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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, IL, and the Howard
Hughes Medical Institute and Department of Cellular and Molecular
Medicine, University of California San Diego, La Jolla, CA.
Selectins are carbohydrate-binding adhesion molecules that play
important roles in control of leukocyte traffic. Glycosyltransferases involved in selectin ligand biosynthesis include the
Selectins are a family of carbohydrate-binding
adhesion molecules that mediate the earliest steps of leukocyte
interaction with the vessel wall.1 Although the
carbohydrate ligands for selectins have not been definitively
characterized at the structural level, some of the glycosyltransferases
responsible for selectin ligand biosynthesis have been identified.
Among these is FucT-VII, an More recently, mice deficient in C2GlcNAcT-I
(C2GlcNAcT-I We have therefore analyzed C2GlcNAcT-I Mice
Cell lines and transfectants
Isolation of neutrophils and generation of Th1 cells Neutrophils were isolated from bone marrow of both C2GlcNAcT-I / and WT mice by double discontinuous
gradients (density of 1.083 and 1.119 g/L; Sigma Chemical, St Louis,
MO). Cells recovered from the lower interface were more than 80%
Gr-1+ and exhibited high side scatter. For CD4 T-cell
activation, CD4 cells were isolated from the spleens of mice by
magnetic bead technology (Miltenyi, Auburn, CA) according to
the manufacturer's instructions. The 24-well tissue culture plates
were precoated with 1 µg/mL each anti-CD3 and anti-CD28. Purified CD4
cells were incubated at 2 × 106 cells/mL on
antibody-coated plates for about 40 hours in the presence or absence of
interleukin-12 (IL-12) (10 ng/mL, Biosource, Camarillo, CA)
plus anti-IL-4 (10 µg/mL 11B11, from American Tissue Culture
Collection, prepared by standard methods), harvested, and replated on
fresh plates with 20 ng/mL IL-2 (Biosource) in the continued presence
or absence of IL-12 plus anti-IL-4. Cells were maintained at about
106 cells/mL, and fresh media and cytokines were added
every 2 days. Except where indicated otherwise, cells were analyzed by
FACS and in rolling assays on day 8.
RT-PCR Semiquantitative RT-PCR for human and murine FucT-VII, C2GlcNAcT-I, or dihydrofolate reductase (DHFR) were carried out exactly as previously described.5,6,14 Primers specific for either human or mouse FucT-VII, primers that are identical for both human and mouse C2GlcNAcT-I, and primers that are identical for mouse and human DHFR were used.Parallel plate attachment and rolling assay The interaction of cells with E-selectin and P-selectin was assayed in a parallel plate flow chamber (Glycotech, Rockville, MD). Monolayers of Chinese hamster ovary (CHO) cells stably transfected with either E-selectin (CHO/E) or P-selectin (CHO/P) were grown to confluence in 35-mm tissue culture plates and served as the rolling substrate. CHO/E cells expressed E-selectin at levels similar to tumor necrosis factor (TNF)-activated human umbilical vein endothelial cells (HUVEC), whereas these CHO/P cells expressed P-selectin at levels 2- to 3-fold higher (data not shown). Neutrophils and activated CD4 cells, prepared as described above, at a concentration of 1 × 106 cells/mL, and transfectants and cell lines at a concentration of 0.5 × 106 cells/mL, were introduced into the flow chamber in a buffer of Dulbecco modified Eagle medium (DMEM) supplemented with 0.1% serum. Shear stress in the flow chamber was controlled by a syringe pump (Harvard Apparatus, Holliston, MA), and except for those shown in Figure 6, were maintained constant at 1.5 dynes/cm.2 Images were obtained using a Nikon Eclipse TE300 inverted microscope. Data analysis was performed using Celltrak software developed by Compix (Cranberry Township, PA), as previously described.3 Briefly, a rolling event is defined as a rolling cell that can be tracked between sequential images separated by a defined time delay, here 2 seconds. The total number of rolling events was collected for 50 sequential images. Data are presented as mean ± SD rolling events or rolling velocity from multiple analyses of the indicated cells.FACS Cells were stained with directly conjugated monoclonal antibody (mAb) 1B11 (glycosylation-dependent epitope of CD43; Pharmingen, San Diego, CA), S11 (CD43, kindly supplied by Dr Tom Waldschmidt, University of Iowa, Iowa City), or Gr-1 (neutrophils/Ly6G) (Pharmingen). For one-color staining with E- or P-selectin chimeras,2 cells were stained with purified E- or P-selectin/IgM chimeras2 (E-RIgM or P-RIgM) (kindly supplied by Dr Lloyd Stoolman, University of Michigan, Ann Arbor) followed by Cy5-conjugated, mouse absorbed goat antihuman IgM (Jackson Immunoresearch, West Grove, PA). For 2-color staining of activated T cells, cells were stained with purified E- or P-selectin/IgM chimeras as above, followed by 1B11-fluorescein isothiocyanate (FITC). All staining was carried out in the presence of 2.4G2/Fc block (kindly supplied by Dr Tom Waldschmidt). Analysis was carried out on a FACScalibur (Becton Dickinson, Mountain View, CA) equipped with Cellquest software.
A survey of multiple hematopoietic cell lines revealed only one
that failed to express detectable C2GlcNAcT-I, as assessed by RT-PCR
(data not shown). This cell line, a murine pre-B-cell line called
300.19,13 also fails to express detectable FucT-VII or
FucT-IV. We and others have previously shown that enforced expression
of FucT-VII in any cell line examined confers the ability to attach and
roll on E-selectin.3,6 The 300.19 cells were therefore
stably transfected with human FucT-VII cDNA, and clones were selected
that expressed levels of FucT-VII messenger RNA (mRNA) approximately
equivalent to myeloid cells (Figure 1A
and data not shown). Analysis of representative clones is shown in Figure 1. The 300.19/FucT-VII cells rolled well on E-selectin, with the
number of rolling events approaching that of the myeloid cell line U937
(Figure 1B). Importantly, the rolling velocity of 300.19/FucT-VII cells
was also quite similar to that of U937 cells (Figure 1C). The 300.19 cells stably transfected with C2GlcNAcT-I alone did not exhibit any
interactions with either E-selectin or P-selectin (data not shown).
When 300.19/FucT-VII cells were stably transfected with C2GlcNAcT-I
cDNA, the number of rolling events did not change (Figure 1B), but the
velocity of these rolling cells was lower (Figure 1C). These data
demonstrate that lymphoid cells can attach and roll on E-selectin in
the absence of C2GlcNAcT-I, and that C2GlcNAcT-I can, in the presence
of FucT-VII, create new ligands, modify existing ligands, or
both.
We therefore examined the rolling of neutrophils from
C2GlcNAcT-I
These results, which were obtained in a standard attachment and rolling
assay at 1.5 dynes/cm2, contrast with those obtained by
FACS analysis of the binding of selectin/IgM chimeras.10
We therefore re-examined the binding of E-RIgM and P-RIgM to
neutrophils from C2GlcNAcT-I The results obtained with neutrophils were extended to analysis of
activated T cells. Previous work has shown that Th1 cells, generated by
activation in the presence of IL-12, have high levels of ligands for
both E- and P-selectin, as assessed both by standard rolling assays and
by staining with E-RIgM and P-RIgM.14,15 CD4 cells were
isolated from WT and C2GlcNAcT-I
As an independent approach toward confirming the crucial role of
C2GlcNAcT-I in P-selectin ligands but not in E-selectin ligands in
activated CD4 cells, we first determined whether the 1B11 mAb, which
has been shown to identify C2GlcNAcT-I-dependent epitopes on myeloid
cells,10 also did so in CD4 cells. Th1 cells from WT and
C2GlcNAcT-I
We therefore used 1B11 staining as a reporter of C2GlcNAcT-I activity
in 2-color FACS analysis of selectin ligand expression, measured using
E-RIgM and P-RIgM binding. In contrast to myeloid cells and cell lines,
staining with E-RIgM or P-RIgM on primary T cells accurately predicts
rolling behavior in this assay4,14,15 (and see below).
Two-color FACS analysis of Th1 cells from WT mice with 1B11 versus
P-RIgM showed concordant staining, across the entire range of 1B11 and
P-RIgM staining (Figure 5A). In contrast, 2-color staining of WT Th1 cells with 1B11 versus E-RIgM displayed a
distinct pattern, with a significant
1B11+/E-RIgM
The above data also suggest that the induction of P-selectin ligands in
developing Th1 cells involves up-regulation of C2GlcNAcT-I in
response to IL-12. In studies to be reported elsewhere, we show that
this IL-12-induced up-regulation of C2GlcNAcT-I, but not the
constitutive low level present in naive CD4 cells, is dependent on
expression of Stat4, whereas FucT-VII induction is largely independent
of Stat4 (S.J. White and colleagues, manuscript submitted). If
C2GlcNAcT-I is not required for formation of E-selectin ligands,
independent control of C2GlcNAcT-I and FucT-VII should permit
independent control of expression of ligands for E-selectin or
P-selectin. Consistent with this idea, and with the data above demonstrating a lack of a requirement for C2GlcNAcT-I in E-selectin ligand formation, we have recently shown that normal IgG plasma cells
express high levels of FucT-VII but little or no C2GlcNAcT-I, and roll
well on E-selectin but poorly or not at all on P-selectin (Underhill
GH, Kansas GS. IgG plasma cells display a unique spectrum of leukocyte
adhesion and homing molecules, submitted for publication). The
E-selectin binding/P-selectin nonbinding phenotype of these plasma
cells is strikingly similar to that of the C2GlcNAcT-I Taken together, these results suggest a new model in which the
independent control of expression of ligands for E-selectin versus
P-selectin in activated CD4+ T cells is accomplished
through independent control of glycosyltransferases that are
differentially required for E-selectin ligands versus P-selectin
ligands. FucT-VII is absolutely required for selectin ligand formation
in T cells, but lower levels are needed for maximal P-selectin ligands
than for maximal E-selectin ligands.4 Quantitative control
of FucT-VII levels will therefore contribute to determining whether
activated T cells express ligands for E-selectin, P-selectin, or
both. The data in the present report indicate that down-regulation or
inhibition of C2GlcNAcT-I would allow for expression of E-selectin ligands but not P-selectin ligands. Other enzymes such as members of
the ST3Gal family of The present results appear to differ somewhat from observations
obtained in vivo (see accompanying report by Sperandio and coworkers28). It is possible that a requirement for
C2GlcNAcT-I exists for rolling on E-selectin only at shear stresses
above the 1.5 dynes/cm2 used in the present studies. To
address this possibility, we analyzed the rolling of WT and
C2GlcNAcT-I
Regarding differences between our results and those of Ellies and coworkers,10 several distinct factors likely play a role. The controlled detachment assay used by Ellies and colleagues bypasses the initial attachment step, and consequently allows for significant interactions with E-selectin at levels of shear considerably higher than in our attachment and rolling assay. We also note that coexpression of C2GlcNAcT-I in stably transfected 300.19/FucT-VII cells decreased the rolling velocity of those cells, without increasing the number of rolling cells. Because a slower velocity indicates a stronger interaction, these observations are consistent with the decrement in rolling on E-selectin seen in the controlled detachment assay in the study by Ellies and associates.10 It is also possible that differences in the nature, presentation, or density of the ligand (E-selectin chimera versus native E-selectin) contribute to the observed differences between the present data and those obtained in a controlled detachment assay. In this regard, the previous studies by Ellies and coworkers10 were performed on E-selectin chimera that we estimate to represent significantly lower densities than is expressed on the stably transfected CHO cells used here, even assuming appropriate orientation of the plate-bound protein. Ligand density is well known to affect both the attachment rate and the number and velocity of rolling cells in these types of in vitro assays. Additionally, presentation of E-selectin in the context of the plasma membrane of living cells may be advantageous compared with molecules immobilized to plastic, particularly those immobilized at low density, such as in the study by Ellies and colleagues. Taken together, the differences inherent in the 2 types of assays, along with the lower densities and different presentations of E-selectin, appear to be sufficient to account for the apparent differences between our results and those of Ellies and coworkers.10 The combined results indicate that formation of at least some
E-selectin ligands does not absolutely require C2GlcNAcT-I, but also
that this enzyme can either modify existing ligands or contribute to
the generation of distinct ligands that are specialized to operate
under conditions of limiting ligand or high shear. The present results
do not allow us to distinguish between the modification of existing
ligands and the generation of novel ones, particularly as the molecular
identity of the relevant E-selectin ligands has not been firmly
established. It is possible that those ligands for E-selectin that
exist in C2GlcNAcT-I If C2GlcNAcT-I is not absolutely required for E-selectin ligand
formation, then why do C2GlcNAcT-I In summary, we have used a well-characterized attachment and rolling assay to show that in transfected cell lines, normal neutrophils and primary T cells, expression of C2GlcNAcT-I is essential for generation of ligands for P-selectin, but is not absolutely required for generation of ligands for E-selectin. These results, in combination with the accompanying report,28 clarify previously discrepant results, and identify C2GlcNAcT-I as the first enzyme selectively required for biosynthesis of only a subset of selectin ligands. Independent control of expression of C2GlcNAcT-I and other enzymes required for selectin ligand formation represents a novel mechanism of regulation of leukocyte traffic.
The authors thank Dr Thomas Waldschmidt, University of Iowa, for provision of reagents and helpful discussions.
Submitted November 17, 2000; accepted February 13, 2001.
Supported by National Institutes of Health grants HL55647 (to G.S.K.), DK48247 (to J.D.M.), and F32CA79130 (to L.G.E.), and by grant 003003N from the American Heart Association (to K.R.S.). G.S.K. is an Established Investigator of the American Heart Association.
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: Geoffrey S. Kansas, Department of Microbiology-Immunology, Northwestern University Medical School, 303 E Chicago Ave, Chicago, IL 60611; e-mail: gsk{at}northwestern.edu.
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C. E. Green, D. N. Pearson, R. T. Camphausen, D. E. Staunton, and S. I. Simon Shear-Dependent Capping of L-Selectin and P-Selectin Glycoprotein Ligand 1 by E-Selectin Signals Activation of High-Avidity {beta}2-Integrin on Neutrophils J. Immunol., June 15, 2004; 172(12): 7780 - 7790. [Abstract] [Full Text] [PDF]
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M. J. Smith, B. R. E. Smith, M. B. Lawrence, and K. R. Snapp Functional Analysis of the Combined Role of the O-Linked Branching Enzyme Core 2 {beta}1-6-N-Glucosaminyltransferase and Dimerization of P-selectin Glycoprotein Ligand-1 in Rolling on P-selectin J. Biol. Chem., May 21, 2004; 279(21): 21984 - 21991. [Abstract] [Full Text] [PDF]
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C. J. Dimitroff, R. J. Bernacki, and R. Sackstein Glycosylation-dependent inhibition of cutaneous lymphocyte-associated antigen expression: implications in modulating lymphocyte migration to skin Blood, January 15, 2003; 101(2): 602 - 610. [Abstract] [Full Text] [PDF]
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M. P. Bernimoulin, X.-L. Zeng, C. Abbal, S. Giraud, M. Martinez, O. Michielin, M. Schapira, and O. Spertini Molecular Basis of Leukocyte Rolling on PSGL-1. PREDOMINANT ROLE OF CORE-2 O-GLYCANS AND OF TYROSINE SULFATE RESIDUE 51 J. Biol. Chem., January 3, 2003; 278(1): 37 - 47. [Abstract] [Full Text] [PDF]
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L. G. Ellies, M. Sperandio, G. H. Underhill, J. Yousif, M. Smith, J. J. Priatel, G. S. Kansas, K. Ley, and J. D. Marth Sialyltransferase specificity in selectin ligand formation Blood, November 15, 2002; 100(10): 3618 - 3625. [Abstract] [Full Text] [PDF]
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 B. Dang, S. Wiehler, and K. D. Patel Increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects results in enhanced leukocyte recruitment under flow conditions J. Leukoc. Biol., October 1, 2002; 72(4): 702 - 710. [Abstract] [Full Text] [PDF]
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E. E. Burch, V. R. S. Patil, R. T. Camphausen, M. F. Kiani, and D. J. Goetz The N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated endothelium via P-selectin in vivo Blood, June 28, 2002; 100(2): 531 - 538. [Abstract] [Full Text] [PDF]
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B. Walcheck, A. Leppanen, R. D. Cummings, R. N. Knibbs, L. M. Stoolman, S. R. Alexander, P. E. Mattila, and R. P. McEver The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin Blood, May 13, 2002; 99(11): 4063 - 4069. [Abstract] [Full Text] [PDF]
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G. H. Underhill, H. A. Minges-Wols, J. L. Fornek, P. L. Witte, and G. S. Kansas IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules Blood, April 15, 2002; 99(8): 2905 - 2912. [Abstract] [Full Text] [PDF]
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K. Uchimura, F. M. El-Fasakhany, M. Hori, S. Hemmerich, S. E. Blink, G. S. Kansas, A. Kanamori, K. Kumamoto, R. Kannagi, and T. Muramatsu Specificities of N-Acetylglucosamine-6-O-sulfotransferases in Relation to L-selectin Ligand Synthesis and Tumor-associated Enzyme Expression J. Biol. Chem., February 1, 2002; 277(6): 3979 - 3984. [Abstract] [Full Text] [PDF]
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Y.-C. Lim, H. Xie, C. E. Come, S. I. Alexander, M. J. Grusby, A. H. Lichtman, and F. W. Luscinskas IL-12, STAT4-Dependent Up-Regulation of CD4+ T Cell Core 2 {beta}-1,6-n-Acetylglucosaminyltransferase, an Enzyme Essential for Biosynthesis of P-Selectin Ligands J. Immunol., October 15, 2001; 167(8): 4476 - 4484. [Abstract] [Full Text] [PDF]
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S. J. White, G. H. Underhill, M. H. Kaplan, and G. S. Kansas Cutting Edge: Differential Requirements for Stat4 in Expression of Glycosyltransferases Responsible for Selectin Ligand Formation in Th1 Cells J. Immunol., July 15, 2001; 167(2): 628 - 631. [Abstract] [Full Text] [PDF]
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M. Sperandio, A. Thatte, D. Foy, L. G. Ellies, J. D. Marth, and K. Ley Severe impairment of leukocyte rolling in venules of core 2 glucosaminyltransferase-deficient mice Blood, June 15, 2001; 97(12): 3812 - 3819. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
1-6-N-glucosaminyltransferase in biosynthesis of ligands for
E-selectin and P-selectin
Top
Abstract
Introduction
1,3-fucosyltransferases FucT-VII and FucT-IV, one or more
sialyltransferases, and at least one O-linked branching enzyme.
Previous studies have shown that core 2 
1-6-N-glucosaminyltransferase (C2GlcNAcT-I; EC 2.4.1.102) is required for functional modification of PSGL-1, the leukocyte P-selectin ligand, but have been ambiguous on whether this enzyme is
involved in E-selectin ligand formation. Using an attachment and
rolling assay under defined shear flow in vitro, this study shows that
C2GlcNAcT-I
