| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Section of Pulmonary and Critical Care Medicine, Departments of Medicine, Pharmacological and Physiological Sciences, Pediatrics, Anesthesia, and Critical Care, and Committees on Clinical Pharmacology and Cell Physiology, Division of Biological Sciences, University of Chicago, Chicago, IL 60637; and
Department of Chemistry, University of Illinois, Chicago, IL 60607
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PLA2 is the rate-limiting enzyme involved in the conversion of membrane phospholipids to arachidonic acid (AA) and lysophospholipids, which are readily metabolized to inflammatory mediators such as leukotriene C4 and platelet-activating factor (PAF) (12). Multiple forms of mammalian PLA2 have been identified. The type IIa 14-kDa secretory PLA2 (sPLA2) is well characterized and known to exist in both an extracellular form in inflammatory fluids (13, 14) and a cell-associated form (15, 16). The cytosolic 85-kDa type IV PLA2 (cPLA2) is structurally distinct. Unlike the 14-kDa PLA2, cPLA2 exhibits a preference for AA in the sn-2 position of phospholipid and is regulated by physiological intracellular Ca2+ concentrations and phosphorylation (17, 18, 19). An 80-kDa calcium-independent cytosolic PLA2 identified in P388D1 macrophages possibly serves as a housekeeping enzyme involved in the remodeling of membrane phospholipids (20). Although inflammatory cells contain multiple structurally distinct forms of PLA2, deletion of cPLA2 by homologous recombination results in substantially attenuated leukotriene C4 synthesis and Ag-induced bronchial hyper-reactivity in mice (21).
The objective of this study was to examine the relationship between cPLA2 activation and eosinophil adhesion to ICAM-1 or VCAM-1 as well as surface expression and conformational changes in Mac-1 and/or VLA-4 in activated human eosinophils. We confirmed in our system that IL-5-induced eosinophil adhesion to ICAM-1 was CD11b/CD18 dependent, and spontaneous eosinophil adhesion to VCAM-1 was VLA-4/CD29 dependent. We found that 1) cPLA2 inhibition, but not sPLA2 inhibition, blocked eosinophil adhesion to Ig supergenes; 2) both IL-5-induced adhesion to ICAM-1 and VLA-4 adhesion to VCAM-1 correlated to time-dependent cPLA2 phosphorylation and increased cPLA2 activity; 3) the PAF receptor antagonist, CV-6209, but neither 5-lipoxygenase (5-LO) nor cyclooxygenase (CO) inhibition, blocked integrin-mediated adhesion of eosinophils; 4) addition of AA to eosinophils after treatment with cPLA2 inhibitors did not reverse the inhibition of adhesion to ICAM-1 or VCAM-1; and 5) blockade of eosinophil adhesion to ICAM-1 or VCAM-1 caused by cPLA2 inhibition was not caused by down-regulation of Mac-1 or VLA-4 expression or to down-regulated expression of Mac-1 epitope for the activation-specific Ab, CBRM1/5. These data suggest that eosinophil adhesion to ICAM-1 or VCAM-1 is regulated by cPLA2 activation and its catalytic product, PAF, and not by AA or its metabolites. Eosinophil adhesion to ICAM-1 and/or VCAM-1 thus is not controlled entirely by Mac-1 or VLA-4 expression and stereology, but requires the phosphorylation and the activation of cPLA2.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cPLA2 inhibitor, arachidonic trifluoromethyl ketone (a-TFMK), and the PAF-receptor antagonist, CV-6209, were purchased from Biomol (Plymouth Meeting, PA). Another, structurally unrelated, cPLA2 inhibitor, surfactin (22), was the gift of Dr. C.-H. Lee (Cheiljedang, Korea). The sPLA2 inhibitor, LY311727, was donated by Dr. Ruth Kramer (Eli Lilly Co., Indianapolis, IN). Zileuton was a gift from Abbott Laboratories (North Chicago, IL). Eosinophil isolation materials were obtained from Miltenyi Biotec (Sunnyvale, CA). Calcium ionophore and indomethacin were purchased from Sigma (St. Louis, MO). AA was purchased from Cayman (Ann Arbor, MI). Polystyrene 96-well microtiter plates were obtained from Costar (Cambridge, MA). Anti-Mac-1 mAb (clone 44) was purchased from Endogen (Woburn, MA). Anti-VLA-4 mAb (clone HP2/1), anti-CD11a mAb (clone 25.3), anti-Mac-1 mAb (clone Bear1), anti-CD29 mAb (Lia 1/2), and anti-CD18 mAb (clone 7E4) were purchased from Immunotech (Westbrook, ME). Mouse IgG1 and FITC-conjugated goat anti-mouse Ig were purchased from Becton Dickinson (Mountain View, CA). The CBRM1/5 mAb against activated Mac-1 was a gift from Dr. T. A. Springer (Harvard Medical School, Boston, MA). mAb 1,1,1 against cPLA2 and rabbit polyclonal antiserum against cPLA2 were obtained as previously described (23). IL-5, soluble ICAM-1, and soluble VCAM-1 were purchased from R&D (Minneapolis, MN). [5,6,8,9,11,12,14,15-3H]AA (sp. act., 100 Ci/mmol) was purchased from New England Nuclear (Boston, MA).
Isolation of human eosinophils
Eosinophils were isolated by a method modified from that reported by Hansel et al. (24). Briefly, 120 ml of whole blood was withdrawn from the antecubital vein and placed into containers containing 2 ml of 1/1000 diluted heparin. Blood was diluted 1/1 with calcium-free HBSS, layered over 15 ml of 1.089 g/ml Percoll, and centrifuged for 20 min at 900 x g. The supernatant and the mononuclear cells at the interface were aspirated carefully, and the inside wall of the tube was wiped with sterile gauze to remove mononuclear cells attached to the wall. To the pellet of granulocytes and erythrocytes, 20 ml of ice-cold sterile water was added and mixed gently for 30 s, after which 20 ml of 2x HBSS was added. If erythrocytes remained, the procedure was repeated. After erythrocyte lysis, granulocytes were washed once in HBSS/0.2% BSA, total cell numbers were counted using a Coulter counter (Hialeah, FL), and neutrophil percentage was calculated by differential counts of Wright-Giemsa-stained cytospin preparations. The supernatant was carefully aspirated, leaving the pellet nearly dry. The pellet was cooled on ice, and 0.65 µl of CD16 beads (Miltenyi Biotec)/million neutrophils was added. Granulocytes were incubated at 4°C for 30 min and then resuspended in 10 ml of HBSS/0.2% BSA. Granulocytes then were passed through a 1 x 10 cm column packed with steel wool and held within a 0.6 Tesla MACS magnet (Becton Dickinson, Mountain View, CA). Cells were eluted with another 30 ml of HBSS/0.2% BSA. Neutrophils binding the Ab-magnetic beads were retained in the magnetized steel wool, while eosinophils passing through the column were collected, washed, and resuspended in HBSS/0.2% BSA. Count and purity were assessed as described above. An eosinophil purity of >99% was routinely obtained. Cells were kept on ice until use.
Cell adhesion assay
The eosinophil adhesion assay was modified from the method described by Nagata et al. (25). Eosinophil adherence was assessed as residual eosinophil peroxidase (EPO) activity of adherent cells. Fifty microliters of soluble human ICAM-1 or VCAM-1 dissolved in 0.05 M NaHCO3 coating buffer (15 mM NaHCO3 and 35 mM Na2CO3, pH 9.2) was added to flat-bottom 96-well microtiter plates and incubated at 4°C overnight. ICAM-1 or VCAM-1 was decanted, and 100 µl/well of neat FBS was added to coated wells; after 60-min incubation at 37°C, the wells were decanted and washed with HBSS before the addition of eosinophils. Eosinophils (1 x 104/80 µl HBSS/0.1% gelatin) were preincubated with different concentrations of cPLA2, sPLA2, 5-LO, CO inhibitors, the PAF receptor blocker, CV6209, anti-CD11a mAb (clone 25.3), anti-CD11b mAb (clone 44), anti-CD18 mAb (7E4), anti-CD29 mAb (Lia 1/2), anti-VLA-4 mAb (HP2/1), or isotype control for 20 min at 37°C. Cells then were added to each well of ICAM-1- or VCAM-1-coated microplates with or without various concentrations of IL-5 and allowed to settle for 10 min on ice. Plates were rapidly warmed to 37°C and incubated for the indicated times. After three washes with HBSS, 80 µl of HBSS/0.1% gelatin was added to the reaction wells, and serial dilutions of the original cell suspension were added to the empty wells to generate a standard curve. One hundred microliters of EPO substrate (1 mM H2O2, 1 mM OPD, and 0.1% Triton X-100 in Tris buffer, pH 8.0) was then added to the 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 (Thermomax, Molecular Devices, Menlo Park, CA). All assays were performed in duplicate. Data storage and analysis were facilitated by the use of computer software interfaced with the reader (Softmax, Molecular Devices). The detection of EPO by this assay was linear between concentrations of 103-104 cells/well, as determined by a standard curve. No EPO activity was detected in the cell-free reaction supernatants (ICAM-1 with or without IL-5 or VCAM-1) following 30-min incubation, confirming that EPO was not present because of spontaneous eosinophil degranulation. Finally, none of the cPLA2 inhibitors inhibited EPO activity.
Immunoblot analysis of cPLA2
Eosinophils (5 x 106/group) were incubated with 5 ng/ml of IL-5 or added to VCAM-1-coated six-well plates for various times, and the reaction was stopped by adding cold stopping buffer (1 mM EDTA in HBSS without calcium). Eosinophils were collected and centrifuged at 400 x g for 10 min. The pellets then were lysed in 400 µl of lysis buffer (20 mM Tris-HCl, 30 mM Na4P2O7, 50 mM NaF, 40 mM NaCl, and 5 mM EDTA, pH 7.4) containing 1% Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 2 mM Na3VO4, and 0.5% deoxycholic acid. After 10 min on ice, the sample was centrifuged at 12,000 x g for 20 min to remove nuclear and cellular debris. The supernatants then were mixed with 10 µl of anti-cPLA2 mAb (Genetics Institute, Cambridge, MA) and shaken for 90 min; 30 µl of protein A-Sepharose beads was added, and incubation was continued for another 30 min. The immunoprecipitated proteins were washed four times with lysis buffer. Afterward, 30 µl of sample buffer was added and boiled for 5 min. The supernatant was collected and saved at -70°C.
Aliquots of immunoprecipitated protein were subjected to SDS-PAGE, using 10% acrylamide gels under reducing condition (15 mA/gel). Electrophoresis was stopped 180 min after the tracking dye had left the gel as described previously (26). Electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semidry system (400 mA, 60 min). The membrane was blocked with 1% BSA for 60 min, then incubated with 1/5000 diluted polyclonal anti-cPLA2 diluted in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) for 60 min. The membranes were washed three times for 20 min each time with TBS-T. Goat anti-rabbit IgG conjugated with HRP was diluted 1/3000 in TBS-T and incubated with polyvinylidene fluoride membrane for 60 min. The membrane was again washed three times with TBS-T and assayed by an enhanced chemiluminescence system (Amersham, Arlington Heights, IL). Autoradiographs were quantitated by densitometric analysis using a Kodak digital science 1D image processing system (Eastman Kodak, Rochester, NY). The results were expressed as the percentage of phosphorylated cPLA2 [(OD of slower migrating bands/OD of both slower and faster migrating bands) x 100].
Assessment of cPLA2 activity in intact eosinophils
The cPLA2 activity in intact eosinophils was assessed by measuring the release of [3H]AA/metabolites from the labeled cells. Radiolabeling of cells with [3H]AA was achieved by including 0.5 µCi/ml [3H]AA in HBSS without calcium for 90 min. Labeled AA that had not been incorporated into cellular lipids was removed by washing the cells three times with HBSS/0.2% BSA. Cells were resuspended in 106/160 µl (HBSS/0.2% BSA) and preincubated with TFMK or vehicle for 20 min at 37°C and then were incubated with or without 5 ng/ml IL-5 for 30 min. Eosinophils were stimulated with 1 µM calcium ionophore for 30 min. The reaction was stopped by centrifugation, and the supernatants were removed and assayed for radioactivity by liquid scintillation counting.
Analysis of surface integrin expression by immunofluorescence flow cytometry
Eosinophils were preincubated with various concentrations of a-TFMK for 20 min and then stimulated by 5 ng/ml IL-5 for 30 min. Thereafter, eosinophils were centrifuged at 400 x g for 10 min, and the pellets were resuspended in PBS/0.5% BSA. Aliquots of 5 x 105 eosinophils were incubated with 10 µl of mAb CD11b (Bear 1), CBRM1/5, or isotype-matched control Ab for 30 min at 4°C. After two washes, the cells were incubated with an excess of FITC-conjugated goat anti-mouse Ig for 20 min at 4°C. The cells were washed twice, resuspended in 1% paraformaldehyde, and kept at 4°C until analyzed. Flow cytometry was performed by FACScan (Becton Dickinson, Mountain View, CA). Fluorescence intensity was determined on at least 5000 cells from each sample. The results were expressed as specific mean fluorescence intensity (control Ab fluorescence subtracted).
Determination of eosinophil viability after inhibition of cPLA2 with a-TFMK or surfactin
To determine whether either a-TFMK or surfactin affected eosinophil viability, trypan blue exclusion was assessed in eosinophils incubated with either inhibitor. Aliquots of 104 eosinophils were incubated for 20 min at 37°C with various concentrations of a-TFMK or surfactin. Eosinophils then were centrifuged at 400 x g, and pellets were resuspended in 10 µl of HBSS. An equal volume of 0.01% trypan blue was added, and viable eosinophils were counted in a hemacytometer.
Statistical analysis
All data are expressed as the mean ± SEM. Differences between groups were assessed by paired t test. Where more than two groups were compared, differences among groups were assessed by one-way ANOVA. Where differences were found, comparisons among groups were made by Fisher’s least protected difference test. Statistical significance was claimed where p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Initial experiments were conducted to determine the
concentration-dependent effect of plated ICAM-1 on eosinophil adhesion.
Eosinophils activated by 5 ng/ml IL-5 adhered to ICAM-1 in a
concentration-dependent manner. At 10 µg/ml ICAM-1, eosinophil
adhesion was substantial (28.7 ± 3.5%) compared with that in
buffer-coated control wells (0.8 ± 0.2%; p <
0.01). Eosinophils that were not activated with IL-5 did not adhere to
ICAM-1-coated plates; nonspecific adhesion was 0.8 ± 0.5% (no
ICAM-1) and 3.8 ± 2.4% with 10 µg/ml ICAM-1
(p < 0.001 vs IL-5-treated eosinophils; Fig. 1
A). On the basis of these
results, 10 µg/ml of ICAM-1 was selected as the optimal concentration
for subsequent experiments. Experiments also were conducted to
determine the kinetics of eosinophil adhesion to ICAM-1-coated plates.
IL-5-induced adhesion was evident at 10 min and plateaued between
15–30 min (Fig. 1B). Consequently, a 30-min incubation time
was used for ICAM-1 adhesion in subsequent experiments. Eosinophil
adhesion was elicited in a concentration-dependent manner by IL-5 (Fig. 1C). At 5 ng/ml of IL-5, eosinophil adhesion was 25.5
± 3.1 vs 4.9 ± 3.2% for buffer-stimulated control (zero point;
p < 0.001). Accordingly, 5 ng/ml of IL-5 was used in
subsequent experiments. The contribution of the leukocyte integrins to
the adhesion of eosinophils to ICAM-1 was also evaluated in inhibition
assays using specific blocking mAb. Adhesion of eosinophils to ICAM-1
was confirmed in our system to be ß2 integrin
mediated, and adhesion of stimulated cells was significantly inhibited
by anti-Mac-1 mAb (clone 44) from 27.1 ± 5.5 to 13.2 ±
2.3% (p < 0.02). Similarly, blockade of the
common ß2-chain mAb anti-CD18 (clone 7E4)
caused a decrease in adhesion to 3.2 ± 1.0%
(p < 0.01 vs control). Neither anti-CD11a
nor the isotype control had a measurable inhibitory effect on
eosinophil adhesion to ICAM-1 (Fig. 1D).
|
2.5 µg/ml
VCAM-1 and required no up-regulating cytokine. Eosinophil adhesion was
26.7 ± 5.8% for plates coated with 2.5 µg/ml VCAM-1 vs
3.1 ± 0.8% for buffer-coated control wells
(p < 0.01; Fig. 2A). This plateau
concentration of 2.5 µg/ml of VCAM-1 thus was selected for all
subsequent experiments. Unlike ICAM-1, eosinophil adhesion to VCAM-1
occurred quickly within 5 min (25.4 ± 4.8 vs 0.6 ± 0.3%
for FBS-blocked buffer-coated controls; p < 0.01; Fig. 2B). Adhesion of eosinophils to VCAM-1 was confirmed to be
ß1 integrin mediated. Eosinophil adhesion to
VCAM-1 was significantly inhibited by anti-VLA-4 mAb (clone HP2/1)
from 29.7 ± 5.3 to 7.9 ± 1.6% (p
< 0.01). Similarly, blockade of the common
ß1-chain mAb anti-CD29 (clone Lia1/2)
caused a decrease in adhesion to 13.8 ± 0.7% (p < 0.01).
The isotype control had no measurable inhibitory effect on eosinophil
adhesion to VCAM-1 (Fig. 2C).
|
a-TFMK, a slow and tight-binding inhibitor, which binds directly
to the active site of cPLA2 (27),
caused concentration-dependent suppression of IL-5-stimulated
eosinophil adhesion to ICAM-1-coated (IC50 = 1.6
µM) or resting eosinophil adhesion to VCAM-1-coated
(IC50 = 2.9 µM) plates (Fig. 3, A and B).
Complete inhibition of eosinophil adhesion to ICAM-1 or VCAM-1 was
achieved at 10 µM (p < 0.001) a-TFMK.
Comparable inhibition of eosinophil adhesion to VCAM-1 or ICAM-1 also
was obtained with a structurally unrelated cPLA2
inhibitor, surfactin, which selectively inhibits
cPLA2 activity through a direct, possibly
covalent, and irreversible binding to the enzyme (22).
Total inhibition of eosinophil adhesion to either ICAM-1 or VCAM-1 was
obtained with 10 µM (p < 0.001) surfactin
(Fig. 3, A and B).
|
, A
and B). These concentrations have been determined previously
to inhibit sPLA2, 5-LO, and CO activities,
respectively (28, 29, 30). The action of
cPLA2 also liberates PAF, which has been shown to
cause eosinophil adherence (31). We addressed this
possibility using the PAF receptor blocker, CV6209 (32).
At 10 µM, CV6209 inhibited IL-5-induced adhesion to ICAM-1 from
25.2 ± 3.4 to 5.6 ± 1.3% (p <
0.01) and spontaneous adhesion to VCAM-1 from 30.8 ± 5.1 to
11.8 ± 2.2% (p < 0.01; Fig. 4, A and B).
|
).
|
Western blot analyses were used to determine whether IL-5 or adhesion to VCAM-1 caused phosphorylation of cPLA2. Phosphorylation of cPLA2 results in an increase in the activity of both recombinant cPLA2 (33) and cellular cPLA2 (18, 26, 34). Although phosphorylation of cPLA2 can occur at several sites (35), only phosphorylation of mitogen-activated protein kinases on serine 505 has been associated with activation of the enzyme. Phosphorylation of the enzyme at serine 505 results in decreased mobility of the protein on SDS-PAGE (33).
Eosinophils were incubated with 5 ng/ml of IL-5 or adhered to
VCAM-1-coated plates at 37°C for various times. Cells then were
lysed, and immunoprecipitated proteins were subjected to SDS-PAGE and
immunoblotting. cPLA2 phosphorylation was
substantially increased with IL-5 stimulation or adhesion to VCAM-1, as
indicated by its slower electrophoretic mobility (Fig. 5, A and C). Fig. 5, B and D, show the relative changes in the two
immunoreactive bands as quantified by densitometry and expressed as a
percentage of phosphorylated cPLA2 (see above).
IL-5-induced cPLA2 phosphorylation was evident at
15 min and progressed with time of eosinophil exposure to IL-5 (Fig. 5B). The kinetics of IL-5-induced
cPLA2 phosphorylation corresponded to eosinophil
adhesion to ICAM-1 (see Fig. 1
B).
cPLA2 phosphorylation with adhesion to VCAM-1
occurred more quickly (Fig. 5D). Phosphorylation was
apparent 5 min after adhesion and persisted up to 30 min, which also
corresponded temporally to the kinetics of eosinophil adhesion to
VCAM-1 (see Fig. 2B). Treatment of the immunoprecipitated
protein with potato acid phosphatase before electrophoresis converted
the protein back to a faster migrating form, which demonstrated that
the slower migration of cPLA2 was the result of
phosphorylation of the enzyme (data not shown).
|
To examine whether the phosphorylation of
cPLA2 corresponded to increased activity of the
enzyme, [3H]AA release was measured after
activation with ionophore in eosinophils pretreated with IL-5.
Incubation of eosinophils with IL-5 alone caused undetectable AA
release; however, IL-5 preincubation augmented ionophore-induced AA
release from 3565 ± 220.6 to 6325 ± 135.0 cpm
(p < 0.05). Preincubation with a-TFMK
inhibited IL-5-primed AA release to the baseline
(p < 0.001; Fig. 6).
|
Exposure of eosinophils to IL-5 caused increased expression of
Mac-1 on the eosinophil surface from 43.7 ± 8.8 to 72.4 ±
15.7 (p < 0.05). However, at concentrations
that blocked IL-5-induced adherence, a-TFMK had no effect on
up-regulation of Mac-1 surface expression (Fig. 7A). We also assessed the
effects of a-TFMK on the expression of the CBRM1/5 epitope, which is
expressed after the activated conformational change of Mac-1. IL-5
induced a 3-fold increase in CBRM1/5 epitope expression on eosinophil
that was not blocked by a-TFMK at concentrations sufficient to block
eosinophil adhesion to ICAM-1 (Fig. 7B).
|
Incubation of isolated eosinophils with either a-TFMK or surfactin
in concentrations 
10 µM did not alter significantly the ability of
eosinophils to exclude trypan blue. In three experiments, control
eosinophils were 97.8 ± 2.3% viable compared with 95.7 ±
2.8 and 92.5 ± 3.4% for eosinophils incubated with 10 µM
a-TFMK or 10 µM surfactin, respectively (p
= NS).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The objective of this investigation was to determine the role of cPLA2 (36) in the regulation of integrin adhesion to VCAM-1 and ICAM-1, which are the major counterligands, caused firm adhesion of eosinophils to endothelium. Studies were performed to assess the relationship between the surface expression and conformation of Mac-1 and VLA-4 and the ability of these integrins to adhere to endothelial counterligands. Our data demonstrated that even in optimal conformation, integrin adhesion depends upon activated (phosphorylated) cPLA2.
By using purified ligands (ICAM-1 and VCAM-1), we first established an
eosinophil adhesion model in vitro. Eosinophils do not spontaneously
adhere to ICAM-1, but require a second signal. IL-5-induced adhesion is
unique to eosinophils, because the receptor is absent on neutrophils
and rarely, if ever, expressed on other leukocytes (3, 8).
We confirmed in the system that we used for these studies that IL-5 was
highly efficacious in the induction of eosinophil adhesion to ICAM-1
and that this adhesion was ß2 integrin
dependent; anti-Mac-1 and anti-CD18 mAb caused marked
inhibition of IL-5-induced adhesion to ICAM-1 (Fig. 1), as has been
suggested previously (3, 37). In contrast to adhesion to
ICAM-1, eosinophils adhere to VCAM-1 spontaneously, and this adhesion
is ß1 integrin dependent; we further confirmed
that anti-VLA-4 and anti-CD29 mAb caused marked inhibition of
adhesion to VCAM-1 (Fig. 2). These results indicated that VLA-4/CD29
was constitutively active, while CD11b/CD18 molecule was expressed in
an inactive state.
Eosinophils treated with selective cPLA2
inhibitors then were used to address the role of
cPLA2 in adhesion to ICAM-1 and VCAM-1. a-TFMK
and surfactin, two structurally and mechanistically different
inhibitors of cPLA2, caused virtually identical
inhibition of eosinophil adhesion to ICAM-1 and VCAM-1 (Fig. 3). Our
data suggest that cPLA2 is a functional mediator
for both ß1 and ß2
integrin-dependent adhesion. Both a-TFMK and surfactin inhibited
eosinophil adhesion in a concentration-dependent manner. Inhibition was
specific; a-TFMK, selectively inhibits cPLA2 with
a 500-fold greater potency than for group IIa
sPLA2 (27, 38). Inhibition of adhesion was not
caused by toxicity of the either inhibitor. Cell viability was always
>92% even at the greatest concentration of inhibitor as assessed by
trypan blue exclusion. Western blots demonstrated that stimulation of
eosinophils by IL-5 or adhesion to VCAM-1 induces a time-dependent
phosphorylation of cPLA2 that is reflected by an
increase in the catalytic activity of cPLA2, and
these cPLA2 phosphorylations correlated
temporally and quantitatively with adhesion to both ICAM-1 and VCAM-1
(Fig. 5). These results further support the role of
cPLA2 activation in ß1
and ß2 integrin-dependent adhesion of
eosinophils. Inhibition of the 14-kDa type IIa
sPLA2, which has been detected in human
eosinophils in the specific granules by immunocytochemistry
(16), with the selective sPLA2
inhibitor, LY311727, caused no blockade of IL-5-induced adhesion to
ICAM-1 or spontaneous adhesion to VCAM-1 (Fig. 4).
We further examined whether cPLA2 activation of
eosinophil adhesion to ICAM-1 or VCAM-1 resulted from synthesis of AA
metabolites or PAF. Exogenously added AA (36) did not
reverse the inhibition of eosinophil adhesion to ICAM-1 or VCAM-1
caused by either surfactin or a-TFMK, suggesting that neither AA nor
its metabolites are involved in eosinophil adhesion (Table I). Using
the CO inhibitor, indomethacin, and the 5-LO inhibitor, zileuton, we
further established that neither CO nor 5-LO metabolites of AA are
involved in integrin-mediated adhesion of eosinophils to VCAM-1 or
ICAM-1 (Fig. 4). We next examined the role of endogenous PAF in
eosinophil adhesion to ICAM-1 and VCAM-1. The PAF receptor antagonist,
CV-6209, inhibited both ß1 and
ß2 integrin-dependent adhesion, suggesting that
cPLA2 catalysis of phosphatidylcholine to PAF
could be an autocrine proadherence factor in eosinophils (Fig. 4). This
supposition is supported by the recent finding that endogenous PAF
produced by IL-5-stimulated eosinophils is also involved in other
effector functions, including eosinophil degranulation
(39). The mechanism by which PAF up-regulates
ß1 and ß2 integrin-mediated adhesion,
however, remains unknown, and we could not distinguish whether the PAF
receptor involved in this process was an intracellular or plasma
membrane receptor.
When eosinophils are activated with IL-5, Mac-1 on their surface develops enhanced ability to bind to ICAM-1. This may be due to changes in the levels of Mac-1 on the cell surface (8) or to conformational changes in Mac-1 as detected by the appearance of activation-associated epitopes (5). We first evaluated whether a correlation exists between cPLA2 inhibition and surface expression of Mac-1. a-TFMK did not block the resulting increase in cell surface Mac-1 expression. We next assessed the effects of a-TFMK on the expression of the activated epitope on Mac-1. It has been shown that CBRM1/5 Ab completely blocks Mac-1-dependent adhesion (5). By contrast, we also found that a-TFMK did not block the expression of the CBRM1/5 epitope on Mac-1.
We note with interest that while IL-5 was needed to prime cells for adhesion of ß2 integrin to ICAM-1 and to cause cPLA2 phosphorylation, VLA-4 adhesion to VCAM-1 occurred with these ligands in their constitutive state (and phosphorylation occurred with greater rapidity). Yet, blockade of cPLA2 or PAF prevented sustained adhesion. This suggests a two-step process by which optimally conformed ligands initiate cPLA2 phosphorylation, which in an autocrine manner then sustains the phosphorylation process. This concept is consistent with our observations in these studies that adhesion cannot occur even with IL-5 up-regulated cells if the cPLA2 phosphorylation or the PAF receptor is blocked.
We conclude that IL-5 up-regulation and conformation change in Mac-1 are not sufficient to cause integrin-adhesion to endothelial counterligands. We further demonstrate that activation of cPLA2 is essential to the formation of stable integrin adhesion for both ß1 and ß2 ligands through a mechanism that does not depend on arachidonate synthesis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address:
3 Abbvreviations used in this paper: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; sPLA2, secretory PLA2; AA, arachidonic acid; PAF, platelet-activating factor; TFMK, arachidonic trifluoromethyl ketone; 5-LO, 5-lipoxygenase; CO, cyclooxygenase; EPO, eosinophil peroxidase.
Received for publication April 7, 1999. Accepted for publication July 6, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  X. Zhu, E. Boetticher, L. Wang, Y. Duan, J. Learoyd, and A. R. Leff Proline-Rich Tyrosine Kinase 2 Regulates Spreading and Migration of Eosinophils after {beta}2-Integrin Adhesion Am. J. Respir. Cell Mol. Biol., September 1, 2008; 39(3): 263 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Johansson, E. A. B. Kelly, W. W. Busse, N. N. Jarjour, and D. F. Mosher Up-Regulation and Activation of Eosinophil Integrins in Blood and Airway after Segmental Lung Antigen Challenge J. Immunol., June 1, 2008; 180(11): 7622 - 7635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Meliton, N. M. Munoz, X. Zhu, and A. R. Leff Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks {beta}2-integrin adhesion in neutrophils J. Leukoc. Biol., February 1, 2008; 83(2): 344 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Barthel, M. W. Johansson, D. M. McNamee, and D. F. Mosher Roles of integrin activation in eosinophil function and the eosinophilic inflammation of asthma J. Leukoc. Biol., January 1, 2008; 83(1): 1 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Leff and N. M. Munoz Future Treatment to Lessen Exacerbations of Chronic Obstructive Pulmonary Disease Proceedings of the ATS, December 1, 2007; 4(8): 659 - 666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhu, J. Learoyd, S. Butt, L. Zhu, P. V. Usatyuk, V. Natarajan, N. M. Munoz, and A. R. Leff Regulation of Eosinophil Adhesion by Lysophosphatidylcholine via a Non-Store-Operated Ca2+ Channel Am. J. Respir. Cell Mol. Biol., May 1, 2007; 36(5): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Meliton, N. M. Munoz, A. Lambertino, E. Boetticher, J. Learoyd, X. Zhu, and A. R. Leff Phosphodiesterase 4 inhibition of {beta}2-integrin adhesion caused by leukotriene B4 and TNF-{alpha} in human neutrophils Eur. Respir. J., November 1, 2006; 28(5): 920 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Barthel, D. S. Annis, D. F. Mosher, and M. W. Johansson Differential Engagement of Modules 1 and 4 of Vascular Cell Adhesion Molecule-1 (CD106) by Integrins {alpha}4beta1 (CD49d/29) and {alpha}Mbeta2 (CD11b/18) of Eosinophils J. Biol. Chem., October 27, 2006; 281(43): 32175 - 32187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Munoz, A. Y. Meliton, A. Lambertino, E. Boetticher, J. Learoyd, F. Sultan, X. Zhu, W. Cho, and A. R. Leff Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces beta2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils J. Immunol., July 1, 2006; 177(1): 574 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sano, A. R. Leff, S. Myou, E. Boetticher, A. Y. Meliton, J. Learoyd, A. T. Lambertino, N. M. Munoz, and X. Zhu Regulation of Interleukin-5-Induced {beta}2-Integrin Adhesion of Human Eosinophils by Phosphoinositide 3-Kinase Am. J. Respir. Cell Mol. Biol., July 1, 2005; 33(1): 65 - 70. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Papi Investigating the steroids and long-acting {beta}2-agonists combination: why do we need more? Eur. Respir. J., April 1, 2004; 23(4): 501 - 502. [Full Text] [PDF]
|
|
|
|
|
|
S. Myo, X. Zhu, S. Myou, A.Y. Meliton, J. Liu, E. Boetticher, A.T. Lambertino, C. Xu, N.M. Munoz, and A.R. Leff Additive blockade of {beta}2-integrin adhesion of eosinophils by salmeterol and fluticasone propionate Eur. Respir. J., April 1, 2004; 23(4): 511 - 517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Myou, X. Zhu, S. Myo, E. Boetticher, A. Y. Meliton, J. Liu, N. M. Munoz, and A. R. Leff Blockade of Airway Inflammation and Hyperresponsiveness by HIV-TAT-Dominant Negative Ras J. Immunol., October 15, 2003; 171(8): 4379 - 4384. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nagase, N. Uozumi, T. Aoki-Nagase, K. Terawaki, S. Ishii, T. Tomita, H. Yamamoto, K. Hashizume, Y. Ouchi, and T. Shimizu A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L720 - L726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhu, B. Jacobs, E. Boetticher, S. Myou, A. Meliton, H. Sano, A. T. Lambertino, N. M. Munoz, and A. R. Leff IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2 J. Leukoc. Biol., November 1, 2002; 72(5): 1046 - 1053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Myou, X. Zhu, E. Boetticher, S. Myo, A. Meliton, A. Lambertino, N. M. Munoz, and A. R. Leff Blockade of Focal Clustering and Active Conformation in {beta}2-Integrin-Mediated Adhesion of Eosinophils to Intercellular Adhesion Molecule-1 Caused by Transduction of HIV TAT-Dominant Negative Ras J. Immunol., September 1, 2002; 169(5): 2670 - 2676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhu, H. Sano, K. P. Kim, A. Sano, E. Boetticher, N. M. Munoz, W. Cho, and A. R. Leff Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils J. Immunol., July 1, 2001; 167(1): 461 - 468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bhattacharya, R. Patel, N. Sen, S. Quadri, K. Parthasarathi, and J. Bhattacharya Dual signaling by the {alpha}v{beta}3-integrin activates cytosolic PLA2 in bovine pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L1049 - L1056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sano, X. Zhu, A. Sano, E. E. Boetticher, T. Shioya, B. Jacobs, N. M. Munoz, and A. R. Leff Extracellular Signal-Regulated Kinase 1/2-Mediated Phosphorylation of Cytosolic Phospholipase A2 Is Essential for Human Eosinophil Adhesion to Fibronectin J. Immunol., March 1, 2001; 166(5): 3515 - 3521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Urasaki, J. Takasaki, T. Nagasawa, and H. Ninomiya Pivotal role of 5-lipoxygenase in the activation of human eosinophils: platelet-activating factor and interleukin-5 induce CD69 on eosinophils through the 5-lipoxygenase pathway J. Leukoc. Biol., January 1, 2001; 69(1): 105 - 112. [Abstract] [Full Text]
|
|
|
|
|
|
N. M. Munoz, G. A. van Seventer, R. T. Semnani, and A. R. Leff Augmentation of LTC4 synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro Am J Physiol Lung Cell Mol Physiol, June 1, 2000; 278(6): L1172 - L1179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Anthonsen, S. Andersen, A. Solhaug, and B. Johansen Atypical lambda /iota PKC Conveys 5-Lipoxygenase/Leukotriene B4-mediated Cross-talk between Phospholipase A2s Regulating NF-kappa B Activation in Response to Tumor Necrosis Factor-alpha and Interleukin-1beta J. Biol. Chem., September 14, 2001; 276(38): 35344 - 35351. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
