By
From the * Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm,
Sweden; and The Burnham Institute, La Jolla, California 92037
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Abstract |
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Recruitment of leukocytes from blood to tissue in inflammation requires the function of specific cell surface adhesion molecules. The objective of this study was to identify adhesion molecules that are involved in polymorphonuclear leukocyte (PMN) locomotion in extravascular
tissue in vivo. Extravasation and interstitial tissue migration of PMNs was induced in the rat
mesentery by chemotactic stimulation with platelet-activating factor (PAF; 107 M). Intravital
time-lapse videomicroscopy was used to analyze migration velocity of the activated PMNs, and
the modulatory influence on locomotion of locally administered antibodies or peptides recognizing various integrin molecules was examined. Immunofluorescence flow cytometry revealed
increased expression of
4,
1, and
2 integrins on extravasated PMNs compared with blood
PMNs. Median migration velocity in response to PAF stimulation was 15.5 ± 4.5 µm/min (mean ± SD). Marked reduction (67 ± 7%) in motility was observed after treatment with
mAb blocking
1 integrin function (VLA integrins), whereas there was little, although significant, reduction (22 ± 13%) with
2 integrin mAb. Antibodies or integrin-binding peptides recognizing
4
1,
5
1, or
v
3 were ineffective in modulating migration velocity.
Our data demonstrate that cell surface expression of 1 integrins, although limited on blood
PMNs, is induced in extravasated PMNs, and that members of the
1 integrin family other
than
4
1 and
5
1 are critically involved in the chemokinetic movement of PMNs in rat extravascular tissue in vivo.
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Introduction |
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Tissue recruitment of circulating leukocytes is a central event in the host defense against infectious and noxious agents. The extravasation process comprises a multistep reaction accomplished through a sequential interaction with vascular endothelium and extravascular matrix components. The initial steps in this process, i.e., rolling along the endothelium and firm adhesion, have been extensively studied both in vitro and in vivo, revealing the function of specific cell adhesion molecules of the selectin and integrin families (1). In contrast, the subsequent event of leukocyte migration in the extravascular tissue in response to a chemotactic stimulus is not well characterized, and the receptor interactions involved in this process are largely unknown.
Several cell surface receptors found on leukocytes recognize and bind extracellular matrix (ECM) components. For
example, all known members of the 1 integrin (VLA, very
late activation antigen) family bind to ECM proteins with
varying affinity for specific ECM components, e.g., fibronectin, collagen, and laminin (2). Although
1 integrins
have a widespread distribution, the expression on leukocytes has repeatedly been shown to be largely restricted to
eosinophils, monocytes, and certain lymphocyte subsets, whereas expression on PMNs is limited (2). However, this
view has been reconsidered in recent years because of data
showing that activated or extravasated neutrophils may indeed express certain
1 integrins that potentially can mediate binding to ECM proteins (6).
The 2 integrins (CD11a-c/CD18) are expressed exclusively on leukocytes and mediate firm adhesion to vascular
endothelium (9). Specific ligand binding has been shown for
fibrinogen and factor X of the coagulation cascade, complement factor C3bi, and intercellular adhesion molecule
(ICAM) 1 (1). Moreover, binding of PMNs to a variety of
biological substrates (e.g., fibronectin, collagen) and nonbiological surfaces (such as plastic) has been reported to be
2
dependent, inasmuch as adhesion can be abrogated by
CD11/CD18-blocking antibodies. Yet another member of
the integrin family that has been demonstrated to bind ECM
components and that is expressed in PMNs is the
v
3 integrin, which binds to vitronectin and fibronectin (10).
The aim of this study was to investigate the role of major
integrin receptors in PMN interstitial migration in vivo. The
potential involvement of the fibronectin-binding integrin receptors in this process was of particular interest because of
their previously documented roles in migration of various cell
types (11). An intravital microscopy model was used for
analyzing leukocyte locomotion in response to local chemotactic stimulation in extravascular tissue of the rat mesentery.
Immunofluorescence flow cytometry revealed increased expression of integrins in extravasated PMNs. This included 1
integrins, which were shown by antibody-blocking experiments to be critically involved in the extravascular PMN migration. However, this process does not seem to engage the
fibronectin-binding receptors
4
1 and
5
1.
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Materials and Methods |
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Surgical Preparation.
Wistar rats of either sex, weighing 200-250 g, were used in the experiments. Anesthesia was induced with equal parts of fluanison/fentanyl (10/0.2 mg/ml; Hypnorm; Janssen-Cilag Ltd., Saunderton, UK) and midazolam (5 mg/ml; Dormicum; Hoffman-La Roche, Basel, Switzerland) diluted 1:1 with sterile water (2 mg/kg intramuscularly). Body temperature was maintained at 37°C by a heating pad connected to a rectal thermistor. Laparotomy was performed, and a segment of the ileum was pulled outside the peritoneal cavity and placed on a heated transparent pedestal to allow microscopic observation of the mesenteric microvasculature. The exposed tissue was superfused with a warmed (37°C) bicarbonate buffer solution equilibrated with 5% CO2 in N2 to maintain physiological pH. The experiments were approved by the regional ethical committee for animal experimentation.Intravital Microscopy.
The exposed rat mesentery was observed through a microscope (Orthoplan; Leitz, Wetzler, Germany) equipped with a water immersion lens (SW × 25, NA 0.60; Leitz). The microscopic image was televised (WV 1050 E/C; Panasonic, Osaka, Japan) and recorded on time lapse video (AG-6010; Panasonic) connected to a time/date generator (WJ-810; Panasonic). Recordings were made at one-seventh normal speed. Analysis of leukocyte migration in the mesenteric tissue was made off-line from the recorded video scenes during playback at normal speed. The migration path of individual leukocytes was drawn on a transparent film placed in front of the monitor for subsequent analysis with a digital image analyzer.Experimental Procedure.
After positioning under the microscope, the exposed mesentery was soaked with 5 ml of buffer solution (37°C) containing platelet-activating factor (PAF; Sigma Chemical Co., St. Louis, MO) at a concentration of 10Antibody Diffusion in the Mesentery.
In separate experiments, the diffusion capability of the Ig molecules in the mesentery was confirmed. Pieces of intact mesenteric tissue (thickness: 20-40 µm) were mounted on plastic rings and placed on top of HBSS-filled wells (400 µl/well) of a 96-well tissue culture plate. 40 µl of FITC-conjugated murine IgG (100 µg/ml) were placed on the surface of the mesentery. The plate was incubated at 37°C for 10 min, and the fluorescence intensity of the IgG content in the upper and lower fluid compartments was measured in a fluorometer (Fluoroscan II; Labsystems Oy, Helsinki, Finland). Over a period of 10 min, an average of 7.5 ± 1.6% of applied antibody diffused through the mesentery per minute and cm2 (n = 3), indicating that there is no significant restriction for diffusion of the antibodies into the mesenteric tissue when being topically administered.Staining of Leukocytes.
Representative samples of the mesentery stimulated with PAF 10
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Immunofluorescence Flow Cytometric Analysis.
Leukocytes collected from rats of the same strain and weight as used in the in vivo experiments were used for analysis of integrin receptor expression. Leukocyte extravasation was induced by intraperitoneal injection of either 3% proteose peptone (Sigma Chemical Co.) or PAF 10Antibodies and Peptides.
The following antibodies were used: mAb HMStatistical Analysis.
Statistical analysis was performed using the Wilcoxon signed rank test for paired observations. The results are presented as mean ± SD for the animals included in each experimental group (n ![]() |
Results |
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Flow cytometric assessment of cell surface
molecule expression on neutrophils that had extravasated
into the peritoneal cavity revealed positive staining for 1
(CD29) and
4 (CD49d) integrin molecules (Fig. 1 and
Table 2). This pattern contrasted to that of blood PMNs
where little or no staining for
1 and
4 was seen (Fig. 1),
indicating that cell surface expression of
1 integrins is induced in conjunction with the extravasation process. Expression of
5 (CD49e) was limited in both cell populations. There was an increased expression of
2 integrins
(CD18) on extravasated PMNs compared to their blood
counterpart, whereas staining for
v
3 (CD51/CD61) was
similarly positive in both PMN populations (Table 2).
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Preincubation of isolated blood PMNs with PAF (107
M) before antibody labeling did not result in
1 or
4 integrin expression dissimilar from that of untreated blood cells
(data not shown).
Topical stimulation of the rat mesentery with PAF (107
M) induced profound adhesion and extravasation of circulating leukocytes. At 30-40 min of chemotactic stimulation, numerous leukocytes (predominantly neutrophils, see
Table 1) were migrating further in the extravascular tissue
(Fig. 2 A). In accordance with the flow cytometric data, immunofluorescent staining of emigrated PMNs in the mesenteric tissue in situ showed surface expression of
1,
4,
and
2 integrin molecules, as illustrated for
4 in Fig. 2 B.
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Fig. 3 A illustrates the frequency distribution of the migration velocity of individual PMNs in response to stimulation with PAF (649 cells in 30 animals total). Among these
cells, the median migration velocity was 15.5 ± 4.5 µm/min
(mean ± SD). The migration velocity was stable over a period of >1.5 h after induction of the chemotactic stimulus.
The role of various integrins in PMN migration was evaluated by topical administration of antibodies to the tissue.
Treatment with anti-1 (mAb HM
1-1) resulted in a pronounced inhibition of PMN locomotion. Migration velocity was reduced by 67 ± 7% (P <0.01; Fig. 4), yielding
a median migration velocity of 4.6 ± 1.3 µm/min (Fig. 3
B). Notably, as evident from Fig. 3 B, the whole population of migrating cells, rather than a certain fraction, was
affected by this antibody treatment. A less pronounced effect was observed with the polyclonal anti-
1 antibody,
which reduced the migration velocity by 32 ± 15% (P <0.01; Fig. 4). No further inhibition was achieved when
the antibody concentration was increased 10-fold. Treatment with two different antibodies against the
2 chain
(CD18) also significantly reduced migration velocity, by 17 ± 14% (mAb CL26) and 22 ± 13% (mAb WT.3) (P
<0.05; Fig. 4). An additive inhibitory effect was observed when anti-
2 mAb was administered together with the
polyclonal anti-
1 serum. This combined treatment reduced
migration velocity by 52 ± 18% (P <0.01; Fig. 4). On the
other hand, coadministration of anti-
2 mAb with the anti-
1
mAb HM
1-1 yielded no further inhibition of migration
velocity above that seen with HM
1-1 alone. The inhibitory effect of the various antibody treatments was observed within minutes after application and persisted throughout the observation period (>40 min) as shown for mAb HM
1-1
(Fig. 5). Purified hamster, mouse, and rabbit IgG isotype
standards did not influence migration velocity (103 ± 11, 95 ± 7, and 99 ± 8%, respectively).
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Despite pronounced upregulation of the integrin 4 subunit (CD49d) on extravasated PMNs, no significant modulatory effect on the migration velocity was observed after
treatment with either of the two anti-
4 mAbs. Antibodies
against
5
1 or
v
3 also did not influence PMN locomotion. Moreover, combined treatment with anti-
2 mAb together with either anti-
4 or anti-
4 plus anti-
5 mAb resulted in no further inhibition of migration velocity above
that obtained with anti-
2 treatment alone (99 ± 4 and 95 ± 10%, respectively, n = 3). The integrin-binding peptides
SLIDIP and RGDGW, which mimic natural ligand binding and block the function of
4
1 and
5
1, respectively,
also had no effect on the migration velocity either in combination (data not shown) or alone (Fig. 4). There was no
difference in the effect for any of the reagents tested when
concentration was raised 10 times (data not shown).
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Discussion |
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Extravasation and tissue accumulation of leukocytes is
one of the key components in the host defense against invading pathogens. After their escape from the blood, the
leukocytes need to migrate in the extravascular tissue, directed by a chemotactic stimulus, to reach the site of injury
or infection. Studies of leukocyte migration in vitro have
indicated the interaction of leukocytic cell surface receptors
with different extracellular matrix components in this process (16). However, interactions with the multitudinous meshwork of biopolymers that characterizes native extracellular matrix and the function in vivo of leukocyte integrins
in the locomotive process have yet to be defined. In this report, we demonstrate that 1 integrins, induced in PMNs in
conjunction with their extravasation, are of critical functional importance for PMN locomotion in extravascular tissue. Our data on a physiological induction of
1 integrin
expression in PMNs agree with recent in vitro and in vivo
findings by Kubes and coworkers (7, 8), and contribute to a
growing body of evidence that
1 integrin expression may
reach significant levels also in neutrophils (6, 17, 18). Also,
the upregulation of
2 integrins on extravasated PMNs is
consistent with the activation-induced upregulation of
2
integrins on the leukocyte surface (19) and the critical role
of this receptor complex in leukocyte adhesion to endothelium and diapedesis through the vessel wall in vivo (20).
A qualitatively similar pattern of PMN 1 integrin expression as obtained with flow cytometric analysis of extravasated PMNs isolated from the peritoneal cavity could
be demonstrated by in situ immunostaining of PMNs migrating in the extravascular tissue of the mesentery. These
findings are also of significance from a methodological
point of view, since they illustrate that antibodies applied
topically to the mesentery indeed do diffuse into the tissue,
and by this route of administration will reach the migrating PMNs (see Materials and Methods). Thus, we may conclude that adequate antibody concentrations were achieved
in the tissue at the level of the migrating cells (no additional
effect was seen when antibody concentration was raised 10 times), and that restrictions in antibody transport could not
account for the lack of effect of some of the reagents used.
Our quantitative measurements of PMN migration in
the rat mesentery in vivo show that 1 and
2 integrins participate in extravascular PMN locomotion, and that they
may cooperate in this process. Blockage of
1 and
2 integrin function impaired the ability of the leukocytes to migrate in the extravascular tissue as indicated by significant reductions in their migration velocity. Anti-
1 antibodies
were clearly more effective in inhibiting PMN migration
than were anti-
2, suggesting a predominant role of
1 integrins in the locomotive process in vivo. The additive inhibitory effect observed when anti-
2 mAb was coadministered with the polyclonal anti-
1 antiserum but not when
combined with the anti-
1 mAb may suggest that a synergistic action of combined anti-
1 and anti-
2 treatment is detectable only when
1 integrins are insufficiently blocked
(as was likely the case when the polyclonal anti-
1 antibody
was used). In contrast to our findings with
2 integrin
blockade, Bienvenu et al. (21), using a similar rat model,
found no inhibition of the extravascular migration with
anti-
2 treatment. Differences in the experimental protocol
(e.g., the antibody concentration used) may explain the discrepant observations. Also, although statistically significant,
the inhibition we found with anti-
2 was limited and may
have been overlooked by these authors.
Although it has been shown for certain leukocyte subtypes that migration on various ECM matrices in vitro requires the function of specific integrin molecules (16, 22),
this report is the first to demonstrate an in vivo role for 1
integrins in the extravascular locomotion of leukocytes.
Even if it can not be deduced which
1 integrins are predominantly involved in the PMN locomotion, our data
suggest, based on use of both monoclonal function-blocking antibodies and integrin-binding peptides, that the fibronectin binding receptors
4
1 and
5
1 do not participate in this process. This finding may seem surprising in
light of the pronounced upregulation of
4 integrins on extravasated PMNs, and the central position being attributed
to fibronectin in various aspects of cell migration (11, 12).
Previous findings have suggested a role for
4
1 and
5
1
in migration of PMN from blood to tissue sites in vivo (17)
or through fibroblast monolayers in vitro (18), seemingly in
disparity with our direct observations in the rat mesentery. Possibly, mAb inhibition of integrin function in these studies interfered mainly with initial adhesion to endothelium
or the fibroblast monolayer and less with the locomotive
function. Interestingly, through direct observation of leukocyte migration in three-dimensional gels, an enhanced
lymphocyte migration after anti-
4 mAb treatment (16), and
reduced PMN locomotion after the gel being supplemented with fibronectin was demonstrated (23). These findings may
suggest that fibronectin-binding integrins (e.g.,
4
1 and
5
1) may support anchoring of the leukocytes to the substrate rather than promote their migratory movement.
Taken together, expression of 1 integrins, limited on
blood PMNs, is induced in this cell population in conjunction with their emigration from blood to tissue. Our data
demonstrate that molecules of this integrin family are critically involved in PMN locomotion in extravascular tissue
in vivo. Hence, in addition to selectin and
2 integrin
functions determining intravascular adhesive events, cell
surface induction and engagement of
1 integrins is suggested to be yet another important physiological mechanism in the multistep process of PMN recruitment to sites
of injury or infection.
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Footnotes |
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Address correspondence to Lennart Lindbom, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. Phone: 46-8-728-7207; Fax: 46-8-332-047; E-mail: lennart.lindbom{at}fyfa.ki.se
Received for publication 11 July 1997 and in revised form 13 March 1998.
This study was supported by the Swedish Medical Research Council (grants 14X-4342 and 04P-10738); the Swedish Foundation for Health Care Sciences and Allergy Research (grant A98110); the IngaBritt and Arne Lundbergs Foundation; and the Karolinska Institutet. E. Ruoslahti was a Nobel Fellow at the Karolinska Institutet when this work was initiated.
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