Department of Physiology, Freie Universität Berlin; and
* Department of Dermatology, Universität zu Köln, Germany
Correspondence: Barbara Walzog, Ph.D., Freie Universität Berlin, Department of Physiology, Arnimallee 22, D-14195 Berlin, Germany.
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion • host defense • inflammation • polymorphonuclear neutrophils
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Adhesion molecules of the integrin family mediate the recruitment of PMN to sites of inflammation by binding to specific ligands and allowing cell-cell and cell-substrate interactions [2 ]. Integrins also transduce signals into the cell, which are thought to control adhesion-related processes, including firm attachment and spreading [3 ]. Furthermore, integrins contribute to the activation of various PMN functions [4 ] and to the induction of PMN apoptosis [5 , 6 ]. Thus, the integrins are good candidates to control PMN in inflammation by integrating adhesion and signaling at the molecular level. However, the molecular sequence of events that mediates these complex functions is poorly understood.
The integrins are heterodimeric molecules consisting of an 
and a
noncovalently bound ß-subunit that span the plasma membrane once
[7
]. Among the integrin family, which is classified
according to the associated ß-subunit, the ß1 (CD29),
ß2 (CD18), and ß3 (CD61) integrins are
expressed on the cell surface of human PMN [2
,
8
, 9
]. Members of the ß2
(CD11/CD18) integrin family represent the most abundant integrins on
PMN, which are designated by the different -subunits as lymphocyte
function-associated antigen 1 (LFA-1; CD11a/CD18), Mac-1 (CD11b/CD18),
and gp150/95 (CD11c/CD18) [2
]. There are currently no
data to show the expression of the fourth ß2 integrin
(CD11d/CD18) on human PMN [10
].
ß2 Integrins (CD11/CD18) mediate PMN adhesion by binding to specific ligands: LFA-1 is critically involved in PMN emigration by binding to the intercellular adhesion molecules 1 and 2 (ICAM-1, -2) on endothelial cells [11 , 12 ], allowing firm adhesion, spreading, and subsequent emigration of the PMN. Mac-1 is also known as a receptor for ICAM-1 [13 ] but several reports suggest a subordinate role in PMN adhesion to endothelial cells compared with LFA-1 [14 , 15 ]. Mac-1 serves as the receptor for complement factor C3bi, fibrinogen, fibrin, and collagens [16 17 18 ]. gp150/95 binds C3bi and fibrinogen as well [19 , 20 ] but the physiological impact of these interactions seems less important due to the low surface expression on PMN when compared to the high abundance of Mac-1 [21 ]. Thus, the ß2 integrins mediate a variety of different cell-cell and cell-substrate interactions of human PMN during the inflammatory response.
Although much progress has been made in understanding the adhesive
functions of the ß2 integrins, the intracellular events
that follow their ligand interactions and allow adhesion-mediated
cellular responses are still incompletely understood. First evidence
for the signaling capacity of the ß2 integrins was
obtained by the finding that tumor necrosis factor
(TNF-)-induced superoxide anion production in human PMN depends on
ß2 integrins [22
]. Subsequently,
activation of different signaling components has been reported upon
ß2 integrin-mediated adhesion including tyrosine
phosphorylation of syk kinase and the src kinases fgr and lyn,
respectively [23
, 24
].
To further elucidate the molecular events that follow extracellular ligand interactions of the ß2 integrins in human PMN, ß2 integrin engagement was induced in this study by adhesion to immobilized fibrinogen or antibody cross-linking of the integrins on the cell surface. Intracellular protein tyrosine signaling after integrin engagement was studied by Western blotting and immunoprecipitation technique, respectively. For control, protein tyrosine phosphorylation was analyzed in PMN derived from CD18-deficient mice and wild-type control animals. The physiological role of integrin signaling was elucidated by studying ß2 integrin-mediated PMN adhesion, shape change, and spreading on immobilized fibrinogen.
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Isolation of murine PMN
Murine PMN were isolated from mutant mice deficient in CD18 or
wild-type control animals of the same genetic background (mixed 129/Sv
and C57BL/6J). All mice have been genotyped by Southern blot analysis
as described previously [26
]. Animals were killed by
CO2 inhalation and bone marrow cells were harvested from
tibias and femurs and incubated overnight in DMEM supplemented with
20% fetal calf serum, 15% cell culture supernatant derived from
Wehi-3b cells (ATCC TIB-68), 1% glutamine, and antibiotics (50 U/mL
penicillin, 50 µg/mL streptomycin) in 5% CO2 at 37°C.
PMN were washed and resuspended in HEPES supplemented with 0.1%
glucose. Before adhesion experiments, PMN were analyzed for expression
of CD18 and Gr-1, a marker of mature PMN, using flow cytometry to
confirm the genotype (data not shown).
Antibodies
The mAb IB4 (mouse anti-human CD18, IgG2a) [16
]
was isolated from hybridoma supernatants (ATCC 10164-HB) by protein
A-Sepharose. Purity was tested by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE); saturating concentration was determined
by flow cytometry. F(ab’)2 fragments of IB4 were prepared
by pepsin digestion followed by protein A-Sepharose purification. The
F(ab’)2 preparations of IB4 showed a uniform molecular
size of about 110 kDa on SDS-PAGE under nonreducing conditions. The
mAbs directed against human CD18 (clone MHM23, IgG1), CD29 (clone K20,
IgG2a), and CD61 (clone Y2/51, IgG1) as well as the fluorescein
isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (F-261) were
obtained from Dakopatts, Glostrup, Denmark. The nonbinding control mAb,
the F(ab’)2 fragments of the secondary polyclonal goat
anti-mouse IgG, the peroxidase-conjugated goat anti-mouse IgG, and the
agarose-conjugated goat anti-mouse IgG were purchased from Sigma,
Deisenhofen, Germany. The anti-phosphotyrosine mAb 4G10 was obtained
from Upstate Biotechnology, Lake Placid, NY. The mouse anti-c-Cbl mAb
(clone 17, IgG1) was obtained form Transduction Laboratories,
Lexington, KY. The polyclonal rabbit anti-c-Cbl antibody was purchased
from Santa Cruz Biotechnology, Santa Cruz, CA. The mouse anti-human
CD61 mAb VI-PL2, the mouse anti-human CD29 mAb MAR4, the phycoerythrin
(PE)-labeled rat anti-mouse CD18 antibody (clone C71/16), and the
FITC-labeled rat anti-Gr-1 antibody (clone RB6-8C5) were obtained form
PharMingen, San Diego, CA.
PMN stimulation by integrin engagement
Integrin engagement was induced by adhesion of PMN to
immobilized fibrinogen or by antibody cross-linking of the integrins on
the cell surface of suspended PMN. For adhesion experiments, 500-µL
aliquots of PMN (5 x 106/mL) in HEPES buffer were
seeded onto Petri dishes (2-cm diameter) coated with human or murine
fibrinogen at a final concentration of 250 µg/mL at 4°C overnight,
followed by two extensive washes. Adhesion was induced at 37°C in the
presence of 1.2 mM Ca2+ and 1 mM Mg2+ alone, or
by additional treatment with 0.2 mM Mn2+ or soluble stimuli
as indicated. In the absence of divalent cations only minimal adhesion
was observed (data not shown). After aspiration of the supernatant, PMN
stimulation was terminated by addition of 90 µL of 1x Laemmli buffer
(2% w/v SDS, 6% v/v 2-mercaptoethanol, 10 % v/v glycerol, and a
trace amount of bromphenol blue in 200 mM Tris-HCl, pH 6.8)
supplemented with 10 mM sodium orthovanadate. For negative control, PMN
were kept in suspension under the experimental conditions used for
adherent cells. The stimulation of suspended PMN was terminated by
addition of one-third volume of 3x Laemmli buffer.
For integrin aggregation by antibody cross-linking, PMN (5 x 106/mL) were incubated with 10 µg/mL of intact primary anti-CD18, anti-CD29, or anti-CD61 mAbs or their F(ab’)2 fragments in HEPES buffer supplemented with 0.25% bovine serum albumin (BSA) and 0.1% glucose for 20 min at room temperature under gentle rotation. After two washes, PMN were suspended in HEPES buffer (5 x 107/mL) supplemented with 0.1% glucose. Integrin aggregation was induced in a final volume of 60 µL at 37°C by treatment of PMN (2 x 106/40 µL) with 20/µL of F(ab’)2 fragments of the secondary antibody in excess at a final concentration of 100 µg/mL. PMN stimulation was terminated by addition of one-third volume of 3x Laemmli buffer. All samples were immediately heated for 6 min at 100°C and subjected to SDS-PAGE.
Immunoprecipitation
PMN (2.5 x 107) were lysed for 10 min on ice
with 500 µL of modified RIPA buffer (50 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 0.1% SDS, 0.5% deoxycholic acid, 100 mM sodium fluoride, 5 mM
diisopropylfluorophosphate, 10 mM sodium vanadate, 2 mM
phenylmethylsulfonyl fluoride, 10 mM tetrasodium pyrophosphate, 10 mM
p-nitrophenyl phosphate, 10 µg/mL antipain, 2 µg/mL
aprotinin, 2 µg/mL chymostatin, 2 µg/mL leupeptin, 1 µg/mL
pepstatin, pH 7.5). Immunoprecipitation of phosphotyrosine residues was
performed in the absence of tetrasodium pyrophosphate. Cell lysates
were precleared by centrifugation (12,000 g, 4°C, 10 min).
The supernatant was subjected to 10 µg of the primary anti-c-Cbl mAb
coupled to 75 µL of agarose-conjugated goat anti-mouse IgG for 1 h at 4°C under gentle rotation. Immunoprecipitates were washed with
lysis buffer twice, eluted by boiling samples in 90 µL of 1x Laemmli
buffer for 6 min at 100°C, and subjected to SDS-PAGE.
Immunodepletion
PMN (4 x 106) were lysed for 10 min on ice
with 80 µL of immunodepletion buffer (50 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 0.05% SDS, 0.5% deoxycholic acid, 1% NP-40, 100 mM sodium
fluoride, 5 mM diisopropylfluorophosphate, 2 mM phenylmethylsulfonyl
fluoride, 10 µg/mL antipain, 2 µg/mL aprotinin, 2 µg/mL
chymostatin, 2 µg/mL leupeptin, 1 µg/mL pepstatin, pH 7.5). Cell
lysates were precleared by centrifugation (12,000 g, 4°C,
10 min). The supernatant was subjected to 10 µg of the primary
anti-c-Cbl mAb coupled to 75 µL of protein G Sepharose for 1 h
at 4°C under gentle rotation. After addition of one-third volume of
3x Laemmli buffer, samples were immediately heated for 6 min at
100°C and subjected to SDS-PAGE.
SDS-PAGE and immunoblotting
Total cell lysates (2.5 x 106/sample) or
eluted immunoprecipitates, respectively, were subjected to SDS-PAGE on
gels containing 10% (w/v) acrylamide under reducing conditions
[27
]. Separated proteins were transferred to
nitrocellulose filters with the use of a semi-dry technique at 150 mA
for 1.5 h. All blots were tested for loading of equal amounts of
protein in each lane by Ponceau S staining. Before incubation for
1 h with a final concentration of 
1 µg/mL of the primary
antibodies in Tris-buffered saline (TBS) supplemented with 0.1% BSA,
filters were blocked by treatment with 3% ovalbumin in TBS for 1 h. After three washes in TBS containing 0.05% Tween-20, filters were
incubated for 1 h with peroxidase-conjugated goat anti-mouse IgG
or anti-rabbit IgG (final dilution 1:1000) in TBS supplemented with
0.1% BSA and subsequently washed as described above. Detection was
performed by chemiluminescence using an ECL kit (Enhanced
ChemiLuminescence, Amersham Life Science, Braunschweig, Germany) and
subsequent autoluminography by exposure to X-ray films (XOMAT-AR,
Kodak, Germany).
Cell surface expression of CD antigens
PMN (5 x 105/100 µL) were incubated with a
saturating concentration of 10 µg/mL of the primary anti-CD18,
anti-CD29, or anti-CD61 mAbs, respectively, for 1 h on ice and
washed twice. After incubation for 1 h with secondary
FITC-conjugated rabbit anti-mouse IgG (final dilution of 1:20) on ice
and in the dark, samples were subjected to flow cytometry (FACScan,
Becton Dickinson). In each sample, 104 cells were counted
and analyzed off-line using CellQuestTM software.
Production of superoxide anions
Production of superoxide anions was measured as superoxide
dismutase-inhibitable reduction of cytochrome c
[28
]. Aliquots of PMN (2 x 105/100
µL) were incubated for 5 min at 37°C. The reaction was started by
addition of a thermoequilibrated solution containing 150 µM
ferricytochrome c (final concentration) and a final
concentration of 100 nM fMLP or vehicle. In parallel samples,
measurements were done in the presence of 600 U/mL superoxide
dismutase. Extinction was determined in triplicate at 550 nm using a
96-well microtiter plate reader (Flow Laboratories, Meckenheim,
Germany). The results were calculated using an extinction coefficient
of 29.5 mM-1 for cytochrome c as specified by
the supplier.
Adhesion assay
PMN (2.5 x 104/100 µL) were seeded onto
96-well microtiter plates coated with human fibrinogen as described
above for integrin engagement. After 30 min, unattached PMN were rinsed
away by washing wells twice with PBS. Adherent cells were fixed with
1% glutaraldehyde in PBS and stained with 0.1% crystal violet. Plates
were photometrically measured at 570 nm after lysis in 0.5% Triton
X-100 overnight at room temperature. Experiments were done in
triplicate. Blanks were measured in the absence of cells to determine
background extinction.
Microscopy
PMN were subjected to morphological analysis 30 min after the
onset of adhesion in the presence of immobilized fibrinogen after the
experimental procedure described for the adhesion assay. PMN were
analyzed before removal of unattached cells on a Nikon microscope using
an HMC 40/0.6 objective.
Reagents
Antipain, aprotinin, BSA, chymostatin, horse heart cytochrome
c, diisopropyl fluorophosphate, deoxycholic acid, human and
murine fibrinogen, N-formyl-Met-Leu-Phe (fMLP), leupeptin,
NP-40, ovalbumin, pepstatin, Percoll, pertussis toxin,
p-nitrophenyl phosphate, phenylmethylsulfonyl fluoride,
Ponceau S, protein G Sepharose, sodium fluoride, sodium-orthovanadate,
tetrasodium pyrophosphate, Triton X-100, tumor necrosis factor
(TNF-), and Tween-20, were obtained from Sigma, Diesenhofen,
Germany. Herbimycin A and genistein were obtained from Calbiochem, La
Jolla, CA. Buffers and Ficoll-Hypaque were obtained from Biochrom,
Berlin, Germany. ECL Western blotting kit (RPN 2106) and
electrophoresis calibration standards for molecular mass determination
were purchased from Pharmacia (Freiburg, Germany). Lethal toxin was a
generous gift of Dr. K. Aktories, Freiburg, Germany.
Statistical analysis
Data shown represent mean ± SD where
applicable. Statistical significance was determined using Student’s
t test; P < 0.05 was considered
statistically significant.
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 View larger version (68K): [in a new window] |
Figure 1. Protein tyrosine phosphorylation upon engagement of ß2
integrins. Anti-phosphotyrosine immunoblot of whole-cell lysates
obtained from human PMN (2 x 106/sample) kept in
suspension or allowed to adhere to immobilized fibrinogen for indicated
times at 37°C in the presence of 1.2 mM Ca2+ and 1 mM
Mg2+. PMN were left untreated or (co-)stimulated by 300
U/mL TNF-, antibody cross-linking of CD18 (X-link), or addition of
0.2 mM Mn2+, respectively. Blots shown are representative
of three independent experiments.
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resulted in protein tyrosine
phosphorylation when PMN were allowed to adhere to immobilized
fibrinogen. The proteins that became tyrosine phosphorylated showed the
same molecular weight when compared to the effect of adhesion alone.
The only exception was a 42-kDa protein that became exclusively
tyrosine phosphorylated upon stimulation with TNF-. Due to its
molecular weight, this protein probably represents MAP kinase, as
suggested by findings of other authors [31
]. All other
proteins showed a more intense phosphorylation upon co-stimulation by
TNF- and adhesion when compared to the effect of adhesion alone,
suggesting a synergistic response. Accordingly, TNF- was a poor
activator of protein tyrosine phosphorylation in suspended PMN. Thus,
substantial tyrosine phosphorylation of most proteins in human PMN
critically required engagement of the ß2 integrins.
Similar results were obtained when PMN were stimulated with other
soluble stimuli (Fig. 2
). Using the bacterial-derived tripeptide fMLP and the cytokines
GM-CSF and IL-8, respectively, a marked tyrosine phosphorylation
occurred in adherent PMN similar to the effect observed in the presence
of TNF-. In contrast, all stimuli revealed a poor effect in
suspended PMN. The only exception was GM-CSF, which induced substantial
tyrosine phosphorylation of a 155-kDa protein in suspended PMN. Thus,
all soluble mediators tested showed a rather small effectiveness in
inducing protein tyrosine phosphorylation in suspended PMN,
demonstrating again that substantial tyrosine phosphorylation seemed to
require the signaling capacity exerted by ß2 integrins
upon extracellular ligand interactions.
 View larger version (98K): [in a new window] |
Figure 2. Effect of soluble mediators on protein tyrosine phosphorylation.
Anti-phosphotyrosine immunoblot of whole-cell lysates obtained from
human PMN (2 x 106/sample) kept in suspension (S) or
adherent to immobilized fibrinogen (A) for 10 min at 37°C in the
presence of 1.2 mM Ca2+ and 1 mM Mg2+. PMN were
left untreated or (co-)stimulated by 300 U/mL TNF-, 100 nM fMLP, 300
U/mL GM-CSF, or 1 µg/mL IL-8, respectively. Blot shown is
representative of three independent experiments.
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. In
contrast, no induction of protein tyrosine phosphorylation was observed
in PMN, which lack expression of CD18 suggesting that protein tyrosine
phosphorylation was dependent on ß2 integrins.
 View larger version (94K): [in a new window] |
Figure 3. Lack of protein tyrosine phosphorylation in the absence of CD18.
Anti-phosphotyrosine immunoblot of whole-cell lysates obtained from
murine PMN (2 x 106/sample) from CD18-deficient or
wild-type control animals that were kept in suspension (S) or allowed
to adhere to immobilized fibrinogen (A) for 10 min at 37°C in the
presence of 1.2 mM Ca2+ and 1 mM Mg2+. Samples
were stimulated by addition of 300 U/mL TNF- or 0.2 mM
Mn2+, respectively. Blot shown is representative of three
independent experiments.
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 View larger version (35K): [in a new window] |
Figure 4. Antibody cross-linking of ß2 integrins (CD18) but not
ß1 integrins (CD29) or ß3 integrins (CD61)
induced protein tyrosine phosphorylation. (A) Anti-phosphotyrosine
immunoblot of whole-cell lysates of human PMN (2 x
106/sample) that were treated with 10 µg/mL of mouse
anti-human mAbs CD29 (K20), CD18 (IB4), or CD61 (Y2/51), respectively,
and stimulated by addition of 100 µg/mL of F(ab’)2
fragments of goat anti-mouse IgG at 37°C for 0.5 min (X-link). The
low-molecular-weight bands are due to binding of the
peroxidase-conjugated anti-mouse antibody to the anti-CD18 antibody
IB4. Blot shown is representative of three independent experiments.
Identical results were obtained when the anti-CD61 mAb VI-PL2, the
anti-CD29 mAb MAR4 or the anti-CD18 mAbs MHM23 and 6.5E were used for
cross-linking (data not shown). (B) Expression of CD29, CD18, and CD61
on the cell surface of PMN as detected by flow cytometry. PMN were
treated with the 10 µg/mL of the anti-CD29, CD18, CD61 mAbs or the
non-binding control mAb (dotted line), respectively, and labeled with
the secondary FITC-conjugated rabbit anti-mouse IgG (final dilution of
1:20). Original fluorescence histograms are shown. Data are
representative of three independent experiments.
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Next, the most prominent protein that became tyrosine phosphorylated upon ß2 integrin-mediated adhesion was identified in human PMN (Fig. 5 ). Western blotting using an anti-c-Cbl antibody revealed that c-Cbl showed the same molecular mass as the 120-kDa protein that became tyrosine phosphorylated upon adhesion to immobilized fibrinogen. Immunoprecipitation of c-Cbl using a specific anti-c-Cbl antibody revealed that the 120-kDa protein was identical to c-Cbl as demonstrated by blotting the c-Cbl precipitates for c-Cbl and phosphotyrosine residues, respectively. This was confirmed by analyzing the phosphotyrosine immunoprecipitates in a Western blot for c-Cbl. Moreover, immunodepletion of c-Cbl from whole-cell lysates resulted in a loss of this protein in the cell lysate, further confirming the specificity of the observed effect.
 View larger version (53K): [in a new window] |
Figure 5. The 120-kDa protein was identical to c-Cbl. Western blots of whole-cell
lysates, c-Cbl immunodepleted cell lysates (ID) and immunoprecipitates
(IP) of c-Cbl, and phosphotyrosine residues using an anti-c-Cbl mAb
(-c-Cbl) or an anti-phosphotyrosine mAb (-P-Tyr), respectively.
Human PMN were kept in suspension (S) or were allowed to adhere to
immobilized fibrinogen (A) for 10 min at 37°C in the presence of 1.2
mM Ca2+, 1 mM Mg2+ and 0.2 mM Mn2+.
For immunodepletion experiments, lysates obtained from adherent cells
were subjected to the protein G-Sepharose-coupled anti-c-Cbl mAb
(AC) or were incubated for control in the
presence of protein G-Sepharose alone (A). Blots shown are
representative of three independent experiments. The polyclonal
anti-c-Cbl antibody gave similar results (data not shown).
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52
and 20% of the values observed in untreated fMLP-stimulated control
PMN (100%). Because the small G-protein rac is a component of the
NADPH-oxidase, this experiment revealed that the treatment of PMN with
LT led to the inactivation of the small G-proteins. In contrast,
inhibition of rac and ras by treatment with 4 µg/mL LT had no effect
on tyrosine phosphorylation, demonstrating that integrin-mediated
signaling was independent of these factors. This was also true for
pertussis toxin (Ptx)-sensitive heterotrimeric G-proteins as shown in
Figure 6B . Treatment of PMN with 300 ng/mL pertussis toxin had no
effect on integrin-mediated signaling, although fMLP-induced superoxide
anion production was reduced to 24% of values seen in untreated
fMLP-stimulated control PMN (100%). Because the fMLP receptor couples
to a pertussis toxin-sensitive heterotrimeric G-protein
[33
], the effect of Ptx on fMLP-induced superoxide anion
production revealed that these G-proteins were successfully inhibited
under the experimental conditions used. Thus, neither the small
G-proteins ras and rac nor pertussis toxin-sensitive heterotrimeric
G-proteins were required to allow protein tyrosine phosphorylation upon
integrin engagement.
 View larger version (25K): [in a new window] |
Figure 6. ß2 Integrin-mediated protein tyrosine phosphorylation was
independent of heterotrimeric G-proteins and the small G-protein ras.
PMN were left untreated for control (-), treated with lethal toxin (LT,
panel A), or pertussis toxin (Ptx, panel B), respectively, for 3 h
at 30°C. Left panels: fMLP-induced production of superoxide anions
measured as superoxide dismutase-inhibitable reduction of cytochrome
c 5 min after addition of 100 nM fMLP. Data represent
superoxide anion production in the presence of the inhibitor in percent
of fMLP-stimulated control (100%). *P <
0.05 versus fMLP-stimulated positive control; n = 4.
Right panels: anti-phosphotyrosine immunoblot of human PMN (2 x
106/sample) left untreated for control (-) or incubated
with 4 µg/mL lethal toxin and 300 ng/mL pertussis toxin,
respectively, before antibody cross-linking of CD18 (X-link) by
treatment with 10 µg/mL of the mouse anti-human CD18 mAb IB4 and
addition of 100 µg/mL of F(ab’)2 fragments of goat
anti-mouse IgG for 0.5 min at 37°C. Blots shown are representative of
three independent experiments.
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 View larger version (61K): [in a new window] |
Figure 7. ß2 Integrin-mediated protein tyrosine phosphorylation was
required for firm adhesion and spreading of PMN. Human PMN were treated
with 30 µM herbimycin A or vehicle for 30 min at 37°C before
induction of adhesion to immobilized fibrinogen for 30 min at 37°C in
the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 1
mM Mn2+. Addition of 10 µg/mL of the anti-CD18 mAb IB4
almost completely abolished PMN adhesion, demonstrating that substratum
interactions were mediated by ß2 integrins (data not
shown). (A) Anti-phosphotyrosine immunoblot of whole-cell lysates of
PMN (2 x 106/sample) 10 min after the onset of
adhesion. Blot shown is representative of three independent
experiments. Similar results were obtained with the tyrosine kinase
inhibitor genistein (data not shown). (B) PMN adhesion to immobilized
fibrinogen after treatment with herbimycin A in percent of untreated
control (100%). Mean ± SD; n = 4;
*P < 0.05 versus control. (C)
Photomicrographs of PMN. Data are representative of three independent
experiments.
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,
fMLP, IL-8, and GM-CSF, respectively, were found to serve as
comparatively weak inductors of tyrosine phosphorylation in suspended
PMN but had strong effects in adherent PMN, suggesting a synergistic
response. Inflammatory mediators are known to activate the binding of
the ß2 integrins and thereby promote adhesion of human
PMN [29
], which may contribute to the observed
enhancement of tyrosine phosphorylation in adherent PMN upon
stimulation by soluble mediators.
The 42-kDa protein that probably represented MAP kinase
[31
] was the only protein that became substantially
tyrosine phosphorylated upon stimulation by soluble mediators. In
contrast, no tyrosine phosphorylation of proteins with a molecular mass
below 53 kDa was observed in human PMN upon engagement of the
ß2 integrins. This finding reveals the diversity of
integrin signaling because ß1 integrins were reported to
trigger activation of MAP kinase in, e.g., fibroblasts
[34
]. In human PMN, there was no signaling at all
detectable upon engagement of the ß1 integrins. Although
we cannot exclude that some protein tyrosine phosphorylation occurred
upon ß1 integrin engagement, which was below the
detection limit of the employed immunoblotting technique used, the
effect of ß1 integrins on the activation of the
intracellular tyrosine signaling cascade in human PMN seems to be
negligible when compared with the effect of engagement of the
ß2 integrins. Among the different leukocyte populations,
this apparent restriction of substantial signaling to the
ß2 integrins, which corresponded to the high abundance of
this molecule on the cell surface seems to be a characteristic feature
of PMN. The integrins 5/ß1 and
v/ß3 have been previously reported to
induce substantial protein tyrosine phosphorylation in murine
macrophages upon antibody cross-linking or upon binding to the
extracellular matrix proteins fibronectin and vitronectin, respectively
[35
].
The fact that ß2 integrins serve as potent signaling
molecules in human PMN, whereas ß1 and ß3
integrins seem to occupy this function in macrophages may show that
these integrins may play different roles in both cell types. This is
consistent with the finding that PMN preferentially interact with the
endothelial cell monolayer via binding of ß2 integrins to
ICAM-1 [14
, 15
, 36
], whereas
VLA-4- (4ß1, CD49d/CD28) VCAM-1 (CD106)
interactions seem to play a critical role in monocyte extravasation
[37
]. Accordingly, emigration of PMN was found to be
severely compromised in CD18 null mice, whereas no reduction of
monocyte extravasation was detectable in the absence of CD18
[38
]. Thus, the observed differences in the signaling
capacity are consistent with the molecular requirements for
extravasation.
Although the role of the leukocyte integrins in mediating adhesion and transducing signals into the cell seems to vary in different leukocyte populations, striking similarities seem to exist between ß1 and ß2 integrins with respect to the signaling pathways employed in leukocytes. In the present study, engagement of ß2 integrin was found to induce tyrosine phosphorylation of c-Cbl. This effect seems to play an important role because firm adhesion, the prerequisite for spreading, was inhibited upon treatment of the cells with herbimycin A, an inhibitor of tyrosine kinases. In the presence of the inhibitor, almost all PMN remained spherical and no shape change occurred. This was in contrast to untreated PMN, which showed a characteristic shape change and spread over immobilized fibrinogen. Thus, the ß2 integrin-mediated phosphorylation of c-Cbl may be critically involved in firm adhesion, subsequent shape change, and spreading of human PMN. Similar results for this role of c-Cbl in adhesion were obtained in murine macrophages upon binding of ß1 and ß3 integrins to fibronectin and vitronectin, respectively [39 ].
The proto-oncogene c-Cbl was observed to be translocated to the cell membrane upon engagement of the ß1 integrins [39 ]. This adhesion-induced redistribution as well as the tyrosine phosphorylation of c-Cbl was found to critically depend on src kinases. The adapter protein c-Cbl was found to form a complex with the src kinase and the phosphoinositol-3-kinase (PI-3-kinase) upon engagement of ß1 and ß3 integrins [35 ]. Accordingly, inhibition of translocation of c-Cbl was observed to result in a reduction of membrane-associated PI-3-kinase activity, which was suggested to be responsible for reduced ß1 integrin-mediated adhesion of murine peritoneal macrophages to fibronectin [39 ]. Similarly, a redistribution of the src kinase fgr to the cytoskeletal fraction was observed in human PMN upon adhesion to immobilized fibrinogen [40 ], suggesting that in addition to tyrosine phosphorylation of c-Cbl, the ß2 integrins may use a signal transduction cascade that is similar to that employed by ß1 and ß3 integrins upon extracellular ligand interactions in macrophages.
The small G-protein rho was previously reported to be involved in the inside-out signaling events that trigger the activation of the ß2 integrins [41 ]. This process, which is thought to induce a conformational change, enhances the binding affinity of the integrins toward their specific ligands [29 ]. In this study, no evidence was obtained that outside-in signaling involves G-proteins. Neither pertussis toxin, which inactivates heterotrimeric G-proteins of the G12/13 and Gq family [33 ] nor lethal toxin from C. sordellii, which inhibits ras and rac [32 ], respectively, affected integrin-induced tyrosine signaling. Thus, these signaling components were not required to allow initial integrin-mediated signal transduction but we cannot exclude that these components are engaged in signaling processes downstream of the observed protein tyrosine phosphorylation events. This is in contrast to the ß1 integrin signaling cascade where activation of ras was identified as a proximal event upon integrin engagement [42 ]. However, the role of ß1 integrin-mediated ras activation for subsequent tyrosine signaling is still controversial [43 , 44 ].
Altogether, the present study shows that ß2 integrins trigger tyrosine phosphorylation of c-Cbl and other proteins upon ligand interaction in human PMN. This effect was restricted to ß2 integrins and was not detectable upon engagement of ß1 or ß3 integrins, respectively, revealing a unique function of the ß2 integrins among the leukocyte integrins expressed on human PMN. The ß2 integrin-mediated signaling was independent of pertussis toxin-sensitive G-proteins and the small G-proteins ras and rac, respectively. The protein tyrosine phosphorylation that was induced upon interactions of the extracellular domain of the ß2 integrin was critical for allowing firm adhesion, shape change, and spreading of PMN. Thus, the present study may suggest a three-step model for the regulation of integrin function in which the initial interaction of the extracellular domain of the ß2 integrins allows intracellular signaling, which in turn may precede firm adhesion, shape change, and spreading of human PMN.
Received August 15, 1999; revised March 8, 2000; accepted March 15, 2000.
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