Greenwood J Howes R Lightman SThe blood

0023 - 6837 / I 4 / 7 001 -0039$03.00/0 L.n sonAronY INvESTIGATIoN

Copyright @ 1994 by The United States and Canadian Academy of Pathology, Inc.

Vol. 70, No. 1, p. 39, 1994 Printed in U.S.A.

The

Blood-Retinal

Barrier

in

Experimental

Autoimmune

lJveoretinitis

Leukocyte Interactions

and

Functional

Damage

J.

GnnpNwooD,

R. Howns,

AND S.

Ltcsrue,N

Department

of

Clinical

Science,

Institute

of Ophthalmology,

Bath

Street, London

ECLV gEL,

UK

BACKGROUND:

In

posterior uveitis the blood-retinal

barrier

(BRB) plays an important role in

the pathogenesis of the disease. However, the morphologic correlate of BRB breakdown and the

route of leukocyte migration remain poorly defined.

EXPERIMENTAL DESIGN: Using an experimental model of autoimmune uveoretinitis in the rat,

we have examined the ultrastructural alterations and leukocyte interactions occurring at the BRB.

By

employing electron-dense tracers, the development

of

BRB breakdown, and the route of extravasation were investigated.

RESULTS: No increase in BRB permeability was found before lymphocytic infiltration. At day 1O

postimmunization with retinal-soluble antigen and beyond, inflammatory cells could be seen within

the retina that was quickly followed by an extensive increase in the permeability of the retinal

vasculature to lanthanum and horseradish peroxidase. Occasionally, horseradish peroxidase re-action product could be seen extending throughout the 'tight junctions' of the retinal endothelia, but not those of the retinal pigment epithelia. Inflammatory cells, particularly mononuclear cells,

were seen forming perivascular cuffs and extending posteriorly towards the outer retina. Retinal

damage was

initially

restricted to the outer nuclear and photoreceptor layers that were

in

close

proximity to these vessels. Leukocytes could be seen adhering to the retinal vessels and penetrating

the endothelial cell cytoplasm close to tight junctions, but were never seen probing the junctions

directly.

At

the retinal pigment epithelium, however, there was

little

evidence of migration into the retina during the early stages of the disease, even though the choroid often became packed

with inflammatory cells. At later stages, oceasional inflammatory cells could be seen between, and

apparently

within,

retinal pigment epithelium cells

in

areas overlying sites of severe choroidal

infiltration.

CONCLUSIONS: The prime site of leukocyte

infiltration

and damage to the BRB in autoimmune

uveoretinitis occurred

at

the level

of

the vascular endothelia and

that

diapedesis takes place

primarily via an intraendothelial process.

Additional key words: Endothelium, Lymphocyte, Migration.

The blood-retinal barrier (BRB) forms a selective

cell-ular interface between the blood and the retinal

paren-chyma and

is

functionally identical

with that of

the blood-brain barrier (BBB). The retinal vascular

endo-thelium and retinal pigment epithelium (RPE) that form

the BRB restrict the movement of polar solutes, and also

play a significant part

in

controlling leukocyte

extrava-sation. Under normal conditions, the level of leukocyte

traffic through the retina is low, but becomes markedly increased during inflammatory diseases

of

the

retina.

This increase

in

cellular

infiltration is

also associated with breakdown of the

BRB

(1) and edema formation

(2). These two separate but related processes play a major part in the pathogenesis of both human posterior uveitis,

and the animal model of this disease, experimental

au-toimmune uveoretinitis (EAU) (3).

Adoptive transfer studies have shown that EAU is a

CD4* T cell-mediated disease and thus is similar in many

respects to the animal model of multiple sclerosis,

exper-imental autoimmune encephalomyelitis (EAE) (4). The inflammatory cells that mediate these diseases must first be recruited from the circulation via interactive

mecha-nisms

with

the vascular endothelium.

This

process is

thought

to

be regulated

by the

level

of

expression of

adhesion molecules on both the leukocyte and

endothe-lium and the degree of avidity between the receptor pairs

(5-9). Evidence also exists for endothelial cell (EC)

GREENWOOD, HOWES, AND LIGHTMAN LesoRlrony ltvpstrcltror.r

vein (HEV)-like endothelia (1, 11-19) in both EAE and

EAU, although

it

is not clear whether this phenomenon

plqys a _{significant role in leukocyte recruitment.}

The retinal vascular endothelii is in close contact with circulating inflammatory cells unlike the RpE which is

separated from the blood

by

the choroidal vessel wall

and Bruch's membrane. As a consequence of this ana_

tomical arrangement,

it

_{is likely that the retinal endo_} thelia is a prime site_of leukocyte recruitment and entry, particularly during the early stages of inflammatory

dis-ease (1, _{3). For circulating leukoiytes to enter the retina} across the retinal vasculature, they must first adhere to

the endothelium followed by diapedesis and migration through

the

extracellular space

of

tne parenchlima.

It

still

remains unclear, however, _{whether leukocvtes mi_}

grate through

the tight

junctions _{between endothelial}

cells _{or via an intracellular route.}

It

_{is possible that the}

path of migration is dependent _{upon the} cell phenotype

and state of cell activation, and that both iniercelluLr

and intracellular routes of diapedesis occur. For leuko_ c.ft9s t_o cross the posterior aspect of the BRB, the RpE,

their flow must

first

be halted by interactions with the choroidal vascular endothelium, followed

by

adhesion and migration out into the extracellula..pu.".

It

_{is only}

when they have crossed the choroidal vasculature thai

they are able

to

interact

with

the RpE. Recruitment,

therefore, at this site is governed not by the RpE, but by

the choroidal endoth_elia. The migration of inflammatory

cells across _{the RPE, and the route they take, has noi}

been _{well documented, although there is sbme ultrastruc_} tural evidence to suggest that migration into the retina from the choroid does occur (10, 14). The mechanisms

involved in inflammatory cell _{extravasation in EAU still}

requires further investigation and the differential role

played by the two barrier sites in the pathogenesis of this

disease need clarifying.

The alteration

in

functional integrity of the BRB, as

a result of both cellular

infiltration

and the release of

vasoactive substances, is a well established phenomenon

that c-linically leads to edema and loss of vision (1b). _The

path by which extravasation of plasma constituents oc_

curs, needs to be defined. _{Unlike that of the BBB, there}

are two major areas of the BRB across which leakage

may occur. Furthermore, the surface area of these t#o

barrier sites _{is collectively much greater than an equiv_}

alent area of cerebral cortex (16),

ind

_{thus has u}

gr""t.,

potential for leakage _{of plasma constituents andidema}

formation. A further question regarding BRB disruption,

as

with that of

the BBB,

is

the

meihanism through

w^high leakage occurs (17). _{Whether this} is via disrupti6n

of

the

tight

junctions, pole formation,

or

transcytosis

remains a contentious issue.

EXPERIMENTAL

DESIGN

, This study was undertaken to investigate the related

phenomena _{of leukocyte extravasatior,}

""rrd BRB break_

down

in

EAU

in

rats and to assess the differential role

ql-ayed by the two sites of the BRB

in

these processes.

The _{_temporal sequence of events}

in

_{rats induced with}

EAU by systemic immunization with bovine retinal S_

antigen was _{determined ultrastructurally with transmis_}

sion and scanning electron microscopy (EM). _Retinal

vascular endothelia were examined for changes

in

mor_

phology,

in

_{particular thickened EC with inireased}

cy_

to^.ljlq _{_otgg"elle} s, and plump morpholo gy characteristic

of _{HEV. The route of leukoiyte} passage-ac.oss _{both the}

retinal

endothelia and

the RpE

_{was investigated Ly}

examining

the

structural interactions between thes-e

cells. Furthermore,

by

using electron-dense vascular

tracers_, the integrity of the two barrier sites and the path through which tracer extravasation occurs was deter_

mined.

RESULTS

AND

DISCUSSION

Llcur

Mrcnoscopv

Toluidine blue-stained sections from the eyes of all

rats were inspected for structural changes. Up io day 10,

all_Tetina appeared histologically

,ror*il

with no

.ig";i

infiltrating-leukocytes. By- _{day-10, a few}

inflam.itory

cells were observed _{particularly in lhe outer nuclear}

ani

photorec.eptor layers,

-t!:

t_qttdr being the known turg"i

of S-antigen-mediated EAU (19). _{Fr6m day 10 onwa"rd,}

there was increased _{extravasation of inflammatory cells}

within the neuroretina with notable perivascular

*fn"g

particularly of the venules, and,ru*eious cells ."igraii;E

throughout the parenchyma (Fig. 1o). The

irfl";;;;r;

cells were predominantly mononuclear and could be seei tracking from retinal vessels into the outer retina

(Fij

1b). As the disease progressed, _{destruction of the outei}

retinal layers occurred particularly

at

points

in

close

proximity to retinal vessels (Fig. 1). Focal accumulation

of inflammatory cells in the choiiocapillaris became more

evident _{with time but with limited e,oiderrce of migraiion}

of cells across _{the RPE. The overall} progression _{of the}

9]:"1.^q was. generally similar to that repoited previously (11, 19) with increasing outer retinal destruction and

thl

formation of

vitritis

and

a

subretinal

..rohu.-orr"gi"

exudate. The disease finally became quiescent _{in the 4"th}

week _{postimmunization (PI), leavingsevere destructi,on}

of the outer layers of the retina, a limited focal inner

layer damage, and little evidence of further

i"nam-aiory

cell infiltration.

ElncrnoN

MrcRoscopy: _{MoRpnor,ocy oF} THE

RnrrNer,

EC

,qNo RpE

_

Th"

injected control animals displayed normal retinal

pC m9rylology throughout the uasc.rja. bed during lhe

4-week _{PIperiod, with no extravasation of inflammitory} celts. _{Similarly, the structure of the RpE was consisteni}

with_ normal _{perfusion-fixed noninjected animals. In}

EAU-induced rats, the retinal EC and

RpE

remained

structurally unchanged during the first I0 to 12 days pI.

.t'rom day 12 onward during

the

active phase

of

the

disease process _{(11), alterations}

in

_{these cells became}

more evident, although large-scale changes d.id not occur

until there was substantial inflammatory cell infiltration

and parenchymal damage. Occasionally raised, bulbous

endothelia could be identified

with

both transmission (FiS. 2a) _{and scanning electron microscopy} (Fig. 2b),

pqrticularly

in

areas

of

extensive cellulaf

infiltiation.

This phenomenon has previously been described in EAE

(12,.7.3,?-0-,.21), _{multiple sclerosis (22),EAIJ (1, 11), and}

uveitis (23), with the implication that these endoihelia

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Vol. 70, No. 1,

la

I.994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

x.b

Ftc. 1. a, Toluidine biue-stained resin section of retina, day 21 PI'

Substantial perivascular cuffing of inflammatory cells (,L) and destruc-tion of underlying outer retina (orrou). Note paucity of leukocytes in choroid. b, Toluidine blue-stained resin section from day 18 postim-munization. Inflammatory cells can be seen migrating into the outer retina from retinal vessel (white atow) causing localized damage' A

few inflammatory cells can be seen in the photoreceptor layer' Adjacent RPE cells contaln numerous cellular inclusions. Figure 1o, x100; b'

x220.

FIc. 2. o, Transmission electron micrograph (?8i14) of retinal

ves-sel, day 21 PI. Raised, bulbous endothelial cell with microvilli (lorge

.'....

\

s:

t-\

arrow) lying over area of large-scale leukocy'te infiltration. HRP

reac-tion product can be seen in BM (srnoll arrow) and' extending into parenchymal extracellular space. b, Scanning electron micrograph (SEM), day 12 PI. Two adherent inflammatory cells can be seen

surrounded by piump, protruding endothelial cells (bloc& arrows). Oc' casional iong microvilli projecting from the EC can be seen (white orrou). Figure 2o, x3,000; b, x2,500.

FIc. 3. o, SEM, day 12 PI. Flattened inflammatory cell adhering to

retinal vessel wall. b, Higher power of o showing microvilli at the junction between ECs (arrorus). Figure 3o, x7,270; b, x3,340'

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42 GREENWOOD, HOWES,

AND LIGHTMAN Llsonerony _{ItvnsrtclrroN}

resemble those of the HEV. Whether these endothelia

also express the addressin adhesion molecules associated with HEV, as has been demonstrated

in

EAE (12, IB),

remains to be established.

In

some large vessels of the retina, especially the arteries, raised, ridge-like endothe_

lia

appear

to

be

a

normal feature and- should

not

be

mistaken for HEV-like endothelia. An additional

poten-tial difficulty in interpreting vessel morphology is derived

from the method of fixation used. In this study, the tissue

was fixed by perfusion through the ascending aorta to

p-reserve _{vessel tone, obtain rapid fixation, and maintain}

the structural interactions belween the retinal EC and

leukocytes (13).

In

_{previous comparable studies with}

EAU, immersion fixation _{was used (10, 11, 14) which} does not sustain vessel tone and fixes the vasculature in a 'collapsed' state. Contrary to the state of endothelial preservation,

the

_{RPE appears}

to

_{be better presewed}

with immersion fixation as this method avoids the

vac-uolation of the junctional region between the cells seen

in

_{perfusion fixed material. Although these differences}

in-fixation technique are _{not critical, they must be con_}

sidered _{when comparisons are made between different}

studies.

In those areas where leukoclte adhesion and infiltra_

tion was greatest, _{the endothelia generally remained flat}

but. thickened, displaying increased amounts of cytosol

and a .dramatic upregulation

in

the levels

of

cyttsohc

organelles _{such as rough endoplasmic reticulum, ribo_}

sosmes, and _{vesicular-like profiles. This activation of the}

endothelia has been reported in both EAE (24) and EAU

(10), and

is

likely

to

be

a

consequence

of

increased

endothelial cell metabolism and protei., synthesis.

Cy-tokine activation of retinal endothelia can lead to uprl_ gulation and increased expression

of

molecules

of

im-munologic significance, such intracellular adhesion mol_

99yle-1 (25), major histocompatibility complex class

II

(26), transforming growth

fictor-p

as

well

as

the

in_

greeseq production of extracellular matrix proteins (g).

In EAU, the structural changes have been quantified and

have demonstrated a clear correlation between

endothe-Iial cell thickness

in

capillaries, venules, and veins and

the severity of the disease (11).

With

_{scanning EM, the}

luminal surface of the endothelial vessels exhii*bited sub-stantial numbers

of

microvilli

that

could be seen to

delineate the contact points between cells (Fig. 3)

in

a

similar fashion

to

that

in

EAE

(13, 21). Occisionally, very long processes _{could also be seen emanating from}

the endothelia _{(Fig. 2b). As the} disease _{progressedio the}

point

of

maximal leukocyte extravasition and tissue

damage, there was evidence of endothelial cell necrosis

and death.

The RPE maintained

its

structure during the early

stages

of the

disease. During

this

period,

the initial

infiltration _{of inflammatory cells into the photoreceptor}

layer was accompanied

by the

appea.a.rce

of

cellular

inclusions, such as phagocltosed malerial and large dark

electron-dense bodies, in the adjacent RpE (Fig. tA). R,

the disease progressed, the RPE persisted

u."u

-orro-layer, with only rare focal signs ofhypertrophy in areas

where there was severe infiltration

ofthe

clioioid. Once

destruction of the photoreceptor layer and formation of

a subretinal exudate had occurred, the RpE remained as

a

qell-presewed monolayer

with

apical microvilli ex_

tending

into

the

exudate.

At

this

stage, there was a

reduction in the number of cytosolic org:anelles which is

likely to result from the cessation of ph"agocltosis _{of rod}

outer segments and their subsequent degradation.

PpRiraslsrr,rry oF THE

BRB

ro

ELEcTRoN_DENsE

TnacnRs

The BRB maintained

its

structural integrity to both HRP and _{lanthanum during the}

first

_{few d'ays of onset}

ofthe disease, being consistent with previous studies (t).

As the numbers of leukocytes interacting with the retinal

E_C and migrating across increased, theie was a detecta_ ble increase in the permeability to tirese tracers. previous

work has demonstrated that this increase in BRB perme_ ability occurs concomitantly

with

leukocytei extravasa_

tion

(1).

In

_{both EAU and EAE}

it

_{is incieasingly clear}

that

these

two

events are inextricably linked

(Zl_Sl)

despite earlier opinion

that

leakage acrors the barrier

occurs before leukocybe infiltration (92, Ag). Leakage of

tracer was nearly always restricted to areas of inflam_ mation and associated almost exclusively with the retinal vasculature and not the RPE.

HRp

could be seen ex_

tending along

the

basement membrane and

into

the extracellular space (Fig. _{). Unlike a previous study in} which abnormalities of the EC tight junctions were not found (14), we have confirmed _{ttrai tightlunction disrup_}

tion does occur (1, 10, 84) as occasionattv

Hnp

*". ."",

alongthe entire length of a'tight' junction (Fig. ad). The

lack

of

finding many junctions hiled

with

t-"racer

is

a

consequence _{of the tortuosity ofjunctions and the diffi_}

culty of obtaining, in a single plane, a complete junction from luminal

to

abluminal side. Moreovei,

it

is

also a

function of their relative infrequency in that opening of

these junctions is likely to occui in a precise and punciate manner (17) with a substantial capacity to reseal. Junc_

tional disruption remains the most likely route through which

initial

_{extravasation occurs, although leukocfre}

penetration may also carry through very small amounts of tracer (27, 28). _{Differential permeability to tracers}

of

different molecular weight (1), which haj been used to distinguish the route of BBB breakdown after

hyperos-molar disruption (3b), is indirative of small pores foiming

through the junctions. Had extravasation occurred

bi

pinocytosis and vesicular transport, as has previouslj, been suggested

in

EAE

(24, eO;,

this

size distinction would not have been apparent. This does not, of course,

exclude _{the possibility that vesicular transport occurs at}

a later stage of the disease, _{although whether}

it

plays a

role in net transfer remains questionable (37).

Occasion-ally, tracer-filled vesicular-like profiles could be

identi-fi-ed (Fig. 4b), but these were mostly associated with the abluminal plasma membrane

that

have been shown to be a normal feature

of

central nervous system (CNS)

endothelia (37).

In

addition, vesicular-like profiles, thai

were largely devoid

of

tracer did become-

a

prevalent feature of _{the activated endothelia during the}

develop-ment of the disease resembling those seen

in

EAE (24),

3"{

T.u"V other pathologic conditions of the CNS (aA),

including the retina (39).

The factors that are responsible for inducing

VOI. ?0, NO. 1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

Frc. 4. TEM, day 21 PI. o, Lymphocyte adhering to large retinal vessel EC. HRP reaction product can be seen filling the BM and extracellular space (orror^us). b, Retinal capillary with HRP reaction product inBM (large arrow) and extracelluiar space. HRP-5lled ablu-minal invaginations can be seen (small arrows)- c, Mononuclear cells adhering to wall of large retinal vessel and in perivascular region with extravasated HRP in BM (orror.o)' d, HRP extending the length of a

tight junction (arrow) and filling the BM and abluminal pits

(arroru-h.eads). a, x8,000; b, x10,000; c, x2,500; d, x20,000.

GREENWOOD, HOWES, AND LIGHTMAN LlsoReronv lNvnstrclrtox

those implicated

in

disruption

of

the

BBB

(17, 40).

During many disease processes of the CNS, there ls the

release of a wide spectrum of compounds with a potential

for acting upon the cells of the blood-CNS _{barrieis. Many}

ofthese agents have been shown to cause opening

ofth!

barrier, although the cellular mechanisms througi which they operate are poorly defined.

It

has been p6stulated

that many of these act by altering intracellular calcium

leading to induction of pinocltosis or alterations in the

binding capacity of the tight junctions. Compounds such

as _{histamine, bradykinin, arachidonic acid and}

its

me-tabolites, the eicosinoids, have long been known to bring

about. changes

in

permeability

but

more recently,

thI

cybokines such as interleukin-1 and tumour neirosis factor, have also been _{implicated in BRB disruption} (41,

42). "Ihe cytokines, that are produced within fhe retina

in EAU (43), are known to induce leukocvte infiltration

(41), _{but whether the increase}

in

_{vesicuiar activity}

re-ported is a direct or indirect consequence of this

phenom-enon requires further analysis.

The physical disruption caused

by

leukocltes pene-trating the BRB may also give rise

to

smali transient

leaks in barrier integrity. In the present study, no tracer

was recorded filling the space between the invading leu_

kocyte and the endothelial membrane as has

been"dem-onstrated

in

EAE (27, 28). This route of extravasation,

however, remains

a

distinct

possibility, _{although the}

endothelial cell is thought to seal once the leukoclte has

migrated through (13), especially if diapedesis is t-hrough

and not between the EC.

_

At

the RPE,

virtually

no leakage

of

HRp

could be

detected, even _{when inflammatory cells were}

in

_close

proximity

in

both the photoreceptor layer and the cho-roid.

In

only

a

single section, where there was total destruction

of

the

photoreceptor layer, could

a

small amount of HRP be detected

filling

the spaces between

the apical microvilli.

It

was not cleir, however, whether this had diffused across _{the RPE or from nearby retinal} vessels. _{The smaller tracer lanthanum was also mostly}

excluded by the RPE.

It

could often be seen extending

through the junction _{between apposing RpE up to, bul} not.beyond,,the apical

tight

junctions

(Fig.5).

_Very

rarely, and

in

minute amounts, lanthanum was seen

between the apical microvilli beyond the tight junctions

indicating a small but detectable _{disruption to the RpE}

tight junctions. These small permeability changes found

at the RPE in EAU are compatible with previous reports

of minimal damage _{to the RPE barrier} (10).

Lpuxocyrn IxrpnncrroNs wrrH

THE BRB

In_the early phase of the disease, where tissue damage

was localised to occasional perivascular _{regions and the}

photoreceptor layer underlying these areas, infiltrating

cells, predominantly mononuclear, were found

in

th6 surrounding tissue and adhering to the luminal wall of

the vessel (Fig. 4a and _{c). The majority of these cells}

were lymphocytic in appearance with a smaller

percent-age

of

monocl'tes

and

occasional polymorphonuclear cells. These inflammatory _{cells, especially those that} were spherical or ovoid in shape, were often attached to

the EC luminal membrane by what appeared to be fairly

tenuous and infrequent connections (Fig. 4a andc).

Wit[

scanning EM, inflammatory cells could be seen adhering

to the vessel wall of veins and venules in large numberi (Fig. 6o) and occasio,nally within microvessels _Gig. 6b).

Mononuclear cells adhering to the vascular endothlehum

could often be seen probing into the endothelial cell in close proximity

tq

but not

into, the tight

junctions

between _.apposing ECs (Figs. 7, 8, g, and fb).

thi.

p"rr-etration into _{the body of the endothelial cell. whicli did}

not

disrupt

the

endothelial cell membrane. has been

previously described for lymphocytes in

Oln

(fS).

Sim-ilarly, the direction of penetration sometimes appeared

to bisect- the plane of a junction _{between two overiapping}

endothelial cells in a manner described by Raine

"{ "t.

ii

EAE

(13) and Wekerle

et

al.

(aa)

witir

myelin basic

protein-specific T cell line lymphocyte migration through brain endothelial cell monolayers in

uitri.

^

Despite the interpretational difficulties imposed by a

2-dimensional image,

it

appeared that in

-os[

case., lhe migratory route was

not via

a junction,

but

in

close

proximity to

it.

The route that leukocytes take through

the _{CNS vascular barrier remains a iontentious issrie.}

Despite growing evidence

that

in

CNS inflammation,

diapedesis _{can occur through endothelial cells (11, 13,}

27, 44), _{there remains a} degree of scepticism especially

from those working outside the CNS. A reason for this could be

that this

route of passage _{may be unique to}

endothelia of the CNS, wheri celli are attached to orr"

another by tight junctions. The strong adhesive

proper-ties

of

these junctions _{may be suffrciently great that}

penetration is more easily accomplished by the leukocyte

taking an _{intracellular, rather than} an intercellular

paih-way. This would involve invagination of the fluid plasma

membrane _{of the EC at the point of penetration of the}

leukoclte pseudopodium (Fig. T,

8,

and 9), either by

phagocytic

type

mechanisms,

or by

electrostatic and

mechanical forces. The invagination would continue un_

til

the

cell becomes _{attenuated and}

the luminal

_and

ablum-inal _{plasma membrane abut each other (Fig. Td}

arld 8/). Once this has occurred, pore formation betiveen

the two membranes could develop, allowing the

unhin-dered passage of the leukocyte into the periviscular space

(Fig. 10d and e). A further advantage ofthis route wbuld

be the_increased degree

of

control over diapedesis

af-forded by the endothelial cell.

It

is already clear that the

recruitment of inflammatory cells by both cerebral and

retinal

endothelia can

be

regulated

by

their

level of

expression

of

adhesion molecules

that

can be induced

and upregulated

by

cytokines (E-7,

g,

25, 4E). Indeed

treatment of EAE animals with an antibody that blocks

the very late antigen-4/vascular cell adhesibn

molecule-1 _{T]hesion pairing has been shown to prevent leukocyte}

infiltration and paralysis (46). However, the endotheiial

cell may al,so play an additional role by _actively

facilitat-ing migration by forming pores at the point of leukocvte

penetration, a process

that is

likely

fo

involve the

ie-arrangement of the cytoskeleton. This hlpothesis, how_

ever,

will

require careful examination

in

order to

estab-lish the route of extravasation and the role of the EC in

diapedesis.

.

In

some cases, mononuclear cells could be seen

par-tially or _{completely surrounded by endothelial cells (Figs.}

VOI.70, NO. 1,

1994

BLOOD'RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

,,\$u

Frc. 6. SEM, day 12 PI. a, Large vessel (probablv vein) with

nu-merous inflammatory cells adhering to the vascular wall' Smaller vessel

(probably arteriole) with ridge-like endothelia devoid of inflammatory cells (asierisk). b, Capillary with spread, migrating inflammatory cell in lumen (arrow\. Figure 6o, x1,090; b, x2'500.

Frc. 7. TEM, day 18 PI. o and b, Lymphocytes adhering to retinal

:r!:i*': {: ;l

EC and projecting pseudopodia into the EC at points close to tight junctionJ (imall arrows). Lanthanum coats the surface of the cells

-(open

arrows). c and d, Higher powers of o and b showing lymphocytes probing into EC (arrowheads\ in close association wittr tight junctions Tarrori). Figure 3o, x15,000; b, x20,000; c, x25,000; d,x50,000.

i::' ."

GREENWOOD, HOWES, AND LIGHTMAN

8b

L,leonltonv Invrstrclrron

\i':\l:'.

...$.j..\;. . \4. _t. .-. .

';{i\l$i,' .'..,r'.ili

r ia:-::..,.. 1-..-_ :. .'

Sl'i :..:' ..-i.1:\l)..' l.i _{':.i;i\...::r-i.l\.i\.s\at}:ir:l.)r"i: ;.,:'r'

FIc. 8. TEM of mononuclear cells probing endothelial cells of ret_

inal vessels, day 12 PI. b and.c, Higher power micrographs of o showing mononuclear cell pseudopodia penetrating EC in association with anJ at a distance _{from_tight junctions (arrowl). An increase in EC rough} endoplasmic reticulum, ribosomes, and vesicular profiles (arrowh.eaii

can be seen. _{d, Mononuclear cell with pseudopod.ia inserted into EC} (arrowhea.d,) _{with no associated tight junction. e and}

f, Mononuclli

cell penetrating deep into EC.near tightjuncti on (arrow)' cau*i.rg .euere EC attenuation (arrowhea.ds-). _{Figure gL, xa,g00; b and c, xZA,}}O;}

i,

Vol.70, No. 1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

-&

Frc. 9. a and b, TEM of serial sections through migrating mono-nuclear cell at junctional region (arrow). Figure 9o, x7,000; b, x8,000. Day 12 PI.

Ftc. 10. o, b, and c, TEM of serial sections through migrating

mononuclear cell, day 12 PI. Inflammatory cell process is penetrating through the EC close to, but not through, the junction (arrows). d,

Higher power micrograph of b showing a hole in EC close to the

junction (small atow) with intact BM. Increased expression of rough endoplasmic reticulum (atowheads) and vesicular profiles (open

ar-roir.,) in EC. . e, Higher power micrograph of c showing mononuclear cell penetrating EC and BM close to the EC junction (small arrow). Dense accumulation of actin-like filaments can be seen in the leading

edge of migrating ceII (large orroru). Figure 10o to c, x8,000; d, x20,000;

1le

t :. :.. :,. ..' :.'ii''.i.r.y.-1y'11r.:1

\\

\:

l 1a

1.,

W\\\RN\\\N\.S\\\\,\{i' ..is\f:iw

FIc. 11. TEM of a mononuclear cell surrounded by EC, day 12 PI.

o, Part of the inflammatory cell remains within the vessel lumen (open

arrow), but is not close to any EC tight junctions (arrows). b, Serial section from same block as o showing an external part of the mono-nuclear cell in continuity with the enclosed part demonstrating that the path of entry is intraendothelial and is not associated with any tight junction. c and d, Higher powers of o showing EC tight junctions (arrows). EC (E and open atows), inflammatory cell (I). e, Higher power from b of part of mononuclear cell protruding into lumen. The

EC (E) at this point is devoid of tight junctions. Figure 11o and b,

[image:10.595.51.549.37.657.2]

x6,000; c and d, x25,000; e, x17,000.

FIG. 12. TEM of mononuclear cell almost completely surrounded by EC at point removed from the tight EC junction (anow). Adherent spherical mononuclear cell attached by tenuous contact point. Day 12

PI. x8,000.

Frc. 13. TEM of inflammatory cell migrating through EC and basement membrane. Some lanthanum can be seen between migrating cell and EC. Day 18 PI. x8,000.

-*s;a*i$''s-*--'r,'if -:r {\.\.:ii\ii.*N$'liaa\i\\-\')'.1l:\\:\\

T

Vol. 70, No. 1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS

49

FIG. 14. TEM of lymphocyte in the process of extravasation' day

ra PrI

",

^S".1i;

ilit*gt, two retinal microvessels showing adherent

iii

-igt"ti"c

cells. L-anthanum can be seen coating the luminal

;;;;;.ilMigtating

Ivmphocvte near the tight junction (anow)'

.o"t"a *itft hnthinum. Figure 14o, x3,900; b' x8'000'

FIc. 15. TEM of inflammatory cell ('L) migratin-g through gap in

tft. gM;iri"h is filled with HRP (arrows)' Dav 21 PI' x4'000'

^

Ftd.- ro. f:Orr'f of RPE. o, Inflammatory cells packed within choroid

""a "ittti"g-g;.tt't t t"-bt"tt.

(l)' Separatio:r-of the layers of Bruch's

[image:11.595.53.562.56.684.2]

50 GREENWOOD, HOWES, AND LIGHTMAN LesoRltoRY INVESTIGATI0N

purified bovine retinal S-antigen (54). The antigen was

emul-sified

in

complete Freund's adjuvant (1:1; Gibco, Paisley,

United Kingdom), and enriched with 2.5 mg/mI of

mycobacte-rium tuberculosis (strain H37Ra). Each animal was injected

with 100 prl containing 50 pg of S-antigen; 50 pl into the footpad

and 50 pl into the base ofthe tail. In addition, each rat received

5 x 10e killed Bordetella Pertussis organisms given in 300 pl of

phosphate-buffered saline intraperitoneally. Control animals

(N

:

10) were injected intraperitoneally

with

complete Freund's adjuvant alone, with or without killed pertussis

orga-nisms.

Ur,rnlsrRucruRAl MoRpHor,ocv

Animals were terminally anaesthetized with pentobarbitone

(50 to 60 mg/kg intraperitoneally, and the thorax opened and

a cannula inserted via the left ventricle into the proximal

ascending aorta. The animals were then killed by perfusing the vasculature with one-half strength Karnovsky's fixative (2%

formaldehyde;2% glutamldehyde; 0.2 u sodium cacodylate; 6.5

mu calcium chloride) at atate of 25 ml/minute. The descending aorta was tied off and afber 3 minutes, the flow rate of fixative

was reduced to 12 ml/minute. After 15 minutes of fixation, the eyes were removed and placed in fixative at

4'

C overnight.

Each retina was then prepared for electron microscopy by an

incision through the sclera behind the ciliary body that was

then extended 360 degrees. The cornea and lens were removed,

and the posterior eye cups washed in cacodylate buffer and embedded in 3% agar just before setting. For transmission EM, 100-pm sagittal sections of the embedded posterior eye cup were cut at the level of the optic nerve on a vibroslice (Campden

Instruments, United Kingdom). For scanning EM, 200- to 500-trrm sections were cut from the same eye.

All

sections were

postfixed

in

1% osmium tetroxide

for

t

hour washed and

dehydrated through ascending concentrations of ethanol. The

100-1cm sections for TEM were flat embedded in resin between

two aluminium foil-coated glass slides. After the resin had set,

the slides were separated and small blocks of retina cut from clearly defined areas of the flat 100-prm sections, and glued to

larger trimmed resin stubs. Thick sections were cut and stained

with toluidine blue and viewed under the light microscope.

Thin sections were cut from selected areas, placed on copper grids, and counterstained with uranyl acetate and/or lead

cit-rate and observed on an Hitachi H600 transmission electron microscope.

Sections for scanning EM were dehydrated in graded ethanol

and critical point dried with COz. They were then stuck to

stubs and sputter coated with 20 nm of gold before observation on an Hitachi 5520 scanning electron microscope.

PrR[.rnlsrr,rty STUDIES wITH TRAcER

Rats in each group were terminally anaesthetized with

pen-tobarbitone (50 to 60 mg/kg intraperitoneally). For the HRP

study, the anti-histamine, diphenhydramine was injected

intra-peritoneally (0.5 m/kg). After 10 minutes, 50 mg of HRP (Sigma

type

II,

Sigma, Dorset, United Kingdom) in 200 pl of saline

was injected intravenously. After 5 minutes, the thorax was opened and the animals were killed by perfusing the vasculature

with one-half strength Karnovsky's fixative (2% formaldehyde;

2% glutanldehyde; 0.2 tu sodium cacodylate; 6.5 ml,t calcium

chloride) at a rate of 25 ml/minute. The eyes were removed and cut into 100-pm sections as described above. The sections were then incubated with diaminobenzidine for 10 minutes to

produce the electron-dense HRP reaction product. After

thor-and EAE (13, 21). These cells lay between the retinal EC

and basal lamina causing

lifting

of the endothelial cell.

This phenomenon may explain the raised appearance of

some

of

the

cells

in

the

SEM micrographs (Fig. 2b).

Alternatively, inflammatory cells could also be seen mi-grating through the EC and basal lamina together,

with-out causing separation (Figs. 10, 13 and 14).

Inflamma-tory cells that had already entered the perivascular region

were also detected penetrating the basement membrane

through relatively small gaps and migrating further into

the

parenchyma (Fig. 15).

This

process

of

migrating through

the

extracellular space

is

thought

to

involve different adhesion molecule pairings to those involved in the binding to, and migration across, the EC (47).

Fur-thermore, it often overlooked that activated lymphocytes

are also induced

to

secrete enzymes that are capable of

degrading the basal lamina and extracellular matrix (48,

49). More recently, the degradation products from these

enzymes have been visualized

in

EAE, generating new

data regarding the mechanisms of lymphocyte migration

(50).

At

the posterior barrier, the choroid was frequently found

to

be

full of

inflammatory cells

but

with

little evidence of migration through the RPE. The presence of inflammatory cells at this site may be due to the release

of inflammatory and chemotactic factors from the

dam-aged overlying retina inducing leukocyte recruitment and

accumulation in the choroidal extracellular space.

Cyto-kines released from

the

retina could

bring

about the

induction and upregulation

of the

requisite adhesion

molecules on the choroidal endothelia, thus initiating the recruitment of circulating inflammatory cells. This would imply that either the RPE barrier has become permeable

to such factors, or that

it

has been stimulated to secrete

them from their basal surface. There is increasing

evi-dence that under certain conditions, RPE cells are

ca-pable of secreting cybokines such as interleukin-6 (51), interleukin-8 (52), and tumour necrosis factor-a (53) which could lead

to

the

recruitment

of

inflammatory cells

in

the absence

of

disruption of the RPE barrier. Migration from the choroid into the retinal parenchyma

appears to be limited by the RPE during the early and

mid stages of the disease, but becomes more noticeable

when Iarge scale

retinal

damage and detachment has

occurred.

In

areas of severe

infiltration

and destruction

of the choroid, inflammatory cells could occasionally be

seen between the layers of Bruch's membrane (Fig. 16o),

between the membrane and the RPE, and between

ad-jacent RPE cells. There was also evidence of

inflamma-tory cells apparently

within

RPE cells (Fig. 16b and c)

which would suggest an intracellular route.

At

this site,

however,

it

was

difficult

to

ascertain

the

direction of

leukocybe migration.

METHODS ANrulr.s

Female Lewis rats

(N

:

54; 150

to

200 gm) were used

throughout. The EAU group (N

:

44) was immunized with

PI. b, Inflammatory cell (.L) engulfed by RPE cell (R) close to junction (straight atow) which has characteristic rows of mitochondria on either

side. The choroid (C) is clear of inflammatory cells. A small amount of

HRP reaction product (curued atow) can be seen between apical microvilli. The photoreceptor layer has been destroyed and a dark

inclusion body (white arrow) can be seen in the RPE. Day 26 PI. c,

Inflammatory cell (.L) apparently within RPE cell (.R) close to junction

Vol.70, No. 1,

1994

BLOOD-RETINAL BARRIER IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS 51

ough washing, they were then osmicated and prepared for transmission EM as described above. For the lanthanum study,

the thorax was opened and the animals were sacrificed by perfusion fixation with one-half strength Karnovsky's fixative

containing 1% lanthanum nitrate for 15 minutes, washed in

distilled water, and the retina prepared for transmission EM

as described above,

Acknowledgenxents: We would like to thank the

Lev-erhulme Trust and Moorfields Eye Hospital Locally

Or-ganized Research Scheme for supporting this work.

Date of acceptance: July 12, 1993.

Address reprint requests to: Dr. J. Greenwood, Department of Clin-ical Science, Institute of Ophthalmology, Bath Street, London ECIV gEL UK.

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