Intravascular release of intact cellular
fibronectin during oxidant-induced injury of the
in vitro perfused rabbit lung.
J H Peters, … , L A Sklar, C G Cochrane
J Clin Invest.
1986;
78(6)
:1596-1603.
https://doi.org/10.1172/JCI112752
.
Fibronectin (Fn) is produced by cells in blood vessels at inflammatory sites in vivo. Fn
release into the circulation thus may be a marker for vascular injury. In support of this, we
found that oxidant-induced vascular injury of isolated perfused rabbit lungs caused elevated
circulating Fn levels. Western blot analysis indicated that Fn released from the injured blood
vessels was intact, dimeric, and possessed electrophoretic mobility identical with Fn
produced by fibroblasts. Unlike Fn isolated from rabbit plasma, Fn derived from lung
perfusate or produced by fibroblasts reacted with antibodies raised to a synthetic peptide
containing sequences from the extra type III Fn domain that is transcribed in fibroblasts but
not hepatocytes. Vascular injury by protease was also associated with intravascular release
of Fn, but with cleavage. Oxidant-induced vascular injury causes release of tissue-derived
Fn, which can be distinguished from plasma Fn by its size and content of antigenic
determinants of the extra type III domain.
Research ArticleFind the latest version:
http://jci.me/112752/pdf
Intravascular
Release of Intact Cellular
Fibronectin
During Oxidant-induced
Injury of the
In Vitro Perfused
Rabbit Lung
John H. Peters, Mark H. Ginsberg, Benjamin P. Bohl, Larry A.Sklar, and Charles G. Cochrane
DepartmentofImmunology, Scripps Clinic and ResearchFoundation,LaJolla, California92037; and PulmonaryDivision, DepartmentofMedicine, University of California, San Diego, SanDiego, California92103
Abstract
Fibronectin (Fn) is produced by cells in blood vessels at inflam-matorysites in vivo. Fn release into the circulation thus may be amarker
for
vascular injury. In supportof
this, we found thatoxidant-induced
vascularinjury of isolated
perfused rabbit lungs caused elevatedcirculating
Fnlevels. Western blot analysisin-dicated
that Fn releasedfrom
theinjured blood vessels was intact, dimeric, and possessed electrophoretic mobility identical with Fn produced by fibroblasts. Unlike Fn isolated from rabbitplasma,
Fnderived
from lungperfusate
or produced byfibroblasts
reacted
with antibodies
raised to asynthetic
peptidecontaining
sequences
from
the extratype III Fndomain
thatis transcribed
in
fibroblasts
but nothepatocytes.
Vascular injury by protease was alsoassociated with intravascular
releaseof
Fn, but withcleavage. Oxidant-induced
vascularinjury
causesreleaseof
tis-sue-derived Fn, which
canbedistinguished from plasma
Fnby
its size
andcontentof
antigenic determinants
of the extra type IIIdomain.
Introduction
Oxidants accumulate
atsites of tissue inflammation
involving
neutrophils (1-4).
The ability
ofantioxidants
toblock
physiologic
and
morphologic
disruption
atthesesites
suggeststhat
oxidantscontribute
toneutrophil-associated
tissueinjury (4-6).
Oxidants probably cooperatewith other
inflammatory
effector
agents,however,
including
proteases, tocausetissue
injury
invivo
(2,
3, 7,
8).
Inorder
to test theeffects of antioxidants
andantipro-teases
in
experimental
animals
andhumans, effector-specific
biochemical markers
of tissueinjury
areneeded.Ideally,
thesemarkers should
besoluble and
present in bloodand/or
otheraccessible
body
fluids.
Fibronectins
arelarge
glycoproteins
found inplasma,
oncellsurfaces,
andin
extracellular
matrices.By binding
othermac-romolecules
aswell
ascells, they
serve topromote
anchorage
ofcells
tosubstrata(9,
10).
Fibronectinsarecomposed
of subunitsof variable
primary
structure(average
molecularweight
250kD),
which
aredisulfide-linked
toform dimers
ormultimers(9-13).
The
predominant
fibronectin
inblood(plasma fibronectin)
isAddress
reprint
requests to Dr. Peters, Department ofImmunology,
IMM12,Scripps Clinic and Research
Foundation,
10666 NorthTorrey
PinesRoad,LaJolla,CA 92037.
Thisispublication4142IMM from the Research Institute of
Scripps
Clinic.
Receivedfor publication24December1985 andin revised
form
7July1986.
secreted by hepatocytes, whereas cellular fibronectin is secreted by a
variety of cultured cells including
endothelial cells andfi-broblasts (14-16).
Despite extensive
physical and immunologicsimilarities,
the two classesof fibronectin
differ in electrophoreticbehavior, solubility, and biologic
activities(14,
17, 18).Primary
structural
differences between
plasma andcellular fibronectins have beenfound
bypeptide
mapping (19) and immunologictechniques (20). Recently,
adifference region
encoding forex-actly
one90-amino acid
type III structural repeat was identifiedin
messenger (m)RNAfrom
humanfibroblasts and two human tumorcelllines, but could
notbe
detectedin
humanliver
mRNA (13, 21, 22).Since
plasmafibronectin is
synthesized by hepa-tocytes,it
is
likely
that the extra type III repeatis
aunique domain
of cellular fibronectins (12, 21, 22).
Fibronectin
accumulates atsites of injury
and
inflammation
in vivo
(23-27) andis produced by cells in blood
vessel walls atthese
sites
(28-31).
Wetherefore asked if cellular fibronectin
might accumulate in the
vascular compartment andin other
fluids
incommunication with sites of
inflammatory
tissue
injury.
Wealso asked
if
mediators of inflammatory tissue injury,
in-cluding
oxidants and
proteases, could promote insitu release of
cellularfibronectin.
Wechose toperform
ourstudies in
aninvitro perfused
lung,since this model
provides
anabundantly
vascular
tissue
that can beobserved for
bothphysiologic
andbiochemical changes
underdefined conditions.
Methods
Isolatedperfusedrabbit lung. Lungs from 2- to 2.5-kg New Zealand white rabbits wereisolatedand perfused aspreviously described (32)
withthe following modifications: a small animal ventilator (Harvard
Instruments,Millis, MA) andahumancoronaryperfusionpump (Olson
Medical Products, Ashland, MA) were used toprovide ventilation and
perfusion, respectively. 50 mg/kg ketamine (Parke-DavisCo., Morris
Plains, NJ) and 10 mg/kg zylazine (Miles Laboratories, Shawnee, KA) weregiven intramuscularlyasanesthesia fortracheotomy. After estab-lishment of ventilation(15%
02,
5%C02,80%N2,respiratory rate 20,tidal volume 10ml/kg,positive end-expiratorypressure1cmH20),32.5
mg/kg sodium pentobarbital (Western Medical Supply, Arcadia, CA) wasgivenpriortothoracotomy.Allanimalswereanticoagulated with 1000 unitsofheparin (Elkins-Sinn,CherryHill, NJ) injected into the
rightventricle 3minpriortopulmonaryarterycannulation. Theperfusing bufferwas amodified Krebs-Ringerbicarbonate buffer containing 2%
bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO) warmedto37.5'Cwith pH adjustedtobetween 7.3 and7.4after equil-ibration with the ventilatinggas.Afterperfusion with 900 mlof the perfusion bufferto washawayblood elements, eachpair of lungswas
suspended froma manual balance inawarmed humid chamberand
perfused throughthe pulmonaryarteryat arateof125 ml/min with perfusate flowing bygravityfromthe left atriumintoa 105-ml recir-culating reservoir. After -30 minof recirculation (15mintoprepare
chamberandtubingand 15 minofequilibration), lung weightand
pul-monaryartery pressure(measured witha watermanometer)were mon-itoredand recorded every 10 minfor90 minof continuous perfusion
J.Clin.Invest.
C) TheAmericanSocietyfor Clinical
Investigation,
Inc.0021-9738/86/12/1596/08 $1.00
and ventilation. Initial perfusate pH averaged 7.36 and didnotdiffer significantly between experimental groups. pHat90min averaged 7.47 and also didnotdiffer between groups.
Injury
models
Oxidant injury. Lung vasculature was exposed to superoxide anion
(O-) by
introducing
thesubstrate7H-imidazo(4,5-d)pyramidine
(purine) (Sigma Chemical Co.) into the perfusion fluid reservoirataconcentration of2 mM at 15min, followed by xanthine oxidase (from bovine milk; Calbiochem-Behring Corp., La Jolla, CA) at afinal concentration of 0.005 U/mlat20 min into the experiment.Exposureof blood vesselstohydrogenperoxide(H202)wasachievedbyconstantinfusion (Harvard infusion pump, Harvard Instruments) of H202 into the perfusate reservoir
at arateof 11 nmol/mlrecirculating buffer/min beginningat20 min andcontinuing through the conclusion of the experimentat90 min. Catalase (from bovine liver; Sigma Chemical Co.)wasaddedtothe
res-ervoirat10 min(priortooxidant)at afinalconcentration of 700 U/ml inthoseexperiments where itwasutilizedto consumeH202in perfusion fluid. H202 and O- were measuredfluorimetricallyin l-mlaliquotsof perfusion fluid taken attimepoints, using240Mgparahydroxyphenyl acetic acid, 40 Mg horseradishperoxidase (HRP),1and 40 ,g superoxide dismutase (all from Sigma Chemical Co.)(33). The assay hasalower limit of detection of -1 MMH202(equivalentto2
,M
O-)in perfusion fluid.Inlungsinjured by H202infusion,noH202accumulation could be detected in thecirculation throughout 90 min ofperfusion despite sig-nificant signs of vascular dysfunction. When lungswereremoved from the perfusion circuit, however, H202 concentration increased linearly with time and was stable for 30 minfollowingcompletion of infusion. Similarly,neither O-norits immediate dismutationproductH202could be detected in the perfusateat anytimepoint during experiments in which xanthine oxidase andpurine(XO/P)wereaddedtogenerate O2-intravascularly. After removal from the lungcirculation,however,aliquots ofperfusionfluid generated O-at rates commensuratewith enzyme and substrateconcentration. Thesefindingswereunaffected bythe presence
orabsence ofperfusatecellsin the samplebeingassayed.
Protease injury.Beginningat 20
min,
N-tosyl-L-phenylalanine chlo-romethyl ketone(TPCK)-treatedtrypsin(Sigma Chemical Co.) diluted in normal saline was constantly infusedat7.7U/mlpermin into the reservoir through the endof the 90-min recirculation. To analyze delivery of proteasetothelung vasculature, samples ofperfusatewereassayed fortrypsin activitybyfollowingthechange in absorbanceovertimeat405 nmwhen samplesorstandardswereincubated with the substrate N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitro-anilide hydrochlo-ride (Kabi Diagnostica, Stockholm, Sweden). Lungs infused with trypsin showedlinearincreasesinperfusate trypsin activityovertime. The average concentration oftrypsinin perfusate changed linearly from 7±4U/ml
at30 min (10 min after onset ofinfusion)to356±35U/mlat90 min. The doses of oxidants and trypsin used had been determined in pre-liminaryexperiments to produce significant increases in lung weight and lavageprotein concentration compared with controls within a 90-min recirculation, consistent with vascular dysfunction.
Preparation
offluid
samples. 2.5-ml samples of perfusion fluid were taken from the reservoir at 0, 30, 60, and 90 min. At the completion of each90-min experiment, a narrow bore tube was quickly inserted into therightmainstembronchus and the lower lobe waslavagedwith 5 ml of ice-cold modified Gey's buffer-carbonate. The average volume recov-ered was 2.8 ml and did not differ significantly between experimental groups. Samples of perfusate and bronchoalveolar lavage (BAL) were1. Abbreviationsused in this paper: BAL,bronchoalveolarlavage; ED,
extra type III domain; ELISA, enzyme-linked immunosorbent assay; GO/G, glucose oxidase-glucose; HRP, horseradish peroxidase; PAP, pulmonary artery pressure; PMSF, phenylmethylsulfonyl fluoride;
SDS-PAGE,
sodiumdodecyl
sulfatepolyacrylamide gel electrophoresis;
TBS, Tris-bufferedsaline; TLCK,
N(alpha)-p-tosyl-L-lysine
chloromethyl
ke-tone;TPCK, N-tosyl-L-phenylalanine chloromethyl ketone;
XO/P,
xan-thine
oxidase-purine.
immediately centrifuged
at11,600
rpm for 8s.Cellpellets
wereresus-pendedin2% acetic acid forcountsand differentialsby
hemocytometer.
Supernatants
weresnap-frozenand storedat-70'C
pendingassay for fibronectinby enzyme-linked immunoassay
(ELISA).Priortofreezing, fluids fromtrypsin-injured lungs
tobeassayedby
ELISAwereimme-diately (within
30s)
made 0.2 mM with lima beantrypsininhibitor (LBTI) (WorthingtonBiochemicalCo., Freehold, NJ) (100-foldmolar excess overmaximumtrypsinconcentrations reached inperfusate)and 6 mMN(alpha)-p-tosyl-L-lysine
chloromethylketone (TLCK) (Sigma ChemicalCo.) (3,000-foldmolarexcess). 90-min perfusate samplesso treated hadactivity
in thetrypsin
assayapproximating
backgroundac-tivity
insamplesfrom controllungsatthesametimepoint (2.4±0.6 U/ ml ininhibitedsamples comparedwith 2.0±0.2U/ml
in controlsamples). Perfusate and BAL supernatantswerepreparedforpolyacrylamide
gel electrophoresisinthe presence ofsodiumdodecylsulfate
(SDS-PAGE)
by boiling
for 4 min withanequal
volume of 10% SDS in Laemmli samplebuffer(0.5MTrispH6.8) preheatedto100IC withorwithout 3% betamercaptoethanol(34).
Preliminary experimentshad shown that immediateboiling
inSDS haltedprogressive proteolysisof fibronectin in tissue fluidstowhich proteases had been added.Therefore,fluids from lungs injuredwithtrypsinwereboiled in SDSimmediately (within30s)
after removal from thelungcirculation. Fluids from control andox-idant-injured lungs, however,could be boiled in SDSat alater time after
freezing
without effectontheintegrityoffibronectin.Preparation
oflung
tissuesamples.Attheconclusion of each exper-iment small sectionswerecutfrom the middle ofonelower lobe and placedin 10% neutral-buffered formalin for light microscopy. Addition-ally, apieceoflungparenchyma fromalower lobewasimmediately
clampedwith tongsprecooledinliquid nitrogen.The frozenlungwas
homogenizedfor 30sinaliquidnitrogen-cooled 3-ml Tefloncartridge usingaMikro-dismembrator(B.BraunInstruments, Melsungen,Federal RepublicofGermany), yielding -50 mg of frozen powder. 500 Ml of boiling samplebufferwerethen addedtothe frozen lung powder derived fromhomogenization, and theresulting suspensionwasvortexedand boiled foranadditional4min. Insolublelungmaterial was thenpelleted by centrifugation, and samplesof the supernatant subjected to SDS-PAGE.
Examination
offibronectins
byWesterntransfer. Electrophoresiswasperformedinlow percentageorgradient SDS-polyacrylamide gels.The proteins in the gelswereelectrophoretically transferredtonitrocellulose sheets(Millipore Corp., Bedford, MA) (35).After transfer, the sheets
wereblocked with 3% gelatin in Tris-buffered saline pH 7.4 (TBS) for 30 minutes and then soaked overnight in 1% gelatin in TBScontaining goat antifibronectin antibodies. Afterwashingin TBS containing 0.05% Tween 20, the sheetsweresoaked for 90 min in 1% gelatin in TBS containing HRP-conjugated swine antibodiestogoatIgG. The sheets
werewashedpriortovisualization of immunoreactive protein bandsby incubation with 0.05% H202 and 0.5 mg/ml HRP ColorDevelopment Reagent (Bio-Rad Laboratories, Richmond, CA) in TBS containing 17% methanol for 30min. Because the BSA in the perfusion buffer contained
tracesofbovine IgG with which the swine antibodies reacted, these an-tibodieswereabsorbed with bovine IgG (Calbiochem-BehringCorp.) immobilizedonSepharose beads priortousein staining Western transfers in which samples of whole perfusate (rather than isolatedfibronectins)
wereexamined.
Isolation
offibronectins.
Fibronectin was purified from rabbitplasma, pooled lung perfusate, human plasma, fetal bovine serum, and condi-tioned media (Dulbecco's modified Eagle's medium containing 10% fi-bronectin-depleted fetal bovine serum) from confluent GM-1380 human fibroblasts and RAB-9 rabbit fibroblasts by affinity chromatographyongelatin Sepharose(36).
To compareelectrophoretic mobility of Fns contained in small
sam-ples of lung perfusate, lavage, and tissue, distortions in electrophoretic mobility duetootherproteins was minimized by isolating fibronectin using small nonlimiting quantities of gelatin agarose beads (Pierce Chemical Co., Rockford, IL)(14). 100 Ml of perfusate or lavagewere
shaken with 40 Ml ofpacked gelatinbeads for15 minat roomtemperature,
2mMEDTA prior to elution with 100
,d
of sample buffer. Lung tissue fibronectin was isolated by first shaking -20 mg oflyophilizedpowder (2) from perfused (bloodless) rabbit lung tissue with 1 ml of 2 molar urea, 10mg/ml porcine intestinal heparin (Sigma Chemical Co.), and 2 mMphenylmethylsulfonylfluoride (PMSF) in PBS at room temperature for 4 h (37). The supernatant from the resulting suspension was diluted 10-foldwith PBS and shaken with gelatin agarose beads prior to washing and elution with samplebuffer.Synthesis
ofhumanfibronectin
extradomainpeptide. The 90 amino acid extra type III domain of human fibronectin contains a region oc-curring 36 to 60 amino acids from its amino terminal end in which there is a relative absence of sequence homology with other type III repeats (13). The 19 amino acid peptide ELFPAPDGEEDTAELQGGC was thereforesynthesized using solid phase methodology on peptide synthe-sizer (model 438, AppliedBiosystems, Foster City, CA) (38). The N-terminal 17aminoacidsof the peptide represent a region of the extra domain running45 to 61 amino acidsfrom its amino terminus. The carboxy terminalglycineand cysteine were added for linkage to the solid phase. Thepeptidemigrated >90% in a single peak when subjected to highperformanceliquid chromatography using a C18 column (Vydac, Western Analytical Products, Temecula, CA). To confirm amino acid composition,thepeptidewashydrolyzed in 6 Nhydrochloricacidfor 24hat 1100C,dried overnight, and then applied to an amino acid an-alyzer column (Beckman 121-M, Beckman Instruments, Inc.,Fullerton, CA). The individual amino acid contents conformed 99.9% with the desiredpeptide composition.Antifibronectin
antibodies. Antibodiestorabbit plasmafibronectin wereraised in a goat by weekly multiple intradermal injections of 100 g ofpurified
antigen
emulsifiedinFreund'sadjuvant
(Difco Laboratories,
Detroit, MI). Antibodiestothe extra type III domain ofhuman fibronectin weresimilarlyraised ingoats by weekly intradermalinjectionsin adjuvant of 150 ,ugofthe extradomainpeptide (ED peptide)coupled to keyhole limpethemocyanin (Pacific Bio-Marine, Venice, CA) by the glutaral-dehydemethod(39). Antibodytitersspecificfor the EDpeptidewere checked by ELISA (40).
Toinsurethat theanti-ED peptide antibodieswere in fact reactive withnonhepatic (cellular)but nothepatic (plasma) fibronectin, equal quantities of fibronectin purifiedfrom humanplasmaandGM-1380 cultured humanfibroblast media(containing fibronectin-depleted fetal bovineserum)were
subjected
toSDS-PAGE withWesterntransfer. Pro-teinconcentrations weredetermined withBCAprotein
assayreagent (Pierce Chemical Co., Rockford, IL) usingBSA asastandard.Staining wasachieved withgoatanti-EDpeptide antibodies,goat anti-EDpeptide
antibodies
preincubated
with EDpeptide,
orgoat anti-rabbitplasma
fibronectinantibodies
(crossreactive
withhumanfibronectin).
As seeninFig. 1, theanti-EDpeptide antibodies didnot reactwithfibronectin isolatedfromhumanplasma,butdidreact
specifically
withfibronectin isolatedfromhuman fibroblastconditionedmedia.Measurement
offibronectin
influid samples.
Aquantitative
non-competitive
ELISAwas constructedusing
rabbitplasma
fibronectin standards (41). Wellsof microtiterplates
were first coated with goat antirabbit plasma fibronectin antibodiesimmunopurified
asdescribed(11)
anddilutedin0.1MNaHCO3. Next, standardsandsamples,
both dilutedinperfusion
buffer(2% BSA)
wereapplied. Finally,
immuno-purifiedgoat antirabbitplasmafibronectin antibodiesconjugated
toal-kalinephosphatase (from bovine intestinal mucosa;
Sigma
Chemical Co.)withglutaraldehyde
(41)wereapplied.
p-Nitrophenyl
phosphate
(Sigma Chemical Co.)at 1
mg/ml
in 1 MdiethanolaminepH
9.8was utilizedassubstrate forcolordevelopment. Asigmoid
standardcurve wasestablishedbytherelationshipbetween thelogof theconcentration of rabbitplasma fibronectin standardsorsample
dilutions and the ab-sorbancereadingat405 nM after 120 min of colordevelopment.
Measurement and characterization
of
totalBAL protein. Total protein inBALsampleswasmeasuredbythemethod ofLowryetal.(42), using
BSA as astandard.Additionally,2
,ul
ofBALfluid from eachexperiment
weresubjectedto
SDS-PAGE
usinga5-20%SDS-acrylamide gradient under bothreducing
andnonreducing
conditions. Visualization ofre-sulting proteinbandswasachievedbyCoomassie Brilliant Blue
staining.
PLAS CELL PLAS CELL PLAS CELL
AntiPeptide Blocked Anti-Plasma Anti-Peptide Fibronectin
Figure 1.Antibodiesto the extra domain peptide react specifically withhumancellularfibronectin.Equal amounts (0.5
Mg)
of fibronectin purified by gelatin affinity chromatographyfrom human plasma and GM-1380 humanfibroblastmedia(containing fibronectin-depleted fe-talcalfserum) weresubjectedto SDS-PAGE (reducing conditions) and Westerntransferinpairedlanes. Staining wasaccomplishedwith (from left to right) anti-ED peptide serum, anti-ED peptide serum preincubated with 100$&g/ml
ED peptide, and anti-plasma fibronectin antibodies.Data
presentation and statistics. Values were expressed as
mean±SEM. Statisticalanalyses were performed by use of the Student's two-tailed t test. P values<0.05 weresignificant.
Results
Effect
of
oxidant orprotease treatment on
fibronectin
concentra-tionsin
perfusion fluid
and alveolar
lavage.
Fibronectin was not
detected in the
BSA-containing
perfusion buffer
by either ELISA
or Western
transfer
prior
tocirculation
through rabbit lungs.
However,
lowlevels
(0.3
to1.0
Ag/ml)
of rabbit
fibronectin
ac-cumulated in
therecirculating perfusate during
the30-min
equilibration period prior
toexperimental
observation. Over the
course
of the
ensuing
90 min,
perfusion
fluid of
oxidant-injured
lungs accumulated
significantly
greaterlevels of fibronectin
thanuninjured
controls
orcatalase-protected lungs (Table I).
Accel-erated
ratesof accumulation of fibronectin in
perfusion
fluid
of
oxidant-injured lungs began
in the
period
between 30 and 60
minutes
(between 10 and
40 minafter addition of oxidant
toperfusion fluid).
Edema
formation,
measured
asincrease in
lung
weight,
generally began
tooccur at -30
min.Fig.
2plots both
perfusion
fluid fibronectin concentration
(measured
every30
min)
andlung
weight gain (measured
every 10min)
as afunction
of time.
In contrast to
the oxidant
injury model, continuous
trypsin
infusion caused edema formation between 60 and 90
min(40-70
minafter
onsetof
infusion).
Perfusate fibronectin
levelsmea-sured
by ELISA decreased
throughout
thetrypsin infusion,
in-cluding
theperiod
of edema formation
(Fig. 2).
Fibronectin retrieved
by lavage
of
thealveolar
spaceat 90 minwaselevated
inoxidant-treated
groupscompared
with
con-trols.
Catalasepretreatment of
perfusion
fluid
prior
toaddition
of oxidants
prevented
the
increase in BALfibronectin associated
with oxidant
exposure(Table I).
Physical
characteristics
offibronectin
inperfusate, lavage,
and
lung
tissueafter
oxidant
orprotease
treatment.Fibronectin
in
perfisate
of lungs treated with oxidants
wasintact anddimeric
(510 kD)
(Fig.
3A).
Underreducing
conditions
thesubunits of
Table L Fibronectin Levels in
Perfusate
and LavageFluid ofIsolatedPerfused
Lungs*Group Perfisate Lavage
Wg/mi pg/mi
Controls 1.5±0.3 0.04±0.01
n=9
H202
10.5±1.9t
0.41±0.12t
n=6
H202 + catalase 1.4±0.3 0.06±0.02 n =4
XO/P
10.9±1.7t
0.28+0.13t
n =4
XO/P + catalase 1.6±0.2 0.07±0.02 n=4
Trypsin 0.1
±0.0t
0.07±0.01 n=6*Samplesfor assay were takenafter90 minof experimental observa-tion.FibronectinwasmeasuredbyELISA,
using
rabbit plasma fibro-nectin standards.t
P<0.05compared
with controlgroup.subunits of fibronectin
extractedfrom normal rabbit lung tissue
(Fig.
3B). Bothmigrated
assingle
bands (-260 kD) incon-ventional
lowpercentage
gels. These bands were of a higher apparent molecularweight than either of the bands formed
by thesubunits of rabbit plasma fibronectin
(210 and 240 kD).Rabbit plasma fibronectin diluted in perfusion buffer
andsub-jected
to2
mMH202
for 90 min showed
no changein
electro-phoretic behavior
(notshown).
In order to resolve small differ-ences inelectrophoretic mobility between subunits, samples
offibronectin
were alsosubjected
toprolonged electrophoresis
un-der
reducing
conditions. Duration of electrophoresis
wascali-brated by
theposition of prestained molecular weight standards
(Bethesda Research
Laboratories,
Bethesda,
MD). Under theseconditions,
thesubunits of perfusate fibronectin
resolved into a doubletpossessing electrophoretic mobility identical
with thatof fibronectin isolated from
the media of rabbitfibroblasts which
had been grown
in fibronectin-depleted
media (seeMethods)
(Fig. 3 C). Treatment of
the lungvasculature
with eitherO2-
orH202
causedintravascular release
offibronectin
ofsimilar
sub-unit composition.
Fibronectin in
theperfusion
fluid of alltrypsin-treated
lungs wasprogressively cleaved
over time. Asshown
in Fig. 4, theCOMArokn-)0-o
HA,(k-6)o.--o
Xi(Oa-41o--orA+C~tafrseEn-)vXO/P+Cta0-.
(-.j~~~~~~~~~~~~~I
~
~
4wE 3
.Io 4-
t
fibronectin
that accumulated in the30-min period
of recircu-lationprior
totime=0min,
wasintact
and ofhigher
molecular
weight
than
plasma
fibronectin.
At later timepoints,
itwasre-placed
with
cleavage
products
concomitant withadministration
of
trypsin. Lavage
fluid fromprotease-injured lungs
also con-tainedfibronectin
fragments,
and thesecomigrated
withfrag-mentsin
perfusion
fluid andlung
tissue extracts taken at thecorresponding timepoint.
Incontrast, intact fibronectin
was de-tected inBAL andlung
tissue extracts fromoxidant-injured lungs
(not shown).
The
fibronectin that accumulated
atlow levels in control
and
catalase-protected lungs
wasphysically
indistinguishable
from oxidant-released fibronectin
when examined withWestern
transfer
using
increased quantities
of isolatedantigen
and/or
antifibronectin
antibody
concentrations(not
shown).
Effect
ofproteolysis offibronectin
onmeasurementbyELISA.
Serialthreefold dilutions
(10
to0.014ug/ml)
ofpurified
rabbitplasma
fibronectin
ineitherperfusion buffer, perfusion
buffer
containing
LBTI andTLCK,
orperfusion
buffercontaining
trypsin
alonewerekept
at4°C
for2 h. Afterincubation, samples
containing trypsin
alone
werethen also
treated withLBTI and
TLCK. The final concentrations of
trypsin, LBTI,
andTLCK
were
equal
tothose achieved
ininhibited perfusate (see
Prepa-ration of Fluid
Samples) samples
fromtrypsin-treated lungs
at 90min. ELISA-derived fibronectin
concentrations insamples
incubated
withtrypsin
andinhibitors averaged 97%, whereas
concentrations in
samples containing uninhibited trypsin
av-eraged
43% of values fordilutions
offibronectin
inperfusate
alone.
Western
transfer examination of thesamples confirmed
that
fibronectin
exposed
totrypsin
without inhibitors
waspro-teolytically degraded.
In contrast,fibronectin
insamples
con-taining
notrypsin
orinhibited trypsin
remained intact(not
shown).
Electrophoretic examination of perfusion fluid
fibronectin
using
anti-extradomain antibodies. Although fibronectin in the
circulation
ofoxidant-treated lungs possessed
electrophoretic
mobility consistent
with cellularfibronectin,
wesought to con-firm thisdesignation
by
testing its reactivity
with anti-EDan-tibodies.
Antibodies
tothe human ED peptide stainedfibronectin
from
perfusion
fluid ofoxidant-injured rabbit lungs but
notequal
quantities
offibronectin
isolated from rabbitplasma.
Staining
wasprevented when
anti-peptide
serum waspreincubated with
ED
peptide. Only
the upperband
ofthe perfusate fibronectin
doublet (detected
withanti-plasma fibronectin antibodies)
was stainedby
antiserum to the EDpeptide.
In contrast,dimeric
perfusate fibronectin
of uniformmobility
wasdetected byboth
rrpsunn-6 o-*o
0 30 60 90 0 30 60 90 0 30 60 90 0 30 60 90
Time (minutes)
Figure
2.Temporal relationship be-tween perfusatefibronectin concentra-tion measuredbyELISA and increase inlung
weightfor(startingfromleft) uninjuredcontrol lungs, lungs exposed toH202withorwithout catalase pro-tection, lungsexposedtoO- withorA B
20oK-I I
200K- 68K-
97K- 68K- 43K-
26K-
97K-1 2 3 1 2 3
97K-
68K-
43K-
26K-1 2
Figure 3.Comparison of fibronectins. Rabbit fibronectins from differ-entsources weresubjectedtoSDS-PAGE andWesterntransferand
stainedusingantirabbit plasma fibronectin antibodies. Samples in (A)
werenonreduced;samples in (B) and (C)werereduced.(A)Equal
vol-umes(10 Ml) of perfusion fluid obtainedat90 min from representative
lungsinjuredwithH202 (lane 1)or O2 (lane 2),andrabbitplasma
fi-bronectin diluted in perfusion buffer (final fifi-bronectin concentration
of 10
Ag/ml)
(lane 3)weresubjectedto3-20% gradient SDS-PAGE without prior isolation of fibronectin.Inadditiontothepredominantdimeric formof fibronectin (shown here),veryfaintstaining of mate-rialwithmobility consistentwithmultimericand/ormonomeric
fibro-nectinwasoccasionally observedwhennonreduced perfusatewas
sub-jectedtoWesternblotting. (B) Similar quantities of fibronectinwere
isolatedongelatinagarosebeads from(1)aheparinureaextract(see Methods)ofuninjured perfused (bloodless)lung tissue,(2) perfusion
fluidobtainedat90min fromalunginjuredbyH202infusion, and
(3)rabbitplasma.Lung tissue fibronectinwaspreparedby absorbing 1 mlof diluted heparinureaextract(see Methods)with40 of gelatin
beads, eluting with 100 ofsample buffer, and subjecting20 Mlof eluatetoelectrophoresis. Fibronectinwassimilarly isolatedfrom
per-fusionfluidexceptthat100 Mlwereabsorbed and 20 Mlofeluatewere
subjectedtoelectrophoresis. Approximately 400ngof rabbitplasma
fibronectinwas runinlane3.(C) Similar quantities of gelatin-isolated
fibronectinsobtainedfromrabbit fibroblastmedia(containing fibro-nectin-depleted fetal bovineserum) (lane 1) andfrom perfusion fluid
fromanH202-injuredlung(lane2)weresubjectedtoprolonged
elec-trophoresisina5%gel.
PLAS PERFUSATE BALLUNG FN 0 30 60 90 90 90
Minutes
Figure 4.Fibronectin samplesinperfusate,lavage froma
representa-tivetrypsin-injured rabbitlung. From lefttoright: plasma fibronectin (50 ng), serial perfusate samples (10 Ml),BAL(20 Ml),and SDS-extract
oflungtissue(5 ul ofsupernatantfroma 100-mg/ml suspensionof
lung tissuehomogenatein sample buffer). Fluid sampleswereboiled
inSDS immediatelyafterremovalfromthelung,orinthecaseof
lung tissue,after homogenizationinaliquid nitrogen-cooled cartridge
PAGEand Western transfer with staining by anti-rabbit plasma
fibro-nectin antibodies.
controls,butonly significantlysointheH202-injurygroup.
Pre-treatmentofperfusion fluid with catalaseprevented disruption of all three parameters of vascular functionby eitherH202or
O2. Thegrouptreated with catalasepriortoXO/P showed a small decrease in PAPcomparedwithbaseline.
SDS-PAGEof BAL fluids with elevatedprotein
concentra-a
PLAS PERF PLAS PERF PLAS PERF
b
PERF
r-typesofantibody under nonreducing conditions (Fig. 5). Anti-bodiestothe EDpeptide also specifically stained higher molec-ularweight subunitsof the intactfibronectinthataccumulated
atlow levels inperfusionfluidofuninjuredlungs(not shown).
Electrophoretic
examinationof rabbit fibroblast
fibronectin
using anti-extra domain
antibodies.
Cellularfibronectin
isolated fromrabbit fibroblast mediawasheterodimeric withtwomajor subunitpopulationsthatcomigrated with those ofrabbitlung perfusate fibronectin.Likeperfusate fibronectin, only the slower migrating subunit population stained with anti-ED antibodies (Fig. 6).Effects
of oxidant
orprotease treatment on parametersof
lung
vascularfunction.
Alllungssubjected
tothe cited doses of injuriousagentswereobservedfor90 minofrecirculation and ventilation. Net increase in lungweight overthis period andBALprotein concentrationweresignificantly increasedinlungs
treated with oxidantsorproteasecomparedwithcontrols (Table II). Increase inpulmonaryarterypressure(PAP)overbaseline wasalsogreaterinthe oxidant and protease-treatedgroupsthan
1 2 3 4 5 6 1 2 3 4
Figure 5.Antibodiesto extradomainpeptidereactspecificallywith perfusatefibronectin.Equalamountsof fibronectin isolatedfrom
rab-bitplasmaorperfusionfluidobtainedfromH202-injuredlungsat90
minweresubjectedtoprolonged5% SDS-PAGE andWestern transfer
inparallel. (a)Afterelectrophoresisunderreducingconditions, elec-trophoreticallytransferred fibronectinswerestained withantipeptide
serum(lanes Iand2), antipeptideserumpreincubatedwithpeptideat
750 Mg/ml (lanes3 and4),orantirabbitplasmafibronectinantibodies (lanes5 and6). (b)Perfusate fibronectin underreducing(lanes Iand 2)andnonreducing (lanes3 and4)conditions. Stainingwasachieved
withantipeptideserum(lanesI and3)orantiplasmafibronectin
anti-bodies(lanes2and4).
1600 Peters, Ginsberg,Bohl,Sklar,and Cochrane
200K-
WFigure 6.Anti-ED antibodies reactwith slower-migrating subunits of fibronectin from rabbit lung perfusate and rabbit fibroblast media. Perfusate fi-bronectin (0.25 and 0.15 ug in lanes I and2,respectively), 200K- andfibroblast fibronectin(50
and30 Ml ofgelatin bead eluate in lanes 3 and 4, respectively) were subjected to prolonged electrophoresis in a 5% gel (re-ducing conditions)followedby Westerntransfer.Stainingwas
achieved with anti-ED antibod-ies in lanes 1 and 3 and anti-plasmafibronectin antibodies 97K- in lanes 2 and 4. Isolation of
fi-11223~4~5
bronectin
from the media (con-taining fibronectin-depletedfe-talbovine serum) of confluent rabbit fibroblasts was accomplished by mixing 100
M1
of packed gelatin agarose beads with 4 ml of media fol-lowed by elution with 100ulofsamplebuffer. To examine the possi-bilitythatfibronectin detected in fibroblast media might represent a mixture of rabbit cellular fibronectin and residual bovine fibronectin,4ml of media not previously exposed to fibroblasts was subjected to affinity-isolationas described above. In lane 5. 30
Mi
of the resulting eluate was subjected to electrophoresis and Western transfer with staining by antiplasma fibronectin antibodies (identical conditions as lanes 2 and 4).tion
uniformly revealed
that thepredominant protein
bandcomigrated with
theBSA in the
perfusion fluid
(not shown).Cellular composition
ofperusate
and
lung
tissue. In allex-periments,
the
majority of nonerythrocytic
cellsidentified by
hemocytometer
inperfusion
fluid
samples were mononuclear with large round nuclei.Wright-stained
smears of these cells revealed thatthey
possessed granular cytoplasmand
weresimilar insizetopolymorphonuclear leukocytes also
detected inper-TableII.Evidenceof Vascular Dysfunction*
Change in Change in BALprotein Group lungweight PAP concentrationt
g cmH20 mg/mi
Controls 0.5±0.2 1.2±0.3 0.4±0.1 n=9
H202 13.5±2.1 § 8.0±+.1§ 2.5±0.7§ n=6
H202+catalase 0.5±0.1 0.01±0.5 0.7±0.1
n =4
XO/P 6.9±0.9§ 2.4±0.5 1.1±0.3§
n=4
XO/P+catalase 0.85±0.3 -0.6±0.9§ 0.4±0.1
n=4
Trypsin 12.9±3.5§ 3.1±0.9 5.4±2.0§ n=6
*Changesin lungweightandPAPrepresent differences between mea-surements at0 and90 min ofexperimentalobservation. Valuesare positiveunlessotherwiseindicated.
tBAL wasobtained after 90 min of experimental observation. §P < 0.05 compared with the control group.
fusate.
Although
total cell number inperfusion
fluidwasnotedto
increase
overtime
in allexperiments, significant
differences incountsdid
notoccurbetweenexperimental
groupsattime
points.
Attime
=90min,
circulating
mononuclear andpoly-morphonuclear
cellcounts were76.7±10.4
and4.3±0.5
X103
cells/ml
innine
lungs
of controlanimals,
66.3±12.9
and4.3±0.6
X
103
cells/ml
in
six
H202-treated lungs,
88.0±19.9
and3.7±0.7
X
l03
cells/ml
in four catalase-protected H202-treated
lungs,
82.4±14.1
and2.2±0.8
X103 cells/ml
in fourXO/P-treated
lungs, 70.7±23.5
and2.4±0.8 cells/ml
in fourcatalase-protected
XO/P-treated
lungs,
and84.9±8.0
and1.9±0.7
X103
cells/ml
in
six
trypsin-treated lungs.
No
significant
differences inmorphology
orcellularity
could bedetected between
oxidant-injured
anduninjured lungs
by
light
microscopy.
In contrast,frequent thinning, elongation,
and ruptureof
alveolarseptaewerenoted inproteaseinjured lungs
(not
shown).
Discussion
Thedata
presented
in thisstudy
show that oxidant andprotease-induced
vascularinjury
are each associated with characteristiceffector-specific
changes
inpulmonary
tissue fibronectin in vitro. Intactfibronectin dimers
arereleasedinto blood vessels ofisolated
perfused
rabbit lungs inresponse tolevels ofcirculating
oxidants that
lie within the consumptive capabilities oflung
antioxidant defenses.
These levels of oxidants neverthelesscause vasculardysfunction
(evident
aslung weight gain),
and thispro-cess correlates
temporally
with fibronectin release. Whensub-jected
toelectrophoresis,
subunits of the released fibronectinmigrate
moreslowly than
those of rabbitplasma
fibronectin. Thisis typical
ofhuman, chicken,
rat, and hamster cellularfi-bronectins
incomparison with
theirplasma
counterparts(14,
17,
43).
Additionally,
the released fibronectin shows similarelectrophoretic mobility
to fibronectin extracted from normal rabbitlung
tissueperfused
free of blood elements. Finally, theperfusion
fluid fibronectin
possesseselectrophoretic
mobility
which is identical with rabbit fibroblast-derived cellular
fibro-nectin. Therefore,
the fibronectin released by theoxidant-injured
vasculature of the in
vitro
perfused lungappearsbyelectropho-retic comparisons
tobe cellular fibronectin.Toconfirm the designation of
perfusate
fibronectinascellularfibronectin
weexploited
the known differences inprimary
struc-ture
between non-hepatic
(cellular) and hepatic (plasma)fibro-nectins
(13). Thefeasibility
of this approachwasrecently dem-onstratedby Hynes (personal
communication) who showed thatantibodies
to afusion protein containinganextra typeIIIrepeat ofratfibronectin reacted with subunits ofratcellular butnotrat
plasma
fibronectin. Inagreementwith Hynes' findings, wewereableto prepareantibodiesto anamino acid sequence within the extra type III domain of human fibronectin that reacted
exclusively
with human and rabbit cellular, but notplasma fi-bronectins. The reactivity of these antibodies with rabbitlung
perfuisate
fibronectin identifies this proteinascellular fibronectin. Becausefluid
accumulationby
theoxidant-injured
lungs
(measured
asweight
gain) accounted for less than a20% addi-tional contraction of the recirculating volume, the greaterthan sixfoldaverage increase in fibronectin concentration achieved in the circulation of these lungs compared with uninjured lungs mustbe attributed tointravascular release rather than concen-tration. The mechanism of vascular release of intact cellularfibronectin
inresponsetooxidants remains
unclear.Fibronectin
could be dislodged from extracellular sites or released by cells
in
proximity tothe vasculature.Becausevarious mononuclear cells (10) as well as
polymor-phonuclear
leukocytes
(44, 45) havebeen shown to synthesizefibronectin, differences
in perfusateconcentrations of fibronectin between oxidant and control groups couldconceivably
arise fromdifferences
incellularcomposition
ofperfusate or tissue. How-ever,significant
differences
in
circulating cells were not apparentbetween these groups.
Nor were therediscernible
differences intissue cellularity
asjudged
by lightmicroscopy. Endothelial cells,the
major
cell group in contact with the circulation in our model, secreteintact
cellularfibronectin dimers
into cell culture medium when grown intissue culture(15,
16). Additionally, these cells have been shown to produce fibronectin in response to tissue injury andinflammation in
vivo (28-31). These facts, takentogether with
our data, suggest that endothelial cells are apo-tential
site of
releaseof fibronectin
inresponse to oxidant stress. In contrast to the results obtained withoxidants,
protease-induced
vascular injuryof
the perfusedlung was associated withdecreased levels of circulating fibronectin
measured byimmu-noassay
(ELISA).
However, Westernblotting revealed that thesamples
subjected
toELISA
did
notcontain
intact fibronectin. Whereasperfusate
samples taken prior to addition of proteasecontained fibronectin with electrophoretic mobility
consistentwith intact
cellularfibronectiin,
samples at latertime
pointscon-tained
fibronectin
that had been
proteolytically degraded. Since
proteolysis was
shown
tolead
todecreased
measured values byELISA,
fibronectin
may havebeenproteolytically
released
from
vessels, but not detected by
immunoassay
due todiminished
antigenicity.
The
increased fibronectin levels in the lavage fluids from
lungs
with
signs
of
vascular
dysfunction (increased
lung
weight
and
lavage
protein)
could
reflect
movementof fluid and
proteins
from the
circulation into the alveoli and/or
localrelease of
fi-bronectin from alveolar
tissue
orcells. Since the
predominant
protein that
wasdetected in the
airspaces
of
injured lungs
comi-grated
with
perfusion fluid
BSA in
SDS-PAGE,
itis
likely
that
fluid
andproteins, including fibronectin,
movedfrom
thecir-culation
toairspaces during
vascular
injury
in
this
model.The
association of oxidant
exposurewith intravascular
re-lease of cellular
fibronectin
in the in
vitroperfused
rabbit
lung
raises the
possibility
that
cellular fibronectin could
representasoluble
markerin blood
orother accessible tissue fluids for
ox-idant-induced
injury.
Total
plasma
fibronectin levels have been
noted
toincrease in
ratsduring
experimental systemic
inflam-matory
joint
disease
(46)
and
during
exposuretohyperoxia (47).
Because
leukocyte-associated
inflammation
andhyperoxia
both
involve
generation
of oxidants
atsites of tissue
injury (1-6, 48,
49), the associated
accumulation
of
circulating
fibronectin in
these models could in part
reflect,
asinourmodels,
releaseof
fibronectin by
oxidant-injured
vasculature. Accumulation of
such
cellular
fibronectin
in theplasma
of intact animals would
notbe
expected
toreflect
injury
toanyspecific
organ,since
cells(endothelial cells, fibroblasts,
etc.)
thatproduce
cellularfibro-nectin
areubiquitous. However,
detection of cellular fibronectin
in
organ-specific
fluid
(BAL,
synovial fluid, Urine)
could
poten-tially
point to local organinjury.
Forexample,
inrecentexper-iments
we havecaused oxidant-induced
injury
of
lung
tissueinintact rabbits
by instillation of
glucose
oxidase andglucose
(GO/G)
intothe lowerairways.
This hasuniformly (in
sevenof
seven
rabbits)
led
tohistologic
evidence of
injury
to thepul-monary
parenchyma
(including
focal
disruption
of
alveolarstructure as well as hemorrhage) 4 h posttreatment. Instillation
of
catalasewith
the GO/G hasuniformly prevented histologic signs of injury (6 out of 6 rabbits), indicating that H202produced
by the
GO/G is
necessary for injury to occur. Using Westernblot analysis,
wehave detected intact cellular fibronectinin
the BALof
allof the rabbits treated with GO/G but none ofthe
rabbits
treated with GO/G plus catalase. TheED-containing
fibronectin
subunits in
the BAL of the rabbits with pulmonarytissue injury
caused by GO/G comigrate during electrophoresiswith
the slower migrating subunits of the cellular fibronectin inperfusate of isolated rabbit
lungsinjured by oxidants as describedin
the presentstudy (Peters and Cochrane, manuscript inprep-aration). This
result alsoconfirms
the capacity of oxidants totrigger
the appearanceof
cellularfibronectin
in bodyfluids notlimited
toin vitro
perfused organ systems.In
summary, in this study we have shown that oxidant andprotease-induced
vascular injury are each associated withchar-acteristic alterations in
fibronectinderived from nonhepatic tis-suein
vitro.
Nonhepatic tissue fibronectin (cellular fibronectin)is therefore
recognized as an in situ target of these two majorclasses
of inflammatory effector
agents. Additionally, we havedescribed
amethod for
the assay of tissue fibronectin to theexclusion of
plasma
fibronectin.
Thus, the use of tissuefibro-nectin
as a probefor
effector-specifictissue injury in vivo is nowfeasible.
Acknowledgments
Wegratefully acknowledge the superb technical assistance of Catherine Case. We thank Dr. Erkki Ruoslahti, Dr. Roger Spragg, Bill Loomis, andSusan and David Revak for theirhelpful discussions.We also thank Dr.Michael
Buchmeier
forpeptide
synthesis,
Ward Coppersmith for construction of the lungperfusion
chamber, and Monica Bartlett and Lynn LaCivita fortypingthemanuscript.Supported by U. S. Public Health Service grants HL 23584 (University ofCalifornia, San Diego, Specialized Center of Research), and HL 28235, and TheCouncil for Tobacco Research.
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