* Department of Physiology and Cellular Biophysics and Department of Pathology, Columbia University College of Physicians
and Surgeons, New York 10032;
Department of Medicine, Division of Rheumatology and Immunology, Medical University of
South Carolina, Charleston, South Carolina 29425-2229; § Department of Medicine, Beth Israel Hospital, New York 10004; and ¶ Athena Neurosciences, South San Francisco, California 94080
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Abstract |
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Chemoattractants differ in their capacity to
stimulate neutrophils to adhere to and to migrate
through matrices containing fibrin. Formyl methionyl
leucyl phenylalanine (fMLP) stimulates neutrophils to
adhere closely to, but not to migrate into, fibrin gels.
Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We
report that 5
1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response
to these chemoattractants. fMLP, but not LTB4, activated neutrophil
1 integrins, as measured by binding of
mAb 15/7 to an activation epitope on the
1 integrins.
Antibodies or peptides that block
5
1 integrins prevented fMLP-stimulated neutrophils from forming
zones of close apposition on fibrin and reversed fMLP's
inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that
block
1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or
to migrate through fibrin gels. These results suggest that
chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general
message that induces neutrophils to crawl and a specific
message that prepares neutrophils to stop when they
contact appropriate matrix proteins for activated
1 integrins.
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Introduction |
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LEUKOCYTE chemotaxis is regulated by the interactions of soluble or surface-bound chemoattractants/chemokines with cognate receptors on the leukocytes. These interactions generate intracellular signals that activate one or more of the leukocyte's adhesion-promoting receptors, thereby enabling these cells to adhere to or migrate through endothelia, epithelia, and extracellular matrices.
Neutrophils (polymorphonuclear leukocytes, PMN)1 express a number of different adhesion-promoting surface
receptors, including 1 and
2 integrins.
2 integrins assume an "activated" conformation when chemoattractants, chemokines, cytokines, or growth factors bind to
specific receptors for these substances on PMN (Diamond and Springer, 1994
; Premack and Schall, 1996
). Activation
increases the capacity of
2 integrins to bind cognate
ligands on cells or matrix proteins, thereby regulating
PMN adhesion to and migration through endothelia (Smith,
1993
; Springer, 1995
), epithelia (McCormick et al., 1995
),
layers of synovial fibroblasts (Gao et al., 1995
; Gao and Issekutz, 1996
), and extracellular matrices (Wright et al., 1988
;
Loike et al., 1991
, 1992
, 1995
). The central roles played by
2 integrins in PMN adhesion and chemotaxis in vivo are
illustrated by the multiple derangements of PMN function
in humans with the inherited disorder leukocyte adhesion
deficiency type 1, in which there is partial to complete absence of
2 chains (Anderson and Springer, 1987
), and in
mice rendered functionally or genetically deficient in
M
2
(CD11b/CD18) integrin (Tang et al., 1997
).
PMN also express 1 integrins, primarily
3
1,
5
1, and
6
1, but also very low levels of
4
1 (Gao et al., 1995
;
Gresham et al., 1996
) that participate in PMN adhesion,
migration, and phagocytosis. For example, C5a, a cleavage
product of the fifth component of complement, and PMA
stimulate
5
1-dependent PMN adherence to fibronectin
(Bohnsack et al., 1995
). Chemoattractant-activated
1 integrins work in concert with
M
2 (CD11b/CD18) integrins to mediate phagocytosis of particles coated with C3bi
by PMN (Pommier et al., 1983
; Wright et al., 1984
; Brown,
1992
).
1 integrins also mediate chemotaxis of platelet activating factor-stimulated rat PMN (Werr et al., 1998
).
We reported previously (Loike et al., 1995) that different chemoattractants specify qualitatively distinct PMN
responses when PMN contact specific matrix proteins. For
example, PMN stimulated with formyl methionyl leucyl
phenylalanine (fMLP) or tumor necrosis factor-
form
zones of close apposition on fibrin and do not migrate through fibrin gels, whereas PMN stimulated with leukotriene B4 (LTB4) or interleukin 8 (IL-8) form zones of
loose apposition on fibrin and migrate efficiently into and
through fibrin gels (Loike et al., 1995
). All of these chemoattractants activate PMN
2 integrins (Diamond and
Springer, 1994
; Premack and Schall, 1996
) and induce
PMN to migrate efficiently through three-dimensional matrices composed of Matrigel or collagen I (Loike et al.,
1995
). Antibodies that block the ligand-binding domains
of
2 integrins inhibit PMN migration through all matrices
tested (i.e., collagen I, Matrigel, and fibrin), in response to
chemoattractants. Therefore, it seemed unlikely that the
different effects of fMLP and LTB4 on PMN chemotaxis
through fibrin gels could result from small differences in
the effects of these chemoattractants on
2 integrins.
1 integrins regulate the activity of
M
2 integrins on
PMN (Brown, 1992
) and monocytes (Pommier et al., 1983
;
Wright et al., 1984
), and of
III
3 integrins on platelets
(Loike et al., 1993
). We reasoned that fMLP, but not
LTB4, might activate one or more PMN
1 integrins and
that signals generated by the interaction of activated
1 integrins with ligands on fibrin might affect PMN chemotaxis. To test this hypothesis we examined the effects of
fMLP and LTB4 on activation of PMN
1 integrins, and of
antibodies and peptides that block
1 integrins on fMLP-
and LTB4-stimulated PMN adhesion to and migration
through fibrin gels. We report here that fMLP, but not
LTB4, activates
1 integrins on PMN, and that the interaction of activated
1 integrins with fibrin alters the quality of
2 integrin-dependent adhesion to, and migration
through, fibrin gels.
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Materials and Methods |
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Reagents
Rhodamine-conjugated polyethylene glycols of 3.5 kD (Rh-PEG 3.5 kD)
and 10 kD (Rh-PEG 10 kD) were prepared as described (Loike et al.,
1993, 1995
). Sources of antibodies and peptides were as follows: mouse
anti-
1 (P4C10) and the peptides GRGDSP and GRGESP were from
GIBCO BRL. Mouse anti-human
x (LeuM5) was from Organon-Teknika Inc. Mouse anti-human
M
2 (MAC-1) was from Upstate Biotechnology Co. Mouse anti-human
5 (SAM1), rat anti-human
6
(GoH3), mouse anti-human
4 (HP2/1), and mouse anti-human
3 integrin (SZ21) were from Immunotech. Phycoerythrin-conjugated F(ab)2
anti-mouse IgG was from Jackson ImmunoResearch. Mouse anti-
3
(PPM6/13) was from Biosource International. Mouse anti-
3 (MAB1957z)
was from Chemicon International. Alexa 488-conjugated F(ab)2 anti-
mouse IgG was from Molecular Probes. LTB4, fMLP, PMA, thrombin,
and Ficoll-Hypaque were from Sigma Chemical Co. Mouse anti-chicken
1 integrin (CSAT) and mouse monoclonal anti-human
1 integrin (AiiB2) were generous gifts from Dr. Clayton Buck (University of California, San Francisco, CA). Mouse mAb 15/7, which recognizes an activation epitope on human
1 integrins (Bohnsack et al., 1995
), was from
Athena Neurosciences. Mouse mAb IB4, which blocks the ligand-binding
domains of human
2 integrins (Wright et al., 1983
), was a generous gift
from Dr. Samuel D. Wright (Merck, Rahway, NJ). PPACK was from Calbiochem-Novabiochem, Matrigel from Becton Dickinson, and collagen I
from GIBCO BRL. Purified fibronectin was from Vitex International. Fibrinogen was from American Diagnostica Inc. Fibrinogen uncontaminated by Factor XIII, fibronectin, and vitronectin, a generous gift of Dr.
Jeffrey Weitz (MacMaster University, Hamilton, Ontario, Canada), was
prepared from fibrinogen obtained from Enzyme Research Labs FIBI. It
was first adsorbed with gelatin-agarose to remove fibronectin and then
passed over an affinity column to remove Factor XIII. The fibrinogen was
precipitated with 25% ammonium sulfate, dialyzed against 150 mM NaCl,
20 mM Tris (pH 7.4), adsorbed with an antibody to human vitronectin
linked to Affi-gel, and dialyzed. PAGE analysis showed the resulting fibrinogen to be free of fibronectin or Factor XIII. Western blot analysis revealed no vitronectin (data not shown).
Preparation of Boyden-type Chemotaxis Chambers
Gels, ~1 mm thick, composed of fibrin, Matrigel, or collagen type IV,
were formed in cell culture inserts (pore sizes 3 or 8 µm) from Becton
Dickinson as described (Loike et al., 1995). Fibrin gels were gently
washed with PBS to remove any residual PPACK.
PMN Adhesion and Closeness of Apposition to Fibrin-coated Surfaces
Fibrin/fibrinogen-coated surfaces were prepared as described (Wright et al.,
1988; Loike et al., 1992
, 1993
, 1995
) and PMN adhesion was measured by
phase-contrast microscopy. Close apposition of PMN to fibrin/fibrinogen-coated surfaces was defined as exclusion of Rh-PEG 10 kD from zones of
contact between PMN and fibrin/fibrinogen measured by fluorescence microscopy as described (Loike et al., 1993
).
PMN Migration
PMN were prepared as described (Wright et al., 1988) from fresh heparinized blood from healthy adult donors after informed consent. PMN used
in these experiments were >95% pure as determined by Wright-Giemsa
staining (Wright et al., 1988
). 106 PMN in 100 µl of PBS supplemented
with 5.5 mM glucose and 0.1% human serum albumin (PBSG-HSA) were
placed in the upper compartment of each insert and incubated for 0-6 h at
37°C in a humidified atmosphere containing 95% air/5% CO2. At the
times and concentrations specified, chemoattractants, antibodies, and/or
peptides were added to the top and/or bottom compartments in 500 µl of
PBSG-HSA. At the end of incubations, chambers were shaken to dislodge
PMN from the lower surface of the inserts. The medium in each lower compartment was collected and its content of PMN was determined using
a Coulter counter (Loike et al., 1995
). Unless otherwise indicated, all values reported are the average of six different samples from at least three independent experiments.
Flow Cytometric Analysis
PMN (105 cells/200 µl of PBSG-HSA) were incubated in suspension at
37°C for 30 min in the presence or absence of fMLP (107 M) or LTB4
(10
7 M), transferred to 96-well polystyrene tissue culture microtiter plates (Corning), incubated for 30 min at 4°C in 200 µl PBSG-HSA containing the indicated primary antibody (2 µg/ml), washed three times with
PBSG-HSA at 4°C, further incubated for 30 min at 4°C with either Alexa
488-conjugated or phycoerythrin-conjugated rabbit anti-mouse F(ab')2 in
200 µl of PBSG-HSA, washed three times again with PBSG-HSA at 4°C,
and resuspended at 4°C in 300 µl PBS containing 2% BSA and 0.3 mg/ml
propidium iodide to determine cell viability. The contribution of dead
cells (usually <2%) was removed from the final data analysis. The mean
fluorescence intensity of 3-5 × 103 cells was determined using a Becton
Dickinson FACSCalibur®.
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Results |
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PMN Chemotaxis through Matrigel and Fibrin Gels
PMN chemotax through three-dimensional gels composed
of reconstituted basement membrane proteins containing
collagen IV, laminin, and fibronectin (Matrigel; Fig. 1), or
collagen I (Loike et al., 1995) in response to a gradient of
fMLP or LTB4. In contrast, PMN chemotaxis through fibrin gels or plasma clots is dependent upon the specific
chemoattractant used. fMLP-stimulated PMN do not migrate through fibrin gels or plasma clots, whereas LTB4-stimulated PMN do (Fig. 2 A; Loike et al., 1995
). Checkerboard analyses confirmed that PMN migrate through
these gels in response to a chemoattractant gradient (Loike
et al., 1995
). Placement of equimolar concentrations of
both fMLP and LTB4 into the bottom chambers inhibited
PMN from migrating through fibrin gels (Fig. 2 A; Loike
et al., 1995
), confirming that fMLP's effect is dominant
over LTB4's effect.
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Commercial fibrinogen contains small amounts of fibronectin and vitronectin. To test whether matrix components other than fibrin are responsible for inhibiting migration of fMLP-stimulated PMN through fibrin gels and
plasma clots, we performed additional experiments using
fibrin gels formed from purified fibrinogen that contained no detectable fibronectin, plasminogen, Factor XIII, or vitronectin. PMN stimulated with LTB4, but not
with fMLP, migrated through gels formed from purified
fibrinogen (Fig. 2 B). Moreover, collagen I gels (60 µg/
insert) each containing 10 µg of purified fibronectin did
not affect the migration of either fMLP- or LTB4-stimulated PMN, whereas the addition of fibrinogen to such
gels blocked migration of fMLP-stimulated PMN (data not shown). These results are consistent with reports (Asakura
et al., 1997; Farrell and al-Mondhiry, 1997
; Suehiro et al.,
1997
; Miettinen et al., 1998
) that fibrin(ogen) contains sequences that are ligands for
1 integrins, and confirm that
fibrin is the matrix component that inhibits migration of
fMLP-stimulated PMN.
Effects of Antibodies against 1 and
2 Integrins on
PMN Chemotaxis through Fibrin Gels and Matrigel
To examine the roles of 1 and
2 integrins in PMN migration through Matrigel (Fig. 1), or fibrin (Figs. 2 A and 3),
we added antibodies that block
1 or
2 integrins to the
upper compartment of Matrigel or fibrin-coated inserts together with PMN and measured the number of PMN that
migrated into the lower compartment in response to fMLP
or LTB4. As expected, mAb IB4, directed against
2 integrins (Wright et al., 1983
), blocked PMN migration
through Matrigel (Fig. 1) or fibrin gels (Fig. 2 A) in response to LTB4. Antibody IB4 also blocked fMLP-stimulated PMN migration through Matrigel (Fig. 1), and did
not alter fMLP's inhibitory effect on PMN chemotaxis
through fibrin gels (data not shown). These results are
consistent with previous reports (Diamond and Springer, 1994
; Springer, 1995
; Premack and Schall, 1996
) that anti-
2 integrin antibodies block PMN migration through endothelia and through gels formed by a variety of extracellular matrix proteins.
In contrast, mAbs AiiB2 (Bohnsack et al., 1990) and
P4C10 (Carter et al., 1990
), which block the common
chain of
1 integrins (CD29), had no effect on fMLP- or
LTB4-stimulated chemotaxis through Matrigel (Fig. 1), or
on LTB4-stimulated PMN migration through fibrin gels
(Fig. 2 A). However, these same anti-
1 chain antibodies reversed fMLP's inhibitory effect on PMN chemotaxis
through fibrin (Fig. 3 A).
|
Control experiments showed that CSAT (Lallier and
Bronner-Fraser, 1991), a mAb that binds to chicken but
not human
1 integrins, SZ21, an antibody against
3 integrins (Lawson and Maxfield, 1995
), and PM6/13, another
antibody against
3 integrins (Patel et al., 1998
), did not
alter the inhibitory effect of fMLP on PMN migration through fibrin gels (Fig. 3 A and data not shown). These
antibodies also did not affect migration of LTB4-stimulated PMN through fibrin gels (data not shown).
Among the antibodies directed against the chains of
1 integrins, only those directed against
5 chains were effective in reversing fMLP's inhibitory effect on PMN migration through fibrin gels (Figs. 2 B and 3 A). Neither antibodies against
4 chains nor antibodies against
6 chains
of
1 integrins affected migration of fMLP- or LTB4-stimulated PMN through fibrin gels (Fig. 3 A) or Matrigel (Fig.
3 B and data not shown).
To confirm that 1 integrins directly interact with fibrin(ogen), we examined the effects of anti-
1 integrins
on the migration of fMLP-stimulated PMN through gels
formed of purified fibrinogen, lacking detectable levels of
fibronectin, vitronectin, plasminogen, or Factor XIII. Both
antibodies directed against
1 and
5 chains of
1 integrins
(Fig. 2 B) reversed fMLP's inhibitory effect on chemotaxis
through these gels. These results are consistent with reports (Asakura et al., 1997
; Farrell and al-Mondhiry,
1997
; Suehiro et al., 1997
; Miettinen et al., 1998
) that fibrin(ogen) contains sequences that are ligands for
1 integrins.
The peptide GRGDSP blocks the interaction of 1 integrins with RGD ligands on matrix proteins (Pierschbacher
and Ruoslahti, 1987
). Like antibodies against
1 integrins,
addition of GRGDSP peptide to the medium allowed
fMLP-stimulated PMN to migrate through fibrin gels
(Table I). Control experiments showed that GRGESP peptide, which does not block binding of
1 integrins
to fibronectin or other RGD-containing matrix proteins
(Pierschbacher and Ruoslahti, 1987
), did not reverse the
inhibitory effect of fMLP on PMN migration through fibrin gels (Table I). Neither peptide affected the number of
PMN that migrated through fibrin in response to LTB4
(Table I) or through Matrigel in response to fMLP (Fig. 3 B).
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Effects of Antibodies against 1 and
2 Integrins
on Adhesion of Chemoattractant-stimulated PMN
to Fibrin
PMN were incubated in control medium or in medium
containing fMLP or LTB4 and allowed to adhere to fibrin-coated 96-well plates. In the absence of chemoattractant
<1% PMN adhered to fibrin (data not shown). Over 40%
of fMLP-stimulated PMN and ~50% of LTB4-stimulated PMN adhered to fibrin. mAb IB4, which blocks the ligand-binding sites of three different 2 integrins (
L
2,
M
2,
and
X
2; Wright et al., 1983
; Loike et al., 1991
), inhibited
adhesion of fMLP- or LTB4-stimulated PMN to fibrin by
75-80% (Fig. 4). In contrast, mAb AiiB2, which blocks the
ligand-binding sites of
1 integrins (Bohnsack et al., 1990
),
had no significant effect on the number of fMLP-stimulated PMN that adhered to fibrin, and enhanced by ~25% adhesion of LTB4-stimulated PMN to fibrin (Fig.
4). These experiments show that
2 integrins are the primary PMN surface receptors that mediate adhesion of
chemoattractant-stimulated PMN to fibrin.
|
fMLP, but Not LTB4, Activates PMN 5
1 Integrins
The findings presented above indicate that 1 integrins,
and specifically
5
1 integrins, mediate the qualitatively
distinct effects of fMLP and LTB4 on PMN adhesion to,
and migration through, fibrin gels. To determine whether
fMLP and LTB4 differentially affect the activation of
1
integrins we used mAb 15/7, which recognizes a conformationally determined epitope on activated
1 integrins
(Bohnsack et al., 1995
). PMN incubated for 30 min with
fMLP exhibited a 10-22-fold increase in binding of mAb
15/7 (Fig. 5 J), compared with unstimulated PMN (Fig. 5
B), whereas PMN incubated for the same length of time
with LTB4 (Fig. 5 F) showed little change over unstimulated PMN (Fig. 5 B) with respect to binding of mAb 15/7.
Control experiments showed that surface expression of
1
integrins was stimulated approximately twofold by LTB4
(Fig. 5 G), and approximately threefold by fMLP (Fig. 5
K), whereas
2 integrin surface expression was stimulated
approximately fivefold by LTB4 (Fig. 5 H) and approximately ninefold by fMLP (Fig. 5 L). Other studies showed
that the extent of expression of the epitope for antibody
15/7 on
1 integrins was dependent upon the dose of fMLP
used to stimulate the PMN, and that 5 × 10
6 M fMLP induced maximal expression of this epitope (not shown). In
contrast, LTB4 concentrations 10-50-fold higher (i.e., 10
6
to 5 × 10
6 M) than those used in the experiments described in Fig. 5 did not increase expression of the 15/7
epitope on
1 integrins (data not shown).
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Effects of Antibodies against 1 Integrins on Closeness
of Apposition of fMLP- and LTB4-stimulated PMN
to Fibrin
We have used exclusion of Rh-PEG 10 kD from zones of
contact between chemoattractant-stimulated PMN and fibrin-coated surfaces as a measure of the closeness of apposition of PMN to the underlying substrate (Loike et al.,
1995). Previously, we reported an inverse correlation between the formation of zones of close apposition between
chemoattractant-stimulated PMN and fibrin gels and the capacity of PMN to migrate through these gels (see Fig. 7
in Loike et al., 1995
). In the present experiments we used
exclusion of Rh-PEG 10 kD to test whether antibodies
and peptides that block
1 integrins, and that facilitate migration of fMLP-stimulated PMN through fibrin gels (Figs.
2 and 3 A and Table I), affect the closeness of apposition
of these cells to fibrin. Antibodies against the
chain of
1
integrins, or against the
5 chain of
5
1 integrins (not
shown), reduced the percentage of fMLP-stimulated PMN
that excluded Rh-PEG 10 kD from zones of contact with
fibrin from 80% to 20-30% (Fig. 6), and reduced the percentage of LTB4-stimulated PMN that excluded Rh-PEG
10 kD from these contact zones from 20% to <2% (Fig.
6). These experiments together with those shown in Fig. 5
show there is a direct correlation between the capacity of
fMLP or LTB4 to activate PMN
1 integrins and the capacity of these chemoattractants to promote close apposition between PMN and fibrin (as measured by exclusion of
Rh-PEG 10 kD), and to inhibit PMN migration through fibrin gels (Fig. 3 A).
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|
Effects of Phorbol Esters on PMN Adhesion and Migration
Tumor-promoting phorbol esters, like ligands that bind to
PMN and macrophage fibronectin receptors, activate M
2
(CD11b/CD18) for phagocytosis of C3bi-coated particles
(Wright and Silverstein, 1982
), and promote formation of
zones of close apposition between phorbol ester-stimulated PMN and fibrinogen-coated surfaces (Table II).
However, phorbol ester-stimulated PMN do not migrate
into fibrin gels, even when treated with antibodies against
5
1 integrins (data not shown). These findings suggested
that phorbol esters activate
M
2 integrins for close apposition to fibrin independently of
1 integrins. To test this
prediction, PMN were incubated with or without antibodies against
1 integrins, allowed to adhere to fibrin- or fibrinogen-coated surfaces in medium containing PMA, and
then were incubated with Rh-PEG 10 kD. 77% of PMA-treated PMN formed zones of close apposition on fibrin
even when they had been treated with antibodies against
1 integrins. In contrast, <15% of the PMA-stimulated
PMN that adhered to these surfaces formed zones of close
apposition when treated with antibodies against
2 integrins (Table II). This experiment shows that when suitably
activated,
2 integrins are capable of mediating close apposition between PMN and fibrin-coated surfaces in the
absence of
1 integrin ligation.
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Discussion |
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The different effects of fMLP and LTB4 on PMN adhesion to and chemotaxis through fibrin gels appear to be a
consequence of qualitative differences in the effects of
these chemoattractants on the activity of 1 integrins. That
is, fMLP activates
1 integrins (Fig. 5 and Table III), stimulates PMN to adhere closely to fibrin(ogen) (Fig. 6 and
Table III; Loike et al., 1995
), and inhibits PMN chemotaxis through fibrin gels (Fig. 2 and Table III; Loike et al.,
1995
). In contrast, LTB4 neither activates
1 integrins
(Fig. 5 and Table III) nor induces PMN to adhere closely
to fibrin(ogen) (Fig. 6 and Table III; Loike et al., 1995
),
and stimulates PMN to migrate through fibrin gels (Fig. 2
and Table III; Loike et al., 1995
). To our knowledge, this is
the first demonstration that signals initiated by two chemically distinct chemoattractants with their respective seven
membrane spanning/heterotrimeric G protein-coupled receptors exert different effects on the activation state of a specific
1 integrin, and regulate PMN migration.
|
Fibrin(ogen)-containing Matrices Exert a Specific Effect
As shown in Fig. 2, fibrin(ogen) is unique among the matrix and plasma proteins tested in arresting the migration
of fMLP-stimulated PMN. This is particularly notable in
the case of fibronectin, a well-recognized ligand for 5
1
integrins. The failure of fibronectin to induce migration
arrest suggests that fibrin(ogen) has heretofore unrecognized properties, independent of its ability to bind
5
1 integrins, that are important in its ability to cause migration arrest.
Relationship between Closeness of Apposition, Tightness of Adhesion, and Cell Migration
DiMilla et al. (1993) and Palecek et al. (1997)
reported
that smooth muscle cells migrate optimally on fibronectin-coated surfaces when their integrins bind to these surfaces
at intermediate strengths. Weber et al. (1996)
reported an
inverse correlation between the strength of adhesion of
chemokine-stimulated monocytes to surfaces coated with
the 120-kD RGD-containing fibronectin fragment and the
capacity of these cells to migrate across filters coated with
this fibronectin fragment. The findings of Keller et al.
(1979)
and of Wilkinson et al. (1984)
, and those reported in Fig. 6, demonstrate an inverse correlation between
closeness of apposition of PMN to surfaces coated with
proteins that express ligands for PMN receptors and the
ability of PMN to migrate on or through matrices containing these proteins. Thus, it seems likely that loose versus
close apposition between cells and matrix protein-coated
substrates reflects weak versus strong adhesion, respectively, between the cells and the substrate.
PMA Bypasses 1 Integrins in Stimulating PMN to
Adhere Closely to Fibrin
Antibodies against 2 integrins reduced adhesion, inhibited close apposition between PMA-stimulated PMN and
fibrin (Table II), and blocked PMN migration through fibrin (data not shown). Antibodies against
1 integrins had
no effect on any of these parameters (Table II and data
not shown). These results demonstrate that the interaction
of activated
2 integrins with fibrin is both required and
sufficient for PMA-stimulated PMN to form zones of close
apposition on fibrin (Table II), and that PMA bypasses the
requirement for engagement of activated
1 integrins by
matrix proteins for PMN to form zones of close apposition
on fibrin.
Pathways by Which fMLP and LTB4 Activate
1 and
2 Integrins
Although the signal transduction pathways by which chemoattractants regulate PMN 1 and
2 integrins remain to
be elucidated, our findings lead us to make three suggestions regarding the organization of these pathways.
First, antibodies that activate 1 integrins do not promote adhesion of unstimulated PMN to fibrin, or inhibit
LTB4-stimulated chemotaxis of PMN through fibrin gels
(unpublished data). These results suggest that signals initiated by both fMLP receptors and activated
1 integrins are
required to inhibit chemotaxis of fMLP-stimulated PMN
through fibrin gels.
Second, the finding that fMLP and PMA have similar effects on PMN adhesion to and migration through fibrin
gels might suggest that the interaction of activated 1 integrins of fMLP-stimulated PMN with fibrin activates protein kinase C, and that this is the mechanism by which
fMLP signals
2 integrins to bind closely to fibrin. However, Laudanna et al. (1996)
reported that calphostin C, a
protein kinase C inhibitor, blocks adhesion of PMA-stimulated, but not of fMLP-stimulated, mouse lymphocytes
transfected with fMLP receptors, to VCAM-1-coated
surfaces. (Adhesion of chemokine-stimulated lymphocytes to VCAM-1 is mediated by activated
4
1 integrins.) Laudanna et al. (1996)
identified rho as a key participant
in fMLP- and IL-8-mediated activation of
4
1 integrins in
mouse lymphocytes. This finding suggests to us that rho
acts downstream of G
i in activating
1 integrins. The report of Caron and Hall (1998)
that rho participates in coupling CR3 (CD11b/CD18) to the actin cytoskeleton suggests that rho also affects
2 integrin-mediated functions.
Whether PMN LTB4 receptors activate rho is unknown and should be investigated.
Third, binding of fMLP to its receptor activates Gi
(Laudanna et al., 1996
). The specific G
activated by
LTB4 in PMN has not been reported. Pertussis toxin,
which inactivates G
i, blocks most effects of LTB4 and of
fMLP on human PMN. Thus, the finding that fMLP activates
1 integrins (Fig. 5 J) while LTB4 does not (Fig. 5 F)
suggests that binding of LTB4 to its receptor activates G
subunits other than, or in addition to, G
i and that this difference in G
subunit utilization is responsible for the divergent effects of fMLP and LTB4 on
1 integrin activation (Fig. 5), and on closeness of PMN adhesion to fibrin
(Fig. 6). Indeed, Arai and Charo (1996)
have shown differential utilization of G
subunits after MCP-1 or IL-8 stimulation of MCP-1 or IL-8 receptor transfected HEK293 cells, and Yokomizo et al. (1997)
have demonstrated that
pertussis toxin treatment does not ablate Ca2+ increases
stimulated by LTB4 in LTB4 receptor-bearing CHO cells.
Proposed Mechanisms by Which fMLP Inhibits PMN Chemotaxis through Fibrin Gels
Our studies suggest at least three distinct mechanisms by
which fMLP could inhibit PMN migration through fibrin
gels. First, the combined strengths of adhesion of activated
1 and
2 integrins to fibrin could be sufficient to immobilize PMN on fibrin. Our unpublished finding that antibodies that activate
1 integrins do not inhibit migration of
LTB4-stimulated PMN through fibrin gels casts doubt on
this combined-strength-of-adhesion hypothesis as an explanation for the inhibitory effect of fMLP on PMN chemotaxis through fibrin.
Second is the possibility that binding of fMLP or LTB4
to its cognate receptors directly and differentially activates
2 integrins for strong or weak adhesion, respectively. According to this hypothesis, fMLP-activated
1 integrins
play no role in inhibiting chemotaxis of fMLP-stimulated
PMN through fibrin. However, since activated
1 integrins
mediate outside-in signaling, RGD peptides and antibodies against
1 integrins reverse fMLP's inhibitory effect on
PMN migration through fibrin by stimulating
1 integrins
to signal trans-dominant negative (Diaz-Gonzalez et al.,
1996
) effects on
2 integrins. Against this hypothesis are
the findings that antibodies against the
6 chains of
1 integrins (Fig. 3), and antibodies that activate
1 integrins (unpublished data), do not reverse fMLP's inhibitory effect
on PMN chemotaxis through fibrin.
Third, and we think most likely, is that the capacity of
fMLP to promote close adhesion to, and to block migration through, fibrin gels is mediated by a cascade of signals
(diagrammed in Fig. 7), in which the interaction of activated 1 integrins with the fibrin matrix causes trans-dominant activation of
2 integrins. This mechanism is consistent with previous studies (Pommier et al., 1983
; Wright et
al., 1984
; Brown, 1992
) showing that interaction of PMN
or macrophages with RGD-containing matrix proteins activates
M
2 integrins for phagocytosis of C3bi-coated particles.
As shown in Fig. 7, we suggest that the interaction of
LTB4 or fMLP with their respective PMN receptors generates a "common" signal that activates 2 integrins for
loose adhesion to fibrin (Fig. 7, A and B, and D and E, respectively). In addition, we propose that fMLP receptors
(Fig. 7 C) also signal activation of
5
1 integrins (Figs. 5 J
and 7 E). We further suggest that binding of activated
5
1
integrins to fibrin matrices clusters these integrins, thereby
generating an outside-in signal that activates
2 integrins
(Fig. 7 F), for close apposition between PMN and fibrin-coated substrates (Fig. 7 G).
Close apposition reflects tight adhesion (Keller et al.,
1979; Wilkinson et al., 1984
; DiMilla et al., 1993
; Palecek
et al., 1997
), presumably mediated by the coupling of
2 integrins to the cytoskeleton. We do not know whether tight
adhesion causes, or is merely associated with, cessation of
migration. In either case, PMN cease migrating (Figs. 2 A
and 3 A, and Table III). We propose that antibodies and
peptides that block the interaction of activated
5
1 integrins with fibrin (Figs. 2 and 3, and Table I) inhibit these
outside-in signals, thereby blocking trans-dominant activation of
2 integrins for close apposition to fibrin (Fig. 6 and
Table II) and allowing PMN to migrate through fibrin.
The interaction of LTB4 with its receptor also generates
a signal that stimulates 2 integrins for loose apposition.
However, LTB4 does not activate
1 integrins (Fig. 5 F).
Therefore, these
1 integrins do not bind to the matrix, do
not generate outside-in signals, and therefore do not initiate trans-dominant activation of
2 integrins for close apposition (Fig. 7, A-C), or cessation of migration.
What Characterizes the Sessile State?
Further work is needed to determine whether cessation of
migration is merely a function of strong adhesion between
PMN and fibrin or whether it reflects reorganization of the
PMN cytoskeleton as observed by Dustin et al. (1997) in
antigen-sensitized T lymphocytes. They found that these
cells become immobilized when they encounter MHC
class II molecules containing a peptide antigen recognized by the T lymphocytes' antigen receptors. They identified
changes in microtubule organization of these sessile T
lymphocytes that distinguish them from their randomly
migrating brethren. We suspect that PMN that adhere to
fibrin after fMLP stimulation will exhibit similar changes
in cytoskeletal organization.
Why Are There So Many Different Chemoattractants for PMN?
Our findings suggest that the availability of many different chemoattractants (e.g., fMLP, LTB4, IL-8, C5a, etc.) serves two complementary functions. First, they provide redundancy, thereby assuring that pathogenic microbes are detected rapidly by the innate immune system. Second, they reflect the need to direct PMN to different tissue sites and to prepare them for interactions with many different types of ligands.
Chemoattractant-encrypted Stop Signals Provide a Gradient-independent Mechanism for Leukocyte Accumulation at Specific Anatomic Sites
Our findings also suggest an alternative to the notion that
leukocyte accumulation at a specific anatomic site in vivo
requires the presence of a gradient of chemoattractant/
chemokine emanating from that site. While there is no
doubt that gradients of chemoattractants/chemokines are
formed in vitro (Keller et al., 1979; Wilkinson et al., 1984
; Huber et al., 1991
; Campbell et al., 1996
, 1997
; Foxman et al., 1997
; Palecek et al., 1997
), they may be difficult to maintain in vivo in the face of the perturbing effects of muscular contraction and variations in blood and lymph flow.
Leukocytes in the vascular system begin to enter specific
tissue compartments when they encounter a chemoattractant/chemokine. We suggest that once within this tissue
compartment leukocytes migrate randomly in response to
a relatively uniform concentration of matrix-bound chemoattractant/chemokine. When in the course of this random walk they encounter extracellular matrix proteins or
cells that express ligands for a specific activated 1 integrin, they adhere strongly and become sessile. By regulating activation of specific receptors and adhesive strengths,
concentrations of chemoattractants/chemokines well below those required to saturate or desensitize chemoattractant/chemokine receptors can mediate a stochastic process
by which leukocytes accumulate at specific anatomic sites
and form highly ordered structures (e.g., granulomas, germinal centers). According to this model, leukocytes accumulate at specific anatomic sites by a process that is similar in principle to the accumulation of flies on fly paper.
Foxman et al. (1997) showed that multiple chemoattractants/chemokines can work in combination to elicit migration patterns that cannot be achieved by a single chemoattractant/chemokine. The mechanisms we and they
have described are complementary. These mechanisms are
likely to be of special importance within tissue compartments where overlapping fields of chemoattractants/chemokines/cytokines surely occur, and where cells migrate in
stepwise fashion from one anatomic site to another (e.g.,
T cell movement in lymph nodes from T cell-rich paracortical zones to germinal centers; Garside et al., 1998
), PMN
accumulation at foci of bacterial infection, or of immune-complex deposition (Wilkinson et al., 1984
). The essential
point of the findings reported here is that by endowing leukocytes, and probably all migrating cells, with a modest
number of receptors for different chemoattractants, chemokines, and cytokines, nature has made optimal use of
instructive and selective mechanisms to achieve a level of
organizational specificity that would otherwise require
substantially more genetic information.
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Footnotes |
---|
Received for publication 18 May 1998 and in revised form 8 January 1999.
Address correspondence to Dr. John D. Loike, Department of Physiology
and Cellular Biophysics, Columbia University College of Physicians and
Surgeons, 630 W. 168th St., Mail hub #22, New York, NY 10032. Tel.:
(212) 305-1510. Fax: (212) 305-5775. E-mail: jdl5{at}columbia.edu
We thank Drs. A.R. Horwitz and Sally Zigmond for helpful discussions, Eugene Butcher and Ellen Foxman for suggestions about the manuscript, Eric Brown for sharing unpublished data, and one of the reviewers for particularly thoughtful suggestions.
This study was supported by National Institutes of Health grant AI20516, Bristol-Myers Squibb Research, and a generous gift of the late Samuel W. Rover.
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Abbreviations used in this paper |
---|
C3bi, cleaved b fragment of the 3rd complement component; C5a, a fragment of the cleaved 5th complement component; fMLP, formyl methionyl leucyl phenylalanine; HSA, human serum albumin; IL-8, interleukin 8; LTB4, leukotriene B4; PMN, polymorphonuclear leukocytes; Rh-PEG, rhodamine-conjugated polyethylene glycol.
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References |
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