1 Section of Pulmonary and
Critical Care Medicine, We examined the
effect of eosinophil ligation to cultured human umbilical vein
endothelial cells (HUVECs) in augmenting the stimulated secretion of
leukotriene (LT) C4 and eosinophil
peroxidase (EPO). The effects of adhesion were compared before and
after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to
HUVECs augmented EPO release caused by
formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 ± 15.3 (BSA control) to 778 ± 225 ng/106 cells for eosinophils
exposed to interleukin-1
eosinophils; adhesion molecules; leukotriene
C4; eosinophil
peroxidase
EOSINOPHIL MIGRATION to the conducting airways is
associated with increased bronchoreactivity in human asthma. This
process is regulated by the sequential binding of adhesion molecules on both the capillary endothelium in the conducting airways [e.g., intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)] and the eosinophil cell surface (11, 16, 23,
24). Prior investigations have shown that selective blockade of either
endothelial surface adhesion molecules (7, 8) or integrins on the
eosinophil cell surface (7) prevents migration of eosinophils into the
conducting airways.
Circulating eosinophils, even in atopic asthmatic subjects, are
relatively quiescent before transmigration and do not secrete substantial quantities of bronchoactive eicosanoids or granular proteins (27). Because eosinophil transmigration (8, 11, 16, 23, 24)
and adhesion begins at the endothelial surface, this study was
undertaken to determine whether adhesive ligation of very late
antigen-4 (VLA-4), a
We found that both Isolation of Peripheral Blood Eosinophils
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-treated HUVECs (P < 0.05) and also caused a twofold
increase in stimulated LTC4 secretion (P < 0.05). To determine
whether augmented secretion resulted directly from adhesive ligation,
studies were also performed with paraformaldehyde-treated HUVECs;
stimulated secretion of LTC4 from
eosinophils was comparable to that for living HUVECs. Our study is the
first demonstration that adhesion to HUVECs through ligation to
4- or
2-integrin on the eosinophil
surface causes augmentation of stimulated secretion of both EPO and
LTC4 and that blockade of adhesion
molecules on either eosinophils or HUVECs prevents the priming effect
on eosinophil secretion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1-integrin, and/or
2-integrin with the homologous
endothelial surface counterligands that occurs in the first stage of
inflammatory diapedesis and transmigration caused priming of stimulated
eosinophil secretion. We thus hypothesized that eosinophil binding to
human umbilical vein endothelial cells (HUVECs) would cause augmented
secretion of both leukotriene (LT) C4 and granular protein in
stimulated human eosinophils. In this study, eosinophils purified by
negative immunomagnetic separation were allowed to adhere on cultured
monolayers of interleukin (IL)-1
-treated HUVECs, which upregulate
both ICAM-1 and VCAM-1 on the endothelial surface (3, 4, 6, 7).
Comparisons were made to adhesive interactions between human
eosinophils adhering to HUVECs not treated with IL-1
, and the effect
of monoclonal antibodies (MAbs) directed against
4- and
2-integrins on the eosinophil
surface or anti-VCAM-1 and anti-ICAM-1 on the endothelial surface was examined. Additional studies were performed with
paraformaldehyde-treated endothelial cells to ensure that augmented
secretion related specifically to adhesive augmentation (14) rather
than to metabolic or secretory functions of HUVECs.
4- and
2-integrins caused adhesive
upregulation of stimulated eosinophil secretion and that blockade of
either eosinophils or endothelial cell surface adhesion molecules prevented the priming effect on eosinophil secretion. Our data are the
first demonstration that the process of eosinophil adhesion at the
endothelial surface likely is linked directly to the priming of
eosinophil secretion of granular protein and bronchoactive LTs during
the cellular transmigration that occurs in human asthma.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
Preparation of HUVECs
Purified HUVECs were studied and characterized for factor VIII expression as previously described (13). Immediately after delivery, the umbilical cord was treated with a 1 mg/ml collagenase solution, and the cells were cultured on a 0.2% gel-coated cell culture flask in medium 199 supplemented with 20% FCS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 200 mM l-glutamine, 25 mM HEPES, 50 µg/ml of endothelial cell growth factor, and 100 µg/ml of heparin. The isolated cells were maintained for 3-4 days at 37°C in a 5% CO2 humidified atmosphere until microscopically confluent. After initial seeding with 0.05% trypsin, HUVECs were washed and resuspended in complete medium 199. The cell mixture was centrifuged at 350 g for 10 min, and the supernatant was discarded. The cell pellet was resuspended in complete medium 199, incubated for an additional 3-4 days at 37°C in a CO2 incubator, and grown to confluence. After 2-3 passages, the cells were replated on 96-well microplates and harvested 2-3 days later. HUVECs were treated with either buffer or 10 IU/ml of IL-1Immunofluorescence Analysis
Passage 3 HUVECs were treated with either buffer or 10 IU/ml of IL-1Inactivation of HUVECs by Paraformaldehyde
To demonstrate that adhesive ligation rather than activated secretion by HUVECs was the cause of augmented secretion from eosinophils, experiments were performed in HUVECs that were first fixed in a paraformaldehyde solution to cause cell death. Monolayers of HUVECs cultured from four different donors were pretreated with IL-1Adhesion Assay and Blockade of Adhesion
Purified human peripheral blood eosinophils from 10 separate eosinophil isolations were resuspended in HBSS containing 5% FCS. For cells treated with an MAb, 106 eosinophils were incubated with either buffer control, 20 µg/ml of anti-VLA-4, or 20 µg/ml of anti-CD18 for 20 min before exposure to either BSA control or treated HUVEC-coated microplate wells. Aliquots of eosinophils (5 × 104 cells/well) were transferred onto the plate and allowed to adhere for 5, 10, 15, and 30 min at 37°C. Eosinophil adhesion was terminated by removal of nonadherent eosinophils by a plate-inversion technique and washed two times with 200 µl of HBSS-FCS solution. Quantification of adhesion of eosinophils to HUVECs was determined as a function of intracellular eosinophil peroxidase (EPO) content, which was developed specifically for assays required in these studies (19). Adherent eosinophils were lysed with 1% Triton X-100, absorbance at 492 nm was measured every 6 s for 1 min, and maximal uptake (Vmax) for each experimental well was calculated by interpolation between successive points (18 s) with Softmax version 2.01 (Molecular Devices, Menlo Park, CA). Standard curves were generated at the same time by adding a known number of eosinophils (1 × 103 to 5 × 104 cells) to untreated microplate wells. Samples were assayed in triplicate, and the number of adherent cells was calculated from standard curves fitted by linear regression (19). The time at which the adhesion was greatest was used in all subsequent experiments (see RESULTS).Identical experiments were performed in six separate eosinophil isolations, allowing cells to adhere to paraformaldehyde-fixed HUVECs at 0, 5, 10, 20, 30, and 45 min at 37°C. Adherent eosinophils were lysed and quantified in an analogous manner as above as a function of EPO content. Eosinophil binding to paraformaldehyde-fixed HUVECs was validated under epifluorescent microscopy (Axiophot, Zeiss). Preliminary experiments were conducted to assess the time at which eosinophil adhesion was maximal for both untreated and paraformaldehyde-fixed HUVECs.
Indexes of Eosinophil Activation
EPO release by kinetic assay. EPO content was assessed as previously described (20, 22) Briefly, isolated eosinophils (n = 5 isolations from 5 separate donors) were resuspended in HBSS buffer, pH 7.40, and 105 cells were pipetted onto either BSA-, HUVECs alone (no IL-1LTC4 secretion by enzyme immunoassay.
The secretion of LTC4 caused by
activation with buffer or fMLP+CB after eosinophil binding to treated
HUVECs or BSA was measured by enzyme immunoassay (EIA; Cayman Chemical,
Ann Arbor, MI). Isolated human peripheral blood eosinophils
(n = 5 isolations from 5 separate donors) were resuspended in HBSS buffer containing 0.1% gelatin. Eosinophils (105
cells · 100 µl1 · well
1)
were loaded onto ten 96-well microplates coated with either BSA or
HUVECs and allowed to adhere for 5 min (see
results). The identical intervention
as for blockade of adhesion molecules was used. After exposure, the
treated cells were activated with either buffer or fMLP+CB for 30 min,
and the reaction was terminated by centrifugation at 500 g for 10 min. Immediately, the
supernatant from 10 wells was collected, snap-frozen in liquid
nitrogen, and stored at
70°C until assayed. Duplicate
samples (50 µl) were pipetted onto the microplate wells coated with a
mouse anti-rabbit MAb provided with the EIA kit (Cayman Chemical).
Acetylcholinesterase-linked LTC4
tracer (50 µl) and LTC4
antiserum (50 µl) were added, and the samples were incubated for 18 h
at room temperature. Each well was aspirated dry and rinsed five times
with ready-made wash buffer (EIA kit, Cayman Chemical). Optimum
development was obtained after the addition of 500 µl of Ellman's
reagent to each well and 5 µl of tracer to the total activity wells.
This assay typically develops in 60-90 min, and microplate reading
was performed on a microplate absorbance spectrophotometer (Thermomax,
Molecular Devices) at 405 nm. The final concentration of each sample
was calculated from standard curves fitted by four-parameter analysis (Softmax version 2.01 software, Molecular Devices), and the
concentration of LTC4 is expressed
in picograms per 106 eosinophils
(pg/106 cells) (19, 20, 30).
Drugs and Suppliers
R15.7 (a CD18 antibody), anti-Analysis of Data
Data are expressed as means ± SE for all groups studied. The degree of activation was assessed by comparison of maximal EPO release or LTC4 secretion in the same cell isolation before and after each intervention with Student's t-test. When multiple comparisons of paired data within a single experimental procedure were made, a Bonferroni correction was applied. In experiments requiring multiple group comparisons, two-way analysis of variance was used. When differences were observed between groups, comparisons were made by Dunnett's test. Significance was claimed when P < 0.05. ![]() |
RESULTS |
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Analysis of Cell Survival
Isolated human peripheral blood eosinophils were 98 ± 5.4% pure, and treated cells utilized in all experimental groups studied remained >98% viable as assessed by trypan blue exclusion analysis. Visually confluent monolayers of cultured HUVECs were readily distinguished with phase-contrast microscopy and remained stably adherent. Microscopic examination with Wright-Giemsa stain revealed vacuolation in 2 of 36 eosinophil isolates, indicating probable activation during or before isolation. These isolated cells were excluded prospectively from all subsequent protocols.Validation of Upregulated Surface Ligands of HUVECs
Flow cytometric analysis of unstimulated HUVECs confirmed that ICAM-1 is constitutively expressed, with a mean fluorescence intensity (MFI) of 24.9 ± 6.93 vs. 5.35 ± 0.16 for IgG1 isotype control (P = 0.03; Fig. 1A). Coincubation with IL-1
|
Time Course of Eosinophil Binding to HUVECs
Maximal adhesion occurred at 5 min and was sustained forBlockade of Adhesion With MAb Directed Against Eosinophil Surface Ligands
Blockade of the
|
Effect of Adhesive Ligation to HUVECs on Eosinophil Secretion of Granular Protein
fMLP+CB caused comparable EPO release at 30 min for cells exposed to either BSA (403 ± 15.3 ng/106 cells) or HUVECs alone [401 ± 77 ng/106 cells; P = not significant (NS)]. Ligation of eosinophils to IL-1
|
Effect of Ligation to HUVECs on LTC4 Secretion
Baseline secretion (before activation) of LTC4 was insignificant in all treatment protocols (Fig. 4). For BSA-exposed eosinophils, LTC4 concentration in the supernatant after 30 min was 1.81 ± 1.13 pg/106 cells, 2.71 ± 0.22 pg/106 cells for eosinophils exposed to HUVECs without IL-1
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Addition of the anti-VLA-4 MAb blocked the augmented
LTC4 secretion caused by activated
eosinophils exposed to IL-1-treated HUVECs to 208 ± 51 pg/106 cells
(P < 0.05 vs. wells receiving no
MAb; see above; Fig. 4). As for EPO release, augmented
LTC4 secretion caused by
stimulated adhesive eosinophil binding to IL-1
-treated HUVECs was
also blocked to baseline after pretreatment with the anti-CD18 MAb (327 ± 72 pg/106 cells;
P < 0.05 vs. wells receiving no
MAb). Pretreatment with both anti-CD18 and anti-VLA-4 MAbs had no
further inhibitory effect on the augmented secretion of
LTC4
(P = NS vs. anti-CD18 or anti-VLA-4 MAb; Fig. 4; also see Fig. 2).
Blockade of Adhesion With MAb Directed Against Paraformaldehyde-Fixed HUVECs
Binding of eosinophils to IL-1
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DISCUSSION |
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This study was undertaken to examine the hypothesis that adhesion of
eosinophils at the endothelial surface also is a process by which
stimulated secretion of bronchoactive substances is primed. Prior
investigations (2, 15, 19, 22) have demonstrated that eosinophils
adhere through the 1-surface
ligand VLA-4 to the extracellular matrix protein fibronectin (FN).
Adhesion of eosinophil VLA-4 to FN required ~60 min and was decreased
at 120 min. This was associated with augmented stimulated secretion of eosinophils caused by fMLP at both 60 and 120 min but not at earlier times. A prior investigation has also demonstrated augmented superoxide anion generation from stimulated eosinophils after exposure to exogenous soluble VCAM-1 (21).
In this study, we examined the effect of adhesion of VLA-4 and
2-integrin on eosinophils to
VCAM-1 and ICAM-1 on living HUVECs. Studies were conducted to determine
whether the degree of adhesion was related to priming of stimulated
eosinophil degranulation and/or secretion of
LTC4. Additional experiments were
performed with paraformaldehyde-fixed HUVECs to assess whether the
augmented stimulated eosinophil secretion was specifically mediated by
adhesive ligation (14) rather than to stimulation of secretory
augmentative substances from viable HUVECs. Although some adhesion of
eosinophils to HUVECs was observed in the "basal" (i.e.,
untreated) state, the greatest adhesion occurred for HUVECs that were
first pretreated with IL-1
, which is known to upregulate VCAM-1, the
specific endothelial surface ligand for VLA-4 (7, 26). Adhesion to IL-1
-treated HUVECs (living and paraformaldehyde fixed) was
augmented nearly twofold at 5 and 30 min, respectively, and diminished
slightly thereafter (Figs. 1 and 5). Blockade of adhesion below
baseline levels (Fig. 2) after the addition of the MAb against the
surface ligand suggests that isolates of peripheral cells even from
mildly atopic donors have some intrinsic adhesive capacity.
We also found that adhesion to IL-1-treated HUVECs caused
augmented secretion of granular protein (EPO) and
LTC4 after stimulation with
fMLP+CB. Secretion was comparably less for untreated eosinophils incubated with either BSA control or HUVECs with no IL-1
(Figs. 3
and 4). This augmented secretion caused by adhesive ligation of human
eosinophil VLA-4 and
2-integrin
to IL-1
-treated HUVECs occurred at 5 min (Fig.
1B); by contrast, Muñoz et al.
(19) and Neeley et al. (22) have shown that eosinophil
binding to FN through VLA-4 requires 60-120 min. Accordingly, both
binding and priming of eosinophil secretion caused by VLA-4 have
substantially different kinetics for different counterligands. Priming
of eosinophil secretion occurs almost instantaneously even in the
presence of blockade of all
2-integrins, whereas adhesion
alone requires 60 min for VLA-4 and the matrix protein FN.
It is interesting to note that adhesion (Figs.
1B and
5A) and stimulated secretion of EPO
(Fig. 3A) and
LTC4 (Figs. 4 and 5B) caused by exposure to
IL-1-treated HUVECs all were blocked to basal levels (i.e.,
comparable to control or non-IL-1
-treated HUVECs) after pretreatment
with an MAb directed against surface adhesion ligands on eosinophils
(
4- or
2-integrin) or on HUVECs (ICAM-1 or VCAM-1). Combined administration of an MAb directed against
both ligands together thus had no incremental inhibitory effect. The
mechanism by which blockade of one ligand on the eosinophil surface
causes blockade of the augmenting effects of the other was not defined
in this study, but steric interference among these macromolecules is a
possibility. Hence blockade with either MAb could cause maximal
inhibition of adhesion and corresponding adhesive priming.
It is important also to specify some other limitations of our findings. All studies were performed in vitro, and it is not possible to extrapolate these data to events of cellular migration in human asthma. Molecular adhesion of eosinophils to endothelium in vivo occurs under conditions of flow and shear stress (1, 10, 18) not replicated in these studies. Nonetheless, these dynamic events may have relatively little effect on the experimental conditions observed in these studies because the ligands in these investigations are active only after firm ligation to the endothelial surface occurs (25, 28).
Blockade of VLA-4 was effected through an
4-chain MAb. Recently, an
4
7
ligand has been identified (9). However, its specific role in
eosinophil function remains to be established. A study in vitro (9) has
shown that mucosal addressin adhesion molecule-1 (MadCam-1), expressed
specifically by gut endothelial cells, is a preferential ligand for
4
7.
Although
4
7
can also bind to VCAM-1, this cell-cell ligation requires greater
integrin activation than binding instantaneously to MadCam-1 (25). In these studies, we elicited eosinophil secretion with the formylated tripeptide fMLP, a chemotactic agent that was shown to cause synthesis and release of LTC4 (20, 30),
eosinophil cytotoxic granular proteins (20, 29), and metabolic burst
activity (21). Muñoz et al. (19) have previously shown that
activation with platelet-activating factor results in eosinophil
secretion of bioactive metabolites that contracts explanted human
airways, largely through the activation of eosinophil 5-lipoxygenase
and secretion of LTC4, and the
secretory process is blocked by relatively selective inhibitors of
phospholipase A2 (30). However,
the actual trigger causing eosinophil activation in human asthma
remains unknown. Thus the priming process caused by eosinophil ligation
to HUVECs cannot yet be related to a specific stimulus in vivo that
provokes eosinophil secretion that is presumed to occur in the human
asthmatic state.
Finally, as for other studies, the precise mechanism by which ligation of eosinophil integrins to HUVECs augments LTC4 secretion was not established. We did establish, however, that priming was caused directly as the consequence of adhesive ligation rather than by secretory products from activated HUVECs. Paraformaldehyde-fixed HUVECs caused similar upregulation of eosinophil secretion to living cells, and this was attenuated by ligand-specific blockade (Figs. 2 and 5B).
Our data demonstrate that eosinophil adhesion to IL-1-treated HUVECs
in vitro is associated temporally with increased augmentation of
stimulated secretion of both granular protein and
LTC4 in concentrations substantially greater than those required to cause contraction of human
bronchial airway explants (19). Because adhesion and augmented
secretion for cells killed with paraformaldehyde and washed after
upregulation with IL-1
was comparable to that for living cells
before treatment with paraformaldehyde for FACScan, it thus
is unlikely that substances secreted from HUVECs have a role in
augmented eosinophil secretion of
LTC4. Stimulated
LTC4 secretion from eosinophils
adhering on monolayers of paraformaldehyde-fixed IL-1
-treated HUVECs
did not differ from eosinophils exposed to buffer-treated
IL-1
-treated HUVECs. These data further confirm that
augmentation of stimulated eosinophil secretion is related solely to
the process of eosinophil binding at the endothelial surface.
Augmentation occurs much more rapidly than that previously reported to
be caused by VLA-4-FN ligation (19, 20, 22) and appears to be mediated
mutually by both
1- and
2-integrins on the eosinophil surface.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-46368; NHLBI Specialized Center of Research Grant IP50-HL-56399; National Institute of Allergy and Infectious Diseases Grant AI-32654; and Astra Zeneca, Inc.
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FOOTNOTES |
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H. Sano is an Astra Traveling Fellow.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. R. Leff, The University of Chicago, Section of Pulmonary and Critical Care Medicine, Dept. of Medicine, MC6076, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: aleff{at}medicine.bsd.uchicago.edu).
Received 16 February 1999; accepted in final form 2 June 1999.
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