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(1,3)-Fucosyltransferase VII-Dependent Synthesis of P- and E-Selectin Ligands on Cultured T Lymphoblasts1
*
Department of Pathology and
Howard Hughes Medical Research Institute, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(1,3)-fucosyltransferase FucT-VII regulates the synthesis of
E-selectin ligands and sialylated Lewisx-related epitopes
(sLex-related epitopes) in human T lymphoblasts. The
current report shows that production of both P- and E-selectin ligands
is FucT-VII dependent, but peak synthesis of each occurs at different
levels of fucosyltransferase activity in intact cells. In brief,
FucT-VII mRNA levels were higher in cultured T lymphoblasts expressing
sLex-related epitopes and both selectin ligands than in
cells expressing P-selectin ligands alone. However, synthesis of the
epitopes and both selectin ligands required the FucT-VII enzyme in
transfected Molt-4 cells. In contrast, neither constitutive nor
transfection-enhanced levels of the FucT-IV enzyme generated active
P-selectin ligands in these lines. In addition, targeted deletion of
the FucT-VII gene in mice markedly inhibited the synthesis of both P-
and E-selectin ligands during blast transformation in vitro. Finally,
the optimal synthesis of active P-selectin ligands occurred at lower
level of FucT-VII activity than required for synthesis of equally
active E-selectin ligands in both cultured T lymphoblasts and FucT-VII
transfectants. Consequently, the FucT-VII enzyme is essential for the
synthesis of both P- and E-selectin ligands by T lymphoblasts, and its
activity determines whether P-selectin ligands are expressed alone or
in conjunction with E-selectin ligands and sLex-related
epitopes on human T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fucose residues are essential for the function of most endogenous
selectin-ligands (11). At least five (1, 3)-fucosyltransferases
synthesize sialyl-Lewisx (sLex)-related
epitopes3 in mammalian cells
(12, 13) and support the synthesis of selectin-ligands in mammalian
transfectants (14, 15, 16). However, FucT-IV and FucT-VII are the leading
candidates for control of ligand synthesis on leukocytes. FucT-IV,
originally named ELFT (17), was the first leukocyte-associated enzyme
linked to selectin-ligand synthesis. Goelz et al. cloned the gene from
an HL-60 library based on its ability to generate an epitope
(C2E5) associated with E-selectin binding
activity on myeloid cells. Overexpression of the gene in some CHO
strains conferred E-selectin binding activity in static adhesion
assays, indicating that the enzyme was capable of ligand synthesis in
suitable host cells. However, subsequent studies showed that E-selectin
ligand synthesis by this gene product was strain specific in CHO cells,
raising the possibility that other (1, 3)-fucosyltransferases
contributed to ligand synthesis in leukocytes (18).
Sasaki et al. (19) and Natsuka et al. (20) subsequently reported
cloning of a gene encoding a distinct (1, 3)-fucosyltransferase
termed FucT-VII. This gene conferred E-selectin binding activity and
high levels of sLex-related epitope expression when
transfected into mammalian hosts including the Namalwa human
B-lymphoblastic cell line. Competitive RT-PCR analysis confirmed the
presence of high concentrations of specific mRNA for the gene in
myeloid cell lines and neutrophils. The murine homologue of the gene is
strongly expressed in the bone marrow and in the high endothelial
venules of lymph nodes, two major sites of selectin-ligand synthesis in
vivo (21). Maly et al. (22) recently confirmed that this enzyme
participates in selectin-ligand synthesis through targeted disruption
of the murine FucT-VII gene. FucT-VII-deficient animals fail to express
binding sites for the selectins on circulating leukocytes. They also
show markedly diminished selectin-mediated adhesion during leukocyte
rolling in cremasteric venules, neutrophil recruitment into the
peritoneum, and lymphocyte homing into lymph nodes.
Recently, Knibbs et al. (23) reported that induction of the FucT-VII
enzyme mediates the synthesis of E-selectin ligands on human T
lymphoblasts used for the adoptive immunotherapy of cancers. These
investigators found that both the FucT-VII and FucT-IV gene products
synthesized E-selectin ligands in the Jurkat cell line. However, only
transfection of the FucT-VII gene product generated active ligands in
association with sLex epitopes. Furthermore, FucT-VII mRNA
levels and enzymatic activity correlated with de novo synthesis of
E-selectin ligands on cultured T lymphoblasts, whereas FucT-IV levels
did not. These findings implied that the FucT-VII gene product was the
essential (1, 3)-fucosyltransferase during synthesis of E-selectin
ligand by T lymphoblasts.
The current study provides definitive proof that the FucT-VII enzyme drives the synthesis of both P-selectin and E-selectin ligands on T lymphoblasts. Evidence is also presented that synthesis of P-selectin ligands occurs at lower levels of FucT-VII activity than synthesis of either physiologically active E-selectin ligands or sLex-related epitopes. Consequently, the level of FucT-VII activity attained in individual cells determines whether P-selectin ligands are synthesized alone or in combination with E-selectin ligands and sLex-related epitopes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The FucT-transfected T-lymphoblastic cell lines were grown in
RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented
with 10% FCS and G418 (Life Technologies) to maintain the transfected
phenotype. Human T lymphoblasts were derived from PBMC isolated by
Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) gradient
from healthy volunteers. These cells were activated on
plate-immobilized anti-CD3 and expanded in IL-2-supplemented
serum-free or serum-supplemented medium as described previously (23)
and further specified in the text. Murine T lymphoblasts were derived
from pooled splenic and lymph node lymphocytes by CD3 cross-linking
(18-h) followed by expansion in IL-2-supplemented AIM-V (Life
Technologies) with diminishing concentrations of FCS (from 10% to 2%
over 4 days). The serum reduction protocol resulted in the best
combination of cell growth and selectin-ligand synthesis in normal
mouse lymphocytes. The resulting CD8- dominant populations
generated from the (1, 3)-fucosyltransferase-deficient (22) and
wild-type animals did not differ substantially with regard to the
levels of CD3, CD18, CD49d, CD44, CD45, or CD62L (data not shown).
Flow cytometry and FACScan analysis
Cell surface marker expression was determined by indirect immunofluorescence and flow cytometry as previously described (23). Anti-CDw65 Ab (Immunotech, Westbrook, ME) was used at a dilution of 1/25. CSLEX1 (American Type Culture Collection (ATCC), Manassas, VA) and HECA452 (ATCC) Abs were used at 10 µg/ml or at saturating concentrations of nude mouse ascites. The anti-Lewisx (CD15) Ab (MMA, Becton Dickinson, San Jose, CA) was used at 10 µg/ml. The isotype- and species-matched control Abs were used at concentrations equal or greater than the test reagents. The FITC- and PE-conjugated secondary Abs (Immunotech) were used at saturating concentrations, generally 1/50–1/100 dilutions of the commercial stock solution. Analysis was conducted with a Becton Dickinson FACScan and Winlist Listmode Analysis program (Verity Software, Topsham, MA).
Selectin chimera binding assays
Culture supernatants containing the murine selectin:human IgM chimeras (selectin chimeras) were prepared by transfection of subconfluent COS cell monolayers using the expression vectors and technique described previously (21). Cells were stained in 96-well microtiter plates by combining 5 x 105 cells with the selectin chimera culture supernatants in a total volume of 50–100 µl for 30 min at 4°C. Following incubation, the cells were washed once in 100 µl of DMEM, resuspended in 100 µl of PE-labeled goat anti-human IgM secondary Ab (Sigma, St. Louis, MO) and incubated for 20 min at 4°C. The cells were pelleted and resuspended in fresh DMEM or Dulbecco’s PBS (Life Technologies) immediately before FACS analysis. Nonspecific staining was established with a murine CD45:human IgM chimera (CD45-chimera) or by addition of EDTA (10 mM) to staining reactions conducted with the selectin chimeras. The level of binding in these controls was approximately the same.
Two-color flow cytometry and FACScan analysis
These assays determined the distribution of carbohydrate epitopes and selectin chimera binding sites on individual T lymphoblasts. A double-indirect staining procedure was developed to optimize the signal strength for both markers. The assays were conducted in round- or V-bottom microtiter plates at 4°C as follows. Cells (5 x 105) were incubated with 4–10x saturating concentrations of CSLEX1 ascites, washed once, reacted with a FITC-conjugated monoclonal rat anti-mouse IgM (PharMingen), and washed again. The cells were then incubated with the selectin chimera, washed once, reacted with a biotinylated monoclonal mouse anti-human IgM (Zymed Labs, South San Francisco, CA), washed once and reacted with PE-conjugated avidin (Molecular Probes, Eugene, OR). After the final incubation, the cells were pelleted and resuspended in fresh buffer just before analysis. The secondary reagents for each marker reacted with their primary targets exclusively. Nonspecific interactions were determined using pooled mouse IgM and EDTA inhibition of selectin chimera attachment. Analysis was performed on two-parameter histograms constructed from 4–10 x 104 cells using the Winlist analysis program.
Parallel plate flow assays
The assay was performed as described previously (23) on either
confluent monolayers of CHO cells stably transfected with human
selectins or immobilized murine selectin chimeras at 
1–1.5
dynes/cm2. The chimeras were immobilized on 35-mm petri
plates, precoated with µ-chain-specific anti-human IgM (Sigma),
by 2–24 of h incubation with 1 ml of the culture supernatants at
37°C.
HECA452 and CSLEX1 inhibition assays
The cells (5 x 105 cells/ml) were preincubated
with pooled IgM, CSLEX1, HECA452, or CSLEX1 + HECA452 for 30 min
(using nude mouse ascites at 4–10 times the dilution required for
saturation or the purified IgM fractions). In the chimera attachment
assays, the cells were washed once and then reacted with the murine
selectin chimeras as described above. In the parallel plate flow
system, the cells were used without the wash step to maintain high
concentrations of the Abs throughout the adhesion assay.
RNA preparation and Northern blot analysis
Total RNA preparation and Northern blot analysis were conducted as previously reported (23).
Transfection of T-lymphoblastic cell lines
The FucT-IV- and FucT-VII-transfected lines were constructed using the expression vectors and technique described previously (23).
Fractionation of cell line based on expression of P-selectin ligands and CSLEX-1 epitopes
Standard panning techniques were modified for this purpose. In
brief, 100-mm tissue culture plates were incubated overnight at 37°C
with either goat anti-human IgM (Sigma) or goat anti-murine IgM
(Biosource International, Camarillo, CA) (10 µg/ml, 4
ml/plate). After three washes in Dulbecco’s PBS, the P-selectin
chimera (5 ml/plate of diluted culture supernatant) or the CSLEX-1 mAb
(1–10 µg/ml purified mAb) were added to the anti-human IgM-
and anti-murine IgG-coated plates, respectively, for 2 h at
37°C. The plates were washed and used immediately for
fractionations.
The transfectants were panned first on the immobilized CSLEX-1 Ab.
Cells (25 x 106) were added to each plate at room
temperature for 30 min. Unbound cells (undetectable to moderate levels
of the CSLEX-1 epitope) were removed with three 10-ml washes in RPMI
medium + 10% FCS (RPMI+). The bound cells (high levels of the CSLEX-1
epitope subpopulation C in Fig. 6
and Table II) were removed via
trituration with RPMI+. The cells with undetectable to moderate levels
of the CSLEX-1 epitope were washed, resuspended in RPMI+, and added to
the P-chimera plates (up to 25 x 106 cells/plate).
After a 30-min incubation at room temperature, the unbound cells
(undetectable to low levels of the CSLEX-1 epitope, undetectable to low
levels of P-selectin ligands subpopulation A in Fig. 6 and Table II)
were harvested with Dulbecco’s PBS (three washes). The bound cells
(undetectable to moderate levels of the CSLEX-1 epitope, high levels of
P-selectin ligands subpopulation B in Fig. 6 and Table II) were
harvested with 10 ml of Ca2+ Mg2+-free PBS
containing 0.02% EDTA (three washes).
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The method detailed by Knibbs et al. (23) was used without modification.
Reverse transcriptase-polymerase chain reaction
The primer sequences and technique have been described (24, 25). In brief, 12 µg of total RNA were isolated as described above and treated with 6 U of amplification grade DNase I before reverse transcription. DNase I was heat inactivated, and reverse transcription was conducted using 400 U of Superscript II Reverse Transcriptase (Life Technologies) per reaction. The cDNA products were serially diluted in separate reaction vials. The forward and reverse primers for FucT-VII, FucT-IV, or ß-actin were combined with Taq polymerase (Life Technologies) and placed in a thermocycler for 40 cycles. PCR reaction products and a 100-bp ladder (Life Technologies) were electrophoresed in 1.4% agarose gels and visualized by staining with ethidium bromide.
Fucosyltransferase knockout animals
Targeted deletion of the murine FucT-VII gene is described by Maly et al. (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first experiments compared the expression of
sLex-related epitopes and selectin-ligands on polyclonal T
lymphoblasts grown from PBMC under culture conditions resulting in
markedly different levels of FucT-VII mRNA (Fig. 1). Lymphoblasts grown in the
IL-2-supplemented, serum-free medium XVIVO15 expressed the highest
levels of the sLex-related epitopes (Fig. 1A)
and FucT-VII mRNA (Fig. 1D). The FI of the CSLEX1 and
HECA452 epitopes varied over a 2–3-decade range, with a significant
percentage of the blasts above 100 FI units (50% of the population
in the experiment shown). T lymphoblasts expanded in XVIVO15 produced
the greatest number of adhesion events on CHO-selectin transfectants at
physiologic levels of linear shear stress (Fig. 1C). The
"velocity histograms" revealed a broad range of rolling velocities,
suggesting a nonuniform distribution of ligands within the polyclonal T
lymphoblast population.
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30% of
the blasts over 100 FI units. As with the blasts grown in XVIVO15
alone, discrete "subpopulations" of epitope expression developed in
some cultures (Fig. 1
A). However, addition of FCS
consistently reduced the overall expression of the
sLex-related epitopes within the population and the
percentage of cells over 100 FI units when compared with XVIVO15 blasts
from the same donor. The XVIVO+FCS blasts also showed 30% fewer
interactions with E-selectin, 15% fewer interactions with P-selectin,
and reduced but detectable levels of FucT-VII mRNA compared with
XVIVO15 blasts. The combination of RPMI and HAB (RPMI + HAB) resulted
in the lowest expression of the sLex-related epitopes with
<5% of the population over 100 FI units. These blasts showed very few
interactions on E-selectin, 50% fewer interactions with P-selectin
(compared with XVIVO15 blasts from the same donor), and virtually no
detectable FucT-VII mRNA by Northern analysis. Thus, the overall
activity of ligands for both E- and P-selectins in cultured polyclonal
T lymphoblasts paralleled, to a degree, the level of FucT-VII mRNA and
the expression of sLex-related epitopes in the population.
However, avid P-selectin ligands were synthesized without FucT-VII mRNA
detected by Northern analysis under some culture conditions. Low concentrations of FucT-VII mRNA detected when T lymphoblasts express P-selectin ligands without E-selectin ligands
The lack of FucT-VII mRNA in Northern blots from the RPMI + HAB
blasts raised the possibility that this enzyme was not required for
synthesis of P-selectin ligands in human T cells. However, a previous
study from our laboratory showed that a low level of FucT-VII enzymatic
activity is detected in human T lymphoblasts cultured from lymph node
cells in RPMI + HAB (23). Northern analysis did not detect FucT-VII
mRNA in 7 µg of purified mRNA in this study, implying that it was a
rare transcript and beyond the sensitivity of the method. Consequently,
RT-PCR was conducted to establish which fucosyltransferases are
transcribed in lymphocytes cultured from peripheral blood (Fig. 2).
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A) and RPMI + HAB (Fig. 2B) mediums. However, the signal disappeared completely when
the reverse-transcribed and ß-actin-normalized material from the
latter was diluted 1/125 before amplification. The FucT-VII gene
product was still detected in the former at this dilution. Furthermore,
the band intensity at the 1/125 dilution of the XVIVO15 extract was
similar to the band intensity in the 1/5 dilution of the RPMI + HAB
extract. The FucT-IV mRNA was detected in the 1/5 dilutions and lost in
the 1/125 dilutions of both extracts. These findings are in general
agreement with Northern analyses from T lymphoblasts grown from lymph
nodes in XVIVO15 and RPMI + HAB serum (23). Thus, the FucT-IV and
FucT-VII genes are transcribed in cultured peripheral blood T
lymphoblasts synthesizing selectin-ligands. The FucT-VII mRNA level is
lowest in the cell population synthesizing P-selectin ligands alone and
highest in the population synthesizing ligands for both selectins along
with high levels of the sLex-related epitopes. The FucT-VII gene product drives synthesis of P- and E-selectin ligands in transfected Molt-4 cells
The RT-PCR experiments demonstrated that both the FucT-VII and
FucT-IV genes are transcribed in normal T lymphoblasts that synthesize
P-selectin ligands in the absence of sLex-related epitopes
and functional E-selectin ligands. The next experiments asked whether
either of these gene products generated P-selectin ligands in
transfected human T lymphoblasts. In our previous study (23), both gene
products synthesized E-selectin ligands when transfected into the
Jurkat cell line. However, P-selectin ligands were not detected in
these cells presumably due to their low levels of
UDPGal:GalNAc1-Ser/Thr ß(1, 3)-galactosyltransferase (Ref. 26 and
our unpublished observations). This defect limits production of
the core 2 precursors known to be essential for the synthesis of
P-selectin ligands (16). Consequently, the current study conducted
transfections in the Molt-4 cell line. This line constitutively
expressed the FucT-IV-dependent epitope CDw65 but not the
FucT-VII-dependent sLex-related epitopes (Fig. 3A). Furthermore, it showed
minimal interactions with the murine selectin chimeras (Fig. 3B) and the human selectin-transfected CHO cells under shear
stress (Fig. 3C).
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). The plasmid used for the transfections
contained both the FucT-VII and genticin resistance genes. Transfection
was performed at a plasmid:target cell ratio of 3 x
105:1 followed by selection in genticin-containing medium.
The uncloned genticin-resistant strain selected for study expressed
sLex-related epitopes over a broad range, implying that the
FucT-VII activity within individual cells varied over a broad range
within the population. This strain expressed the
sLex-related epitopes over a 3-decade range with 50% of
the population over 100 FI units. The distribution of
sLex-related epitopes in the transfectant was similar to
that observed in polyclonal T lymphoblasts cultured in XVIVO15 + FCS.
These transfectants bound both the murine E- and P-selectin chimeras
and rolled efficiently on human selectin-transfected CHO cells. FucT-IV transfected Molt-4 cells behaved differently. They increased synthesis of the VIM2 (CDw65) epitope but did not express sLex-related epitopes or show rolling activity on the CHO transfectants. A small increase in the attachment of the E-selectin chimera (four- to fivefold above the EDTA control) was noted, but no binding sites for the P-selectin chimera were detected. The fucosyltransferases produced identical results when transfected into the CEM cell line (data not shown). Thus, the FucT-VII enzyme generated sLex-related epitopes and ligands for both P- and E-selectin that function at physiologic levels of shear stress when transfected into T-lymphoblastic cell lines other than Jurkat. However, the FucT-IV enzyme alone, even when expressed at substantially higher levels than normally found in cultured T lymphoblasts, did not produce functional P-selectin ligands.
Peak synthesis of P-selectin ligands occurs at lower levels of sLex-epitope expression than peak synthesis of E-selectin ligands in cultured T lymphoblasts
The experiments in Fig. 1 suggested that the peak synthesis of
P-selectin and E-selectin ligands occurred at different levels of
FucT-VII activity in T lymphoblasts. The next series of experiments
investigated this hypothesis at the single-cell level. A previous study
found that FucT-VII enzymatic activity in extracts of T lymphoblasts is
directly proportional to the mean levels of CSLEX1 and HECA452
epitopes/cell in the population (23). Consequently, expression of these
epitopes provides an indirect measure of FucT-VII activity in
individual cells. Furthermore, preincubation with saturating
concentrations of the CSLEX1 Ab did not block the attachment of the
P-selectin chimera and had a minor effect on attachment of the
E-selectin chimera (Table I). Therefore,
the relationship between FucT-VII activity and selectin-ligand
synthesis was investigated by multiparameter flow microfluorometry
using the CSLEX1 epitope as an indirect measure of the former and
attachment of selectin chimeras as a direct measure of the latter
(Figs. 4–6).
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. The
mean intensity of selectin chimera staining is plotted as a function of
the mean intensity of CSLEX1 staining on T lymphoblasts cultured in
several different media in Fig. 5.
Relatively few T lymphoblasts cultured in XVIVO15 + FCS (Fig. 4a) reacted with the CD45-IgM chimera or the polyclonal
murine IgM control. A subset of CSLEX1 negative cells interacted with
both selectin chimeras (Fig. 4, b and c,
quadrant 1). However, the maximum number of
E-selectin-binding sites/cell rose in concert with CSLEX1 expression
whereas, the maximum number of P-selectin-binding sites per cell did
not increase over the same range (Fig. 4, b and
c; compare quadrants 1 and 2;
Fig. 5). The mean number of P-chimera-binding sites per cell rose
slightly as the intensity of CSLEX1 increased in the population. This
reflected an increase in the overall percentage of cells with at least
some detectable P-selectin binding sites. The maximum number of
P-selectin binding sites/cell, however, was essentially the same in the
CSLEX1 negative and positive "subpopulations" (Fig. 4c,
quadrant 1 vs quadrant 2). Although
the percentages of positive cells varied, the same relationship between
selectin-ligand synthesis and CSLEX1 expression was observed on T
lymphoblasts grown in RPMI + HAB (Fig. 5a), XVIVO15 + HAB
(Fig. 5b) and XVIVO15 + FCS (Fig. 5c).
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P- and E-selectin ligand synthesis in Molt-4 cells required
insertion of the FucT-VII gene. Insertion of the FucT-IV gene using the
same expression vector did not result in synthesis of P-selectin
binding sites (Fig. 3). Thus, changes in FucT-VII activity alone are
responsible for selectin-ligand synthesis in the transfectants.
Furthermore, the broad range of CSLEX1 expression in the uncloned
transfectants mimicked the behavior of cultured T lymphoblasts and
implied that FucT-VII enzymatic activity varied considerably among
cells in the population. Consequently, the transfectants provided a
direct test of the hypothesis that maximal P-selectin ligand synthesis
in intact cells occurs at lower levels of FucT-VII activity than
maximal synthesis of E-selectin ligands.
The FucT-VII-transfected Molt-4 cells reacted minimally with the
control reagents (Fig. 6a).
Both selectin chimeras bound to CSLEX1-negative FucT-VII transfectants
with attachment of P-selectin substantially higher than E-selectin in
this region (Fig. 6, b and c, quadrant
1). E-chimera attachment increased in parallel with the
level of CSLEX1 expression whereas P-chimera attachment increased
relatively little over the same range (quadrant 1 vs
quadrant 2 in Fig. 6, b and c).
Again, the slight increase in the level of P-selectin attachment
observed at high levels of CSLEX1 expression reflected a drop in the
percentage of cells with low numbers of binding sites. The maximal
number of sites per cell was the same in CSLEX1-negative, low and high
subpopulations. Thus, the FucT-VII-transfected Molt-4 cells and the
cultured T lymphoblasts showed similar relationships between CSLEX1
expression and synthesis of selectin binding sites.
The relationship among FucT-VII enzymatic activity, CSLEX1 expression,
and selectin-ligand synthesis was explored directly by measuring 1,3
FT activity in cell-free extracts from three subpopulations of
transfected Molt-4 cells (Table II and Fig. 6). The transfectants were first
separated into subpopulations expressing different levels of the
CSLEX-1 epitopes as described in Materials and Methods. The
CSLEX-1 epitope high fraction (subpopulation C) was not further
fractionated. The subpopulation with undetectable to moderate levels of
CSLEX-1 epitopes was further subdivided into those with undetectable to
low levels of P-selectin ligands and those with high levels of ligand
(subpopulations A and B, respectively). The approximate distributions
of the three subpopulations, based on the range of P-selectin chimera
and/or CSLEX1 staining in each (Table II), are overlaid on the two
parameter histograms in Fig. 6, b and c.
Subpopulation A expressed low levels of the P-selectin ligand on 31%
of cells, low levels of the CSLEX1 epitope on 5% of cells, and no
detectable 1,3 FT activity in a cell-free extract. Subpopulation B
expressed high levels of the P-selectin ligand on 91% of cells, low
levels of the CSLEX1 epitope on 35% of cells and 106 ± 0.4
pmol/h/109 cells of 1,3 FT activity in a
cell-free extract. Subpopulation C expressed high levels of the
P-selectin ligand on 95% of cells, high levels of the CSLEX1 epitope
on 91% of cells, and 314 ± 1.2 pmol/h/109 cells of
1,3 FT activity in a cell-free extract. Thus, 83% of the maximal
P-selectin ligand expression/cell occurred in subpopulation B, which
contained 25% (or less based on MFI) of the maximal CSLEX1
expression per cell and 30% of the maximal FucT-VII-dependent
1,3 FT activity in extracts. The mean level of P-selectin
ligands/cell rose by 1.2-fold in subpopulation C. In contrast, the
mean levels of E-selectin ligands per cell (compare MFI for E-chimera
in B and C, Fig. 6b), CSLEX1 per cell,
and FucT-VII dependent 1,3 FT activity in extracts rose by at least
threefold in subpopulation C.
T lymphoblasts tether and roll on E-selectin in the presence of high concentrations of the HECA452 and CSLEX1 Abs
The CSLEX1 and HECA452 Abs did not effectively inhibit selectin
chimera attachment to cultured T lymphoblasts (Table I). This
observation was surprising in view of reports that the mAbs react with
E-selectin ligands on myeloid cells (27, 28, 29, 30) and some lymphocytes (2, 31). Studies reporting inhibitory activity with these mAbs generally
used static adhesion assays; therefore, the mAbs were tested using
cultured T lymphoblasts and selectin-transfected CHO cells in the
parallel plate flow chamber at 1–1.5 dynes/cm2 (Table III). High concentrations of the two Abs
combined did not significantly reduce the number of T lymphoblasts
interacting with immobilized human E- or P-selectin under shear. A 30%
increase in the rolling velocity on both E- and P-selectin was detected
when using T lymphoblasts with very high levels of
sLex-related epitopes. The Ab mixture did not alter the
rolling velocity of T lymphoblasts on P-selectin when using cells with
relatively low levels of sLex-related epitopes on the
surface. These experiments used high titer ascites as a source for the
mAbs. However, 500 µg/ml of ammonium sulfate-precipitated and
column-purified Abs behaved similarly (L. M. Stoolman and R.
Craig, unpublished observations).
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Studies with human cells suggested that the FucT-VII enzyme was
necessary for the synthesis of both E- and P-selectin ligands in T
lymphoblasts. The next experiments used mice with gene-targeted
deletion of the FucT-VII locus to provide additional support for this
hypothesis. T lymphoblasts were cultured from mice homozygous for the
FucT-VII null allele and from wild-type controls homozygous for the
normal allele. Splenic lymphocytes from 2–3 animals in each group were
pooled, activated on plate-immobilized anti-CD3, and expanded in
culture medium supplemented with IL-2. The cell populations were CD8
dominant and did not differ substantially in the levels of CD3, CD18,
CD49d, CD44, CD45, or CD62L (data not shown). The WT lymphoblasts bound
well to both murine (Fig. 7, a
and b) and human (Fig. 7c) selectins. The
FucT-VII KO lymphoblasts, in contrast, showed no interactions with
fluid phase murine chimeras, immobilized murine chimeras or human
selectin-transfected CHO cells under the assay conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rossiter and colleagues reported that T lymphoblast clones derived from skin-infiltrating T cells coexpressed E- and P-selectin ligands more frequently than clones derived from the peripheral blood of the same individual (32). The peripheral blood clones frequently expressed P-selectin ligands without either E-selectin ligands or sLex-related epitopes. These findings imply that the metabolic changes responsible for synthesis of E-selectin ligands on skin-infiltrating T cells are maintained during cloning and differ from the requirements for the synthesis of P-selectin ligands. The current study shows that modulation of FucT-VII mRNA levels and enzymatic activity alone produces similar phenotypic differences in polyclonal T lymphoblast populations. Consequently, differences in FucT-VII transcription or mRNA stability following T cell activation may determine whether P-selectin ligands are expressed alone or in conjunction with E-selectin ligands on cloned T cells.
The precise contribution of the FucT-IV enzyme to selectin-ligand synthesis in T lymphoblasts remains unclear. FucT-IV is active in cultured T lymphoblasts and related cell lines (13); therefore, the current study does not rule out a requirement for FucT-IV in the synthesis of FucT-VII-dependent ligands. However, the FucT-IV enzyme does not synthesize detectable E- or P-selectin ligands at the relatively low levels of constitutive enzyme activity found in the Molt-4 cell line. Similar findings have been reported in other leukocytic human cell lines (25). In addition, transfection of the FucT-IV gene into Molt-4 increased expression of the FucT-IV-dependent epitope CDw65 by more than 10-fold, but the transfectants showed only modest interactions with E-selectin and none with P-selectin. Furthermore, the FucT-IV remaining in T lymphoblasts cultured from FucT-VII-deficient animals did not synthesize ligands we could detect either in fluid phase or under shear. Of course, more sensitive assay techniques may detect selectin-binding sites on cells expressing relatively low levels of the FucT-IV in the absence of FucT-VII. Nonetheless, T lymphoblasts with FucT-IV alone clearly bind to E- and P-selectin less efficiently than cells expressing both FucT-IV and FucT-VII.
Several lines of evidence suggest that FucT-IV may contribute more to selectin-ligand synthesis in neutrophils than in lymphoid cells. Neutrophil rolling and recruitment occur at reduced but readily detectable levels in FucT-VII-deficient mice (22), implying that FucT-IV in neutrophils synthesizes active ligands for one or more of the selectins in the absence of FucT-VII. Knibbs et al. (23) confirmed this possibility in vitro, showing that the FucT-IV gene product generated active E-selectin ligands in the Jurkat cell line when raised to levels normally found in myeloid cell lines. The K562 and BJAB lines described by Wagers et al. (25) behaved similarly. Together with the findings in the current study, these observations suggest that the contribution of FucT-IV to selectin-ligand synthesis in leukocytes is lineage dependent, reflecting, in part, differences in the maximal level of enzyme activity attained in each lineage. Consequently, inhibition of FucT-VII alone may have a more profound impact on the selectin-dependent recruitment of T cells than of neutrophils.
The CSLEX1 and HECA452 Abs did not efficiently block selectin-ligand function on cultured normal T lymphoblasts whether the epitopes were expressed at low or high levels on the cell surface (Tables I and III). The parallel plate flow system used cells at low concentrations (5 x 105/ml) and kept them in suspension throughout the assay to minimize agglutination. Under these conditions, concentrations up to 500 µg/ml for each Ab caused a 30% increase in rolling velocities but no significant change in the number of adhesive interactions on either E- or P-selectin. Partial inhibition of murine E-selectin (<30%) and P-selectin (<25%) chimera attachment with the HECA452 Ab was observed. However, the CSLEX1 Ab did not inhibit chimera attachment and a mixture of both Abs was no more effective than HECA452 alone. The mAbs, particularly HECA452, inhibited E-chimera attachment to the transfected Molt-4 cells more effectively than to cultured normal T lymphoblasts. Whether this reflects differences in the availability, affinity, or structure of the ligands in the two populations remains to be determined. However, it underscores the importance of conducting studies on normal cells as well as cell lines.
Previous static adhesion assays conducted in microtiter plates showed significantly higher levels of inhibition with both Abs. The CSLEX1 Ab inhibited neutrophil and HL-60 attachment to immobilized E-selectin (27, 33). The HECA452 Ab partially inhibited the attachment of PBL to E-selectin under similar conditions (2). One cannot exclude the possibility that the density or structure of the relevant carbohydrate moieties on cultured T lymphoblasts differs from those on PBL, neutrophils, and myeloid cell lines. However, it is also possible that adhesion in static and flow assays depends on different sets of selectin "ligands." In standard flow assays, the initial interaction or tether must form quickly (in seconds) since the interaction occurs under linear shear stress. Static assays, on the other hand, allow cells to interact with substrate for 30–60 min before shear is applied (e.g., the wells are washed), thus detecting both rapidly and slowly formed attachments. Consequently, flow assays are biased toward selectin-ligand interactions with rapid on times, while static assays may detect contributions from a greater variety of ligands.
Whatever accounts for the difference between static and flow assays, the current results imply that most HECA452 and CSLEX1 epitopes on cultured T lymphoblasts are not required for interactions with immobilized selectins initiated under flow conditions. Consequently, one cannot assume that glycoconjugates carrying these epitopes in cell-free extracts, such as PSGL-1 (31, 34), are essential for T lymphoblast tethering and rolling on E-selectin under shear. It remains possible that the sLex-related epitopes detected in cell-free extracts contribute to E-selectin binding activity but are sequestered or modified such that they react poorly with Abs on intact cell. Alternatively, glycoconjugates expressing these epitopes may attach to E-selectin through other, nonreactive oligosaccharide chains. Comparative analysis of carbohydrate structures and E-selectin binding activities is needed to determine which of the putative ligands (16, 35, 36, 37, 38) carries oligosaccharides that mediate adhesive interactions under shear. Despite the uncertainty regarding the contribution of sLex-related epitopes to ligand function, there is no doubt that the synthesis of active E-selectin ligands parallels expression of the epitopes over a broad range. Therefore, the CSLEX1 and HECA452 mAbs remain useful markers of E-selectin binding activity in human leukocytes.
In conclusion, the synthesis of selectin-ligands involves several protein "acceptors," multiple glycosyltransferases and, for P-selectin ligands, tyrosine sulfation as well. The induction of ligand synthesis during T cell proliferation involves changes at multiple steps in the synthetic pathways. However, the pivotal role of FucT-VII in the synthesis of P- and E-selectin ligands suggests that blockade of its transcription and/or activity provides a novel approach to treatment of selectin-dependent immune lesions.
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2 Address correspondence and reprint requests to Dr. Lloyd M. Stoolman, Department of Pathology, University of Michigan, Room 4224, Medical Sciences Building 1, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: sLex-related epitopes, epitopes expressed by the sialyl-Lewisx tetrasaccharide and related structures (including the CSLEX1 and HECA452 epitopes); FucT-IV, (1,3)-fucosyltransferase IV; FucT-VII, (1,3)-fucosyltransferase VII; CHO, Chinese hamster ovary; PE, phycoerythrin; FI, fluorescence intensity; MFI, mean fluorescence intensity; HAB, human AB serum; 1,3 FT, (1,3)-fucosyltransferase; KO, knockout.
Received for publication February 23, 1998. Accepted for publication July 30, 1998.
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(1,3)-fucosyltransferases which synthesize sialyl Le(x) and sialyl Le(a), the carbohydrate ligands for E- and P-selectins,in human malignant cell lines. Cancer Res. 53:5559.(1,3)-fucosyltransferase, FucT-VII, in leukocyte adhesion to E-selectin. Blood 88:2125.(1,3)-fucosyltransferase gene (ELFT). J. Biol. Chem. 269:1033.(1,3)-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes. J. Biol. Chem. 269:14730.-1,3-fucosyltransferase capable of synthesizing the sialyl Lewis x
determinant [Published erratum appears in 1994 J. Biol.
Chem. 269:20806]. J. Biol. Chem. 269:16789.
(1,3)-fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands. J. Biol. Chem. 271:8250.(1,3)-fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 86:643.[Medline]
T cells. J. Immunol. 159:3574.[Abstract]
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= mean velocity (± SE) for "rolling"
events under 100 µm/s. D, Representative Northern
blots showing the steady state levels of FucT-VII and ß-actin mRNA in
the T lymphoblast populations. The images were acquired using the
PhosphorImager (Molecule Dynamics, Sunnyvale, CA) from blots probed
with 32P-labeled cDNAs as described previously (23).
