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*
BHF Cardiovascular Medicine Unit, Imperial College School of Medicine at the National Heart and Lung Institute, Hammersmith Hospital;
Electron Microscopy Unit, Royal Brompton and Harefield National Health Service Trust; and
University College London Medical School, London, United Kingdom; and
§
Centocor, Malvern, PA, 19355
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 have been implicated in this
process, and in vitro studies have identified
vß3 as a heterotypic ligand for PECAM-1.
In the present study we have addressed the roles of these molecules by
investigating and comparing the effects of PECAM-1 and
vß3 blockade on leukocyte migration in
vivo. For this purpose we have examined the effects of neutralizing Abs
directed against PECAM-1 (domain 1-specific, mAb 37) and
ß3 integrins (mAbs 7E3 and F11) on leukocyte responses in
the mesenteric microcirculation of anesthetized rats using intravital
microscopy. The anti-PECAM-1 mAb suppressed leukocyte
extravasation, but not leukocyte rolling or firm adhesion, elicited by
IL-1ß in a dose-dependent manner (e.g., 67% inhibition at 10 mg/kg
37 Fab), but had no effect on FMLP-induced leukocyte responses.
Analysis by electron microscopy suggested that this suppression was due
to an inhibition of neutrophil migration through the endothelial cell
barrier. By contrast, both anti-ß3 integrin mAbs, 7E3
F(ab')2 (5 mg/kg) and F11 F(ab')2 (5 mg/kg),
selectively reduced leukocyte extravasation induced by FMLP (38 and
46%, respectively), but neither mAb had an effect on IL-1ß-induced
leukocyte responses. These findings indicate roles for both PECAM-1 and
ß3 integrins in leukocyte extravasation, but do not
support the concept that these molecules act as counter-receptors in
mediating leukocyte transmigration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lß2, CD11a/CD18) and
Mac-1 (Mß2,
CD11b/CD18) (6, 7, 8, 9), and members of the Ig superfamily,
such as ICAM-1 and ICAM-2 (10, 11), platelet-endothelial
cell adhesion molecule-1
(PECAM-1)3 (12, 13), and junctional adhesion molecule (14). Despite
increased in vitro and in vivo evidence for the involvement of these
molecules in leukocyte transendothelial cell migration, their relative
importance in leukocyte emigration in vivo is still unclear. Among the molecules listed above, PECAM-1 (CD31) is one of the very few that appears to specifically mediate leukocyte transmigration and has thus been the subject of much interest in the context of leukocyte emigration in the last 10 yr. PECAM-1 is a 130- to 135-kDa membrane glycoprotein, with an extracellular portion composed of six Ig-like conserved units of the C2 subclass, a single transmembrane portion, and a short cytoplasmic tail (15). It is expressed on the surface of platelets, most subsets of leukocytes, and endothelial cells, where it is concentrated at intercellular junctions (12). PECAM-1 has important signaling properties; it is capable of both initiating and responding to cellular activation, for which it is dependent on its cytoplasmic tail (16, 17, 18, 19). Although PECAM-1 has been implicated in numerous biological functions, there is now ample evidence, from both in vitro and in vivo investigations, demonstrating an important role for this molecule in the control of leukocyte transmigration, a function for which its expression profile and binding/signaling properties are well suited (12, 20, 21, 22, 23). Furthermore, recent studies with PECAM-1-deficient mice have shown a defect in neutrophil migration through the perivascular basement membrane (24), in agreement with previous findings using an anti-PECAM-1 Ab (23), although in the former study the overall number of recruited leukocytes was normal.
PECAM-1 is known to support both homotypic and heterotypic cellular
interactions, although the relative importance of these molecular
events in the regulation of leukocyte migration in vivo remains
unclear. In vitro, Liao et al. (25) demonstrated that
different domains of PECAM-1, supporting homotypic or heterotypic
interactions, mediated leukocyte transendothelial cell migration or
leukocyte interaction with extracellular matrix components,
respectively. Abs that inhibit PECAM-1 homotypic interactions have also
been shown to suppress leukocyte migration in vivo (26, 27). Possible roles for PECAM-1-dependent heterotypic
interactions in leukocyte migration have not been addressed however. In
this context, a number of putative PECAM-1 ligands have been reported,
including heparan sulfate proteoglycans (28), an as yet to
be characterized 120-kDa glycoprotein expressed on activated T
lymphocytes (29), ADP-ribosyl cyclase (CD38)
(30), and the integrin
vß3 (31, 32). Of these reported heterotypic ligands, the
ß3 integrin,
vß3, is perhaps the
most intriguing, because, like PECAM-1, it has been implicated in
leukocyte transmigration (33).
The integrin vß3
shares a common subunit with the platelet molecule
IIbß3 (GPIIb/IIIa),
although its expression profile is very different.
vß3, while being
expressed at low levels by platelets, is expressed at higher levels by
smooth muscle cells, endothelial cells, monocytes, and neutrophils and
at several million copies per cell by osteoclasts
(34, 35, 36). In many cell types it is physically associated
with another protein within the cell membrane, integrin-associated
protein (IAP, CD47), which is known to modulate a number of its
functions (37, 38). Neutralizing Abs recognizing
vß3 or IAP reduce
leukocyte migration through endothelial cell monolayers in vitro
(33, 39). As two independent groups have reported that
PECAM-1 and vß3 act as
counter-receptors (31, 32), but other groups have
challenged the validity of such an interaction (40), the
aim of the present study was to compare the effects of blocking PECAM-1
or vß3 on leukocyte
migration in vivo. For this purpose we chose to investigate and compare
the effects of Abs directed against PECAM-1 or
ß3 integrins in two rat models of inflammation,
namely leukocyte migration through IL-1ß- and FMLP-activated
mesenteric venules, previously shown to be PECAM-1-dependent and
independent reactions, respectively (23). We hypothesized
that if the molecules PECAM-1 and
vß3 acted as
counter-receptors in leukocyte transmigration, blocking Abs directed
against these molecules would exhibit similar effects on leukocyte
migration in models of inflammation in vivo. Our findings, however,
demonstrate that blockades of PECAM-1 and
vß3 exert very
different inhibitory effects on leukocyte migration in vivo, arguing
against the possibility that these molecules act as counter-receptors
in the regulation of leukocyte migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Sprague Dawley rats (200–250 g) were purchased from Harlan-Olac (Bicester, U.K.).
Reagents
Reagents were purchased as follows: Hypnorm (fentanyl-fluanisone mixture) from Janssen-Cilag (High Wycombe, U.K.), sodium pentobarbitone from Rhône Mérieux (Harlow, U.K.), osmium (VIII) oxide from Johnson Matthey PLC (Royston, U.K.), and sodium cacodylate from Agar Scientific (Stanstead, U.K.). All other reagents were purchased from Sigma-Aldrich (Poole, U.K.).
Monoclonal Abs
The anti-PECAM-1 mAb 37 (murine IgG
1) has been developed,
and its specificity for domain 1 of PECAM-1 and its cross-reactivity
with rat cells have been confirmed, as recently reported
(41). The nonbinding isotype-matched control mAb 125
(murine IgG1) was produced in a similar manner. These Abs were used
either as whole Ab or Fab. Fab were prepared by papain digestion and
were purified by protein A affinity chromatography (see Ref.
41 for detailed description). The
anti-ß3 integrin mAbs used were mAb 7E3
(murine IgG
1) and mAb F11 (murine IgG1). 7E3 is an anti-human
ß3 integrin mAb that recognizes the platelet
integrin, IIbß3, and
the vitronectin receptor,
vß3, with equal
affinity and has been shown to exhibit some cross-reactivity with rat
cells (42, 43). Ab F11 is a specific function-blocking
anti-rat ß3 integrin mAb (44).
These Abs were used either as whole Ab or F(ab')2 for in
vivo experiments. F(ab')2 fragments were prepared
by pepsin digestion as detailed previously (45). Either
murine anti-rat MHC class I mAb (IgG1, Harlan Sera-lab, Sussex,
U.K.) or mouse myeloma IgG1, MOPC-21 (Sigma-Aldrich), was used as
control mAb. A chimeric version of 7E3 (c7E3), in which the constant
regions of the parent murine Ab have been replaced with the constant
regions of human and 
IgG chains, was used for flow cytometry
studies, with a human LFA-3-human Fc chimera used as the control
molecule (a gift from Biogen, Cambridge, MA).
Flow cytometry
Blood was collected from untreated animals by cardiac puncture and was anti-coagulated with lithium heparin (10 IU/ml). Aliquots were incubated at 4°C with primary Ab (10 µg/ml) for 30 min, followed by FITC-conjugated goat anti-human Fc-specific IgG secondary Ab (Dako, Ely, U.K.) for 30 min. Erythrocytes were lysed using FACS lysing solution (Becton Dickinson U.K., Oxford, U.K.). Samples were analyzed using an EPICS XL flow cytometer (Beckman Coulter, High Wycombe, U.K.). Neutrophils were identified by characteristic forward and side scatter profiles.
Platelet aggregation
Male rats were treated by i.v. injection of anti-ß3 mAb, 7E3 F(ab')2, and blood was harvested 6, 24, or 48 h later. Blood was anti-coagulated with sodium citrate (0.32% final concentration), and platelet-rich plasma was prepared by centrifugation at 600 x g for 6 min. Once the platelet concentration had been adjusted to 2–3 x 105 platelets/µl by addition of platelet-poor plasma, platelet aggregation in response to ADP (10 mM) was measured using a PAP-4 aggregometer (Biodata, Horsham, PA). Inhibition of ADP-induced platelet aggregation was calculated by comparison with control samples.
Intravital microscopy
Male rats were prepared for intravital microscopy as previously described (46). Briefly, following sedation with i.m. Hypnorm (0.1 ml), animals were anesthetized with i.v. sodium pentobarbitone (30 mg/kg loading dose followed by 30 mg/kg/h). The animals were maintained at 37°C on a custom-built heated microscope stage. Following midline abdominal incision, the mesentery adjoining the terminal ileum was carefully arranged over a glass window in the microscope stage and pinned in position. The mesentery was kept warm and moist by continuous application of Tyrode’s balanced salt solution. Mesenteric venules (20–40 µm in diameter) were viewed on an upright fixed-stage microscope (Axioskop FS, Carl Zeiss, Welwyn Garden City, U.K.) fitted with water immersion objectives. Images were captured with a video camera (C5810-01, Hamamatsu Photonics U.K., Enfield, U.K.) for viewing on a monitor (PVM-1453 MD, Sony U.K., Weybridge, U.K.) and storage by videocassette recorder (AG-MD830E, Panasonic U.K., Bracknell, U.K.). As the resolution of intravital microscopy does not allow definitive distinctions to be made between different subpopulations of leukocytes, all responses are quantified in terms of leukocyte behavior. Hence, rolling leukocytes were defined as those cells moving slower than the flowing erythrocytes, and rolling flux was quantified as the number of rolling cells moving past a fixed point on the venular wall per minute, averaged for 4–5 min. Firmly adherent leukocytes were defined as those that remained stationary for at least 30 s within a 100-µm segment of a venule. Extravasated leukocytes were defined as those in the perivenular tissue adjacent to, but remaining within a distance of 50 µm of, a 100-µm vessel segment under study. In some experiments the mesentery was excised at the end of the quantification to allow preparation for electron microscopy as detailed below.
In experiments studying IL-1ß-induced leukocyte responses, male rats were sedated with i.m. Hypnorm, after which test Ab, control Ab, or vehicle was given by i.v. injection. Fifteen minutes later, recombinant rat IL-1ß (10 ng in 5 ml of sterile saline; gift from Dr. K. Vosbeck, Ciba-Geigy, Basel, Switzerland) or vehicle was given by i.p. injection. Four hours later, the mesenteric tissue was exteriorized and prepared for intravital microscopy, as described above. In each animal multiple vessel segments from multiple vessels were quantified. In experiments studying FMLP-induced leukocyte responses, the mesenteric tissue was prepared for study, and once a suitable vessel had been selected for observation, baseline readings were taken. The rats were then treated i.v. with test or control Ab, and after 15 min, FMLP (10-7 M final concentration) was applied topically to the mesenteric tissue in the superfusion buffer. Leukocyte responses within the chosen vessel were quantified for 1 h, during which the topical application of FMLP was maintained.
Electron microscopy
In selected experiments following the dynamic quantification of leukocyte responses the portion of mesentery containing the test vessels was excised and fixed in buffer containing 2.5% glutaraldehyde (50 mM sodium cacodylate, 4 mM HCl, and 0.18 mM CaCl2). Samples were then postfixed in 1% osmium VIII oxide and, following dehydration in methanol, were embedded in araldite resin before sectioning. Thin sections (1 µm) were stained with toluidine blue to allow location of venules. Ultrathin sections (0.1 µm) were mounted on copper mesh grids and stained with uranyl acetate and lead citrate before viewing on a transmission electron microscope (Hitachi 7000, Hitachi U.K., Hayes, U.K.). The total number of leukocytes associated with each vessel was counted, and their positions, according to the following classification, were noted: A, within lumen of the venule; B, crossing the endothelium; C, between the endothelium and perivascular basement membrane; D, crossing the basement membrane; and E, outside the venule, but within 50 µm of it. For each venule, the fraction of leukocytes that had crossed the endothelium but were still inside the basement membrane was calculated according to the following equation C/(C + D + E). In each series of experiments, tissue samples from at least three animals were analyzed, and from each animal at least three vessels were studied in detail.
Peritoneal recruitment
Following sedation with i.m. Hypnorm, male rats were given test or control Ab (5 mg/kg) by i.v. injection. Fifteen minutes later, recombinant rat IL-1ß (10 ng in 5 ml of sterile saline) or vehicle was given by i.p. injection. Four hours later, animals were humanely killed, and the peritoneal cavity was washed with normal saline containing 0.25% BSA and heparin (2 IU/ml). Total cell counts were performed using Kimura stain (47). Differential cell analysis was determined in exudate smears prepared in a cytocentrifuge (Cytospin-3, Shandon, Runcorn, U.K.) and stained with May-Grünwald/Giemsa stains. A minimum of four animals were used for each group.
Statistical analysis
Data are presented as the mean ± SEM unless stated otherwise. Statistical significance was assessed using one- or two-way ANOVA or unpaired Student’s t test as appropriate. A p value <0.05 was considered significant. Analysis was performed using Prism 3.0 for Windows (GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study we demonstrated that an anti-PECAM-1
polyclonal Ab suppressed leukocyte migration through IL-1ß-activated
rat mesenteric venules by blocking the response at the level of the
perivascular basement membrane (23), observations that
suggested a novel in vivo role for PECAM-1 and have subsequently been
supported by the findings of Duncan et al. in PECAM-1-deficient mice
(24). However, as both in vitro and in vivo studies have
identified domain 1 of PECAM-1 as a key component of the molecule in
leukocyte transendothelial cell migration (25, 26), we
sought to extend our earlier observations by studying the effect of a
domain 1-specific anti-PECAM-1 mAb on IL-1ß-induced leukocyte
migration through rat mesenteric venules, as studied by intravital
microscopy. Intraperitoneal administration of recombinant rat IL-1ß
(10 ng) 4 h before exteriorization of the mesenteric tissue
resulted in a significant increase in leukocyte rolling flux (data not
shown), leukocyte firm adhesion, and leukocyte transmigration compared
with responses detected in rats treated with i.p. saline (Fig. 1
). Intravenous pretreatment with domain
1-specific anti-PECAM-1 mAb 37 inhibited the cytokine-induced
transmigration of leukocytes through the vessel wall in a
dose-dependent manner (52 and 68% inhibition at 5 and 10 mg/kg,
respectively; p < 0.05), but had no effect on
leukocyte rolling flux (data not shown) or leukocyte firm adhesion
(Fig. 1A). A similar specific inhibitory effect of the
Ab on leukocyte transmigration was detected when Fab of the Ab was used
(67% inhibition at 10 mg/kg; p < 0.01; Fig. 1B). In contrast, an isotype-matched control Ab, mAb
125, used as a whole molecule or a Fab, had no effect on the leukocyte
responses quantified (Fig. 1).
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85%), as shown in Fig. 2, A
and B. Interestingly, in
contrast to our previous findings with an anti-PECAM-1 polyclonal
Ab (23), there was no evidence of neutrophil trapping
between the endothelial cell layer and the perivascular basement
membrane, in the IL-1ß-stimulated vessel walls of animals treated
with mAb 37 (Fig. 2C). These results suggest that the
domain 1-specific anti-PECAM-1 mAb 37 blocks neutrophil
transmigration by reducing their ability to penetrate the endothelial
cell layer, as opposed to the basement membrane.
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To compare the functional roles of PECAM-1 and ß3 integrins in leukocyte migration in vivo, the aim of the following experiments was to extend the studies described above by investigating the effects of two different anti-ß3 integrin Abs on IL-1ß-induced leukocyte responses. The Abs used were mAbs F11 and 7E3.
mAb F11 is a murine anti-rat ß3 mAb
(44). Ab 7E3, however, is a murine anti-human
ß3 Ab, and although in vitro it has been shown
to bind to rat aortic smooth muscle cells (42, 43),
initial experiments were conducted to investigate the cross-reactivity
of this Ab with rat molecules before its use in vivo. Platelet
aggregation was measured ex vivo 6, 24, and 48 h after i.v.
administration of 7E3 F(ab')2 (4 mg/kg). This
treatment significantly reduced ADP-induced platelet aggregation both 6
and 24 h after Ab treatment (Fig. 3A). Additionally, 7E3 was
found to bind to rat leukocytes, as shown by whole blood flow cytometry
(Fig. 3B). Because leukocytes do not express
IIbß3, these results
suggest that 7E3 binds to rat leukocytes via an interaction with
vß3, although the
possibility of its binding to platelets adherent to leukocytes or to
Mac-1 (7E3 has been reported to bind to an activated state of Mac-1
(48, 49)) cannot be excluded.
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). Abs
7E3 and F11 were also without an affect on leukocyte rolling flux and
firm adhesion (results not shown). Similarly, neither Ab was able to
inhibit the recruitment of leukocytes into the peritoneal cavity of
rats 4 h after i.p. administration of IL-1ß (data not shown).
Having observed differing effects of PECAM-1 or
ß3 integrin blockade on IL-1ß-induced
leukocyte responses, the studies were extended to a different model of
leukocyte migration for further analysis of the roles of these
molecules.
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In the following series of experiments the effects of anti-PECAM-1 mAb 37 and anti-ß3 integrin mAbs 7E3 and F11 on leukocyte responses elicited by FMLP were examined. Topically administered FMLP (10-7 M) induced a time-dependent increase in leukocyte firm adhesion and transmigration over the 1-h in vivo test period. Significant levels of firmly adherent leukocytes were detected 10 min postapplication of the chemoattractant, with the response reaching a maximal level within 30 min of FMLP administration. As expected, the time course of leukocyte transmigration was slightly slower than that observed for firm adhesion, in that significant levels of extravascular leukocytes were first detected at around 20–45 min postapplication of FMLP, a response that continued to increase for the duration of the 1-h in vivo test period.
Fig. 5 shows that in agreement with our
previous findings (23), blockade of PECAM-1 had no effect
on FMLP-induced leukocyte responses. In contrast, however, the
anti-ß3 integrin mAbs 7E3 and F11
selectively suppressed FMLP-induced leukocyte transmigration, while
having no effect on leukocyte firm adhesion (Figs. 6 and 7).
With respect to mAb 7E3, however, there was a trend toward increased
leukocyte firm adhesion, although this did not achieve statistical
significance. In these studies both Abs were used as
F(ab')2, with 7E3 F(ab')2
and F11 F(ab')2 reducing FMLP-induced leukocyte
transmigration at 60 min by 38% (p < 0.05)
and 46% (p < 0.01), respectively. At the
doses used, none of the Abs used in the present study had an effect on
circulating leukocyte numbers (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3. In
common with PECAM-1, in vitro studies have implicated
vß3 in mechanisms of
leukocyte motility and transmigration (27, 33, 50), but to
our knowledge no studies have investigated the role of this integrin in
leukocyte transmigration in vivo. The aim of the present study was to
directly investigate and compare the roles of
vß3 and PECAM-1 in
leukocyte migration through stimulated rat mesenteric venules, as
observed by intravital microscopy. Because we had no access to a
selective vß3
inhibitor suitable for studies in rats, we analyzed the effect of an
anti-PECAM-1 mAb in comparison with the effects of two
anti-ß3 integrin mAbs. The results of this
in vivo study confirm a role for PECAM-1 in leukocyte transmigration
and provide the first demonstration that Abs against
ß3 integrins can exert a specific effect on the
migration of leukocytes through vessel walls. Of importance, however,
our results demonstrate a clear difference in the inhibitory effects of
the anti-PECAM-1 Ab and the anti-ß3
integrin Abs. Specifically, while PECAM-1 blockade suppressed
IL-1ß-induced leukocyte transmigration,
anti-ß3 integrin Abs inhibited leukocyte
transmigration induced not by IL-1ß, but by the chemoattractant FMLP.
These findings, while confirming roles for both PECAM-1 and
ß3 integrins in leukocyte transmigration,
suggest that PECAM-1 and
vß3 do not necessarily
act as counter-receptors to facilitate leukocyte migration in
vivo. PECAM-1-neutralizing Abs have proved effective at reducing leukocyte accumulation in a number of models, including leukocyte accumulation in inflamed rat peritoneum, rat lung, and murine peritoneum (21, 22). Although studies such as these have established a role for PECAM-1 in leukocyte recruitment to sites of inflammation in vivo, relatively few investigations have addressed the precise stage of leukocyte migration at which PECAM-1 is involved. In this context we have previously demonstrated that a polyclonal anti-PECAM-1 Ab inhibited leukocyte migration through IL-1ß-activated rat mesenteric venules by suppressing the movement of leukocytes through the perivascular basement membrane (23), findings supported by recent observations in PECAM-1 knockout mice (24). The present study used the same model of IL-1ß-induced leukocyte recruitment into rat mesenteric venules to investigate the effect of a domain 1-specific anti-PECAM-1 mAb 37. Interestingly, the results demonstrated that mAb 37 blocked leukocyte transmigration by interfering with the movement of neutrophils through the endothelium, rather than through the perivascular basement membrane. These findings support the in vitro and in vivo observations of Liao et al. (25, 26) by demonstrating that PECAM-1 can mediate the migration of leukocytes through both endothelial cells and the perivascular basement membrane in vivo; the former response is mediated primarily by domain 1 of the molecule.
In vitro studies have shown that the different domains of PECAM-1
have distinct roles for ligand binding and receptor function. Domains 1
and 2 of PECAM-1 have been linked with PECAM-1-mediated homotypic
interactions (40) and appear to be critical for the
movement of leukocytes through endothelial cell monolayers in vitro
(25, 26, 51). By contrast, domain 6 may be required for
heterotypic interactions and the migration of leukocytes through the
basement membrane (25). However, a proposed model in which
domain 1 blockade is associated with disruption of homotypic
interactions and suppression of leukocyte migration in vivo is not
necessarily supported by the findings of the present study. The domain
1-specific Ab used in our experiments, mAb 37, has been shown to block
PECAM-1-dependent heterotypic, but not homotypic, interactions and had
no effect on TNF--induced leukocyte accumulation in human skin
transplanted onto SCID mice (41). In this latter study,
however, Abs that interfered with PECAM-1-dependent homotypic
adhesion (as assayed by L cell aggregation), blocked TNF--induced
leukocyte accumulation in vivo. Although those findings demonstrate
that there is a good association between the ability of domain
1-specific Abs to disrupt PECAM-1 homotypic interactions and their
ability to suppress cutaneous leukocyte migration in the human
skin/SCID mouse model, it does not rule out a functional role for other
PECAM-1-dependent mechanisms in leukocyte migration in vivo. One
important point may explain the difference between the in vivo results
obtained with mAb 37 in our study and those conducted using human
skin/SCID mice. In the in vivo model used in the latter investigation,
only transplanted human endothelial cell PECAM-1 is susceptible to Ab
blockade by mAb 37, because mAb 37 does not cross-react with murine
PECAM-1 on infiltrating leukocytes. In contrast, in the present study
as mAb 37 cross-reacts with rat PECAM-1, the Ab can bind to both
endothelial cell and leukocyte PECAM-1 molecules, thus enhancing its
potential to suppress PECAM-1-mediated adhesion events and, hence,
PECAM-1-dependent leukocyte transmigration.
The present study as well as using a combination of intravital and electron microscopy to demonstrate that an anti-domain-1 PECAM-1 mAb (37) can specifically reduce the ability of neutrophils to penetrate the endothelium also reaffirms that the requirement for PECAM-1 in leukocyte transmigration is stimulus specific (23, 52). In agreement with our previous observations (23), PECAM-1 blockade had no effect on rapid leukocyte migration through rat mesenteric venules induced by the chemoattractant FMLP. This finding supports our previous explanation that leukocyte chemoattractants, such as FMLP, may stimulate leukocyte migration by directly acting on leukocytes to activate their integrins and perhaps mobilize their granular proteases. Such a chemoattractant-induced response may bypass the need to activate endothelial cells and hence trigger leukocyte transmigration by PECAM-1-independent mechanisms (23).
The integrin vß3 is
expressed by endothelial cells, neutrophils, monocytes, and, to a
lesser degree, platelets (34, 36). Neutralizing Abs
against it and its membrane-associated protein, CD47 (IAP), have proven
effective at reducing leukocyte transmigration in vitro (39, 39). Because
vß3 has been proposed
as a ligand for PECAM-1 (31, 32), we examined its role
in leukocyte migration in vivo. Hence, we investigated the effects of
two anti-ß3 integrin mAbs, namely 7E3 and
F11. mAb 7E3 is a murine anti-human ß3
integrin Ab (36). Before its use in our rat models of
inflammation, we first examined the ability of this Ab to cross-react
with rat molecules. In this context, 7E3 was found to inhibit rat
platelet aggregation ex vivo and to bind to rat neutrophils, as
determined by flow cytometry. F11 is a specific anti-rat
ß3 integrin mAb (44).
In contrast to our findings with the anti-PECAM-1 mAb,
anti-ß3 integrin Abs had no effect on
leukocyte migration through IL-1ß-activated rat mesenteric venules,
but selectively suppressed the transmigration phase of FMLP-induced
leukocyte emigration. The mechanism by which ß3
integrins may be regulating FMLP-induced leukocyte migration in vivo is
at present unclear, although a number of possible mechanisms can be
proposed. The ß3 integrin,
vß3, has been
implicated in a number of cellular responses intimately associated with
the process of leukocyte transmigration. Specifically, it has been
shown to regulate leukocyte motility on endothelial cell-associated
adhesion molecules, such as VCAM-1 and ICAM-1, by modulating the
functions of leukocyte integrins such as
4ß1 and
Lß2 (LFA-1) (33, 50). Such
vß3-mediated
down-regulation of integrin-dependent firm adhesion may provide a
mechanism by which firmly adherent leukocytes become deadherent,
allowing them to engage in the process of transmigration
(33). With respect to the transmigration phase, there is
some evidence suggesting that
vß3 may have a
regulatory role on the activity of leukocyte proteases
(53), and through its ability to interact with
extracellular components, such as vitronectin and fibronectin, it may
mediate leukocyte migration across and through the perivascular
basement membrane (54, 55). Hence, in our experimental
system, FMLP, through its ability to directly stimulate neutrophils,
may initiate a cascade of integrin activation events that induces
ß2 integrin-mediated firm adhesion followed by
vß3-mediated leukocyte
deadhesion, triggering the transition from firm adhesion to
extravasation. Furthermore, it is unclear at present whether
vß3 is involved in
leukocyte migration through the endothelium or the perivascular
basement membrane, an aspect of leukocyte extravasation that is
currently under investigation in our laboratory. The lack of effect of
the anti-ß3 integrin mAbs on
IL-1ß-induced leukocyte transmigration may suggest that in response
to an endothelial cell-activating factor, the activation of adhesion
pathways involving molecules such as PECAM-1 overrides the
vß3-dependent
mechanisms. If the results obtained with the
anti-ß3 Abs were predominantly due to
suppression of vß3,
then they would suggest that while PECAM-1 and
vß3 are both involved
in leukocyte transmigration in vivo, these molecules do not necessarily
act as counter-receptors in the process of leukocyte emigration.
However, although our observations may support a role for
vß3 in leukocyte
transmigration, the possible role of alternative ligands with which the
Abs 7E3 and F11 could interact needs to be considered.
As indicated above, both 7E3 and F11 bind to rat
IIbß3 (present study
and Ref. 44), and
IIbß3 blockade on
platelets could contribute to our in vivo observations. Because both
platelets and neutrophils can bind to fibrinogen, via
IIbß3, and Mac-1,
respectively, platelets adherent to the endothelial surface may
facilitate the adhesion of neutrophils and their subsequent
recruitment, using fibrinogen as a bridging molecule. Not only has this
bridging phenomenon been shown in vitro under flow conditions
(56), but additionally, 7E3 has been shown to inhibit
binding of cell surface Mac-1 to fibrinogen (57). However,
if platelet IIbß3 were
contributing to leukocyte recruitment in our model, one or both
anti-ß3 Abs would be expected to reduce
firm adhesion as well as transmigration, which they did not. Although
the present results do not rule out a role for
IIbß3, it seems
unlikely that the inhibitory effect of the
anti-ß3 integrin Abs is as a result of
inhibiting platelet
IIbß3; it is more
likely due to a blockade of
vß3 on leukocytes.
This is supported by two lines of evidence. First,
vß3 neutralizing Abs
can inhibit leukocyte transmigration in vitro (33), and
second, a recent study has shown that even in the absence of platelets,
the ß3 integrin-blocking Ab fragment, c7E3 Fab
(abciximab, ReoPro), can reduce leukocyte accumulation in an ex vivo
model of myocardial ischemia (58).
In addition to binding the ß3 integrins
(IIbß3 and
vß3), 7E3 has been
shown to bind to an activated form of the ß2
integrin, Mac-1 (48, 49), although its affinity for Mac-1
has been estimated to be 20- to 50-fold lower than that for
IIbß3
(57). Mac-1 is present on neutrophils and monocytes and
has been shown to be involved in leukocyte transmigration (6, 8). Although it is possible that the reduction in leukocyte
transmigration seen in 7E3-treated animals in the present study is the
result of inhibition of this ß2 integrin, two
lines of evidence argue against this. First, if inhibition of Mac-1
were a significant confounding factor, then suppression of leukocyte
firm adhesion would also have been detected. Anti-Mac-1 mAbs have been
shown in vitro to reduce FMLP-induced leukocyte adhesion under both
static and flow conditions (59, 60). In the present study
7E3 had no effect on FMLP-induced firm adhesion. Second, an additional
anti-ß3 integrin mAb (F11), which to our
knowledge does not bind Mac-1, produced results identical to those
for 7E3.
This study is the first to demonstrate roles for both PECAM-1 and
ß3 integrins, most likely
vß3, in leukocyte
transmigration in vivo. The findings further demonstrate that the roles
of these molecules in leukocyte migration are divergent with respect to
stimulus specificity, suggesting differential requirements for PECAM-1
and ß3 integrins in the regulation of leukocyte
transmigration in response to different inflammatory mediators.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sussan Nourshargh, BHF Cardiovascular Medicine, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, U.K. W12 0NN.
3 Abbreviations used in this paper: PECAM, platelet-endothelial cell adhesion molecule; IAP, integrin-associated protein; c7E3, chimeric 7E3.
Received for publication January 24, 2000. Accepted for publication April 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrins. Circulation 98:1085.Mß2 integrin I domain recognized by anti-IIbß3 monoclonal antibody 7E3 and its involvement in leukocyte adherence. J. Biol. Chem. 273:20372.vß3 and 4ß1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1. Eur. J. Immunol. 27:3242.[Medline]
vß3. Cell 85:683.[Medline]
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