Physiol Rev
83: 309 –336, 2003; 10.1152/physrev.00023.2002.
Unique Structural Features That Influence
Neutrophil Emigration Into the Lung
ALAN R. BURNS, C. WAYNE SMITH, AND DAVID C. WALKER
Department of Medicine, Section of Cardiovascular Sciences, The DeBakey Heart Center at Baylor College of
Medicine and the Methodist Hospital and Department of Pediatrics, Section of Leukocyte Biology, Baylor
College of Medicine, Houston, Texas; and Department of Pathology, University of British Columbia,
Vancouver, British Columbia, Canada
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I. Introduction
II. Alveolar Wall
A. Cellular anatomy and organization of the alveolar wall
B. Extracellular matrix of the alveolar wall
III. Neutrophil Migration Across Vascular Endothelium
IV. Role of Endothelial Adhesion Molecules in Neutrophil Transendothelial Migration
A. PECAM-1
B. JAMs
C. VE-cadherin (cadherin-5)
D. CD99
V. Neutrophil Migration Across Basement Membrane
A. Role for proteases?
B. Preexisting holes in the basement membrane
VI. Fibroblasts: a Source of Contact Guidance and Stimulation for Neutrophil Migration
VII. Role of Integrins in Neutrophil Migration Through Extracellular Matrix
VIII. Neutrophil Migration Across Alveolar Epithelium
IX. Conclusions and Future Directions
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Burns, Alan R., C. Wayne Smith, and David C. Walker. Unique Structural Features That Influence Neutrophil
Emigration Into the Lung. Physiol Rev 83: 309 –336, 2003; 10.1152/physrev.00023.2002.—Neutrophil emigration in the
lung differs substantially from that in systemic vascular beds where extravasation occurs primarily through
postcapillary venules. Migration into the alveolus occurs directly from alveolar capillaries and appears to progress
through a sequence of steps uniquely influenced by the cellular anatomy and organization of the alveolar wall. The
cascade of adhesive and stimulatory events so critical to the extravasation of neutrophils from postcapillary venules
in many tissues is not evident in this setting. Compelling evidence exists for unique cascades of biophysical,
adhesive, stimulatory, and guidance factors that arrest neutrophils in the alveolar capillary bed and direct their
movement through the endothelium, interstitial space, and alveolar epithelium. A prominent path accessible to the
neutrophil appears to be determined by the structural interactions of endothelial cells, interstitial fibroblasts, as well
as type I and type II alveolar epithelial cells.
I. INTRODUCTION
The migration of neutrophils from blood into tissue
at sites of inflammation has been modeled as a series of
sequential adhesive steps proceeding from tethering on
endothelium under shear conditions in postcapillary
venules (244). This model has been investigated in a
variety of vascular beds (147) and in vitro with monolayers of endothelial cells in parallel plate flow chambers
(244). The tethering event in this model depends on adwww.prv.org
hesion molecules in the selectin family, E-selectin and
P-selectin on the endothelium and L-selectin on the neutrophil, as well as ligands for the selectins expressed on
both cell types. These adhesion molecules are necessary
to efficiently initiate the cascade of adhesive steps ultimately leading to firm attachment of the neutrophils to
the endothelium. Many of the molecular and functional
aspects of this model have been recently reviewed and are
not repeated here (144, 244).
In the lung, a principal site of leukocyte emigration in
0031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
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neutrophils at the alveolar walls (51, 52, 102, 115, 180,
289). The role of mechanical factors in the initial sequestration of neutrophils in the alveolar capillaries is supported by evidence that neither L-selectin nor ␤2-integrins
are required (51, 64, 132). In contrast, both selectins and
␤2-integrins are required for the localization of neutrophils in postcapillary venules at sites of inflammation.
The events following the initial sequestration of neutrophils within alveolar capillary beds are apparently influenced by adhesion molecules. For example, simple
systemic activation of neutrophils by intravenous injection of chemotactic factors [e.g., interleukin (IL)-8 or C5a]
results in rapid (⬍1 min) neutropenia with massive sequestration of neutrophils within alveolar capillaries. This
event is not dependent on L-selectin or ␤2-integrins, but
the retention times within this capillary bed are influenced by these adhesion molecules (51, 132), and this
adhesion is likely an interaction of leukocyte adhesion
molecules and endothelial adhesion molecules. Blockade
of the adhesive mechanism (e.g., using blocking monoclonal antibodies) results in release of neutrophils from the
lungs (51, 58, 59, 64, 158). Mediator-induced decreases in
deformability are temporally correlated with upregulation
of adhesivity of ␤2-integrins (e.g., both occurring within
⬃1 min of exposure to IL-8), allowing both physical trapping and sticking to the vascular wall within the alveolar
capillary bed. A similar phenomenon occurs in the liver
where sequestration apparently does not depend on adhesion, but retention and liver injury are heavily dependent on ␤2-integrins (120).
Pulmonary infection or injury often results in pronounced emigration of neutrophils from these capillary
beds into the alveolar space. Doerschuk et al. (54) demonstrated a number of years ago that this process can
occur through at least two distinct pathways. One pathway
reguires ␤2-integrins, whereas the other does not, and the
distinction depends on the stimulus (54, 96, 126, 137, 180,
185, 203, 205, 234). There is also evidence from studies in
the peritoneal cavity that macrophages are essential for
production of a stimulus that elicits ␤2-integrin-independent neutrophil emigration (173). Furthermore, even
within the pathway requiring ␤2-integrins, 20 –30% of the
migration appears to be independent of ␤2-integrins. Studies of intercellular adhesion molecule (ICAM)-1, a major
ligand for ␤2-integrins, reveal some interesting differences
with regard to the stimuli inducing the pulmonary inflammation (32). In response to intratracheal Escherichia coli
lipopolysaccharide (LPS), a stimulus that induces ␤2-integrin-dependent neutrophil emigration, ICAM-1 expression on capillary endothelial cells and alveolar type II
cells was increased. In response to Streptococcus pneumoniae, a stimulus that induces ␤2-integrin-independent
migration, ICAM-1 was only increased on alveolar type II
cells but not on capillary endothelial cells.
The mechanisms accounting for these apparently dis-
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response to inflammation is the alveolar capillary bed. A
distinctive characteristic of this bed is the complex interconnecting network of short capillary segments where
the path from arteriole to venule crosses several alveolar
walls (often ⬎8) and contains often ⬎50 capillary segments (9, 53, 102, 151, 248). Compared with blood in the
large vessels of most vascular beds, the blood in this
complex network contains ⬃50-fold more neutrophils
and manyfold more lymphocytes and monocytes (56).
Videomicroscopic study (82, 201) of these vessels revealed that the transit of neutrophils through this network
required a median time of 26 s and mean time of 6.1 s. The
increased transit time was due primarily to the time neutrophils spent stopped within this vascular network. In
contrast, the transit times of red blood cells (RBC) ranged
from 1.4 to 4.2 s. The longer time required for the neutrophils to pass through this bed apparently accounts for
their increased concentration.
Recruitment of neutrophils into the lungs through the
alveolar capillary network contrasts with the recruitment
of neutrophils through postcapillary venules at sites of
inflammation in a number of important ways. The tethering mechanisms required to capture neutrophils from
flowing blood in larger vessels are apparently not necessary in the alveolar capillary bed. The diameters of spherical neutrophils (6 – 8 ␮m) are larger than the diameters of
many capillary segments (2–15 ␮m), and ⬃50% of the
capillary segments would thereby require neutrophils to
change their shape to pass through (55, 82, 104, 166).
Given the large number of capillary segments through
which a neutrophil must pass (often ⬎50), most neutrophils must change shape during transit from arteriole to
venule. Morphometric analysis of neutrophils in the alveolar capillary beds revealed significant deviation from
spherical shape (55, 82). Computational models of the
capillary bed describing flow, hematocrit, pressure gradients, and the effects of deformation on the capillary transit times of neutrophils support the concept that the
structure of the capillary bed and the deformation of
neutrophils are critical under normal conditions (110).
During inflammation, much of the sequestration and
infiltration occurs through vessels so narrow that physical
trapping is sufficient to stop the flowing neutrophil (51,
53, 56, 61, 82, 99, 150, 231). A number of investigations
have provided evidence that activation of neutrophils further contributes to the failure of transit through the alveolar capillary bed (63, 91, 92, 106, 115, 187, 194, 199, 278,
290), and changes in mechanical properties after activation may be of major importance. Binding of mediators
such as chemotactic factors (e.g., the complement fragment C5a) to neutrophil receptors induces a transient
resistance of the cells to deformation (27, 35, 60, 62, 69,
115, 289). Because neutrophils must deform to pass
through the capillary bed, leukocyte activation by inflammatory mediators could effect further concentration of
NEUTROPHIL EMIGRATION IN THE LUNG
II. ALVEOLAR WALL
A. Cellular Anatomy and Organization
of the Alveolar Wall
Understanding how neutrophils move from alveolar
capillary lumen to alveolar airspace requires that we
know something about the anatomy and organization of
the lung. For our purposes, the lung can be thought of as
having two parts: the central airways (trachea, bronchi,
and terminal bronchioles) and the peripheral parenchyma
(respiratory bronchioles, alveolar ducts, and alveoli). In
adult human lung, there are 300 million alveoli, and each
alveolus is ⬃0.25 mm in diameter. The walls between
alveoli have a single capillary network that is in contact
with alveolar air on both sides. Each alveolus contains
⬃1,000 capillary segments, and the entire capillary network is connected to 300 million arterial end branches
and an equal number of veins (103, 284). By transmission
electron microscopy, a cross-sectional view of the alveolar wall (Fig. 1) reveals one side of the wall is relatively
thin [ⱕ0.2 ␮m in humans and even less in small mammals
(0.1 ␮m)], consisting of an endothelium joined to an overlying epithelium through a shared basement membrane.
The other side of the wall is much thicker and more
variable in its dimensions depending on the nature of the
interstitial components (extracellular matrix, fibroblasts,
and less frequently mast cells and pericytes) that lie between the capillary endothelium and alveolar epithelium
(284).
In humans, estimates of the alveolar surface area are
impressive at 100 m2 (nearly the size of a tennis court)
with the capillary endothelial surface being smaller, by
⬃10 –20%, than the alveolar surface (284). Two types of
epithelial cells cover the alveolar surface. Type I pneumocytes are squamous epithelial cells that send out extensive cytoplasmic sheets that penetrate pores of Kohn
(holes within the alveolar wall connecting one alveolus to
another) and cover portions of neighboring alveoli. Type
II pneumocytes are cuboidal epithelial cells that synthesize and secrete the pulmonary surfactant lining the alve-
FIG. 1. Transmission electron micrographs of peripheral canine lung showing
the structural organization of the alveolar wall. In A, three capillary segments
(Cp) in cross-section are visible. A fibroblast (Fb) in the alveolar wall shows several cytoplasmic projections extending
into the alveolar interstitium. Note the
minimal tissue barrier between the alveolar space (AS) and capillary lumen in
the thin wall (*) compared with that in
the thick wall (**). A portion of the alveolar wall is enlarged in B. Collagen 8 and
elastin (E) are evident in the interstitium
of the thick wall. The endothelial basement membrane (**) is less compact (i.e,
less electron dense) compared with the
epithelial basement membrane (*). Note
how the fibroblast forms electron-dense
focal adhesions (white arrows) with both
endothelial and epithelial basement
membranes. Bar is 2 ␮m in A and 500 nm
in B.
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tinct pathways for neutrophil emigration from alveolar
capillaries are poorly understood. In vitro studies of neutrophil locomotion indicate that different chemotactic
factors may select different pathways. For example, neutrophil emigration through human umbilical vein endothelial cell monolayers is not blocked by antibodies to ␤2integrins if the stimulus is IL-8 or leukotriene (LT) B4,
but transendothelial migration in response to the chemotactic tripeptide [N-formyl-methionyl-leucyl-phenylalanine
(fMLF)] is ␤2-integrin dependent (179). Clear evidence for
such chemotactic factor selection in vivo has not been
published.
The pathways recruited by different stimuli may also
depend on the route of exposure to the alveolar capillary
bed. For example, unilateral intrabronchial instillation of
hydrochloric acid in rat lungs induces ␤2-integrin-independent sequestration and emigration in alveolar capillaries. Neutrophil sequestration also occurs in the contralateral lung, and the retention of neutrophils there is ␤2integrin dependent (180). Intratracheal E. coli LPS results
in ␤2-integrin-dependent emigration, while intravenous
administration results in sequestration that is ␤2-integrin
independent (54, 69).
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B. Extracellular Matrix of the Alveolar Wall
The extracellular matrix (ECM) of the alveolar septum has two principle components: a basement membrane which underlies alveolar epithelial and capillary
endothelial cells and an interstitial matrix which is maintained by fibroblasts. When viewed by electron microscopy (Fig. 1), the alveolar epithelial basement membrane
(ABM) is organized as follows: immediately beneath the
epithelium lies an electron translucent layer referred to as
the lamina rara externa (LRE). Adjacent to this layer lies
the basal lamina proper, which is referred to as the lamina
densa (LD). The LD contains proteoglycans (perlecan,
heparan sulfate, and chondroitin sulfate), fibronectin, entactin, laminin, and type IV collagen; the globular protein
core of perlecan is embedded in the LD, while its heparan
sulfate side chains are positioned within the LRE. Beneath the LD lies the lamina fibroreticularis (LF) in which
type VII collagen-anchoring fibrils link the LD to the fibrillar (e.g., type III collagen) ECM. In general, the capillary
endothelial basement membrane (CBM) is less compact
than the ABM, and the layer lying between the capillary
lamina densa and the endothelium is referred to as the
lamina rara interna (LRI) (212, 223, 225, 264, 265).
Microdomains exist within the basement membrane
as defined by marked differences in chemical composition. For example, at the ultrastructural level, there are
distinct differences between the ABM and CBM in the
distribution of electron-dense ruthenium red anionic
sites. Based on selective enzyme degradation, ruthenium
red appears to react with heparan sulfate proteoglycan,
and the LRI of the adult rat lung has one-fifth the number
of ruthenium red anionic binding sites compared with the
LRE. Interestingly, this difference is not observed in neonatal rats; hence, the diminished reactivity of the LRI
appears to be an age-acquired phenotype (26, 264, 265). In
the ABM of adult rats and rabbits, differences in microdomains between type I and II cells have been reported.
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Specifically, there are significantly fewer sulfated molecules beneath type II cells (223). In vitro experiments
suggest sulfation of basement membrane components is a
critical determinant of type II cell responses to growth
factors that regulate proliferation and differentiation
(225).
It is generally accepted that alveolar epithelial and
endothelial cells synthesize and maintain the basement
membrane. However, in vitro studies suggest fibroblasts
contribute to the process by reorganizing the collagen
matrix or by supplying soluble factors (79). In vitro studies of neonatal lung mesenchymal cells suggest these cells
may be the primary producers of entactin during lung
development (228) and that extracellular entactin is fairly
stable, a finding consistent with the absence of intracellular staining for entactin in adult rodent tissues (23).
The interstitial matrix of the lung is comprised of a
fiber system (collagen and elastin) embedded within a
proteoglycan gel (Fig. 1). Collagen fibers resist tensile
stresses while elastin provides tissue elasticity and recoil.
Collagen and elastin are embedded in a hydrated porous
proteoglycan gel that resists compressive forces. While
hoops of elastic fibers are prominent around alveolar
openings and alveolar ducts (191, 212), they also form a
reticulum surrounding and enclosing each alveolus (176).
Type III fibrillar collagen is found within alveolar walls
and appears aggregated at the entrance rings to alveolar
ducts while the distribution of type I collagen is more
irregular and less prominent (204).
III. NEUTROPHIL MIGRATION ACROSS
VASCULAR ENDOTHELIUM
Much of what we know about neutrophil transendothelial migration comes from in vivo studies of postcapillary venules in the systemic circulation and from in vitro
models of neutrophil trafficking using endothelial cells
derived from large veins (e.g., umbilical veins). Hence,
generalizations in the literature regarding neutrophil
transendothelial migration may not be appropriate to the
lung. The primary reason for this is because, as discussed
in section I, the principal site for neutrophil emigration in
the lung is the alveolar capillary bed. What follows is an
overview of issues surrounding our current understanding of neutrophil transendothelial migration. Whenever
possible, data gathered from studies in the lung are presented and compared with data obtained in nonpulmonary tissues.
It is generally accepted that neutrophils migrate
across the endothelium by penetrating junctions that lie
within the intercellular cleft. This paradigm is based on
studies of nonpulmonary tissues and originated with
Julius Arnold (1875), who used the injection of cinnabar
to demonstrate the borders of endothelial cells. He sug-
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olar surface (135). Type II cells also possess the capacity
to proliferate and make more type II cells, or differentiate
and replace injured type I pneumocytes (1). The cell
volume of a squamous type I cell is approximately twice
that of a type II cell while its alveolar surface is 50 times
that of a type II cell. Expressed as a percentage of the
total number of cells within the alveolar wall, type I
pneumocytes account for 8%, type II pneumocytes 16%,
capillary endothelial cells 30%, interstitial cells 36%, and
alveolar macrophages 10%. Hence, epithelium and endothelium make up about one-fourth each of the alveolar
wall. Interestingly, the surface covered by one type I cell
is 4,000 –5,000 ␮m2 compared with ⬃1,000 ␮m2 for an
endothelial cell, which means endothelial cells are ⬃4
times more numerous than epithelial cells (284).
NEUTROPHIL EMIGRATION IN THE LUNG
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FIG. 2. Neutrophils migrate preferentially at tricellular corners
where the borders of three endothelial cells converge. In A, human
neutrophils can be seen adhering to and migrating across a human
umbilical vein endothelial cell (HUVEC) monolayer that has been
treated with interleukin-1 (IL-1; 10 U/ml for 4 h). When viewed in the
light microscope, endothelial borders appear black after silver staining
(tricellular corners are indicated with arrows). One neutrophil is sitting
on the body of the endothelial cell (1), another on top of a tricellular
corner (2), and a third is transmigrating at a tricellular corner (3); a
portion of the neutrophil is beneath the monolayer (arrowhead). In B,
immunofluorescence staining for ZO-1 (a tight junction protein) reveals
discontinuous staining at tricellular corners (arrows). C is a scanning
electron micrograph showing a human neutrophil fixed in the act of
migrating across an IL-1-treated HUVEC monolayer. D is a scanning
electron micrograph of a rabbit neutrophil fixed in the act of migrating
across the pulmonary microvascular endothelium after intrabronchial
instillation of Streptococcus pneumoniae (108 organisms, 2 h). In both
panels, the borders of three endothelial cells (1, 2, and 3) converge on
the trailing uropod (tail) of the migrating neutrophil at a tricellular
corner. Bar is 5 ␮m. [A and D from Burns et al. (33), copyright CRC
Press, Boca Raton, FL, 2001.]
ICAM-2 (CD102) is constitutively expressed on vascular
endothelial cells (45) and is a ligand for leukocyte ␤2integrins CD11a/CD18 and CD11b/CD18 (145, 249, 291). In
vitro, ICAM-2 appears to play a role in neutrophil (116)
and eosinophil (83) transendothelial migration. However,
prolonged (24 h) exposure of HUVEC monolayers to IL-1␤
or tumor necrosis factor (TNF)-␣ decreases ICAM-2 surface expression by 50%, and its association with cell
borders is lost (170). In our laboratory, loss of ICAM-2
from HUVEC borders is evident as early as 4 h after
stimulation with IL-1␤ (Burns, unpublished observations).
Hence, if ICAM-2 participates in neutrophil arrest on cell
borders, its actions may be limited to the initial phase of
an inflammatory response.
Does ICAM-2 play a role in neutrophil emigration in
the lung? Based on light microscopic immunohistochemistry of mouse lung, Gerwin et al. (83) reported ICAM-2
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gested leukocytes move across blood vessel walls (diapedesis) by passing between endothelial cells at either
points of dense staining, “stigmata,” or circles of stain,
“stomata” (13). Nearly 100 years later, with increased
resolution, transmission electron mirographs taken by
Marchesi and Florey seemed to confirm Arnold’s observation that interendothelial junctions were the main sites
through which neutrophils (and other leukocytes) pass
from blood to tissue (162, 163). Since firmly adherent
neutrophils can migrate quickly (⬍2 min) across the endothelium (29, 250), it has been suggested that endothelial
junctions must open and close rapidly during the migration process (39).
Cytokine-activated human umbilical vein endothelial
cell (HUVEC) monolayers are a commonly used in vitro
model for studying neutrophil trafficking (29, 31, 50, 108,
245). In a manner similar to Arnold’s use of cinnabar (13),
we have used silver staining to reveal the presence of
endothelial borders as an aid in determining the site of
neutrophil diapedesis (Fig. 2). We find that ⬎70% of migrating neutrophils cross the endothelium at tricellular
corners where the margins of three endothelial cells converge (29). Under physiological flow conditions (2 dyn/
cm2), rolling neutrophils arrest (become firmly adherent)
on cytokine-activated endothelium, and the distance an
arrested neutrophil moves before it migrates across the
endothelium is only 5.5 ⫾ 0.70 ␮m (under static conditions in the absence of flow, the distance is similar 6.8 ⫾
0.9 ␮m) (89). This distance is less than one neutrophil cell
diameter, suggesting the surface properties of endothelial
borders and tricellular corners favor neutrophil arrest.
Using immunofluorescence microscopy, we (29) and
others (18, 107) have noticed gaps (0.25–2 ␮m across) in
endothelial border staining patterns for tight junction proteins (occludin, ZO-1, and ZO-2) at tricellular corners (Fig.
2). Assuming an upper gap value of 2 ␮m for each corner
and 5 corners/endothelial cell, tricellular corners comprise ⬍10% of the available border area. Because neutrophil transmigration on IL-1␤-treated HUVEC monolayers
occurs exclusively at cell borders, mathematical probability predicts that ⬍10% of the neutrophils should migrate at tricellular corners if the process is random (29).
This value is significantly less than observed values
(⬃70%), suggesting neutrophil migration is not a random
process. Moreover, a comparison across species (human,
dog, and mouse) reveals the preference for tricellular corners is widespread (77, 64, and 47%, respectively). Although the percentage of neutrophils migrating
at corners is significantly different between species
(human ⬎ dog ⬎ mouse), in each case the number migrating at corners greatly exceeds that predicted by random chance (i.e., ⬍10%) (33).
With respect to leukocyte emigration, ICAM-2 and
P-selectin (CD62P) are the only adhesion molecules to
show preferential expression on endothelial borders.
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FIG. 3. Transmission electron micrograph of a canine neutrophil
(PMN) within a capillary segment (Cp). Note how the neutrophil has
deformed and assumed an elongated “sausage” shape to pass through
the narrow capillary segment. Two type II pneumocytes (*) can be seen
on the thick side of the alveolar wall. Bar is 2 ␮m.
larger vessels in a rabbit model of streptococcal pneumonia (Fig. 2). Of 28 neutrophils observed in the process of
transmigration, 14 (50%) were observed crossing at tricellular corners, 7 (25%) at bicellular borders, and 7 (25%)
were crossing transcytotically (i.e., through the endothelial cytoplasm) (33). Unfortunately, the small size and
segmented nature of the capillary bed makes it difficult to
determine the site of neutrophil migration using SEM.
However, using transmission electron microscopy and
serial section reconstruction of pulmonary capillaries, we
reconstructed the migration paths of five neutrophils
(275). Three were migrating at capillary tricellular corners, one was migrating between two endothelial cells,
and another was migrating transcytotically through the
endothelial cytoplasm.
Again, as found in vitro using HUVEC monolayers,
the marked preference for tricellular corners (⬃50% in
large vessels and ⬃60% in capillaries) in the pulmonary
circulation is far greater than that predicted by chance
(⬍10%). Importantly, freeze-fracture electron microscopy
shows that alveolar capillary endothelial tight junctions
are discontinuous at tricellular corners (276). We made
similar observations in our HUVEC monolayers (31),
which leads us to hypothesize that preferential migration
at tricellular corners allows neutrophils to pass around
rather than through endothelial tight junctions. While
early studies suggested neutrophil transendothelial migration is associated with widespread proteolytic degradation of endothelial junctions (5, 50), subsequent studies
established that widespread degradation is a technical
artifact and unrelated to the migratory process (31, 177)
(see sect. IV).
In the absence of proteolysis, lateral displacement of
endothelial junctional complexes (tight and adherens
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staining in alveolar capillaries and large pulmonary vessels. Using an ovalbumin model of allergic inflammation,
they found that mice deficient in ICAM-2 showed prolonged eosinophil accumulation in the lung interstitium.
Although this finding reveals an essential role for ICAM-2
in eosinophil emigration during allergic inflammation,
similar studies with neutrophils are lacking and will require further investigation.
In vitro, using HUVEC monolayers, it is very clear
that P-selectin can play an important role in recruiting
neutrophils to endothelial borders (30). Although not expressed constitutively on the endothelial surface, P-selectin is stored in endothelial cytoplasmic granules known as
Weibel-Palade bodies. Histamine and thrombin trigger
rapid (minutes) fusion of Weibel-Palade bodies with the
endothelial surface. This results in preferential P-selectin
upregulation along endothelial borders where, under
physiological flow conditions, it mediates neutrophil rolling on endothelial borders. Of significance to the lung,
scanning electron microscopic observations of large pulmonary vessels show that after histamine infusion, leukocytes are captured along endothelial borders (30). This is
consistent with intravital observations showing selectinmediated leukocyte rolling in pulmonary arterioles and
venules (134). However, neutrophils do not roll in capillaries (134), the principal site of neutrophil emigration,
and current evidence does not favor a role for P-selectinmediated capture in pulmonary capillaries. Using confocal microscopy, Yamaguchi et al. (292) report that Pselectin is absent from rat pulmonary microvessels.
However, this result should be interpreted with caution
since the level of P-selectin expression may be so low as
to exceed the limit of immunohistochemical detection.
Weibel-Palade bodies have been observed in alveolar capillaries of rats (125) and humans (279), but they appear to
be far more numerous in larger pulmonary vessels (236).
Whether capillary Weibel-Palade bodies contain enough
P-selectin that can be mobilized to the endothelial surface
to influence neutrophil localization at borders remains to
be determined.
With the exception of one report, precise information
concerning the site of neutrophil migration across the
alveolar capillary endothelium is lacking. Moreover, the
conventional model of neutrophil tethering, rolling, and
arrest on inflamed endothelium is not applicable to lung
capillaries. Approximately 50% of the capillary segments
are narrower than the mean diameter of a spherically
shaped neutrophil. This size discrepancy forces neutrophils to deform and assume a “sausage” shape as they
enter and squeeze through narrow capillary segments (55,
102) (Fig. 3). For this reason, in the pulmonary circulation, neutrophil tethering and rolling on inflamed endothelium is restricted to larger diameter pulmonary arterioles and venules (133, 134, 136). We have used scanning
electron microscopy (SEM) to examine some of these
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The inability of ICAM-1 or PECAM-1 to induce an
increase in [Ca2⫹]i may relate to the type of antibody used
or the species/tissue being examined. In a separate study,
endothelial calcium increases were detected after antibody ligation of PECAM-1, even with Fab fragments, and
the authors noted the effect was specific for certain antibodies (93). In vitro experiments with rat brain microvessel endothelial cells show cross-linking ICAM-1 results in
Rho activation, phosphorylation of cytoskeletal proteins
and transcription factors, and ICAM-1 association with
the cytoskeleton (11, 70). Similarly, in cultured human
pulmonary microvascular endothelial cells treated with
TNF-␣ for 24 h, ICAM-1-dependent neutrophil adhesion or
ICAM-1 cross-linking induces cytoskeletal remodeling
and phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK). Interestingly, in this model,
the majority (60%) of adherent neutrophils localize to
endothelial cell borders, and this response is attenuated
by pretreating the endothelium with SB203580, an inhibitor of p38 MAPK (280 –282). Although this finding suggests ICAM-1 plays a role in regulating neutrophil migration toward endothelial junctions, one must keep in mind
that these are cultured cells treated with TNF-␣ for 24 h to
increase ICAM-1 surface expression. We know from ultrastructural immunogold studies in mice that pulmonary
capillary endothelial cells express very little constitutive
ICAM-1 (32, 121). In humans, ICAM-1 is not detectable on
alveolar capillaries by immunohistochemical staining; it is
detectable on larger veins and arteries (75). Moreover, in
mice, 4 h after airway instillation of S. pneumoniae, large
numbers of neutrophils are present in the alveolar airspace, yet capillary endothelial ICAM-1 surface expression is not increased (32). In rabbits, 2 h after instillation
of S. pneumoniae, 75% of the migrating neutrophils adhere and crawl through endothelial borders (33). Although the level of ICAM-1 expression on rabbit pulmonary capillaries was not determined, there is no reason to
think it will be different from that in mice and humans.
The fact that neutrophils can migrate into alveolar space
using ICAM-1-independent mechanisms should not be
taken to imply that ICAM-1 signaling is not important;
rather, ICAM-1 is unlikely to be the only mechanism for
recruiting neutrophils to endothelial borders.
Endothelial cytoskeletal rearrangements appear to
be necessary for leukocyte transmigration. In a study of
human microvascular endothelial monolayers, disruption
of endothelial microfilaments by cytochalasin B or latrunculin A inhibits monocyte transmigration (128). On
HUVEC monolayers, inhibition of myosin light-chain kinase (MLCK) diminishes neutrophil transmigration and
inhibits endothelial F-actin formation, myosin filament
formation, and myosin light chain (MLC) phosphorylation
(101, 222). As mentioned in the preceding paragraph,
neutrophil adhesion to human pulmonary microvascular
endothelial cells (HPMVEC) treated with TNF-␣ also
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junctions) would seem necessary for neutrophil diapedesis to occur at tricellular corners. Freeze-fracture observations on pulmonary capillaries show the size of the
tricellular pore created by the tight junction discontinuity
is only 27 nm in diameter (276). Transmission electron
microscopic observations suggest that neutrophils migrate through pores that are 1–2 ␮m wide (31, 163). From
in vitro and in vivo studies (33), it is also clear that a
significant fraction (25–30%) of neutrophils migrates between pairs of adjacent endothelial cells (i.e., bicellularly). Even though tight junction fibrils between pairs of
adjacent endothelial cells can be discontinuous, gaps between these fibrils are typically too small (3– 6 nm) to
allow neutrophils to pass (237, 238). Hence, we hypothesize that lateral displacement of junctional complexes is
necessary to create a pore wide enough (1–2 ␮m) to
accommodate a transmigrating neutrophil.
There is good evidence that tight junctions can slide
within the membrane. In a study of mechanically stretched
trachea, freeze-fracture electron microscopy shows the
height of the epithelial tight junction complex shortens by as
much as 25%, and small gaps appear in the fibrils (33).
Moreover, during an acute inflammatory response to cigarette smoke exposure, plasma exudate expands tracheal
epithelial tricellular corners, seemingly driving the tight
junctions apart (33). The idea that junctional complexes
“slide” apart during neutrophil diapedesis gains support
from studies on VE-cadherin, a transmembrane protein involved in the formation of endothelial adherens junctions.
Shaw et al. (232), using HUVEC monolayers transfected with
green fluorescent protein-tagged VE-cadherin and real time
imaging, obtained compelling evidence that adherens junctions open and close (much like a curtain) during leukocyte
diapedesis. VE-cadherin and its role in leukocyte transendothelial migration are described in detail below (see sect. IVC).
Displacement of junctional complexes may require
the neutrophil to signal the endothelium. Neutrophil adhesion is known to induce a coordinate increase in endothelial cytosolic free calcium, phosphorylation of
serine-19 and threonine-18 on endothelial myosin regulatory light chains, and isometric tension generation by
endothelial monolayers (80, 101, 109, 222). Neutrophilmediated outside-in endothelial signaling likely involves
the same molecules that mediate neutrophil adhesion to
the endothelial surface. On HUVEC monolayers (pretreated with LPS or histamine to upregulate surface adhesion molecule expression), monoclonal antibodies directed against E-selectin, P-selectin, and vascular cell
adhesion molecule (VCAM)-1 induce transient increases
in intracellular Ca2⫹ concentration ([Ca2⫹]i) and alterations in F-actin distribution. Interestingly, antibody
ligation of platelet/endothelial cell adhesion molecule
(PECAM)-1 or ICAM-1 appeared to have no effect, not
even after application of a secondary antibody to induce
cross-linking (156).
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Physiol Rev • VOL
FIG. 4. Transmission electron micrograph of a canine alveolar capillary (Cap) showing numerous caveolae (arrows) within the endothelial
cytoplasm. As, airspace. Bar is 500 nm.
detected within 15–30 min (81, 255). The localized release
of VEGF at sites of firm neutrophil adhesion would provide specificity to the process by restricting caveolar fusion and transendothelial pore formation in the vicinity of
the neutrophil. While the mechanism remains speculative,
the circumstantial evidence is compelling and warrants
further investigation.
IV. ROLE OF ENDOTHELIAL ADHESION
MOLECULES IN NEUTROPHIL
TRANSENDOTHELIAL MIGRATION
Although much is known about the role adhesion
molecules play in sequestering neutrophils in pulmonary
capillaries, much less is known about their role in regulating neutrophil migration across the endothelium. Most
studies focus on the importance of interendothelial clefts
as primary routes for neutrophil migration. Support for
this notion stems from findings that PECAM-1 (CD31),
junctional adhesion molecules (JAMs), VE-cadherin, and
CD99 localize to interendothelial clefts and antibodies
raised against these molecules can inhibit (anti-PECAM-1,
anti-JAM-1, anti-CD99) or enhance (anti-VE-cadherin) leukocyte transendothelial migration (4, 17, 49, 90, 140, 141,
154, 167, 181, 183, 184, 195, 226). Although these studies
suggest these molecules function as “gate keepers” regulating neutrophil transendothelial migration, specific studies addressing their role in neutrophil emigration in the
lung are scarce.
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leads to endothelial cell stiffening and F-actin reorganization. Interestingly, unlike HUVEC monolayers, endothelial
stiffening does not appear to rely on MLCK or MLC phosphorylation (280). This may reflect phenotypic differences
between HUVEC and HPMVEC. Alternatively, activation
of MLCK and MLC phosphorylation is triggered by neutrophil transendothelial migration but not adhesion to the
endothelial surface. In any event, it is clear that adhesive
interactions between leukocytes and endothelial cells
trigger endothelial cytoskeletal changes. Because tight
junction transmembrane proteins (occludin and claudin)
and adherens junction transmembrane proteins (VE-cadherin) are anchored to the cytoskeleton through accessory cytoplasmic linker proteins (e.g., ZO-1, ZO-2,
catenins) (78, 117, 118, 252, 253), current evidence suggests outside-in signaling and cytoskeletal remodeling following endothelial adhesion molecule ligation is a mechanism for displacing endothelial junctions.
A puzzling observation from the streptococcal pneumonia studies (33, 275) is that a significant fraction (⬃20% in
capillaries and ⬃25% in larger pulmonary vessels) of neutrophils migrate directly through endothelial cytoplasm (i.e.,
transcytotically). Studies in skin (73, 105) and spinal tissue
(157) also document a transcytotic route for neutrophils in
venules which in one case (73) appears to be the sole route
across the endothelium. The regulatory mechanism(s) behind transcytotic migration in the lung (and elsewhere) is
unknown. However, neutrophil-derived vascular endothelial
growth factor (VEGF) may play a critical role in determining
whether neutrophils emigrate through interendothelial clefts
or transendothelial pores.
While VEGF functions as an endothelial growth and
survival factor, it also increases endothelial permeability
(189). Topical administration or subcutaneous injection
of VEGF in animals induces endothelial gaps (transcellular pores) and increased permeability within 1–10 min (72,
213). Pores large enough to accommodate red blood cells
and platelets have been described (72). The mechanism
behind VEGF-induced gap formation may be related to
caveolar fusion (74). Endothelial caveolae are micropinocytotic vesicles ⬃60 nm in diameter. Dvorak and colleagues (66, 267) believe fusion between adjacent caveolae gives rise to larger structures known as vesicuolovacuolar organelles (VVOs). The high-affinity VEGF
receptor-2 (FLK-1, KDR) localizes to caveolae (74) and
VVOs (71). In response to VEGF, transcytotic increases in
macromolecular permeability are mediated through
caveolae (74), and transendothelial membrane-lined
pores develop through VVO fusion with the apical and
basal endothelial surfaces (66). These observations are
important and relevant to neutrophil migration in the lung
for two reasons. First, pulmonary capillary endothelial
cells contain large numbers of caveolae (95, 220) (Fig. 4).
Second, ⬎70% of human neutrophil VEGF localizes to
specific granules and, after stimulation, VEGF secretion is
NEUTROPHIL EMIGRATION IN THE LUNG
A. PECAM-1
Physiol Rev • VOL
tibody ligation, a situation that potentially mimics neutrophil PECAM-1 engagement with endothelial PECAM-1
(68). Hence, it is not clear if sufficient PECAM-1 remains
on the neutrophil surface to engage with endothelial PECAM-1 and form an effective zipper during neutrophil
transendothelial migration.
There is only one published study demonstrating a
role for PECAM-1 in neutrophil emigration in the lung. In
a rat model of IgG immune complex deposition, neutrophil emigration into the alveolar airspace was inhibited
75% using a polyclonal rabbit antibody to human PECAM-1 which recognizes rat PECAM-1 (266). Although
this study did not determine the site (endothelium or
basement membrane) at which migration was inhibited,
in vitro studies show this antibody blocks human neutrophil transmigration at the level of the endothelium (184).
However, this does not mean we can conclude PECAM-1
is important for neutrophil migration across the alveolar
capillary endothelium. In a separate study in rat mesentery activated by IL-1␤ (4 h), the same rabbit polyclonal
antibody had no effect on neutrophil transendothelial
migration, but migration across the venular basement
membrane was inhibited by 50% (269). Hence, while antibody inhibition of PECAM-1 can interfere with neutrophil emigration in the lung, the role PECAM-1 plays in
neutrophil migration across the alveolar capillary endothelium and basement membrane remains unclear and
more studies are needed.
Given the ability of antibodies and soluble recombinant PECAM-1 to inhibit leukocyte emigration, it was
surprising to find that mice with a targeted deletion in
PECAM-1 show only a mild and transient defect in leukocyte emigration. Specifically, neutrophil and monocyte
migration across inflamed endothelium appears normal,
whereas neutrophils, but not monocytes, experience a
delay in crossing the basement membrane (65, 260). The
delay appears to be temporary as neutrophil emigration
into the peritoneal cavity is reduced at 4 h after IL-1
stimulation, but not at 24 h (65). The delay also appears to
be cytokine specific, since it occurs in response to IL-1␤
but not TNF-␣ (260). In an attempt to explain discrepancies between PECAM-1 inhibition studies and PECAM-1
knock-out studies, it has been argued that PECAM-1deficient mice exhibit compensatory changes in adhesion
molecule usage. Normally, PECAM-1 blockade inhibits
leukocyte transendothelial migration by 80 –90%. Hence,
molecules and mechanisms accounting for residual migration (10 –20%) might be amplified in the knock-out
animal. Alternatively, some unknown molecule(s) mediate leukocyte transendothelial migration in the knock-out
(182). If a compensatory change exists, it must account
for the temporary nature of the delay at the basement
membrane, explain why neutrophils and not monocytes
experience this enhanced delay, and provide insight into
why this delay is cytokine specific. Until discrepancies
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PECAM-1, a 130-kDa transmembrane protein and
member of the immunoglobulin (Ig) superfamily, is expressed on endothelial cells, leukocytes, and platelets. In
human placental blood vessels, ultrastructural immunogold labeling showed PECAM-1 localizes to basolateral
membranes of interendothelial clefts (17). The extracellular region of PECAM-1 is organized into six globular
domains that engage in homophilic interactions (3) as
well as heterophilic interactions with glycosaminoglycans
(GAGs) (283). A signaling function for PECAM-1 is suggested by recent studies showing it becomes phosphorylated on tyrosine residues Y663 and Y686 and associates
with at least four cytoplasmic signaling molecules (SHP-1,
SHP-2, SHIP, and phospholipase C-␥1) (98, 119, 202).
PECAM-1 may also influence the endothelial cytoskeleton
as recent data suggest it functions as a scaffolding molecule for ␤- and ␥-catenins, accessory cytoplasmic molecules that link transmembrane proteins to the cytoskeleton (114).
The evidence that PECAM-1 plays a critical role in
leukocyte emigration originated with the observation that
anti-PECAM-1 antibody or soluble recombinant PECAM-1
blocks monocyte and neutrophil transendothelial migration (184). Subsequent studies established that globular
domains 1 and 2 are necessary for leukocyte migration
across inflamed endothelium while domain 6 regulates
migration across the subendothelial basement membrane
(148, 149). It has been suggested that homophilic interactions between leukocyte PECAM-1 and endothelial PECAM-1 may occur during transendothelial migration. This
“zipper” hypothesis proposes that endothelial permeability is preserved during leukocyte migration because as the
leading edge (pseudopod) of the neutrophil unzips interendothelial junctions, junctional contacts are replaced by
endothelial PECAM-1 interactions with neutrophil PECAM-1 (250). Although this is an attractive hypothesis, it
assumes a sufficient level of PECAM-1 on the neutrophil
surface exists to engage in homophilic interactions with
the endothelium. Resting neutrophils express relatively
low levels of PECAM-1 compared with the high levels
(⬎106 copies/cell) found on endothelial cells (48). In a
pertioneal model of inflammation in mouse, PECAM-1 is
downregulated on emigrated neutrophils. In vitro, mouse
neutrophil migration across TNF-␣-treated (4 h) HUVEC
monolayers is associated with a loss of PECAM-1 from the
neutrophil surface, and PECAM-1 downregulation appears to require more than just chemotactic stimulation,
since neither TNF-␣ nor fMLF alone altered PECAM-1
expression (36). The latter observation contrasts with a
separate study on human neutrophils showing treatment
with IL-8, fMLF, or phorbol 12-myristate 13-acetate (PMA)
alone downregulates PECAM-1 surface expression. PECAM-1 downregulation was also seen after PECAM-1 an-
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between PECAM-1 inhibition and deletion are resolved,
the certainty with which we attribute a role for PECAM-1
in leukocyte transendothelial migration must be questioned.
B. JAMs
1. JAM-1
In mice, JAM-1 localizes to epithelial cells, endothelial cells, and megakaryocytes. It received its name based
on its predominant expression in epithelial and endothelial intercellular clefts. In humans, JAM-1 localizes to
epithelial and endothelial cells as well as hematopoietic
cells (neutrophils, monocytes, lymphocytes, and erythrocytes) (161, 192, 288). Comparisons between human
JAM-1 and mouse JAM-1 show 68% identity and 79% similarity over the entire protein amino acid conservation in
the putative transmembrane, and cytoplasmic tail region
is 84% identical and 92% similar (288). A functional role in
maintaining epithelial and endothelial barrier function
was suggested by the ultrastructural immunogold finding
that JAM-1 is positioned apically within the epithelial
intercellular cleft of mouse duodenum, colocalizing with
tight junctions (167). Comparable immunogold studies of
endothelial cells are lacking.
While dual label confocal microscopy and optical
sectioning suggest JAM-1 colocalizes with endothelial
tight junction proteins (AF-6 and cingulin) (67, 167), these
findings must be interpreted with caution. The practical
Physiol Rev • VOL
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JAMs belong to a novel subset of Ig molecules that
belong to the larger cortical thymocyte Xenopus (CTX)
family (16). The JAM family has three members (JAM-1,
JAM-2, and JAM-3), and full-length amino acid sequences
for these molecules suggest they exist as transmembrane
proteins with two globular extracellular domains, a single
membrane-spanning segment, and a short intracellular
tail. Simultaneous discovery of these molecules by different laboratories has resulted in confusing JAM nomenclature when comparing human and mouse systems. Presently, mouse JAM-1 is equivalent to human JAM-1, mouse
JAM-2 is the homolog of human JAM-3, and mouse JAM-3
is equivalent to human JAM-2, which is also known as
VE-JAM-2 (16). After transfection into Madin-Darby canine kidney (MDCK) II cells, JAM proteins are enriched at
cell-cell borders of transfected cells and not at borders of
transfected and nontransfected cells, suggesting JAMs
engage in homophilic adhesive interactions (16, 186). At
least for JAM-1, homophilic adhesion appears to involve
JAM-1 cis-dimerization and trans-interaction of the NH2terminal domains with JAM-1 dimers on the adjacent cell
(19, 46, 130). What follows is a description of each JAM
member and a determination as to whether it might play
a role in neutrophil emigration in the lung.
resolution limit for the confocal microscope in the z-axis
is 0.5 ␮m, and endothelial cell borders can be as thin as
0.2 ␮m (31). Hence, while merged fluorescence images of
thin samples (e.g., endothelia) give the appearance of
colocalization, in reality the molecules may be separated
along the z-axis by as much as 0.5 ␮m. Accurate spatial
localization of JAM-1 within the interendothelial cleft will
require ultrastructural immunogold techniques. The anticipated result is JAM-1 will localize to endothelial tight
junctions, since biochemical findings from immunoprecipitation and glutathione S-transferase (GST) pull-down
experiments (using epithelial cells and endothelial cells)
show the cytoplasmic PDZ-binding domain of JAM-1 interacts with junctional proteins ZO-1 and AF-6 (20, 67).
By Northern blot analysis, JAM-1 expression is high
in murine lung tissue. At the light microscope level, frozen
sections of human lung show JAM-1 staining in both
bronchi and alveolar epithelial cells (154). Although staining in pulmonary capillaries was not reported, reliable
discrimination of epithelial and endothelial JAM-1 staining is difficult at the level of the light microscope, owing
to the thinness (ⱕ0.2 ␮m) of the alveolar wall and juxtapositioning of epithelial and endothelial cells. Clearly, this
is another example where immunogold electron microscopy studies are needed to determine whether alveolar
capillary endothelial cells express JAM-1 and if it localizes
to the intercellular cleft.
Studies showing human JAM-1 localization to endothelial cells and leukocytes raise the possibility that homophilic interactions between endothelial and leukocyte
JAM-1 may facilitate neutrophil transendothelial migration. The observation that JAM-1 remains on the neutrophil surface after activation with interferon (IFN)-␥, LPS,
or formyl peptide (288) is consistent with the idea that it
might engage endothelial JAM-1 during diapedesis. While
mouse leukocytes do not express JAM-1, anti-murine
JAM-1 monoclonal antibody, BV11, inhibits spontaneous
and chemokine-induced [monocyte chemoattractant protein (MCP)-1 or MCP-3] human monocyte migration
across cultured mouse endothelial monolayers. BV11 also
inhibits human monocyte migration across LPS-treated
cultured mouse endothelium (167). In vivo, BV11 partially
inhibits monocyte emigration in a mouse model of skin
inflammation (167) and monocyte and neutrophil emigration in a mouse model of cytokine-induced meningitis
(49). Interestingly, BV11 did not inhibit leukocyte influx in
a mouse model of infectious meningitis, implying fundamental differences in the mechanisms underlying leukocyte migration in the two models (i.e., cytokine-induced
vs. organism-induced inflammation) (142). Hence, a role
for JAM-1 in neutrophil emigration may vary depending
on the nature of the inflammatory insult. In certain inflammatory settings, neutrophil diapedesis may be JAM-1 independent.
A new level of complexity was added to the JAM-1
NEUTROPHIL EMIGRATION IN THE LUNG
Physiol Rev • VOL
likely related to hypoxic pulmonary vasoconstriction (88,
100, 152), it remains to be determined whether, under
conditions of reduced or no blood flow, redistribution of
JAM-1 or PECAM-1 away from interendothelial clefts is a
mechanism for downregulating neutrophil emigration in
the lung.
2. JAM-2 and JAM-3
In mice, Aurrand-Lions et al. (15) identified a novel Ig
superfamily molecule whose expression was restricted to
endothelial cells, particularly endothelial cells in high
endothelial venules, lymphatic vessels in lymphoid organs, and vessels within the kidney. The molecule was
termed JAM-2 because it shares 31% sequence identity
with JAM-1 at the protein level. Moreover, it localizes to
cell-cell contacts and decreases paracellular permeability
when transfected into Chinese hamster ovary cells. When
transfected into epithelial cells (MDCK) expressing tight
junctions, immunofluorescence staining for JAM-2 codistributed with ZO-1 and occludin (15). Simultaneously, in
another laboratory, Arrate et al. (14) cloned human
JAM-3, a novel Ig superfamily molecule with 32% identity
to human JAM-1 at the protein level. By Northern analysis,
human JAM-3 is present in lung tissue, but the highest
levels occur in kidney, brain, and placenta (14). Arrate et
al. (14) suggest mouse JAM-2 isolated by Aurrand-Lions et
al. (15) is the homolog of human JAM-3. If this is the case,
then differences in tissue distribution exist between species because mouse JAM-2 is poorly expressed in lung and
absent in brain. Hence, based on tissue distribution, although there may be a role for human JAM-3 in neutrophil
emigration in the lung, a role for murine JAM-2 seems
unlikely.
During this time, human JAM-2 was isolated and its
protein sequence found to be 35% identical to human
JAM-1 and mouse JAM-1 (41). Northern blot analysis revealed human JAM-2 is highly expressed in heart and
poorly expressed in lung. Human JAM-2 shares 36% protein sequence homology with human JAM-3, and its sequence is identical to another human molecule, VE-JAM,
which localizes exclusively to endothelial borders and is
expressed prominently in high endothelial venules (HEV)
(195). Subsequently, mouse JAM-3 was identified and the
coding sequence suggested it was the mouse equivalent of
human JAM-2 (16). Mouse JAM-3, like human JAM-2, is
poorly expressed in lungs and shows stronger expression
in heart (16). Hence, given the poor levels of expression
for human JAM-2 and mouse JAM-3 in lung, these molecules seem unlikely to play important roles in neutrophil
emigration from the pulmonary microvasculature. In humans, a more likely role for JAM-2 is in lymphocyte
trafficking across HEV, since human JAM-2 is a counterreceptor for human JAM-3 and activated lymphocytes
express human JAM-3 (14). A similar situation may exist
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story when Ostermann et al. (192) reported that human
JAM-1 is a heterophilic ligand for the leukocyte ␤2-integrin CD11a/CD18 (LFA-1). Deletional analysis suggested
the membrane proximal domain 2 of JAM-1 was responsible for adhesive interactions with LFA-1. Using blocking
antibodies, the authors found IL-8-stimulated neutrophil
migration across resting HUVEC monolayers was largely
mediated by LFA-1 (80%) and ICAM-1 (45%). Pretreatment
of HUVEC monolayers with anti-JAM-1 alone or in combination with anti-ICAM-1 reduced neutrophil migration
by ⬃60%. Because LFA-1 binds to the membrane proximal
domain 2 region of JAM-1, there is a possibility for homophilic interactions between endothelial JAM-1 domain
1 and neutrophil JAM-1 domain 1. Hence, complex interactions between homophilic binding of endothelial JAM-1
to endothelial JAM-1, heterophilic binding of neutrophil
LFA-1 to endothelial JAM-1 and homophilic binding of
neutrophil JAM-1 and endothelial JAM-1 may provide another molecular zipper for transendothelial migration
(192).
Finally, both JAM-1 and PECAM-1 have been suggested to also play negative roles in leukocyte transendothelial migration. Endothelial cultures treated for 8 –24 h
with a combination of TNF-␣ and IFN-␥ show substantial
loss of these molecules from the intercellular cleft; treatment with either cytokine alone has little or no effect.
While the bulk of the evidence suggests the loss is due to
redistribution from cleft to cell surface (193, 215), internalization and decreased synthesis may also be involved
(211). Conceivably, loss of JAM-1 and PECAM-1 cleft
molecules may shut down the inflammatory response by
preventing leukocyte migration. Indeed, using static assays, where neutrophils are allowed to settle and adhere
to endothelial cells in the absence of flow, a marked
reduction in neutrophil (and monocyte) migration across
TNF-␣- and IFN-␥-treated HUVEC monolayers has been
noted (211, 233). However, this reduction was not seen
when the assay was performed under flow conditions (1
dyn/cm2) (233). Surprisingly, under flow conditions, antibody against PECAM-1 inhibits monocyte transmigration
by 66% when the monolayer is costimulated with TNF-␣
and IFN-␥ (no data given for neutrophils) (233). This
observation suggests PECAM-1 participates in transmigration even when not associated with interendothelial
clefts, indicating a potential signaling function (190). The
issue of flow and its ability to affect neutrophil transmigration is an interesting consideration for the lung. Because leukocyte emigration occurs preferentially in alveolar capillaries, flow may be temporarily reduced or
absent in select capillary segments as marginated neutrophils deform to pass through the microvasculature (102).
Moreover, in a rabbit model of streptococcal pneumonia,
this effect is enhanced as capillary blood flow to the
pneumonic regions decreases 20 –50% over the first 8 h
(57). Although the mechanism of reduced blood flow is
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in the mouse, since HEVs express all three JAM family
members (16) and mouse JAM-3 is a counterreceptor for
mouse JAM-2, which is expressed on lymphocytes (and
monocytes) (B. Imhof, personal communication).
C. VE-cadherin (Cadherin-5)
Physiol Rev • VOL
D. CD99
As this review was being written, a new interendothelial cleft molecule was identified that has the potential
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VE-cadherin belongs to the cadherin family, and its
expression is restricted to endothelial intercellular clefts.
Conceptually, in the presence of calcium, cadherin monomers become rigid and competent to undergo cis-dimerization. Trans-dimerization follows when cadherin dimers
on adjacent cells engage in homophilic interactions (129,
200, 251). In general, the cadherin cytoplasmic tail binds
␤- or ␥-catenin, which in turn links to ␣-catenin. Catenins
link cadherins to the cytoskeleton, and these interactions
are critical to the formation of adherens junctions, beltlike structures that lie within the intercellular cleft below
the level of the tight junction (251).
VE-cadherin plays a critical role in maintaining the
paracellular barrier properties of the endothelium. Measuring Evans blue dye extravasation in heart and lungs,
Corada et al. (38) found that a rat monoclonal antibody
against mouse VE-cadherin (BV13) induced a concentration- and time-dependent increase in vascular permeability. In vitro experiments suggest the antibody affects VEcadherin specifically as no change was observed in the
endothelial border staining patterns for a variety of other
molecules (PECAM-1, JAM-1, and two tight junction
markers, ZO-1 and cingulin). In the lung, 7–9 h after
antibody administration, electron microscopy showed
neutrophils adhering to denuded capillary basement
membranes and migrating across the alveolar wall (38). In
a mouse model of thioglycollate-induced peritonitis, VEcadherin antibody administration accelerated neutrophil
emigration into the peritoneal cavity (90). These studies
establish a clear role for VE-cadherin (adherens junctions) in maintaining the integrity of the endothelium, but
are adherens junctions really barriers to leukocyte transendothelial migration?
As discussed above, neutrophil migration in the lung
(at least in response to S. pneumoniae) occurs preferentially (50%) at tricellular corners where the margins of
three endothelial cells converge and tight junctions are
inherently discontinuous (33). Immunofluorescence images of HUVEC monolayers suggest adherens junctions
are also discontinuous at tricellular corners (29). Hence,
tricellular corners may provide the neutrophil with a portal for moving around (rather than through) both adherens junctions and tight junctions. Still, during streptococcal pneumonia, ⬃25% of the migrating neutrophils move
between pairs of adjacent endothelial cells (bicellular
migration) where adherens junctions appear to have
fewer constitutive gaps (33). Whether bicellular migration
involves adherens junction proteolysis has been the subject of much debate.
Initially, Dejana et al. (46), using an in vitro model of
leukocyte trafficking, reported that neutrophil adhesion
and migration induced widespread proteolytic cleavage of
the adherens junction complex. Subsequently, this was
shown to be an artifact arising from postfixation proteolysis (177). Allport et al. (6) renewed the debate with a
migration study of monocytes and differentiated U937
leukocyte cells interacting with HUVEC monolayers under flow conditions. Using immunofluorescence microscopy, they observed focal disruptions in VE-cadherin border staining at the site of leukocyte migration and
suggested this was not due to artifactual postfixation
proteolysis, since monocytes have significantly lower protease levels than neutrophils and differentiated U937 cells
lack fixation-resistant elastase (6). However, these cells
still contain other proteolytic enzymes and since a conventional fixation procedure was employed, rather than a
modified one that minimizes postfixation proteolysis (31),
the results are inconclusive. In a subsequent study, Shaw
et al. (232) overcame fixation issues by using HUVEC
monolayers transfected with VE-cadherin coupled to
green fluorescent protein (GFP) and real-time fluorescence microscopy (232). Under flow conditions (1.5 dyn/
cm2), leukocytes (monocytes and neutrophils) could be
seen migrating through preexisting gaps (holes) in VEcadherin-GFP border fluorescence, and some gaps were
located at tricellular corners. At other times, a de novo
gap appeared at the site of leukocyte arrest on the endothelial surface. Migrating leukocytes appeared to push
VE-cadherin aside as they moved across the monolayer
and then, within 5 min, the displaced material diffused
back to refill the junction. The opening and resealing of
adherens junctions is likely mediated through catenin
interactions with the cytoskeleton (251).
In summary, despite the obvious contribution VEcadherin makes to the maintenance of the pulmonary
microvascular integrity, in vitro studies suggest VE-cadherin is not a significant barrier to neutrophil transendothelial migration. Widespread proteolytic cleavage of the
adherens junction complex is not required for neutrophil
emigration. Instead, neutrophils trigger a reversible lateral displacement of the VE-cadherin complex enabling
the endothelium to reseal after the migration event. This
“curtain” effect also seems to operate at tricellular corners where small constitutive discontinuities in the adherens junction complex are expanded to accommodate
migrating neutrophils.
NEUTROPHIL EMIGRATION IN THE LUNG
V. NEUTROPHIL MIGRATION ACROSS
BASEMENT MEMBRANE
A. Role for Proteases?
Following diapedesis across the alveolar capillary
endothelium, neutrophils must cross the subendothelial
basement membrane to gain access to the interstitial
space. Whether this process involves proteolytic degradation of the basement membrane is controversial. Observations of leukocyte migration have not provided evidence of significant disruption of endothelial basement
membranes (112, 113, 160, 164, 231). Neutrophil elastase
and gelatinase B each has a high capacity for extracellular
matrix degradation. Delclaux et al. (47) report that in
response to formyl peptide, neutrophil migration across
Matrigel (a matrix derived from mouse sarcoma) is inhibited by 50% using gelatinase B or elastase inhibitors.
However, this observation is difficult to interpret, since
Matrigel by itself is capable of stimulating elastase and
gelatinase release (178). The stimulatory effect of Matrigel contrasts with a report showing that fibronectin associated with subendothelial basement membrane acts as a
protective substrate and inhibits neutrophil activation
(168). Similarly, it has been reported that neutrophil conPhysiol Rev • VOL
tact with type IV collagen (a major constituent of the
alveolar capillary basement membrane) (10, 127, 221, 293)
inhibits subsequent neutrophil activation by a variety of
mediators (25). This effect appears to be mediated
through the ␣3-chain of type IV collagen, and this may
decrease the potential for damage as neutrophils traverse
the capillary wall (178).
The endothelium may be another determinant that
regulates the neutrophil’s proteolytic arsenal. Mackarel et
al. (159) found that neutrophil migration across cultured
human pulmonary endothelium, and its associated subendothelial basement membrane was not affected by inhibitors of elastase or gelatinase. Similarly, Huber and Weiss
(111) reported that neutrophil migration across human
umbilical vein endothelial cells and their subtending basement membrane was not dependent on neutrophil elastase or cathepsin G and was resistant to inhibitors directed against neutrophil collagenase, gelatinase, and
heparanase. Moreover, scanning electron microscopic examination of the transmigrated basement membrane
showed no evidence of holes, even though basement
membrane barrier function (as measured by the passage
of monastral blue) was compromised. The presence of the
endothelium was critical as denuded basement membranes did not support neutrophil migration. Because
endothelial cells synthesize serine and metalloproteinases
and are able to degrade basement membrane components, it was suggested that the endothelium prepared the
basement membrane for neutrophil invasion (111). In any
event, these studies provide compelling evidence that
neutrophil proteases are not required for passage across
the basement membrane. Recent in vivo studies uphold
this concept by showing neutrophil emigration at inflammatory foci is normal in mice genetically deficient in
elastase (22) or gelatinase B (24).
B. Preexisting Holes in the Basement Membrane
So how do neutrophils penetrate the basement membrane if they don’t use proteases? Is there a mechanical
explanation? Walker and colleagues (21, 274) believe that
in vivo the basement membrane is perforated with preexisting holes and neutrophils gain access to the interstitial
space by migrating through these holes (Fig. 5). The next
few paragraphs examine the evidence that holes in the
basement membrane are portals for neutrophil emigration.
In rabbits and mice, the majority (75– 80%) of capillary endothelial tight junctions are located at the intersection of thick and thin walls of the alveolar septum (274).
Since the majority of neutrophil migration in the alveolar
capillaries appears to occur at tricellular corners (275), it
follows that thick walls are the principal site into which
neutrophils migrate. Pericyte profiles are clustered at or
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to regulate leukocyte trafficking in the lung. The molecule
is CD99, a heavily O-glycosylated 32-kDa type I transmembrane protein whose previous expression had been noted
only on hematopoietic cells. Schenkel et al. (226) report
that CD99 is expressed at endothelial cell borders, and a
monoclonal antibody (hec2) against CD99 blocks monocyte migration across resting or cytokine-treated (IL-1␤ or
TNF-␣) HUVEC monolayers by ⬎90%. Anti-CD99 does not
affect monocyte adhesion, and blocking CD99 on the
monocyte is just as effective as blocking CD99 on the
endothelium. Additional studies with CD99 transfectants
suggest monocyte transendothelial migration likely involves homophilic interactions between endothelial CD99
and monocyte CD99. Interestingly, anti-CD99 causes migrating monocytes to arrest within the intercellular cleft,
at a point distal to the blockade effect seen with domain
1 or 2 PECAM-1 antibodies which prevent monocytes
(and neutrophils) from entering the interendothelial cleft
(149, 184). Whether CD99 regulates neutrophil transendothelial migration is unknown, but Schenkel et al. report
(data not shown) that neutrophils express CD99. The
distribution of CD99 within the vasculature is largely
unknown, but hec2 antibody reacts with human arterial
and venous endothelium (umbilical cords) as well as microvessels (dermis). Although it remains to be seen
whether CD99 is expressed in the pulmonary vasculature,
further investigation is warranted given the potential of
this molecule to regulate leukocyte trafficking.
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BURNS, SMITH, AND WALKER
near the intersection of the thin and thick wall. Endothelial tight junctions are associated with pericyte profiles in
a nonrandom fashion, and ⬃40% of the pericyte profiles
around alveolar capillaries are within 0.5 ␮m of endothelial tight junctions (242, 274). In other tissues, pericytes
surround capillaries and make numerous contacts with
the endothelium (209, 239, 241, 261). Others (77, 270 –273)
have shown that numerous cytoplasmic interdigitations
occur between endothelial cells and pericytes in immature capillaries of human granulation tissue. These observations suggest there are holes in the basement membranes of endothelial cells through which contacts are
made with pericytes.
Using transmission electron microscopy and serial
section reconstruction, Walker and colleagues determined that fibroblasts are also clustered at the intersection of thick and thin alveolar walls. Fibroblast extensions
were observed penetrating the endothelium through slitlike holes (0.43–1.0 ␮m in length and 0.16 – 0.48 ␮m in
width), and all holes were located at intersections of thick
Physiol Rev • VOL
and thin walls. During streptococcal pneumonia in rabbits, migrating neutrophils appeared to pass through
small holes in the basement membrane, as evidenced by
their hourglass-shaped appearance. Importantly, neutrophils penetrated the basement membrane at the same loci
where holes occupied by fibroblasts were observed in
control rabbits (274). Collectively, these observations
support the concept that basement membrane holes occupied by fibroblasts are avenues through which neutrophils migrate into the extracellular compartment of the
alveolar wall.
Before a neutrophil could pass through a hole in the
basement membrane, the fibroblast extension would have
to be withdrawn or displaced. Fibroblast retraction can
be triggered by mechanical stress as shown by experiments in dermal explants subjected to sinusoidal stretching. Stretching causes stellate fibroblasts with long cytoplasmic processes to become spherical and lose contact
with the ECM . The ECM relaxes, and interstitial pressure
becomes more negative, which results in a rapid (5–10
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FIG. 5. Summary of fibroblast (red)
relationships with type I (green) and type
II (purple) pneumocytes, capillary endothelial cells (yellow), and pericytes (orange) in human and rabbit alveolar walls.
In rabbit and human lung, type I pneumocytes extend up the lateral surface of
type II pneumocytes. In rabbit, interdigitations occur between types I and II
pneumocytes immediately below the
tight junctions (1), while in the human
they only occur on the basal surface of
the type II pneumocyte (2 and 8). In human lung, extensions of the subtending
fibroblast may cross the epithelial basement membrane at the margin of the type
I pneumocyte extension lying below the
type II pneumocyte so that all three cells
may make contact (2). In both human
and rabbit lung, cytoplasmic extensions
of the type II pneumocyte may penetrate
the basement membrane to invaginate
the fibroblast (3). In human lung, a single
cytoplasmic extension of the fibroblast
may penetrate the basement membrane
to invaginate the type II cell cytoplasm
(4). Sometimes in human lung, the fibroblast makes simple contacts through apertures in the basement membrane in areas where the type II cell membrane is
simple (5) or infolded (7). Cytoplasmic
extensions of type I pneumocytes in human lung penetrate the basement membrane to contact the fibroblast (10). As
observed in rabbits, human fibroblasts
may also contact a capillary pericyte
through an aperture in its basement
membrane (9) or the endothelial cell in a
similar fashion (11). In summary, 1, 3, 9,
and 11 occur in rabbit, whereas 2 and 11
occur in human lung. Our unpublished
evidence suggests contact 10 may also
occur in rabbits.
NEUTROPHIL EMIGRATION IN THE LUNG
VI. FIBROBLASTS: A SOURCE OF CONTACT
GUIDANCE AND STIMULATION FOR
NEUTROPHIL MIGRATION
As neutrophils pass through the endothelial basement membrane, they emerge into the thick wall interstitium. Within the interstitium two types of fibroblasts exist. The first type of fibroblast is intimately associated
with fibrous connective tissue elements and oriented parallel to the epithelium, whereas a second type is stellate
and oriented perpendicular to the epithelial cells of the
alveolar walls (Figs. 1 and 5). The latter has been characterized as a myofibroblast (122, 123) and is thought by
some to be capable of contraction (124, 143). It is the
myofibroblast that Walker et al. (274) describe as having
cytoplasmic extensions that penetrate the endothelial
basement membrane.
Sims (240) demonstrated that fibroblasts within the
alveolar wall are connected by adherens-like junctions on
cytoplasmic extensions of the fibroblasts. Adherens-like
junctions also occur between fibroblasts and the basement membranes of type I and II epithelial cells (123, 240,
274). Walker and co-workers (21) determined that individual myofibroblasts provide a physical bridge from preexisting holes in the endothelial basement membrane,
through the interstitium, to similar holes in the basement
membrane of epithelial type II pneumocytes, simultaneously keeping all three cells in physical contact. Taken
together, these observations define a reticulum of fibroblasts within the alveolar wall that simultaneously link the
endothelium to the epithelium at morphologically distinct
points through apertures (holes) in the respective basement membranes (Fig. 5).
Several other studies (2, 26, 265) demonstrated discontinuities in the basement membranes of rat type II
pneumocytes through which extensions of cytoplasm
contact interstitial fibroblasts. Through morphometric
analysis they estimated these contacts occur with a frePhysiol Rev • VOL
quency of 0.5 and 0.7 per pneumocyte, respectively. In
rabbit lung, Walker et al. (274) reported similar round
holes that range in diameter from 0.14 to 0.7 ␮m in the
basement membranes of type II pneumocytes at points of
contact with fibroblasts. Using serial sections from three
type II pneumocytes, Walker et al. (274) showed not only
does the basement membrane of each pneumocyte have
multiple holes (Fig. 5), but also each pneumocyte is connected to more than one fibroblast.
During streptococcal pneumonia in rabbits, migrating neutrophils are always in close contact with alveolar
wall fibroblasts (21). Morphometric estimates suggest
that on average, 30% of the neutrophil surface is close
enough (ⱕ15 nm) to a fibroblast surface to engage in
adhesive interactions. The idea that this close association
is an adhesive contact is supported by the presence of a
layer of dense fibrillar cytoplasm along areas of neutrophil contact with fibroblasts. In spite of the mild edema
that occurs in the alveolar walls of S. pneumoniae-infected rabbit lung, ⬃70% of the surface area of migrating
neutrophils is close enough to either extracellular matrix
elements or fibroblast plasma membranes to be adherent.
Although in vitro experiments suggest chemokines (e.g.,
IL-8) bind to ECM proteoglycans like heparin and heparin
sulfate (50a, 138a), and promote neutrophil haptotaxis
(directed migration in response to an immobilized attractant) (216a), evidence showing that such a mechanism
operates in vivo is lacking. Moreover, ECM elements (collagen, elastin, etc.) are not physically arranged in a manner that provides a continuous and unique structural
“bridge” between holes in the endothelial and epithelial
basement membranes. Hence, the ECM may play more of
a “hand-hold” role than a directional role. Conversely,
because fibroblasts connect points of neutrophil entry
into the interstitium to points at which neutrophils leave
the interstitium (21), we believe they play a unique directional (guidance) role in neutrophil migration.
Although the mechanisms of neutrophil adhesion are
not completely understood, several in vitro studies found
that PMA-activated human neutrophils show enhanced
CD18-dependent adhesion to skin and lung fibroblasts
(86, 235). Armstrong and Lackie (12) found activated
rabbit neutrophils can both adhere to and travel along the
surface of cultured chicken fibroblasts. Burns et al. (28)
determined that canine neutrophils adhere to cultured
canine lung fibroblasts in the presence of platelet-activating factor (PAF), but not IL-8. The combination of PAF
and IL-8 allowed neutrophils to adhere and crawl on the
fibroblast surface. Fibroblasts treated with TNF-␣ secrete
IL-8 protein into the medium, and secreted IL-8 was chemotactic for neutrophils (28). Collectively, these findings
suggest lung fibroblasts can function not only as an adhesive substrate for neutrophils, but also as a source of
stimulation for neutrophil migration. Burns et al. (28)
found neutrophil crawling was completely inhibited by
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min) rise in tissue edema (165). Whether mechanical
stress plays a role in fibroblast retraction during neutrophil emigration in the lung is unknown, but tissue edema
frequently accompanies lung injury (87, 258). In the event
that fibroblasts retract, many of the exposed holes would
still be too small to allow a neutrophil to pass. Estimates
from electron microscopy suggest the hole must be at
least 1 ␮m in diameter to permit neutrophil emigration
(274). In the absence of proteolysis, the neutrophil would
have to mechanically enlarge the hole, and this seems
feasible since the basement membrane is thought to be
somewhat elastic (76). An example of this elasticity can
be found in earlier observations by Majno and Palade
(160) showing red blood cells assuming deformed shapes
as they pass through the basement membrane in vivo.
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VII. ROLE OF INTEGRINS IN NEUTROPHIL
MIGRATION THROUGH
EXTRACELLULAR MATRIX
The adhesive properties of various ECM elements
affect both the direction and rate of leukocyte locomotion
(139). For example, fibronectin and proteoglycans (e.g.,
heparin) are widely distributed throughout the interstitium, either in association with collagen fibers or free in
the matrix (84, 224, 227, 256). In vitro, the addition of
heparin, fibronectin, or laminin to type I collagen gels
decreases neutrophil motility. It seems fibronectin and
laminin coat the collagen and create a substrate that
favors adhesion over motility. Heparin causes collagen
fibers to aggregate, and this increases the mesh size of the
gel to the point that neutrophil motility is compromised
(138). While elastin is probably not a good substrate for
cell attachment because of its highly hydrophobic nature
(44), in vivo studies suggest microfibrillar proteins associated with elastin (40) support adhesion of migrating
cells to elastic fibers (198).
Clearly, adherence to ECM is important for neutrophil localization to inflammatory sites. Integrins are the
major class of cell adhesion receptor that mediate cell-
Physiol Rev • VOL
matrix interactions. ECM ligands for integrins include
fibronectin, fibrinogen, laminin, collagens, entactin, tenascin, thrombospondin, and vitronectin (8, 97, 219). The
ligand for many integrins is an RGD (Arg-Gly-Asp) sequence contained in many extracellular matrix components including fibronectin, laminin, and the collagens
(214, 218, 219). While leukocyte ␤2-integrins CD11b/CD18
and CD11c/CD18 are known to bind fibrinogen following
leukocyte activation (7, 155), recent studies suggest ␤2integrins are not essential for interactions with other ECM
molecules. In vitro, activated CD18 null neutrophils adhere to fibronectin and vitronectin through ␣5␤1 and ␣v␤3,
respectively, while adhesion to endothelial laminin 10 is
through ␣6␤1 (243). In rat mesentery, after chemotactic
stimulation, ␤1-integrins were found to be more critical
than ␤2-integrins in supporting neutrophil motility in the
interstitium. Specifically, with the use of antibodies or
peptides recognizing various integrins, ␤1-integrin blockade inhibited neutrophil migration velocity by 70%; ␤2integrin blockade inhibited migration velocity by only
20%. Additional experiments determined that neutrophil
locomotion was critically dependent on ␣2␤1 (70%), but
not ␣4␤1 or ␣5␤1 (286, 287).
While ␤1-integrins have limited expression on circulating neutrophils, expression levels increase markedly
after transendothelial migration (131, 217, 286, 287). Antibody cross-linking experiments suggest ligation of ␤2integrin provides a signal for ␤1-integrin upregulation
(285). In a similar context, PECAM-1 ligation has been
shown to generate an inside-out signal to activate ␤1-, ␤2-,
and ␤3-integrins (reviewed in Ref. 182). Taken together,
the process of neutrophil adhesion and transmigration
appears to be associated with ␤1-integrin activation.
With respect to the lung, ␤1-integrins clearly play a
role in neutrophil emigration. In rats, neutrophil emigration into the alveolar airspace is partially (20%) CD18
dependent. Antibody blocking studies suggest the CD18independent component to this migration is largely
(⬃80%) mediated by the ␤1-integrins ␣4 and ␣5 (34). In
mice, while neutrophil emigration following LPS treatment is largely (80%) CD18 dependent, migration into the
airspace still requires ␤1-integrins, particularly ␣5 and ␣6.
In a situation where neutrophil emigration is entirely
CD18 independent (e.g., instillation of KC, the murine
ortholog of human IL-8), antibody inhibition of ␤1-integrin
or specific ␣-subunits (␣2, ␣4, ␣5, and ␣6) blocks neutrophil emigration into the alveolar airspace. Ultrastructural
examination of lung tissue 4 h after KC stimulation and
␤1-integrin blockade suggests inhibition of neutrophil emigration is not at the level of the capillary endothelium,
but rather in the migration from interstitium into alveoli
(210). While it was suggested ␤1-integrin blockade interferes with neutrophil adhesion and migration on extracellular matrix and/or fibroblast surfaces, one needs to con-
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anti-CD18 monoclonal antibody. In contrast, neutrophil
adhesion was only partially (70%) CD18 dependent (28).
While the identity of the CD18-independent component of
adhesion is unknown, the ␤1-integrin family of adhesion
molecules is a likely candidate. In a slightly different
model that examines neutrophil migration through a human lung fibroblast barrier, Shang and Issekutz (23) identified a role for CD18, as well as ␣4-, ␣5-, and ␣6␤1-integrins. With the use of a cocktail of blocking antibodies to
these molecules, neutrophil migration was inhibited by
60%. Further inhibition (75%) in migration was seen when
an antibody to the neutrophil ␣9␤1-integrin was added to
the cocktail (229).
Neutrophil adhesion to cultured lung fibroblasts is
also partially (50%) dependent on fibroblast ICAM-1, a
ligand for CD18 (28). The identity of other fibroblast
ligands has not been determined, but apparently, it reportedly does not involve extracellular matrix proteins or
blood proteins since PMA-mediated neutrophil adhesion
to lung fibroblasts does not require serum, albumin, type
I or type IV collagen, or fibronectin (235). More recently,
it has become apparent that cytokines and mast cell products will induce VCAM-1 expression on human lung fibroblasts (172, 230, 246). While ␤1-integrins ␣4 and ␣9 are
known to bind VCAM-1 (230, 257), it remains to be determined whether VCAM-1 expression on lung fibroblasts
mediates ␣4- and ␣9-dependent neutrophil adhesion and
motility.
NEUTROPHIL EMIGRATION IN THE LUNG
VIII. NEUTROPHIL MIGRATION ACROSS
ALVEOLAR EPITHELIUM
Compared with what we know about the regulation
of neutrophil migration across the endothelium, we really
know very little about migration across the alveolar epithelium. Basic information is lacking regarding the behavior of tight junctions and adherens junctions during neutrophil transepithelial migration in the lung. It will be
important to determine if neutrophil transepithelial migration is associated with junctional proteolysis or
whether the junctions slide apart like a curtain (232) to
accommodate the migrating neutrophil. The migration
mechanisms for crossing the epithelilum could turn out to
be very different from those used to cross the endothelium. It is important to remember that unlike crossing the
endothelium where the movement is in the apical to basal
direction, neutrophils are now moving in a basal to apical
direction as they cross the epithelium. For example,
ICAM-1 is expressed in abundance on the apical surface
of the alveolar epithelium, and it would not be surprising
to find that it serves as an adhesive ligand for CD18dependent neutrophil adhesion (262, 263), functioning as
an adhesive tether to retain neutrophils at specific locations (196). However, because ICAM-1 is not expressed on
the basolateral aspect of alveolar epithelial cells (32), it is
unlikely to play a role in neutrophil transepithelial migration in the physiologically relevant basal to apical direction.
Much of what we do know about neutrophil transepithelial migration comes from in vitro studies using intestinal epithelial monolayers. Studies of neutrophil migration across intestinal epithelial monolayers suggest
neutrophils take a paracellular route as they migrate in
the basal to apical direction, and this involves focal disruption of epithelial tight junctions. The notion that neu-
Physiol Rev • VOL
trophil transepithelial migration is associated with tight
junction disruption is largely based on studies showing
reversible loss of epithelial barrier function as measured
by tracer and flux studies (reviewed in Ref. 196). However, this claim is not supported by morphological evidence, since freeze-fracture images of epithelial tight
junctions appear normal during neutrophil transmigration
(188), just as they do in endothelial cells (31, 33). Migration across the epithelium does not appear to depend on
neutrophil oxidant production or protease release (196).
Nash et al. (188) conclude that if junctional abnormalities
exist, they must be highly focal.
During neutrophil migration across intestinal epithelial monolayers, neutrophils tend to accumulate in clusters beneath the epithelium at so-called invasion sites (39,
188, 197). The presence of nonrandom invasion sites and
general preservation of tight junction morphology during
neutrophil transepithelial migration can be explained if
neutrophil migration occurs preferentially at epithelial
tricellular corners. Intestinal epithelial tight junctions are
known to be discontinuous at tricellular corners (247),
and intestinal epithelial cells used for neutrophil migration studies (e.g., T84 cells) have on average six corners
per cell (85). Hence, there are six nonrandom sites associated with each cell where tight junctions are discontinuous, and these sites could allow neutrophils to migrate
around rather than through tight junctions without compromising tight junction barrier function. Despite numerous reviews and articles on neutrophil transepithelial migration, the issue of tricellular corner migration has been
largely ignored.
Earlier in this review, we postulated that neutrophils
find their way to type II pneumocytes by crawling along
fibroblasts in the interstitial space. The neutrophil crawls
into the basal lateral space of the type II pneumocyte by
passing through a hole in the basement membrane that
was once occupied by a type II pneumocyte contact with
an underlying fibroblast (Fig. 5). From here, neutrophils
appear to preferentially enter the alveolar lumen adjacent
to type II pneumocytes (33, 42, 274). In fact, in a study of
dog lung, Damiano et al. (43) report that all observed
cases of neutrophil egress from the interstitium were
associated with type I/type II junctions. When viewed
from the alveolar surface (33, 42), the site at which the
neutrophil emerges is at an epithelial tricellular corner
where the border of two type I pneumocytes meet the
border of a type II pneumocyte (Fig. 6). Just as in endothelial cells, freeze-fracture images of tricellular corners
between type I and II pneumocytes show tight junction
discontinuities (Walker, unpublished observation). Tricellular corner discontinuities are a common feature for a
variety of epithelia including tracheal (276, 277), intestinal
(247), nasal (171), bladder (268), and caput epididymal
(254). In the lung, the type I/II corner discontinuity pro-
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sider the effects this treatment might have on other cell
types.
For example, fibroblast adhesive contacts with collagen are critically dependent on ␤1-integrins. Fibroblasts
are able to exert tension on the ECM, and antibodies
against ␤1-integrins interfere with this adhesive interaction. ␤1-Integrin blockade decreases connective tissue fiber tension, and this increases negative interstitial pressure, which results in rapid edema formation (206, 207).
Hence, although anti-␤1-integrin antibody clearly inhibits
neutrophil emigration, the effect may be indirect. ␤1-Integrin blockade may adversely affect the organization of the
interstitial matrix and render it unsuitable for neutrophil
motility. Interstitial chemotactic gradients may be diluted
and fibroblast networks disrupted (retracted) resulting in
diminished contact guidance for neutrophils.
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FIG. 6. Type I pneumocyte borders converge with type II pneumocytes
at tricellular corners, the preferred site for neutrophil emigration. In A,
scanning electron microscopy of rabbit lung reveals numerous microvillar
projections on the surface of a type II pneumocyte (*). On type I pneumocytes, microvilli are largely confined to the cell borders (arrows). B is a
cut-away diagram based on freeze-fracture replicas of lung tissue. The
cut-away shows the arrangement of epithelial tight junctions (orange) at a
tricellular corner where two type I pneumocytes (yellow) converge on a
type II pneumocyte (turquoise). The tight junction complex shows increased depth at the corner. A discontinuity (arrows) in the junctional
complex exists because it is not possible to make a three-sided junction
(see text for details). C is a scanning electron micrograph showing neutrophil transepithelial migration in rabbit lung 4 h after tracheal instillation of
Streptococcus pneumoniae. A neutrophil (arrow) is emerging onto the
alveolar surface at an epithelial tricellular corner where the borders of two
type I pneumocytes converge on a type II pneumocyte. Bar is 5 ␮m. [C from
Burns et al. (33), copyright CRC Press, Boca Raton, FL, 2001.]
vides a natural path through which the neutrophil can
migrate around rather than through the tight junction.
This may be particularly important for transepithelial migration, since epithelial tight junctions are more substantial (deeper), particularly at tricellular corners (Fig. 6),
than those found on capillary endothelial cells. The increased tight junction complexity may partially account
for the observation that the alveolar epithelium is 10 times
less leaky than the alveolar capillary endothelium (259).
Studies in the intestinal epithelium suggest CD11b/
CD18 and CD47 play critical roles in neutrophil transmigration (reviewed in Ref. 196). Neutrophils from patients
with leukocyte adhesion deficiency (LAD) failed to migrate across an intestinal epithelial cell monolayer. The
dependency on CD18 was mimicked by antibody against
CD11b/CD18 but not against CD11a/CD18 or CD11c/
CD18. In the lung, CD18 is not always required for neutrophil emigration into the alveolar airspace. For example, in response to S. pneumoniae, neutrophil emigration
in the lung is CD18 independent (54). An autopsy performed on an LAD patient suffering from a complete
deficiency of CD11/CD18 showed emigrated neutrophils
in the lung (94). Hence, unlike the intestinal epithelium,
CD18 is not absolutely required for migration across the
alveolar epithelium. CD47 is expressed on all hematopoietic cells and most other cell types (169, 208). In vitro,
antibody blockade of epithelial or leukocyte CD47 inhibits neutrophil migration across intestinal epithelia, collagen-coated filters, and endothelia (37, 153). In the neutrophil, CD47 is stored in secondary specific granules, and its
translocation to the cell surface appears to facilitate neutrophil migration. Transfection of CD47 into CD47-deficient epithelial cells (Caco-2) also results in enhanced
neutrophil transepithelial migration. Exactly how CD47
regulates neutrophil migration is not clear, but it has been
proposed that in its activated state, CD47 induces downstream tyrosine kinase-linked signaling events which result in enhanced neutrophil migration (153). Although
specific experiments demonstrating a role for CD47 in
neutrophil migration across the alveolar epithelium are
lacking, a recent study suggests CD47 is important for
monocyte emigration in the lung. Using an in vitro model
of monocyte migration across cultured alveolar epithelial
cells, Rosseau et al. (216) found transepithelial migration
largely depends on CD11b/CD18 and CD47. The additional
engagement of ␤1-integrins (␣4, ␣5, and ␣6) was required
for optimal migration (216).
In the lung, JAM-1 is present on alveolar epithelial
cells (154). Whether JAM-1 plays a role in human neutrophil migration across the alveolar epithelium is unknown.
On the basis of findings with a human T84 colon cancer
epithelial cell line, a role for JAM-1 in regulating interepithelial tight junction assembly has been suggested by Liu
and colleagues. They identified a panel of monoclonal
antibodies against the extracellular portion of human
NEUTROPHIL EMIGRATION IN THE LUNG
Physiol Rev • VOL
FIG. 7. Fibrin exudates erupt into the alveolar space during streptococcal pneumonia in rabbit lung. Four hours after tracheal instillation
of Streptococcus pneumoniae, scanning electron microscopy reveals
fibrin strands emerging into the alveolar space through macroscopic
discontinuities (pores) in the alveolar wall (A). B shows how fibrin
strands organize into a reticulum that functions as an adhesive substrate
for neutrophils as they emerge into the alveolar lumen. Bar is 5 ␮m.
phil emigration. Under conditions where serous exudates
and leukocytes are spewed into the alveolar lumen, neutrophil migration from blood to airspace may bypass the
adhesion mechanisms discussed in this review.
IX. CONCLUSIONS AND FUTURE DIRECTIONS
With respect to inflammation, the pulmonary circulation is very different from the systemic circulation. Capillaries, not venules, are the primary site for neutrophil
emigration. The molecular paradigm explaining how adhesion molecules regulate neutrophil tethering, rolling,
arrest, and transmigration in the systemic circulation
does not apply to the pulmonary capillary bed.
During inflammation in the lung, neutrophils sequester in capillaries through a process involving mechanical
and adhesive changes not only in the neutrophil, but also
in the endothelium. Mechanical changes in the cytoskeleton of activated neutrophils cause them to stiffen and
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JAM-1 that inhibit epithelial barrier repair after disruption
by calcium depletion. Immunofluorescence microscopy
suggests these anti-JAM-1 antibodies inhibit both occludin and JAM-1 in reassembling tight junctions. Although
this study is important because it provides the first direct
evidence for JAM-1 having an important role in interepithelial junction assembly, it is important to note that these
antibodies do not inhibit human neutrophil migration
across T84 epithelial monolayers, human microvascular
endothelial cell monolayers, or HUVEC monolayers (154,
233). Because domains 1 and 2 of JAM-1 recognize JAM-1
domain 1 and leukocyte CD11a/CD18, respectively (192),
and Liu and colleagues did not map the epitope sites (i.e.,
domain 1 or 2) recognized by their anti-JAM-1 antibodies,
it is difficult to interpret the lack of inhibition seen in
neutrophil transmigration. Simultaneous antibody blockade of both domains 1 and 2 may be necessary to block
neutrophil transepithelial migration.
Finally, in closing, it is necessary to point out that
neutrophil emigration in the lung is not always an ordered
process regulated by adhesion molecules. In his monograph on the lung, William Snow Miller (176) suggests
fibrinogen exudates and their conversion to fibrin strands
are common features of lobar pneumonias. As far back as
the 1920s, using a light microscope, Miller reported that
early inflammatory stages of acute lobar pneumonia begin
with engorgement of blood capillaries followed by a serous exudate into the alveoli in which epithelial cells,
leukocytes, and red blood cells are present (174, 175).
Nearly 80 years later, using a scanning electron microscope, we have confirmed Miller’s observation and are
able to show fibrin strands erupting into the alveolar
space through macroscopic discontinuities in the alveolar
wall. The strands organize into netlike structures (Miller
described these as broad sheets of fibrin) providing an
adhesive substrate for emigrated neutrophils (Fig. 7). Because fibrin is also an adhesive substrate for bacteria,
neutrophil adhesion and migration into fibrin gels is critical for bacterial killing. In vitro, complement fragment
C5a and LTB4 appear to enhance neutrophil migration
into fibrin gels and bacterial killing (146). While the origin
of the alveolar wall discontinuities is unknown, Miller
suggests the serous exudate accumulates in the restricted
space beneath the epithelium (i.e., interstitium) and the
cells are pushed off by the increased pressure. Support
for this “pressure” mechanism can be found in a transmission electron micrograph showing large numbers of
neutrophils simultaneously exploding into the alveolar
lumen through a macroscopic discontinuity in the alveolar wall (Fig. 8). Preexisting holes in the basement membrane might be involved in this process, since the holes
represent the path of least resistance for movement of
serous fluids and cells from blood to airspace. Clearly, we
have overlooked (ignored?) Miller’s observations in our
rush to explain how adhesion molecules regulate neutro-
327
328
BURNS, SMITH, AND WALKER
lodge within narrow capillary segments. Depending on
the stimulus, the leukocyte ␤2-integrin CD18 may or may
not be required for neutrophil emigration. The molecular
mechanisms regulating the migration of the neutrophil
across the endothelium are beginning to be understood.
Conceptually, it is easy to understand how cytoskeletal
remodeling in both the neutrophil and endothelium are
required for neutrophil transendothelial migration between endothelial cells. The neutrophil inserts a pseudopod into the interendothelial cleft, signaling ensues, the
endothelial cytoskeleton remodels, and interendothelial
junctions separate. However, this mechanism does not
explain how a significant amount (20% in capillaries and
25% in larger vessels) of neutrophil migration in the lung
occurs through direct penetration of the endothelial cytoplasm, nor does it explain why neutrophils show a
preference for endothelial tricellular corners where the
margins of three endothelial cells converge. What is the
mechanism that mediates neutrophil localization at tricellular corners? What factors (e.g., stimulus, adhesion molecules, surface topography) favor paracellular migration
over transcellular migration?
Compared with what we know about neutrophil inPhysiol Rev • VOL
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FIG. 8. Macroscopic discontinuities (pores) in the alveolar wall
provide another mechanism for neutrophil emigration into the alveolar
space. Four hours after tracheal instillation of Streptococcus pneumoniae, transmission electron microscopy reveals a discontinuity
(pore) in the alveolar capillary wall through which multiple neutrophils
emerge simultaneously into the alveolar space (As). The margins of the
pore are identified by arrows. Capillary lumens (*) appear empty because the tissue was fixed by vascular perfusion. Bar is 10 ␮m.
teractions with endothelial cells, we know even less about
neutrophil migration through the interstitium and across
the alveolar epithelium. Studies of neutrophil migration
through extracellular matrices suggest there is a role for
adhesion molecules, particularly ␤1-integrins. Conversely,
neutrophil-mediated proteolysis of the matrix does not
seem to be required for migration. In the lung, a neutrophil travels a remarkably short distance (typically ⬍1 ␮m)
in going from blood to airspace. It shows a preference for
emigration into the alveolus by migrating at tricellular
corners where the margins of two type I pneumocytes
converge on a type II pneumocyte. Interstitial fibroblasts
may play a key role in directing neutrophils to these
preferred epithelial migration sites because single fibroblasts form cellular bridges between capillary endothelia
and type II pneumocytes. Fibroblasts make intimate contacts with these cells through preexisting holes in the
basal lamina. There is morphological and in vitro evidence that after migrating through holes in the endothelial
basal lamina, neutrophils adhere to and crawl on interstitial fibroblasts. By way of its unique position within the
alveolar wall interstitium, the fibroblast guides the neutrophil to the space between the type II pneumocyte and
its underlying basement membrane. Preferential migration across the epithelium occurs at tricellular corners
where type I and II pneumocytes meet because epithelial
junctions are inherently discontinuous at these sites. This
schema for neutrophil migration across the alveolar wall
is summarized in Figure 9.
From the preceding statements, it can now be appreciated that neutrophil emigration in the lung is significantly regulated by its unique microanatomy. Future insights into molecular mechanisms regulating neutrophil
emigration in the lung will benefit from careful consideration of the anatomical issues. If we hope to learn more
from in vitro models, we must move in a direction that
incorporates the unique positioning of fibroblasts within
the matrix in ways that allow for the formation of intimate
contacts with endothelial and epithelial cells through
holes in the basement membrane. We must also pay attention to the pathways (paracellular and transcellular)
taken by neutrophils as they cross the endothelium and
epithelium. Although it is likely the pathway taken will be
stimulus dependent, it is also likely the pathway will
change during the course of the inflammatory episode.
For example, mild inflammation may begin in a regulated
fashion with paracellular neutrophil emigration, but as
the inflammatory process intensifies and chemokine concentrations shift, transcellular migration may be favored.
Only by understanding these types of issues can we hope
to design effective therapeutics for treating acute and
chronic inflammatory diseases like adult respiratory distress syndrome, emphysema, multiorgan failure, ischemia/reperfusion injury, graft versus host disease, and
autoimmune disease.
NEUTROPHIL EMIGRATION IN THE LUNG
329
This work was supported by National Institutes of Health
Grants AI-46773, HL-42550, and HL-66569 and by Medical Research Council of Canada Grant MRC 5–91232.
Address for reprint requests and other correspondence: A. R.
Burns, Dept. of Medicine and Pediatrics, Sect. of Cardiovascular
Science, Baylor College of Medicine, Rm. 515B, One Baylor Plaza,
Houston, TX 77030 (E-mail: [email protected]).
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