1 Applied Immunobiology Research Group, Department of Surgery, University of
Newcastle, The Medical School, Newcastle upon Tyne NE2 4HH, UK
2 Institute of Pharmacy, Chemistry and Biomedical Science, University of
Sunderland, Sunderland SR1 3SD, UK
* Author for correspondence (e-mail: j.a.kirby{at}ncl.ac.uk)
Accepted 12 May 2003
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Summary |
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Key words: Heparan sulphate, Inflammation, Chemokine, IFN-, TNF-
, RANTES
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Introduction |
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Previous work has shown that a number of important pro-inflammatory
cytokines, including IFN-, some interleukins and most chemokines
(Lortat-Jacob et al., 1995
;
Morita et al., 1994
;
Proudfoot et al., 2001
;
Rot et al., 1996
) interact
specifically with HS. This interaction is often focused at specific sequence
domains within the HS oligomer which are characterised by extensive
N-sulphation. Generation of these sequence domains is regulated during HS
biosynthesis in the Golgi apparatus
(Gallagher, 1997
). The HS
copolymer is attached to a core protein via a tetrasaccharide linkage and
extended by alternate transfer from UDP-sugar donors of N-acetyl-D-glucosamine
and D-glucuronic acid monomers. As the chain extends, it is potentially
modified by the addition of sulphate residues at the amino group of
glucosamine (N-sulphation) and at a range of hydroxyl groups (O-sulphation)
within the disaccharide unit. Additionally, glucuronic acid may be modified by
epimerisation of carbon 5 (C-5) to form iduronic acid. The O-sulphation and
epimerisation modifications are dependent on the initial replacement of the
N-acetyl group of glucosamine with an N-sulphate group
(Unger et al., 1991
); for
example, the C-5 epimerase activity requires N-sulphation of an adjacent
glucosamine (Li et al., 1997
).
The family of enzymes responsible for this important regulatory reaction are
the N-deacetylase/N-sulphotranferases (NDSTs), of which there are four known
human homologues (NDST-1, -2, -3 and -4). Whilst these enzymes share the same
basic function they do appear to have subtly different activities, with NDST-3
and -4 being expressed at low levels and with a restricted distribution
(Aikawa et al., 2001
).
The interaction between certain cytokines and sulphated domains on HS are
ionic in nature, forming between basic amino acid sequences on the protein and
anionic, sulphated domains on the GAGs
(Lortat-Jacob et al., 1995;
Morita et al., 1994
;
Pye et al., 1998
). This
interaction has a number of important functions, including protection,
concentration and presentation of cytokines which are produced in small
quantities by a small number of cells within the microenvironment. This
process is believed to be of particular importance for presentation of
chemokines on the surface of endothelial cells
(Tanaka et al., 1996
) and for
stabilisation of the concentration gradients necessary for leukocyte
chemotaxis (Adams and Lloyd,
1997
; Ali et al.,
2002
; Appay and Rowland-Jones,
2001
; Patel et al.,
2001
; Witt and Lander,
1994
).
Heparin is structurally similar to HS, but is more uniformly sulphated
along the GAG chain making it the most negatively charged molecule in the
body. In many cases the amino acid sequences required for protein interaction
with heparin have been defined as XBBXBX or XBBBXXBX, where B represents a
basic residue (Hileman et al.,
1998). The heparin binding properties of many chemokines,
including the prototypical CC-chemokine RANTES (CCL5), which contain the
XBBXBX motif within their primary sequence have been extensively studied
(Kuschert et al., 1999
). It
has been shown that RANTES shows selectivity with regards to GAG binding, with
its affinity for heparin being three orders of magnitude greater than for
chondroitin sulphate; site-directed mutation has shown that the XBBXBX motif
on RANTES is a principal site for heparin binding
(Kuschert et al., 1999
;
Proudfoot et al., 2001
).
Previous work has shown marked differences in the expression of NDST
transcripts between both tissues and different species
(Aikawa et al., 2001;
Habuchi et al., 2000
;
Shworak et al., 1999
).
However, few studies have compared the levels of NDST expression between
individual cell types or studied how expression of these molecules is
modulated by external stimuli. Given the important function of chemokine
binding to endothelial HS during inflammation and the dependence of this
process on the extent of sulphation of the GAG molecule, this study was
designed to investigate the role of modulation of HS sulphation during
endothelial inflammation. Northern blotting and RT-PCR analysis were used to
study the expression of individual NDST transcripts and a range of functional
assays were used to investigate how changes in NDST expression correlated with
sequestration of RANTES by endothelial cells.
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Materials and Methods |
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RNA extraction and purification
Cell lines were grown to confluency and total RNA extracted using RNAzol B
reagent (AMS Biotech). Adherent cell lines (HMEC-1, A549, Hep G2 and HEK 293)
were grown in 75 cm2 flasks and were lysed in situ with 3 ml RNAzol
reagent preheated to 70°C. Some HMEC-1 cultures were stimulated with 100
U/ml of both TNF- and IFN-
for 4 or 16 hours before the
extraction of RNA. Suspension cell lines (HMC-1 and U937) were grown to
confluency in 15 ml of media, pelleted and lysed with 3 ml RNAzol B reagent
preheated to 70°C. Oligotex mRNA mini kits (Qiagen) were then used to
purify the mRNA from the total RNA extracted using the RNAzol B reagent. RNA
was quantified by measurement of absorbance at 260 nm and was stored at
-20°C.
Northern blotting
RNA samples were electrophoresed in formaldehyde denaturing agarose gels
and blotted onto Hybond XL nitrocellulose (Amersham Pharmacia). The amount of
RNA loaded onto the gels was either 100 µg of total RNA or 1 µg of mRNA.
The NDST-1 probe was prepared by random prime synthesis with 20 ng of PCR
product as template and [-32P]dCTP (Amersham Pharmacia) for
labelling. An antisense RNA probe for NDST-2 was prepared by transcription
with T3 RNA polymerase from 1 µg of a linearised NDST-2 pBluescript II
KS-clone; this probe was labelled with [
-32P]UTP (Amersham
Pharmacia). Excess nucleotides were removed from the probes by purification
using Sephadex G-50 columns (Amersham Pharmacia).
Reverse transcription coupled PCR
First strand cDNA was synthesised using 0.5 µg of mRNA. Fragments of
NDST-1 and -2 were initially amplified from cDNA using the proof reading
polymerase pfu (Stratagene) for subsequent sequence verification. The
exon-spanning primer sequences are shown below:
NDST1-forward (5'-CACACAGAACGAACTACGC-3')
NDST1-reverse (5'-CCCGTTGATGATCTTGTCC-3')
NDST2-forward (5'-GCCTCCAGTTCCACCTC-3')
NDST2-reverse (5'-CGACGAAGAACTGGTCC-3')
Amplifications were performed in a DNA thermal cycler (Hybaid) for 35 cycles under the following conditions: 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds. For the semi-quantitative analysis of expression, the amplification was dropped to 27 cycles and housekeeping transcripts of glyceraldehyde 3-phosphate dehydrogenase (Gap-DH) were analysed for comparison.
Antibodies and flow cytometric analysis
An anti-HS monoclonal antibody (F58-10E4; Seikagaku Corp) was used for
primary cell surface labelling followed by secondary labelling with a
FITC-conjugated goat anti-mouse polyclonal antibody (GAM-FITC; Becton
Dickinson) before flow cytometric analysis. Expression of the promiscuous
Duffy antigen receptor for chemokines (DARC; CD234) was also measured by
immunofluorescence.
HMEC-1 cells were grown to confluence in 75 cm2 flasks and
stimulated with 100 U/ml of both TNF- and IFN-
in MCDB-131
medium; control cells were unstimulated. The cells were stimulated for periods
between 16 and 72 hours prior to harvesting by incubation at 37°C for 10
minutes with 5 ml PBS containing 3 mM EDTA; in some cases cytokine-stimulation
was performed for 24 hours in the presence of cycloheximide at 10 µg/ml
(Sigma). The cells released from a single culture flask were split into four
aliquots. Primary antibody was added to three of these at a final
concentration of 5 µg/ml, with the fourth being used for an isotype-matched
(IgM) primary and secondary antibody control. The tubes were incubated at
4°C for 1 hour, washed again with PBS, resuspended in 50 µl of 10
µg/ml secondary antibody in PBS and incubated for 30 minutes at 4°C.
The cells were then washed again and analysed by flow cytometry (FACsort;
Becton Dickinson); the signal from control immunofluorescence did not change
from autofluorescence levels. Data analysis was performed using FCS Express
(De Novo software) and Excel (Microsoft).
GAG labelling and purification
GAGs were purified by incubating HMEC-1 cells for 24 hours with 2.5 µCi
[3H]glucosamine (Amersham). Cells were washed in PBS then
stimulated with cytokines as above for 0, 4 or 16 hours. Cells were incubated
for 1 hour in 0.1 N NaOH at 37°C after which they were neutralised with
sodium acetate. The cell extracts were loaded onto a DEAE Sepharose column,
washed with 0.3 M NaCl to remove contaminating proteins, and then eluted in
1.5 M NaCl (Esko, 2002). The
relative amount of GAG present in each sample was estimated and the
incorporated tritium measured by scintillation counting.
Western blotting to detect HS proteoglycans
This study was performed using methodology described previously
(Olofsson et al., 1999).
Briefly, cells were detached from confluent 75 cm2 flasks by
washing with PBS containing 3 mM EDTA and then incubation for 10 minutes at
37°C in 5 ml of the same solution. The cells were then washed by
centrifugation in PBS and resuspended in a solution containing 10 mU/ml
heparatinase (Seikagaku Corp), 0.5 U/ml chondroitinase ABC (Seikagaku Corp),
100 mM NaCl, 1 mM CaCl2, 0.1% Triton X-100, 50 nM 6-aminohexanoic
acid, 20 µg/ml leupeptin, 2.5 µg/ml pepstatin A, 1 mM PMSF, and 50 mM
Hepes at pH 7.0 before incubation at 37°C for 3 hours. The samples were
run under non-reducing conditions by SDS-PAGE and transferred to hybond-P
membranes (Amersham Pharmacia). In order to get good separation of all
proteins, the samples were run on both 12% and 8% acrylamide gels.
Immunoblotting was performed with mouse antibody 3G10 (Seikagaku Corp.)
followed by goat anti-mouse immunoglobulin conjugated to horseradish
peroxidase (Pharmingen). The 3G10 antibody is specific for a neo-epitope
generated on HS 'stubs' which remain attached to proteoglycan core proteins
after heparitinase digestion (David et
al., 1992
). Bound antibody was detected using the ECL substrate
(Amersham Pharmacia). Following primary development the blots were stripped
and re-probed with a mouse antibody specific for
-tubulin (Sigma) to
control for equal protein loading.
Measurement of RANTES binding
The HMEC-1 cell line was grown to confluency in MCDB-131 medium in chamber
slides (Falcon). The cells were stimulated for 16 hours with fresh media
containing 100 U/ml IFN- and 100 U/ml TNF-
either with or
without 100 mM sodium chlorate; none of these treatments was toxic during this
period. Chlorate is a reversible inhibitor of the enzyme ATP sulphurylase,
which produces the 3'-phosphoadenosine-5'-phosphosulphate
substrate required for sulphotransferase activity
(Safaiyan et al., 1999
).
Further cells were treated with 10 mU of heparitinase (Seikagaku Corp) for 3
hours at 37°C to deplete cell surface HS. Control cells were grown in
medium alone. After incubation, the cells were fixed in cold methanol and the
chamber well was removed from the slide. The cells were rehydrated with PBS
and were covered with 150 µl PBS containing 200 ng of RANTES (Peprotech)
for 16 hours. After washing, the cells were incubated with 10 µg/ml of
mouse anti-RANTES (Caltag) for 3 hours at 4°C before incubation for 1.5
hours with 10 µg/ml GAM-FITC (Becton Dickenson). The slides were washed and
mounted using Vectorshield (Vectorlabs) before analysis by scanning laser
confocal microscopy using Comos v7 software (Biorad). Control preparations
were routinely incubated with and without RANTES to distinguish between
exogenous and endogenously produced chemokine; an isotype-matched primary
control antibody (IgG2b) produced no significant immunofluorescence.
Radioligand binding experiments
The binding of 125I RANTES to HMEC-1 cells was assessed in 12
well plates. Each well was seeded with 1x104 cells, which
were grown to confluency. The cells were then washed and treated with either
100 U/ml of both TNF- and IFN-
, 100 U/ml of TNF-
and
IFN-
in 100 mM sodium chlorate, or medium only. The cells were all left
for a further 4 or 16 hours before fixation in cold methanol for 10 minutes,
washing with PBS and incubation for 2 hours at 37°C in PBS containing 377
pM 125I-RANTES and 300 nM of unlabelled RANTES. The cells were
washed twice with PBS to remove any unbound ligand. The cells were then lysed
by incubation at 37°C for 1 hour in a solution of 0.1 M NaOH and
transferred to test tubes; the radioactivity was then measured by gamma
counting (Clinigamma, Wallac UK).
Chemotaxis assay
HMEC-1 monolayers were propagated on 24-well format transwell membranes (3
µm pore; Falcon plastic) by seeding each with 4x105 cells
in supplemented MCDB-1 medium and culturing for 72 hours; some monolayers were
activated with TNF- and IFN-
for 100 U/ml for the final 24
hours. The monocyte cell line THP-1 (ATCC TIB 202) was cultured in RPMI1640
medium supplemented in 10% foetal calf serum and activated with 100 U/ml
IFN-
for 24 hours to enhance their chemotactic response (data not
shown). For chemotaxis assays the lower compartment of each transwell chamber
was filled either with normal medium or medium supplemented with RANTES at 10
ng/ml and the chambers were cultured for 30 minutes to allow chemokine
distribution within the monolayer. One million THP-1 cells were then added to
the upper compartment of each chamber and the system was incubated for 90
minutes. After this time the upper surface of each membrane was wiped clear of
excess cells and the membranes were fixed in methanol at -20°C before
staining with Haematoxylin and mounting; the mean number of migrant cells per
high power field was determined by microscopic examination of the lower
surface of each filter.
Statistical analysis
All groups were compared with the corresponding control using Student's
two-sample t-test. All groups were measured as triplicates and
P values <0.05 were considered significant.
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Results |
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Fig. 1 shows the northern blots for NDST-1 and -2 (Fig. 1A and B, respectively); the data and nature of the cells is summarised in Table 1. The pattern of gene expression differed between all the cell types except the two epithelial cell lines (HEK-293 and A549), which showed broadly similar transcript expression. Neither NDST-1 nor NDST-2 could be detected in the resting HMEC-1 cell line by northern blotting.
|
|
Semi-quantitative RT-PCR analysis of the HMEC-1 cell line
The low detectable levels of NDST expression in the HMEC-1 cell line led to
further investigation by semi-quantitative RTPCR. It is possible that levels
of expression may be tightly regulated in order to respond more efficiently to
external stimuli. In order to test this hypothesis the HMEC-1 cell line was
stimulated with the pro-inflammatory cytokines TNF- and IFN-
.
The level of expression of NDST-1 and -2 was then examined by
semi-quantitative RT-PCR. Expression of the chemokine RANTES was used as a
positive control as this transcript is known to be upregulated under these
conditions (Mantovani et al.,
1997
). Fig. 2 shows
the level of expression of NDST-1, -2 and RANTES following stimulation with
the pro-inflammatory cytokines TNF-
and IFN-
for 4 or 16 hours
(Fig. 2A and B, respectively).
By 4 hours post-stimulation there was a significant downregulation of both
NDST-1 (P<0.05) and NDST-2 expression (P<0.01), with
upregulation of the chemokine RANTES (P<0.001) providing a
positive control. After 16 hours the situation was reversed with NDST-1 now
showing significant upregulation (P<0.01) and NDST-2 also showing
an increase, although this did not reach significance (P<0.08).
Once again, RANTES expression was significantly higher than the control
(P<0.005).
|
Analysis of HMEC-1 phenotype
The monoclonal antibody 10E4 was used to define the abundance of sulphated
domains within HS on the endothelial cell surface. The specificity of this
antibody was demonstrated by David et al.,
(David et al., 1992) who
showed that binding to HS was completely abrogated by selective desulphation
to produce an N-desulphated HS molecule which retained O-sulphated groups.
Antibody binding was restored following re-N-sulphation of the molecule, but
no binding was observed after re-N-acetylation. HMEC-1 cells were analysed by
FACS to observe the levels of cell surface HS sulphation on resting cells and
after stimulation for 16, 24, 48 and 72 hours with the cytokines TNF-
and IFN-
. Fig. 3A shows
representative flow cytometric histograms which demonstrate an increase in
binding of the 10E4 antibody, and hence in expression of N-sulphated epitopes,
with time following cytokine stimulation of the cells. Importantly, no
increase in the binding of 10E4 antibodies to cytokine-stimulated cells was
observed in the presence of cycloheximide, indicating a requirement for
protein synthesis (data not shown). The summary data in
Fig. 3B indicate little 10E4
antibody binding above isotype control levels at time 0 but an increase in
median fluorescence was observed after cytokine-stimulation for 16 hours and
it continued to increase for 72 hours (P<0.005). The cells
expressed very low levels of cell surface DARC; this was not increased
following cytokine stimulation of the cells (data not shown).
|
Analysis of cell surface GAG turnover by cytokine-stimulated HMEC-1
cells
A pulse-chase experiment was performed to measure the rate of GAG turnover
in order to detect any effect of treatment of HMEC-1 cells with
pro-inflammatory cytokines. HMEC-1 cells were cultured overnight with
[3H]glucosamine. The cells were then washed, returned to unlabelled
media and allowed to grow for a further 4 or 16 hours under normal conditions
or in the presence of TNF- and IFN-
. The GAG complement of the
cells was then purified and the amount of incorporated tritium was measured.
Fig. 4 shows that the amount of
radiolabelled glucosamine falls over time, indicating GAG turnover. However,
after 4 and 16 hours the level of incorporation was the same for both
unstimulated and cytokine-stimulated cells (P>0.05), suggesting
that the rate of turnover remained the same under both conditions. This
suggests that changes in cell surface sulphation occur as a consequence of
modification of newly synthesised HS; there appears to be no enhanced shedding
of GAGs by the cytokine stimulated cells.
|
Quantification of HS proteoglycans by western blotting
Increases in the abundance of sulphated HS epitopes on the surface of
cytokine-stimulated HMEC-1 could occur as a consequence of increased synthesis
of HS proteoglycans. In order to investigate this, a series of quantitative
western-blotting experiments was performed using the 3G10 antibody, which
recognises residual unsaturated glucuronate stubs on all HS proteoglycan core
proteins (David et al., 1992).
Fig. 5 shows a composite of
blots run on both 8 and 12 percent acrylamide gels. The HS stub-bearing core
proteins are visible as multiple bands between 200 kDa and 35 kDa and appear
to be predominantly a mixture of glypicans and syndecans. There was no
increase in the density of these bands relative to the corresponding control
-tubulin band following stimulation with TNF-
and IFN-
.
Indeed, the bands at 90 kDa and 78 kDa showed a small reduction in intensity
after 16 hours. The absence of an increase in the quantity of
proteoglycan-associated HS stubs following cytokine stimulation of the HMEC-1
cells indicates that the increase observed in HS sulphation (10E4 antibody
binding) occurs at the level of the individual GAG molecules.
|
Examination of the binding of exogenous RANTES to HMEC-1 cells
As RANTES is known to bind to sulphated domains on HS, a series of
experiments was performed to determine whether treatment of endothelial cells
with pro-inflammatory cytokines resulted in an increased potential for RANTES
sequestration by HS molecules following upregulation of NDST-1 and -2.
Fig. 6 shows the results from
an immunofluorescence scanning laser confocal microscope study of the binding
of exogenous RANTES to resting, cytokine-stimulated and resting HMEC-1. Little
surface-bound RANTES was observed following chemokine addition to unstimulated
HMEC-1 (Fig. 6A). However,
following stimulation with TNF- and IFN-
for 16 hours a greater
amount of the added chemokine was bound to the cell surface
(Fig. 6B); at this time,
endogenously produced RANTES made a minimal contribution to cell-surface
chemokine expression (Fig. 6C).
Cells stimulated with TNF-
and IFN-
in the presence of chlorate
(an inhibitor of GAG sulphation) also bound minimal quantities of exogenous
RANTES (Fig. 6D). Removal of HS
from cytokine-stimulated cells by treatment with heparitinase also reduced
subsequent RANTES binding to background levels (data not shown). Construction
of a X-Z image through Fig. 6B
(shown in Fig. 6E) shows that
exogenous RANTES was bound on the apical surface of cytokine-stimulated
HMEC-1. Mean results from semi-quantitative analysis of cell-associated
fluorescence are shown in Fig.
6F; this graph shows a 2.8-fold increase in RANTES binding to
cytokine-activated cells (P<0.01) which cannot be accounted for by
the detection of RANTES produced endogenously by the activated cells.
Furthermore, blockade of GAG sulphation reduced RANTES binding by
cytokine-activated cells to levels associated with resting cells
(P>0.05); this excluded the possibility that the increase in
chemokine-binding was caused by upregulated expression of any known or unknown
specific chemokine receptors.
|
Radioligand quantification of RANTES binding to HMEC-1 cells
A further series of experiments was performed to investigate the binding of
RANTES to endothelial cells; ligand binding was performed at physiological
salt concentrations to prevent disruption of ionic protein-GAG interactions.
Fig. 7 shows that stimulation
of the cells for 16 hours with TNF- and IFN-
produced a 40%
increase in RANTES binding (P<0.002); this increase was not
observed for cells stimulated with pro-inflammatory cytokines in the presence
of chlorate. Again this shows that the binding of RANTES to HMEC-1 endothelial
cells increases after stimulation with pro-inflammatory cytokines and suggests
that this increase is dependent on GAG sulphation rather than binding to any
specific receptor.
|
Transendothelial leukocyte chemotaxis
A series of experiments was performed to assess the potential of RANTES to
induce vectorial migration of monocytes across monolayers of resting or
cytokine-activated HMEC-1 cells. It was found that neither resting nor
activated endothelium supported efficient leukocyte migration in the absence
of exogenous RANTES applied to the basal surface of the monolayer. However,
the addition of RANTES caused a significant increase in migration across both
resting and cytokine-activated endothelial cell monolayers. Importantly,
comparison of the chemotactic response across resting and cytokine-activated
HMEC-1 showed that the activated monolayer supported the migration of 3.4-fold
more leukocytes per unit area (Fig.
8; P<0.0001).
|
![]() |
Discussion |
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Northern analysis of members of the NDST family showed clearly, and for the
first time, that the pattern of NDST expression varies between different cell
types, with a panel of 6 resting cell lines showing marked differences in
their relative expression of NDST-1 and -2. In this study the gels were loaded
with 100 µg of total RNA or 1 µg of mRNA; these relatively large amounts
of RNA were used in order to detect low levels of transcription. For the
NDST-2 blot, mRNA was used in order to reduce background binding to ribosomal
RNA, which masked the transcripts when large amounts of total RNA were used.
Similar patterns of NDST-1 and -2 expression were observed for the lung and
kidney epithelial cells, but it cannot be concluded that this observation
extends to all epithelial cells. Despite the high level of RNA loading,
neither NDST-1 nor -2 was observed in preparations from resting HMEC-1
endothelial cells. Significantly, RT-PCR failed to detect expression of NDST-3
and -4 in any cell line. This is consistent with a previous study which showed
that the expression of these two genes is highly restricted
(Aikawa et al., 2001).
It is of interest that the transcript for NDST-1 was approximately 9 kb in
length compared to a coding sequence of 2649 bp
(Dixon et al., 1995); this
suggests that the transcript contained extensive untranslated regions (UTRs).
This is consistent with the large 3'-UTRs of up to
5 kb observed
during the sequencing of NDST-1 from a placental cDNA library
(Dixon et al., 1995
). The
coding sequence for NDST-2 is 2652 bp
(Humphries et al., 1998
), but
longer transcripts were again observed, indicating large UTRs. These UTRs may
play a role in the regulation of message stability and expression. Indeed, a
recent report by Grobe and Esko (Grobe and
Esko, 2002
) has highlighted a mechanism for post-transcriptional
regulation of NDST expression in which the 3'-UTRs contain cryptic
translation codons that might reduce the rate of translation. Three bands were
observed on the NDST-2 blots, which is consistent with previous studies
showing multiple NDST-2 transcripts
(Kusche-Gullberg et al., 1998
;
Orellana et al., 1994
;
Toma et al., 1998
). The
observed high level expression of NDST-2 in the liver and mast cell lines is
again consistent with results from a previous study
(Orellana et al., 1994
). The
differing pattern of expression of mRNA encoding NDSTs between the cell lines
is suggestive of variations in NDST function resulting in a change in the
extent of N-sulphation within HS. This may result in differences in the
potential of HS to bind extracellular proteins, leading to tissue-specific
differences in protein function.
The failure of northern blotting to detect NDST expression in HMEC-1
endothelial cells suggests that the messages encoding these enzymes are
normally expressed at a low level or are turned over rapidly, allowing
detection only by RT-PCR. This observation is surprising, as it is known that
HS constitutes up to 50% of the total GAG expressed by endothelial cells
(Cockwell et al., 1996).
However, the endothelium functions as the interface between blood and the
tissues and must be able to respond dynamically to its microenvironment in
order to regulate the passage of cells into sub-endothelial tissues during
inflammatory immune responses (Rix et al.,
1996
). To achieve this it would be valuable for the endothelium to
have a mechanism that enables rapid alteration of its potential to bind a wide
range of pro-inflammatory factors in order to present them at the cell
surface. Regulation (up or down) of the extent of HS sulphation within the
cellular microenvironment would provide a candidate mechanism for increasing
cytokine presentation during episodes of inflammation, and could allow the
effect of these cytokines to be localised.
Analysis of the upstream sequence between bases -1 and -1000 of all four
members of the NDST family (AliBaba 2.1;
http://www.gene-regulation.com)
was performed to detect potential transcription factor binding sites. Five
putative Sp-1 binding sites were identified in the region -1 to -300 and
another at position -970 of the NDST-1 sequence. Analysis of the NDST-2
sequence predicted five Sp-1 sites between -1 and -300, two between -400 and
-500 and a further two between -800 and -900. These sites were not identified
in the sequences of either NDST-3 or -4. The transcription factor Sp-1 is
known to be induced by TNF- and plays an important role in promoting
the expression of vascular cell adhesion molecule-1 (VCAM-1)
(Neish et al., 1995
) and
intercellular adhesion molecule-1 (ICAM-1)
(Voraberger et al., 1991
) by
pro-inflammatory cytokine stimulated endothelial cells. This suggests a
potential mechanism by which NDST-1 and -2 could be upregulated by
pro-inflammatory cytokines.
Evidence presented in the current study implicates NDST upregulation as an
important component of the activation of endothelial cells during episodes of
inflammation. Stimulation of the endothelial cell line HMEC-1 with the
pro-inflammatory cytokines TNF- and IFN-
produced significant
changes in the level of expression of NDST-1 and -2, which correlated with
changes in structure of HS on the cell surface. Interestingly, changes in the
level of expression of NDST appeared to be bi-phasic following the stimulation
of endothelial cells with pro-inflammatory cytokines. Initially the levels
fell slightly, but significant increases in expression were observed after 16
hours; the expression of sulphated 10E4 epitopes on the cell surface was
elevated after 16 hours and continued to increase for up to 72 hours. A
previous study has shown that IFN-
reduces the incorporation of
sulphate into HS immediately after stimulation
(Praillet et al., 1996c
),
whilst the presence of IFN-
has been shown to increase the relative
amount of HS compared to other GAGs, such as chondroitin sulphate
(Praillet et al., 1996a
;
Praillet et al., 1996b
).
Differences between the binding of the 10E4 antibody to resting and
cytokine-activated endothelial cells could not be explained by differential
shedding of GAGs as no significantly enhanced loss of radioactivity was
observed following stimulation of cells that had been pre-labelled with
[3H]glucosamine.
Flow cytometric data showed that pro-inflammatory cytokine-stimulation
produced a change in the epitopic structure of HS at the endothelial cell
surface which is likely to reflect an increase in the overall level of N- and,
ultimately, O-sulphation. Previous studies have shown that overexpression of
NDSTs can result in the production of HS with increased N- and O-sulphation
(Cheung et al., 1996;
Pikas et al., 2000
),
suggesting a direct correlation between NDST expression and the level of HS
sulphation. It is possible that these changes are not limited to HS; indeed, a
recent study has shown that over-sulphation of HS in cells which express high
levels of NDST-1 is associated with decreased sulphation of chondroitin
sulphate (Bengtsson, 2003
). A
previous study has also shown that treatment of endothelial cells with
TNF-
or IFN-
can increase the incorporation of sulphate into HS
within a similar timescale to that observed in the current work, but the
mechanism for this was not defined (Klein,
1992
). Further work will be required to characterise the HS
species produced by cytokine-stimulated HMEC-1, but it is now apparent that
the change in sulphation correlates with an increased ability of the cells to
bind the chemokine RANTES. This is consistent with a demonstration that the
extent of HS sulphation varies between endothelial cells from different sites,
with the highly sulphated HS expressed by bone marrow endothelium having a
high affinity for the chemokine SDF-1
(Netelenbos, 2001
).
The binding of RANTES to HMEC-1 is restricted to cell surface GAGs as these
cells do not express specific receptors for this chemokine. Indeed, the only
specific chemokine receptor that has been demonstrated on endothelial cells is
CXCR4 (Volin et al., 1998),
which binds the chemokine SDF-1; the RANTES receptors CCR2 and CCR5 are
restricted to leukocytes (Mack et al.,
1999
; Qin et al.,
1998
; Sorensen et al.,
1999
), and DARC was expressed at a constant low level. As RANTES
is known to bind to highly sulphated domains on HS
(Mbemba et al., 2001
), it is
most likely that the increased binding of this chemokine observed after
endothelial cell activation is a specific consequence of a
chlorate-inhibitable, heparitinase-sensitive, NDST-mediated increase in cell
surface expression of sulphated HS. This observation has implications for
previous studies that have attempted to measure cell surface chemokine
binding. Variable results from these studies
(Ali et al., 2000
;
Appay et al., 1999
;
Mbemba et al., 2001
) could
simply reflect natural changes in the extent of sulphation of the HS expressed
by particular cell types.
In separate studies our group has shown that the sequestration of
chemokines to cell surface GAGs can increase the biological activity of these
factors and is an essential prerequisite for chemokine-mediated migration of
leukocytes across cell monolayers (Ali et
al., 2002; Ali et al.,
2000
). The current study demonstrated that treatment of the HMEC-1
model endothelium with TNF-
and IFN-
does not increase
transendothelial migration of THP-1 monocytes, but cytokine-activation does
synergise with subendothelial RANTES to induce a greatly enhanced chemotactic
response. It is likely that the activation of endothelial cells by TNF-
and IFN-
is necessary for efficient chemokine sequestration and
presentation by the endothelium, leading to the induction of leukocyte
migration from the apical to the basal surface of the endothelium. Indeed, the
reduced potential of undersulphated HS to bind chemokines to the surface of
resting endothelium could prevent non-specific inflammation from occurring as
a consequence of inappropriate sequestration of circulating chemokines by
normal vascular endothelial cells.
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Acknowledgments |
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References |
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