From the Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts 02215
Circulating leukocytes are thought to extravasate from venules through open interendothelial
junctions. To test this paradigm, we injected N-formyl-methionyl-leucyl-phenylalanine
(FMLP) intradermally in guinea pigs, harvesting tissue at 5-60 min. At FMLP-injected sites,
venular endothelium developed increased surface wrinkling and variation in thickness. Marginating neutrophils formed contacts with endothelial cells and with other neutrophils, sometimes
forming chains of linked leukocytes. Adherent neutrophils projected cytoplasmic processes into
the underlying endothelium, especially at points of endothelial thinning. To determine the
pathway by which neutrophils transmigrated endothelium, we prepared 27 sets of serial electron microscopic sections. Eleven of these encompassed in their entirety openings through
which individual neutrophils traversed venular endothelium; in 10 of the 11 sets, neutrophils
followed an entirely transendothelial cell course unrelated to interendothelial junctions, findings that were confirmed by computer-assisted three-dimensional reconstructions. Having
crossed endothelium, neutrophils often paused before crossing the basal lamina and underlying
pericytes that they also commonly traversed by a transcellular pathway. Thus, in response to
FMLP, neutrophils emigrated from cutaneous venules by a transcellular route through both endothelial cells and pericytes. It remains to be determined whether these results can be extended
to other inflammatory cells or stimuli or to other vascular beds.
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Introduction |
Among the earliest and most important events in acute
inflammation is the emigration of neutrophils and
other inflammatory cells from the blood into the tissues by
way of postcapillary venules and small veins (1, 2). This
process, which has been the subject of intense investigation
for more than a century, has been resolved into successive
phases of leukocyte margination, rolling, adhesion, and finally, transmigration across endothelium (3). In recent
years, much has been learned about the molecular events
responsible for neutrophil attachment to venular endothelium. It is now clear that chemical mediators generated
during inflammation induce, redistribute, or increase the
binding avidity of complementary "adhesion" molecules
present on both endothelial cells (ECs)1 and inflammatory
cells; many such adhesion molecules have been identified
including L-, P- and E-selectins, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1), and several integrins (3). It is thought that
members of the selectin family are largely responsible for
the early, loose tethering of rolling neutrophils to endothelium, and that subsequent firm adhesion requires activation
of the
2 (CD18) integrin family for binding to EC counterreceptors such as ICAM-1.
In contrast to the numerous studies of leukocyte rolling
and adhesion, very little attention has been paid in recent
years to the pathway by which adherent inflammatory cells
cross endothelium. It is widely believed that this issue was
settled definitively in the 1960s. At that time, several
groups of electron microscopists described adhesion of inflammatory cells, particularly neutrophils but also other
granulocytes and monocytes, to venular endothelium at
sites of inflammation (4). They noted that adherent inflammatory cells extended pseudopod-like processes into
the underlying ECs. In some instances, emigrating leukocytes appeared to be present in large EC vacuoles, apparently at a distance from the nearest interendothelial junction (7). This finding raised the possibility that leukocytes
had been engulfed or phagocytosed by ECs and that neutrophils emigrated through endothelium rather than passing between adjacent ECs. However, interpretation was appropriately cautious because it was recognized that leukocytes that appeared distant from junctions in single images
might nonetheless have extended into and extravasated
through intercellular junctions that would only have become evident at deeper levels of sectioning.
It was agreed that serial electron microscopic sections
would be needed to determine if neutrophils or other leukocytes exited venules by passing between or through ECs.
However, preparation of numerous consecutive thin sections is a formidible task even with modern equipment and
was especially difficult with the technology available in the
early 1960s. Nonetheless, at least two groups attempted this
approach. In studies of leukocyte emigration in inflamed
skin, Hurley and Xeros (6) performed limited numbers of
serial sections that showed "penetration is occurring
through, or immediately adjacent to, a junction between
endothelial cells". In a second study Marchesi prepared
what Hurley (2) described as "semi-serial" sections of neutrophils emigrating from mesenteric venules and reported
that "in most of them passage through an intercellular junction could be demonstrated" (5). After the publication of
these papers, opinion, which had up until that time been
divided, crystallized around the position that leukocytes emigrated from venules by passing through open inter-EC
junctions. Thus, Hurley wrote in 1983, "It appears almost
certain that all neutrophils, eosinophils and monocytes escape in this way [between endothelial cells] but the work
necessary to produce and examine serial sections of large
numbers of emigrating leucocytes has prevented conclusive
proof of this hypothesis" (2). Since that time, the consensus
view has only rarely been challenged (10).
Over the past 35 yr, there have been substantial improvements in all aspects of electron microscope technology. For
this reason and because our own studies led us to doubt the
consensus model of leukocyte transmigration, we reinvestigated the pathway by which neutrophils emigrated across
venular endothelium in the setting of acute inflammation. To
this end, we made extensive use of serial electron microscopic
sections accompanied by computerized three-dimensional
reconstructions. We report here that in response to the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophils emigrate from inflamed
venules primarily by a transendothelial pathway and that,
after crossing the endothelial barrier, they often traverse
underlying pericytes, also by a transcellular route.
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Materials and Methods |
Experimental Design.
Acute inflammatory lesions were induced in the flank skin of female Hartley guinea pigs (700-995 g;
Elm Hill, Chelmsford, MA) with FMLP. Animals were anesthetized intramuscularly with 10 mg/kg ketamine and 20 mg/kg xylazine. Hair was clipped from the flanks and various concentrations of test and control substances were injected intradermally in
a randomized pattern at 1-60 min before killing. FMLP (Sigma
Chemical Co., St. Louis, MO) was dissolved in DMSO to provide a stock solution of 4.38 mg/ml (10
2 M); this was diluted in
HBSS such that the 0.2-ml injection volumes contained 8.76 ng-
87.6 µg FMLP (10
7-10
3 M).
Most experiments were performed with 10
5 M FMLP, although no morphological evidence of cellular injury was detected
with concentrations >10-fold higher. To determine the effective
concentration of FMLP present in skin at various times after intradermal injection in a volume of 0.2 ml, we performed clearance studies with [3H]FMLP (NET563, 80 Ci/mmol; DuPont
New England Nuclear Research Products, Boston, MA) diluted
400-fold in cold 10
5 M FMLP. The blebs raised at injection sites
were outlined individually with a magic marker and squares of
full-thickness skin corresponding to the initial bleb were harvested at time 0 and at intervals up to 2 h. Harvested skin was
weighed, minced, dissolved in tissue solubilizer (Solvable; Packard Instrument Co., Inc., Meriden, CT), and diluted 10-fold in
formula 989 liquid scintillation counting solution (Packard Instrument Co.) for beta counting. Clearance data were fit to a two
component exponential decay curve (KaleidaGraph, v. 3.0.8; Synergy Software, Reading, PA). Based on these data, and assuming
that 1 g of tissue corresponds to a volume of 1 ml, we calculated
the actual concentration of FMLP present in skin to be as follows:
time 0, 2.5 × 10
6 M; 10 min, 1 × 10
6 M; 60 min, 2.5 × 10
7 M.
Control sites were injected with HBSS alone or with HBSS
supplemented with DMSO in concentrations corresponding to
those present in FMLP injections. All animal protocols were approved by the Beth Israel Hospital Institutional Animal Care and
Use Committee (Boston, MA).
Tissue Processing.
Animals were killed by C02 narcosis. Skin
test and control sites were excised and fixed by immersion for 4 h
in freshly prepared 2.0% paraformaldehyde-2.5% glutaraldehyde-
0.025% calcium chloride in 0.1 M sodium cacodylate buffer, pH
7.4. Tissues were postfixed for 2 h in 1.5% sym-collidine-buffered osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Spurr
resin as previously described (14, 15)
Alternatively, tissues were fixed by perfusion. Animals were
anesthetized with ketamine and xylazine. The thoracic cavity and
pericardium were opened and a blunt-tipped 19- gauge needle with attached PE-50 tubing was inserted into the apex of the left ventricle. After incising the right atrium to permit outflow, 250 ml
(equivalent to approximately three blood volumes) of fixative was
injected while maintaining normal systemic arterial pressure of
80-94 mm Hg. The entire procedure was complete within 15 min. Experimental and control skin sites were then excised and
fixed for an additional 4 h in fixative before processing as above.
Preparation of Serial Thin Sections.
To follow the pathway of
leukocyte diapedesis, we prepared multiple sets of consecutive serial electron microscopic sections. Venules that exhibited emigrating leukocytes were identified by light microscopy in 1 µm
Giemsa-stained sections. Serial 100-nm-thick sections were then
cut, collected on carbon-Formvar-coated single-slot grids, and
viewed in an elecron microscope (CM10; Philips, Eindhoven, The Netherlands). Consecutive sections of individual vessels were photographed, generally at a magnification of 10,000.
Three-dimensional Reconstructions.
Membrane outlines of the
endothelial openings through which neutrophils transmigrated as
well as ECs and neutrophil plasma membranes were traced onto
transparent plastic sheets. Consecutive images were then scanned
into a Power Macintosh (6100/60) computer (Apple Computer Inc.,
Cupertino, CA) using a scanner (Scanjet 4c scanner; Hewlett-Packard Co., Palo Alto, CA) and Deskscan II 2.3 (Hewlett-Packard Co.) software. Color images were converted into 8-bit grayscale
PICT files using Adobe Photoshop (version 3.0.4). Consecutive
grayscale images were subsequently converted into hierarchical
data format (HDF) image files (Transform PPC version 3.3.0;
Fortner Research LLC, Sterling, VA). Finally, the HDF files representing the consecutive serial electron microscopic sections
were reconstructed in three dimensions using software (Slicer
v.1.1b3; Fortner) by volume rendering.
Morphometric and Statistical Analysis.
The extent of "wrinkling"
of luminal and abluminal plasma membranes of venular endothelium
was quantitated by calculating a folding index (16). Statistical analyses
were performed using the nonparametric Kruskal-Wallis ANOVA
test, Dunn's multiple comparisons test or the Mann-Whitney U test.
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Results |
In accord with many previous reports, circulating leukocytes, mostly neutrophils, marginated and became attached
to the luminal surfaces of venular endothelium at skin sites
injected with 10
5 -10
7 M FMLP (1, 2, 4). Both large
venules/small veins (30-80 µm luminal diam) and smaller
venules (10-30 µm diam) of the deep and superficial dermal vascular plexuses were involved (Figs. 1 and 2). Leukocyte margination and attachment first became evident at
~15 min after FMLP injection and persisted through at
least 60 min. Higher concentrations of FMLP increased the
number of venules exhibiting leukocyte diapedesis and
overall reaction intensity, but did not alter the kinetics;
even at the highest concentrations tested leukocyte attachment was not detected before 10-15 min.

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Fig. 1.
Large venule in
guinea pig skin harvested 60 min
after intradermal injection of
10 5 FMLP. Many neutrophils
and a single eosinophil (eos) are
captured at various stages of attachment to and extravasation
across vascular endothelium and
underlying pericytes (p). Two
neutrophils (single joined arrow),
one in the lumen and another
partway across the endothelium,
are tethered together. Another
neutrophil (long arrow) has projected a cytoplasmic process into
an underlying EC. Other neutrophils (arrowheads) and the eosinophil have crossed the EC barrier, but remain superficial to
pericytes, forming dome-like structures that bulge into the vascular lumen. Still another neutrophil (open arrow) that has already crossed the endothelium
has extended a process into the
basal lamina and indents an underlying pericyte. Other neutrophils (some indicated by n) have
crossed both the EC and pericyte
barriers and have entered the
surrounding connective tissues.
L, lumen. Bar, 10 µm.
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Fig. 2.
Progression of neutrophil migration across venular endothelium at 60 min after intradermal injection of 10 5 M FMLP. (A) Three neutrophils in the vascular lumen (L) are tethered together at point contacts; two of these also attach to the endothelium (e) at disparate sites. Three additional
neutrophils (n) have already crossed the endothelial barrier. (B) An adherent neutrophil (n) projects two separate pseudopods into an EC (e). As confirmed in deeper serial sections, the smaller of these (arrowhead) projects into the EC at a point adjacent to the nucleus, whereas the larger (dominant) pseudopod
forms a blunt projection into a thinned region of EC cytoplasm nearby. The EC shows extensive wrinkling of its abluminal surface, except over the nucleus, which is rounded with pinched folds and bulges into the lumen. (C) A chain of three interconnected neutrophils. One of these (elongate cell) has
traversed both endothelium (e), basal lamina, and pericyte (p) layers and extends into the underlying connective tissue while its trailing edge remains
within the vascular lumen. The second neutrophil has formed attachments to endothelium at two discontinuous sites and the third remains in the vascular
lumen with no attachments to endothelium. (D) Higher magnification of a portion of Fig. 1, illustrating two neutrophils and an eosinophil (eos) that have crossed the endothelium but have not penetrated the pericyte (p) layer; each is covered over with a thin overlay of flattened, relatively smooth endothelium that, together with the leukocytes, form dome-like structures that bulge into the vascular lumen. L, lumen. Bar, 3 µm.
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Alterations in Venular Endothelium at FMLP-injected Skin
Sites.
Beginning as early as 5 min after injection of
FMLP, the profile of venular endothelium changed significantly from a "coast of California" to a "coast of Maine"
contour (Table 1). Both the luminal and abluminal plasma
membranes were thrown into thin folds that projected for
varying distances into the vascular lumen and abluminally
into the underlying basal lamina (Figs. 1 and 2). Surface
wrinkling developed before leukocyte adhesion and persisted through the latest times studied (i.e., 60 min). However, portions of ECs in immediate contact with attached
or transmigrating leukocytes generally exhibited less or no
wrinkling (Table 1). In addition, EC nuclei became
rounded with accentuated nuclear folds and projected into
the vascular lumen beneath a thin covering of cytoplasm.
Finally, EC thickness became less uniform after FMLP injection; mean EC thickness changed little, but there was a
significant decrease in median cell thickness and a significant increase in the range of thickness (Table 2). As a result,
zones of extreme EC thinning alternated with zones of increased thickness, the latter being especially prominent
over rounded nuclei that projected into the vascular lumen.
Changes were similar in tissues that were fixed by immersion or perfusion.
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Table 2
Variation in Thickness of Venule EC in Guinea Pig
Skin Measured 60 min after Intradermal Injection of 10 5 M FMLP
or HBSS
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Leukocyte Adhesion and Diapedesis.
Initial contacts between circulating neutrophils and venular endothelium
were focal and often multiple with intervening skip areas
(Fig. 1 and Fig. 2, A and C). Neutrophils also developed contacts with other neutrophils, forming chains of tethered
cells; often more than one of these was in contact with vascular endothelium (Fig. 1 and Fig. 2, A and C).
In agreement with earlier descriptions (4, 8), adherent
neutrophils extended one or more cytoplasmic processes
into the underlying endothelium (Fig. 2 B). These initial
projections showed no particular relation to inter-EC junctions, which remained normally closed, or to zones of cytoplasm that were thick or thin. However, projections that
became dominant and that led to neutrophil transmigration
generally involved zones of thinned endothelium.
Having breached the endothelium, neutrophils sometimes proceeded to cross the underlying basal lamina immediately, and in such instances, it was possible to observe
a single neutrophil whose leading front had advanced into
the interstitium before its trailing edge had left the vascular
lumen (Fig. 2 C). More commonly, however, neutrophils
that had crossed the endothelial barrier were held up for a
time at the level of the basal lamina. Such neutrophils were
covered with a thin rim of overlying EC cytoplasm and
formed dome-like structures that bulged into the vascular
lumen (Fig. 1, and Fig. 2 D). At sites of neutrophil-EC contact, both the luminal and abluminal EC surfaces lost
their wrinkled appearance and exhibited relatively smooth
contours (Fig. 2, A-D; Table 1).
Neutrophil attachment and transmigration did not noticeably impair the patency of larger venules and small veins
(Figs. 1 and 2). However, in smaller venules, marginating
and transmigrating neutrophils sometimes accumulated in
such numbers that their lumens were compromised with
resulting vessel distension and a relative smoothing of EC
contours (not shown).
Pathway of Neutrophil Transmigration across Endothelium as
Determined by Serial Thin Sections and Three-dimensional Reconstructions.
To ascertain the pathway of neutrophil extravasation across venular endothelium, we analyzed 27 sets
of serial sections prepared from skin sites injected with 10
5
M FMLP (effective starting concentration in skin was 2.5 × 10
6 M) at intervals of 15, 30, or 60 min (Table 3). In 11 of
these sets, serial sections encompassed the entire pore
through which individual neutrophils emigrated, and included uninvolved surrounding endothelium on all sides.
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Table 3
Sets of Serial Thin Sections Performed on Guinea Pig
Skin that Encompassed the Pores through which Neutrophils Emigrated
across Venular Endothelium or Pericytes in Response to Locally
Injected FMLP
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Representative serial sections illustrating two such pores
are illustrated in Fig. 3-5, along with a three-dimensional
reconstruction of one. (Journal space limitations precluded
additional illustrations of serial sections and reconstructions,
which are available from the authors.) These demonstrated
definitively that at least 10 of the 11 openings were trans-EC pores, not inter-EC gaps. Certain properties of these 10 pores are summarized in Table 4. In none of the 10 did the
pore involve or abut upon an inter-EC junction. In only
one case (No.11 in Table 3) did a pore extend sufficiently close to an intercellular junction that we could not determine whether it actually extended into that junction.

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Fig. 3.
Transmigration of a neutrophil (n) across a thinned portion of venular endothelium (e) at a skin site injected 60 min earlier with 10 5 M FMLP.
12 of a series of 45 consecutive serial sections (sections 10-19 and 21-22) are illustrated and together encompass in its entirety the transendothelial pore
(F-J) through which the neutrophil is migrating. Maximum pore diameter was 0.75 µm. L, lumen. Bar, 1 µm.
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Table 4
Some Properties of the 10 Definitive Trans-EC Pores
through which Neutrophils Migrated in Response to FMLP
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In 16 sets (Table 3), serial sections began at a point
somewhere within the pore and extended distally to include endothelium beyond the pore, thereby sampling
large portions of an EC opening, but not encompassing it
completely. In none of these sets did the pore involve or
abut on an inter-EC junction.
Neutrophil Passage through Pericytes Underlying Venular Endothelium.
Pericytes provide an extensive, though incomplete, covering around venular ECs, which emigrating
neutrophils commonly encountered after traversing venular
endothelium and basal lamina (17). Unlike venular ECs,
pericytes showed no evidence of plasma membrane wrinkling or other changes in contour in response to FMLP.
Nonetheless, neutrophils passed through pericytes in a
manner analogous to their passage across vascular ECs by
extending processes into and eventually through the pericyte cytoplasm (Figs. 6 and 7). Pericyte cytoplasm adjacent
to sites of neutrophil penetration demonstrated striking cytoskeletal organization such that microfilaments were oriented in a direction perpendicular to the invaginating neutrophil process. Serial sections confirmed these findings. Of
20 sets of serial sections studied, 12 entirely encompassed the pore through which neutrophils crossed pericytes (Table 3) and in at least 8 of these, 12 neutrophils traversed
pericytes by a transcellular route.

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Fig. 6.
Neutrophil (n) that has already crossed endothelium (e) is at an early stage of transmigration through a subendothelial pericyte (p) 60 min after
intradermal injection of 10 5 M FMLP. 8 of a series of 90 consecutive serial sections (sections 60-66 and 68) are illustrated. B-G illustrate the transpericyte pore through which the neutrophil has advanced a cytoplasmic projection. Maximum pore diameter was 0.83 µm. Bar, 1 µm.
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Fig. 7.
Neutrophil (n) migrating through a subendothelial pericyte (p) 60 min after intradermal injection of 10 5 M FMLP. Four (sections 12-14
and 16) of a series of 46 consecutive serial sections illustrate a transcytoplasmic pore in the pericyte through which the neutrophil has extended a blunt cytoplasmic projection. The projecting neutrophil pseudopod is filled with a feltwork of microfilaments. Note also the prominent, horizontally arrayed microfilaments in the immediately adjacent pericyte cytoplasm (C, arrowheads). Bar, 0.1 µm.
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Discussion |
The data presented here and summarized diagramatically
in Fig. 8 provide strong evidence that in response to
FMLP, neutrophils extravasate from dermal venules predominantly by a trans-EC route rather than by emigrating
through inter-EC junctions. These data challenge the long-dominant paradigm that neutrophils emigrate from venules
exclusively by passing between EC, i.e., through EC junctions. It remains to be determined whether these results can
be extended to other inflammatory cells, to other inflammatory stimuli, or to other vascular beds. Nevertheless, our
findings establish the important principle that leukocytes
can extravasate by a trans-EC route. Our data also imply
that venular ECs are joined together by junctions whose
attachments are unexpectedly durable and remain intact,
even in acute inflammation. This is consistent with studies
reporting that openings in vascular endothelium induced
by vasoactive agents, cytokines, or by supraphysiological intraluminal pressure are frequently trans-EC rather than
inter-EC (18, 19).

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Fig. 8.
Schematic diagram
summarizing different aspects of
neutrophil diapedesis in response to FMLP. In A, neutrophil n1 extends a cytoplasmic
process that forms contact with a
thinned portion of EC e1 as in
Fig. 2, A and B; in B-D n1 has
progressed through e1, the underlying basal lamina (bl) and a
pericyte (p1). This pattern of
near-simultaneous trans-migration through endothelium as
well as other components of the
vascular wall is illustrated in Fig.
2 C, but was less common than
that exhibited by neutrophils n2
and n3 that, after crossing the
EC barrier, paused for a time before progressing farther. In A, n2
initially makes contact with the
endothelial surface with two
processes, as in Fig. 2 B; one of
these becomes dominant and
leads the way as the neutrophil begins to traverse EC e2 in B,
again as in Fig. 2 B. Having
crossed the endothelial cell barrier, n2 does not immediately
progress through the basal lamina; instead it remains covered
over for a time (C and D) by a
thin overlying rim of flattened
EC cytoplasm (as in Fig. 1, arrowheads) and Fig. 2 D) before extending a pseudopod through
the basal lamina and underlying
pericyte in D. As it progresses through e2 (C and D), it is embraced by cytoplasmic processes that extend from abluminal portions of both e2 and an adjacent EC, e1 (as in Fig. 5). Transmigration of n3 is similar to that of
n1 and n2, except that n3 persists
for a longer time in the subendothelial space superficial to the
basal lamina. n3 in A corresponds to the neutrophil marked
by a long arrow in Fig. 1; n3 in
C and D models neutrophils illustrated in Fig. 1, arrowheads and
Fig. 2 A and D. Extravasation of
all three neutrophils is transendothelial and inter-EC junctions
remain closed.
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Fig. 5.
Computer-generated three-dimensional reconstruction of the transmigrating neutrophil illustrated in Fig. 4. The panels portray successive
rotations toward the viewer around a horizontal axis at angles of 60°, 120°, 240°, and 300° as indicated. 0° (not shown) would represent a vascular cross
section at right angles to the direction of blood flow and 90° (also not shown) would represent a view looking directly down on the luminal surface. Emigrating neutrophil (n), purple, upper and lower left; in the other panels, the neutrophil was subtracted electronically to visualize the pore that passes cleanly
through the cytoplasm of EC e1 (orange-brown) distinctly apart from the junction of e1 with e2 (yellow). Cytoplasmic arms of both e1 and e2 embrace the
neutrophil luminally and, to a lesser extent, abluminally. L, lumen.
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Fig. 4.
Transmigration of a neutrophil (n) across a thinned portion of venular endothelium at 60 min. after local intradermal injection of 10 5 M
FMLP. 12 sections (numbers 13, 14, 16, 19, 21, 24, 25, 27, 29, and 30-32) of a series of 74 consecutive serial sections are illustrated. Portions of the neutrophil's nucleus (E-G) are included within the pore. The pore passes through a single EC (e1), but a junction of e1 with a second EC, e2, is indicated
(arrow); this junction is intact and maintained a distance of >1 µm from the pore margin at all levels of sectioning. L, lumen. Bar, 1 µm.
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Having traversed the endothelium, some neutrophils
proceeded to cross the underlying basal lamina immediately. More commonly, however, neutrophils paused for a
time between endothelium and basal lamina before proceeding farther. After crossing the basal lamina, extravasating neutrophils encountered pericytes that they frequently
crossed by a transcellular route. This finding is remarkable in that pericytes do not provide a continuous covering
around venules (17). Presumably, therefore, neutrophils
could have migrated around pericytes; that they instead
commonly migrated through pericytes affirms their proclivity for transcellular migration.
It is possible that differences in experimental design contributed to the differences between our results and those of
investigators who formulated the consensus paradigm. Our
work was performed in a single species (guinea pig), in a
single tissue (skin), with a single inflammatory agent
(FMLP), and applies primarily to a single type of inflammatory cell, the neutrophil (a single eosinophil was also encompassed in our serial sections; Fig. 1, and Fig. 2 D). In
contrast, the reports emanating from the early 1960s that
proposed an inter-EC route of inflammatory cell transmigration were performed in the rat (mesentery, skin), rabbit
(ear skin, mesentery), or dog (pancreas) and used different
agents (mechanical trauma, homologous serum, UV light,
ligation of pancreas, heat, nitrogen mustard, or turpentine)
to induce leukocyte diapedesis (4). The possibility remains, therefore, that neutrophils traverse venular endothelium by different routes in different species, in different tissues, and in response to different inciting agents. On the
other hand, there is support in these early studies for the
mechanism we have proposed in that all of the authors
cited reported that neutrophils projected pseudopod-like
processes into ECs (4). More recently, studies of monocyte and neutrophil chemotaxis across an endothelial cell
monolayer have added support for a trans-EC pathway for
inflammatory cell migration. Thus, Migliorisi et al. (20) reported that both adherent monocytes and neutrophils frequently extended pseudopods into the apical plasma membranes of ECs and only rarely projected such processes into
the junctional region between cells.
Other data presented here also have implications for the
pathogenesis of acute inflammation. Among these is the
finding that FMLP induced wrinkling of the EC plasma
membrane as early as 5 min after injection and therefore
before leukocyte attachment (Table 1). These results are
consistent with studies from a number of different laboratories, indicating that ECs as well as other noninflammatory
cells, express functional FMLP receptors that, depending
on context, may be stimulated by FMLP to induce cell contraction, relaxation, and migration (21). Membrane
wrinkling was accompanied by increased variation in EC
thickness with areas of extreme thinning alternating with
areas of increased thickness. Focal EC thinning may have
facilitated leukocyte emigration by shortening the transmigration pathway.
Another finding of interest is the observation that emigrating neutrophils formed attachments not only to venular
endothelium, but also to other neutrophils. These contacts
were frequently maintained as neutrophils crossed endothelium in tandem (Figs. 1, 2). Consistent with this finding,
Alon et al. (24) recently reported that in an in vitro system,
neutrophils and other leukocytes formed "strings of rolling
cells" that were linked together by L-selectin, and that such
bridging favored cell attachment to endothelium. The
linked chains of neutrophils observed in our study may
have resulted from L-selectin bridges.
Finally, our findings have implications for an understanding of the molecular mechanisms that underlie leukocyte emigration from venules. Holding to the long-standing consensus paradigm of leukocyte diapedesis, most
investigators have presumed that leukocytes crossed endothelium through opened EC junctions. Thus, they have
postulated mechanisms that might be expected to cause EC
junctions to open, e.g., leukocyte proteases that cleave cell adhesion molecules, mechanical forces exerted by leukocytes on EC junctions, and signaling events that cause EC
to pull apart. If, however, neutrophils exit venules by passing through, rather than between, ECs, very different
mechanisms may be imagined. These might include rearrangements of cell cytoskeleton that allow neutrophils to
thrust processes into and through ECs and pericytes as well
as molecular events that generate and reseal pores in ECs
and pericyte plasma membranes.
Address correspondence to Harold F. Dvorak, Department of Pathology, Beth Israel Deaconess Medical
Center, 330 Brookline Ave., Boston, MA 02215. Phone: 617-667-4343. Fax: 617-667-2943. E-mail: hdvorak{at}bidmc.harvard.edu
This work was supported by United States Public Health Service National Institutes of Health grants
CA50453, CA58845, HL54465, and AI33372 and by salary support from the Beth Israel Hospital Pathology
Foundation, Inc.
1.
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| Hurley, J. 1983. Acute Inflammation. Churchill Livingstone,
Edinburgh. 157pp.
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Muller, W..
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