CD10/neutral endopeptidase 24.11 in
developing human fetal lung. Patterns of
expression and modulation of peptide-mediated
proliferation.
M E Sunday, … , B Reyes, M A Shipp
J Clin Invest. 1992;90(6):2517-2525. https://doi.org/10.1172/JCI116145.
The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of […]
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CD10/Neutral
Endopeptidase 24.11 in Developing Human Fetal Lung
Patternsof Expression and Modulation of Peptide-mediated Proliferation
Mary E. Sunday,* Ji Hua,* John S. Torday,t Bernadette Reyes,* andMargaretA.Shipp'
*DepartmentofPathology, Brigham& Women'sHospitaland Harvard MedicalSchool, Boston,Massachusetts02115;tDepartment of Pediatrics, UniversityofMaryland MedicalSchool,Baltimore,Maryland21201; and~Department ofMedicine,
Dana-FarberCancer Institute and Harvard MedicalSchool, Boston,Massachusetts02115
Abstract
The cell membrane-associated enzyme CD10/neutral
endo-peptidase24.11(CD10/NEP)functions inmultipleorgan sys-tems todownregulate responses to peptide hormones. Recently,
CD10/NEP was found to hydrolyze bombesin-like peptides
(BLP), which are mitogens for normal bronchial epithelial cells and smallcell lungcarcinomas. Growth of
BLP-respon-sive small celllungcarcinomas was potentiated byCD10/NEP inhibition, implicating CD10/NEPin regulationof
BLP-me-diatedtumorgrowth.BLP arealsolikelytoparticipatein nor-mal lung development because high BLPlevels are found in fetallung,andbombesin inducesproliferationandmaturation ofhumanfetallungin organ cultures andmurine fetallungin utero. Toexplore potentialroles forCD10/NEPinregulating peptide-mediatedhumanfetal lung development,wehave
char-acterized temporal and cellular patterns ofCD10/NEP
ex-pression and effects ofCD10/NEP inhibition in organ cul-tures.PeakCD10/NEP transcriptlevels areidentifiedat 11-13 wkgestation by Northern blots andlocalized toepithelial
cellsandmesenchyme ofdeveloping airways byinsitu hybrid-ization.CD10/NEP immunostaining ismostintensein
undif-ferentiated airway epithelium. Inhumanfetal lung organ cul-tures,inhibitionof CD10/NEP with either phosphoramidonor
SCH32615 increasesthymidine incorporation by 166-182% (P
< 0.025). The specific BLP receptor antagonist,
[Leu'3-psi(CH2NH)Leu1'bombesin abolishestheseeffectson fetal
lunggrowth, suggesting thatCD10/NEPmodulates
BLP-me-diated proliferation. CD10/NEP expression in the growing frontof airwayepithelium and theeffects of CD10 /NEP inhibi-tors inlung explantsimplicatetheenzyme in theregulation of
peptide-mediated fetal lung growth. (J. Clin. Invest. 1992.
90:2517-2525.)Key words: bombesin * common acute
lympho-blastic leukemia antigen*metalloendopeptidase * respiratory
epithelium* growthregulation Introduction
Molecular cloning and expression studies have shown that
CD1O (common acute lymphoblastic leukemia antigen) is
identical to the zinc metalloprotease, neutral endopeptidase
Addresscorrespondenceto Dr.Mary E.Sunday,Department ofPathol-ogy, Brigham & Women'sHospital, 75 Francis Street, Boston, MA
02115.
Receivedforpublication 7November 1991 and in revisedform 22 June 1992.
24.11(NEP,"enkephalinase")(1-4).This cell membrane-as-sociatedenzyme(CD
lO/NEP)'
hydrolyzesanumber of natu-rallyoccurring peptides, including the endogenous opioidpen-tapeptides Met-and Leu-enkephalin, substance P,
neuroten-sin, oxytocin, bradykinin, angiotensins 1 and 2, atrial
natriuretic factor, endothelin and the chemotactic peptide,
fMLP(2, 4-6).CDlO/NEP,which isexpressedonearly
lym-phoid progenitors, neutrophils, and a variety of
nonhema-topoieticcelltypes(5-8),functions inmultipleorgan systems
to downregulate inducedresponses topeptide hormones
(9-16).Forinstance, CD lO/NEP isexpressedathigh levels in the lung (17-19), where it modulates responses to tachykinins
suchassubstancePthatmediateneurogenicinflammation
(9-12, 16).Inhibition of CD lO/NEPdramaticallyincreases both
the binding ofsubstance P to bronchial membranes and the
resulting proinflammatoryphysiologicaleffects(9-11, 16).
Anadditionalfamilyof peptides whoseeffectsare modu-lated by CDlO/NEP are the bombesin-like peptides (BLP) (20). The amphibian peptide bombesin and its homologue
gastrin-releasingpeptide (GRP) arepotentmitogens for
nor-mal bronchial epithelial cells (21), fibroblasts (22-24), and
certain tumor types including small cell lung carcinomas (SCLCs) (25, 26). Inmany SCLCs, anautocrine loopexists
whereby tumor cells secrete BLP, express BLP receptors, and
respond to BLP with increased cellular proliferation (25). CD10/NEP inactivates BLP by cleaving these peptidesat two
sites within the7amino acid (aa) conserved carboxy terminus thatisrequired for biologic activity (27).The growthof
BLP-responsive SCLCs was inhibited by CD1O/NEP and
poten-tiated by CD10/NEP inhibition, implicatingthe enzymeinthe
regulation of BLP-mediated autocrinetumor cell growth (27). BLPhave also been implicated innormal fetal lung
develop-ment (28, 29). GRP mRNAs werefirstdetected in human fetal
lungat9-10wkgestation,peaked at 16-30 wk at levels 25-fold
higherthaninadult lungs, and subsequently declined to near
adult levels by 34wkgestation(28). Insituhybridization analy-sesof GRP mRNAs in human fetallungrevealed a
proximal-to-distalspatial and temporalpatternofgene expression,
appar-ently in parallel withthe growthoftheairways(28). The high
levelsof GRP mRNAs foundduringthepseudoglandular and
canalicular phases of pulmonarydevelopment suggested that BLPmightplay animportant role in this process. This
hypoth-esiswassubsequently confirmedin human and murine fetal lung organ culture studies (29). Bombesin was added to hu-manandmurine fetallung organ cultures and administered to
1.Abbreviations usedinthis paper: BLP, bombesin-like peptide; BN, bombesin; CD1O/NEP, CD10/neutral endopeptidase 24.11; GRP,
gastrin-releasing peptide; PF/C, paraformaldehyde-fixed/cryostat;
PF/PE, paraformaldehyde-fixed/paraffin embedded; SCLCs, small celllung carcinomas.
J.Clin.Invest.
©TheAmericanSociety for Clinical Investigation,Inc.
0021-9738/92/12/2517/09 $2.00
pregnant miceduring the respective time periods in gestation when endogenous fetal lung GRP levels were most elevated (29). Bombesin administration resulted in a dose-dependent
increase in fetal lunggrowth and maturation as assessed by
biochemical and morphologicalapproaches(29). Inaddition, anantibombesin (BN) (mAb)blocked murinefetallung
matu-ration in vivo (30)andin serum-freelung organcultures (29).
Recently CD10/NEP immunoreactivity has been identi-fied in epithelial cells of both fetal and adult lung( 17, 27). Since CD10/NEP modulatesBLPeffects and BLPs stimulate the growth and maturation of fetal lung,wehave analyzedthe
temporal and cellular expression ofCDO0/NEP in human fetal
lung and comparedittothatofBLP.We havealso assessed the
potential role ofCDI0/NEP in fetal lung development by eval-uating the proliferation of human fetal lungin organcultures treatedwith specific CDI0/NEP inhibitors.
Methods
Fetal tissue collection. Human fetal lung tissues were obtained within 1
h after electivetherapeutic abortions up to - 22 wk gestation. The
numbers andgestational ages of the lung samples analyzed were: 1, 8 wk;2, 10wk, 1,I Iwk; 3, 12 wk; 1, 13 wk; 4, 14 wk; 3, 15 wk; 2, 16 wk; 2, 17 wk;2, 18 wk; 2, 20 wk; 2, 21 wk; and 2, 22 wk. Of these, three samples at 14 wkgestation and one sample at 15 wk gestation were used
fororgan cultures.The 8-wk sample and - 50%oftheremaining
speci-mens were usedforhistochemicalanalyses, and the remainder of the samples wereusedforRNAblots.Appropriateinformedconsent was
obtained fromeachpatientandnorecords werekeptofpatients' iden-tification.These studies have been approved in strict accordance with
institutional guidelinesby the HumanEthicsCommittee at Brigham & Women'sHospital withyearly renewalsconsistentwith National
Insti-tutesofHealthguidelines.
RNApreparations and Northern blotanalyses.TotalRNAwas pre-pared bythe methodof Chirgwinetal.(31), electrophoresed through
1.5%(wt/vol)formaldehyde-agarosegels, andtransferredto
nitrocel-luloseby standard methods(32). Gels werestained with ethidium bromidetovisualizethe28Sand18S ribosomalRNAbandsto ensure
intact RNA recovery. GRP mRNAsweredetectedbyhybridizationto aGRPcomplementaryRNA(cRNA) probewhichwaspreparedusing
1-2,goflinearized cDNAtemplate.The650-bpGRP PstIfragment
wasclonedintopGEM3,linearizedwith BamHI, and transcribed with SP6 RNApolymerase(33).The GRP cRNAprobewaslabeled with
[32P]UTP
to aspecificactivityof 108cpm/Ag.CDI0/NEPmRNAs weredetectedbyhybridizationto a 1.6-kb EcoRIfragment fromthe humanCD10/NEPcDNA openreading frame (34) whichwaslabeledwith(32P]dCTPusingthe randomprimingmethod(35).
GRPhybridizationwascarriedoutin 5x standardsaline citrate (SSC) (lxSSC=0.15 MNaCl,0.0 15 M NaCitrate);5XDenhardt's
solution (1X Denhardt'ssolution=0.02%[wt/vol] Ficoll-400,0.02%
[wt/vol] BSA, 0.02%[wt/vol]polyvinylpyrrolidone-40); 50%(vol/ vol)formamide; 50 mMsodiumphosphate, pH6.5; 0.2%(wt/vol)
SDS and denatured salmon sperm DNA(200;ig/ml)at65°Cfor 18 h.
CD1O/NEPhybridization was similarlyperformed at42°C. Filters
werewashedat65°C for 2 x 15 min in 2xSSC/0.2%SDSfollowed by
2 x30 min washes in 0.2xSSC/0.2%SDS,andexposedfor 24-48 hat
-70°Cwith Kodak XAR film withanintensifyingscreen.
Densitometrywascarriedoutbythree-dimensionalintegrationof the area ofwhole bands on autoradiograms using a densitometer (model300A;MolecularDynamics, Sunnyvale, CA).
Insituhybridization. Insituhybridizationofduplicatefetallung
serialsectionswasperformedaspreviouslydescribed(36). Inbrief, 5-Mm sections were placed on coated slides and baked at 65°C
over-night,rehydrated,and treated with4%paraformaldehydein PBSfor 10 min. After washing, slides were sequentially incubated with Triton X-100 (0.3% in PBS) for 15 min,proteinaseK(1 Ag/mlin 100 mM
Tris, 50 mM EDTA, pH 8.0) at 420C for 30min,and 4% paraformal-dehyde for 5 min. Afterwashing,tissueswereacetylated (acetic anhy-dride 0.25% in 0.1 M triethanolamine) for 10 min and prehybridized in 50%formamide/2X SSC ( lXSSC=0.15 MNaCI,0.015MNacitrate) at50C for 20min.Subsequently, sections were hybridized at 50C for 16 h in a sealed moist chamber with 20 Ml of 5 X 104cpm/Ml of
3"S-la-beled antisense cRNA probe in hybridization buffer (50% formamide, 10%dextran sulfate, 2x SSC, 0.5% BSA, 0.5% Ficoll-400, 0.5%
poly-vinylpyrrolidone-360,2.5gg/mlyeasttRNA, and0.5% SDS). Addi-tional serial sectionswereincubated in parallel with an equivalent amountofthecorresponding sense cRNA probe.3S-labeledGRP
anti-senseandsensecRNAprobeswerepreparedaspreviouslydescribed (33). The CD10/NEP cRNA used for in situ hybridization was a
701-bp Clal/Xholfragment derivedfrom the openreading frame( 1). The
CD10/NEP fragmentwassubclonedinto theBluescript vector(Strata-geneInc.,LaJolla,CA), linearizedwithClal,andtranscribed with T3 RNApolymerase(antisense orientation)orlinearizedwith Xhol and transcribed with T7 RNA polymerase (sense orientation).
After hybridization, slideswerewashedin 4X SSC at 42°C for 20 minX3 anddigestedin RNAseA(20Mg/mlin 10 mM Tris, 1 mM
EDTA,50 mMNaCI)at42°C. Subsequently,slides were dehydrated inethanolscontaining0.3 Mammonium acetate,dippedin Kodak NTB-2 photographic emulsion, airdried, and exposed in light-tight boxes with desiccant for 8 d. Slideswerethendeveloped withKodak D-19 and Fix and tissuescounterstained with hematoxylinandeosin. Tissue sectionswereviewed andphotographedusingboth conven-tionalbright-fieldanddark-fieldmicroscopy.Forcomparison
ofproxi-mal to distalairways, wedefined proximal airwaysas bronchi and bronchiolesentirelylinedbycolumnarepitheliumandassociatedwith
closelyapposed mesenchymaltissueincluding desmin-positive cells,
and distalairwaysasbronchioles andprimitive airways lined by mostly
cuboidalepitheliumandassociated withonlyloosemesenchymal
tis-suethat didnotcontaindesmin-positivecells.
Immunoperoxidase analyses. Immunoperoxidase analyses were
performedwithserialsections ofthesamefetal lungs thatwereusedfor
in situhybridization.The human CDIO monoclonalantibody(mAb) J5(a gift from JeromeRitz,Dana-Farber CancerInstitute,Boston,
MA)wasusedaspreviouslydescribed (27).Theaffinity-purifiedIgG fraction ofpolyclonalrabbit anti-NEP (agift from CatherineLucas at
Genentech, Inc.,SanFrancisco, CA)wasusedat aworking dilution of 1:2,000. mAbtocytokeratins (CAM5.2) wasobtained fromBecton Dickinson & Co.(San Jose,CA)and mAbtodesmin(D33)was ob-tained fromDakopatts A/S(Glostrup,Denmark).Both CAM 5.2 and D33wereusedatworkingdilutions of1:100.
Controlsconsisted of serial fetallung sections incubatedinparallel
with:(a) antiserumormAbpreabsorbed withspecificantigen (50yg/ mlsolublerecombinantCD1O/NEP3.4.24.11 [a gift fromBob
Bri-denbaugh,Genentech, Inc.,SanFrancisco, CA]);(b)a1:500 dilution ofpreimmunerabbit serum;or(c)a 1:500 dilution ofanunreactive
myelomaproteinofthesameIgGsubclass astheCD10/NEP monoclo-nalantibody (MOPC21;SigmaChemicalCo.,St.Louis,MO).
Immunoperoxidase analyses were performed as previously
de-scribed, usingthe avidin-biotincomplexmethod(36)without the per-meabilization of tissues with 0.3% TritonX-100.Inbrief,tissueswere
fixedfor 16hin 4%paraformaldehyde, routinelyprocessedinto
paraf-fin,andcut at5Mmontocoatedslides. Tissuesonslideswereblocked
for 20 min with 1:10 dilution of normal goatseruminPBS and
subse-quentlyincubatedat4°Covernightwith theappropriatedilutions of
primary antibodyor antiserum.A biotinylated secondary antibody
(1:200)wasthenappliedtotheslides for 30 min. Afterendogenous peroxidase wasblockedfor 30 min in 0.3% H202/methanol,slides
wereincubatedfor 45 min with the standard avidin-biotincomplex
reagent,developedfor 5 min in diaminobenzidine(0.25gm/100 ml in
PBS),andcounterstained with2%aqueousmethylgreen.
Humanfetal lungorgan cultures.Fragmentsof human fetallung
wereasepticallyexcisedonice and cultured inMEMplus5%fetal calf
serum(Gibco-BRLLifeTechnologies,Inc.,Gaithersburg, MD)alone
100 nM), the specific bombesin receptor antagonist [Leu'3-psi(CH2NH)Leu'4]bombesin (Peninsula Laboratories, Inc., Bel-mont, CA; LI 3BN, 100 nM) (37), the specific CDIO/NEP antagonists SCH32615 (Schering-Plough Corp., Bloomfield, NJ; 5 uM) (38)or
phosphoramidon (cat R6128; Sigma Chemical Co.;5MgM)(27, 39), or bombesin+LI3BN,orSCH32615 +Ll3BN. Cultures were incubated in 5% C02/air at 370C on a rocking platform at three oscillations/min (29, 40). After44 h of culture,[3H]thymidine (DuPont Co., New England Nuclear Research Products, Boston, MA; 4 MCi/ml) was
added to cultured fetal lungs for the last4hbefore tissueswere har-vested.[3H]thymidine incorporation into acid-precipitable counts was determined(22) and normalized for DNAcontent(cpm/MgDNA).
DNAcontent wasdetermined after trichloroacetic acidprecipitation
by the method of Burton (41). Duplicateorquadruplicatesamples were obtained for each culture condition in each of three independent
experiments. Thethymidine incorporationin untreated control
sam-ples from eachexperiment(mean±SE)wasdefinedasbaseline and the percentage change inthymidine incorporationin each of the treated samples determined(mean±SE).The results from the three
indepen-dentexperimentswerepooledfor finalanalysis.
Results
Northern analyses. Toanalyze CD10/NEP transcripts during
fetallung development and compare thetemporal expression ofCD10/NEP to that of the mammalian BLP, GRP, total RNAsfrom fetallung samples ( 10-22 wkgestation)were
pre-pared, blotted, and probed with bothaGRPcRNAandCD 10/ NEPcDNA. GRPtranscripts (- 900-950 bp)were first
de-tectable at 10-12 wk and subsequently increased and peaked between 15-20 wkgestation (Fig. 1 A and [28]). In contrast, the major 5.7- and 3.7-kbCD10/NEP transcripts (1, 2, 13)
were barely detectable at10wkgestation,peaked at 1 1-12wk,
anddeclinedthereaftersuch thattheywerebelow the limits of
detectiononNorthern blotsoftotal RNA at 16 wk(Fig. 1B). GRP andCD1O/NEP transcriptsweresubsequently
quan-titated by densitometry and normalized for the relative amountsof totalRNAby scanningthenegative of the
photo-graph from the corresponding ethidium bromide stained gel
(Fig. 1 C). The normalized densitometric analysis indicates that CD1O/NEP transcripts peaked in fetal lungduring the first trimester and subsequently declined at about the same
time ( 14-15 wk, early second trimester) that GRP transcript
levels rose(Fig. 1 D).
In situ hybridization. In situ hybridization was
subse-quentlyperformedtospecifically determine which cellsin
hu-man fetal lung transcribed CD1O/NEP (Fig. 2). Fetal lung sections were examined by light microscopy to characterize cellular morphology (Fig.2,a, c, e, andg) and by dark-field microscopytovisualize autoradiographic signalsbetter(Fig.2,
b, d,fandh).CD10/NEP transcriptsweredetectable asearly
as8wkgestation (Fig.2,aandb).The strongesthybridization
signals were observed in the most primitive distal airways
where the majority of grainsoverlayepithelial cells (Fig. 2, a andb).However,therewas alsoincreasedgrain density overly-ing airway-associated mesenchymeand weakhybridization
as-sociated with scatteredmesenchymal cells in the loose
connec-tive tissue in betweenairways(Fig.2,aandb).Toconfirm the
specificityof the CD10/NEPhybridization signals,additional
8-wkfetal lung sectionswerehybridized withthe correspond-ing CD10/NEPsenseprobe which is identical, ratherthan
com-plementary, toCD10/NEP transcripts. As shown in Fig. 2,c
and d, noappreciable signalsweredetectedwith thisCD1O/
NEPsense
probe.
Weeks:
1011 12 13
15
16
17 18 20
B
-28S
DENSITOMETRY ON HUMAN FETAL LUNG mRNAs
D
6
C
.
E
a CD1 O/NEP
l.
198 10 12 14 16 18 20 22
WEEKS GESTATION
Figure1.Northern blotanalyses of GRP andCD10/NEP transcripts
in humanfetal lung. Northern blots of total fetal lungRNA(10 g/ lane, 10-20wkgestation)were hybridizedwith thehumanGRP
cRNAprobe (A)orthehumanCD10/NEP cDNA probe (B).
Arrows(right)indicate the expected sizes of the900-950-bpGRP and3.7- and 5.7-kbCD10/NEP transcripts.The28SribosomalRNA
band from the ethidium bromide-stained RNAgelused inAis shown in (C). The negative from this ethidium bromide-stained gelwas
usedtonormalize relative densitometric units of GRP and CD10/ NEPwhichareshown in(D).
By 12 wkgestation,CD10/NEP transcriptswereeasily
de-tectable in the epithelial cells and airway-associated
mesen-chyme of distal less differentiated airways (Fig. 2, eandf).
Therewas atrend towardsstrongerCD10/NEP hybridization
in distal ascompared to proximal airways (data notshown) whereas GRP transcriptswereonly detectable in more differ-entiatedproximal airwaysthathadbeguntobranch(datanot
shownand [28 ]).
At 15 wk(Fig. 2,g andh) andmoreweaklyat 17 wk
gesta-tion (datanotshown), CD10/NEPtranscriptswere
predomi-nantly associated with the distalmostundifferentiatedairways.
Additional sections ofthe same 15- and 17-wk fetal lungs
probed with the controlCD10/NEPsense cRNA were
com-pletely devoid of hybridization signals (data not shown).
CD10/NEP transcripts in 18-21-wk gestation human fetal
lung weredetectedonly in bronchial-associated glands by in
situhybridization (datanotshown).
Immunohistochemicalanalyses. TocorrelateCD10/NEP
transcriptswith cellsurfaceprotein expression,
immunohisto-chemical analyseswereperformedonadditional fetal lung
hybrid-I
ization studies.Initially, immunoperoxidasestudiesweredone
onparaformaldehyde-fixed/cryostat (PF/C) sections with re-agents including anti-CD10/NEP, cytokeratin, and desmin
monoclonal antibodies. Consistent with the previous in situ
studies,therewasCD10/NEPimmunostainingofboth the
epi-thelium andairway-associated mesenchymal tissueat8 wk
ges-tation (Fig. 3 a). The specificity of CD10/NEP immunostain-ingwasconfirmed bydemonstratingthatserial sections incu-bated withCD10/NEPmAb that had beenpreabsorbedwith solubleCD10/NEPhadmarkedlyreducedsignals (Fig. 3b).
To comparethelocalization ofCD10/NEPtothatof other known markers, additional serial sectionswere stained with
anticytokeratinand antidesminmAbs.Cytokeratin
immuno-stainingof thesame 8-wk fetal lungidentifiesairway
epithe-lium inproximal (P) and distal(D)airways (Fig.3c),whereas desminimmunostainingidentifies smooth muscle differentia-tion whichis associated withonlythemoredifferentiated
prox-imalairways(Fig.3d). ComparisonsbetweenFig. 3,a, c,and
d,demonstrate thatproximalmoredifferentiatedairwayshave
weakerCD10/NEPimmunostainingthandistalless
differen-tiatedairways.
In 12-wkPF/Csections,therewasonly weakCD10/NEP
antigen-specific immunostaining ofproximal airway
epithe-lium, with weaktomoderatestaining of distal airways(Fig. 3 e). Both theepithelialcells whichwereidentified
by
cytokera-tin immunostaining and the
mesenchyme
surrounding
des-min-positive differentiated smooth muscle cells expressed
CD 10/NEP(Fig.3g).The
specificity
ofCD10/NEP immuno-stainingat 12 wkwasconfirmedbyincubatingadditional fetal lungsections with theCD1O/NEPmAbfollowingpreabsorp-tion with solubleCD10/NEP(Fig.
3f).
Sinceprevious studies suggestedthat theremightbe
differ-encesbetween theCD1O/NEP immunostaining of PF/C
sec-tions and paraformaldehyde-fixed/paraffin-embedded (PF/
PE)sections(27),wealso evaluatedPF/PE fetal lungsamples
for CD10/NEPexpression (Fig. 4).Boththeanti-CD10 mAb
(Fig. 4,A-J) andananti-CD10 antiserum (datanotshown)
gave similarpatterns ofCDlO/NEP immunostaining. Inthe PF/PEsections theepithelialCD10/NEPstainingwas particu-larly prominent whereas the mesenchymal staining was less pronounced(Fig.4). These resultssuggestthat the methodof tissueprocessingmayaffectCD10/NEP immunoreactivityin
specifictissues (42-44). Consistent withourresultsfrom the
Northernanalysis (Fig. 1), insituhybridization (Fig. 2)and
immunostaining of PF/C sections (Fig. 3), CD1O/NEP
ex-pression in PF/PE sections was mostintense at earlier time
points (12
wk,
Fig.4,AandB).Inlatersamples(15-18 wk),CD10/ NEP expressionwasprimarily restrictedto keratin-posi-tiveepithelial cells in the less differentiated distal airwaysnot yetsurrounded by desmin-positivemesenchymalcells(Fig.4, C-F, and datanot shown). AdditionalPF/PE-fixed samples incubated withCD10/NEP mAborantiserum that had been preabsorbed with solubleCD10/NEP had markedly reduced signals,confirming thespecificityof theimmunostaining(Fig. 4,B, D, and F, and datanotshown).
In previous studies using adult animals, inhibition of
CD1O/NEP potentiated the bronchoconstriction associated with substance P release (9-11, 16). Since substance P is
re-leasedfromnervefibers localizedtothedesmin-positive
mesen-chymal cells surrounding proximal airways, we examined
more maturefetallungspecimensfor additionalCD10/NEP
mesenchymalimmunostaining. PF/Cfetal lung sectionswere
used becausemesenchymalCD10/NEPimmunostainingwas
moreprominentwith this methodoffixation(Fig.3versusFig.
4). TheCD 10/NEPanddesminimmunostainingof serial
sec-tions froma22-wkfetallung isshown inFig. 5.Whereas the CDIO/NEP immunostaining of airway-associated
mesen-chyme inyoungerfetallungswasexternaltoanddistinctfrom
desmin-positive smooth muscle cells (Figs. 3 and 4), the
CD I0/NEP
immunostaining
inthe olderfetal lungs includeddesmin-positivesmooth musclecellssurroundingtheproximal
airways(Fig. 5), aswellasbronchial-associatedglands (data
notshown).
Humanfetal lungorgancultures.Toexplore thepotential
function of CD1O/NEPin developinglung, fetallung organ
cultureswere setup aspreviously described (29) using three
different14-15-wkgestationhumanfetallungs. The 14-15-wk
timepointwaschosen because bothCD10/NEPandGRPare
clearly detectable in humanfetallungduring this interval (Fig.
1).Fetallungorgancultureswereincubated with mediaalone
(Neg),bombesin(BN), thespecific bombesinreceptor
antago-nist Ll3BN,BNplusLl3BN, the specificCD10/NEP
inhibi-torSCH32615 (38), SCH32615 plus L13BN,or anunrelated
CD1O/NEP inhibitor, phosphoramidon (27). Sampleswere
subsequently evaluated for
[3H]thymidine
incorporationwhich was normalized for DNA content (Methods, [29]).
[3H]thymidine incorporation insamplesincubatedwith
me-dia alonewas compared to
[3H]thymidine
incorporation insamples incubatedwith bombesin, LI3BN, SCH32615,
phos-phoramidon,ortherespectivecombinationsand the
percent-Figure2. Insituhybridization of human fetal lung forCD1O/NEPand GRPtranscripts. Each pair of photographs(a,b; cd;ef;gh) represents thesamefield viewed bylight-fieldmicroscopytodemonstratehistology(left: a, c, e, g) and dark-field microscopy to demonstrate
autoradio-graphic grains (right:b, d,fh).(aandb)8-wk gestation human fetal lung probed withCD1O/NEPantisense cRNA. Note hybridization is weak inaproximal (P) airway, more intense in an early distal (D,) airway, and most intense in a further distal (D2) airway. (c and d) 8-wk lung probed with CD10/NEPsensecRNA. Note the minimal background and the absence of a specific hybridization signal. (e andf )12-wkgestation
humanfetal lung probed withCD1O/NEPantisense cRNA with hybridization signals in both proximal and distal airways. (g and h) 15-wk
gestationhumanfetal lung probed withCD1O/NEPantisense cRNA with weak hybridization in one distal
(DI)
airway andstronger hybridiza-tionassociated with another more distal (D2) airway.Figure 3. Immunohistochemical analyses of paraformaldehyde-fixed/cryostat sections of human fetal lung. Human fetal lungs of 8-wk gestation
(a-d)and 12-wk gestation(e-h) were immunostained with the following mAbs: (a and e) Anti-CD1O/NEP mAb J5.CD1O/NEP
immuno-stainingisprominent in airway-associated mesenchyme and scattered mesenchymal cells and is also present in the distal (D) airway epithelium
It"'
~~~~6-I,.
I
Aft. uZ~'
A
.
Figure4.
agechange in thymidineincorporation resultingfrom the addi-tions determined. The results from the threeindependent
ex-periments are pooled and summarized in
Fig.
6. Bombesinsignificantly increased thymidine incorporation in the fetal
lung organ cultures(116%increase,P<0.001)and the addi-tion of LI 3BN to bombesin-treated cultures reduced [3H
H-thymidine incorporation to baseline levels. The CD1O/NEP
inhibitor SCH326 15 alsosignificantlyincreasedthymidine in-corporation in fetal lung organ cultures (166% increase, P
< 0.001).The addition ofL1 3BNtoSCH326 15-treated
cul-tures similarly reduced thymidine incorporation to baseline levels suggesting thatthe SCH32615 effect was mediated by
BLPs.Toobtainadditional evidence that the increased
thymi-dine incorporation in SCH32615-treated fetal lung samples
was a specific consequence of CD1O/NEP
inhibition,
addi-tionalsamplesweretreated withanunrelatedCD10/NEPin-hibitor, phosphoramidon. As shown inFig.6,
phosphorami-don-treated fetallungorgan cultures also
incorporated
signifi-cantlymore[3H]thymidine ( 182% increase, P<0.025).
Discussion
Wehaveevaluated theexpression andfunctionof CD 10/NEP
in human fetal lung because the enzyme regulates
BLP-me-diated growth ofSCLC andBLPs stimulate theproliferation
andmaturation of human and murine fetallungorgancultures (29) and normal pulmonary neuroendocrine cells (45). By in
situhybridization analysis, CD10/NEP transcriptswere
iden-tifiedin epithelial cells, airway-associated mesenchyme, and
scattered mesenchymal cells as early as 8wk gestation. The
patternofCD I0/NEPimmunoreactivitywassimilartothatof
thecorresponding transcripts,withprominent epithelial
stain-ing primarily of distal undifferentiated airways. Consistent
with Northern analyses, CD1O/NEP immunostaining was mostprominent in the first trimesteranddeclined thereafter. Given thetightcorrelation betweenCD10/NEPmRNAlevels,
cellsurface expression andenzymaticactivity( 1,2), these
re-sults arepredictive for highlevelsofcellsurface CD10/NEP
enzymaticactivity in the leastdifferentiated airways in early
fetal lung development. Therefore,theobservation that human
fetal lung proliferation is increasedwhen CD
O0/NEP
isinhib-itedwitheitherSCH326 15orphosphoramidon is of particular
interest. The fact thatabombesin receptor antagonist abolishes theeffects ofCD10/NEPinhibitorsonhumanfetal lung sug-geststhatCD10/NEPisregulatinglocallevelsof endogenous BLPs andthat theenzyme mayfunctiontolimitBLP effects
duringtheearlieststagesoffetal lung development.
W z 100
0
0
Nsg
BN SCH Phos L138N L13BN L13BNA8N +SCH
Figure6. CDI0/NEPinhibition stimulates growth in human fetal lungorgan culture via endogenousBLPs.Lungorgancultureswere setupinduplicatetoquadruplicate using14-15-wk gestation human fetal lung in MEM+5% fetal calfserumalone (Neg)ormedia with bombesin(BN, 100nM), SCH32615 (SCH, 5 MM),
phosphorami-don(Phos, 5 MM),the specific BN receptor antagonist, [Leu'3-psi(CH2NH)Leu'4]BN (L13BN, 100 nM),orBN 100 nM plus L13BN 100 nM,orSCH 5 ,uM plus L13BN 100nM.Thymidine
in-corporation (cpm/Mg DNA)in treated samples is representedas
per-centagechange comparedtountreated control samples (expressed
asmean±SEM).The values shownarethepooledresultsfrom three
independentexperiments.The actualpercentagechanges with respect
tountreated samplesareshown below. P values comparing untreated samplestoexperimental sampleswerecalculated using the unpaired Student'sttest:Neg,0±17; BN,116±22,P<0.001;SCH, 166+40, P<0.001; Phos, 182±59, P<0.025; L13BN, 0±16, P= 1.0;L13BN +BN,-46±21, P=0.140; L13BN+SCH, 1±12, P=0.954.
In ourinitial studies, we demonstrated thatCD1O/NEP wasexpressed by pulmonary neuroendocrine cells and that the enzyme regulated the BLP-mediated autocrine growth of SCLCs. Thepresent study shows thatCD10/NEPis also
ex-pressedby undifferentiated epithelial cells in developing
air-ways and that the enzyme modulates the growth of normal
fetal lung.Therefore,in additiontomodulating the autocrine growth ofneuroendocrine cells, another possible role of the
enzymeistoregulate thepeptide-mediatedparacrine growth of
bronchial epithelial cells. Pulmonary neuroendocrine cells have recently beenimplicated in the paracrine stimulation of adjacent bronchial epithelial cell growth in developing hamster lung (46).
That CD1O/NEP expression peaks in first trimester fetal lung and ismostabundanton epithelialcellslining distal
un-Figure4.Immunohistochemicalanalyses of paraformaldehyde-fixed/paraffin-embeddedsections of human fetal lung. Human fetal lungs of 12
wkgestation (a and b), 15 wkgestation(candd),and 18 wk gestation (e andf) were immunostained as follows: (a, c, and e) Anti-CD10/NEP
mAb,J5.CD1O/NEPimmunostainingis stronger in distal (D) airway epithelium than in proximal (P) airway epithelium and is more intense inearlysamples.(b, d,andf)J5 preabsorbed with solubleCD10/NEP.Preabsorption of J5 with solubleCD1O/NEPmarkedly reduces the
im-munostaining in 12-wk samples(b)and completely eliminates the staining in 15-18-wk samples (d andf). Note that
CD10/NEP
epithelialstainingismoreprominentonPF/PE samples whereas mesenchymal staining is more prominent on PF/C samples (compare Figs. 4 and 3).
Figure5.Immunostainingofparaformaldehyde-fixed/cryostatsections of 22-wk gestation human fetal lung forCD10/NEPanddesmin.
Mag-nification inaand cis 100andinbandd is 200. (a and c)Anti-CD10/NEPmAb, J5. Note prominent immunostaining of mesenchymal
tissue associated withtheproximal airway(L=airway lumen) and weak immunostaining of loose mesenchyme. Arterial smooth muscle (A =artery) is negative forCD10/NEP;(bandd) Desmin mAb. Well-differentiated smooth muscle cells associated with the proximal airway (L)
arepositively stainedfor desmin. Note that desmin-positive smooth muscle cells in the proximal airways of 22-wk fetal lung areCD10/NEP
differentiated airways is of interest because CD10/NEPis also expressed byimmature progenitors of another cellular lineage.
CDlOwasoriginallyidentifiedas a cell surface glycoprotein on normal and malignantlymphoidprogenitors that were either uncommitted or committed to only the earliest stages of B or T
cell differentiation (reviewed in reference 8). CD10-positive
lymphoid progenitors areabundant early in fetal
hematopoi-eticdevelopment butdeclinein number with subsequent fetal maturation andbirth (8). Recent studies indicate that CD 10/ NEPalso modulates the growthof lymphoid progenitors rais-ing the possibility that the enzyme may function in a similar
capacityindifferent organs (47).SinceCD 10/ NEP is
predomi-nantly expressed by earlylymphoid progenitors in fetal bone marrow and liver and by undifferentiated epithelial cells in fetal lung, there may be common regulatory elements control-ling expression ofthe enzyme indifferenttissues during fetal development. Ofnote, several 5'alternativelyspliced CD 10/ NEPuntranslated region cDNA sequences have been
identi-fied, raising the possibility that unique regulatory elements
control CD1O/NEP expression at different developmental stages or indifferentcell types(3).
CD10/NEPmay havedifferentroles in early and late stages
ofpulmonarydevelopment, dependingupon thespecificcell typesexpressing the enzyme (16-19) and thelocalization of
relevantCD10/NEP peptide substrates. Forthisreason, the
CDI0/NEPimmunostainingofdesminpositive mesenchymal
cellssurrounding proximal airwaysin olderfetallungsisalsoof
interest. CD10/NEP has previously been identified on cul-turedfibroblasts (8)andmultipletumorsofmesenchymal
ori-gin (48). The enzyme may also modulate peptide-mediated
proliferationof pulmonary mesenchymal cells since BLPs are
knownmitogensforpulmonaryfibroblasts(24, 29). Since
ad-ditionalpulmonarypeptidessuchassubstancePand
endothe-lin are alsoCD10/NEPsubstrates and fibroblastmitogens (49, 50),the enzyme mayregulatefibroblastproliferationmediated
bymultiple pulmonary
peptides.
CD10/NEPis also knowntomodulatesubstanceP-mediated bronchoconstrictionin adult
animalmodels(9-11, 16). Substance P, which isreleased by
nervefiberslocated inmesenchyme surroundingproximal
air-ways,presumably acts on CD10/NEPpositivesmooth muscle elements.
Our resultsindicatethatCD10/NEPisprimarilyexpressed
by undifferentiated bronchial epithelium in developing
air-ways and that the enzyme modulates the growthofhumanfetal
lung. Thesestudiesprovide additional insightinto the roleof
the enzyme inregulating pulmonary peptide-mediated
prolifer-ative responses.
Acknowledgments
Wethank theDepartment ofGynecological PathologyatBrigham&
Women's Hospital for their assistancewith tissue collection, Sherry
Graham andAmyCard for excellenttechnicalassistance,and Dr.
Ger-aldinePinkus forreviewingtheimmunoperoxidaseslides.
This workwassupported byNational Institutes ofHealthgrants R01-HL-44984 (M.E.Sunday)andROI-CA55095(M.A.Shipp).
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