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A microscopic in vitro study of neutrophil diapedesis across the blood-

brain barrier

M. von Wedel-Parlow and H.-J. Galla

Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany

The cerebral microcapillary endothelium forms a highly important barrier between the blood and the interstitial fluid of the

brain (blood-brain barrier) that controls the passage of molecules and cells in and out of the central nervous system (CNS).

Several CNS diseases include leukocyte extravasation through the endothelium via two mechanistically distinct routes, the

paracellular and the transcellular pathway. The visualization of a leukocyte migrating through endothelial cytoplasm very

close but distinct from the junctional area requires advanced ultrastructural technical settings. Scanning electron

microscopy in combination with multi-color confocal laser scanning fluorescence microscopy and scanning force

microscopy are unique tools to study the diapedesis route in detail. This review highlights the advantages of these

visualization methods used to monitor leukocyte transmigration in a newly established in vitro model of the inflamed

blood-brain barrier consisting of primary cultured porcine brain capillary endothelial cells which express a tight

endothelial barrier even under inflammatory conditions.

Keywords blood-brain barrier; transmigration; scanning electron microscopy; confocal laser scanning microscopy;

scanning force microscopy

1. Introduction

The blood-brain barrier (BBB) plays the predominant role in actively transporting nutrients to the brain and controls the

passage of xenobiotics and pathogens, thus guaranteeing the homeostasis of the central nervous system (CNS) and

proper neuronal function. Brain capillaries are lined with a single endothelial cell layer [1] forming strands of

continuous tight junctions which seal the intercellular cleft between adjacent cells [2].

However, several CNS diseases are associated with BBB dysfunction involving an immense infiltration of leukocytes

like neutrophils [3]. The transmigration of these immune cells across endothelial barriers is a central component of both

the innate and adaptive immunity and occurs in a complex multistep process including tethering, selectin-dependent

rolling on the endothelial surface [4-5], chemokine-dependent activation, flattening and integrin-mediated firm adhesion

[6-7]. After neutrophil polarization via the actin cytoskeleton they undergo lateral migration on the endothelial surface

searching for optimal transmigration sites [8-9] and finally diapedese into the perivascular tissue (Fig.1).

While the knowledge on this sequential set of events has recently undergone additional refinements and is now

widely elucidated, the molecular mechanisms of the last step, the eventual diapedesis, are still under debate [10].

Classically, leukocyte extravasation has been assigned to the paracellular migration pathway requiring a transient

disruption of intercellular junctions. Nevertheless, the opposing concept of one cell passing through the body of another

cell (transcellular) has already been discussed for a long time [11].

chemokine

gradient cytokines

junctional

disassemblyparacellular transcellular

cytokines

chemokines

vasoactive

factors

proteases

?blood

brain

Fig. 1 Neutrophil adhesion and migration mechanisms at the BBB endothelium. Neutrophils in the circulation slow down by

binding to chemokines presented on the endothelial surface and become activated by further luminal mediators causing cytoskeleton

rearrangements and the release of vasoactive factors which increase endothelial permeability. Some chemokines attract neutrophils

by chemotaxis. Activated neutrophils firmly adhere to the endothelium and initiate intracellular signalling pathways which regulate

junctional integrity. Depending on endothelial barrier integrity neutrophils diapedese para- or transcellular.

However, the lack of evidence for transcellular migration in in vitro settings impedes acceptance and mechanistic

investigations of this pathway. Furthermore, transcellular events appear in very close proximity to the junctions and

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

©FORMATEX 2010 1161

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thus might be mistaken for paracellular migration [12-13]. Thus, the visualization of a leukocyte migrating through

endothelial cytoplasm very close but distinct from the junctional area requires advanced ultrastructural technical

settings. Due to the lack of an appropriate BBB in vitro model providing in vivo-like properties even under

inflammatory conditions, no studies have been able to demonstrate transcellular migration events in cultured cerebral

microvascular endothelial cells yet. Based on a well-characterized BBB model of primary porcine brain capillary

endothelial cells [14] optimal experimental conditions providing an inflamed endothelium with uncompromised in vivo-

like barrier function were identified.

Previous morphological findings revealed that porcine brain capillary endothelial cells (PBCEC) grown in 55 nM

hydrocortisone (HC) containing serum-free medium develop extrusions, so called marginal folds, at their cell borders

[15] which are a hallmark of cerebral endothelial cells in vivo.

Transmigration of immune cells requires a dynamic endothelial cytoskeleton, which facilitates tethering of

neutrophils on the luminal endothelial surface by translocating cell adhesion molecules to the cell surface under

inflammatory conditions [16-17]. Thus, an inflammatory response of the primary cell culture was induced by the

cytokine tumor necrosis factor-α (TNF-α) which initiates and regulates the expression of adhesion molecules as for

example selectins at the endothelial luminal surface to enable the extravasation of leukocytes from the vasculature into

the inflamed tissue. The E-selectin staining shown in Figure 2A [18] provided clear evidence that inflammation in HC-

cultured PBCEC was successfully induced by the application of 2 ng/mL TNF-α. Furthermore, a widening of

transendothelial pores is a necessary pre-requisite for non-paracellular migration [19]. Our inflammation model reports

on the induction of cytoskeletal dynamics as a reorganization of the filamentous actin in PBCEC treated with 2 ng/mL

TNF-α was observed. Fibrous actin is redistributed from the perijunctional rings to the cytosol and filigree-like actin

strands cover the cell nuclei (Fig. 2B [20]). Thus, although HC is well-known to be an anti-inflammatory compound,

the potency of TNF-α is strong enough to dominate the cellular response in endothelial cells In general high

concentrations of cytokines can induce the redistribution of tight junction associated proteins and influence the

architecture of the cytoskeleton [21]. This phenomenon would adulterate the in vitro findings especially if the BBB

model is used as a tool to study leukocyte trafficking (under activated conditions). However, as shown in Figure 2C

[18], incubation of PBCEC with 2 ng/mL TNF-α did not affect the in vivo-like distribution of the tight junction-

associated protein zonula occludens-1. Therefore, this model allows a morphologic study of the process of

transendothelial migration of neutrophils by employing different visualization methods presented in this review.

25 µm

CA

25 µm

Fig. 2 Morphology of TNF-α-treated PBCEC cultured with hydrocortisone. Cerebral endothelial cells were cultured on filters

inserts with nanoporous polycarbonate membranes (A;C) or gelatine coated polystyrene coverslips (B). Culture media was

exchanged to serum free media supplemented with 55 nM hydrocortisone after 5 days in culture. 48 h later cells were incubated with

2 ng/mL TNF-α for 4 h before cells were stained for E-selectin (A), fibrous actin (B) and zonula occludens-1 (ZO-1) (C) [18; 20].

2. Scanning electron microscopy

Scanning electron microscopy represents an adequate tool to study the ultractructure of cell surfaces with high

resolution. Electron beams scan the surfaces achieving resolutions between less than 1 nm and 20 nm revealing

potential morphological changes of the inflamed cell monolayers upon interaction with neutrophils.

As shown in Figure 3 [18] scanning electron micrographs of endothelial surfaces clearly demonstrate, that a TNF-α-

induced inflammation leads to the formation of microvilli-like structures on PBCEC (Fig. 3B) but does not affect the

typical cylinder-like continuous structure of the marginal folds observed in un-inflamed PBCEC (see arrows on Fig.

3A). However, PBCEC which were additionally allowed to interact with neutrophils expressed rather vesicle-like,

irregular marginal fold structures (Fig. 3C) and build up microvilli-like structures all over the endothelial surface (Fig

3D). This indicates an involvement of these vesicular structures in the neutrophil extravasation process.


1162 ©FORMATEX 2010

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Fig. 3 Scanning electron micrographs demonstrating the impact of neutrophil incubation on the morphology of inflamed

PBCEC. Arrows depict marginal folds and the general surface structure of the PBCEC monolayer. (A) Un-stimulated PBCEC. (B)

TNF-a stimulated PBCEC without contact to neutrophils. (C and D) TNF-a activated PBCEC after incubation with neutrophils. All

PBCEC were cultured with 55 nM HC. TNF-a application (2 ng/mL) 4h before neutrophil incubation; neutrophil incubation time: 2h;

Bars: A and C 2 µm, B and D 1,5 µm. [18].

More detailed scanning electron microscopic images of the neutrophil-endothelial contact zone clearly revealed that

the vesicle-like structures of the endothelial cell margins interact with the membrane protrusions of transmigrating

neutrophils (arrows in Figure 4 [18]). This proves the active involvement of the endothelium. The vesicular material

seems to be entrained by the invading neutrophil as its shape points to the interstitium.

The high resolution images furthermore disclose the similarity of the vesicular bulges of the immune cells and the

endothelial cells. First hints for transcellular migration events were given by the observation of membrane interactions

of both cell types not only involving the tight junctional regions but rather being distributed over the whole PBCEC

surface.

Fig. 4 Scanning electron micrographs showing the adhesion and transmigration of a neutrophil across inflamed PBCEC. B,

D and F show enlarged sections of boxed parts of the original images (A, C and E). Arrows show contact areas of neutrophils with

the PBCEC surface. PBCEC cultured with 55 nM HC; TNF-a application (2 ng/mL) 4h before neutrophil incubation; neutrophil

incubation time: 2h; Bars: B, D, F 0.5 µm; A, C 1.5 µm; E 3 µm. [18].

Indeed, further morphological studies represent strong indications for immune cell migration across PBCEC

following a transcellular path though the endothelial cell bodies. Figure 5 [18] indicates that, in addition to the

junctional areas (see arrows), the neutrophil membrane interacts with the PBCEC surface at the center of the endothelial

cell. As the vesicular material remains on the endothelial membrane at the point of invasion, it could contribute to

barrier restoration after a completed diapedesis event and might promote the expeditious resealing of the cell body to

minimize any permeability increase during transmigration.



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Fig. 5 Scanning electron micrographs showing a transcellular migration event of a neutrophil across inflamed PBCEC. Arrows

show contact areas of the flattened neutrophil with the EC surface. Note the distance of the neutrophil to the cell junctions indicating

transcellular migration. PBCEC cultured with 55 nM HC; TNF-a application (2 ng/mL) 4h before neutrophil incubation; neutrophil

incubation time: 2h; Bars: A 5 µm, B 3 µm. [18].

3. Confocal laser scanning microscopy

Whether the involved microvilli-like structures which connect PBCEC with the immune cells originate from the

endothelium or the leukocytes is not answerable by SEM analyses. Only a differential staining of the individual cell

types provides information about the respective contributions. Thus, the two cell types needed to be discriminated by

multi-color confocal laser scanning fluorescence microscopy (CLSM) which allows to distinguish different cell types

by individual fluorescent labels. This was accomplished by staining living PBCEC monolayers with the red membrane

dye DiI and neutrophils with the green fluorescent dye calcein AM. After the staining procedure the immune cells were

allowed to interact with PBCEC before the fixation for subsequent fluorescence microscopy analyses.

Beside the advantage of individual morphological analysis CLSM provides insights into intracellular events when

optical xz- or yz-sections are recorded that cut through the cytoplasm.

Figure 6A [20] shows the PBCEC monolayer shortly before an adherent neutrophil, which is located on the

endothelial surface next to the cell margins (arrow), starts its transmigration. The optical sections through the same field

of view in z-direction clearly reveal a deformation of the cell surface at the position of the neutrophil. The endothelial

cell starts to embrace the leukocyte beginning from the adhesion point (arrow) and the PBCEC layer already provides

access of the neutrophil to the basolateral cell side. Additionally to Figure 6A also Figure 6B [20] clearly shows a

neutrophil captured during its transcellular diapedesis through one single endothelial cell. This image depicts vesicular

structures at the immersion point of the neutrophil which were already visible in the electron microscopic studies (Fig.

3-5). CLSM analysis now clearly reveals that these bulges definitely originate from the endothelial membrane since

they bear the same dye the endothelial membrane was labeled with. Closer examination of the fluorescent images

discloses that no dye deriving from the neutrophils is found within the vesicles. The optical xz- and yz-sections of this

image show that the extravasation has already proceeded so far that the endothelial cell layer partially covers the

luminal side of the neutrophil. At the position of the neutrophil the endothelium is detached from the substrate and

develops an excavation in which the neutrophil is situated just beneath the basolateral cell membrane (yz-section,

arrow). The PBCEC barrier is still unclosed suggesting that the extravasation process is not yet completed. The

occurrence of partially interconnected bulges at the luminal cell surface surrounding the immersion spot is also clearly

indicated in Figure 6C [20]. These endothelial derived vesicles project into the interstices (arrows in the optical z-

sections).

Since not only tight junctions but also adherens junctions contribute to the barrier function of cerebral endothelial

cells the adherens junctions protein VE-cadherin which is known to provide a barrier for transmigrating neutrophils [22]

was immunocytochemically stained. As shown in Figure 6D [23], PBCEC incubated with neutrophils show a distinct

and continuous VE-cadherin expression at the cell borders. The optical z-sections show the engulfment of the neutrophil

by the VE-cadherin stained PBCEC.



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Fig. 6 Confocal laser scanning microscopy analysis of PBCEC-neutrophil interactions. Neutrophils were captured during

transcellular extravasation through the endothelium. Optical xy-sections are arranged in the upper panel whereas the xz- and yz-

sections are arranged in the lower panel. (A-C) CLSM images of a confluent PBCEC layer stained heterogeneously with 3 µg/mL DiI

(red). Neutrophils were stained with 2 µM calcein AM (green). (A) Arrow in the xy-section indicates transcellular migration. Side

views show the firm adhesion of the neutrophil and the arrow indicates the initiation of neutrophil embracement by the endothelium.

(B) Advanced transcellular migration of a neutrophil partially covered by the endothelium (arrow in the optical yz-section).

Development of vesicular structures at the neutrophil immersion site (arrow in the top view). (C) Arrows indicate interconnected

bulges surrounding the diapedesis spot and projecting into the interstitium. (D) Merged CLSM image of (i) PBCEC

immunofluorescently labelled with anti-VE-cadherin (green) and (ii) neutrophils immunofluorescently labelled with anti-pig

granulocytes (red). Optical xz- and yz-sections show the engulfment by the VE-cadherin stained endothelium. [20; 23].

4. Atomic force microscopy

Electron and confocal laser scanning microscopy studies only allow the visualization of fixed cell preparations. To

overcome this issue and to investigate ultrastructural changes of living organisms atomic force microscopy (AFM)

based on short-range van der Waals interactions [24] represents a great tool. This method does neither require fixation

of the cells nor a vacuum environment which might influence the sample causing artefacts. Thus, biological processes

can be recorded in situ in ambient air or a liquid environment. In contrast to SEM and CLSM techniques AFM

applications are able to provide three-dimensional information about the cell sample in atomic resolution. Therefore,

this non-destructive method is an adequate tool to study the morphology of the endothelial surface as well as the

adhesion and diapedesis of neutrophils over time in situ providing additional information about inflammatory processes.

Figure 7 [20] shows an AFM image of a PBCEC monolayer after its incubation with neutrophils. The endothelial

nucleus and the cell margins are clearly visualized. The right arrow in Figure 7A points at an adhered neutrophil. Its

dimensions indicate that it is captured during the diapedesis stage. The immune cell sticks to the membrane of one

single endothelial cell while the cell margins remain virtually unaffected (left arrow).

Figure 7B proves the adhesion to only one endothelial cell and indicates that the cell margins are slightly deformed

but remain intact (arrows).

Usually a leukocyte passage across activated endothelia requires less than two minutes [25]. The AFM image of

Figure 7C has been captured approximately nine minutes after Figure 7A and clearly shows the disappearance of the

neutrophil (arrow) and the remaining vesicular structures on the PBCEC surface.

These AFM studies support the concept of transcellular migration of neutrophils across cerebral microvascular

endothelial cells. Thus, this study evidently demonstrates that the AFM technique is a non-invasive tool not only to

characterize the morphology of the luminal surface of a living endothelial cell layer during the interaction of PBCEC

and neutrophils but also to monitor the adhesion and diapedesis process in situ under physiological conditions. Repeated

AFM imaging of the same field of view contribute significantly to our understanding about the transmigration pathway

as described above.



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Fig. 7 AFM images of an inflamed PBCEC monolayer during transcellular extravasation of a neutrophil. (A) The image shows an

adhered neutrophil (right arrow) embraced by marginal folds at the cell junctions (left arrow). (B) Enlarged section of image A

clearly indicating the integrity of the marginal folds beside the neutrophil (arrows). The left part next to the immersion point appears

already elevated; the neutrophil is captured directly during the passage across the endothelium. (C) Same position as image A 9

minutes later. The image reveals the disappearance of the neutrophil and shows the cell margins. The integrity of the cell layer is

clearly demonstrated. The former position of the neutrophil is marked by a dashed white circle. [20].

5. Conclusions

The BBB in vitro model applied in this study very closely mirrors the situation in vivo and avoids most of the typical

cell culture derived artefacts. In order to describe leukocyte trafficking across the endothelial monolayer high resolution

imaging of the cell morphology provides important details. A combination of three different microscopic techniques

helped to develop a comprehensive understanding of leukocyte transmigration. SEM studies allow an ultrastructural

analysis of the cell surface but these studies miss the ability to analyse intracellular processes and to distinguish

different cell types. Therefore, CLSM approaches represent an adequate tool in addition to the SEM surface analyses.

To However, these techniques only show the cellular morphology during transendothelial migration in fixed cell

preparations. Non-invasive AFM analyses help to overcome this issue and have been successfully established as a tool

to study the interactions between immune cells and the endothelial barrier cells. Thus, the BBB model has been

successfully adapted to the biophysical techniques allied in this study to allow measurements under chemically defined

and physiological conditions.

Acknowledgements The support by Sabine Hüwel for continuous and expert help with the cell cultures as well as a very strong

contribution of Dr. Olympia Ekaterini Psathaki to cell isolation, electron microscopic imaging and ECIS-recordings is gratefully

acknowledged.

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