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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-04-1007.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Cellular and Molecular Medicine,
Glycobiology Research and Training Center, and Howard Hughes Medical
Institute, University of California, San Diego; Cardiovascular Research
Center and Department of Biomedical Engineering, University of Virginia
Health Sciences Center, Charlottesville, VA; Department of Biomedical
Engineering and Microbiology-Immunology, Northwestern University
Medical School, Chicago, IL.
Selectin ligands are glycan structures that participate in
leukocyte trafficking and inflammation. At least 6 ST3Gal
sialyltransferases (I-VI) have been identified that may contribute to
selectin ligand formation. However, it is not known which of these
sialyltransferases are involved in vivo and whether they may
differentially regulate selectin function. We have produced and
characterized mice genetically deficient in ST3Gal-I, ST3Gal-II,
ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in
selectin ligand formation, there was no finding of leukocytosis with
these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was
found to play a role in the synthesis of selectin ligands. In vitro
rolling of marrow-derived neutrophils on E- or P-selectins presented by
Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV.
However, in a tumor necrosis factor Leukocytes in the bloodstream use a carbohydrate
adhesion system involving the selectins for cell tethering and rolling
on the vascular endothelium (reviewed in Lowe1 and
Springer2). This lectin-ligand system is essential for the
subsequent transmigration of adherent cells through the endothelium,
thereby contributing to the development and function of leukocytes at
secondary lymphoid organs and sites of inflammation. The expression
pattern of the 3 selectin molecules E, L, and P reflects the
physiologic role that each plays in vivo. E- and P-selectin are induced
on the vascular endothelium during inflammation, whereas P-selectin is also found on platelets, and L-selectin is expressed on the surface of
leukocytes. Absence of these molecules, singly or in combination, yields defects to varying degrees in leukocyte homeostasis,
trafficking, and innate immune responses during
inflammation.3-7
The carbohydrate (glycan) ligands of the selectins are less well
defined. The prototypical selectin ligand structure is a terminal
tetrasaccharide (Sia
Six genes have been identified in the mammalian genome that encode
Golgi-resident sialyltransferases that form Sialyltransferase function may also be influenced by competition in the
Golgi with other glycosyltransferases that operate in concurrent
biosynthetic and branching steps, potentially affecting the formation
of downstream terminal branch structures. For example, it is known that
ST3Gal-I effectively competes with C2GlcNAcT-I for the same substrate,
and thereby reduces core 2 O-glycan branch formation in
vivo.19 Such characterization of genetically altered mice
has revealed the essential and modulatory role of specific glycosyltransferases and their glycan products in leukocyte-endothelial recognition by the selectins.20 We have furthered this
line of investigation by producing mice deficient in
ST3Gal-II and -III genes using Cre-loxP
mutagenesis and comparing selectin ligand formation among these mice as
well as those already available lacking ST3Gal-I and
ST3Gal-IV.19,21 Our findings show that only ST3Gal-IV of
the 4 analyzed plays a substantial role in selectin ligand formation in vivo.
Tissue Northern
Gene targeting and mutant mouse production
Hematology Blood was collected from the tail vein of anesthetized mice into EDTA (ethylenediaminetetraacetic acid) microtubes (Becton Dickinson, Mountain View, CA) and analyzed with a CELL-DYN 3500 calibrated with normal mouse blood. Differential blood counts were also performed on Wright-Giemsa-stained blood smears.Isolation of peripheral blood leukocytes and flow cytometry Blood from wild-type or mutant mice was collected as above into lithium heparin microtubes (Becton Dickinson) and diluted 1:1 in phosphate-buffered saline (PBS). An equal volume of 2% dextran T500 (Pharmacia, Uppsala, Sweden) in PBS was added, and the cells were incubated at 37°C for 10 minutes. The upper layer containing peripheral blood leukocytes (PBLs) was removed and washed in PBS. Red blood cells were lysed with PharM lyse (Pharmingen, San Diego, CA), and PBLs were resuspended in PBS containing 0.1% bovine serum albumin (PBS/BSA). Cells were incubated with 0.5 µg/mL Fc block (Pharmingen) prior to antibody, immunoglobulin M (IgM)-chimera, and lectin staining. Ricinus communis agglutinin-I (RCA-I), Erythrina crystagalli lectin (ECA), or Peanut agglutinin (PNA; all from Vector Laboratories, Burlingame, CA) were incubated with PBLs in combination with antibody Gr1 (Pharmingen) for 10 minutes prior to analysis by flow cytometry on a FACScalibur (Becton Dickinson). Mouse P- and E-selectin cDNAs were linked to the CH2, CH3, and CH4 domains of human IgM to construct P- and E-selectin IgM chimeras.9 Supernatants from transfected COS cells were diluted 1:20 for P-selectin IgM or 1:30 for E-selectin IgM chimeras in PBS/BSA. An antihuman IgM fluorescein isothiocyanate (FITC) antibody (Sigma Chemical, St Louis, MO) was added at 1:1000 for 15 minutes, and labeled selectin chimeras were added to PBLs in the presence of Gr1 for 10 minutes prior to flow cytometry. In other experiments, PBLs from wild-type littermates were treated with Arthrobacter ureafaciens neuraminidase (Sigma) in 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 140 mM NaCl, pH 7.0, for 1 hour at 37°C prior to incubation with selectin chimeras.In vitro rolling assay Monolayers of Chinese hamster ovary (CHO) cells stably transfected with either human P- or E-selectin24 served as the rolling substrate in a parallel plate flow chamber (Glycotech, Rockville, MD). Bone marrow neutrophils, prepared as previously described,24 were introduced into the flow chamber at a concentration of 1 × 106 cells/mL. Wall shear stress was maintained at 1.5 dynes/cm2, and images were obtained with a Nikon Eclipse TE300 inverted microscope (Nikon, Melville, NY). Rolling events, defined as a rolling cell that can be tracked between sequential images separated by a time delay of 2 seconds, were measured and analyzed as described.25Antibodies and cytokines The P-selectin monoclonal antibody (mAb) RB40.34 (rat IgG1, 30 µg/mouse)26 was used to block P-selectin-dependent leukocyte adhesion and rolling in vivo. The rat antimouse E-selectin mAb 9A9 (rat IgG1, 30 µg/mouse)27 was used to block E-selectin function in vitro and E-selectin-dependent rolling in vivo. For the in vivo model, recombinant murine tumor necrosis factor (TNF-) (500 ng/mouse; R&D, Minneapolis, MN) was diluted in 0.3 mL normal saline and
injected intrascrotally 2 hour prior to the experiment.
Intravital microscopy and cremaster muscle preparation. Mice were anesthetized with an intraperitoneal injection of ketamine (125 mg/g body weight, Ketalar; Parke-Davis, Morris Plains, NJ), xylazine (12.5 mg/g body weight; Phoenix Scientific, St Joseph, MO), and atropine sulfate (0.025 mg/g body weight; Elkins-Sinn, Cherry Hill, NJ). Mice were then placed on a heating pad to maintain body temperature. Intravital microscopy experiments were conducted with a microscope (Axioskop; Zeiss, Thornwood, NY) equipped with a saline immersion objective (SW 40/0.75 numerical aperture) and connected to a charged-coupled device (CCD) camera (model VE-1000CD; Dage-MTI, Michigan City, IN) and a video recorder (Panasonic, Secausus, NJ). After tracheal intubation, the left carotid artery was cannulated for systemic administration of anesthetics and mAbs and for taking blood samples during the experiment.The cremaster muscle was prepared as described
earlier28 and superfused with thermocontrolled (35°C)
bicarbonate-buffered saline. Systemic blood samples (10 µL) were
taken after each mAb injection and stained with Kimura to assess
systemic white blood cell counts. Leukocyte rolling was observed in
venules with diameters ranging from 20 µm to 45 µm. Microvessel
diameters, lengths, and rolling leukocyte velocities were measured by
using a digital image processing system.29,30 The number
of rolling cells was counted in each 100-µm segment of postcapillary
venules. Centerline blood flow velocity was measured by using a dual
photodiode and a digital online cross-correlation program (Circusoft
Instrumentation, Hockessin, DE) and converted to mean blood flow
velocity by multiplying with an empirical factor of
0.625.31 Wall shear rates ( Statistics Statistical analysis was performed by using Sigma-Stat 2.0 software package (SPSS Science, Chicago, IL). Average vessel diameter, leukocyte rolling, leukocyte rolling velocities, and wall shear rates between groups and treatments were compared with the one-way analysis of variance (ANOVA) on ranks (Kruskal-Wallis) with a multiple pairwise comparison test (Dunn test). Leukocyte rolling between untreated and antibody-treated groups was compared with Student t test or by the Wilcoxon rank-sum test as appropriate. Statistical significance was set at P < .05, indicated by asterisk (*).
Sialyltransferase tissue distribution and targeted gene disruption The ST3Gal family of sialyltransferases appears to consist of a total of 6 genes in mammals. All encode type II transmembrane proteins residing in the Golgi apparatus and bearing a common sialylmotif that is essential for donor substrate binding and catalytic activity.32 ST3Gal-I to -IV RNA expression is broadly distributed with variations in levels observed among distinct tissues (Figure 1B). Multiple RNA transcripts are noted in some cases, as has been described.12 Although the patterns of RNA expression were different for each ST3Gal gene studied, all tissues surveyed expressed multiple ST3Gal sialyltransferases.The production and initial characterization of ST3Gal-I- and
ST3Gal-IV-mutant mice has been described.19,21 Both are
fertile and without overt developmental and morphologic abnormalities. Herein, we have similarly generated mice lacking functional ST3Gal-II and -III sialyltransferases by Cre-loxP gene targeting to
produce deletions of either the large sialylmotif or transmembrane
domain, respectively (Figure 2A,B,E,F).
The loxP-flanked and deleted alleles of each gene were confirmed by
Southern blot analysis of embryonic stem cell DNA (Figure 2C,G).
Correctly targeted embryonic stem (ES) cells were used to
generate mutant mice bearing the deleted allelic structures (Figure
2D,H), as previously described.22 Heterozygous
ST3Gal-IIwt/
Sialyltransferase mutations result in increased  -linked galactose
residues present among various glycan classes. Lectins that bind
-linked galactose were used to assess the loss of ST3Gal function
among myeloid and lymphoid cell types. These studies revealed
differential increases in the exposure of -linked galactose on
specific peripheral blood leukocytes among all 4 homozygous mutant
genotypes (Figure 3A). Binding of the
RCA-I lectin, which has a preference for unsialylated terminal
galactose on type II and type III glycans,33 was increased
on the surface of neutrophils among mice homozygous for deletions in
the genes encoding ST3Gal-I, -II and, -IV. Increased binding to ECA
lectin, which is specific for unsialylated type II
chains,34 occurred to a substantial extent only among
cells homozygous for the ST3Gal-IV deletion. PNA lectin binding, which
primarily discriminates between sialylated and unsialylated type III
glycans,35 revealed an increase in unsialylated
Gal1-3GalNAc- among neutrophils from mice homozygous for deletions
in ST3Gal-I, ST3Gal-II, and ST3Gal-IV and CD8+ T cells from
mice homozygous for deletions in ST3Gal-I and ST3Gal-IV. PNA can also
recognize unsialylated type I chains to some extent,36 and
the increase in binding to CD8+ T cells from mice
homozygous for the ST3Gal-III deletion may reflect this additional
binding specificity. Neutrophils from ST3Gal-III-mutant mice did not
show any binding changes with the use of RCA-I, ECA, or PNA lectins,
suggesting the possibility that ST3Gal-III is not expressed in
Gr1+ cells, or perhaps that other sialyltransferases may
fully compensate for ST3Gal-III deficiency. In addition to providing
data on the cell types in which these ST3Gal sialyltransferases operate
in vivo, the results obtained reflect closely the described substrate specificity and preferences from in vitro enzymatic studies that have
been described.12
Hematologic findings in ST3Gal deficiencies Hematologic analyses of all 4 mutations bred to homozygosity revealed erythroid profiles within normal limits, as compared with wild type littermates (Table 1). Although the numbers of lymphocytes in circulation were also normal, we noted a consistent increase in circulating monocytes and a likely decrease in eosinophils in ST3Gal-I-deficient mice. However, none of the ST3Gal deficiencies resulted in leukocytosis. Interestingly, both ST3Gal-I and ST3Gal-IV deficiencies resulted in thrombocytopenia. In the absence of ST3Gal-IV, exposure of galactose occurs in a manner among some plasma constituents that triggers asialoglycoprotein receptor (ASGPR) clearance mechanisms and thereby reduces levels of von Willebrand factor and platelets in circulation.21
P- and E-selectin ligand deficiency on Gr1+ leukocytes from ST3Gal-IV-mutant mice The binding of selectin chimera immunoglobulin (IgM) Fc fusion proteins was used in flow cytometric analyses as a measure of selectin ligand levels on the cell surface.9 When compared with Gr1+ cells, which are primarily mature neutrophils, from wild-type littermates, P-selectin immunoglobulin chimera binding was
reduced by approximately 50% among ST3Gal-IV-deficient samples with
mean peak fluorescence intensity measurements (MFIs) of 207 and 97, respectively (Figure 3B). A slightly greater reduction in E-selectin binding to approximately 40% of control levels was found (MFI, 96 and
37, respectively). However, neither of these reductions in E- and
P-selectin ligand levels were as great as those observed in the absence
of the glycosyltransferase C2GlcNAcT-I11 (Figure 3B).
Interestingly, a slight but reproducible increase of approximately 20%
in P-selectin chimera binding was observed in ST3Gal-I-deficient mice.
In contrast to these changes, no alterations in selectin ligand levels
were observed among Gr1+ cells from ST3Gal-II- and
ST3Gal-III-deficient mice.
Multiple sialyltransferases in E- and P-selectin ligand formation The possibility that only ST3Gal-IV contributes to E- and P-selectin ligand formation was investigated in vitro by using neuraminidase (sialidase) treatment of intact cells, followed by flow cytometry as described above. Sialidase treatment of ST3Gal-IV-deficient Gr1+ cells further reduced P- and E-selectin-immunoglobulin chimera binding, suggesting that other sialyltransferases, in addition to ST3Gal-IV, are also involved in E- and P-selectin ligand formation (Figure 4).
Reduced rolling of ST3Gal-IV  / neutrophils, we used a parallel plate
attachment and rolling assay, as previously described in studies of
mice with C2GlcNAcT-I deficiency.24 Transfected CHO cells
used as a monolayer in this assay express E-selectin at levels
approximating those found on human umbilical vein endothelial cells
activated by TNF, whereas P-selectin-expressing CHO cells express
levels 2 to 3 times above that.24 The assay was carried
out in a flow chamber with a wall shear stress maintained at 1.5 dynes/cm2 as previously described.25
Consistent with a partial effect on P-selectin ligands measured by flow
cytometry, bone marrow-derived ST3Gal-IV-deficient neutrophils showed
a 50% ± 16% reduction in rolling on P-selectin expressed by CHO
cells (Figure 5A).
ST3Gal-IV/ neutrophils showed a 22% ± 9%
reduction in rolling on E-selectin in this assay (Figure 5B), also
consistent with the flow cytometry assay.
Leukocyte rolling in vivo is impaired in ST3Gal-IV-deficient mice Intrascrotal injection of TNF- leads to the expression of
E-selectin and enhances the expression of P-selectin on venular endothelial cells of the cremaster muscle.37 To compare in
vivo rolling with the above results from the flow chamber and cytometry experiments, we analyzed leukocyte rolling in the TNF--pretreated cremaster muscle. We studied leukocyte rolling in 23 venules of 8 TNF--treated mice deficient in ST3Gal-IV and compared the results with rolling in 19 venules of 5 littermate control mice. Hemodynamic and microvascular parameters for both groups indicate similar vessel
diameters, centerline velocities, and wall shear rates (Table
2). The results indicated a reduction in
the number of rolling cells per 100-µm vessel length in
ST3Gal-IV-deficient mice compared with wild-type littermates when
treated with the blocking P-selectin mAb RB40.34 (Figure
6A). The reduction involving E-selectin-mediated rolling in ST3Gal-IV/ mice was
consistent with the reduction in E-selectin-mediated rolling of
ST3Gal-IV/ leukocytes in the flow chamber (Figure 5).
In contrast, blocking E-selectin with mAb 9A9 in vivo, which leads to
P-selectin-mediated rolling, revealed a similar number of rolling
cells per vessel length in both groups (Figure 6A).
Leukocyte rolling velocities in TNF-
Recognition of selectin ligands by leukocytes and endothelial cells of the vasculature forms the basis for a substantial component of the inflammatory response and contributes to leukocyte homeostasis. Although single genes exist to produce each of the selectins, the formation of selectin ligands requires the orchestrated action of many distinct glycosyltransferase genes that encode enzymes operating in the secretory pathway, primarily within the Golgi apparatus. Changes in the normal expression profile of glycosyltransferases in various cell types can alter glycan branching and influence terminal modifications in the Golgi, thereby providing multiple regulatory points in selectin ligand formation that can act to partition the physiologic activities of selectins.11,38 Among the 19 sialyltransferase genes found in the mammalian genome to date, 6 are ST3Gal sialyltransferases that may operate either singly or in combination in producing selectin ligands.12,39-41 We have rendered mice deficient in 4 of these 6 candidates: ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV, and have analyzed the relative contribution of each to selectin ligand formation among neutrophils. All ST3Gal deficiencies studied result in exposure of galactose termini; however, each ST3Gal operated to a different degree among leukocyte cell types studied. None of the ST3Gal deficiencies caused leukocytosis, which is a phenotype found in glycosyltransferase-mutant mice with severe deficiencies of selectin ligands.9,11,42 We have noted a concurrence of our in vivo findings with data acquired
from in vitro enzymatic analyses that proposes ST3Gal-I predominantly
sialylates type III glycan chain termini (Gal ST3Gal-II deficiency results in increased PNA binding to peripheral blood neutrophils, as well as increased binding of RCA and to a lessor extent ECA. Unlike ST3Gal-I, no change in PNA binding to CD8+ T cells was observed. ST3Gal-II prefers glycolipid substrates, and, although studies have indicated that selectins can recognize glycolipids,44 no evidence of a deficiency in selectin ligands on neutrophils was found in the absence of ST3Gal-II. These findings do not preclude the possibility that glycolipids bear selectin ligands, and perhaps in some cases among tumor cells.45 ST3Gal-III levels have been associated with the formation of
sLex in lung carcinoma.16 However, ST3Gal-III
prefers to sialylate type I (Gal ST3Gal-IV deficiency was unique among the 4 ST3Gal
sialyltransferase mutations studied by substantially reducing the
formation of selectin ligands on circulating neutrophils. However, this reduction was only partial when compared with C2GlcNAcT-I-deficient neutrophils. In addition, a further reduction in selectin binding to
ST3Gal-IV-deficient neutrophils was noted following neuraminidase treatment in vitro. This finding indicates the likelihood that other
sialyltransferases are involved in selectin ligand formation in vivo.
It is also possible, although perhaps unlikely, that this finding
reflects conformational alterations in glycoproteins occurring on
de-sialylation that alter E- and P-selectin-IgM chimera binding
independent of the role of We have investigated the role of ST3Gal sialyltransferases in selectin ligand formation by first applying flow cytometry as a screen to detect changes in selectin ligand expression levels. This screen has been found to be a valuable initial approach as flow cytometric findings of decreased selectin ligands are found associated with defects in neutrophil rolling in vitro on synthetic and cell-based selectin substrates.9,11 However, neutrophil rolling and recruitment in vitro and in vivo can provide functional data regarding alterations observed in selectin ligand expression. We observed a substantial decrement in rolling on cell monolayers bearing P-selectin, as well as E-selectin, using bone marrow-derived neutrophils. These findings are similar in scope to the flow cytometric results and indicate a functional role for ST3Gal-IV in selectin ligand formation. The number of neutrophils rolling per length of inflamed vascular
endothelium following TNF- The initial step of leukocyte tethering to the endothelium during
inflammation is largely dependent on P-selectin interactions. No effect
of ST3Gal-IV deficiency was found on E- or P-selectin ligands in this
process. Because PSGL-1 is the major ligand for P-selectin and in vivo
rolling is markedly reduced in PSGL-1-deficient mice,48
these data suggest that ST3Gal-IV does not contribute to functional
selectin ligands on PSGL-1 in vivo. In contrast, we found that
E-selectin-dependent leukocyte rolling velocity was increased in
ST3Gal-IV Other sialyltransferase mutations not yet produced or examined may also be informative in resolving the degree of contribution to selectin function by sialic acid linkages. Of the 6 ST3Gal sialyltransferases identified thus far, and the 2 remaining to be analyzed in this manner for selectin ligand formation, ST3Gal-V bears a strong glycolipid substrate preference, like ST3Gal-II, but specifically generates the ganglioside GM3.13 In contrast, ST3Gal-VI is similar to ST3Gal-IV with specificity for type II glycan chains.14 We would therefore hypothesize that ST3Gal-VI will be found to play a substantial role in selectin ligand formation in vivo. Selectin expression and selectin ligand formation provide multiple points of regulation pertaining to cell type communication during leukocyte homeostasis and innate immune responses. Distinct physiologic outcomes emerge from the characterization of mice inheriting genetic deficiencies of various selectins and glycosyltransferases operating in selectin ligand formation, including FucT-VII, C2GlcNAcT-I, and FucT-IV.5,9-11,25,48-50 We have herein provided evidence of a functional segregation involving ST3Gal sialyltransferase activity in the formation of selectin ligands in vivo. Among ST3Gal-I, -II, -III, and -IV sialyltransferases, only ST3Gal-IV provides a substantial degree of selectin ligand formation in vivo. Our data suggest that ST3Gal-IV contributes to the characteristic slow rolling velocity observed for E-selectin-mediated rolling during inflammation without substantially affecting E-selectin-mediated capturing of leukocytes. These findings reveal a substantial degree of specificity among ST3Gal sialyltransferases in vivo in the formation of selectin ligands on neutrophils.
We thank John Lowe for providing the selectin chimera supernatants, Dietmar Vestweber for providing monoclonal antibody RB40.34, and Barry Wolitzky for providing monoclonal antibody 9A9.
Submitted April 1, 2002; accepted June 2, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2002-04-1007.
Supported by the National Institutes of Health program project grant PO1-HL57345 (J.D.M.), HL58710 (G.S.K.), HL54136 (K.L.), and F32CA79130 (L.G.E.). M. S. is supported by a stipend from the German Research Foundation (DFG) SP 621/1-1. J.D.M. acknowledges support as an Investigator of the Howard Hughes Medical Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jamey D. Marth, Howard Hughes Medical Institute, 9500 Gilman Dr 0625, CMM-W Building, Room 333, University of California San Diego, La Jolla, CA 92093; e-mail: jmarth{at}ucsd.edu.
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Top
Abstract
Introduction
allele, LE-110
(5'-CCAGCCAGCAGAGGATCTGATAC) and LE-115
(5'-CGCAGGGGGCGTTTCTAGAC) to detect the 450-bp ST3Gal-III wild type
allele, and LE-110 and rlox to detect the 300-bp ST3Gal-III 
w) were estimated as 2.12 (8 vb/d), where vb is the
mean blood flow velocity, d is the diameter of the vessel, and 2.12 is
a median empirical correction factor obtained from velocity profiles
measured in microvessels in vivo.
) and control mice (
)
treated with either P-selectin blocking mAb RB40.34 or E-selectin
blocking mAb 9A9. Data are presented as the mean ± SEM. *
indicates significant difference; P < .05. (B-D) Cumulative
velocity distribution for ST3Gal-IV