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Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden
Correspondence: Joachim Werr, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail: joachim.werr{at}fyfa.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • leukocytes • adhesion • extravasation • migration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits [2
,
7
, 8
]. Several studies have shown that all
leukocyte subsets, with the exception of PMN, constitutively express
ß1 integrins that mediate cell binding to different
extracellular matrix components, including collagens, fibronectin, and
laminin [7
, 9
]. However, limited expression
of ß1 integrins has been identified on circulating PMN
also [4
, 10
11
12
], and accumulating data
indicate that ß1 integrins can be rapidly upregulated on
PMN under certain circumstances in the inflammatory process
[3
, 6
, 13
, 14
].
Thus, although expression of ß1 integrins in circulating
PMN is relatively insensitive to chemoattractant stimulation
[10
], increased expression may be induced in conjunction
with transendothelial migration [3
, 6
,
13
]. We recently demonstrated that ß1
integrins are upregulated in extravasated human PMN and that PMN
migration in extravascular tissue is largely dependent on the function
of 2ß1 integrin [15
].
Together, these findings indicate that expression of ß1
integrins, although sparse on circulating PMN, is induced in
association with the extravasation of these cells and that members of
the ß1 integrin family play an important role in PMN
recruitment to extravascular tissue. ß2 Integrins (CD11/CD18) are of fundamental importance for PMN firm attachment to the endothelium in inflamed tissue, and they have been shown to play critical roles in several steps of PMN activation also [2 ]. There is a growing body of evidence that activation of one family of adhesion molecules may regulate the activity of another and that ß2 integrins are involved in such mechanisms of receptor crosstalk [16 17 18 ]. In light of these findings and the fundamental role that has been attributed to ß2 integrins in PMN activation, this study was aimed at exploring the possibility that signaling by ß2 integrins, consequent to ligand binding, may be involved in emigration-associated upregulation of ß1 integrins on PMN. Through antibody cross-linking of CD18, it was revealed that ß2 integrin engagement triggers induction of ß1 integrin surface expression in PMN. Furthermore, this mode of activation was shown to induce ß1 integrin-dependent PMN adhesion to matrix proteins and to stimulate PMN motility in collagen gels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 (CD49b)
integrin subunit, L25.3 (Pharmingen) reacting with the 4
(CD49d) subunit, mAb16 (Pharmingen) reacting with the 5
(CD49e) subunit, mAb13 (Pharmingen) against the common ß1
(CD29) integrin chain, and IB4 (courtesy of Dr. Samuel Wright, The
Rockefeller University, New York) against the common ß2
(CD18) integrin chain. All antibodies employed were mouse
immunoglobulin G (IgG), except mAb13, a rat IgG.
Isolation of human PMN
Human PMN were isolated from whole blood by single-step density
centrifugation over Polymorphprep (Nycomed Pharma AS, Oslo, Norway).
Briefly, blood (5 ml) was layered onto 3 ml of the separation medium
and centrifuged at room temperature at 500 g for 30 min. The
PMN-rich band was collected and washed twice in ice-cold Hanks’
balanced saline solution (HBSS). Contaminating erythrocytes were lysed
by 20 sec hypotonic shock, and PMN were washed and resuspended in
culture medium [modified Eagle’s medium (MEM); Life Technologies,
Gaithersburg, MD] again at concentrations indicated.
PMN activation and CD18 cross-linking
Isolated PMN (1x106 cells/ml) were incubated with
10 µg/ml of anti-CD18 mAb IB4 at 4°C for 40 min, washed twice at
150 g for 7 min, and resuspended in ice-cold MEM.
Cross-linking of surface-bound IB4 was then induced by incubating the
cells with goat anti-mouse F(ab')2 fragments (Jackson
ImmunoResearch Laboratories, West Grove, PA; final dilution, 1:20) at
conditions indicated below for the different experimental procedures.
In those cases where N-formyl-methionyl-leucyl-phenylalanine
(fMLP; Sigma, St. Louis, MO) was used to activate PMN, either in
combination with CD18 cross-linking or alone, the chemoattractant was
present throughout all steps of incubation at a final concentration of
10-6 M. Herbimycin A (Sigma) was used as a
tyrosine kinase inhibitor at a final concentration of 30 µM. PMN were
pretreated with herbimycin A for 10 min at 37°C before exposure to
antibodies and/or fMLP, and the substance was present at the same
concentration during all the following incubation steps.
Immunofluorescence flow cytometric analysis
Incubation of PMN with anti-CD18 mAb (IB4), with or without fMLP
(10-6 M), was performed according to the
protocol above. Ligation of CD18 was induced by incubation with goat
anti-mouse F(ab')2, first at 4°C for 20 min and then at
37°C for specified time periods (5, 20, and 30 min). PMN were then
fixed in 4% paraformaldehyde (Sigma) at room temperature for 20 min,
washed twice, and resuspended in HBSS at 4°C to yield a final
concentration of 20 x 106 cells/ml. The fixed PMN
were incubated with saturating concentration of mouse IgG (Pharmingen;
final dilution, 1:20) at room temperature for 30 min to prevent binding
of integrin antibodies to cell surface-bound F(ab')2
fragments. PMN were then incubated with fluorescein isothiocyanate
(FITC)-conjugated anti-ß1 integrin mAb (mAb13), rat IgG
(10 µg/ml), or FITC-conjugated, isotype-matched control antibody for
20 min at 4°C in the dark. A rat IgG (mAb13) was chosen for detection
of ß1 integrin in an effort to minimize binding to the
cell surface-bound, goat anti-mouse F(ab')2 fragments. PMN
were washed twice and analyzed on a FACSort flow cytometer (Becton
Dickinson, Mountain View, CA). Gating was based on forward and
side-scatter parameters, and purity of analyzed cells was assured with
a neutrophil-specific marker for CD16 (antibody DJ130c; Dako, Glostrup,
Denmark). Fluorescence intensity of 104 cells was analyzed.
The relative mean fluorescence intensity (MFI) was calculated as the
mean fluorescence of PMN treated with specific antibody divided by the
mean fluorescence of the nonspecific, isotype-matched control antibody.
Laser-scanning confocal microscopy
ß1 Integrin expression in activated PMN was
detected also with laser-scanning confocal microscopy.
Anti-CD18-treated PMN, with or without fMLP
(10-6 M), were incubated with goat anti-mouse
F(ab')2 for 20 min at 37°C. PMN were then fixed in
paraformaldehyde and treated identically to the protocol described
above for immunofluorescent fluorescein-activated cell sorter (FACS)
analysis, using anti-ß1 integrin antibody mAb13 for
detection of ß1 integrin expression. PMN were viewed in a
laser-scanning confocal imaging system (Insight Plus, Meridian
Instruments, Okemos, MI) under normal transmitted and laser-emitted
fluorescent light. Correction for unspecific antibody binding and
background fluorescence was made by comparing specific mAb fluorescence
with that of samples treated with irrelevant antibody at the same
concentration and incubation time.
PMN adhesion assay
Plastic tissue culture dishes (96-well) were coated overnight at
4°C with 10 µg/ml of fibronectin (Sigma), rat collagen (type I),
recombinant human intercellular adhesion molecule-1 (ICAM-1; R&D
Systems, Wiesbaden-Nordenstadt, Germany), or bovine serum albumin (BSA;
Sigma); diluted in phosphate-buffered saline (PBS) collagen (type I);
and purified from rat-tail tendons, according to standard procedures, a
generous gift from Dr. Björn Öbrink (CMB, Karolinska
Institutet, Stockholm, Sweden). The dishes were washed twice with PBS.
Cross-linking of CD18 was induced by incubating IB4-treated PMN with
goat anti-mouse F(ab')2 for 20 min at 4°C. PMN suspended
in MEM (100 µl), was added to the wells (105 cells/well)
together with anti-integrin antibodies and incubated at 37°C in
humidified air for 20 min. Antibodies P1E6, L25.3, mAb13, and mAb16
were used at a final concentration of 20 µg/ml and IB4, at a final
concentration of 10 µg/ml. In addition, IB4 (10 µg/ml) was added to
all PMN suspensions subjected to CD18 cross-linking to prevent cell
adhesion via nonbound CD18 molecules exposed on the cell surface. After
the incubation period, the wells were washed twice with cold PBS to
remove nonadherent cells, and PMN adhesion was quantified by assaying
myeloperoxidase (MPO) activity in the remaining cell sample.
MPO assay
The PMN-specific enzyme MPO was quantified as previously
described by Suzuki et al. [19
]. In brief,
PMN were lysed by addition of 100 µl 0.5% hexadecyltrimethylammonium
bromide in 50 mM potassium phosphate buffer (pH 6.0) to each well. The
enzyme activity in samples from each well was determined
spectrophotometrically (Titertec Multiscan MCC, Flow Laboratories,
McLean, VA), as the change in absorbance at 650 nm that occurs in the
redox reaction of H2O2-tetramethylbenzindine
catalyzed by MPO. For calculation of a standard curve, a known number
of PMN in suspension were serially diluted, lysed, and analyzed for MPO
activity.
PMN invasion into gels of collagen (type I)
Gels were formed in 24-well culture dishes (250 µl/well) by
mixing 8.5 vol of rat collagen solution at a concentration of 1.5 mg/ml
with 1 vol of x10 MEM and 0.5 vol of 4.4% NaHCO3
solution. fMLP at a final concentration of
10-7 M was added to the gel prior to
polymerization. CD18 cross-linking was performed by incubating
IB4-treated PMN with goat anti-mouse F(ab')2 at 4°C for
20 min. PMN (0.5x106) were suspended in 200 µl MEM
containing 10-9 M fMLP, placed on top of the
polymerized gels, and incubated in humidified air for 30 min at 37°C.
PMN invaded the gel along the established chemotactic gradient with a
directional movement toward the bottom of the well. Anti-integrin mAbs
were added to the cell suspension at the same concentrations as those
used in the adhesion assay described above. As indicated in the
protocol above used for PMN adhesion, anti-CD18 mAb IB4 was added to
all PMN suspensions subjected to CD18 cross-linking to prevent cell
adhesion via nonbound CD18 molecules exposed on the cell surface. Four
experiments were run in duplicate gels, and 10 randomly chosen
microscopic fields (with a defined area of 0.0625 mm2) were
analyzed in each gel with a Leitz Orthoplan microscope equipped with a
water immersion lens (Leitz UOx55W, NA 0.80). PMN invasion into the
gel was quantified by counting all cells detected within the gel when
focusing down through the entire gel thickness.
Statistical analysis
Data are presented as means ± SD. Statistical
significance was calculated using the Mann-Whitney test for independent
samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.5 times that for cells treated with
fMLP only. As shown in Figure 4
, adhesion to fibronectin and collagen
of PMN subjected to CD18 cross-linking was inhibited to a significant
extent by anti-ß1 integrin antibodies. However, PMN
adhesion to ICAM-1 and BSA was not affected by antibodies against
ß1 integrins. Thus, CD18 cross-linking in PMN resulted in
increased, integrin-dependent adhesion to fibronectin and collagen but
not to ICAM-1 and BSA. Taken together, these data suggest that
cross-linking of CD18 provoked PMN adhesion to fibronectin and collagen
via ß1 integrin receptors by mechanisms dependent on
CD18-mediated PMN activation but independent of direct adhesive
interactions of CD11/CD18 with these matrix proteins. In contrast,
adhesion to ICAM-1 or a BSA-coated surface relied on ß2
integrin function and was not influenced by antibody cross-linking of
CD18.
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4 and 5 integrin and to collagen via
2 integrin4 integrin subunit (Fig. 5a
), adhesion to fibronectin of PMN subjected to CD18 cross-linking
and fMLP stimulation was reduced to 74 ± 8% of control. A more
pronounced inhibition of PMN adhesion was seen after blockage of the
5 subunit (55±6% of control). Blockade of the
4 and 5 subunits resulted in reduced
adhesion closely similar to that seen after blockage of the common
ß1 chain (42±12% and 45±13% of control,
respectively), whereas blockade of the 2 subunit proved
ineffective in modulating PMN adhesion to fibronectin. Conversely, PMN
adhesion to collagen appeared to be mediated predominantly via the
2ß1 integrin (Fig. 5b)
. Similarly,
inhibition of ß1 or 2 was effective in
blocking adhesion (36±10% and 42±11% of control, respectively), and
no additional inhibition occurred after combined treatment (40±10% of
control). These data suggest that PMN adhesion to fibronectin and
collagen subsequent to CD18 cross-linking is mediated to a major extent
via specific ß1 integrins. Apparently, ß1
integrin-dependent adhesion to fibronectin predominantly involves
5ß1 and to a lesser extent,
4ß1, whereas adhesion to collagen is
mediated via 2ß1. As evident from Figure 5a
and 5b , significant adhesion of PMN to fibronectin and collagen
remained after function inhibition of ß1 integrin
receptors. Because the experiments were performed in the presence of a
saturating concentration of antibodies against CD18, the remaining
adhesion has to be interpreted not to be dependent on ß1
or ß2 integrin receptors, suggesting PMN adhesion being
mediated by pathways other than those studied here. Notably,
ß1 integrin receptors were not involved in PMN adhesion
to BSA (Fig. 5c)
.
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2 integrin subunit or the
common ß1 integrin chain resulted in largely reduced
migration, whereas blockade of the 4 and
5 subunits was without effect on the parameters studied.
These data show that ligation of CD18, physiologically induced by PMN
adhesion to the collagen gel or artificially induced by antibody
cross-linking, is a prerequisite for the
2ß1 integrin-dependent PMN migration into
the gel. Supposedly, engagement of CD18 triggers upregulation of
ß1 integrin expression, as demonstrated above, which then
becomes indispensable for PMN migration within the gel.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The upregulation of ß1 integrins in PMN subjected to combined fMLP stimulation and CD18 cross-linking was blocked by exposure of PMN to herbimycin A, indicating involvement of tyrosine phosphorylation in this process. ß2 Integrin ligation and fMLP stimulation have been described to activate tyrosine kinases in PMN [24 , 25 ], and integrin upregulation has been shown to correlate with tyrosine kinase activity [26 ]. In contrast, activation of PMN with phorbol ester, which is known to bypass protein phosphorylation, results in limited, if any, upregulation of ß1 integrins [10 ]. Collectively, these findings suggest that tyrosine phosphorylation is a necessary yet not sufficient step for ß1 integrin upregulation in PMN. Recently, elevated intracellular Ca++ levels and activation of protein kinase C were shown to act synergistically in the activation of ß1 integrins in granulocytic HL 60 cells [27 ]. Also, CD18 cross-linking has been shown to induce Ca++-dependent responses in PMN that are not altered by herbimycin A [23 ].
The functional state of ß1 and ß2 integrins
in PMN subjected to CD18 cross-linking was investigated in PMN adhesion
and migration assays. Previous studies have clearly shown a fundamental
role of ß2 integrins in PMN adhesion to various
substrates and PMN migration under different conditions
[16
, 28
29
30
]. Consistent with these
studies, we observed that fMLP-stimulated PMN adhesion to collagen,
fibronectin, ICAM-1, and BSA to a major extent was ß2
integrin-dependent inasmuch as adhesion was abrogated with antibodies
against CD18. We also found that PMN adhesion to collagen and
fibronectin, but not to ICAM-1 and BSA, was enhanced by antibody
cross-linking of CD18. Because in this situation PMN adhesion via CD18
was prevented, and what is more, the experiments were performed in
excess of anti-CD18 antibodies, we conclude that PMN adhesion to
fibronectin and collagen after CD18 cross-linking was mediated via
mechanisms distinct from the ß2 integrins. These adhesion
pathways were specific for the matrix proteins because adhesion to
ICAM-1 and BSA after CD18 cross-linking did not differ from that of
CD18 mAb treatment. A similar pattern to PMN/matrix adhesion was
observed with regard to PMN migration in collagen gels. Thus, PMN
adhesion to fibronectin, collagen, and migration in collagen after CD18
cross-linking was dependent on ligation of the ß2
integrins but independent of their physical binding capacity. As shown
here, one major adhesive mechanism induced by ß2 integrin
ligation was via upregulation of functional ß1 integrins.
Function-blocking antibodies against ß1 integrins reduced
adhesion to collagen and fibronectin of PMN subjected to CD18
cross-linking by 50%. ß1 Integrin-dependent adhesion
to fibronectin was mediated predominantly by
5ß1 and to a lesser extent by
4ß1. PMN adhesion to fibronectin has been
shown previously to occur via the 4ß1 and
5ß1 integrins [3
,
12
, 31
]. ß1 Integrin-dependent
PMN adhesion to collagen, alternatively, was found to be entirely
dependent on the 2ß1 integrin, which, to
our knowledge, is the first demonstration of
2ß1 integrin-mediated adhesion of PMN to
collagen.
As previously observed in several studies and also shown here, PMN
adhere more readily to fibronectin than to collagen. We have previously
shown that PMN migration in an extracellular matrix (ECM) environment
can be inhibited through function blockade of the
2ß1 integrin but not the
4ß1 and 5ß1
integrins [15
]. Combined, these findings indicate that
ß1 integrins can mediate adhesive and motile cell-matrix
interactions. Differences in conformational regulation of
ß1 integrin receptors evoked by ligand binding
[32
] may explain differences in adhesive and dynamic
functions among these receptors.
In accordance with previous studies using similar migration models, we
show that blockage of ß2 integrin function resulted in an
almost complete inhibition of PMN migration into collagen gels,
apparently as a consequence of the inability of the cells to adhere to
the gel. However, after antibody cross-linking of CD18, PMN migrated
into the gel in numbers comparable with what was seen for cells not
treated with antibodies, indicating that ß2 integrin
ligation is a prerequisite for PMN invasion into the gel. Again, in
this situation, the ability of CD18 to directly interact with the
matrix proteins was impeded because of anti-CD18 mAb treatment. Our
finding that PMN migration into gels after CD18 cross-linking was
comparable to that for untreated PMN and was similarly dependent on the
2ß1 integrin suggests that
ß2 integrins play a critical role in PMN motility by
regulating the expression and function of other integrin receptors
(e.g., ß1 integrins as shown here) rather than by
mediating a direct physical binding to the substrate. These findings
are supported by observations of crosstalk between ß1 and
ß2 integrins in human T cells [33
] and
similar studies suggesting that integrin-receptor functions are
significantly modulated by mechanisms related to cell-surface receptor
crosstalk [34
]. Summarizing available data on
ß1 integrins in PMN, it becomes clear that the expression
and function of these receptors are transient and may undergo rapid
modulation during the process of extravasation and migration in
extravascular tissue. We have shown that ß2 integrins,
recognized as the most abundant integrin receptors in PMN and involved
in various steps of PMN activation, also initiate intracellular
signaling events, which fundamentally alter the state of expression and
function of ß1 integrins in PMN. Thus, our findings
suggest a mechanism of integrin receptor crosstalk in PMN where
ligation and engagement of ß2 integrins, through binding
to counterreceptors on endothelium and ECM, trigger upregulation of
ß1 integrins, which then may act in concert with the
ß2 integrins to optimize PMN motility in extravascular
tissue.
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ACKNOWLEDGEMENTS |
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Received December 3, 1999; revised March 20, 2000; accepted April 14, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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