thought to be mainly involved in the biosynthesis of
Gal14GlcNAc units in N-glycans and in lactose synthesis
through binding with -lactalbumin in the mammary
gland.33 Our
previous and present studies demonstrated that 4GalT-I is also
responsible for the biosynthesis of core 2 O-glycans, given that core
2 O-glycan biosynthesis was reduced by more than 80% in the
erythrocytes25 and
leukocytes of 4GalT-I–deficient mice. Given that selectin ligands
such as sLex and 6-sulfo sLex are mainly expressed on
the core 2 branch of
O-glycans,3
selectin ligand expression would be expected to be reduced in
4GalT-I–deficient mice. As shown in
Figure 4, the binding of
4GalT-I–deficient neutrophils and monocytes to P-selectin was
reduced in these mice, though PSGL-1 expression was not affected. Moreover,
4GalT-I–deficient mice showed selectin-deficient phenotypes,
including peripheral leukocytosis and impaired acute and chronic inflammatory
responses. These results clearly indicate that the biosynthesis of selectin
ligands is greatly impaired in 4GalT-I–deficient mice.
Seven 4GalT genes have been isolated, and 6 of them are
candidates for the biosynthesis of O-glycans and selectin
ligands.23 Using
purified 4GalT enzymes and synthetic oligosaccharide acceptors, Ujita et
al demonstrated that 4GalT-IV synthesized the core 2 branch most
efficiently, whereas 4GalT-I was involved in the biosynthesis of the
core 4 branch in O-glycans
biosynthesis.34,35
These findings are not consistent with our results. The N-acetyl
lactosamine extension activity in core 2 and core 4 O-glycans was
compared among the 4GalTs using various synthetic acceptors in their in
vitro experiments. However, the in vivo situation could be different from that
of the in vitro study, which used large amounts of purified 4GalTs and
synthetic oligosaccharide acceptors instead of glycoproteins. Because the
expression level of each 4GalT gene differs according to cell
type, the biosynthesis of the core 2 branch of O-glycans could be
carried out by different 4GalT genes in different cell types.
We examined the biosynthesis of core 2 O-glycans in leukocytes, where
selectin ligands are synthesized. Therefore, 4GalT-I definitely
contributes to and plays a major role in the biosynthesis of the core 2 branch
of O-glycans and selectin ligands in living leukocytes.
Given that approximately 20% of the core 2 branches were galactosylated by
a -1,4 linkage and that P-selectin bound weakly to neutrophils and
monocytes in 4GalT-I–deficient mice, other 4GalT
gene(s), such as 4GalT-IV, must be involved in the biosynthesis
of the core 2 branch of O-glycans and selectin ligands. Recently, it
has been reported that 4GalT-IV is involved in the biosynthesis of
6-sulfo sLex, an L-selectin
ligand.36 To
identify the other 4GalT gene or genes responsible for
selectin-ligand biosynthesis, mice deficient in those genes will have to be
analyzed.
Peripheral leukocytosis and normal lymphocyte homing to PLNs in
4GalT-I–deficient mice
4GalT-I–deficient mice exhibited peripheral leukocytosis and
neutrophilia similar to those seen in selectin- and
selectin-ligand–deficient mice, strongly suggesting that
4GalT-I–deficient mice are impaired in selectin-dependent
endothelial cell adhesion. A comparison of the 4GalT-I–deficient
mice with selectin-deficient mice and other glycosyltransferase-deficient mice
is shown in Table 1. E- and
P-selectin double-deficient mice exhibit severe leukocytosis and
neutrophilia,30
whereas P-selectin single-deficient mice show mild neutrophilia without
leukocytosis37 and
L-selectin and E-selectin single-deficient mice show normal
hematology.38,39
Fuc-TIV/VII double-deficient mice also show severe leukocytosis and
neutrophilia,14 but
those in core 2 6GnT-deficient mice are
milder.11 The
leukocytosis and neutrophilia of 4GalT-I–deficient mice were
intermediate, comparable to those of core 2 6GnT-deficient mice, which
show a complete deficiency of core 2 O-glycans and a partial
deficiency of selectin
ligands.11
4GalT-I–deficient mice also showed considerable deficiency in core
2 O-glycans biosynthesis and a partial deficiency of selectin
ligands. Therefore, deficiency in the biosynthesis of core 2
O-glycans causes moderate leukocytosis and neutrophilia.
Normal hematopoietic activity of the bone marrow cells
(Figure 5C) and extramedullary
hematopoiesis in the spleen and liver (data not shown) suggest that leukocyte
production was increased in the periphery in 4GalT-I–deficient
mice, though increased hematopoiesis in the bone marrow cannot be ruled out
completely. Subclinical infection will induce cytokine production and cause
extramedullary hematopoiesis, similar to those observed in E- and P-selectin
double-deficient
mice.30,40
Our 4GalT-I–deficient mice, however, were kept under specific
pathogen-free (SPF) conditions, and no pathologic features, including the
ulcerative cutaneous infection seen in the E- and P-selectin double-deficient
mice, were observed. Therefore, the basal expression of E- and P-selectins and
their oligosaccharide ligands is indispensable for the regulated production of
leukocytes in the periphery, independent of infection. Altered leukocyte
homeostasis might be caused by several mechanisms, as discussed
previously.30 One
possibility is that tissue leukocytes are reduced because of a defect in
selectin-dependent endothelial cell adhesion, causing an elevation of
leukocytes in the blood. Another possibility is that neutrophilia may be
caused by an increase in neutrophil half-life in the circulation, as reported
in P-selectin–deficient
mice.41 Homeister
et al14 reported
that extreme leukocytosis in Fuc-TIV/VII double-deficient mice is caused by
decreased neutrophil turnover and increased neutrophil production in the
periphery. Because epithelial cell proliferation is enhanced in
4GalT-I–deficient
mice,20 leukocyte
proliferation might be augmented.
Lymphocyte homing to PLNs is mediated by L-selectin on lymphocytes and
L-selectin ligands on the HEVs of PLNs. Indeed, lymphocyte homing is impaired
in L-selectin–deficient
mice39 and in
Fuc-TVII–deficient
mice.13 However,
the weight of the PLNs and the in vivo lymphocyte homing activity were
comparable between 4GalT-I–deficient and control mice
(Figure 5D and data not shown).
These results suggest that lymphocyte homing to PLNs was not affected in
4GalT-I–deficient mice, as is also observed in core 2
6GnT-deficient
mice.11 Taken
together, these observations indicate that core 2 O-glycans are
dispensable for the biosynthesis of L-selectin ligands.
Reduced acute and chronic inflammatory responses in
4GalT-I–deficient mice
Cell adhesion through selectins and their oligosaccharide ligands is a
prerequisite for leukocyte accumulation at inflammatory sites. Zymosan-induced
inflammation is a model in which acute inflammation is mainly mediated by
neutrophils. Zymosan-induced neutrophil recruitment is reduced in E- and
P-selectin double-deficient
mice27 and fully
compromised in Fuc-TIV/VII double-deficient
mice.14 These
results indicate that E- and P-selectins and sLex play essential
roles in zymosan-induced inflammation. In 4GalT-I–deficient mice,
neutrophil recruitment was fully reduced, and ear swelling was partially
reduced, suggesting that the selectin ligands on neutrophils were greatly
compromised in 4GalT-I–deficient mice.
CHS and DTH responses are commonly used as a model of chronic inflammatory diseases.32 These responses consist of the initial sensitizing phase and the subsequent elicitation phase. A sensitizing allergen such as TNCB or mBSA applied epidermally is captured by Langerhans cells in the skin. Langerhans cells then migrate to draining lymph nodes and present the allergen to naive T cells in the sensitizing phase. The elicitation phase occurs when epidermal cells encounter the same allergen to which they have been previously exposed. The primed CD8+ Tc1 cells and CD4+ Th1 cells mount CHS and DTH responses, respectively, at the site of challenge and secrete cytokines and chemokines. These cytokines and chemokines recr
-1,4-galactosyltransferase-I–deficient mice
;n = 6) and
; n = 6) at 2 to 3 months of age were measured by an automatic cell
counter. (B) Numbers of neutrophils, lymphocytes, and monocytes from
