Effects of polymorphonuclear leukocyte transmigration on the barrier function of cultured intestinal epithelial monolayers

Effects of polymorphonuclear leukocyte

transmigration on the barrier function of

cultured intestinal epithelial monolayers.

S Nash, … , J Stafford, J L Madara

J Clin Invest.

1987;

80(4)

:1104-1113.

https://doi.org/10.1172/JCI113167

.

We describe a model to study the effects of polymorphonuclear leukocyte (PMN)

transmigration on the intestinal epithelial barrier. Human PMN were induced to transmigrate

across high resistance monolayers of a cultured human intestinal epithelial cell line (T84

cells) by chemotactic gradients produced by formyl methionyl leucyl phenylalanine (FMLP).

With maximal transmigration monolayer resistance decreased by 48 +/- 12.6% in 15 min

and by 83 +/- 1.6% in 60 min. This response was dependent on the size of the FMLP

gradient and the density of PMN transmigration. The decrease in resistance correlated with

number of PMN migrating across monolayers, and was accompanied by increases in flux of

paracellular tracers. Macromolecular tracer studies localized the leak sites to foci at which

PMN impaled the epithelium. Removal of the chemotactic gradient led to restoration of

baseline resistance within 18 h. PMN transmigration across intestinal epithelial monolayers

occurs via intercellular occluding junctions and may be associated with a reversible

increase in epithelial permeability.

Research Article

Find the latest version:

Effects

of Polymorphonuclear

Leukocyte

Transmigration

on

the

Barrier

Function

of

Cultured Intestinal

Epithelial Monolayers

Shirin Nash, Joan Stafford,and James L. Madara

Departments ofPathology (GastrointestinalPathology), Brigham andWomen'sHospital andHarvard MedicalSchool, andthe Harvard Digestive Diseases Center,Boston,Massachusetts02115

Abstract

We describe a model to study the effects of polymorphonuclear

leukocyte (PMN) transmigration on the intestinal epithelial

barrier. Human PMN were induced to transmigrate across

high

resistance monolayers of a cultured human intestinal

epi-thelial cell

line

(T84

cells)

by chemotactic

gradients produced

by

formyl

methionyl leucyl phenylalanine

(FMLP).

With

maximal

transmigration monolayer resistance decreased by

48±12.6% in 15 min and by 83±1.6% in 60 min. This response

was dependent on the size of the

FMLP

gradient

and the

den-sity

of PMN

transmigration.

The decrease

in

resistance

corre-lated

with number of PMN

migrating

across

monolayers,

and

was

accompanied

by increases in flux of paracellular

tracers.

Macromolecular tracer studies localized the leak sites

to

foci

at

which PMN

impaled

the

epithelium.

Removal

of

the

che-motactic gradient led to restoration of baseline resistance

within

18

h.

PMN

transmigration

across

intestinal

epithelial

monolayers occurs via intercellular

occluding junctions

and

may be associated with

a

reversible increase in

epithelial

per-meability.

Introduction

Migration

of

polymorphonuclear leukocytes (PMN) across

in-testinal

epithelium

occurs

in

a

variety of idiopathic

and

infec-tious

inflammatory

diseases afflicting

the small

intestine

and

colon.

This

histologic

finding,

which

may lead

to

the

accumu-lation of

neutrophilic

exudate

within

dilated

crypts (crypt

ab-scesses),

is utilized

by

pathologists

to

determine the

degree

of

"activity"

of the

inflammatory

process

(1,

2).

Mucosal

prepa-rations from patients with inflammatory

bowel

disease may

show decreased

epithelial barrier

function,

as

assessed

by

resis-tance

measurements,

even

in the absence of detectable

ulcer-ation (3). This

decrease in

epithelial

barrier

function

in

inflam-matory

diseases is probably

due to the

additive

effects of

many

interwoven

events.

One

factor

that

might contribute

to

intes-tinal

epithelial barrier

dysfunction

is

the

impalement

of the

epithelium

by

transmigrating

PMN. In

these

experiments,

we

have

utilized

electrophysiologic, morphologic,

and

tracer

tech-niques

to

examine the

effects of

human PMN

transmigration

on

the

barrier

function

of

monolayers

composed of human

Address reprintrequeststoDr. Nash, Departmentof

Pathology,

Brigham and Women'sHospital, 75 FrancisStreet, Boston, MA

02115.

Receivedfor publication4March 1987 andin

revisedform

4May

1987.

intestinal epithelial cells

_(Tff

cells). We have previously shown

that these monolayers exhibit stable, high resistance to passive

ion flow and consist of confluent polarized columnar cells (4,

5). Our current studies show that in the presence ofan optimal

transepithelial chemotactic gradient, substantial PMN

trans-migration

across

this

epithelium is accompanied by large, but

reversible, barrier defects. In contrast, in the presence of only a

small number of transmigrating PMN, the epithelium exhibits

a

remarkable

ability

to

maintain

barrier

integrity. This system

may serve as a

useful

model of intestinal inflammation, in

which

interactions between epithelial cells and inflammatory

cells, such

as

PMN,

can

be

better characterized.

Methods

Cell culture. T84cells were passaged and grown on 1.98

cm2

ring

mounted, collagen coated, permeable supports after the method of

Dharmsathaphorn as previously described (4-6). 200 monolayers composed ofcells from passages 16-47 were used in these experiments. Transepithelial resistances developed by these monolayers varied

be-tween200and2,400 ohms cm2 with themajority being>500 ohms

cm'.

We

intentionally

utilized

monolayers

withvariable

resistances,

since others havesuggestedthat thedensityofchemotactic migration

of PMNacrossepithelialmonolayersmay bedirectly affected bythe baseline resistance of the monolayer (7, 8). Sinceourexperimental

system allowsustorecord serial resistancereadingsfrom individual monolayers (see below),weweredirectly abletotestthis hypothesis.

Wealso utilized 12monolayers ofahighresistance Madin-Darby

caninekidney (MDCK)' cell line to determine whether the major

findingsof thisstudywererestrictedto

T84

intestinal monolayers,or

were more broadlyapplicable. MDCK monolayersweregrown in

Dulbecco-Vogt modified Eagle's mediumsupplementedwith 1%

pen-icillin-streptomycin, 1%glutamineand 10% fetal calf serum,pH7.4. Subcultures andmonolayersweremadeasfor

_T&

cells.These

mono-layers developed resistances ranging from 536 ohms cm2 to 1,834

ohms cm2 witha meanof1,128 ohms cm2.

PMN isolation. PMN wereroutinelyisolated fromanticoagulated

(sodiumcitrate 13.2 g, dextrose11.2 g, in 500 ml water,pH6.5)whole blood collectedbyvenipuncturefrom normal donors ofbothsexes(9).

The PMN collection procedure was approved bythe Brighamand

Women'sHospitalHumanSubjects Committee (protocol 85-1465-1).

Platelet-richplasmawasremovedaftercentrifugation (1,000g, 3min)

and thecellpelletdiluted 1:3 with Hanks' balanced salt solution

with-outCa2+ andMg2+ (M.A.

Bioproducts, Walkersville, MD)

at37°C.

PMNwereseparatedbycentrifugation (500g, 40min)on

lymphocyte

separationmedium

(Litton

Bionetics,

Kensington, MD),

followed

by

dextran sedimentationat37°Candhypotonic lysisofcontaminating

redbloodcells.PMN(95%pure)with 98%

viability

by dyeexclusion

were suspended in tissueculture medium (described above) at 107

PMN/mloffluid,heldat4°C,andusedfor

experiments

within 1 h after isolation.

1.Abbreviations usedinthispaper:EM,electron

micrograph;

FMLP, N-formylmethionyl leucyl

phenylalanine; HRP,

horseradish peroxi-dase;MDCK,

Madin-Darby

canine

kidney.

J.Clin.Invest.

© The AmericanSocietyfor ClinicalInvestigation, Inc.

0021-9738/87/10/1104/10

$2.00

Induction

of

PMNtransmigration. Ring-mounted monolayers

wereelevated on glass beads in plastic tissue-culture wells to allow

mixing of solution under thering-monolayerassembly.Monolayers

wereoriented such that theapical("mucosal")aspect of the cells faced

the upper compartment. PMNweresuspendedinafinal volume of 1

mlandappliedtotheapicalsurface ofmonolayersand chemotactic

gradients were establishedacrossmonolayers by adding

N-formyl-methionyl-leucyl-phenylalaline (FMLP) (Sigma ChemicalCo., St. Louis, MO) to the basal ("serosal") compartment. Incubation was

carriedout at370Cina95%air/5% CO2 atmosphere.

Todetermine if the functional effects of PMN

transmigration

on

epithelialbarrier functionwerereversible,weremovedthe chemotac-ticgradientinasubset ofexperimentsand allowed recoveryto occur

forvarying time periods.

Todetermineif theeffects ofPMNtransmigrationonepithelial barrierfunctionwereindependentof the direction of PMNmovement

wereversed thedirectionofthechemotacticgradient byaddingFMLP

10-7

Mtothe

apical

compartmentof those

monolayers

that had been used for theresealing experiments. Resistance measurements were

madeafter incubationof monolayersat370Cfor 1 h.

Ussing chamber studies. Transepithelialresistance of

_Tg4

mono-layers wasdeterminedin low turbulenceUssing chambers,modifiedto

acceptring-mountedmonolayersaspreviouslydescribed(4-6).Each

reservoir was connected via agarbridgestovoltage sensitive calomel electrodes andtoAg-AgClcurrentpassingelectrodes.Voltage

deflec-tions elicited bythe passage of100 uA currentpulseswereusedto

determinetransepithelialresistance. We havepreviouslydetermined that the current voltagerelationshipis linear uptothe 100 MA range

(datanotshown). Afterequilibration, resistancewasmeasured and recorded asthe absolute resistance minus the fluidresistance. The

resistance ofthecollagen-coated filter (NucleporeCorp., Pleasanton, CA) (<3 ohms

cm2)

wasneglected.Ina setof control

_Tg,

monolayers,

wedetermined thatwecouldseriallyremoveandreinsert monolayers intoUssingchambers without affecting baselineresistance (see

Re-sults).Wewerethus abletotakeinitial and postexperimental

resis-tancereadingsonthesamemonolayer. Other data (seeResults)

veri-fiedthatexperimentally modified barrier functiondoesnotchangefor

atleast 4 hafterremovalofexperimental conditions.Therefore,itwas

appropriatetoobtain functional data(resistance response)on

experi-mentaltissuesby suchUssingchamber studies.

Transepithelial fluxratesof mannitol and inulin were carried out

on

_Tg4

monolayers under experimental conditions and compared with

fluxratesobtained fromcontrol monolayers of comparablebaseline resistanceaspreviously described(4).

Morphologic techniques. At the end ofexperiments, portions of

monolayers were processed for thick and thin sections and forfreeze fractureaspreviouslydescribed (4, 5). Half of most monolayers was

utilized to quantitate PMNtransmigration (chemotaxis assay, see

below).

Horseradish peroxidase (HRP), (Sigma type 2; 40,000 molecular

weight),was used to assess the permeability ofoccluding junctions to macromolecules. HRP 0.5% in tissue culturefluidwasapplied to the

apicalaspectof experimentalandcontrol monolayers for thelast20

min ofhour-longchemotaxisexperiments.Control monolayers had

eithernoFMLPin the basal compartment with PMNontheapical sideor,alternatively,noFMLPin the basal compartment and also no

PMN in the apical compartment. Tissues were removed from the

chemotaxis chambers, fixed and processed for peroxidase localization

(10)and electronmicroscopy.

Chemotaxisassay. Wetook advantage of the presence of

endoge-nousperoxidase inPMNbut not in

_Tg4

cells andstained portions of monolayers for peroxidase localization by the method of Graham and

Karnovsky (10). Monolayerswerethen dehydrated through graded

alcohols and mountedasflat sheets on glass slides for quantitation of

transmigrated PMN.Using the light microscope, the surface of each

monolayer

wasidentified

_by

_{the presence of}rare

_{contaminating}

red

blood cells that also containendogenous peroxidaseandareuniformly

stained bythe above method. The base ofthe

monolayer

wasidentified by locating the pores in the Nuclepore filter. PMN between these two

surfaces(i.e., subjunctional PMN) exhibited granular cytoplasmic

stainingandwerecounted andexpressedasthechemotactic response

in PMN/cm2 of monolayer. 1-Mm sections confirmed that PMN

counted by this methodwerelocated between

_T8

cells and the

under-lyingcollagen-coatedfilter andrepresented PMN that hadmigrated

through

Tg,

cellmonolayers.

Weexcluded thepossibilitythat significantnumbers of

transmi-gratingPMN maymigrate through Nucleporefilters into the serosal

compartmentbyperformingPMNcounts onthe fluid from this

com-partment. Fluidfromtheserosal compartment wasaspirated, spun

down, andresuspended in 100 lambda of tissue culture fluid. Cell

counts were attempted usingahemocytometer, and noPMN were

present in this compartment(n= 14).

Statistical methods. Functionalandnumericalmorphologic data

wereexpressedas meansandSEMsforeachexperimentalandcontrol

group,respectively. Statistical differences between groups were deter-mined by Student's t test. The relationship between baseline

resis-tancesandresistance and chemotactic responseswasdeterminedby linearregression.

Results

Monolayers that

were

removed from and reinserted into

Uss-ing chambers

at

intervals of 15-60

min,

maintained resistance

values similar to

their own baseline value (decrease of

7±4.21%

with reinsertion). Thus, in all experiments, each

monolayer could reasonably serve as its own control, and

resis-tance

readings

were

recorded before and after

PMN

transmi-gration.

FMLP

dose response. Dose-response studies were carried

outfor 2 h with

varying

concentrations of FMLP

added

to

the

basal compartment

and

107

PMN

added

to

the

apical

com-partment of the

monolayers. Maximal

resistance and

chemo-tactic

responses occurred with gradients of 10-

Mto

l0-7

M

FMLP

(96.8±2.22%

and

81.2±15.0%

resistance fall,

respec-tively;

and 13.2±2.7 X

104/cm2 and 13.0±16.8

X

104/cm2

PMN

chemotactic

response,

respectively, Fig. 1). Higher

or

lower

concentrations of FMLP diminished both responses.

Furthermore, the resistance response correlated well with the

chemotactic

response

(r

=

0.96, Fig. 2). When data from

indi-vidual

monolayers were analyzed, it became clear that ablation

of

monolayer resistance (decrease of

>

90%) occurred when

the number of

transmigrating PMN

reached a density of

ap-proximately 10

X

104

PMN/cm2 (Fig. 3).

PMN

dose response. PMN dose-response studies were also

carried

out

by varying the number of PMN applied to the

apical

surface of the

Tg4

monolayers in

the presence of a

stan-dard FMLP concentration in the basal compartment

(10-7

M).

A

large resistance response (83.6±1.6% decline) was achieved

with

107

PMN (Fig. 4). This represented an initial

PMN:T84

cell

ratio

of

10:1 and

produced

a

chemotactic response of

13.4±1.06

X

104 PMN/cm2 (Fig. 4). Lower concentrations of

PMN

lead to lesser resistance and

chemotactic responses and

103

PMN

in the apical compartment (PMN:T84 ratio of

1:1,000) produced neither resistance nor chemotactic

re-sponses

(Fig. 4).

transepi-L _{aol 1 50}

12.0

LZ50:

V'I~~~~~~~~~~~~

3~~~-60

Q,

0 1°9 1o1F-5L]8 10-7 1o-6

[FULPI,M

Figure 1.Effects of varying transepithelial gradients of the

chemotac-tic peptideFMLPonresistance andchemotacticresponsesof

_Tg

in-testinal cell monolayers.Confluent

T84

monolayers (7-12 d) on ring-mounted Nuclepore filters (5 uM pore) were elevated on glass beads

in plastic tissue-culturewells. Monolayers were oriented such that

the

apical

aspect of the cells facedthe uppercompartment.

107

PMN wereapplied to the apical surface and a chemotactic gradient was es-tablished across the monolayer withvaryingdoses of FMLP in the

basal compartment.Chemotaxiswasallowedtoprogressat

370C

in

a95%air/5% CO2atmospherefor2h.Resistance response (percent fall in monolayer resistance from itsownbaseline)andchemotactic

response (PMNX104

transmigration/cm2)

areexpressedasI±SEM

from three to sevenexperimentsfor each bar. Maximal resistance andchemotacticresponses occurred with chemotacticgradientsof

10-7

Mand

10-6

M.Weused 10-7MFMLP for

subsequent

studies.

thelial

chemotactic gradient of

1o-7

M FMLP.

Both

resistance

and chemotactic responses began

early.

Monolayer resistance

fell

by

48±12.6% by 15

min,

reaching

a maximum

of

83.6±1.6%

at

the end

of

1 h

(Fig. 5).

Chemotactic

responses of

transmigrated

PMN

were

1.7±0.6

X

104/cm2

at 15

min,

and

13.4±1.06

X

104/cm2

at

the end

of

1

h

(Fig.

5).

Mannitol-inulinflux. To better characterize the

size

of

the

epithelial

barrier defect

produced by

transmigration of PMN

through

T84

monolayers,

we

performed transepithelial

manni-tol and inulin flux studies

on

monolayers

incubated

for

1

h

with

107

PMN

on

the

apical surface

in the presence of a

trans-epithelial chemotactic gradient

of

10-7

M

FMLP.

Results

were

compared

to

those

obtained from studies

performed

on

con-trol monolayers.

The

latter

were

matched

to

experimental

monolayers for baseline resistance.

As

shown in Table I,

che-motaxis

was

associated with

significant

increments in

manni-tol and inulin flux of

approximately

threefold

greater values

than those of controls. The flux

rates

of

mannitoland inulin

did

not

change

over

the 80

min

during

whichthe flux

studies

were

performed indicating

that the observed

alterations were

t- 100/

|_ 80 * Figure 2. Correlation between

resistance response

(i±SEM)

Et 60- and chemotactic response

(I±SEM)

formonolayersused

tZ

40

in dose responseexperiments.

1, - / slope= 0.14 Theresistance response reflects

Q

20

09interpt=-0693

the number oftransmigrating

PMN.Resistancefallofmore

0 5 10 15 20 than 50% from baseline

corre-CHEMOTACTICRESPONSE lateswith a density of 5X 104

PMN(x iO4)Tronsmigrof/on/cm2 PMN/cm2.

R_

'ZrooO

80

tr}t

20^

0 4 6

8 0 2 1 1 2 2 2

8 10 12 14 16 18 20 24

CHEMOTACTIC RESPONSE PMN(X10<}) rnsmigrution/cm2

Figure 3. Correlation between resistanceresponseand chemotactic

responseof individual monolayers. Differingresponses were

achieved by varyingthesize of the chemotactic gradient.Progressive

increaseinmonolayer resistanceresponse(i.e., falling resistance)

rea-sonablyparallelsanincreasing densityoftransmigratingPMN.

Monolayer resistanceappearstobe completelyablated (> 90% fall)

when densities of migratingPMNreach -

10'

PMN/cm2.

not

rapidly reversible.

To ensurethat the increase in

perme-ability of

thesetwo

extracellular

spacemarkerswasnotdueto

gross

disruption of the monolayers,

we performed similar

mannitol flux

studies

on

monolayers in which

we

mechani-cally disrupted either 10

or

20%

of

the

surface

area(i.e., 90or

80%

ofmonolayer

was

composed of

normal

epithelium).

Such

manipulations resulted in enhanced mannitol

flux rates

ex-ceeding control values by

at least 10-fold (over threefold

greater

than

PMN

transmigration

induced changes). The

ef-fects

of

PMN

transmigration

on

permeability

thusarenot

at-tributable

to

generalized monolayer disruption.

ResealingofT84 cell junctions following PMN

transmigra-tion. To determine

whether

_Tg4

monolayers could

reseal after the PMN-induced

resistance

drop,

we

incubated

13

mono-layers for 2, 4,

or

18 h in normal media

after removal

of

experimental conditions (i.e., I07

PMN

in

aFMLP gradient of

_1

I-ti 1001'

1i

75i

LL,

E 50r

tz

I5

PM/v:484 1:1000

PM/V I03

1:10 1:1 10:1

105 106 107

150

cZS

Jj1:o...

0....

i2

scO

....

.1

1o1

Figure 4. Effect ofvaryingdensities ofappliedPMNonsubsequent

resistance and chemotactic responses in the presence ofa

transepi-thelial chemotacticgradientof10-7MFMLP.PMN

suspensions

of

I03

(PMN:T84 ratio of

1:1000);

I01 (PMN:Tg4

ratio of

1:10); 106

(PMN:Ts

ratio of1:1);107

_(PMN:Tg4

ratio of10:1)cellswereused.

Noresistanceorchemotactic responseswerenotedat

PMN:T84

cell

ratiosof1:1000andmaximal responses occurredatratiosof 10:1. Resistance andchemotacticresponsesagain

parallel

oneanother.

Re-sistance and chemotactic responses represent i±SEM from 3to22

100 i5.0

iao

75

50~~~~~~~~~~~~~~~~~.

25-0 15 30 45 60 120

MINUTES

Figure5. Time course ofchemotacticand resistance responsesacross

Tg4

monolayers. I07PMN wereplacedin the presence ofa l-7M

gradientof FMLP. Maximal responseswereachievedat60 min. The

resistance responseoccurredrapidlyascomparedtothechemotactic response.Thiswould beanticipatedsince resistance isasensitive index ofbarrierfunction that isinordinately affected bythe presence of a relatively smallsubpopulationof enhancedpermeabilitysites (12). Results represent i±SEM from 5 to 22experiments.

Io-'

Mfor

30

min).

As

shown

in

Fig. 6, monolayer

resistances recovered tobaseline values by 18 h.

PMN

transmigration

from

"serosal"

to

"mucosal"

direc-tion.

To determine

if PMN

transmigration

inducedeffectson

epithelial barrier functionwere

independent

of the direction of

PMNmovement, we

added Io-7

M FMLPtothe

apical

com-partment

of monolayers

that had resealed

(thus trapping

PMN

in

the

subepithelial space) and

reincubated them for 1 h. As

shown in

Fig. 7,

a

large

resistance

responsewas

obtained

con-firming that the

effects of

PMN

transmigration

also

occur when PMNtrapped

in

the

subepithelial

spaces

of monolayers

were allowed to

remigrate into

the

apical

compartment

(i.e.,

from

serosalto

mucosal

direction).

Effect

of

epithelial permeability

on

PMN

transmigration.

Others have

reasonably proposed that the

degree of

PMN

che-motaxis

that occurs across an

epithelium would

be

in

part

determined by the

resistance

of that

epithelium since gradients

of chemotactic

agents

might

be

better detected if

junctions

were more permeable (7, 8). To test

if

the extent

of

PMN

transmigration through

T84

monolayers in the

presence

of

a

10-7

M

FMLP gradient varied with

the

baseline

resistance of

the

monolayer,

we

compared

resistance and

chemotactic

re-sponses

of individual monolayers

against

their baseline

resis-tances. For

these

experiments, monolayers

were

selected in

which

baseline resistance ranged

from

125 to 2,400ohm

cm2.

Inexperiments run

for

2, 1, or0.5 h

(107

PMN in a

107-M

FMLP

gradient),

the correlation

coefficients

betweenbaseline

Me80 1

_ 60

0 - 48

80 K

sS

t

_~~~~~~~~.

E.

,

Y--

~~~~~~.

a

Q...:--.,....

...

S

t-'':,

~~~.

t

%

~~~~~~~..

2

8.---

,~~~.-.

.. .. .

..

60 ...

..-HOURS

Figure6. Time course of

T8.

monolayer resealing after removal of the chemotactic gradient. In these experiments monolayers were ex-posed toconditions of

"maximal"

chemotaxis for 30

mmn

before

re-movingthegradient. Underthese conditionsresistanceresponsesof

75% were obtained and did not change for periods up to 4 h after

gradientremoval. However,by 18 h complete resealing had occurred

asevidenced by loss of the resistance response. Resistance response at 0 hrepresents that obtained at the end of 30

mmn

ofchemotaxis. Results represent

i±SEM

from threeexperimentsfor eachbar except at 2 hwheren = 2.

resistances and resistance response were r = -0.2; r = 0.16, and r

=-0.3,

respectively. In experiments run for 1, 0.5, and 0.25 h,the correlation coefficients between baseline resistance and chemotactic response were r = -0.2, r = -0.3, and r = -0.2.Thus, both resistance response (on a basis of percent-age of baseline value) and chemotactic response are indepen-dent ofthe baseline resistance values of any given monolayer.

Effiects

of chemokinesison the epithelial barrier. To

deter-mine whether the apparent effects of PMN transmigration on monolayer permeability could be due, in part, to FMLP-elic-ited enhanced random migration (i.e., chemokinesis) of PMN rather than chemotaxis, we incubated six monolayers with both FMLP

l0-7

M and

107

PMNon the apical surface (i.e., noFMLP gradient) for 1 h and measured resistance and che-motactic responses occurring under such conditions. The re-sults (Fig. 8) indicate that these conditions produced only triv-ialdifferences from controls in both resistance and chemotac-tic responses. Such results from experiments in which PMN andFMLP were incubated together while overlying

Ts~mono-layers also indicate that the resistance response observed dur-ing chemotaxis is not due to

_{FMILP-stimulated}

release of PMN secretory products.

Effect

ofpreexposing monolayers to FMLP. To rule out the

possibility that FMLP gradients induced alterations in

epithe-Table

L

Effect

of

PMNTransmigration on

Transepithelial

Resistance

and

on

Mannitol (r

= _3.6

_A)

_{and Inulin (r}

= _11-15

A)

_Flux

Postexperiment

Monolayers Baseline R PostexperimentR Postexperiment mannitol inulin

ohms cm2 ohmscm2 flux

Mmol/h

cm2 flux

jsmol/h

cm2

Controls (n= 5) 1,257.4±359.5 1,238.8±285.7* 87.6X

10-4±36.40

5.0X

l0-4+1.2§

Experimental(n = 5) 1,280.8±321.5 353.4±154.0 318.8 X

_10-4+132.3

14.3X

10-4+5.9

a

~~~~~~~~

%..

OF-80k

60H

40

20h

0-... ...

...

18 19

HOURS

Figure 7. Effects of PMN

transmigrationfrom "serosal"

to"mucosal" direction. In these experiments, monolayers which had returned to their ownbaseline resistance values by 18h (see Fig. 6) were

rein-cubatedfor 1 h with FMLP in

theapicalcompartment. Under these conditions,

resis-tanceresponse of>50% was

achievedin 1 h.

lial

cells that then stimulated PMN to transmigrate, we

incu-bated

six monolayers with FMLP

(10-v

M) in the basal

com-partment

with tissue

culture

fluid

in the

apical

compartment

for

1

h.

Subsequently,

the FMLP was replaced by tissue culture

fluid

and PMN

IO'

were

added

to

the

apical

compartment for

an

additional

hour, and

resistance

and

chemotactic responses

were measured (Fig. 8). Again, both of these responses were

trivial when compared with those

obtained

under

optimal

chemotactic

gradients.

Morphologic correlates ofPMN

transmigration.

PMN were

highlighted

by

localizing

PMN

endogenous

peroxidases

in en

face

mounted

monolayers,

thus

allowing

an

overall

view of

the

distribution of transmigrating

PMN. 15

min after the onset of

the

standard

experiment (107

PMN

in

a

transepithelial

gra-dient of

10-'

M

FMLP),

a

speckled

pattern

of

PMN

impale-ment

sites

was noted

(Fig.

9). The

density

of

transmigrated

PMN

progressively increased

with time and

by

1 h

60-90%

of

the

monolayer surface

was

densely

stained

(Fig.

9

b, c).

This

speckled

pattern

of

PMN

transmigration

sites

was not

ob-served in

separate

experiments in

which

a

gradient

of FMLP

(l0-7

M)

was

used

to

drive

PMN

(107)

across

bare

collagen-coated filters.

The latter

displayed

a

diffusely homogeneous

staining

pattern.

This

observation

suggests

that the

distribu-..] --

-C6~~ ~ ~ ~ ~ ~ ~~6

25

Chemnotoxis Chremohinesis FMLP

Pre-Exposure

Figure 8.Compari-sonofresistance and chemotactic response of

Tu4

monolayers incubatedunder (a)chemotactic conditions

(l107

PMN

apical; 10-7M FMLP basal X 1 h; (b) chemokinetic conditions(107

PMN/1l0-7

M FMLP combined on apical side X1 h); (c) FMLP preexposure(T84cellsincubated in presence of10-7MFMLP gra-dient for 1 h, gragra-dient removed and107PMNapplied apically for an

additional hour). Resistance and

"chemotactic"

responses are

attrib-utable to the presence ofachemotactic gradient.

tor+

:l X.

54p

} 4

.4

4

i ^

s : *. ... -9~'

b $.

....

at

24

Figure 9. Effect of timeonchemotactic responseasdisplayed by light microscopic analysisofmonolayersexamineden

face. T84

monolayers stained forperoxidase localizationto

highlight

PMN

granules (see Methods). Panel showsspeckledpattern of PMN within

monolayers

(I07

PMNincubated witha

lO-'

MFMLP

gradient);

re-sponsesat(a)15 min;

(b)

30 min; and(c)60 min. PMN aggregates becomelarger andmoredensewithtime until themonolayeris

dif-fusely

penetrated byPMNat60 min. Arrowheads indicate individual PMN whilearrowsindicate the

5-MM

pores in themonolayers (X

800).

tion of

PMN

transmigration

sites within

the

epithelium

is

not

entirely

random.

1-Mm

sections of monolayers from standard

experiments

(15-60 min) showed foci

where PMN

accumulated both

be-tween

individual T84 cells and

between the

monolayer

and

the

underlying collagen-coated

filter

(Fig. 10).

PMN

adjacent

to

the

apical

surface of

the

Ts

cellswere

rarely

seen and PMN below the

surface of

the

collagen table

werealsorare.

Exami-nation

of

monolayers

harvestedas

early

as 15 min

after

the FMLP

gradient

was

imposed,

also

confirmed

that PMN

associ-ation with

T84

cell

surfaces

was an

extremely

rareor

short lived

rp

I&I.-.,W. .3 'n.

A,

IA

F.4

AMC&

Figure 10. 1 umsection of

T84

monolayerafter PMNtransmigration.

PMN havemigratedinto the monolayer(I07PMN

IO-'

MFMLP

gradientX I h) andcanbeidentified

(arrowheads)

between

T84

cells and beneathT84cells and theunderlying layerofcollagenand Nu-clepore filter. Theintegrityof themonolayerappears intact(X1,280).

event even

early

in the course of the PMN

transmigration

response.

Extensive

sampling

demonstrated occasional foci in

which three

to seven PMN

appeared tightly apposed

to each

other

and

to

neighboring T84

cells within thezoneof the

oc-cluding junction.

Evenmore

infrequent

wasthe presence ofa

single

PMN

migrating through

a

junctional

site. These

images

of

PMN

migrating

across

junctions

waspresent in

only

10%

of

sections examined (15

of 150

sections),

even

though

these

sec-tions

were

obtained from blocks

originally

identified as

har-boring

areas rich in

transmigrating

PMN

by

localization of

endogenous peroxidase

activity.

Given the vast number of PMN

that

cross

the

epithelium

but

the

infrequency

of

captur-ing

an

image

of

PMN

crossing

the

junction,

the

transjunc-tional

migration

event must occur very

rapidly (seconds

to

minutes).

Analysis of thin sections confirmed the

presence

of clusters

of

PMN

both under the

monolayer

and

within

the

monolayer

in the

subjunctional

space. When PMN were

captured in the

process

of migrating

across

the

junction

(Fig.

11), they

ap-peared

to

do

so

by

extending

thin

cytoplasmic pseudopods

acrossthe

occluding

junction

and

separating

adjacent

T84 cells.

When clusters

of 4-10

PMN were

identified in

a

transjunc-tional location

(Fig. 12),

there

was

close

apposition

between

cell membranes

of T84 cells and PMN. Both cell

types

inter-digitated

by

means

of

cytoplasmic pseudopodia.

Alterations in

the

cytoskeleton

of both T84 cells and

PMN were present at

sites of

intimate

contact

between the plasma membranes of

these

two

cell

types.

These cytoskeletal alterations consisted

chiefly

of

a

dense

cortex

of intermediate

and

microfilaments

adjacent

to

the

plasma membranes

(Fig.

12,

inset).

In

horseradish peroxidase (HRP) experiments,

tracerwas

applied

to

the

apical

surface for the last 20

min

of standard

hour-long experiments (I07

PMN

apical with

a

FMLP gradient

of

10-'

M).

The

latter

allowed us to

detect

sites

of

transjunc-tional permeation since paracellular

spaces

underlying

such

sites

would be labeled

with

tracer. In

eight experimental

mono-layers,

HRP

leaks appeared

to

be

preferentially

associated with

foci of

transmigrating

PMN

(Fig.

13

A).

Atother sites

contain-ing subjunctional PMN,

HRP

labeled the

paracellular spaces

but

was

generally

excluded

atthe level

of

the

occluding

junc-tion

(Fig.

13

B).

These

findings

support the proposal that en-hanced

permeability is attributable

to a subset

of junctions

that are

being

actively invaded by

PMN. Comparable HRP leaks were generally not present in control monolayers

al-though

in one control

monolayer

paracellular labeling did occurthatis

unexplained.

/4

4~~ ~~~~~~~~~~~~4.

r

.

> .

P

M~~~~~~~~~~~~~~~~~~~~~~~i-

t~~~~~~~~M

"N~~~~~~~~~~

FXur 11...Elcto micrgrp of PM inetn th mooae

itheia dicotnut (roh ads).PM mgraio isfro "mco sal" to "srsl compartments (A.: X;1100...

usn.

h e ft n .O n

'

^~~~~~~~~~~~~~~O

Figuretw Electron

micrograph

ofPMN

indenting

themonolayer

andpassing

single

file

through

a

junctional

impalementsite.Tranv

migration

occursbyextensionofpseudopodia

through

thesite of

ep-ithelial

discontinuity (arrowheads).

PMNmigration is from

"muco-sapial "_compartments

_(a

_x

_M1,000).

_o

We

also

examined

T84

occluding

junction

fine

structure

using

the

freeze

fracture

technique. Occluding junction

mor-phology,

asassessed

by

freeze

fracture,

was

indistinguishable

between

control and experimental monolayers

indicating

that

junctional

abnormalities,

if

present,

are

_highly

focal.

(We

have

previously

described

T84

cell

tight junction

structure

in

de-tail

[4]).

Effiects

of

PMN

transmigration

on

_{permeability of}

mono-layers of

another

cell line. When

IO'

PMNwere

_placed

in the

apical

compartment and driven

across MDCK

monolayers

witha

transepithelial gradient

of

10'7

M

FMILP,

mean

resis-tance

fall

was

67.1±6.45%.

Control MDCK

monolayers

with-outa

chemotactic

gradient

showed

instability

in

resistance,

in

contrast to

_{T84 monolayers, (mean}

fall in

monolayer

resistance

of

32.1±9.4%)

but still had

significantly

higher

resistances than

experimental monolayers (P

<

_0.01).

Chemotactic

responses

for

experimental

MDCK

monolayers

were I

1±0.

12 x

104

PMN/cM2

vs.

0.3±0.12

x 104

_PMN/CM2

_{for controls}

_(P

... ., ..._f3, . .1 ': : j .;f

Figure 12. EM depicting animpalementsite at whichanaggregate of opment of focal cortical densities in PMN andT84cells is indicated

PMNhavesuccessfullyseparated

T&4

cells.PMNpseudopods are in- by arrowheads (- X8,500) and those on the left side of the EM are

timatelyassociated with each other and withpseudopodsof sepa- better seen in the inset. PMNmigrationis from mucosal to serosal

ratedT84cells(arrows). Cytoskeletal rearrangements with the devel- compartments. (- x25,000).

Discussion

We

show that in

responseto a

chemotactic

gradient,

human

PMN can

be

induced

to

migrate

across

monolayers composed

of

a

human

intestinal

epithelial

cell line

(T84

cells).

Due to

limitations ofexperimental design,

our

data

largely

are

derived

from

experiments

in

which

transmigration proceeded

in a

mucosal

to

serosal

direction.

Howeverwe

have

been able

to

reproduce

our

major resistance findings

under

conditions of

serosal

to

mucosal

transmigration (see Results).

The

migratory

pathway consists of

the

occluding

junction

and

paracellular

space

and thus is similar

to

that taken

during

PMN

migration

across

natural endothelia

and

epithelia (7, 8,

1

1).

A

prerequi-site

of this

migratory

process

is

the

opening

of epithelial

inter-cellular

occluding

junctions

and,

indeed,

substantial

perme-ability abnormalities,

as

assessed

by resistance, flux, and

ultra-structural

tracer

studies,

may occur

with PMN

transmigration.

In contrast to

the

major resistance effects which, given

the nature

of this

parameter, might

be

produced by focal (4, 12)

permeability abnormalities,

measurement

of the

transjunc-tional flux of the

paracellular

tracers

mannitol

and

inulin

showed

only threefold increases. Given

the

magnitude of

the

epithelial impalement

by PMN, it would

not

have been

sur-prising

if

fluxes

of these markers had

been much greater. For

instance, mechanical disruption of

only 10%

of

theepithelial

cells in

an

otherwise

unmanipulated

monolayer

caused

man-nitol

and inulin fluxes

to

increase

10-fold.

Hence

it

appears

that under conditions

producing

dense

PMN

transmigration

and

substantial

resistance decreases,

junctional

permeability

defects

atany

given

time

may

be

highly

focal. Further evidence

for

this

conclusion

include the fact that (a)

we were

unable

to

detect structural abnormalities in

occluding

junctions using

freeze fracture techniques; (b)

HRPtracer

studies

demonstrate

that

although macromolecular permeability defects in

the

junctional

barrier do

exist, they

are

confined

to

foci with

transmigrating

PMN;

and

(c) mannitol

and

inulin

permeabili-ties

are not

differentially

enhanced in

accordance with their

differing

molecular

dimensions.

Such

presumptive

focal

defects in the

junctional

perme-ability

barrier

seen

with dense

PMN

transmigration

could

As

By

..f

k",.

In..

.X&&

tz.Kcit

By. *

-'I

cP

A~~~~~~~.~A

'4t.89 *,8

PSr*4t,:

+~~~~~~~~~~~~~~~~~~

i

,

_ar,..

Figure

13.EMderived from

monolayers exposed

tomucosalHRP

during

PMN

transmigration. (A)

Atasite of active

junctional

impa-lement

by

PMN,HRP

diffusely

labels the

paracellular

spaces

(black

stain)

andispresentboth between

adjacent

PMN

(arrows)

and

be-tween PMN and

epithelial

cells

(arrowheads). (-

X

6,000). (B)

Ata

interpret

our data as

_providing

evidence that both of these

phenomena

may be

occuning

to some extent. Incontrast to

PMN

transmigration

across

bare,

collagen-coated filters,

PMN

migrationacross

T84

monolayersis not

entirely

random. One

speculative

but

logical

basis for this observation is that foci existin a

_{given monolayer}

atwhich thejunctionalbarrierto

permeation by

FMLPis

_relatively

poorandthuschemotactic

gradients

are more

readily

detected.

Our

data

also indicate

that

resealing

of

sites

of

junctional

impalement by

PMNdoesoccur. When PMN transmigration is

driven under conditions where

the

initial

_PMN:Tb4cell

ratio

is

1:o1,

marked

resistance

decrements ensue; but if the

che-motactic

gradient

is

removed,

the

monolayers

can

completely

reseal

within

18 h.

This

contrasts with the 5 d it takes new

monolayers

plated at

confluent density

to develop stable

resis-tances

(4,

5).

Evans etal.

(13)

have also demonstrated that the

resistance

decrease

associated

with

migration

of peritoneal

ex-udate cells

across

MDCK monolayers

canbereversed if

con-ditions

that

stimulate

migration

are removed.Similarly, Milks

N4,

-W7*

* u

PMN

''Heffi- 6

09~~~e

site where PMNtransmigrationhaspreviouslyoccurred, HRPagain

diffuselystains the paracellular spaces but appearstobeexcludedat

this timefrommostof theoverlyingintercellularoccluding junctions (arrowheads).PMNmigrationisfrom mucosaltoserosaldirection.

(- X

5,300)-et

al.

(14)

have also

recently shown that

a

renal cell

line that

forms low resistance monolayers

may

reseal

after

PMN

trans-migration.

However,

not

only

can

monolayers reseal within

hours

after

a

transmigrating "stampede" of

PMN,

but

individ-ual

junctions

appear to be

able

to

either reseal short-term

(sec-onds-minutes)

or not

lose their barrier

capability

at all

during

more

limited

degrees

of

monolayer

impalement. This

latter

conclusion

can

be derived

from

circuit

model

analysis of

the

resistance

response

occurring in experiments

where the initial

PMN:T84

ratio

was

1:10.

In these

experiments,

only

trivial

resistance decrements

were noted although - 103 PMN had

crossed

the monolayer

at

widely distributed sites. Knowing

the

dimensions of

the PMN pseudopods that cross the

occluding

junction

(as

assessed

by

electron

microscopy

of thin sections),

the

resistance of

our

Ringer's

solution

and the

depth of the

T84

occluding junctions (4),

we can

then

calculate the expected

resistance

response

for

1O'

invaded

but unsealed

junctions

(see

Appendix).

As

this

analysis

shows,

103

unsealed

transjunc-tional

impalement

sites, each

having

the cross-sectional

di-",.3w--..

lf-.,

I I

.'Awt" %.

I

.1.

;-I

II

io _4.

84

mension of

a PMN

pseudopod,

should

dramatically

reduce

monolayer resistance.

In

striking

contrast to

this prediction,

resistance

was

observed

to

decrease from

a meanof

1,100 ohm

cm2

to _

1,045

ohm

cm2.

Such data indicate that short-order

restitution/maintenance of the junctional barrier

to

the

flow

of

even

ions

can

be achieved

in states in

which submaximal

transjunctional

migration

of PMN occurs.

In

patients with intestinal inflammatory diseases,

PMN epithelial

transmigration

may

be used

as astructural marker to

judge the

severity of disease activity (1, 2). Often,

but not

exclusively,

PMN

migrate

across crypt

epithelium

and the

ac-cumulation of these cells in the

crypt

lumen

represents the classic

"crypt

abscess"

(1, 2).

Such directional movement

of

PMN

implies the

presence

of

a

chemotactic gradient,

and

in-deed chemotactic factors have been identified in the stool of

patients with ulcerative colitis (15)

and

chemotactic peptides,

specifically

FMLP

are

known

to

be

synthesized

and

released

by

bacteria such as

Escherichia

coli (16).

We have

reviewed

slides

from

biopsies

and resected

specimens of patients

with

inflammatory

bowel

disease and counted the number of

trans-migrated

PMN/number

of

crypt

epithelial

cells. Such

analysis

shows that

ratios of

at

least

1:1 are

achieved in

crypt

abscesses.

This would correspond

to a PMN

transmigration density of

106

PMN/cm2

of

crypt

epithelium and would thus be within

the

range at

which

we

observe

maximal alterations in

perme-ability.

We

speculate

that

the increased intestinal

permeability

described

in

patients with active

inflammatory

bowel

disease

(3)

might,

at

least in

part,

result

from

PMN

transmigration.

Appendix

Calculationof the volume(V)of"pores"inthe intercellularoccluding junctionsofourmonolayers.(i)This pore volume may be viewedas

being equivalent to the volume ofRinger's solution present in the pores andcanbe calculatedusingOhm's law foran

_electrolytic

con-ductor R=Lp/A(17).R= 1,100ohmcm2(meanresistance of

mono-layers used inexperimentswith

101

PMN).L = 0.5

AM

(depth of

junctionalporesatthelevelof the intercellular

occluding junction

(4).

p=54 ohmcm(specific resistivityofourRinger's solution).

1.1 X 103ohmcm2=

[5

X

10-5]

cmX54 ohmcm

A

cm2

Acm2= [5

X10-5]cm

X54 ohmcm

1.1 X 103 ohm cm2

2.7X 10-3

1.1 X 103

A=2.45X10-6 cm2

junctionalpore volume=2.45X10-6cm2X[5X

10-5]

cm =2.45X5 x 10-"1 cm3

= 122.5 3

Calculation of volume(V)ofaPMNpseudopodof diameter 1,uM

(measured by

ultrastructural

analysis)

V=

_3.14(0.5)2

X0.5

,u3

=3.14X0.125

A3

=0.3925M3

Inexperiments in which

101

PMN wereused, chemotactic response

showed 1,000PMNhad crossed theT84celljunctions(Fig. 4).After

passage of 1,000 PMN, the junctional pores, if unsealed, have

ex-panded suchthat the new junctional pore volume (V)

V= 122.5 A3+I.3925M3X 1,000]

= _{122.5IA + 392.5} A3

=515M3

Areaof these pores (Acm2)=

515

3

0.5 i

= 1030M 2

Expected resistance of the monolayer (with unsealed impalement

sites)after passage of1,000PMN

_ [5X l0-5]cm X54 ohm cm 10.3X

10-6

cm2

2.7X

lo-'

ohm cm2

10.3 X 10-6 cm2

=260 ohm

Conclusion. Observed resistance of monolayers used in

experi-ments with

101

PMNafter passage of 1,000 PMN shows only a 5% fall

from a baseline of 1,100 ohm cm2(Fig. 4), whereas by the above

calculation it should be 260 ohm. Hence, in order to maintain mono-layer resistance of about 1,045 ohm cm2 after passage of 1,000 PMN, rapid resealingof PMN impalement sites must occur.

Acknowledaments

The authors thank Dr. Michael Gimbrone for helpful discussions of

thesedata; Ms. Meryl Green and Virginia Hamel for their efforts in the preparation of this manuscript and Ms. Susan Carlson for expert tech-nical support.

This workwassupported byNational Institutes of Health grant

DK-35932 and byagrant from the National Foundation for Ileitis and Colitis. Dr. Nash is supported by U. S. Public Healthtraininggrant

HL-07066-11.Dr.Madarais supported in part by the American

Gas-troenterologicalAssociation/RossResearch Scholar Award.

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