(Journal of Leukocyte Biology. 2000;68:251-259.)
© 2000
by Society for Leukocyte Biology
Parallel induction of epithelial surface-associated chemokine and proteoglycan by cellular hypoxia: implications for neutrophil activation
Glenn T. Furuta*,
,
Andrea L. Dzus*,
Cormac T. Taylor* and
Sean P. Colgan*
* Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Womens Hospital; and
Combined Program in Pediatric Gastroenterology and Nutrition, Childrens Hospital, and Harvard Medical School, Boston, Massachusetts
Correspondence: Sean P. Colgan, Ph.D., Brigham and Womens Hospital, Center for Experimental Therapeutics and Reperfusion Injury, Thorn 704, 75 Francis Street, Boston, MA 02115. E-mail:
colgan{at}zeus.bwh.harvard.edu
 |
ABSTRACT
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Neutrophil-induced damage to the protective epithelium has been
implicated in mucosal disorders associated with hypoxia, and such
damage may be initiated by epithelial-derived chemokines. Because
chemokines can bind to membrane proteoglycans, we hypothesized that
chemokines may associate with epithelial surfaces and activate
polymorphonuclear neutrophils (PMN). Epithelial hypoxia
(pO2 20 torr) resulted in a time-dependent induction of
interleukin-8 (IL-8) mRNA, soluble protein, as well as surface protein.
Such surface IL-8 expression was demonstrated to be dependent on
heparinase III expression, and extensions of these experiments
indicated that hypoxia induces epithelial perlecan expression in
parallel with IL-8. Finally, co-incubation of post-hypoxic epithelia
with human PMN induced IL-8-dependent expression of the PMN
ß2-integrin CD11b/18. These data indicate that chemokines
liberated from epithelia may exist in a surface-bound, bioactive
form and that hypoxia may regulate proteoglycan
expression.
Key Words: interleukin-8 mucosal disorders hypoxia
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INTRODUCTION
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The pathological hallmark of many acute and chronic intestinal
diseases is the accumulation of polymorphonuclear leukocytes (PMN)
adjacent to crypt epithelial cells of the intestine, termed crypt
abscesses [1
]. Significant tissue damage brought about
by PMN migration across intestinal epithelium has been demonstrated in
a variety of diseases including Crohns disease, ulcerative colitis,
hemorrhagic shock, and reperfusion injury [2
,
3
]. Studies of human mucosae in such diseases suggest
that PMN transepithelial migration predates focal breakdown of the
epithelial surface [2
], and that defective epithelial
barrier function also predates structural discontinuities in the mucosa
[4
]. At present, the pathophysiological factors that
lead to the formation of such inflammatory responses are poorly
understood.
A common feature of many disease processes is tissue hypoxia. Recent
studies have shown that hypoxia induces a number of genes through
diverse mechanisms and likely contributes significantly to inflammatory
processes [5
]. Tissue hypoxia may orchestrate leukocyte
recruitment through induction of chemotactic cytokines (chemokines),
polypeptides that function as leukocyte activators and chemoattractants
in vitro and in vivo [6
].
Chemokines can function as soluble mediators or can specifically bind
to polysaccharide components of cell-surface and extracellular-matrix
proteoglycans [7
]. Proteoglycan binding sites on
chemokines are generally distinct from the receptor binding site,
utilize basic amino acid residues (lysine, arginine, or histidine) for
binding, and proteoglycans can bind multiple chemokine molecules
[8
, 9
]. Some studies have suggested that
chemokine activity is enhanced when bound to matrix components,
particularly the proteoglycan heparan sulfate [7
,
10
], possibly through oligomerization of the chemokine
[8
]. Proteoglycan structure can vary significantly, even
between tissues, and it has been hypothesized that these structural
differences may account for tissue localization of specific chemokines
and, as a result, specific leukocyte populations [11
].
The chemokine interleukin-8 (IL-8) has an established role in mucosal
disease [12
], and intestinal epithelia are primary
sources of IL-8 [13
]. Mechanistic in vitro
studies suggest that epithelial-derived IL-8 serves as a recruitment
signal for PMN. For example, apical colonization of epithelia with
pathogenic bacteria induces the basolateral release of IL-8 and
imprints the extracellular matrix with chemokine [14
].
This insoluble, matrix-associated chemokine serves as a haptotactic
recruitment signal for PMN to the basolateral epithelial surface
[14
, 15
]. Less is known, however, about the
subsequent steps in PMN transmigration (i.e., after initial
PMN-epithelial contact). PMN adhesive interactions to an as yet
unidentified epithelial ligand are mediated primarily by CD11b/18,
whereas transmigration through the epithelial paracellular space is
predominantly CD47-dependent [16
]. This epithelial
paracellular pathway provides a unique environment for leukocyte
movement. Unlike the thin, flat substrate of the vascular endothelium,
transmigration through columnar epithelium mandates that PMN move
greater than two cell lengths through the paracellular space. It is
possible, therefore, that epithelia orchestrate PMN movement through
the paracellular space by providing chemokine in a surface-expressed
fashion. At present, this pathway has not been studied. We demonstrate
here that epithelial cells pre-exposed to cellular hypoxia express
functional IL-8 in a surface-expressed manner. Such induction of
surface chemokine required the parallel activation of both IL-8 release
and the surface up-regulation of perlecan. Thus, under defined
conditions, the epithelial cell surface may provide a haptotactic
substrate for PMN movement.
 |
MATERIALS AND METHODS
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Cell culture
T84 intestinal epithelial cells (passages 6785) were grown and
maintained as confluent monolayers on collagen-coated permeable
supports as previously described in detail [17
].
Monolayers were grown on 0.33-, 5-, or 45-cm2
ring-supported polycarbonate filters (Costar, Cambridge, MA), as
indicated, and utilized 612 days after plating as described
previously [18
].
Epithelial cultures were exposed to hypoxia as previously described
[15
]. Briefly, growth media was replaced with fresh
media equilibrated with hypoxic gas mixture and cells were placed in
the hypoxic chamber (Coy Laboratory Products, Ann Arbor, MI). Oxygen
concentrations were as indicated in torr (normoxia equal to
pO2 of 150 torr) with the balance made up of nitrogen,
carbon dioxide (constant pCO2 35 torr), and water vapor
from the humidified chamber. These culture conditions are non-toxic to
epithelia [15
].
Quantitation of IL-8
Supernatants from normoxic and hypoxic epithelial monolayers
were quantitated for IL-8 secretion with the use of a capture sandwich
enzyme-linked immunosorbent assay (ELISA) and an IL-8 standard curve as
described before [15
]. Values are reported as IL-8 per
106 cells in a 1-mL volume of conditioned supernatant.
Detection of surface-associated IL-8 or surface-associated heparan
sulfate was determined using intact monolayers of normoxic or hypoxic
T84 cells or by flow cytometry of cells that were lifted from the
permeable support. Briefly, confluent T84 cells were grown to
electrical confluence on permeable supports, incubated in normoxia or
hypoxia as indicated. Analysis of surface chemokine or proteoglycan on
intact cell monolayers was performed by ELISA on insert-grown cells
(0.33 cm2) as described previously [19
]
(values are reported as IL-8 per 106 cells relative to a
standard curve). For assessment by flow cytometry, intact monolayers (5
cm2) were transferred to a solution of modified Hanks
balanced salt solution (HBSS; without Ca2+ or
Mg2+) containing 5 mM
ethyleneglycol-bis(ß-aminoethyl ether)-N,
N, N, N-tetraacetate (EGTA, Sigma
Chemical) at 37°C. Monolayers were agitated for 2 h at 37°C
and vigorously washed to remove cells, as described elsewhere
[14
]. Cells elicited in this fashion were cooled to
4°C, blocked with 100 µL media containing 10% bovine calf serum
(BCS) for 1 h at 4°C, followed by addition of rabbit anti-human
IL-8 polyclonal antibody (20 µg/mL in media, Endogen, Boston, MA) or
control rabbit serum (Sigma, 1:500 dilution in media) for 2 h at
4°C. Cells were extensively washed with cold HBSS followed by
addition of fluorescein-conjugated goat anti-rabbit secondary Ab
(1:1000 in media, Cappel, West Chester, PA) and analyzed by flow
cytometry. Control cells to determine background were incubated with
only the secondary antibody. Cells were analyzed for surface expression
of IL-8 or CD11b, as indicated, by flow cytometry as described before
[20
] with a FACScan flow cytometer (Becton Dickinson
Immunocytometry Systems, Mountain View, CA) and CellQuestTM
software.
Transcriptional analysis
The transcriptional profile of epithelial cells exposed to
ambient hypoxia was assessed in RNA derived from control or hypoxic
epithelia (T84 cells at 6 or 18 h hypoxia) using quantitative
genechip expression arrays (Affymetrix) [21
]. Reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA
levels was performed using DNAse-treated total RNA as previously
described [22
] using primers specific for IL-8 (5-ATG
ACT TCC AAG CTG GCC GTG GCT-3 and antisense primer 5-TCT CAG CCC
TCT TCA AAA ACT TCT C-3, 289-bp fragment), epican (5-GCA GAA TGT
GGA CAT GAA GA-3, and antisense primer 5-ATG CTA AAA AAG ATT CGC
AAT G-3, 150-bp fragment), perlecan (5-CTG CCC CTC GTA GGC ACC-3
and antisense primer 5-CAC TCC CAT CCA ACC TGC-3, 231-bp fragment),
syndecan-1 (5-GAA CCA AAT CTG GAA GCC AA-3 and antisense primer
5-GAC ACA GGC ACG ACT GCT T-3, 182-bp fragment), or control
ß-actin [22
] (5-ATG ACT TCC AAG CTG GCC GTG GCT-3
and antisense primer 5-TCT CAG CCC TCT TCA AAA ACT TCT C-3, 661-bp
fragment). Each primer set was amplified using 25 cycles of 94°C for
1 min, 60°C for 2 min, 72°C for 4 min, and a final extension of
72°C for 7 min. The PCR reactions were then visualized on a 1.5%
agarose gel containing 5 µg/mL of ethidium bromide.
Chemokine assessment in biotinylated plasma membrane fractions
Epithelial cells were grown to confluence on 45-cm2
permeable supports, and exposed to hypoxia or normoxia as indicated.
Plasma membranes were isolated using nitrogen cavitation (200 psi, 8
min, 4°C) as previously described [16
]. Plasma
membrane fractions were labeled with biotin (1 mM in HBSS) as
previously described [16
] and re-pelleted by
ultracentrifugation to remove excess biotin. Recombinant human IL-8
(100 ng/mL) was directly biotinylated [1 mM NHS-biotin (Pierce
Chemical, Rockford, IL) in HBSS] and excess biotin was removed by
multiple washes on a 5-kDa cut-off membrane filter (Amicon, Beverly,
MA). Fractions were pre-cleared with 50 µL pre-equilibrated protein-G
Sepharose (Pharmacia, Uppsala Sweden). Immunoprecipitation of IL-8 was
performed with rabbit polyclonal anti-IL-8 followed by addition of 50
µL pre-equilibrated protein-G Sepharose and overnight incubation.
Washed immunoprecipitates were boiled in non-reducing sample buffer
[2.5% sodium dodecyl sulfate (SDS), 0.38 M Tris pH 6.8, 20%
glycerol, and 0.1% bromophenol blue], resolved by non-reducing
SDS-polyacrylamide gel electrophoresis (PAGE; 18% polyacrylamide gel),
transferred to nitrocellulose, and blocked overnight in blocking
buffer. Biotinylated proteins were labeled with streptavidin-peroxidase
(Pierce Chemical) and visualized by enhanced chemiluminescence (ECL;
Amersham, Arlington Heights, IL).
Antisense oligonucleotide treatment of cells
Electrically confluent T84 cells grown as monolayers on
permeable supports were washed three times in warm T84 cell media. IL-8
phosphorothioate antisense oligonucleotide (40 base oligonucleotide)
derived from the 5 untranslated sequence of exon 1
[23
], purchased from Oncogen Sciences (Cambridge, MA),
were diluted to 200 nM in serum-free media containing
N-[1-(2,3-dioleyloxy)propyl]-N, N,
N-trimethylammonium chloride (DOTMA, Lipofectin solution at
10 µg/mL, GIBCO) and applied to cells (150 µL) to the basolateral
surface only. Serum-containing media were used in the opposing well
(i.e., apical surface). Cells were incubated in normoxia or hypoxia, as
indicated, for 24 h followed by addition of 100 µL
serum-containing medium to the basolateral surface. Cells were allowed
to incubate for an additional 24 h, then used as described above
to assess IL-8 secretion or PMN transmigration. Controls consisted of
media only or media containing DOTMA without antisense
oligonucleotides.
Heparinase treatment of intact epithelia
Monolayers of the human intestinal epithelial cell line T84 were
grown as inverted monolayers on permeable supports. Confluent
monolayers were exposed to hypoxia (pO2 20 torr, 48 h), EGTA-elicited (as described above), and incubated in the presence
or absence of heparinase III (1 U/mL, Sigma) for 2 h at 37°C.
Monolayers were extensively washed at 4°C, and processed for flow
cytometry as described above.
Immunoprecipitation of biotinylated heparan sulfate
T84 cells were grown to confluence on 5-cm2
polycarbonate rings, exposed to experimental hypoxia conditions, as
indicated, washed with HBSS followed by labeling of extracellular cell
surface proteins with biotin (NHS-Biotin, 1 mM, Pierce), as described
previously [24
]. Unbound biotin was quenched with
NH4Cl (50 mM) in HBSS. Labeled T84 cells were lysed with
lysing buffer (150 mM NaCl, 25 mM Tris, 1 mM MgCl2, 1%
Triton X-100, 1% Nonidet P-40, 5 mM EDTA, 5 µg/mL chymostatin, 2
µg/mL aprotinin, and 1.25 mM PMSF, all from Sigma). Cell debris was
removed by centrifugation (10,000 g, 5 min). Lysates were
pre-cleared with 50 µL pre-equilibrated protein G-Sepharose
(Pharmacia) for 2 h. Immunoprecipitation of heparan sulfate was
performed by addition of anti-heparan sulfate monoclonal antibody
(clone A7L6, rat IgG2a from Upstate Biotechnology, Lake Placid, NY) for
2 h followed by 50 µL pre-equilibrated protein G-Sepharose
overnight on an end-over-end rotator. Washed immunoprecipitates were
boiled in non-reducing sample buffer (2.5% SDS, 0.38 M Tris, pH 6.8,
20% glycerol, and 0.1% bromophenol blue), separated by SDS-PAGE (10%
linear gel) under non-reducing conditions and transferred to
nitrocellulose through the use of standard protocols. Biotinylated
proteins were labeled with streptavidin-peroxidase and visualized by
enhanced chemiluminescence (ECL; Amersham). Resulting heparan sulfate
bands were quantified from scanned images using NIH Image software
(Bethesda, MD).
PMN transepithelial migration
PMN transmigration into and across confluent intestinal
epithelial monolayers was examined as previously described in detail
[25
, 26
]. Briefly, human PMN were isolated
from normal human volunteers by a gelatin sedimentation technique
[27
] and suspended in modified HBSS (without
Ca2+ and Mg2+, with 10 mM HEPES, pH 7.4, Sigma)
at a concentration of 5 x 107/mL. T84 monolayers,
plated in the inverted position to allow PMN interaction with the
physiologically relevant basolateral surface, were washed free of media
with HBSS (containing Ca2+ and Mg2+).
Transmigration assays were performed by the addition of PMN to the
upper chambers after chemoattractant (1 µM fMLP) was added to the
opposing (lower) chambers. At time 0, 1 x
106 PMN were added and transmigration was allowed to
proceed for 2 h at 37°C. In subsets of experiments, PMN were
pre-incubated with anti-IL-8 R mAb (anti-CXCR-1, clone 42705.111,
subclass IgG2a from R & D Sysytems, Minneapolis, MN) at indicated
concentrations for 30 min at 4°C before addition to epithelia. All
experiments were performed at 37°C. PMN migration into epithelial
monolayers was quantified by assaying for the PMN azurophilic granule
marker myeloperoxidase (MPO) as described previously
[26
]. After each transmigration assay,
nonadherent/loosely adherent PMN were extensively washed from the
surface of the monolayer and PMN cell equivalents, estimated from a
standard curve, were assessed as the number of PMN associated with the
monolayer. Previous studies have clearly demonstrated that the
morphological location of monolayer-associated PMN under these
conditions is within the epithelial paracellular space and not adherent
to the permeable support or the apical membrane surface
[25
, 28
].
PMN CD11b/18 expression
The influence of epithelia on PMN CD11b/18 was assessed by flow
cytometry during co-incubation of PMN with intestinal epithelial cells.
Briefly, 2.5 x 106 PMN were co-incubated with
1.25 x 107 T84 cells (pre-exposed to hypoxia or
hypoxia with added antisense oligonucleotides) for 20 min at 37°C.
Cells were immediately washed in HBSS, and incubated with anti-CD11b
monoclonal antibody (clone 44a, subclass IgGsa obtained from American
Type Tissue Collection) followed by FITC-conjugated secondary antibody.
CD11b/18 was analyzed on PMN from co-incubations by gating flow
cytometer on PMN.
Data presentation
ELISA data were compared by analysis of variance (ANOVA) or by
Students t test. Values are expressed as the mean and
SEM of n experiments. Flow cytometry data were
expressed as mean fluorescence intensity (MFI) and analyzed by the
Kolmogorov-Smirnov (K-S) two-sample test.
 |
RESULTS
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Induction of soluble and surface IL-8 by hypoxia
Epithelia are demonstrated sources of bioactive chemokines
[13
]. Here, we explored the possibility that hypoxia
induces epithelial surface IL-8. T84 cells grown on permeable supports
were exposed to ambient oxygen tension (pO2 150 torr,
72 h) or hypoxia (pO2 20 torr, 1272 h). Cell culture
supernatants were harvested, pooled, and assayed for IL-8 by capture
ELISA, and washed monolayers were examined for expression of IL-8 by
cell ELISA. As shown in Figure 1A
, and consistent with previous data [15
], hypoxia
induced a time-dependent increase in soluble IL-8 production
(P < 0.001 by ANOVA), with maximal production at
48 h. Similarly, as shown in Figure 1B
, monolayers grown in this
fashion revealed a parallel increase in surface-bound
(monolayer-associated) IL-8 (P < 0.01 by ANOVA) and
mRNA induction by hypoxia (maximal at 12 h hypoxia, see Fig. 1C
).

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Figure 1. Hypoxia induces soluble and monolayer-associated IL-8, and IL-8 mRNA.
Monolayers of the human intestinal epithelial cell line T84 were grown
on polycarbonate permeable supports. Confluent monolayers were exposed
to hypoxia (pO2 20 torr, 048 h). (A) Soluble supernatants
were collected from the basolateral surface and assayed for IL-8 by
capture ELISA. (B) Washed monolayers were assessed for
monolayer-associated IL-8 by ELISA. Results represent the mean ±
SEM for 1012 individual monolayers in each condition.
Values are reported as IL-8 per 106 cells relative to a
standard curve. (C) RT-PCR was used to determine IL-8 message levels in
response to hypoxia. After exposure to indicated periods of hypoxia,
total RNA was isolated, DNAse I treated, and amplified by RT-PCR using
IL-8 specific primers. As indicated, lanes represent (from left to
right); normoxia, 2-, 6-, 12-, and 24-h exposure to hypoxia,
respectively. In the bottom panel, corresponding ß-actin mRNA
expression are shown to demonstrate equivalent loading. Data shown are
representative of three experiments.
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|
It has been previously demonstrated that soluble chemokines bind to
cell matrix proteins, and that such bound chemokine can serve as a
recruitment signal for PMN [14
, 15
]. Thus,
it is possible that the cell-associated IL-8 demonstrated in Figure 1B could reflect matrix-associated IL-8. To circumvent this potentially
confounding issue, cells pre-exposed to hypoxia were elicited from
permeable supports using EGTA and examined for surface-bound chemokine
by flow cytometry. As shown in Figure 2
, hypoxia induced a time-dependent induction (P <
0.01 by ANOVA) of surface-bound IL-8 examined under these conditions,
with maximal expression at 48 h of hypoxia (MFI of 130.5 vs. 8.9
for 48 h hypoxia and control normoxia, respectively,
P < 0.01). It is important to note that control
normoxic cells expressed significant cell-surface IL-8 (MFI of 8.9 vs.
2.2 for normoxia and background control, respectively,
P < 0.05).

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Figure 2. Evaluation of epithelial membrane-associated IL-8. Monolayers of T84
cells were grown on polycarbonate permeable supports. Confluent
monolayers were exposed to hypoxia (pO2 20 torr, 048 h,
as indicated). (A) Surface IL-8 was assessed from EGTA-elicited
epithelial cells by flow cytometry. Also indicated is the secondary
antibody only control (2° only). (B) Plasma membrane preparations
were generated from epithelial cells pre-exposed to indicated hypoxia.
Membranes were biotinylated, re-pelleted, and IL-8 was
immunoprecipitated from solubilized fractions. Washed
immunoprecipitates were boiled in non-reducing sample buffer, resolved
by non-reducing SDS-PAGE (18% polyacrylamide gel), transferred to
nitrocellulose, and blocked overnight in blocking buffer. Biotinylated
proteins were probed with streptavidin-peroxidase and visualized by
enhanced chemiluminescence (ECL). Also indicated is a biotinylated IL-8
standard for reference. Results are representative of four experiments
(A) and three experiments (B).
|
|
As further verification for hypoxia-elicited induction of surface
expressed chemokine, plasma membrane fractions from control normoxic
cells, and cells pre-exposed to hypoxia (24 or 48 h) were probed
for IL-8 through the use of immunoprecipitation of biotinylated
protein. As shown in Figure 2B
, a clear induction of IL-8 is evident in
plasma membrane fractions from hypoxic cells (densitometry values of
31.5 ± 8.8 and 63.2 ± 14.5 relative densitometry units for
24 and 48 h hypoxia, respectively, compared to 0.6 ± 2.2 for
controls, P < 0.05, n = 3
experiments). Similar analysis of biotinylated recombinant IL-8 (100 ng
total protein) treated in the same manner revealed a corresponding band
at a similar molecular weight. These data indicate the likelihood that
a fraction of hypoxia-elicited IL-8 remains tightly associated with the
epithelial plasma membrane.
Influence of IL-8 antisense oligonucleotides on cell-surface
chemokine
To better define the role of hypoxia in induction of IL-8
surface-bound chemokine, IL-8 mRNA was blocked in hypoxic cells with
antisense oligonucleotide probes. As we have demonstrated previously
[15
], the conditions for loading oligonucleotides do not
influence cell viability (judged morphologically and assessed by
transepithelial resistance, a sensitive measure of epithelial
viability, data not shown). As shown in Figure 3
, hypoxic epithelia incubated with IL-8 antisense oligonucleotides
for 48 h result in significantly decreased surface IL-8 (MFI of
620.5 vs. 12.8 for 48 h hypoxia and 48 h hypoxia pre-loaded
with IL-8 anti-sense oligonucleotides, respectively, P < 0.001). As a control, pre-loading epithelia with a scrambled
oligonucleotide did not influence surface IL-8 expression (data not
shown). No differences were observed between control normoxia cells and
48 h hypoxia pre-loaded with IL-8 anti-sense oligonucleotides (MFI
of 9.3 vs. 12.8, respectively, P = not significant).
These data indicate that IL-8 antisense oligonucleotides prevent
hypoxia-induced surface IL-8.

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Figure 3. Pre-loading epithelia with IL-8 antisense oligonucleotides (ASO) blocks
hypoxia-induced surface IL-8. Monolayers of T84 cells were grown on
polycarbonate permeable supports. Confluent monolayers were pre-loaded
with IL-8 ASO (200 nM) or mock treated with DOTMA followed by exposure
to hypoxia (pO2 20 torr, 48 h, as indicated). Surface
IL-8 was assessed from EGTA-elicited epithelial cells by flow
cytometry. Also indicated is the secondary antibody only control (2°
only) and the normoxic epithelial control (CTL). Results are
representative of three experiments.
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|
Role of heparan sulfate in cell-surface IL-8
Chemokines such as IL-8 bind with high specificity to the
polysaccharide components of cell-surface and extracellular-matrix
proteoglycans, particularly heparan sulfate proteoglycans
[7
]. Thus, we determined whether hypoxia-induced
cell-surface IL-8 was diminished by enzymatic degradation of heparan
sulfate on intact cells. As shown in Figure 4
, heparinase III significantly decreased hypoxia-induced IL-8
expression (MFI of 54.6 vs. 280.5 in the presence and absence of
heparinase III treatment, respectively, P < 0.025).
Similar treatment of normoxic cells (Fig. 4
, CTL) also decreased
surface IL-8 staining (MFI of 3.4 vs. 3.9 for normoxic control treated
with heparinase and 2° Ab only, respectively, P = not
significant), suggesting that baseline surface expression of IL-8 in
normoxic cells is likely mediated by heparan sulfate. These data
suggest that IL-8 binds to a surface component consistent with heparan
sulfate.

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Figure 4. Heparinase treatment of epithelia decreases surface expression of IL-8
elicited by hypoxia. Monolayers of the human intestinal epithelial cell
line T84 were grown on polycarbonate permeable supports. Confluent
monolayers were exposed to hypoxia (pO2 20 torr, 48 h), EGTA-elicited (as described above), and incubated in the presence
or absence of heparinase III (1 U/mL) for 2 h at 37°C.
Monolayers were extensively washed at 4°C, and assessed for
surface-expressed IL-8 by flow cytometry. Also indicated is the
secondary antibody-only control (2° only) and the normoxic epithelial
control (exposed to similar conditions of heparinase, CTL). Results are
representative of three experiments.
|
|
Hypoxia induces epithelial proteoglycans; role for intracellular
cAMP
Increased cell-surface IL-8 expression may be only in part
explained by hypoxia-induced IL-8. For instance, conditions other than
hypoxia which induce soluble IL-8 (e.g., phorbol myristic acetate, 10
ng/mL) [15
] do not result in increased surface IL-8
(data not shown), suggesting additional factors in this observation. It
is possible, therefore, that hypoxia could influence expression of
surface proteoglycans, providing a dual mechanism for increased surface
IL-8 accumulation (i.e., parallel induction of both IL-8 and
proteoglycan). A transcriptional profiling approach was adopted to
identify induction of proteoglycans that might contribute to
surface-expressed chemokine. Microarray analysis [29
]
was utilized to broadly screen hypoxia-regulated genes in RNA derived
from intestinal epithelia (T84 cells). This approach identified a
time-dependent induction of the epican (3.2- and 5.4-fold increase over
control normoxia at 6 and 18 h hypoxia, respectively) and perlecan
(1.6- and 3.3-fold increase at 6 and 18 h hypoxia, respectively),
but no influence on syndecan (0.9- and 1.1-fold increase at 6 and
18 h hypoxia, respectively). RT-PCR analysis was employed to
verify these microarray results (Fig. 5A
) and revealed a time-dependent induction of epican and perlecan
mRNA expression, but no obvious change in syndecan expression.
Immunoprecipitation of perlecan from surface biotinylated preparations
was performed on T84 cells exposed to hypoxia (048 h), conditions
that result in increased surface IL-8 expression (Fig. 1
and 2)
. As
shown in Figure 5B
, increased expression of two proteins consistent
with perlecan (50 and 90 kDa) [30
] were observed with
increasing time of hypoxia. Both the
50- and
90-kDa proteins were
present in control normoxic cells, but significantly induced by hypoxia
at 3648 h (determined by densitometry, increase of 3.7 ± 0.9
and 4.2 ± 1.1 for the 50- and 90-kDa proteins, respectively,
P < 0.02 for both). The
68-kDa protein associated
with 24- and 36-h hypoxic cells was inconsistently observed.

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Figure 5. Hypoxia induces mRNA and surface expression of proteoglycans. T84
monolayers were cultured to confluency on 5-cm2 permeable
supports and exposed to indicated periods of hypoxia (pO2
20 torr). (A) Total RNA was isolated and examined for perlecan, epican,
and syndecan transcript by RT-PCR. ß-Actin transcript was used as an
internal control. (B) Cells were cooled to 4°C and labeled (apical
and basolateral) with biotin followed by immunoprecipitation of heparan
sulfate using clone A7L6 mAb. Immunoprecipitates were subjected to
SDS-PAGE on a 10% polyacrylamide gel followed by Western blotting,
incubation with streptavidin-peroxidase, and development by enhanced
chemiluminescence. Prominent 50- and 90-kDa bands are observed (open
arrows). The 68-kDa protein associated with 24 and 36 h hypoxic
cells was inconsistently observed. Results are representative of three
experiments.
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We and others have previously defined a role for intracellular cAMP in
regulation of gene expression by hypoxia [22
,
31
32
33
]. This cAMP influence maps to genes that bear a
cAMP response element (CRE) [22
, 34
]. Our
analysis revealed that the cloned perlecan gene [30
]
bears a previously unappreciated CRE (TGACGTGG). Similar analysis of
epican did not reveal such a CREB binding site (data not shown).
Decreased intracellular cAMP is commonly associated with cellular
hypoxia [35
]. Moreover, previous studies have determined
that elevation or maintenance of cAMP levels in hypoxic cells can
prevent induction of CRE-bearing gene products [22
,
31
, 36
]. Thus, we determined whether
co-treatment of epithelia with hypoxia and the adenylate cyclase
agonist forskolin reversed the influence of hypoxia on induction of
heparan sulfate. As shown in Figure 6
, ELISA analysis of surface perlecan revealed that increasing
concentrations of forskolin resulted in decreased hypoxia-elicited
heparan sulfate expression (64 ± 13% decrease with 10 µM
forskolin, P < 0.025). Similar to previous results
with rat glomerular epithelia [37
], forskolin suppressed
T84 cell surface expression of perlecan in normoxic cells (45 ±
7% decrease with 10 µM forskolin, P < 0.05). These
data indicate that hypoxia induces epithelial perlecan in a
cAMP-regulated fashion.

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Figure 6. Hypoxia-induced surface expression of perlecan is diminished by the
cAMP agonist forskolin. Monolayers of the human intestinal epithelial
cell line T84 were grown on polycarbonate permeable supports. Confluent
monolayers were exposed to normoxia (pO2 150 torr, 48 h) or hypoxia (pO2 20 torr, 48 h) in the presence or
absence of indicated concentrations of the adenylate cyclase agonist
forskolin. Washed monolayers were assessed for monolayer-associated
perlecan by ELISA. Results represent the mean ± SEM
for 810 individual monolayers in each condition.
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Bioactivity of surface-expressed IL-8
We next defined whether hypoxia-induced, epithelial surface IL-8
was functional. To do this, two experimental approaches were used.
First, we determined whether hypoxia influenced PMN accumulation within
epithelial monolayers, and whether anti-IL-8 receptor antibodies would
influence such responses. As shown in Figure 7A
, and consistent with previous studies [15
],
exposure of T84 epithelial monolayers to hypoxia and subsequent
assessment of PMN accumulation within washed monolayers revealed
significantly increased PMN migration into epithelial monolayers
(4.9 ± 0.88 x 104 for normoxic controls vs.
13 ± 1.20 x 104 PMN for hypoxia,
P < 0.001). Previous morphological studies with this
model system have demonstrated that PMN accumulate within the
paracellular space of epithelia [25
, 38
].
Pre-incubation of PMN with anti-IL-8 receptor antibodies resulted in a
concentration-dependent inhibition of PMN accumulation in monolayers
pre-exposed to hypoxia (ANOVA, P < 0.01) but not
normoxia (ANOVA, P = not significant).

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Figure 7. Functional assessment of hypoxia-induced surface IL-8. (A) Monolayers
of the T84 cells were grown as inverted monolayers (i.e., basolateral
surface upward) on permeable supports. Confluent monolayers were
exposed to normoxia or hypoxia (pO2 20 torr, 48 h) and
reoxygenated with buffer containing PMN (1 x
106/monolayer) pre-incubated with indicated concentrations
of anti-IL-8 R mAb (CXCR-1). PMN were allowed to migrate for 2 h
at 37°C. Transmigration was quantitated by assaying for
monolayer-associated (the number of PMN that remain firmly associated
with washed monolayers) PMN myeloperoxidase. Results represent the
mean ± SEM for seven to nine individual monolayers in
each condition. (B) Monolayers of T84 cells were grown on polycarbonate
permeable supports. Confluent monolayers were pre-loaded with IL-8 ASO
(200 ng/mL) or mock treated with DOTMA followed by exposure to hypoxia
(pO2 20 torr, 48 h). Cells were EGTA-elicited and
co-incubated with PMN (5:1 epithelia-to-PMN ratio) for 15 min at
37°C. Washed co-incubations were assessed for PMN CD11b/18 (Mac-1) by
gating on PMN with flow cytometry. Also indicated is the secondary
antibody-only control (2° only) and baseline PMN Mac-1 expression
(basal). Results are representative of three experiments.
|
|
As a second analysis, we determined whether co-incubations of PMN and
epithelia bearing surface IL-8 would influence PMN CD11b/18, because
IL-8 is known to up-regulate this molecule on PMN [39
].
Epithelia were pre-exposed to conditions that increase surface IL-8 (48
h hypoxia) or to similar conditions in the presence of antisense IL-8
(to block increased surface IL-8, see Fig. 3
). Cells were lifted from
permeable support membranes (see Materials and Methods) and incubated
with PMN at a 5:1 epithelia-to-PMN ratio. CD11b/18 expression on PMN
was examined by flow cytometry (gating on PMN). As can be seen in
Figure 7B
, CD11b/18 expression was induced by more than 10-fold by
incubation with epithelia pre-exposed to hypoxia (MFI of 11.9 vs. 160.7
for basal and epithelial-exposed PMN, respectively, P < 0.01). These results represent a shift in magnitude approximating
that induced by 10 ng of recombinant human IL-8 (MFI 145.6 ,
n = 4, P < 0.01 compared to
unactivated PMN). Moreover, this shift was significantly diminished
when PMN were incubated with cells pre-exposed to the combination
hypoxia and IL-8 antisense oligonucleotides (MFI of 58.9 vs. 160.7 in
the presence and absence of IL-8 antisense oligonucleotides,
respectively, P < 0.025), indicating that induction of
PMN CD11b/18 is, at least in part, dependent on surface IL-8.
Experiments addressing whether epithelia and/or PMN release soluble
IL-8 during such co-culture revealed no detectable IL-8 in co-culture
supernatants (examined by ELISA, lower limit of detection <10 pg/mL,
data not shown). Taken together, these experiments indicate that
hypoxia-elicited, epithelial-associated IL-8 modulates PMN-epithelial
interactions and that surface-associated chemokine may activate PMN in
this setting.
 |
DISCUSSION
|
|---|
Chemokine and growth factor binding to extracellular matrices
provides a mechanism for recruitment and activation of leukocytes at
tissue sites. Many cell types also express surface proteoglycans,
although less is understood regarding their role in binding chemokines
and whether such cell surface chemokine is functional in the setting of
complex tissue architechture. We report here that epithelial hypoxia
activates parallel induction of IL-8 and proteoglycan mRNA and surface
expression. Extensions of these initial findings reveal that
hypoxia-elicited induction of surface-tethered chemokine functionally
recruits and activates PMN localized within epithelial paracellular
space.
Leukocyte recruitment to tissue sites is highly ordered. After
extravasation from the vascular space, leukocytes migrating within
mucosal tissue respond to locally generated signals for successful
transit to the epithelium. These signals may exist in soluble forms
(e.g., fMLP) or may present themselves bound to matrix proteoglycans
(e.g., chemokines). Given its high negative charge, heparan sulfate
readily interacts with proteins and such interactions are increasingly
implicated in a variety of physiological processes [10
].
Previous studies, for instance, have indicated that chemokine
bioactivity in vitro may not be predictive in
vivo [11
], suggesting that additional molecules
contribute to chemokine function. A likely mechanism is the selective
association of chemokines with tissues specialized to recruit specific
leukocyte subpopulations. We have previously demonstrated that
epithelial hypoxia activates basolateral release of IL-8, and that IL-8
binds to epithelial extracellular matrix (i.e., is detected in
acellular monolayers) [15
]. The findings of chemokine
binding to proteoglycans are not unique. A number of studies have
directly demonstrated chemokine binding to glycosaminoglycan
subpopulations on numerous cell types [7
,
9
]. In some cases, proteoglycan-bound chemokine may show
enhanced bioactivity relative to the soluble forms of the molecule
[7
], and in one published finding, the chemokine
interferon-
-inducible protein-10 (IP-10) was demonstrated to use
proteoglycans as a signal-transducing receptor [40
].
Similarly, nuclear magnetic resonance analysis of IL-8-IL-8 receptor
complexes revealed that the heparin-binding residues of IL-8 are
exposed, thus allowing for the distinct possibility that surface
proteoglycans can present chemokine in a form that binds to the
receptor [41
]. Our studies indicate that epithelial
surface-bound IL-8 recruits PMN to the basolateral surface of intact
epithelia and that such surface-tethered chemokine provides a pathway
for induction of PMN CD11b/18 expression. We do not know, however,
whether chemokine binding to surface proteoglycan enhances chemokine
activity. Accurate estimates of chemokine concentrations expressed on
the cell surface are difficult to obtain with this model, limiting the
direct comparison of PMN CD11b/18 activation by soluble IL-8. Thus, at
the present time, we do not know whether IL-8 binding to epithelial
surface heparan sulfate enhances IL-8 bioactivity.
Previous studies indicate that hypoxia induces chemokine generation in
endothelia [42
] and in epithelia [15
]. At
present the mechanism(s) of such activation are not certain, but likely
involves activation mediated by the transcription factors cAMP response
element binding protein (CREB) [22
] and nuclear
factor-
B [22
, 42
]. Indeed, our analysis
of the cloned IL-8 gene [23
] revealed that it too bears
a CRE (TTTCGTCA) 150 base pairs upstream from the TATA signal in the
human IL-8 gene. Consistent with this observation, it was recently
revealed that cellular hypoxia induces activation of a number of genes
bearing CREB binding sites at or near the promoter [22
,
34
], including E-selectin [32
], tumor
necrosis factor
[22
, 31
], COX-2
[43
], and MHC class II [22
,
31
]. A similar hypoxia-induced activation pattern was
observed with perlecan, which also bears a CRE (TGACGTGG) upstream of
the transcription start site [30
]. Moreover, perlecan
has been demonstrated to be cAMP responsive [37
]. Of
note, transcriptional analysis also revealed that epican is prominently
induced by hypoxia (Fig. 5)
, although mechanisms of such induction
remain unclear. Taken together, these studies indicate the likelihood
that hypoxia activates a cAMP-regulated IL-8 and perlecan expression in
epithelia.
A strong correlation between reperfusion injury and the acute
inflammatory response exists [3
]. The direct role of PMN
has been implicated in tissue damage resulting from reperfusion injury,
although mechanisms remain only partially understood
[3
]. In the intestine for instance, PMN accumulation at
the level of the epithelium has been shown to play a central role in
mucosal injury during intestinal reperfusion injury
[44
45
46
]. In disorders such as Crohns disease,
significant evidence exists that ischemia may substantially contribute
to tissue damage [47
48
49
]. Thus, it seems likely that
intestinal ischemia and PMN-epithelial interactions may be related
events. Here, we demonstrate that hypoxia-induced surface IL-8
functionally activates PMN CD11b/18 expression. This observation has
significance for a number of reasons. First, CD11b/18 has been
extensively studied as an important adhesion molecule for PMN adhesion
to, and transmigration across intestinal epithelia [50
],
and that hypoxia enhances PMN transmigration. Second, although we have
previously demonstrated that PMN transmigration across epithelia
pre-exposed to hypoxia is CD11b/18-dependent [15
], the
ligand for CD11b/18 on epithelia remains elusive. Third, the
proteoglycan heparin (a glycosaminoglycan structurally related to
heparan sulfate) is a demonstrated ligand for PMN CD11b/18
[51
], thus it is tantalizing to speculate that heparan
sulfate may also serve as a surface ligand for PMN CD11b/18. Using
monoclonal antibodies directed against perlecan, we have not observed
inhibition of PMN transmigration (data not shown). However, this
approach is limited by the fact that these monoclonals may not bind to
the perlecan epitope necessary to block function.
In conclusion, epithelial exposure to hypoxia activates the
cAMP-regulated induction of both heparan sulfate and IL-8, resulting in
the expression of a surface-bound form of chemokine that functionally
activates PMN migration and CD11b/18. Thus, these studies contribute to
the growing body of evidence that cAMP-responsive genes may play an
important role in mediating crosstalk between hypoxia and inflammatory
events.
 |
ACKNOWLEDGEMENTS
|
|---|
This work was supported by National Institutes of Health Grants
DK-50189, HL-60569, DK-02564, DK-02682, project 3 of PO-1 DE 13499, and
by a grant from the Crohns and Colitis Foundation of America.
Received January 12, 2000;
revised March 31, 2000;
accepted April 10, 2000.
 |
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