Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo.

0.88 MB·English

by  Coisne, Caroline

Coisneetal.FluidsandBarriersoftheCNS2013,10:7
FLUIDS AND BARRIERS
http://www.fluidsbarrierscns.com/content/10/1/7
OF THE CNS
STUDY PROTOCOL Open Access
Live cell imaging techniques to study T cell
in vitro
trafficking across the blood-brain barrier
in vivo
and
Caroline Coisne, Ruth Lyck and Britta Engelhardt*
Abstract
Background: The central nervous system (CNS) is animmunologically privileged site to which access for circulating
immune cellsis tightly controlled by the endothelial blood–brain barrier (BBB) located inCNS microvessels. Under
physiological conditions immune cell migration across the BBB is low.However, inneuroinflammatory diseases such
as multiple sclerosis,many immune cells can cross theBBB and cause neurological symptoms.Extravasation of
circulating immune cellsis a multi-step process thatis regulated by thesequentialinteractionof different adhesion
and signaling molecules on theimmunecells and on theendothelium. The specialized barrier characteristics ofthe
BBB, therefore, imply theexistence of unique mechanismsfor immunecell migration across the BBB.
Methods and design: An in vitro mouse BBB model maintainingphysiological barrier characteristics ina flow
chamber and combinedwith high magnificationlive cell imaging, has been established. This model enables the
molecular mechanisms involved in themulti-step extravasation of T cellsacross thein vitro BBB, to be definedwith
high-throughput analyses. Subsequently these mechanisms have been verified in vivo using a limited number of
experimental animals and a spinal cord window surgical technique. The window enables live observation of the
dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions
using real time epifluorescenceintravital imaging. These in vitroand in vivo live cell imaging methods have shown
that the BBB endothelium possesses uniqueand specialized mechanisms involved in themulti-step T cell migration
across this endothelial barrier under physiological flow.The initial T cell interaction with theendothelium is either
mediated byT cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4+ T cells
crawl over long distances against the direction of flow to find the rare sitespermissive for diapedesis through the
endothelium.
Discussion: The sequential useof in vitro and in vivo live cell imaging of T cellsinteractingwith the BBBallowsus
to delineate thekinetics and molecular determinants involved in multistep extravasation ofencephalitogenic T cells
across the BBB.
Keywords: BBB, Immune cell trafficking, Live cell imaging, CD4 T cells, CD8 T cells, α4-integrins
Background that hold the specific molecular keys to breach the BBB
The endothelial blood–brain barrier (BBB) protects the and to enter the perivascular or leptomeningeal spaces
central nervous system (CNS) from the constantly chan- [1]. Mechanisms operating at the BBB are, therefore, in-
ging milieu in the vascular compartment by strictly con- strumental in controlling immune cell migration into
trolling the movement of molecules across its interface. the CNS. Whereas under physiological conditions the
Thus, the BBB also establishes the border between the number of immune cells crossing the BBB is low, during
immune system and the CNS. Immunosurveillance of CNS inflammation such as in multiple sclerosis (MS) or
the CNS is achieved by allowing defined immune cells its animal model, experimental autoimmune encephalo-
myelitis (EAE), a high number of immune cells enter the
CNS parenchyma causing inflammation, edema and de-
*Correspondence:[email protected]
TheodorKocherInstitute,UniversityofBern,Bern3012,Switzerland myelination [2]. Interestingly, even in the inflammatory
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state the BBB still controls immune cell migration into flow conditions that occur in vivo. Thus verification of
the CNS. This is exemplified by the fact that in MS and in vitro findings in experimental animals in vivo is advis-
EAE,myeloidcellsandactivatedmemory/effectorTcells able to overcome limitations of the in vitro system.
preferentially cross the BBB. Thus, the kinetics and mo- Microscopic access to the CNS microcirculation for live
lecular interactions occurring between circulating im- cell imaging has been achieved by the development of
mune cells with the BBB are crucial in the pathogenesis sophisticated cranial and spinal cord window surgical
ofEAE andMS. preparations [7,8]. A cranial window allows direct
In general, the multi-step recruitment of circulating visualization of the leptomeningeal and cortical grey
immune cells across the BBB is regulated by the sequen- matter microcirculation whereas a spinal cord window
tial interaction of various adhesion or signaling mole- provides access to the leptomeningeal and spinal cord
cules on the leukocyte and on the endothelial cell white matter microcirculation [9,10]. We have pioneered
surfaces [3,4]. First, the interaction of adhesion mole- the use of epifluorescence intravital microscopy (IVM)
cules from the selectin family with their cognate carbo- of the spinal cord white matter microvasculature in the
hydrate ligands induces the rolling of the immune cell mouse to investigate in real time the molecular mechan-
along the endothelial cell surface at reduced velocity. isms involved in the multistep extravasation of CD4+
Next, chemokines displayed on the endothelial surface encephalitogenic T cells across the BBB in vivo [9,10].
bind to their respective G-protein coupled receptors These Tcells induce experimental autoimmune enceph-
(GPCRs) on the leukocyte. This triggers the activation of alomyelitis (EAE), an animalmodel for multiplesclerosis
integrins on the immune cell surface via a conform- (MS). Blocking Tcell adhesion to the BBB by the func-
ational change. Activated integrins bind to their endo- tional blocking of α4-integrin, inhibits the development
thelial ligands of the immunoglobulin superfamily and of EAE and is used as a therapeutic approach for the
mediate the firm arrest of the immune cell on the endo- treatmentofMS[10,11].
thelial surface. The arrested immune cell polarizes and Our current insight into the molecular mechanisms
starts crawling on the endothelial surface in search of a involved in immune cell trafficking into the CNS relies
site permissive for diapedesis. Successful migration of a on studies performed with CD4+ Tcells in EAE. Accu-
circulating immune cell across the endothelial cell wall, mulating evidence suggests, however, that CD8+ T cells
therefore, requires the productive interaction of the im- are also critically involved inthe pathogenesis of MS. In-
mune cell with the endothelial cells at each step of the deed, CD8+ Tcells accumulate within active MS lesions,
multi-step recruitment cascade [4]. Because the BBB often outnumbering CD4+ Tcells [12]. Therefore, in this
endothelium is highly specialized, unique dynamics and study protocol we present our investigation on the
molecular mechanisms are required for immune cell mi- multi-step recruitment of CD8+ T cells across inflamed
grationinto theCNS. spinal cordmicrovesselsduringEAEinvivo.
Recently available sophisticated live cell imaging tech- Theaim here istodescribe theinvitro andinvivo live
nologies combined with in vivo surgical window pre- cell imaging approaches we have used to study the dy-
parations that overcome anatomical barriers and with namics and molecular mechanisms involved in the
BBB models in flow chambers in vitro, have provided multi-step T cell migration across the inflamed BBB in
powerful tools to study the cellular and molecular the context of the animal model of MS. We will high-
mechanisms involved in immune cell migration under light the suitability of our in vitro imaging system of the
physiological and pathological conditions. Combining BBB under flow for investigating the molecular mechan-
both techniques in the same laboratory ensures that the isms involved in mediating shear-resistant T cell arrest
number ofanimals usedisminimized. versus T cell crawling or T cell diapedesis across the
Advantages for experiments with in vitro BBB models BBB. In addition we will describe experimental proce-
are high resolution imaging of the endothelium, easy dures and results of studying the migration of CD8+ T
molecularandbiochemicalmanipulations,lessvariability cells across the inflamed BBB by means of intravital
and, last but not least, the possibility of a high through- fluorescence videomicroscopy (IVM)ofthespinalcord.
put ofexperimentalconditions. Using in vitro BBB mod-
els established from different genetically-modified mice, Methods and design
we defined the endothelial cell adhesion molecules me- LivecellimagingofTcellrecruitmentacrosstheBBB
diating post-arrest T cell interactions and especially T invitro
cell crawling against flow on the BBB [5]. As these find- CD4+Tcells
ings were confirmed by others in vivo [6], the in vitro In this study we used the encephalitogenic proteolipid
flow chamber approach has proved meaningful. Never- protein (PLP) specific CD4+ Th1 effector/mem-
aa139-151
theless, the limitations of this experimental approach are ory Tcell line, SJL.PLP7, that has been described in de-
the absence of blood viscosity and of pathophysiological tail before [13]. Tcells were used 3 days after the third
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or fourth re-stimulation with the PLP peptide at limited to an area of 0.28 cm2 by a custom-made silicon
aa139-151
aconcentration of0.5×106cells permL. ring with a diameter of 0.6 cm (Figure 1D). Endothelial
cells are stimulated with recombinant murine tumor
InvitroBBBmodels necrosis factor alpha (TNF-α 10 ng/mL, PromoKine,
The polyoma middle T oncogen immortalized mouse Vitaris) for 16 to 20 h prior to the experiment. For
brain endothelioma cell line (bEnd5) was described in optimal imaging quality, the culture dish has a hydro-
detail before [14,15]. The cells were used between pas- philic foil-like base and excellent optical properties
sages 18 and 25 and cultured for at least 3 days on (μ-dish35mm-low,ibidiVitaris,Baar,Switzerland).To allow
laminin-coated surfaces (Roche, Basel, Switzerland). The fordifferentialinterferencecontrast(DIC)imaging,which
isolation and culture procedures of primary mouse brain relies on glass or a specific DIC-compatible plastic, the
microvascular endothelial cells (pMBMECs) have also field of view (FOV) is covered with glass (Figures 1B
been described in detail before [15-17]. These cells were and C). The flow channel is formed from a central rect-
cultured on Matrigel-coated surfaces (BD Biosciences, angular cut-out in a removable silicon mat. The height
Allschwil, Switzerland) and used as primary cells (pas- of the flow channel is defined by the thickness of the
sage=0) 5–7 days after plating. The yield of pMBMECs silicon mat and the mat is fitted onto the lower surface
fromonemousebrainsufficestoseed3wellswithasur- of the flow chamber which has inlet and outlet tubes
facearea of0.3cm2each. (Figure 1B). Stable mounting of the chamber on the
endothelial monolayer is achieved through two integrated
Invitrolivecellimaging magnets (Figure 1C) exerting positive magnetic pull to-
All animal experiments for in vitro and in vivo experi- wardsametalring,whichisplacedontotheoutersurface
ments were performed in accordancewith the legislation of the base of the culture dish. After the silicon ring is
on animal welfare of the Swiss government and removedfromtheculturedish,theinlettubingoftheflow
approved by the Kanton Bern, Switzerland. To limit the chamber is filled with migration assay medium (MAM)
numbersofmiceneeded tobesacrificedfortheisolation (5% calfserum,10mMHepesinDMEMwithglutamine)
of pMBMECs, we have developed a small custom-made and the flow chamber is placed on the endothelial cells.
flow chamber (Figure 1). Growth area of pMBMECs is Flow is applied by connecting the outlet tubing to a
Figure1Theinvitroflowchamber.Theflowchamberisshownfromtheside(A),fromthebase(B)andfromthetop(C).Whitearrowsin
panelAshowtheinletandoutlettubes.BlackarrowsinpanelsBandCshowthefieldofview.Arectanglewithinthethinsiliconmatvisiblein
panelBsurroundstheinflowandtheoutflowandrestrictsmediumflowtoasmallchamber2mmwideand0.25mmhigh.Whitearrowsin
panelCshowthemagnetsembeddedintotheflowchambertofixthechamberviaametalringopposedonthebaseoftheculturedish.The
cloningringshownwithadiameterof0.6cminimageDrestrictsthesurfaceareaofbrainendothelialcellsto0.28cm2.Scaleisincm.
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syringeautomaticallydrawnupbyaprecisionpump(Har- AnalysisofdynamicTcellinteractionswiththebrain
vard Apparatus, Holliston, MA, USA). The flow rate is endothelium:Tcellarrestandmigratoryphenotype
calculatedaccordingtotheformula: The dynamic interaction of Tcells with the endothelium
is evaluated by assigning a migratory phenotype to each
(cid:1) (cid:3)
Tcell.Tothisend,eacharrestedTcellisassignedadigit
τ dyne=cm2 ¼ð3(cid:2)μ(cid:2)QÞ=ð2(cid:2)a2(cid:2)bÞ
shortly after the accumulation phase (as an example: see
Additional file 3: Movie 3, at time point 8 min 20 sec).
[18] with μ (dynamic viscosity)=0.083 dyne*sec/cm2 The behavior of each individual T cell is analyzed
(DMEM,5%calfserumat37°C[19]); throughout the complete movie and then accordingly
Q (flow)=variable value to be controlled by the pump assigned to one category. Tcells that continuously crawl
incm3/sec; are categorizedas“Crawling”.Tcellsthatdiapedese after
having crawled to the site of diapedesis are categorized
a.(half heightofchamber)=0.125 mm; as “Crawling/Diapedesis”, T cells that detach from the
b.(widthofchamber)=2mm. endothelium are categorized as “Detachment”. T cells
that do not crawl are categorized as “Stationary”
Aspiration of T cells from a reservoir via the inlet (Figure 2A). When dynamic T cell behavior with
tubing is performed at 1.5 dyne/cm2 until the T cells pMBMECsisimagedathigherresolution,additionalcat-
appear in the field of view. T cell interaction with egories can be defined. For example, we added a cat-
the endothelial surface occurs during the accumula- egory “Crawling/partial diapedesis” describing T cells
tion phase, which is started by a reduction of flow which crawled and started but did not completely dia-
to 0.2 dyne/cm2. This allows settling of the T cells pedese duringtheobservationtime (Figure 2B).Arrested
on the endothelial surface, which due to the size of the Tcells that enter or leave the FOV during the recording
flowchamberonlyoccursunderreducedshearconditions. time are excluded from the evaluation. The categories
Theaccumulation phaseisterminated after 4 min as illu- arethenexpressedas%ofarrestedTcells.Todetermine
strated in Movie 1 (12 images/min, Additional file 1) their crawling velocities and crawling distances, all T
and Movie 2 (3 images/min, Additional file 2); or cells categorized as “Crawling” or “Crawling/Diapedesis”
after 8 min in Movie 3 (3 images/min, Additional file 3) are tracked manually, using ImageJ software (National
by increasing the flow to 1.5 dyne/cm2, thus mimick- Institute of Health, Bethesda, MD, USA) using the man-
ing more closely physiological flow conditions within ualtrackingandchemotaxis plugins.
CNS post-capillary venules. Image recording in time-
lapse mode is started at the beginning of the accumu- Invitrolivecellimagingallowsadetailedanalysisofthe
lation phase and continued for 15 to 30 min. dynamicbehaviourofTcellsadherentonthesurfaceof
BBBendothelialcells
Different in vitro BBB models are available for studying
Microscopicequipmentforcomputer-controlledinvitrolive the cellular and molecular mechanisms of Tcell migra-
cellimaging tion across the BBB. We compared the migration of
For microscopic imaging, the assembled flow chamber encephalitogenic T cells across the polyoma middle T
is placed on the stage of an inverted microscope oncogene-immortalized brain endothelial cell line,
(AxioObserver.Z1, Carl Zeiss, Feldbach, Switzerland) bEnd5, to primary mouse brain microvascular endothe-
equipped with a temperature-controlled chamber (37°C). lial cells (pMBMECs) in a static two chamber-based
Image acquisition is performed by computer control assay as described by Röhnelt and colleagues in 1997
using the AxioVision 4 software (Carl Zeiss) at a rate of [20].AlthoughTcelladhesiontobothinvitroBBBmod-
3 or 12 images per min and with a 10-fold (Objective els was comparable, Tcell diapedesis across bEnd5 was
EC “Plan-Neofluar” 10x/0,3 Ph1 M27) (Additional file 1: 4.5 fold more efficient when compared to migration
Movie 1), 20-fold (Objective LD “Plan-Neofluar” 20×/0,4 across pMBMECs within 6 h [15]. This suggests that
Korr Ph2 M27) (Additional file 3: Movie 3) or 40-fold pMBMECs, but less so bEnd5, provide a strict barrier
(Objective LD “Plan-Neofluar” 40×/0,6 Korr Ph2 for Tcell diapedesis as observed in vivo. Since the bar-
M27) (Additional file 2: Movie 2) magnification using rier characteristics of pMBMECs more closely resemble
a monochrome CCD camera (AxioCam MRmRev, the integrity of the BBB in vivo, it is likely that barrier
Carl Zeiss). The size of the image (FOV) acquired characteristics influence the cellular and molecular path-
with the camera depends on the microscope magnifi- ways of Tcell migration across the BBB in vitro. There-
cation and is 653 μm × 869 μm for 10-fold magnifi- fore we continued to study the molecular mechanisms
cation, 438 μm × 329 μm for 20-fold and 215 μm × involved in this process by employing pMBMECs
162 μm for 40-fold. derived from mice deficient for intercellular cell
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Figure2ThemigratoryphenotypeofTcells.RepresentativeexperimentsofTcellinteractionswithTNF-αstimulatedpMBMECsinvitrounder
flowconditionsduringaperiodof15minutes(2A)orfor3differenttimeperiodsof10,15or20minutes(2B).ThebehaviorofeacharrestedT
cellwasanalyzedbyeyeinanofflineanalysisofthetime-lapsevideosandassignedtoonecategoryandexpressedinpercentageofinitially
arrestedTcells.ArrestedTcellsthatcrawledintooroutoftheFOVduringtherecordingtimewereexcludedfromtheanalysis.“Crawling”:Tcells
thatpolarizedandcrawledatleasttwoTcelldiameterdistancebutdidnotdiapedeseacrosstheendothelium.“Crawling/partialdiapedesis”:T
cellsthatpolarized,crawledandstartedbutdidnotcompletediapedesisduringtheindicatedtimeperiod.“Crawling/Diapedesis”:Tcellsthat
polarizedandcrawleduntiltheyfinallycrossedtheendothelialcellmonolayer.“Detachment”:Tcellsthatdetachedduringtheevaluationperiod.
“Stationary”:Tcellsthatdidnotpolarizeandremainedstationary.2A:Experimentimagedwith10xobjective.Atotalof64cellswerecategorized.
2B:Experimentimagedwith20xobjective.Atotalof37cellswerecategorized.
adhesion molecule (ICAM)-1 and ICAM-2 (ICAM-1/ monolayer (Additional file 1: Movie 1, Additional file 2:
ICAM-2 dKO) and pMBMECs derived from wild type Movie 2). The velocity of crawling on TNFα-stimulated
(wt) mice. There was a dramatic reduction of Tcell dia- pMBMECs is about 4 μm/min, and preferentially against
pedesis across either ICAM-1 KO or ICAM-1/ICAM-2 the direction of flow [15]. Evaluation of the dynamic be-
dKO pMBMECs when compared to wt pMBMECs [5]. havior of T cells while adherent to the endothelial sur-
A disadvantage of the static two-chamber setup is that it face is qualitatively and quantitatively analyzed such that
does not discriminate between the involvement of endo- all arrested Tcells are counted and set to 100% and the
thelial ICAM-1 in T cell adhesion to the BBB versus T 4 categories “Crawling”, “Crawling/Diapedesis”, “Detach-
cell diapedesis across the BBB. Therefore, we extended ment” and “Stationary” are expressed as fractions of
ourexperimentalportfoliotoaninvitrolive cellimaging initially-arrested Tcells. Figure 2A shows one represen-
method that enables visualization of the multistep Tcell tative experiment using encephalitogenic CD4+ Th1 T
extravasation across in vitro BBB models under condi- cells and TNFα-stimulated pMBMECs over an observa-
tionsofphysiologicalflow. tion period of 15 min. In this experiment, 64% of Tcells
Although flow chambers are commercially available, continuously crawled, 27% crawled and diapedesed, 1%
wedevelopeda small-sizedflow chamber(Figure 1)suit- detached from the endothelium and 7% remained sta-
able for a small area of cultured brain endothelial cells tionary without crawlingduringtheobservation period.
and for the low number of pMBMECs obtained from To determine how recording time affects the dy-
each isolation procedure. Using this flow chamber we namic interaction of T cells with pMBMECs under
visualized the dynamic behavior of encephalitogenic flow in vitro, we analyzed the migratory phenotype
CD4+ T cells while adherent on the apical surface of at three time points: 10, 15 and 20 min (Movie 3
pMBMECs. Whereas many Tcells arrest on the surface shows 20 min, Additional file 3). As shown in
under low shear stress, non-bound T cells are readily Figure 2B, 10 min recording resulted in 62% con-
washed away when the shear stress is increased to tinuously crawling T cells, whereas 20 min recording
physiological conditions. However, most of the T cells reduced this to 43%. This reduction was offset by an
which resist detachment after increase of shear remain increase in the fraction of T cells that completely
adherent throughout the remaining observation period. diapedesed across the monolayer from 11% at
These Tcells polarize within seconds and start crawling 10 min to 43% after 20 min. Thus, recording times
on the endothelial surface. Crawling occurs either con- must be carefully chosen and strictly maintained
tinuously throughout the complete recording period, or during an experimental series to allow for compar-
is followed by diapedesis across the endothelial able data analysis.
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Employingthisinvitrolive cellimagingsetup,wehave scoring system as follows: 0, healthy; 0.5, limp tail; 1,
compared T cell interactions on pMBMECs with those hind leg parapesis; 2, hind leg paraplegia; and 3, hind leg
on bEnd5 cultures [15]. Thisshowed that Tcells need to paraplegiaandincontinence. Mice suffering from clinical
crawl long distances on pMBMECs, preferentially scores 0.5 (limp tail) to 2 (hind leg paraplegia), with a
against the direction of the flow, to find sites permissive body weight of at least 15 g were used as recipients for
for diapedesis. However, they readily cross a monolayer IVMexperiments.
of bEnd5 cultures [5]. This supports the suggestion that
the integrity of in vitro BBB models impacts on T cell
CD8+Tcellisolation
migrationacrosstheBBB.Tcellcrawlingagainstthedir-
CD8+ Tcells were prepared from Tcell receptor (TCR)
ection of the blood flow is a unique behavior of ence-
transgenic C57BL/6 mice in which CD8+ T cells
phalitogenic T cells when crossing inflamed spinal cord
recognize the immunodominant MHC class I (H-2Kb)
microvessels during the onset of EAE in vivo [6]. Hence,
epitope of chicken ovalbumin (SIINFEKL). Spleen and
our in vitro live cell imaging setup can be used to study
lymph nodes were collected from OT-I mice, cut in
the cellularandmolecularmechanismsinvolvedinTcell
piecesanddigested30minat37°Cin5mLRoswellpark
migration into the CNS. To this end we analyzed the
memorial institute (RPMI) medium supplemented with
role of endothelial ICAM-1 and ICAM-2 in this process.
DNAse I (0.2 mg/mL; Boehringer Manheim, Germany)
Using pMBMECs from wt and ICAM-1/ICAM-2 dKO
and liberase CI (0.4 mg/mL; Roche Applied Sciences,
mice, we found that whereas Tcell arrest on pMBMECs
Switzerland). Afterwards, digested organs were crushed
is mediated by endothelial ICAM-1 and VCAM-1,endo-
between 2 sterile glass slides. The resulting cell suspen-
thelial ICAM-1 and ICAM-2 are essential for T cell
sion was then filtered through a sterile 100 μm-nylon
polarization and crawling on brain endothelium under
mesh and centrifuged for 10 min at 250 g. Cells, 7.5×
flowinvitro [5].
106 per 60 mm diameter petri dish, were plated in cul-
Combining in vitro pMBMEC preparations from
ture medium (RPMI-1640 supplemented with 10% FBS,
genetically-modified mice with live cell imaging under
2 mM L-glutamine, 1 mM sodium pyruvate, 100 U peni-
flow, can identify cellular and molecular mechanisms
cillin-streptomycin, 0.05 mM 2-mercaptoethanol) and
involved in the multi-step Tcell migration into the CNS
50 μg SIINFEKL peptide (OVA- peptide ; Peptides
in the context of neuroinflammatory diseases. Observa- 257-263
international,Louisville,KY,USA)wereadded.Cellsuspen-
tions made in vitro [5] can be verified in vivo [6]. This
sionswereincubatedat37°Cin7%CO for5days.Onday
experimental setup can provide valuable insights into 2
4,IL-2(5ng/mL;R&DSystems,Abingdon,UK)wasadded
the molecular mechanisms directing transcellular or
overnight in each dish. Then freshly-activated live CD8+
paracellular diapedesis of T cells across the BBB. It can
OT-I T cell blasts were isolated by Nycoprep 1.077 A
also be used to study the multi-step migration of other
(Axis-Shield,Dundee,UK)densitygradientcentrifugation.
immune cell subsets such as neutrophils, monocytes or
CD8+Tcells acrosstheBBB.
FluorescentlabelingofTcells
Livecellimagingofimmunecellrecruitmentacrossthe After 3 to 4 days in culture, OT-I T cells were labeled
BBBinvivo:Intravitalfluorescencevideomicroscopy(IVM) with 2.5 μM Cell Tracker™ green (CMFDA; Molecular
Recipientmiceandinductionofactiveexperimental probe, Oregon, USA) in culture medium (RPMI-1640
autoimmuneencephalomyelitis supplemented with 10% FBS, 2 mM L-glutamine, 1 mM
C57BL/6 female mice, 8–12 weeks old, with an approxi- sodiumpyruvate,100Upenicillin-streptomycin,0.05mM
mate body weight of 20 g were used in accordance with 2-mercaptoethanol) for 45 min at 37°C in the dark. Cells
the local government legislation on animal welfare and weresubsequentlywashedbyaddingfreshcompletewash
experimentation. EAE was induced by sub-cutaneous buffer (HBSS supplemented with 5% FCS and 25 mM
immunization with 200 μg of myelin oligodendrocyte HEPES) and centrifuged 10 min at 250 g. Excess dye was
glycoprotein peptide (MOG ) in incomplete removed from the Tcells by plating 5×106 fluorescently
aa35-55
Freund’s adjuvant (IFA; Santa Cruz, USA) supplemented labeled cells in a 100 mm petri dish in10 mL culture
with 4 mg/mL nonviable, desiccated Mycobacterium tu- mediumfor30minat37°C.Celltracker™green-labeledT
berculosis (H37RA; Difco Laboratories, Detroit, USA) cells were directly used for IVM or stored in complete
exactly as described before [10]. On day 1 and 3 post- mediumat37°Cand7%CO upto6hbeforeuse.Inpar-
2
immunization, 300 ng pertussis toxin from Bordetella allel with the spinal cord window microsurgery, 5-6×106
pertussis (LuBioScience, Lucerne, Switzerland) per of Cell trackerTM green-labeled immune cells were col-
mouse were injected intra-peritoneally. Assessment of lectedandcentrifugedfor10minat250g.The cell pellet
clinical disease score and weight of mice with active was then re-suspended in a small volume of NaCl 0.9%
EAE was evaluated twice a day using a four-point isotonic solution. Cells were counted and the volume of
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NaCl 0.9% isotonic solution was adjusted to obtain a cell trauma to the microvasculature and underlying spinal
suspension of 4×106 cells in 300 μL. The Tcell suspen- cord parenchyma. The preparation was then covered
sionwasfilledintoa1mL-syringereadyforinjectioninto with a transparent plastic membrane to prevent dehy-
themousecirculation. drationandaccess ofambientO tothe exposed tissue.
2
Microsurgicalpreparationofthespinalcordwindow Intravitalfluorescencevideomicroscopy(IVM)
Micewereanaesthetizedbysubcutaneousinjectionofketa- The animal remaining within the stereotactic head
mine-hydrochloride/xylazine(100mg/kgand5.6mg/kgre- holder was transferred to the stage of the inverted fluor-
spectively), followed by a subcutaneous injection of escence microscope (Figure 3). IVM was performed by
acepromazine (1.5 mg/mL). Throughout the experiment, epi-illumination techniques using a custom-made
anaesthesia of the animals was carefully monitored and, if Mikron IVM500 microscope (Mikron Instruments, San
necessary,ahalfdosewasinjectedtomaintaindeepanaes- Marcos, CA, USA) coupled with a 50 W mercury lamp
thesia.DuringthesurgicalprocedureandIVMexperiment, (HBO 50 microscope illuminator, Zeiss, Switzerland)
bodytemperaturewasmaintainedbyplacingtheanimalon attached to combined blue (exciter 455DF70, dichroic
a thermo-controlled heating pad to prevent hypothermia 515DRLP, and emitter 515ALP) and green (exciter
which would influence blood supply to the brain and the 525DF45, dichroic 560DRLP, and emitter 565ALP) filter
hemodynamicparametersofthecirculation. blocks. The microscope is connected to a low-
Under the stereomicroscope, the right common ca- light-imaging silicon-intensified target (SIT) camera
rotid artery was catheterized in the direction of the aor- (Dage-MTI Inc., Michigan city, IN, USA) coupled with a
W
tic arch for systemic infusion of fluorescently-labeled T Trinitron color video monitor (Sony, Switzerland) and
cells and 1% tetramethylrhodamine isothiocyanate a videotimer (MicroImage Video Systems, Boyertown,
(TRITC)-conjugated Dextran used as a plasma marker. USA). For later real time off-line analysis, images
Afterwards, the animal was turned to the prone position were recorded using a digital videocassette recorder
and the head was placed in a stereotactic holder. The (VCR) (Figure 3). Observations were made using a
midline skin of the neck was incised for 2–3 cm and the ×4,×10 and×20 long-distance objectives (Zeiss,
paravertebral musculature was dissected from the cer- Switzerland), resulting in×80, ×215 and×440 mag-
vical spine processes and retracted laterally by the use of nifications, respectively.
4–0 threads, exposing the vertebral lamina. A laminec- First, the microvasculature of the spinal cord was
tomy was then carried out from C7 to C2 and the dura observed within the green light epi-illumination (×4 ob-
mater over the spinal cord was removed avoiding any jective) by intra-carotid injection of the pre-warmed
Figure3Experimentalsetupoftheintravitalfluorescencevideomicroscopyworkstation.Theanimalpreparationunderanesthesiais
placedunderanepifluorescencemicroscope,coupledwithamercurylampconnectedtoalow-light-imagingsilicon-intensifiedtarget(SIT)
camerathatincludesanimageprocessor,associatedvideotimer,adigitalvideocassetterecorder(VCR)andavideomonitor.Forlateroff-line
analysis,realtimevideoswererecordedusingadigitalvideocassette.A:Evaluationoftheinitialcontactfraction(%)ofOT-ICD8+Tcellswiththe
postcapillaryvenules(20–60μmdiameter)ofthespinalcordmicrovasculatureofmicewithEAEB:Showstheevaluationofcaptureandrolling
fractions(%)ofOT-ICD8+Tcellswiththepostcapillaryvenules(20–60μmdiameter)ofthespinalcordwhitemattermicrovasculatureofmice
afflictedwithMOG -inducedEAE.
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fluorescent plasma marker TRITC-conjugated Dextran culture supernatants. Endotoxin levels, determined using
(1%, MW=155,000; Sigma-Aldrich, Switzerland) in 0.9% Endosafe test (Charles River Laboratories, Sulzfeld,
isotonic NaCl. The spinal cord is divided into two parts Germany), were below detection level. Endotoxin-free rat
by the middle dorsal vein, delineating a top and bottom IgG2bwasusedasisotypecontrol.
half of the entire window. On both sides, capillaries and
post-capillary venules draining into the middle dorsal QuantitativeanalysisoftheIVMdata
vein can readily be visualized. Between 4 and 6 stepwise Initial contact of circulating T cells within post-
FOVs per animal could be delineated on each side of the capillary venules of the spinal cord white matter in
spinal cord window (10× objective). Using the blue light mice with active EAE. From each observed post-
epi-illumination (10× objective), 4×106 Cell Tracker™ capillary venule (diameter=20–60 μm), the percentage
green-labeled activated OT-I CD8+ T cells were slowly of T cells initiating contact with the BBB endothelium
infused in 3 aliquots of 100 μL and were directly as observed by IVM was determined at the time point
observed within the spinal cord microcirculation where of cell injection. The total number of T cells was
they initiated contact with the inflamed spinal cord injected in 3 aliquots and 1 FOV was visualized for
white matter endothelium. For each injection of 100μL, each injection. Thus the initial interaction of circulating
a different FOV was recorded for a minimum of one mi- T cells could be analyzed in a substantial number of
nute in order to observe sufficient CD8+ Tcells interact- spinal cord post-capillary venules per animal. The num-
ing with the endothelium for later off-line analysis. After ber of T cells (> 10 cells/min) rolling along the vessel
the infusion of each aliquot, the arterial catheter was wall or captured (abruptly arrested without any prelim-
flushed with60to80μLpre-warmedisotonic0.9% NaCl inary rolling step) was counted per post-capillary ven-
to guarantee all cells were injected. At different time ule, and related to the total number of fluorescent
points after cell injection (10 min, 30 min and 1 h), all circulating T cells (total cellular flux, TFx) passing
fields of view of the spinal cord window were sequen- through the vessel during one minute. The rolling frac-
tially scanned and recorded for further evaluation of the tion (RF) or the capture fraction (CF) was calculated
number of permanently adhering fluorescent CD8+ T and the total initial contact fraction (ICF) calculated
cells. At the end of the recording period, animals were from the sum of RF and CF (summarized in Table 1).
sacrificed. Both rolling and capture events were confirmed by cal-
culating the critical velocity in μm.s -1 (V ). V is the
crit crit
velocity of an idealized cell traveling along, but not
TargetingofcellsurfaceadhesionmoleculesonBBB interacting with the vessel wall. It can be derived from
endothelium the parabolic velocity profile of the circulation in the
In order to evaluate the involvement of a specific adhe- microvessel, as follows:
sion molecule or itsligandinTcelltraffickingacrossthe
spinal cord microvasculature endothelium in vivo, acti- Vcrit ¼Vblood(cid:3)ðDL=DVÞ(cid:3)½2(cid:4)ðDL=DVÞ(cid:5)
vated T cells or the BBB endothelium were pre-treated
with function-blocking antibodies. To this end, 4×106 in which D and D correspond to the diameter (mm)
L V
Cell TrackerTM green-labeled CD8+ Tcell blasts in 300 of the leukocyte and the diameter of the post-capillary
μL isotonic 0.9% NaCl solution were incubated with venule, respectively, and V corresponds to the
blood
120 μg blocking monoclonal antibody (mAb) directed mean blood flow velocity (summarized in Table 1). Any
against a specific adhesion molecule for 20 min prior to leukocyte circulating below V was considered as an
crit
their injection into the bloodstream. The use of interacting cell rolling along the vessel wall, whereas
antibodies in vivo requires endotoxin-free antibody any cell traveling above V was defined as a non-
crit
preparations and appropriate isotype controls. Using interacting cell [21,22]. Statistics using Mann–Whitney
non-blocking antibodies from the same isotype as the U-Test to compare 2 variables and Kruskall-Wallis to
blocking mAb, ensures against nonspecific side effects compare more than 2 variables were then performed.
mediated by the Fc portions of the immunoglobulins.
Control antibodies specific for molecules expressed on Firm adhesion of Tcells within inflamed spinal cord
the surface of circulating immune cells or on the BBB post-capillary during EAE. Firmly adherent T cells
endothelium,whichdonotinterferewithTcelltrafficking were identified as fluorescent cells that stick to the
are preferable over non-binding irrelevant isotype control vessel wall without movingordetaching.Trapped Tcells
antibodies remaining in the circulation. In this study, rat- within the capillary network, were defined as cells that
anti-mouseα4-integrin(PS/2),ratanti-mouseα4β7integ- do not move and clearly obstruct the capillary lumen,
rin (DATK-32), and rat anti-mouse β7 integrin (Fib 504) resulting in blood flow stasis. The permanent adhesion
were used and obtained from serum-free hybridoma of Tcells at 10 min, 30 min and 1 h after infusion was
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Table1Parametersanalyzedbyintravitalmicroscopy(modifiedfrom[22])
Parameter Unit Formula
TotalCellularFlux(TFx) min-1 NumberofTcellsthatpassthroughavesselduringagivenobservationperiod
InitialContactFlux(ICFx) min-1 Numberofimmunecellsthatinteract,eitherbyrollingorgettingcapturedto,withthevesselwall
duringanobservationperiod
InitialContactFraction(ICF) % ICF=ICFx/TFx×100
RollingFlux(RFx) min-1 Numberofimmunecellsthatrollalongthevesselwallduringanobservationperiod
RollingFraction(RF) % RF=RFx/TFx×100
CaptureFlux(CFx) min-1 Numberofimmunecellsthatarecapturedtothevesselwallduringanobservationperiod
CaptureFraction(CF) % CF=CFx/TFx×100
FirmAdhesion/FieldOf none NumberoffirmlyadherentTcellsperonefieldofview(FOV)withinthespinalcordwindow
View(FOV)
expressed as the number of adherent and trapped Tcells spinal cord vascular system by injection of TRITC-dex-
per field of view (FOV) observed with the×10 objective tran, OT-I T cells were systemically infused via the
[23]. As 4–6 FOVs could be identified on each side of right carotid artery and their interaction with the spinal
the spinal cord window, all calculations of firmly ad- cord microvasculature observed and recorded in real
herent T cells per FOV from different mice were time (Figure 3, Additional file 4: Movie 4 and Additional
grouped to calculate the mean +/− standard deviations file5:Movie 5). The initial contact (rolling and capture)
foreachanimal.StatisticsusingMann–WhitneyU-Testto and firm adhesion of OT-I T cells to the spinal cord
compare2variablesandKruskall-Wallistocomparemore vasculature were evaluated by off-line frame-by-frame
than2variablesarethenperformed. video analysis. The following conditions were studied:
rat IgG2b used as a control antibody, PS/2 (anti-α4
Contributionofa4β1-versusa4β7-integrinintheinteraction subunit), DATK-32 (anti-α4β7 integrin) and Fib 504
ofCD8+TcellswiththeinflamedBBBinvivo (anti-β7 subunit). Upon systemic infusion, activated
Blocking Tcell entry into the CNS with the humanized OT-I T cells were observed to pass through the spinal
anti-α4 integrin antibody, natalizumab, has proved effi- cord microvessels and initiate contact with the inflamed
cient in the treatment of relapsing-remitting multiple CNS endothelium (Additional file 4: Movie 4). Contact
sclerosis [11]. However, natalizumab is associated with initiationwasmediatedeitherbyOT-ITcellsrollingwith
an increased risk for developing progressive multifocal reduced velocity along the vascular wall or to a lesser
leukoencephalopathy, a fatal disease of the CNS caused degree by capture, i.e. an abrupt arrest of the CD8+ T
by JC virus infection of oligodendrocytes [24]. This ob- cells on the vascular wall. Pre-treatment of OT-I Tcells
servation suggests that therapeutic targeting of α4-integ- with either isotype control mAb or blocking antibodies
rins may ultimately impair immunosurveillance of the against α4-, β7- or α4β7- integrins showed no effect on
CNSbycytotoxic CD8+Tcells. their intrinsic abilities to initiate contact with the
To investigate if CD8+ Tcells use molecular mechan- inflamed BBB endothelium (Figure 4A), either by rolling
isms similar to CD4+ Tcells to migrate across the BBB or capture to the spinal cord microvasculature wall
in vivo, we studied the interaction of CD8+ OT-I Tcells (Figure 4B). To determine whether the initial contact of
with the inflamed spinal cord white matter microvascu- OT-I T cells resulted in arrest and firm adhesion to the
lature in C57BL/6 mice during EAE to determine if inflamed microvasculature (Additional file 5: Movie 5),
CD8+ T cells also use α4β - but not α4β7-integrins to the number of OT-I Tcells permanently adhering within
1
adhere to the inflamed BBB as previously shown for microvessels at different time points (10 min,30 min and
CD4+ T cells [25,26]. The purity of CD8+ OT-I T cell 1 h) after T cell infusion for each tested condition was
preparations was confirmed by FACS staining, which measured(Figure5).Tenminutesafterinfusion,theinhib-
demonstrated that 95% of the OT-I Tcell blasts stained itionofα4-integrinsresultedina50%reductionofthefirm
positive for CD8, an acceptable purity for performing adhesion of OT-I T cells to the microvasculature when
IVM (data not shown). Prior to their infusion into the compared to IgG2b isotype control treatment, whereas
circulation of the recipient mouse, fluorescently-labeled blocking of α4β7- or β7-integrins only reduced the adhe-
OT-I Tcell blasts were pre-treated with integrin block- sionofOT-ITcellsby30%.Thesedatasuggestedthatboth
ing or control antibodies (480 μg Ab/ 4 × 106 OT-I T α4-integrins mediate OT-I adhesion to the inflamed spinal
cells/ 400 μl with the exception of DATK-32, which cord microvasculature. Interestingly, the involvement of
was used at 960 μg/ 4 x 106 OT-I T cells/ 400 μl due α4-integrins in mediating OT-I T cell adhesion to the
to its low affinity). Following the visualization of the inflamed BBB was only transient, as at the later times
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Figure4QuantificationofOT-ICD8+Tcellinteractionswiththespinalcordmicrovasculatureinvivo.A:Evaluationoftheinitialcontact
fraction(%)ofOT-ICD8+Tcellswiththepostcapillaryvenules(20–60μmdiameter)ofthespinalcordmicrovasculatureofmicewithEAE.Each
dotrepresents1venule.Allvaluesshowthemedianwiththeinterquartilerangeofn=22analyzedpost-capillaryvenulesfrom3miceforrat
IgG2bcondition,n=18analyzedpost-capillaryvenulesfrom5miceforanti-α4β7condition,n=18analyzedpost-capillaryvenulesfrom6mice
foranti-β7conditionandn=23analyzedpost-capillaryvenulesfrom4miceforanti-α4condition.B:Showstheevaluationofcaptureand
rollingfractions(%)ofOT-ICD8+Tcellswiththepostcapillaryvenules(20–60μmdiameter)ofthespinalcordwhitemattermicrovasculatureof
miceafflictedwithMOG -inducedEAE.N=22analyzedpost-capillaryvenulesfrom3miceforratIgG2bcondition,n=18analyzedpost-
35-55
capillaryvenulesfrom5miceforanti-α4β7condition,n=18analyzedpost-capillaryvenulesfrom6miceforanti-β7conditionandn=23
analyzedpost-capillaryvenulesfrom4miceforanti-α4condition.StatisticalsignificancewasdeterminedbytheMann–WhitneyU-Test.
adhesion of OT-I T cells was no longer inhibited by the Statisticalanalysis
presenceofα4-integrinblocking antibodies.Atthesetimes All statistical analysis were performed using the Graph-
there were lower numbers of OT-I cells firmly adhering Pad Prism software (version 5.00, GraphPad Software,
undercontrolconditions.Theseresultssuggestthatduring CA,USA).Dataarepresentedasmeanvalues+/−stand-
EAE, activated CD8+ T cells interact with the inflamed ard deviation (SD). Mann–Whitney U-Tests were used
BBB. In contrast to CD4+ T cell blasts, CD8+ T cells are for comparisons between different data sets. Asterisks
able to initiate contact and to maintain stable adhesion to indicate significant differences (*P<0.05, **P<0.01
theinflamedBBBindependentofα4-integrins[10,25]. and ***P<0.005).
Figure5QuantificationofOT-ICD8+TcellfirmadhesiontothepostcapillaryvenulesofthespinalcordmicrovasculatureofC57BL/6
miceduringEAE.PermanentlyadheringOT-ITcellswerecounted10min,30minand1houraftercellinfusion.Eachdotrepresentsthe
numberofadherentOT-ITcells/fieldofview(FOV).Thenumbersofmiceanalyzedatt=10minforeachconditionwasn=8forratIgG2b,n=
6foranti-α4β7,n=6foranti-β7andn=8foranti-α4.Att=30,n=8forratIgG2b,n=6foranti-α4β7,n=6foranti-β7andn=4foranti-α4.
Attimet=1h,thenumberofmicewasn=7forratIgG2b,n=5foranti-α4β7,n=5miceforanti-β7andn=5foranti-α4.Dataare
presentedasmeanvalues+/−standarddeviation(SD).Mann–WhitneyU-Testwasusedforcomparisonsbetweendifferentdatasets.Asterisks
indicatesignificantdifferences(*P<0.05,and***P<0.005),n.s.:notsignificant.