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2-Integrin-Mediated Adhesion of Eosinophils to Intercellular Adhesion Molecule-1 Caused by Transduction of HIV TAT-Dominant Negative Ras1
* Section of Pulmonary and Critical Care Medicine, Departments of Medicine and
Neurobiology Pharmacology and Physiology and Committees on Molecular Medicine, Clinical Pharmacology, and Cell Physiology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-integrin adhesion,
neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to
VCAM-1. Thus, a substantially different signaling mechanism was
identified for 1-integrin adhesion. We conclude that
H-Ras-mediated activation of ERK is critical for
2-integrin adhesion and that Ras-protein functions as
the common regulator for cytokine-, chemokine-, and G-protein-coupled
receptors in human eosinophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Integrins are a diverse family of 
heterodimetric transmembrane
adhesion receptors that are important for firm adhesion and subsequent
transendothelial migration of leukocytes (5, 7). The
2-integrin subfamily consists of four
integrins, CD11a (LFA-1), CD11b (Mac-1), CD11c, and CD11d, which share
a common 2 subunit (CD18) and are exclusively
expressed on leukocytes (8). The Mac-1 (CD11b/CD18)
molecule as well as the very late antigen-4
(VLA-4)4 (CD49d/CD29)
molecule of the 1-integrin subfamily
contribute to eosinophil adherence to endothelium through binding to
the endothelial ligands ICAM-1 and VCAM-1 (9, 10), in
which bidirectional (inside-out and outside-in) transmembrane signal
transduction is involved.
Recent studies indicate that cytosolic phospholipase
A2 (cPLA2)
phosphorylation is essential for integrin-mediated eosinophil
adhesion (11, 12). We also have reported
previously that pharmacological inhibition of
cPLA2 prevents Ag and IL-5-induced eosinophil
migration and airway hyperresponsiveness in immune-stimulated guinea
pigs (13). However, the process by which
cPLA2 is activated to cause integrin-mediated
adhesion remains unknown. H-Ras has been reported to cause
attenuation of IL-5-mediated survival of eosinophils in vitro at 16 h
(14). H-Ras also has been reported to enhance both
1- and
2-integrin-mediated adhesion in
nondifferentiated cell systems (15, 16). By contrast,
H-Ras has been reported to dowregulate 1-,
2-, and 3-integrin
adhesion (17, 18).
To assess the mechanism of 2-mediated integrin
adhesion in human eosinophils, we transduced dominant negative (dn)
H-Ras protein using an HIV-TAT protein vector into fully differentiated
human eosinophils and examined the mechanism of chemokine, cytokine,
and chemoattractant peptides in the induction of adhesion. To examine
the role of Ras in eosinophil adhesion, we constructed a dnRas plasmid
(pTAT-dnRas) containing six His residues, 11 amino acids of TAT, an N17
dn H-Ras, and purified TAT-dnRas fusion protein (Fig. 1
A). The role of H-Ras in
adhesion was then determined in TAT-dnRas-transduced eosinophils
stimulated by the cytokine IL-5, the chemokine eotaxin-1, or the
chemoattractant fMLP. We further determined the involvement of H-Ras in
the regulation of Mac-1 expression, conformational change to the
activated state, and lateral clustering of integrin on the eosinophil
surface. Our data demonstrate that Ras is a common regulator of
extracellular signal-regulated kinase (ERK) phosphorylation, which
changes 2-integrin into its active
conformation and activates lateral movement of integrin to achieve
critical clustering for adhesion mediated by cytokines, chemokines, and
chemoattractant substances.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-5, eotaxin-1, soluble human ICAM-1, and soluble human VCAM-1 were purchased from R&D Systems (Minneapolis, MN). Eosinophil isolation materials were obtained from Miltenyi Biotec (Sunnyvale, CA). fMLP was purchased from Sigma-Aldrich (St. Louis, MO). Polystyrene 96-well microtiter plates were obtained from Costar (Cambridge, MA). H-Ras cDNA (17N mutant) in pUSEamp, GST-Ras binding domain (RBD), and anti-Ras mAb (clone RAS10) were obtained from Upstate Biotechnology (Lake Placid, NY). pcDNA3.1/CT-GFP-TOPO vector was purchased from Invitrogen (Carlsbad, CA). T4 ligase was purchased from Fermentas (Hanover, MD). BL21(DE3)pLysS cells were obtained from Novagen (Madison, WI). Ni-NTA columns were purchased from Qiagen (Valencia, CA). A 10,000 MWCO Slide-A-Lyzer dialysis cassette was purchased form Pierce (Rockford, IL). PD98059 and ERK1/2 Ab were purchased from New England Biolabs (Beverly, MA). Phosphorylation-specific ERK1/2 polyclonal Ab, AgeI, and SphI were obtained from Promega (Madison, WI). Anti-CD11b mAb (clone 44) was purchased from Endogen (Woburn, MA). Mouse IgG was purchased from BD Biosciences (Mountain View, CA). Donkey anti-rabbit IgG conjugated with HRP was purchased from Amersham (Arlington Heights, IL). The CBRM1/5 mAb against activated CD11b was a gift from Dr. T. A. Springer (Harvard Medical School, Boston, MA). pTAT was a gift from Dr. S. Dowdy (University of California at San Diego, La Jolla, CA).
Generation of TAT fusion protein
A cDNA fragment encoding dn H-Ras was amplified by PCR from the H-Ras cDNA (17N mutant) in pUSEamp with the following primers (sense, 5'-ATC ATG ACA GAA TAC AAG CTT GTG GTG GT-3'; antisense, 5'-AGG AGA GCA CAC ACT TGC AGC TCA TG-3'). The amplified PCR products were inserted into a pcDNA3.1/CT-GFP-TOPO vector. After insertion of H-Ras 17N cDNA, the end of dnRas cDNA had a TAA stop codon. Site-directed mutagenesis was conducted to create an AgeI cutting site in pcDNA3.1-dnRas. The mutagenized primers were as follows (mutagenized bases are identified by lowercase letters): 5'-GCT CGG ATC CAC TAG TCC AGa ccG GTG GAA TTG CCC TTA TC-3' (sense) and 5'-GAT AAG GGC AAT TCC ACC ggt CTG GAC TAG TGG ATC CGA GC-3' (antisense). The mutated plasmids were digested with AgeI/SphI and ligated into an AgeI/SphI digested pTAT vector (kindly provided by Dr. S. Dowdy) using T4 ligase.
Purification of TAT fusion proteins was performed by a modified method described by Nagahara et al. (19). TAT-dnRas was purified by sonication of high-expressing BL21(DE3)pLysS cells in 10 ml of buffer Z (8 M urea, 20 mM HEPES, pH 8.0, 100 mM NaCl). Cellular lysates were resolved by centrifugation, loaded onto 5-ml Ni-NTA columns in buffer Z, washed, and eluted with imidazole in buffer Z sequentially in 100, 250, and 500 mM concentrations. Urea and imidazole were removed from the resultant protein solution using a low-volume 10,000 MWCO Slide-A-Lyzer dialysis cassette (Pierce). Each fusion protein was flash-frozen at -80°C.
Cell adhesion assay
Cell adherence was assessed as residual eosinophil peroxidase (EPO) activity of adherent cells. Fifty microliters of soluble human ICAM-1 (10 µg/ml) or VCAM-1 (5 µg/ml) 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 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. Cells (1 x 104/100 µl HBSS/0.1% gelatin) were added to each well of ICAM-1- or VCAM-1-coated microplates with or without stimulators such as 10 ng/ml IL-5, 100 ng/ml eotaxin-1, 1 µM fMLP, or 1 nM PMA and were allowed to settle for 10 min on ice. Plates were rapidly warmed to 37°C and incubated for 30 min. After wash with HBSS, 100 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. A total of 100 µl of EPO substrate (1 mM H2O2, 1 mM o-phenylenediamine, 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 1.0 x 103 and 1.5 x 104 cells/well, as determined by a standard curve. The effect of TAT-dnRas or PD98059 on integrin-mediated adhesion was tested by preincubation of TAT-dnRas or PD98059 with cells for 30 min at 37°C.
Isolation of human eosinophils
Eosinophils were isolated by a method modified from Hansel et al. (20). The method is based on Percoll centrifugation (density 1.089 g/ml) to isolate granulocytes, hypotonic lysis of RBCs, and, finally, immunomagnetic depletion of neutrophils by a magnetic cell separation system using anti-CD16-coated MACS particles. Eosinophil purity of 99% was routinely obtained, as assessed by Wright-Giemsa staining. Cells were kept on ice until use.
Immunoblot analysis of ERK1/2
Eosinophils (2 x 106/group) were
stimulated with 10 ng/ml IL-5 for 10 min, 100 ng/ml eotaxin-1 for 1
min, 1 nM fMLP for 1 min, or 1 nM PMA for 5 min, and the reaction was
stopped by centrifugation at 12,000 x g for 30 s.
The pellets were then lysed in 80 µl of lysis buffer (20 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, 1 mM
Na3VO4, 1 µg/ml
leupeptin, and 1 mM PMSF). After 20 min on ice, the samples were
centrifuged at 12,000 x g for 2 min to remove nuclear
and cellular debris. The supernatants were then mixed with 14 µl of
6x sample buffer and boiled for 5 min. The samples were collected and
saved at -70°C.
Samples were subjected to SDS-PAGE, using 10% acrylamide gels under reducing condition (15 mA/gel). 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 and then incubated with 1/5000 anti-phosphorylation-specific ERK1/2 Ab or 1/1000 anti-ERK1/2 Ab diluted in TBST overnight. The membranes were then washed three times for 20 min with TBST. Donkey anti-rabbit IgG conjugated with HRP was diluted 1/3000 in TBST and incubated with polyvinylidene fluoride membrane for 60 min. The membrane was again washed three times with TBST and assayed by an ECL system (Amersham).
Analysis of surface integrin expression by immunofluorescence flow cytometry and confocal microscopy
Eosinophils were preincubated with TAT-dnRas or PD98059 for 30 min at 37°C and then stimulated by 10 ng/ml IL-5, 100 ng/ml eotaxin-1, 1 µM fMLP, or 1 nM PMA for 30 min. Thereafter, cells were centrifuged at 400 x g for 10 min, and the pellets were resuspended in PBS with 1% BSA for FACScan analysis or in PBS with 0.1% BSA for confocal microscope analysis. Aliquots of 5 x 105 cells were incubated with 10 µg/ml of mAb directed against Mac-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 (BD Biosciences). Fluorescence intensity was determined on at least 5000 cells from each sample. The results were expressed as the specific mean fluorescence intensity (sMFI) (control Ab fluorescence subtracted).
To confirm the distribution of Mac-1, Mac-1 staining was also performed on cytospin preparations (Cytospin 2; Shandon, Pittsburgh, PA), and fluorescence was analyzed using an Axiovert confocal microscope (Zeiss, Oberkochen, Germany) equipped with an external argon-krypton laser (488 nm).
Ras activation assay
The activated Ras was affinity precipitated following the manufacturer’s instructions (Upstate Biotechnology). Next, the eosinophils (5 x 106/group) were incubated with 100 nM TAT-dnRas for 30 min and then stimulated with 10 ng/ml IL-5 for 10 min, 100 ng/ml eotaxin-1 for 1 min, 1 nM fMLP for 1 min, or 1 nM PMA for 1 min. The reaction was stopped by centrifuging at 12,000 x g for 30 s, and the cell pellets were immediately lysed in Ras affinity precipitation lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM EGTA, 10% glycerol, 1% Nonidet P-40, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 50 mM NaF, 2 mM Na3VO4, and 1 mM PMSF). After 20 min on ice, the sample was centrifuged at 16,000 x g for 10 min at 4°C to remove nuclear and cellular debris. The supernatants were then incubated with GST-RBD for 30 min at 4°C with rocking. The GST-RBD beads were washed three times with lysis buffer. Bound proteins were eluted by boiling in 2x Laemmli sample buffer, separated by SDS-PAGE, and processed for immunoblot analysis using an anti-Ras mAb (clone RAS10).
Statistical analysis
All measurements were expressed as mean ± SEM. Variation between two groups was tested using Student’s t test. Variation among more than two groups was tested using ANOVA followed by Fisher’s protected least significant difference. A value of p < 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To access the efficacy of protein transduction, eosinophils were
incubated with 100 nM TAT-dnRas (Fig. 1
A) for 
30 min, and
samples were analyzed by Western blot. TAT-dnRas in cell lysates was
detected within 5 min of incubation; maximal detection occurred within
20–30 min (Fig. 1B). Consequently, a 30-min incubation time
was used in the subsequent experiments. Eosinophils were 95% viable
during the course of these experiments, as indicated by trypan blue
exclusion. TAT-dnRas transduction into eosinophils inhibited
IL-5-induced ERK1/2 phosphorylation in a time-dependent fashion. ERK1/2
phosphorylation was reduced at 10 min and blocked completely after 15
min (Fig. 1C, upper panel). TAT-dnRas caused
concentration-dependent blockade of IL-5-stimulated ERK1/2
phosphorylation. ERK1/2 phosphorylation was reduced at 30 nM and
blocked completely with 100–300 nM (upper panel). TAT
vehicle control (Fig. 1D, second from right) did
not block ERK phosphorylation. TAT-dnRas alone had no effect on ERK
phosphorylation (Fig. 1D, right). These
results demonstrated that TAT-dnRas functionally suppressed ERK
phosphorylation after transduction into eosinophils using HIV-TAT
protein.
Effect of TAT-dnRas on adhesion of eosinophils to ICAM-1 caused by IL-5, eotaxin-1, fMLP, and PMA
We then examined the role of H-Ras in IL-5-, eotaxin-1-, or
fMLP-induced eosinophil adhesion to plated ICAM-1. Adhesion of
eosinophils to ICAM-1 was previously confirmed to be mediated by
2-integrin (Mac-1) in our system
(12). To examine the potential role of ERK in eosinophil
adhesion, we first used a pharmacological inhibitor of
mitogen-activated protein/ERK kinase (MEK), PD98059. This compound
inhibits ERK1/2 phosphorylation and activation (21, 22).
In preliminary studies, we determined the concentrations of IL-5,
eotaxin-1, fMLP, and PMA causing maximal adhesion of eosinophils to
ICAM-1 (see Materials and Methods). PD98059 blocked adhesion
to eosinophils activated by IL-5, eotaxin-1, and fMLP at their most
efficacious concentrations in a concentration-dependent manner (Fig. 2A).
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A) and 2) dnRas prevents ERK
phosphorylation (Fig. 1, C and D), we next tested
the hypothesis that TAT-dnRas would block eosinophil adhesion to
ICAM-1. Adhesion of nonstimulated eosinophils to ICAM-1 was 3.0 ±
0.8%. Adhesion caused by 10 ng/ml IL-5, 100 ng/ml eotaxin-1, and 1
µM fMLP (as above) was blocked by transduction of 100 nM TAT-dnRas
from 30.6 ± 4.5 to 14.1 ± 1.7% (p <
0.01), from 28.7 ± 1.6 to 5.7 ± 1.8% (p
< 0.01), and from 24.4 ± 2.1 to 12.6 ± 1.7%
(p < 0.01), respectively (Fig. 2B). As a
further control, eosinophils also were pretreated with PMA, which
activates ERK phosphorylation by postmembrane receptor induction of
protein kinase C, a mechanism which bypasses Ras. PMA-induced
eosinophil adhesion to ICAM-1, which is not H-Ras mediated, was not
blocked by PD98059 or TAT-dnRas (Fig. 2).
In contrast to 2-integrin adhesion, which
requires integrin activation, 1-integrin
(VLA-4) adheres to VCAM-1 in its constitutive state (12).
Eosinophil adhesion was 24.0 ± 4.3% for VCAM-1-coated wells vs
2.4 ± 0.5% for buffer-coated control wells (p
< 0.01) (Fig. 3A). PD98059
had no inhibitory effect on eosinophil adhesion to VCAM-1 (Fig. 3A). Accordingly, pretreatment with TAT-dnRas also did not
prevent spontaneous eosinophil adhesion to VCAM-1 (Fig. 3), suggesting
that H-Ras is not involved in VLA-4-mediated eosinophil adhesion (see
Discussion).
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We next examined whether TAT-dnRas suppressed endogenous Ras and
ERK activation caused by IL-5, eotaxin-1, or fMLP. Eosinophils were
pretreated with or without TAT-dnRas for 30 min at 37°C and then
stimulated with 10 ng/ml IL-5, 100 ng/ml eotaxin-1, 1 µM fMLP, or 1
nM PMA. Cell lysates were precipitated by a GST fusion protein
containing RBD of Raf-1 (amino acids 1–149 of Raf-1), which binds only
to GTP-bound (activated) Ras, and were probed by a mAb to Ras. IL-5,
eotaxin-1, or fMLP all caused Ras activation (Fig. 4A). Maximum Ras activation
occurred at 10 min for IL-5 and at 1 min for eotaxin-1, fMLP, and PMA.
PMA, which caused adhesion that was not blocked by TAT-dnRas, caused no
activation of Ras (Fig. 4A). Ras activation caused by IL-5,
eotaxin-1, or fMLP caused ERK1/2 phosphorylation in eosinophils, which
was blocked by TAT-dnRas (Fig. 4B).
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We next examined the role of three previously postulated
mechanisms of integrin-mediated adhesion: 1) up-regulation of surface
integrin molecules (23), 2) the affinity/conformational
changes of the 2-integrin (10),
and 3) the avidity/clustering of surface integrins (24).
Accordingly, we first determined the role of H-Ras in regulating Mac-1
(CD11b/CD18) expression by flow cytometry. Stimulation of eosinophils
with 10 ng/ml IL-5, 100 ng/ml eotaxin-1, 1 µM fMLP, or 1 nM PMA all
caused increased expression of the CD11b -chain of Mac-1 on the
eosinophil surface from 38.6 ± 7.9 to 79.0 ± 13.4 sMFI
(p < 0.01), 61.1 ± 13.4 sMFI
(p < 0.01), 67.0 ± 11.0 sMFI
(p < 0.01), and 98.2 ± 13.3 sMFI
(p < 0.01), respectively (Fig. 5A). Pretreatment of
eosinophils with a concentration of 50 µM PD98059 (Fig. 5A) or 100 nM TAT-dnRas (Fig. 5B), concentrations
which blocked eosinophil adhesion to ICAM-1 (Fig. 2), did not block
up-regulation of Mac-1.
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C). Both TAT-dnRas and PD98059 prevented
the active conformation of Mac-1 caused by IL-5, eotaxin-1, or fMLP. In
contrast, postmembrane-mediated up-regulation of the active
conformational epitope of Mac-1 caused by PMA as measured by CBRM1/5
expression was not attenuated by either TAT-dnRas or PD98059 (Fig. 5C). Involvement of H-Ras in Mac-1 clustering
We further determined the mechanism of the Mac-1 clustering in
eosinophil adhesion using TAT-dnRas. Confocal immunofluorescence
microscopy demonstrated that IL-5, eotaxin-1, and fMLP all caused focal
clustering of Mac-1 on the eosinophil surface, which was blocked by
pretreatment with TAT-dnRas (Fig. 6) or
PD98059 (data not shown). In contrast, PMA caused Mac-1 clustering and
subsequent adhesion to ICAM-1 that was not inhibited by either
TAT-dnRas or PD98059. These data indicate that Mac-1 up-regulation is
not the mechanism by which H-Ras causes adhesion of eosinophils to
ICAM-1. Rather, Ras-mediated adhesion corresponds to active
conformational change and focal clustering of
2-integrin on the eosinophil surface.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-integrin adhesion to
the endothelial counterligand ICAM-1. By transducing dnRas directly
into fully mature human eosinophils using an HIV-TAT fusion protein, we
were able to determine the common pathway for induction of eosinophil
adhesion to Mac-1 caused by cytokine (IL-5), chemokine (eotaxin-1), and
chemoattractant (fMLP) stimulation. TAT-dnRas blocked endogenous Ras
activation and subsequent ERK phosphorylation, as predicted from
pharmacological studies using an inhibitor of MEK (Fig. 4). For IL-5,
eotaxin-1, and fMLP, this substantially attenuated Mac-1 adhesion to
ICAM-1 (Fig. 2). These results indicate that Ras may play a critical
role in eosinophil adhesion caused by inside-out signaling from
cytokine, chemokine, and chemoattractant.
Blockade of ERK phosphorylation caused by dnRas had no effect on
up-regulation of Mac-1 after cellular activation. However, dnRas
blocked both the induction of active conformation of Mac-1 and the
focal lateral clustering of Mac-1 (Fig. 6) as assessed by confocal
microscopy and immunofluorescent staining after activation by IL-5,
eotaxin-1, and fMLP. This is the first demonstration of a common
activation pathway for both induction of the active conformation of
Mac-1 and the clustering distribution of adhesion molecules in
eosinophils. The relative importance of each of these two mechanisms
was not defined in this investigation. It is important to note,
however, that the binding of mature human peripheral blood eosinophils
by VLA-4 to VCAM-1 is regulated independently of Ras (Fig. 3) and that
our data demonstrate unique and separate mechanisms for cell membrane
adhesion of 2- and
1-integrin to their respective counterligands,
ICAM-1 and VCAM-1.
We have shown previously that both 2- and
1-integrin-mediated adhesion depend critically
upon the phosphorylation of the 85-kDa cPLA2
(12) and that eosinophil-mediated adhesion to fibronectin
is critically dependent upon ERK phosphorylation (11).
However, at the cell membrane, neither ERK inhibition nor dnRas caused
any blockade of adhesion of mature human eosinophils to VCAM-1. Thus, a
separate mechanism exists at the cell surface for regulation adhesion
of VLA-4, which is constitutively expressed in its active state, and
for Mac-1 adhesion, which must be activated from its dormant state.
Although the mechanism potentially regulating Mac-1 adhesion appears to
involve Ras activation as a critical pathway, these studies do not
elucidate the mechanism by which VLA-4 adhesion to VCAM-1 is
regulated.
It is important to recognize some limitations of these findings.
Adhesion in vivo occurs during a state of constant flow and is a
multistep process. The extent to which investigations using plated
ligands replicate events in the living state, under conditions of
constant flow and shear stress, cannot be assessed for individually
plated ligands. However, we have shown previously that inhibition
of cPLA2 activity, which is activated by
ERK1/2, prevents eosinophil migration and airway hyperresponsiveness in
guinea pigs (13). We also note that blockade back to
baseline control levels of 2-integrin-ICAM-1
adhesion was complete only for eotaxin-1 (Fig. 2). This may be a
consequence of necessity for examining singly the binding of individual
integrin/Ig-supergene reactions, or it may imply that Ras mediation of
Mac-1 adhesion to ICAM-1 has additional regulatory components.
The sequential events of eosinophil migration and, particularly, the
regulation of integrin avidity still are not fully elucidated, and our
study has not outlined each step of Ras-mediated Mac-1 adhesion.
However, we have demonstrated for the first time two potentially
different mechanisms for regulation of eosinophil adhesion by
2- and 1-integrin in
fully mature human eosinophils by HIV-TAT transduction of a dnRas into
primary isolates of peripheral blood eosinophils. We demonstrate that
Ras-induced adhesion is highly specific for Mac-1 and that
VLA-4-mediated adhesion is regulated in a substantially different
manner at the cell surface. Our data demonstrate that receptor-mediated
activation of 2-integrin may be regulated
substantially and uniquely by Ras protein and that integrin surface
clustering and conformational change rather than increased surface
expression of Mac-1 are the critical steps for adhesion of activated
eosinophils. Our findings suggest that Ras may be the common regulator
for Mac-1-mediated adhesion of eosinophils induced by cytokines,
chemokines, and membrane-activating chemoattractants.
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Acknowledgments |
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Footnotes |
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2 S.M. and X.Z. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Alan R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: aleff{at}medicine.bsd.uchicago.edu
4 Abbreviations used in this paper: VLA, very late Ag; cPLA2, cytosolic phospholipase A2; dn, dominant negative; ERK, extracellular signal-regulated kinase; RBD, Ras binding domain; EPO, eosinophil peroxidase; sMFI, specific mean fluorescence intensity; MEK, mitogen-activated protein/ERK kinase.
Received for publication May 17, 2002. Accepted for publication July 1, 2002.
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2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88:7538.1 and 2 integrin avidity by chemoattractants in eosinophils. Proc. Natl. Acad. Sci. USA 93:10939.1 and 2 integrin-dependent adhesion of human eosinophils. J. Immunol. 163:3423.: implications for leukocyte transmigration. Mol. Biol. Cell 12:3074.This article has been cited by other articles:
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  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]
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 M. Lerm, A. Holm, A. Seiron, E. Sarndahl, K.-E. Magnusson, and B. Rasmusson Leishmania donovani Requires Functional Cdc42 and Rac1 To Prevent Phagosomal Maturation. Infect. Immun., May 1, 2006; 74(5): 2613 - 2618. [Abstract] [Full Text] [PDF]
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),
1 µM fMLP (
), or 1 nM PMA (
) for 30 min. B,
Eosinophils were pretreated with 100 nM TAT-dnRas (
) or 100 nM
TAT-vehicle (
) for 30 min at 37°C and then were stimulated by 10
ng/ml IL-5, 100 ng/ml eotaxin-1, 1 µM fMLP, or 1 nM PMA for 30 min.
Nonstimulated control (Cont.) and stimulated controls for each stimulus
are indicated as open bars and filled bars, respectively. Eosinophil
adhesion to plated ICAM-1 was measured by residual eosinophil peroxide
activity. Each point represents the mean ± SEM of four separate
experiments. *, p < 0.01 vs control for all
comparison.
