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The border-crossing behavior of eosinophils and neutrophils in the lung
Zuurbier, A.E.M.
Publication date
2001
Link to publication
Citation for published version (APA):
Zuurbier, A. E. M. (2001). The border-crossing behavior of eosinophils and neutrophils in the
lung.
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CHAPTER VI
PECAM-1 is Implicated in
Transendothelial Migration of
Neutrophils, but not of Eosinophils
I liuplcr I 7
PECAM-1 is implicated in Transendothelial Migration of Neutrophils,
but not of Eosinophils
Astrid E.M. Zuurbier. Frederik P.J. Mul. Dirk Roos and Peter L. Hordijk
Central Laboratory of the Netherlands Mood Transfusion Service (CLB) and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
PECAM-1 is involved in leukocyte transendothelial migration. We have studied the role of PECAM-1 in
thein-ritro migration of neutrophils and eosinophils across endothelial cell monolayers. Pre-treatment of neutrophils
with an antibody to PECAM-1 (HEC170) blocked transendothelial migration of neutrophils in response to PAF. C5a and 11.-8. but the migration was hardly affected «hen endothelial cells were pre-treated. Moreover, this mAb did not impair migration across epithelial monolayers. In contrast to neutrophils, eosinophil
transendothelial migration was not blocked by HEC170 mAb, irrespective of the eosinophil activation state or the chemoattractants used, although eosinophils express PECAM-1 to the same level as neutrophils. Thus, neutrophil, but not endothelial, PECAM-1 is implicated in transendothelial migration in vitro, suggesting that an interaction between PECAM-1 and an endothelial ligand plays a role in the migration process. This interaction is not involved in eosinophil migration, pointing to a role for neutrophil PECAM-1 in selective neutrophil
extravasation.
Introduction
in lung inflammation and asthmatic reactions, circulating leukocytes, primarily neutrophils and eosinophils, migrate across the lung endothelium and the lung epithelium into the airway lumen. The process of transmigration initially involves selcctin-mcdiated rolling of the granulocytes on the endothelial cell surface and is followed by firm adhesion to and passage across the endothelial cell layer ( 1 ;2). Subsequently, the granulocytes migrate through the interstitial matrix and across the epithelium into the lung lumen.
The endothelium plays an important role in the recruitment of granulocytes. It is the first barrier that has to be crossed and acts therefore as a gate to the underlying tissue. Furthermore, endothelial cells can be activated by inflammatory stimuli, resulting in release of a wide range of proinflammatory mediators (3:4) and increased cell-surface expression of adhesion molecules required for leukocyte transmigration (3), such as E-selcctin, P-selectirr, intercellular adhesion molecule (ICAM)-l, vascular cellular adhesion molecule (VCAM)-I, and platelet/endothelial cell adhesion molecule (PECAM)-l (5:6).
The role of the selectins, ICAM-1 and VCAM-1 has been extensively studied (5). but the precise role of PECAM-1 in transendothelial migration is less well defined. PECAM-1 orCD31 is a
130-kD single-chain molecule containing six lg-like domains, a transmembrane region and a cytoplasmic tail (7:8). It is expressed on the surface of neutrophils (8-10). eosinophils) 1 1 ). platclets(S). monocytes (8). NK cells, subsets of T cells(S). and on continuous endotheha where it is concentrated at the borders between opposing cells (7:12). Depending on the cell type. PECAM-1 is capable of mediating homophilic interactions, i.e. PECAM-1 on one cell binds to PECAM-1 on another cell, or heterophylic interactions, i.e. the ligand for PECAM-1 on the opposing cell is another
I'F.CAM-I is implicated in Trunscndothclial Migration o/ Xeiilrophils
structure (7:8). For instance, it has been shown that ccvß3 mtegnn, which is expressed by endothelial
cells and subsets of T cells, is a heterophylic ligand for 1 (13:14). Other described PECAM-1 ligands are an uncharactcn/ed PECAM-120-kD molecule on activated T lymphocytes (PECAM-15) and CD3S ( PECAM-16). Ligation of PECAM-1 stimulates tyrosine phosphorylation of its cytoplasmic tail, leading to association with a number of signaling proteins, including SHP-2 (7) and activation of the small GTPaseRapl (17).
The role of PECAM-1 in transendothelial migration might be to act as a homophilic adhesion molecule and to direct migration of the cells through the endothelial cell junctions by the formation of a haptotactic gradient of PECAM-1 on the endothelial cell surface. This hypothesis is attractive, because there is indeed a gradient 6f PECAM-1 expression from the apical endothelial surface to the junctions. Within the junction, it is more concentrated at the basal side of the cleft (12). However, PECAM-1 may also act indirectly through activation of integrins by intracellular
signaling(7:9:17). Stimulation of leukocyte PECAM-1 has been shown to cause up-regulation and activation ofß2 integrins on monocytes, neutrophils (9; 1 8) and natural killer cells ( 19) and to increase
adhesive functions of ß, integrins on eosinophils (1 I), T cells (20) and CD34' hematopoietic progenitor cells (21 ).
Several studies have shown that PECAM-1 is required for transendothelial migration of neutrophils in vitro (6:8) and in vivo (8:10:22). However, little is known about the role of PECAM-1 in eosinophil transendothelial migration. In this paper we show that neutrophil, but not eosinophil transendothelial migration is mediated by PECAM-1. although both cell types express PECAM-1 to the same extent. Moreover, we show that neutrophil, but not endothelial, PECAM-1 is invoked in in vitro
transendothelial migration.
Materials & Methods
Reagents
Platelet-activating factor (PAF) and C5a were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant human (rh) IL-5 was bought from Pepro Tech Inc. (Rocky Hill, NJ, USA), and rhlL-8 and bFGF from Boehringer Mannheim (Mannheim. Germany). Eotaxin was derived from Santa Cruz Biotechnology (Santa Cruz, CA, USA ). CD3 1 monoclonal antibody (mAb) ( IgG 1 : HEC170) (23 ) w as isolated from hybridoma supernatant by precipitation with 50% saturated ammonium sulphate and subsequent protem-A affinity chromatography. CD 14 mAb (IgG 1 ; 8G3). human scrum albumin (USA) and fibronectin were obtained from the Central Laboratory of the Netherlands Blood Transfusion Sen ice (CLB) (Amsterdam, The Netherlands). Fluorescently labeled secondary antibody (Ab) to mouse immunoglobulins was from Dako (Glostrup, Denmark). C5a, PAF, IL-5 and IL-8 were dissolved in phosphate-buffered saline, pll 7.4. (PBS) supplemented with 0.5 % (w/v) human serum albumin (HSA) and were stored at-20T. HEPES medium contained 132 mM NaCl. 6.0 mM KCl, I.OmMCaCL, 1.0 mM MgSOj. 1.2 mM Kl LP04. 20 mM FIEPES. 5.5 mM glucose and 0.5 % (w/v) HSA (pH 7.4). Lysis
buffer consisted of PBS supplemented with 0.1 % (v/v) Tween-20 (Merck, Schuchardt, Germany), 0.2 % (wA ) N-acetyl-N,N,N-trimethyl-ammonium bromide (Sigma). 0.2 %(w/v) bovine serum albumin (BSA) (Sigma) and 20 mM EDTA.
Chapter I 7
Cell culture
Freshly isolated, primary HUVECs (24) and HUVEC cell line cells (25) were cultured in HUVEC medium (RPMI 1640 (Gibco Life Technologies. Gaithersburg, MD, USA) supplemented with 10% (v/v) heat-inactivated human serum. 100 U/ml penicillin. 100 ug/ml streptomycin, 2 m M glutamine and 1 ng/ml bFGF) in culture flasks coated with 1 mg/ml fibronectin. The 2 -4th passages of the primary HUVECs and the 10'1' to 30* passage of HUVEC cell line cells were used for sub-culturing on
fibronectin-coated polycarbonate Transwell membranes (3.0 urn pore size, 12 mm diameter; Costar. Cambridge. MA. USA).
The human lung epithelial adenocarcinoma-derived cell line H292 (American Type Culture Collection CRL 1848) (26) was grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated human serum, 100 U/ml penicillin, 100 ug/ml streptomycin and 2 mM glutamine. in uncoated culture flasks. The 4' to 30lh passages of H292 cells were used for the transmigration assay. To study the
granulocyte migration across epithelial monolayers in the physiological direction, H292 cells epithelial cells were routinely cultured on the bottom side of Transwell polycarbonate membranes (3.0 u.m pore size, 12 mm diameter) (Costar, Cambridge, MA), as described (27).
Granulocyte isolation
Blood was obtained from healthy volunteers. Granulocytes were purified from a buffy coat of 500 ml of blood by density gradient centrifugation over isotonic Percoll, as described (28). After lysis of the erythrocytes in the pellet fraction with a cold erythrocyte lysis buffer containing 155 mM NH4CI. 10 mM
K.HCO3 and 0.1 mM EDTA (pH 7.4), the granulocytes were washed twice in PBS. The granulocyte cell suspension consisted for more than 95% of neutrophils. This cell suspension was used for the isolation of eosinophils via the formyl-methionyl-leucyl-phenylalanine (fMLP) method (29). The purity and viability of the eosinophils were more than 95 %. This procedure leads to the isolation of relatively unprimed eosinophils compared to conventionally used isolation procedures with immunomagnetic beads (30).
Transmigration assay
Fresh medium was added to the Transwells 4 hours prior to the start of the transmigration assay, and the Transwells were washed twice with HEPES mediumjust before starting the transmigration assay. The lower compartment was filled w ith pre-warmed HEPES medium w ith or w ithout indi\ idual
chemoattractants. The granulocytes ( 10 ml) were labeled with 4 pg. ml Calcein-AM (Molecular Probes) diluted in HEPES medium for 30 min at 37°C prior to the onset of the transmigration assay (31 ). After labeling, the cells were washed twice with HEPES medium. Where indicated, purified eosinophils (2x10" cells/ml) were primed with 10""' M IL-5 in HEPES medium for 30 min at 37°C and were washed in incubation medium. Monoclonal Abs (10 ug/ml) were added to the granulocytes and endothelial cells 10 mm before the start of the transmigration assay, or the cells were separately pre-incubated with mAbs for 15 min and washed before the onset of the transmigration assay.
Granulocytes were placed in the upper compartment. The Transwells were incubated for either 40 mm (neutrophils) or 1.5 h (eosinophils) at 37°C with 5% C O: and maximal humidity. After the
incubation, the upper and lower compartments were washed with HEPES medium. The fluids were collected and the membranes were excised. The cells in the fluids and the excised membranes were lysed in lysis buffer. The extent of transmigration was quantified by means of fluorescence measurement, i.e. the Icxcls of Calcein-AM in the upper compartment, lower compartment and membrane were measured w ith a spectrofluorometcr (Model RF-540. Shimadzu Corporation. Kyoto, Japan). The percentage of
PFC AM-1 is implicated ui Transctulolliclial Migration oj Seiitropluls
labeled cells that had transmigrated was calculated from the amount o f fluorescence detected in the lower compartment in relation to the fluorescence o f the originally added Calcein-AM-labelcd granulocytes.
FA CS analysis
The expression o f surface antigens on granulocytes was measured by indirect immunofluorescense with flow cytometry. The cells were incubated with the m A b CD31 H E C I 7 0 or with m A b C D 1 4 8G3. as indicated in the text, for 30 min at 4°C. After the cells had been washed with a 30-fold excess o f ice-cold PBS containing 1 % (w/v) BSA and 14 ug/ml azide, binding o f m A b was detected by incubation with PE-conjugated goat-anti-mouse-lg for 30 min at 4°C. The fluorescence intensity o f the cells was measured w ith a flow cytometer (FACScan. Becton Dickinson, San Jose, C A , U S A ) .
Statistical analysis
The results were expressed as the mean ± SEM o f the number o f different experiments mentioned in the legends and analyzed w i t h the Student's t-test. Two-sided p v alues were calculated, and p values exceeding 0.05 were considered not to be significant.
Results
T o investigate the role o f P E C A M - 1 in the transmigration o f granulocytes across monolayers o f endothelial cells, we performed a transmigration assay in the presence o f m A b H E C I 7 0 . As shown in Fig I, m A b H E C I 70 blocked migration o f neutrophils across H U V E C monolayers in response to P A F by 4 7 % , whereas neutrophil migration across H292 bronchial epithelial monolayers, which are P E C A M
-I-deficient, was not affected (Fig. 1 ). The i n h i b i t o r y effect on migration seemed not to be restricted to PAF-driven migration, because transendothelial migration o f neutrophils towards other
chemoattractants. such as C5a and I L - 8 . was also reduced when H E C I 7 0 was added (Fig. 2). Moreover, incubation w i t h CD3 1 m A b H E C 6 5 . w h i c h is directed against domains 3-6 (32), d i d not impair neutrophil transendothelial migration (data not shown). Thus, these results suggest that the first two domains o f C D 3 1 arc implicated in neutrophil transendothelial migration.
Both neutrophils and endothelial cells express P E C A M - 1 . and the molecules on both cell types may play a role in transmigration. To unravel which molecular interaction is blocked by the H E C I 70 m A b , we determined whether endothelial or neutrophil-derived P E C A M - 1 was inhibited in its function after HEC170 m A b treatment. Endothelial monolayers and neutrophils were separately pre-treated w ith H EC 170 m A b and washed. We found that the migration was hardly affected when H U V E C was pre-treated with HEC170, whereas neutrophil prc-trcatment reduced migration by 3 9 % (Fig. 3).
Figure 1. Inhibition of neutrophil migration across monolayers of either 11292 lung epithelial cells or III. 'VECtowards PAF(100nM). 'Hie cells were pre-incubated for 10 min with CD3I mAb HECI70 or with an irrelevant mAb (CD14), and the mAbs remained present during the transmigration assay. The results are shown as percentage inhibition of control
transmigration, i.e. pre-incubated with mAb ('1)14. Data are means ± SEM of five independent experiments. Asterisks indicate significance of difference « ith control migration (** p< t) (II ) .S> 60 E 40 •- 20 h
-•* *
-T
migration across HUVEC migration across H292( 'liiiplcr I 'I
Figure 2. Neutrophil transendothelial
migration in response to C5a (10 iiM) and IL-8 (10 n\l). The cells were pre-incubated for 10
min with an irrelevant mAb (CD14) (open
bars) or with CD31 mAb HFC I 70 [black bars), and the Abs remained present during the
transmigration assay. Data are means of two independent experiments. hepes C5a 50 40 30 -•£ 20 10
—
• •
—
• •
PMN pre-treated HUVEC pre-treatedFigure 3. Inhibition of neutrophil transendothelial
migration towards PAF(100 nM). Either the
endothelial cells or the neutrophils were separately pre-incubated w ith CD31 mAb 1 IECI 70 or with an irrelevant mAb (CD14) for 15 min, and the cells were washed before the onset of the transmigration assay. The results are indicated as percentage inhibition of control transmigration, i.e. cells pre-incubated with mAb CD 14. Data are means ± SEM of four independent experiments. Asterisks indicate significance of difference with control migration (**/;< 0.01).
In contrast to neutrophils, eosinophil transmigration across endothelial monolayers was not blocked by H EC 170 mAb (Fig. 4) irrespective of whether the endothelium or the eosinophils or both were pre-treated with the mAb (data not shown). The proportion of eosinophils associated with the HUVEC monolayer was not affected by H EC 170 mAb either (data not shown). In addition, the lack of inhibition was not due to the fact that the transmigration was assessed through monolayers of endothelial cell-line cells, because H EC 170 mAb did not affect eosinophil migration across monolayers of primary HUVEC either (data not shown). Moreover, priming of the eosinophils did not increase the sensitivity to HECI70 mAb. because migration of IL-5-primed eosinophils was not blocked by HECI70 mAb (Fig 5a & 5b). The type of chemoattractant also seemed not to matter, because CD31 mAb inhibited neither the migration towards PAF/C5anor towards PAForeotaxin (Fig. 5a & 5b). These results were unexpected, because eosinophils express PECAM-I on their surface to the same extent as found on neutrophils (Fig 6).
PEC 'AM-I is implicated in Tratiseiidoiliclnil Migration <>j Neutrophils o ra O) E "o 60 40 — 20 —
* *
Figure 4. Effect oj CD31 niAb HECI 70 on neutrophil migration across monolayers of IH 'VEC towards PAF (100 ii.M) and on eosinophil migration lii response to PAP t IftMl com/lined with ( '5a (10 nM). "1 he cells were pre-incubated for 10 min with
mAb HECI70 or with an irrelevant mAb (CD14), and the mAbs remained present during the transmigration assay. The results are indicated as percentage inhibition of control transmigration, i.e. pre-incubated with mAb CD14. Data are means ± SEM of four independent experiments. Asterisks indicate significance of difference with control migration (** p< 0.01 ).
neutrophil migration
eosinophil migration
hepes PAF/C5a hepes PAF eotaxin
Figure 5. Transendothelial migration oj IL-5-primed eosinophils. The cells were pre-incubated for 10 min with an irrelevant mAb (CD14) {open Inns) or with CD31 mAb IIEC 170 {black bars) and the Abs remained present during the transmigration assa\ (a) Migration in response to the combination of PAF ( 1 pM ) and C5a (10 nM ). Data are means ± SEM of three independent experiments, (b) migration in response to PAF (1 iiM) and eotaxin (100 ng/ml). Data arc means of two independent experiments.
PECAM-1 expression on neutrophils 24 319 PECAM-1 expression on eosinophils
Figure 6. PPCAM-I surface expression measured with
( 'DM mAb HECI 70 (thick line) was compared to a control m. \h tilun line) for neutrophils and for eosinophils h\ flow cytometry. I he mean lluorescense
intensities are depicted in the figure Results are representative of three independent experiments.
( 'hap/er I 'I
Discussion
The role of PECAM-I in neutrophil transendothelial migration was investigated in an in vitro transmigration model, consisting of confluent primary HUVEC monolayers grown on Transwell membranes. To study the role of PECAM-I, cither neutrophils or endothelial cells were pre-treated with a blocking monoclonal antibody to PECAM-I (HEC 170) prior to the transmigration assay. Our results suggest that the first two domains of CD31 are implicated in neutrophil transendothelial migration. The fraction of neutrophils associated with the HUVEC monolayer, i.e. the number of neutrophils bound to the endothelial cells or the Transwell membrane, was not affected by blocking H EC 1 70 mAb (data not shown); thus, the neutrophils were not accumulating abo\c the Transwell membrane. These results suggest that the reduced neutrophil migration was not due to impaired capability of the neutrophils to cross the basement membrane deposited by endothelial cells on the Transwell membrane. Because H EC 170 binds to the first two domains of PECAM-I, it can be concluded that these domains are not implicated in neutrophil passage across this basement membrane. This notion has been confirmed by other investigators, who showed that the first two domains of PECAM-1 are implicated in the passage of leukocytes through endothelial junctions and not in passage through the basement membrane (8; 10).
HEC170 mAb did not block neutrophil migration across H292 bronchial epithelial monolayers, which are PECAM-I-deficient. This indicates that H EC 170 mAb docs not induce signaling in neutrophils leading to reduced motility, and that the HEC170 mAb-sensitive molecular interaction is absent or plays a less prominent role in transepithchal migration. Furthermore, preliminary results obtained with stable H292 transfectants expressing CD31 at the intercellular junctions suggest that neutrophil transepithchal migration is unaffected by CD31 transfection (data not shown). Taken together, these results indicate that CD31 plays a prominent role only in endothelial transmigration, and that the process of transepithelial migration differs from transendothclial migration even when CD31 is present at the intercellular junctions of the epithelial cells. Comparable experiments have been performed by Zocchi et al (33) but with different results. These investigators reported that lymphocyte migration is greatly stimulated when CD31 is transfected in N1H/3T3 fibroblast. This difference might be due to the fact that other cell types were used. The molecular basis underlying lymphocyte transfibroblast migration and neutrophil transepithchal migration surely differs and this may also apply to the role of CD3 1 in these processes. It would be instructive to investigate whether or not lymphocyte migration across CD31 -expressing epithelial cell monolayers is augmented as well, in that case CD3 I on endothelial cells would be m\ ok cd in
lymphocyte, but not in neutrophil, transmigration. Vice versa, we do not expect that neutrophil migration across CD3 1-expressing fibroblast monolayers would be affected because we show in this report that neutrophil CD3 I. but not CD31 expressed on cellular monolayers, is involved in neutrophil
transmigration. We found that only pre-treatment of neutrophils, and not of endothelial cells, with blocking CD31 mAb inhibited migration. This lack of effect of endothelial cell pre-treatment cannot be due to detachment of mAb during washing of the endothelial cells, because this mAb remained bound to endothelial cells after washing for several days (data not shown). Such a lack of effect has been observed before by Muller el al (34). but these investigators attributed this to presumed difficult) in delivering the mAb to the intercellular junctions w here the lion share of the CD31 molecules are expressed. However, we presume that HEC 170 mAb can readily reach the extracellular molecules in the endothelial intercellular junctions, because this has been shown for other mAbs (35). Moreover, in theory one would expect that in this experimental set-Lip (w ith the mAb present during the transmigration assav ). CD3 1 mAb is able to reach those CD3 I molecules that interact mitiallv w ith neutrophils.
EEC AMI is implicated in Transendotlicliul Migration of Xeiiiroplnls
because these molecules must be reached by neutrophils too. In addition, the finding that transfection of CD3 I into epithelial cells did not affect neutrophil transepithelial migration also points to a modest, if any, contribution of CD31 expressed on cellular monolayers to neutrophil transmigration We therefore conclude that domains I and/or 2 of neutrophil, but not endothelial. PECAM-1 are implicated in neutrophil transendothelial migration in vitro.
Up to date, only the homophilic interaction between endothelial PECAM-1 and neutrophil PECAM-1, has been considered to be invoked in transendothelial migration in vitro and in vivo (8;34). However, our results indicate that this interaction plays a minor role in in-viiro neutrophil transendothelial migration. Instead, an interaction between neutrophil PECAM-1 and an. as yet unknown, ligand on endothelial cells seems to play a role in the migration process. This ligand seems to be absent on epithelial cells, because transepithelial migration was not affected by HEC1 70 mAb. Interaction with this ligand on endothelial cells could lead to activation of the neutrophil and result in enhanced migration. The recent publications that ligation of PECAM-1 on neutrophils results in down-regulation of L-selectin and up-regulation and activation of ß2-integrins(18;36) support this hypothesis.
A potential candidate for such a ligand is <xvß3. This integrin binds to PECAM-1 via its second
lg-like domain ( 13) and is expressed by endothelial cells and not by epithelial cells. However, whether or not HEC170 mAb affects the interaction between PECAM-1 and avß3 is unclear. Moreover, it has
recently been reported that, in rat, PECAM-1 and avß3 integrin play different and independent roles in
neutrophil migration (37). Thus, it cannot be deduced from our data whether or not neutrophil PECAM-1 interaction with endothelial avß3 leads to activation of neutrophil integrins, resulting in stimulated
transendothelial migration.
In contrast to neutrophils, eosinophil transmigration across endothelial monolayers was not blocked by HEC170 mAb. These results were unexpected, because eosinophils express PECAM-1 on their surface to the same extent as found on neutrophils. In eosinophils, the interaction between eosinophil PECAM-1 and endothelial a j i , has been shown to cause activation of eosinophil ß,-integrins, leading to firm adhesion of eosinophils to endothelial cells through a4ß , VCAM-1 interaction (11). In theory, treatment of
eosinophils with HEC170 mAb could affect the eosinophil adhesion and migration in two ways: a) it could mimic the interaction with a ligand by cross-linking PECAM-1 ; b) it could block the interaction of PECAM-1 with a ligand. In practice, we did not observe any effect of HEC170 mAb on eosinophil adhesion and migration, although HEC170 mAb bound to eosinophils to the same extent as to neutrophils. Thus, cither PECAM-1 is not involved in eosinophil transendothelial migration or HEC170 mAb does not affect the molecular interaction involved in a PECAM-1-dependent migration pathway.
The reason why eosinophil adhesion and transendothelial migration is not sensitive to HEC170 treatment is obscure. One major difference between eosinophils and neutrophils is that neutrophils migrate more rapidly and more efficiently across endothelial monolayers. Eosinophil migration ma\ be less sensitive to activation by means of PECAM-1 ligation and thus, the PECAM-1-mediated interaction may be of less importance to the migration process of eosinophils The observation that PECAM-1 plays different roles in neutrophil and eosinophil migration confirms the idea that each cell type is differently equipped to reach its destination.
( 'hapter VI
On the basis o f these results it can be concluded that leukocyte P E C A M - I could be implicated m the specificity o f leukocyte recruitment, i.e. endothelial cells may express a ligand that interacts with P E C A M - 1 and selectively activates the migratory machinery o f neutrophils, but not o f eosinophils, resulting in selective neutrophil extravasation. This finding may be o f importance for the understanding o f neutrophill-mediated inflammation, such as inflammation caused by bacteria and chronic inflammation such as chronic obstructive lung disease (COPD).
Footnote
This study was financially supported by the Netherlands Asthma Foundation (gram no. 32.96.43)
References
1. Springer,T.A. 1994. Traffic signals lor lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76:301.
2. Bianchi.E., Bender,J.R., Blasi.F.. and Pardi.R. 1997. Through and beyond the wall: Late steps m leukocyte transendothelial migration Immunol. Today 12:586.
3. Mantovani.A., Bussolino.F.. and Introna.M. 1997. Cytokine regulation of endothelial cell function: From molecular level to the bedside. Immunol Today 5:231.
4 Mul,F.P.J. / Zuurbier.A.E.M.. Janssen,II., ('alalat.J.. Van Wetering.S., Hiemstra,P.S„ Roos.l)., and Hordijk,P.L. 2000. Sequential migration of neutrophils across monolayers of endothelial and epithelial cells. J.Leukoc.Biol. 68:529.
5 Cines,D.B„ Pollak.E.S.. Buck.C , A „ l.oscalzo.J.. Zimmei man.G.A.. McEver.R.P.. Pober.J.S..
W k k . T . M . . Konkle.B.A.. Schwartz,B.S., Barnathan.E.S.. M e d ae.K.R.. Hug,B.A., S c h m i d t . A . M . . and Stern. D.M. 1998. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91:3527.
6. Rival,Y., Del Maschio.A., Rabiet,M.-J.. Dejana.E.. and Duperray.A. 1996 Inhibition of platelet endothelial cell adhesion molecule-1 synthesis and leukocyte transmigration in endothelial cells by the combined action of TNF-a and IFN-y. J.Immunol 157:1233.
7. Newman.P.J. 1997. The biology of PECAM-1../ Clin.lnvest 99:3.
8. MullerAV.A. and Randolph.G.J. 1999. Migration of leukocytes across endothelium and beyond: Molecules involved in the transmigration and fate of monocytes. J.Leukoc.Biol. 66:698.
9. Berman.M.E. and MullerAV.A. 1995. Ligation of platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31 ) on monocytes and neutrophils increases binding capacity of leukocyte CR3 (CD I lb/CD 18). .1.Immunol. 154:299.
10 Christoildou-Solomidou.M., Nakada.M.T.. Williams.J., MullerAV.A., and DeLisser,H.M. 1997 Neutrophil platelet endothelial cell adhesion molecule-1 participates in neutrophil recruitment at inflammatory sites and is down-regulated after leukocyte extravasation. J.Immunol. 158:4872. 11 C hiba.R.. Nakagawa.N.. kurasawa.k.. Tanaka.Y.. Saito.Y.. and Iwamoto.I. 1999. Ligation of CD31
(PECAM-1 ) on endothelial cells increases adhesive function of avßj integrin and enhances ßi integrin-mediated adhesion of eosinophils to endothelial cells. Blood 94:1319.
12. Ayalon.O., Sabanai.H., Lampugnani.M.G., Dejana.E., and Geiger,B. 1994, Spatial and temporal relationships between Cadherins and PECAM-1 in cell-cell junctions of human endothelial cells. J. Cell Biol. 1:247.
13. Piali.l... Hammel.P.. I herck.C„ Bachmann.F.. Gisler.R.H.. Dunon.D.. and Imhof.B.A. 1995
CD31/PECAM-1 is a ligand for a,ß:, integrin involved in adhesion of leukocytes to endothelium. J.Cell Biol. 130:451.
14. Buckley,CD., Doyonnas.R., Newton.J.P., Blystone.S.D., Brown,E.J.. Watt.S.M., and Simmons.D.L. 1996. Identification o f avß3 as a heterotypic ligand for CD31/PECAM- \. J.Cell Sei. 109:437.
15. Prager.E., Sunder-Plassmann.R.. Hansmann.( '., Koch.f '.. Hotter,W .. Knapp.W.. and Stockinger.H. 1996. Interaction ofCD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses. J.Exp.Med. 184:41.
16. Deaglio.S., M o r r a . M . , Mallone.R.. Ausiello.C.M.. Prager.E.. Garbarino.G., Dian/ani.l .. Stockinger.H.. and Malavasi.F. 1998. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor o f CD31 .an lg
I'F.CAM-I is implicated m Tran\endolhclial Migration of Seitlrophils
1 7. Reedquist.K.A., Ross.F... Koop.F.A.. W'olthuis.R.M.F., Zvvartkruis.FJ.T, Van Kooyk,Y., Salmon.M.. Buckley.C.D., and BosJ.L. 2000. The small G l Pa.sc. kap I. mediates CD31-induced integrin adhesion J.Cell Biol 148:1151
18. Elias IIKC.G., Spellberg,J.P., Karan-Tamir.B.. Lin,C--H., \V'ang,Y.-J., McKenna.P.J., Muller.W.A., Zukovvski.M., and A n d r e « . » . P . 1998. Ligation of CD31 PECAM-1 modulates the function of lymphocytes, monocytes and neutrophils. Eur.J.Immunol. 6:1948.
19. Berman.M.E., Xie.Y., and M u l l t r . W . A . 1996 Roles of platelet endothelial cell adhesion molecule-1 (PECAM- 1. CD31 ) in natural killer cell transendothelial migration and ß2 integrin activation. J.Immunol. 156:1515.
20. Tanaka.Y.. Albelda.S.M., Horgan.K.J., \ an Seventer.G.A., Shimi/.u.Y.. Newman.W., Hallam,.!.. Newman.P.J.. Buck.C.A.. and Shaw.S. 1992. CD31 expressed on distinctive T cell subsets is a preferential amplifier of b\ integrin-mediated adhesion. J.Exp.Med 176:245.
2 I Leavesley.D.l., Oliver.J.M., Swart.B.W., B e r n d t . M . C . Haylock.D.M., and Simmons.P.J. 1994 Signals from platelet/endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34+ hematopoietic progenitor cells. J.Immunol. 10:4673.
22. Bogcn.S.. Pak.J.. Garifallou.M., Deng.X.. and Muller.W.A. 1994 Monoclonal antibody to murine PECAM-1 (CD31 ) blocks acute inflammation in vivo. J.Exp.Med. 179:1059
23. Van MounkJ A.. Leeksma.O.C, ReindersJ.H.. De (iroot.P.G.. and Zandbergen-Spaargaren..!. 1985. Vascular endothelial cells synthesize a plasma membrane protein indistinguishable from the platelet membrane glycoprotein IIa. J.Biol.Clwin. 20:1 1300.
24 Brinkman.H.-J.. Mertcns.K.. Holthuis..!.. Z w a r t - H u i n i n k X . A . , G r i j m . K . . and Van Mourik.J.A. 1994 The actuation of human blood coagulation factor X on the surface of endothelial cells: A comparison with various vascular cells, platelets and monocytes. Br.J.Haematol. 2:332.
25. Fontijn.R.. H o p . C . Brinkman.H.-J.. Slater.R.. Westerveld.A., Van Mourik.J.A.. and Pannekoek.H. I 995. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retro\ irus containing human papilloma virus 16 E6/E7 DNA. Exp.Cell Res. 216:199.
26. Banks-Schlegel,S.P., Gazdar.A.F., and Harris.C. 1985. Intermediate filament and cross-linked envelope expression in human lung tumor cell lines. Cancer Res. 45:1187.
27 L i u . L - Mul.F.P.J.. Kuijpers.T.W., Lutter.R., Roos.D., and Knol.F.F. 1996 Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms. Ann.N) Acad.Sei. 796:21.
28 Roos.D.and De Boer.M. 1986. Purification and cryopreservation of phagocytes from human blood. In: Methods Enzymol. Volume 132. Immunochemical Techniques Part.I Phagocytosis and Cell-Mediated Cytotoxicity (G Di Sabato and J Everse, eds.), Academic Press, New York 225.
29. Koenderman.I... Hamclink.M.I... Kok.P.T.M.. Verhoeven.A.J., and Bruijnzeel.P.L.B. 1988 An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals. J.l.cukoc.Biol. 44:79.
30 Blom.M.. Tool.A.T.J.. Mul.F.P.J.. Knol.F..F., Roos.D., and Verhoeven,A.J. 1995 Eosinophils isolated with two different methods show different characteristics of actuation. J.Immunol.Methods 178:183. 31. Akeson.A.L. and VVoods.C.W. 1993. A fluorimetric assay for the quantification of cell adherence to
endothelial cells. J.Immunol.Methods 2:181.
32. Y a n . H . - C . PilewskiJ.M., Zhang.Q., DeLisser.H.M., Romer.L.. and Albelda.S.M. 1995 Localization of multiple functional domains on human PECAM-1 (CD31 ) by monoclonal antibody epitope mapping. Cell Adhes.Commun. 3:45.
33. Zocchi.M.R.. Ferrero.F., Leone.B.F... Rovere.P.. Bianthi.E.. Toninelli.F.. and Pardi.R. 1996. CD31/PECAM-l-driven chemokine-independent transmigration of human T lymphocytes. Eur.J.Immunol. 26:759.
34. Muller.W.A.. Wtigl.S.A.. Deng.X., and Phillips.!).M. 1993. PECAM-1 is required for transendothelial migration of leukocytes. J.Exp.Med. 178:449.
35 Hordijk.P.I... Anthony.F.. Mul.F.P.J.. Rientsma.R.. Oomen.L.OJ.M.. and Roos.D. 1999 Vascular-endothehal-cadherin modulates endothelial monolayer permeability. J.Cell Sei. 112:1915.
36. Pellegatta.F.. Chierchia,S.L., and Zocchi.M.R. 1998. Functional association of platelet endothelial cell adhesion moleculc-1 and phosphoinositide 3-kinase in human neutrophils. J.Biol.Chem. 273:27768. 37. Thompson.R.D., Wakelin.M.W .. I.arbi.K.Y., Dewar.A., Asimakopoulos.G.. Horton.M.A.,
Nakada.M.T.. and Nourshargh.S. 2000. Divergent effects of platelet-endothelial cell adhesion molecule-1 and ß3 integrin blockade on leukocyte transmigration in vivo. J Immunol. 165:426.
