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Section of Allergy and Clinical Immunology, Department of Medicine, University of Wisconsin, Madison, WI 53792
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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+ IL-4 (82.2 ± 4.2% positive cells) >
TNF- (41.8 ± 5.1%) > IL-1ß (20.8 ± 4.7%). IL-4
alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion
to cytokine-treated HPMEC followed the same order as that observed for
VCAM-1 expression. Interestingly, EOS migration across cytokine-treated
HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to,
HPMEC; IL-1ß (21.2 ± 1.4% migration) > TNF- (12.6 ±
2.6%) > TNF- + IL-4 (9.1 ± 2.0%). EOS adhesion was greatest
with TNF- + IL-4-treated HPMEC, was dependent on VCAM-1, and
inhibited with anti-4 integrin mAb (67.7
± 7.5% inhibition, p < 0.0005). In contrast, the
highest EOS migration occurred across IL-1ß-treated HPMEC and was
inhibited by anti-ß2 integrin mAb (40.4 ± 2.5%
inhibition, p < 0.005). Viable HPMEC were required
for EOS migration but not adhesion. Our results suggest that EOS
adhesion and transmigration are differentially regulated by VCAM-1 and
ICAM-1 expression and the interaction of these adhesion proteins with
their respective counterligands, i.e., 4 and
ß2 integrins on EOS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-4, the
expression of ICAM-1 and VCAM-1 is increased (3, 4, 5, 6, 7). It is proposed
that EOS then bind to these endothelial adhesion proteins by their
counterligands: ß2 integrins,
Lß2 (LFA-1) and
Mß2 (Mac-1), for ICAM-1, and
4 integrins, 4ß1 (very late
Ag (VLA)-4) and 4ß7, for VCAM-1 (8, 9).
Since all leukocytes utilize the ß2 integrin/ICAM-1
pathway for adhesion and transmigration (3, 4, 5, 10, 11), selective
recruitment of EOS to the airway occurs by other pathways, such as
4 integrin/VCAM-1 interaction, since the
4 integrin is constitutively expressed on EOS but not on
neutrophils (12, 13). Anti-4 integrin or anti-VCAM-1
mAb have been shown to inhibit EOS, but not neutrophil, adhesion to
cytokine-treated endothelial cells (11, 14). The contribution of
4 integrin/VCAM-1 pathway in EOS transendothelial
migration, however, remains to be fully defined (5, 7, 15, 16).
The importance of the 4 integrin/VCAM-1 interaction and
the contribution of this complex to EOS migration have been largely
derived from HUVEC, a macrovascular, nonpulmonary cell (6, 7, 11, 12, 13, 14, 15).
Since EOS participation in allergic inflammation involves small
vessels, we propose that human pulmonary microvascular endothelial cell
(HPMEC) monolayers are likely to be more relevant than HUVEC as an in
vitro model and to reflect leukocyte trafficking to the lung; this
conclusion is based on information that macro- and microvascular
endothelial cells have considerable heterogeneity in their biologic
properties (17). For example, VCAM-1 is selectively induced by IL-4 in
HUVEC but not microvascular endothelial cells from skin or intestine
(18, 19). Furthermore, although cytokine-regulated adhesion molecule
expression is well documented in HUVEC (20), there is limited
information on adhesion molecule expression by HPMEC (21). This report
determines the cytokine regulation of ICAM-1 and VCAM-1 expression on
HPMEC, the influence of these molecules on EOS adhesion and
transmigration, and the involvement of 4 and
ß2 integrins in these two processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Percoll was purchased from Pharmacia (Uppsala, Sweden).
HBSS, RPMI 1640 medium, PBS, newborn calf serum (NCS), FCS,
trypsin-EDTA, L-glutamine, and penicillin-streptomycin
were obtained from Life Technologies (Grand Island, NY). Plasma
fibronectin was obtained from Armour Pharmaceutical (Tuckahoe, NY).
Recombinant human IL-1ß, TNF-, and IL-4 were purchased from R&D
Systems (Minneapolis, MN). Other reagents were purchased from Sigma
(St. Louis, MO) unless otherwise stated.
Cell culture
HPMEC cryopreserved as tertiary or quaternary cultures were purchased from Clonetics (San Diego, CA). These cells were isolated from the vasculature surrounding the alveolar sacs and were characterized as endothelial cells by Clonetics for acetylated low density lipoprotein uptake, factor VIII-related Ag expression, and positive staining for platelet endothelial cell adhesion molecule (PECAM)-1 (CD31) and Matrigel. Endothelial cell basal culture medium supplemented with 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 12 µg/ml bovine brain extract, and 5% FBS was obtained from Clonetics. To promote HPMEC attachment and growth, all culture surfaces were precoated with 10 µg/ml fibronectin for 1 h at 37°C. Cells were passaged before they reached confluence. The profile of adhesion molecules on HPMEC has been determined to be equivalent between passages 3 and 12 (21). Cells derived from two different donors were used at passages 5 through 9 in this study and found to give equivalent results.
Expression of ICAM-1 and VCAM-1 on HPMEC
The expression of ICAM-1 and VCAM-1 on cytokine-treated HPMEC
was examined by flow cytometric analysis. HPMEC were seeded into
fibronectin-coated six-well tissue culture plates (Corning, Corning,
NY), and preconfluent monolayers were incubated with medium, IL-1ß,
TNF-, IL-4, or TNF- + IL-4 for the indicated times. Cultures were
washed twice with HBSS, and cells were detached with 25 mM
HEPES-buffered PBS containing 10 mM disodium EDTA for 40 min at 37°C.
The cells were washed, resuspended in PBS with 0.2% sodium azide and
2% BSA, and stained with phycoerythrin (PE)-conjugated mouse
anti-human ICAM-1 mAb (clone: LB-2, IgG2b, Becton Dickinson,
San Jose, CA) and/or FITC-conjugated mouse anti-human VCAM-1 mAb
(clone: 1.G11B1, IgG1, Southern Biotechnology Associates, Birmingham,
AL) for 30 min on ice. Mouse IgG2-PE and IgG1-FITC (Becton Dickinson)
were used as isotype controls. Labeled cells were washed and analyzed
on a Becton Dickinson FACScan flow cytometer after propidium iodide
staining. Initial gating was done using forward scatter and propidium
iodide fluorescence to eliminate dead cells and debris. Quadrant lines
were determined from the IgG2-PE and IgG1-FITC fluorescence dot plot.
At least 5000 events were collected for each sample, and the percentage
of cells positive for ICAM-1 and/or VCAM-1 was determined.
IL-4 bioassay
Bioactivity of IL-4 was confirmed by CD23 induction on B lymphocytes (CD19) (22). Briefly, PBMC were separated from healthy subjects using histopaque-1077 (Sigma) and incubated for 48 h at 37°C, 5% CO2, with different concentrations of IL-4 in enriched medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin). Cells were washed and two-color stained with FITC-conjugated mouse anti-human CD23 mAb (clone: B-G6, IgG1, Biosource International, Camarillo, CA) and PE-conjugated mouse anti-human CD19 mAb (clone: 4G7, IgG1, Becton Dickinson). Mouse IgG1-FITC and IgG1-PE (Becton Dickinson) were used as isotype controls. The percentage of CD19-positive cells also expressing CD23 was determined by flow cytometry.
Human subjects
EOS were isolated from the peripheral blood of subjects with allergic airway disease such as allergic rhinitis and mild asthma. Subjects ranged in age from 22 to 60 years, and gender distribution was equal. Immediate hypersensitivity was confirmed by at least one positive skin reaction (>3 mm), by the prick-puncture technique, to extracts of common allergens, including ragweed, house dust mite, grass pollen, cat dander, and dog dander. Except for as needed inhaled ß agonists, subjects were taking no medications at the time of study. Informed consent was obtained before participation in the study, and the study was approved by the University of Wisconsin Human Subjects Committee.
EOS separation
EOS were isolated using negative immunomagnetic bead selection, as previously described (23). Briefly, heparinized blood was diluted with HBSS without Ca2+ and centrifuged for 20 min at 700 x g over 1.090 g/ml Percoll. Plasma, mononuclear cell band, and Percoll were removed, and the RBCs in the pellet were lysed by hypotonic shock. The resulting granulocytes were washed with 4°C HBSS supplemented with 2% NCS (HBSS/NCS). Concurrently, purified anti-CD16 from the mouse myeloma clone 3G8 (a generous gift from Dr. David M. Segal, National Cancer Institute, Bethesda, MD) was incubated with goat anti-mouse IgG-coated magnetic beads (PerSeptive Biosystems, Framingham, MA) for 1 h. The anti-CD16-bound beads were washed and incubated with granulocytes for 40 min at 4°C. Steel wool (size 00) columns prepared in 10-ml syringes were soaked 2 to 4 h in ethanol, washed with HBSS, and filled with 4°C HBSS/NCS. The cells and magnetic beads mixture was filtered through the column in a magnetic field (MACS system, Miltenyi Biotec, Auburn, CA) to remove neutrophils bound to magnetic beads. CD16-negative EOS (>98% purity and >99% viability) were collected, washed, and resuspended in enriched medium.
EOS adhesion assay
EOS adhesion to confluent HPMEC monolayers was determined by measuring the residual EOS peroxidase (EPO) activity of adherent cells, as previously described (24). Briefly, EOS (1 x 105/ml in enriched medium) were placed onto cytokine-treated HPMEC monolayers and incubated for 60 min at 37°C. Plates were vigorously washed with 37°C HBSS. Then 100 µl of enriched medium was added to the reaction wells, and 100 µl of the original EOS suspension was added to empty wells to determine a standard EPO curve. EPO substrate (1 mM H2O2, 1 mM o-phenylenediamine dihydrochloride, and 0.1% Triton X-100 in 55 mM Tris buffer, pH 8.0) was then added to all wells. After a 30-min incubation at room temperature, 50 µl of 4 M H2SO4 was added to stop the reaction. Absorbance was measured at 490 nm in a microplate reader (Bio-Tek Instruments, Winooski, VT). Percent EOS adhesion was calculated from the log-dose response curve.
Transendothelial migration of EOS
HPMEC (2.5 x 105 cells/ml) were cultured on fibronectin-coated transwell inserts (6.5- or 12-mm diameter polycarbonate membrane with 3-µm pores, Costar, Cambridge, MA). Medium was added into the upper compartment only to inhibit the formation of a HPMEC bilayer. HPMEC monolayers formed within 2 days and were confirmed for confluence and cobblestone appearance by Diff-Quik staining (Baxter Scientific Products, McGaw Park, IL). Confluent monolayers were incubated in the presence or absence of cytokines for the indicated times. After cytokine treatment, both upper and lower compartments of the transwell were washed three times with 37°C HBSS. EOS (3–5 x 106/ml) were then added to the upper compartment, and enriched medium to the lower compartment. After a 3-h incubation with 5% CO2 at 37°C, 10 mM EDTA was added in the lower compartment, and plates were gently vibrated to dislodge any migrated EOS that were adherent to the bottom of the filters before the transwell inserts were removed. The number of migrated EOS were counted in triplicate by hemocytometer, and percent migration was determined as: (migrated EOS)/(total EOS added into upper compartment) x 100 (%).
Effect of anti-integrin Ab on EOS adhesion and migration
To define the involvement of 4 and
ß2 integrins in EOS adhesion to, and migration across,
cytokine-treated HPMEC, EOS were preincubated with 1 µg/ml of mouse
anti-human 4 integrin mAb (clone: HP1/2, IgG1, a
generous gift from Dr. Roy R. Lobb, Biogen, Cambridge, MA) and/or 3
µg/ml of mouse anti-human ß2 integrin mAb (clone:
L130, IgG1, Becton Dickinson) for 30 min at room temperature before
adhesion or migration assays were performed. Optimal mAb concentration
was determined by dose-response study. Concentration-matched mouse IgG1
(Becton Dickinson) was used as an isotype control.
Paraformaldehyde fixation of HPMEC
To assess the contribution of endothelial cell viability to EOS adhesion and migration, cytokine-treated HPMEC monolayers were fixed using 1% paraformaldehyde in PBS at ambient temperature for 15 min. After washing the monolayers three times with 37°C HBSS, 1% glycine in enriched medium was added (ambient temperature for 1 h) to quench the residual paraformaldehyde. Plates or transwells were then washed three times before use. As a positive control for EOS migration, RANTES (10 nM, Biosource International) was added to the lower compartment.
Statistics
Data are presented as mean ± SEM, and the groups were analyzed by ANOVA with repeated measures and Scheffe constants, or Student’s t test, for paired comparisons. A p value less than 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-4
HPMEC expression of ICAM-1 was significantly increased (>80%
positive cells) by either 100 pM IL-1ß or TNF- (± IL-4) after a
6-h incubation; this level of expression was maintained when evaluated
at 24 and 72 h (p < 0.0001 vs baseline at
each time point, Figure 1
). The kinetics
and magnitude of VCAM-1 expression, however, were different when the
response to IL-1ß and TNF- was evaluated. Although maximal VCAM-1
expression occurred at 6 h with IL-1ß, only 20.8 ± 4.7%
of the HPMEC expressed VCAM-1 (p < 0.01 vs
baseline); these values returned to baseline levels by 24 h (Fig. 1a). TNF-, in contrast, caused a significant
increase in HPMEC expression of VCAM-1 following 6 h of incubation
(26.5 ± 4.3%, p < 0.01 vs baseline) and reached
peak values at 24 h (41.8 ± 5.1%, p <
0.001 vs baseline, Fig. 1b). Based upon these
experiments, we selected 6 h for IL-1ß and 24 h for TNF-
(± IL-4) for incubation times with HPMEC monolayers.
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c), it acted synergistically with TNF- to
increase VCAM-1 expression to 82.2 ± 4.2% by 24 h
(p < 0.0001 vs baseline; p <
0.005 vs TNF- alone, Fig. 1d). When we compared
the cytokine effect on HPMEC adhesion molecule expression, the order of
potency for VCAM-1 was TNF- + IL-4 > TNF- > IL-1ß >
IL-4; the relative potency for cytokine effects on ICAM-1 expression
was IL-1ß = TNF- = TNF- + IL-4 >> IL-4. Under the conditions
of our experiments, VCAM-1 was always expressed on HPMEC in conjunction
with ICAM-1; that is, no endothelial cell cultures expressed only
VCAM-1 (Fig. 2).
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).
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Maximal EOS adhesion to HPMEC occurred after 60 min of incubation;
3 h was optimal for EOS transendothelial migration (data not
shown). Pretreatment of HPMEC monolayers with IL-1ß or TNF- (±
IL-4) significantly enhanced EOS adhesion (medium: 13.5 ± 1.2%
adhesion; IL-4: 14.6 ± 1.1%; IL-1ß: 27.9 ± 2.2%;
TNF-: 34.3 ± 2.5%; TNF- + IL-4: 40.0 ± 3.0%;
p < 0.0001 for IL-1ß, TNF-, TNF- + IL-4 vs
medium and IL-4, Fig. 4a). The effect of
TNF- + IL-4 on EOS adhesion was significantly greater than that of
either TNF- alone (p < 0.05) or IL-1ß
(p < 0.0001). The order of cytokine potency on
EOS adhesion was: TNF- + IL-4 > TNF- > IL-1ß; this was
the same order of cytokine potency as VCAM-1 expression on HPMEC.
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(±
IL-4) significantly enhanced EOS migration (medium: 1.6 ±
0.4% migration; IL-4: 2.8 ± 0.5%; IL-1ß: 21.2 ±
1.4%; TNF-: 12.6 ± 2.6%; TNF- + IL-4: 9.1 ± 2.0%;
p < 0.0001 for IL-1ß vs medium and IL-4;
p < 0.001 for TNF- vs medium and IL-4;
p < 0.05 for TNF- + IL-4 vs medium, Fig. 4b). The effect of IL-1ß on EOS migration was
significantly greater than that of either TNF- alone
(p < 0.02) or TNF- + IL-4
(p < 0.001). Given these observations, the
order of cytokine potency on EOS migration was: IL-1ß > TNF- >
TNF- + IL-4, the inverse order of potency that is observed for
VCAM-1 expression on, and EOS adhesion to, HPMEC.
Effect of anti-4 and
anti-ß2 integrin mAb on EOS adhesion and
transmigration
To determine the identity and involvement of EOS adhesion
integrins in these processes, EOS were pretreated with
anti-4 and/or anti-ß2
integrin mAb before adhesion to, or migration across,
IL-1ß- or TNF- + IL-4-treated HPMEC. Treatment of EOS
with a concentration-matched IgG1-isotype control did not affect EOS
adhesion or migration (data not shown). EOS adhesion to IL-1ß-treated
HPMEC was significantly, but only partially, inhibited by
anti-4 or anti-ß2 integrin mAb
(p < 0.05 and p < 0.005,
respectively, Fig. 5a);
total inhibition of EOS adhesion to HPMEC required the presence of both
mAb. When TNF- + IL-4-treated HPMEC were evaluated, EOS adhesion was
significantly inhibited by anti-4 integrin
(67.7 ± 7.5% inhibition, p < 0.0005).
Anti-ß2 integrin alone had no inhibitory effect on EOS
adhesion; when the anti-ß2 mAb was combined with
anti-4 integrin, significantly greater inhibition of
EOS adhesion was noted when compared with anti-4
integrin alone (p < 0.05).
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+ IL-4: 63.1 ± 9.8% inhibition, p <
0.05, Fig. 5b). Moreover, the presence of
anti-4 integrin significantly increased EOS
transmigration across IL-1ß-treated HPMEC (p
< 0.05). Furthermore, the addition of anti-4
integrin to anti-ß2 integrin mAb had no additional
inhibitory effect on EOS transmigration than anti-ß2
integrin alone. Effect of paraformaldehyde fixation of HPMEC on EOS adhesion and migration
To assess the contribution of endothelial cell viability to EOS
adhesion and migration, IL-1ß-treated HPMEC were fixed in 1%
paraformaldehyde (15 min). Although HPMEC fixation had no effect on EOS
adhesion (unfixed: 23.8 ± 3.9% adhesion; fixed: 24.0 ±
6.1%, Fig. 6), EOS transmigration was
significantly inhibited in the presence of fixed HPMEC monolayers
(unfixed: 17.2 ± 3.8% migration; fixed: 1.3 ± 0.8%,
p < 0.05). RANTES (10 nM) stimulated significant EOS
transendothelial migration irrespective of HPMEC fixation, suggesting
that the fixed monolayer retained the capacity of EOS diapedesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(± IL-4). However, these cytokines had a marked difference in
their potency to stimulate VCAM-1 expression on, and EOS adhesion to,
HPMEC monolayers: TNF- + IL-4 > TNF- > IL-1ß > medium.
In contrast, when EOS transendothelial migration was evaluated, the
order of cytokine effectiveness on cell movement was reversed from that
observed for VCAM-1 expression and adhesion. These observations
indicate that, under conditions of strong ICAM-1 expression, EOS
adhesion to, and migration across, cytokine-treated HPMEC were
differentially regulated by the expression status of VCAM-1.
To identify the EOS integrins that interact with HPMEC-expressed ICAM-1
and VCAM-1, and how these interactions determine cell adhesion and
migration, we performed experiments to test the hypothesis that these
two HPMEC adhesion proteins modulate different EOS functions. It is
generally accepted that firm adhesion of leukocytes to
cytokine-stimulated endothelial cells is mediated by
4 (4ß1 and
4ß7) and ß2
(Lß2 and Mß2)
integrins (8). The involvement of these EOS integrins in our in vitro
model system was determined using anti-4 and
anti-ß2 integrin mAbs. EOS adhesion to
IL-1ß-treated HPMEC, expressing predominately ICAM-1, was equally,
but only partially, inhibited by either anti-4 or
anti-ß2 integrin mAb. When TNF- + IL-4 were added
to HPMEC, leading to both ICAM-1 and VCAM-1 expression,
anti-4 integrin significantly inhibited EOS
adhesion, while anti-ß2 mAb alone had no effect.
These results confirm the functional importance of the 4
integrin/VCAM-1 pathway for EOS adhesion to HPMEC. When either IL-1ß
or TNF- + IL-4 were used to treat HPMEC, a combination of
anti-4 and anti-ß2 integrin mAbs
was necessary to reduce EOS adhesion to baseline values (EOS adhesion
to nonstimulated HPMEC), suggesting that these mAbs are sufficient to
completely block ß2 integrin/ICAM-1 and 4
integrin/VCAM-1 interactions.
In agreement with studies using HUVEC (5, 16), only
anti-ß2 integrin mAb significantly inhibited EOS
migration across cytokine-treated HPMEC. This finding suggests that the
ß2 integrin/ICAM-1 interaction, but not 4
integrin/VCAM-1, is required for EOS transendothelial migration. Wegner
et al. (25) also reported that ICAM-1 was crucial for EOS infiltration
to the lung in a primate model of asthma. Interestingly, in our study,
anti-4 integrin mAb did not inhibit EOS
transmigration but actually increased cell migration across
IL-1ß-treated HPMEC monolayers. Moser et al. (5) and Ebisawa et al.
(16) also demonstrated that anti-4 integrin or
anti-VCAM-1 mAb failed to inhibit EOS migration across IL-1ß- or
TNF--treated HUVEC. Moreover, Kuijpers et al. (26) reported that
"freezing" ß1 integrin in a state of high avidity by
activating mAb completely inhibited EOS migration across fibronectin or
endothelial cell-coated filters. Therefore, it is likely that, although
the 4 integrin/VCAM-1 pathway is important for selective
EOS adhesion to HPMEC, strong adhesion via this pathway actually
inhibits subsequent ß2 integrin/ICAM-1-dependent
transendothelial migration.
We have reported that rhVCAM-1 alone can activate EOS adhesion and its
respiratory burst (27) while EOS interaction with ICAM-1 requires an
additional costimulus for activation (28). These findings agree with
other reports that Mn2+, platelet activating factor, FMLP,
or granulocyte-macrophage (GM)-CSF can activate both adhesion and
functions dependent on granulocyte ß2 integrin
interaction with endothelial ICAM-1 (29, 30, 31). We now propose that
adhesion of EOS to HPMEC via very late Ag (VLA)-4 and VCAM-1 fulfills
the role of a second stimulus for ICAM-1-dependent EOS migration. In
support of this hypothesis, Weber et al. (32) reported the differential
regulation of ß1 and ß2 integrin avidity of
EOS by chemoattractants such as RANTES and MCP-3 (monocyte
chemoattractant protein-3). Although these chemokines rapidly increased
and then subsequently reduced EOS adherence via 4
integrin and VCAM-1, which was dependent on the actin cytoskeleton,
they increased and prolonged EOS conformational changes of
ß2 integrin, promoting its interaction with ICAM-1. Thus,
these particular chemokines may facilitate migration of EOS by first
adhering these cells to VCAM-1, then quickly detaching them from VCAM-1
and/or increasing the availability of the ß2
integrin/ICAM-1 interaction. Although our model does not involve the
addition of chemokines, we observed a similar interrelationship between
VCAM-1-dependent EOS adhesion and subsequent migration across
cytokine-activated HPMEC monolayers via ICAM-1.
Fibronectin, which coated the surface of the HPMEC culture flask,
has also been reported to be an activator of EOS functions (33, 34). To
define the possible role of fibronectin as an agonist in our system,
each experiment included a control of HPMEC without cytokine treatment
(medium controls in Figs. 2, 4, and 5). These data provided the
baseline determinants of EOS adhesion and migration and would include
any fibronectin activation. In addition, one well of HPMEC was stained
and observed microscopically to confirm monolayer confluence in each
experiment.
Our cytokine effects on adhesion protein expression by HPMEC were
distinct from responses observed with HUVEC; with HUVEC, VCAM-1
expression associated with IL-1ß was greater than that caused by
TNF- and was maintained for more than 24 h (19, 20). Carley et
al. (17) also demonstrated that HPMEC differ from HUVEC with respect to
prostaglandin and plasminogen activator production, the ability of
tube-like structure formation, and the distribution of factor
VIII-related Ag. Although IL-4 selectively induces VCAM-1 expression by
HUVEC or nasal polyp-derived microvascular endothelium (6, 35), it has
no effect on VCAM-1 expression by microvascular endothelial cells from
skin or intestine (18, 19). Likewise, IL-4 alone had no effect on
VCAM-1 expression on HPMEC in our study. The highest expression of
VCAM-1 occurred (82.2 ± 4.2%) when IL-4 was combined with
TNF-. This synergistic effect was also demonstrated in HUVEC culture
(36, 37) and confirms the importance of IL-4 for increased and
sustained VCAM-1 expression.
Finally, we evaluated the requirement of endothelial cell viability for EOS adhesion and transmigration. Our data indicate that EOS adhesion to cytokine-treated HPMEC occurred regardless of HPMEC viability. However, subsequent transmigration was almost completely inhibited when cytokine-treated HPMEC monolayers were first fixed with paraformaldehyde. This finding suggests that viable HPMEC are essential for adherent EOS to migrate in this model. Similarly, Asako et al. (38) and Huang et al. (39) demonstrated that an alteration of the cytoskeleton, or an increase in cytosolic free calcium in endothelial cells, was required for neutrophil migration, but not adhesion. To determine whether fixation "closed" the endothelial cell tight junctions, thus inhibiting EOS diapedesis, RANTES was added to the bottom wells as a chemoattractant. RANTES induced EOS migration across fixed HPMEC, indicating that a fixed monolayer still retained its capacity for EOS diapedesis, and that RANTES-induced EOS migration is independent of HPMEC viability.
In conclusion, EOS adhesion to, and migration across, cytokine-treated
HPMEC appear to be differentially regulated by the expression status of
ICAM-1 and VCAM-1 and by the expression and activation of
4 and ß2 integrins on EOS. The biologic
relevance of these observations is not established but raises a number
of possibilities. Our observations suggest that eosinophil interaction
with VCAM-1 and ICAM-1 are different and inversely related. The
interaction of eosinophil with VCAM-1 is associated with a high level
of adhesion but not transendothelial migration. The interaction with
ICAM-1 appears important for cell migration but not initial adhesion.
However, to direct eosinophils to sites of inflammation, it is
essential that both cell responses are achieved. Specifically how these
two responses are interrelated, if indeed they are, is the next step to
investigate. In that regard, we propose that studies with HPMEC promise
to provide a new insight into how EOS, and other inflammatory cells,
adhere to and migrate across lung microvasculature, accumulate in the
airways, and contribute to allergic inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Julie B. Sedgwick, CSC H6/367 Allergy Unit, University of Wisconsin Hospital, 600 Highland Avenue, Madison, Wisconsin 53792. E-mail address:
3 Abbreviations used in this paper: EOS, eosinophils; HPMEC, human pulmonary microvascular endothelial cells; NCS, newborn calf serum; PE, phycoerythrin; EPO, eosinophil peroxidase.
Received for publication November 10, 1997. Accepted for publication March 20, 1998.
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References |
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4ß7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin. Immunology 89:112.[Medline]
4ß1 (very late antigen-4) dependent. J. Immunol. 146:3419.[Abstract]
receptors (CD23) on normal human B lymphocytes. J. Exp. Med. 165:1459. and interleukin-1ß. Am. J. Respir. Cell Mol. Biol. 13:555.[Abstract]
4ß1 (VLA-4). Immunology 82:222.[Medline]
. J. Immunol. 145:865.[Abstract]
in cultured endothelial cells. J. Clin. Invest. 95:264.
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 Y. Rifai, A. S. F. Elder, C. J. Carati, D. J. Hussey, X. Li, C. M. Woods, A. C. Schloithe, A. C. Thomas, R. D. Mathison, J. S. Davison, et al. The tripeptide analog feG ameliorates severity of acute pancreatitis in a caerulein mouse model Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G1094 - G1099. [Abstract] [Full Text] [PDF]
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 S. K. Mathur, E. A. Schwantes, N. N. Jarjour, and W. W. Busse Age-Related Changes in Eosinophil Function in Human Subjects* Chest, February 1, 2008; 133(2): 412 - 419. [Abstract] [Full Text] [PDF]
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L. Y. Liu, M. E. Bates, N. N. Jarjour, W. W. Busse, P. J. Bertics, and E. A. B. Kelly Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-{alpha} J. Immunol., October 1, 2007; 179(7): 4840 - 4848. [Abstract] [Full Text] [PDF]
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H. Abdala-Valencia and J. M. Cook-Mills VCAM-1 Signals Activate Endothelial Cell Protein Kinase C{alpha} via Oxidation J. Immunol., November 1, 2006; 177(9): 6379 - 6387. [Abstract] [Full Text] [PDF]
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A. M. Brooks, M. E. Bates, R. F. Vrtis, N. N. Jarjour, P. J. Bertics, and J. B. Sedgwick Urokinase-Type Plasminogen Activator Modulates Airway Eosinophil Adhesion in Asthma Am. J. Respir. Cell Mol. Biol., October 1, 2006; 35(4): 503 - 511. [Abstract] [Full Text] [PDF]
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 E. B. Cook, J. L. Stahl, A. M. Brooks, F. M. Graziano, and N. P. Barney Allergic tears promote upregulation of eosinophil adhesion to conjunctival epithelial cells in an ex vivo model: inhibition with olopatadine treatment. Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3423 - 3429. [Abstract] [Full Text] [PDF]
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M. Iikura, M. Ebisawa, M. Yamaguchi, H. Tachimoto, K. Ohta, K. Yamamoto, and K. Hirai Transendothelial Migration of Human Basophils J. Immunol., October 15, 2004; 173(8): 5189 - 5195. [Abstract] [Full Text] [PDF]
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 T. L. Deem and J. M. Cook-Mills Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species Blood, October 15, 2004; 104(8): 2385 - 2393. [Abstract] [Full Text] [PDF]
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J. B. Sedgwick, Y. S. Hwang, H. A. Gerbyshak, H. Kita, and W. W. Busse Oxidized Low-Density Lipoprotein Activates Migration and Degranulation of Human Granulocytes Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 702 - 709. [Abstract] [Full Text] [PDF]
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 M-J. Dallaire, C. Ferland, N. Page, S. Lavigne, F. Davoine, and M. Laviolette Endothelial cells modulate eosinophil surface markers and mediator release Eur. Respir. J., June 1, 2003; 21(6): 918 - 924. [Abstract] [Full Text] [PDF]
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A. Holub, J. Byrnes, S. Anderson, L. Dzaidzio, N. Hogg, and A. Huttenlocher Ligand density modulates eosinophil signaling and migration J. Leukoc. Biol., May 1, 2003; 73(5): 657 - 664. [Abstract] [Full Text] [PDF]
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L. Y. Liu, J. B. Sedgwick, M. E. Bates, R. F. Vrtis, J. E. Gern, H. Kita, N. N. Jarjour, W. W. Busse, and E. A. B. Kelly Decreased Expression of Membrane IL-5 Receptor {alpha} on Human Eosinophils: II. IL-5 Down-Modulates Its Receptor Via a Proteinase-Mediated Process J. Immunol., December 1, 2002; 169(11): 6459 - 6466. [Abstract] [Full Text] [PDF]
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R. B. Berger, N. M. Blackwell, J. H. Lass, E. Diaconu, and E. Pearlman IL-4 and IL-13 Regulation of ICAM-1 Expression and Eosinophil Recruitment in Onchocerca volvulus Keratitis Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 2992 - 2997. [Abstract] [Full Text] [PDF]
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 P S Thomas and G Heywood Effects of inhaled tumour necrosis factor alpha in subjects with mild asthma Thorax, September 1, 2002; 57(9): 774 - 778. [Abstract] [Full Text] [PDF]
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 R. SUZUKI and A. N. FREED Heparin Inhibits Hyperventilation-Induced Late-Phase Hyperreactivity in Dogs Am. J. Respir. Crit. Care Med., January 1, 2002; 165(1): 27 - 33. [Abstract] [Full Text] [PDF]
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S. Esnault and J. S. Malter Granulocyte Macrophage-Colony-Stimulating Factor mRNA Is Stabilized in Airway Eosinophils and Peripheral Blood Eosinophils Activated by TNF-{{alpha}} Plus Fibronectin J. Immunol., April 1, 2001; 166(7): 4658 - 4663. [Abstract] [Full Text] [PDF]
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 W. W. Busse and R. F. Lemanske Asthma N. Engl. J. Med., February 1, 2001; 344(5): 350 - 362. [Full Text] [PDF]
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 S. H. Jacobson, P. Thylen, and J. Lundahl Three monocyte-related determinants of atherosclerosis in haemodialysis Nephrol. Dial. Transplant., September 1, 2000; 15(9): 1414 - 1419. [Abstract] [Full Text] [PDF]
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H. Yamamoto, J. B. Sedgwick, R. F. Vrtis, and W. W. Busse The Effect of Transendothelial Migration on Eosinophil Function Am. J. Respir. Cell Mol. Biol., September 1, 2000; 23(3): 379 - 388. [Abstract] [Full Text]
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H. Tachimoto, M. M. Burdick, S. A. Hudson, M. Kikuchi, K. Konstantopoulos, and B. S. Bochner CCR3-Active Chemokines Promote Rapid Detachment of Eosinophils from VCAM-1 In Vitro J. Immunol., September 1, 2000; 165(5): 2748 - 2754. [Abstract] [Full Text] [PDF]
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H. E. Matheny, T. L. Deem, and J. M. Cook-Mills Lymphocyte Migration Through Monolayers of Endothelial Cell Lines Involves VCAM-1 Signaling Via Endothelial Cell NADPH Oxidase J. Immunol., June 15, 2000; 164(12): 6550 - 6559. [Abstract] [Full Text] [PDF]
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L. H. K. Lim, R. J. Flower, M. Perretti, and A. M. Das Glucocorticoid Receptor Activation Reduces CD11b and CD49d Levels on Murine Eosinophils . Characterization and Functional Relevance Am. J. Respir. Cell Mol. Biol., June 1, 2000; 22(6): 693 - 701. [Abstract] [Full Text]
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S. Shahabuddin, P. Ponath, and R. P. Schleimer Migration of Eosinophils Across Endothelial Cell Monolayers: Interactions Among IL-5, Endothelial-Activating Cytokines, and C-C Chemokines J. Immunol., April 1, 2000; 164(7): 3847 - 3854. [Abstract] [Full Text] [PDF]
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L. Liu, P. Ridefelt, L. Hakansson, and P. Venge Regulation of Human Eosinophil Migration Across Lung Epithelial Monolayers by Distinct Calcium Signaling Mechanisms in the Two Cell Types J. Immunol., November 15, 1999; 163(10): 5649 - 5655. [Abstract] [Full Text] [PDF]
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