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Eosinophils in chronically inflamed human

upper airway tissues express transforming

growth factor beta 1 gene (TGF beta 1).

I Ohno, … , J Gauldie, M Jordana

J Clin Invest. 1992;89(5):1662-1668. https://doi.org/10.1172/JCI115764.

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement

membrane thickening which […]

Research Article

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Rapid Publication

Eosinophils in Chronically

Inflamed Human

Upper

Airway Tissues Express

Transforming Growth Factor ,B1 Gene (TGFB1)

1.Ohno,* R. G. Leat K. C. Flanders,' D. A. Clark,t D. Banwattt J. Dolovich,11 J. Denburg,t C. B.Harley,' J.Gauldie,* and M. Jordana*

*Departments ofPathology, WMedicine,

IlPediatrics,

and'Biochemistry,McMaster University,Hamilton,Ontario L8N-3ZS Canada; and Laboratory ofChemoprevention, National CancerInstitute,National Institutes ofHealth, Bethesda,Maryland20892

Abstract

Transforming growth factor, (TGFj8)is a multifunctional pro-tein which has been suggested to phay a central role in the

pathogenesisof chronicinflammationandfibrosis.Nasal poly-posis is aconditionaffecting theupperairways characterized by the presence of chronic inflammation andvarying degrees of fibrosis. To examine thepotentialrole ofTGFBin the pathogen-esis of thiscondition,weinvestigatedgeneexpressionand cyto-kine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGFjBl cDNA probe, we detected

TGFj81-specific

mRNA in nasalpolyp tissues, as well asinthetissue from apatientwith allergic rhinitis, but notin the normal nasal mucosa. Bythe combination of tissue section staining with chromotrope 2R with in situ hybridization using the same

TGF(#1

probe, we found that - 50%oftheeosinophilsinfiltratingthepolyp

tis-sueexpress the

TGF,81

gene.Inaddition, immunohistochemi-callocalizationof

TGFjS1

wasdetectedassociated with

extra-cellular matrixaswellasin cells in thestroma.These results suggest that in nasalpolyposiswhereeosinophilsarethemost

prevalentinflammatorycell,TGFB1synthesized by these cells maycontribute to the structuralabnormalities such as stromal fibrosis and basement membrane thickeningwhich character-ize this disease. (J. Clin. Invest. 1992. 89:1662-1668.) Key words: nasalpolyposis *inflammation * cytokines * histochemis-try * in situhybridization

Introduction

Nasal polyps are grapelike structures of unknown etiology

which arise from the posterior and sphenoid sinus mucosae

and eventually occlude thenares. Nasalpolyposis occurs as

frequentlyinpatientswith atopyasin thegeneralpopulation. Polyp tissue is characterized bythepresence of chronic inflam-mation in whicheosinophilsrepresentthe mostprevalent

infil-AddresscorrespondencetoManel Jordana, M.D., Ph.D., Department ofPathology,Room 4H-I7A, McMasterUniversity,1200Main Street West,Hamilton,OntarioL8N-3Z5, Canada.

Receivedfor publication5September 1991 and in revisedform 11 December 1991.

trating inflammatory cell (1). Additional features of nasal

polyps includevaryingdegrees of basement membrane thick-eningand fibrosisinthestroma(2,3)which are similar tothose recentlydescribed in asthma (4). The mechanism underlying thesestructural abnormalities isnotyetknown.

Transforming growth factor ,B

(TGF(#)'

is a 25-kD homodi-meric orheterodimeric protein that can mediate a broad

spec-trum ofbiologicalactivities(5).

TGFP

wasinitially recognized by its ability to transform normal ratfibroblastsinvitro(6, 7).

Ithasbeen subsequently shown that this factor is a

chemoat-tractant for fibroblasts (8), stimulates fibroblast proliferation (9), and enhances fibroblast collagen and fibronectin synthesis (10, 11), whileinhibiting collagenasegeneexpression(12), in vitro. Furthermore, the subcutaneous injection of TGF(B into mice causes rapid granulation andangiogenesisat the localsite

(13), and the association between TGF(3 gene expression de-tectedbyinsituhybridization and eithercollagen gene expres-sion or collagen accumulation in the tissue has been docu-mented inbleomycin-induced pulmonary fibrosis (14), experi-mental hepatic fibrosis (15), and systemic sclerosis (16). The purpose of this studywas toinvestigate whethernasal polyp tissues contained cells which express the gene for

TGF(#

andto

detect thelocalization of TGF3 geneproductinthese tissues.

Methods

Tissues. Nasalpolyptissueswereobtained fromsevensubjects

under-goingpolypectomy fornasalobstruction.Inaddition,four inferior tur-binate tissueswereobtained atthetime ofsurgery forsubmucosal resection;oneofthesepatientshadallergic rhinitis. Upon arrivalatthe lab, tissueswererinsed threetimes withHam's F12medium.Asection ofthetissuewasdissectedout,frozeninliquid nitrogen,and storedat

-70'Cfor subsequentRNAisolation.For insituhybridization,asmall piece oftissue (20-30 mg)wasfixed in4%paraformaldehydein 0.01 M PBS (pH7.4) for15 minatroomtemperaturefollowed by70% ethanol andembedded inparaffin. Forimmunohistochemistry, tissueswere

fixed in periodate-lysine-paraformaldehyde (10 mM NaIO4, 75 mM lysine,and 2%paraformaldehyde in 37.5 mM phosphate buffer pH 6.2)overnightat4°Cand thendehydratedthrougha sucrosegradient. Tissueswerefrozeninliquidnitrogen, cut in5-,umthick sections, and placedontoslides whichwerethenkeptat-20°C until warmed up to roomtemperatureforprocessingimmunohistochemistry.

Probe construction andlabeling. Plasmid pTGFB33which contains aporcineTGFB1

(pTGF#1)

cDNAinsert in thevectorpCDV1 was kindly provided byDrs. P.Kondaiah and K. Flanders(National Insti-tutesofHealth, Bethesda, MD)(17).A640-bp SacI/Pvu17IIfragment

1.Abbreviation usedinthispaper:TGFjB1, transforminggrowthfactor ,B1.

J.Clin. Invest.

©TheAmericanSocietyforClinicalInvestigation,Inc. 0021-9738/92/05/1662/07 $2.00

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was cutand used aspTGFflcDNA probe. This probe does not cross-reactwitheither TGF(2or

TGFf33

(Dr. K. Flanders, personal commu-nication).ForNorthernhybridization, labeling ofthe

pTGF#31

cDNA probe wascarried out by random priming using an oligo-labeling kit (Pharmacia, Uppsala,Sweden) with[ca-32PjdCTP(New England Nu-clear, Boston,MA). For in situhybridization,thepTGFf Iprobeand a DNA 1-kb ladder (Bethesda Research Laboratories, Gaithersburg, MD) asnegative control, were labeled using the same kit with"S-dCTP

(NewEnglandNuclear).

RNA isolation and Northern blot analysis. Frozen tissues were ho-mogenizedin 4 Mguanidinium isothiocynatesolution (1 ml/0. 1 g wet tissue), andtotal RNA wasextracted from thesolution by phenol-chloroform extraction (18). 10,goftotal RNAfromeachtissueand from cultured human lung fibroblasts stimulated with IL-awere elec-trophoresed through 1% agarose gelscontaining6%formaldehydeand 0.5 Mg/ml of ethidium bromide, andtransferred ontonitrocellulose (Schleicher& Schuell Inc., Keene, NH)bycapillary blotting. Filters were incubated in prehybridization solution (40% formamide, 0.1% SDS, 2X standardsalinecitrate(SSC)[1XSSC = 150 mMNaCl, 15 mMtri-sodium citrate,pH7.0], 5XDenhardt'ssolution[SOX Den-hardt'ssolution =Ficoll5 g,polyvinylpyrolidone5 g, andBSA 5 g in 500 mlofH20], 1 mMsodium pyrophosphate,10 mMTris-HC1[pH 7.5], and0.1 mg/mlofdenatured salmon spermDNA)at42°C for4-6 hfollowedbyhybridizationin the samesolutioncontaining106cpm/ mloflabeledpTGF-,Bl probeat42°Cfor 16-18 h.After hybridization, filterswerewashedfour timesin 2XSSC and 0. 1% SDSat room temper-aturefor5min,twice in0.1XSSCand0.1%SDSat55°C for30 min. Then, Kodak x-ray film (Eastman Kodak Co., Rochester, NY)was exposedtofiltersat-70°C withintensifyingscreens.

Poly(A)+ RNA was purified by oligo (dT)-cellulose (Pharmacia) chromatography from total RNAisolated fromoneallergic rhinitis tissueandonenasalpolyp tissuesby the lithiumchloridemethod(19), andelectrophoresedandhybridizedasdescribed above.Wecouldnot isolate poly(A)'RNAfrom normal nasal mucosa because normal mu-cosa was sosmall thatwecouldnotget total RNAenoughtoisolate poly(A)'RNAbythismethod.

In situ hybridization. In situhybridizationwasperformedas de-scribedbyTronetal.(20). Sections2-3 ,mthickwere cutandfloated onto3-aminopropyltriethoxysilanecoatedslides. Theyweredewaxed throughxylene, rehydrated throughanethanolseries,andpretreated with carbol chromotrope (1.25% chromotrope 2R in1%phenol)for 30 minat roomtemperature.Slideswerethen immersed inafreshly pre-pared solution of0.25% acetic anhydridein0.1 Mtriethanolamine buffer (pH 8.0)at roomtemperaturefor 10minfollowed by10 minin 0.1 Mglycinein0.1MTris-HCI (pH 7.4),anddehydratedthroughan ethanolseries. Thehybridization solution containedthefollowing: 35S-labeled pTGFBl cDNA probe or DNA ladder (5X105 cpm/30,u1), 0.2XSSC,50%formamide, 0.5XDenhardt'ssolution, 10mM dithio-threitol,salmonspermDNA(1mg/ml)andEscherichia coli tRNA (0.1 mg/ml). 30Mlof this solutionwasappliedtoeachslide, overlaidwitha coverslip,andsealed with rubbercement.Afterincubation ina humidi-fiedchamberat42°Cfor 14-16 h thecoverslipswereremoved and the slides were washed at42°C in lXSSC/50% formamide for 30 min followedbywashes in 0.1XSSCat roomtemperaturefor10 min. Be-foreautoradiography,slideswereimmersed in carbolchromotropefor 30 min to staineosinophils (21). Forautoradiography, slides were dippedinemulsion(KodakNTB-2;EastmanKodak, Rochester,NY) diluted 1:1 with distilledwaterandexposedat4°Cfor 12-14 d.They were thendeveloped (Kodak developer; Kodak Canada, Toronto, Can-ada), fixed (Kodakfixer;KodakCanada)andcounterstained with he-matoxylinorhematoxylin/eosin.

Immunohistochemistry. To examine the distribution of TGFjBl geneproduct in nasal polyptissues, specificantibodies forTGF, Iand theavidin-biotin peroxidase technique were usedaspreviously de-scribed (22). Briefly, after blocking ofendogenous peroxidase in hydro-gen peroxide/methanol, permeabilization with hyalurodinase and

blockingwith normalgoatIgG,sectionswereincubated witharabbit

antibodyraisedto apeptide correspondingtothefirst 30 amino acids of mature TGF(31 (LC 1-30) which was affinity purified against

TGFO31.

Then,sections wereextensivelywashed andapplied with bio-tinylated goat anti-rabbit IgG and avidin-peroxidase complex fol-lowedbyvisualizationwith 3,3'diaminobenzidine (SigmaChemical Co.,St.Louis, MO)andhydrogen peroxideandcounterstainingwith Mayer'shematoxylin.Controls included replacingtheprimary anti-body with normal rabbit IgG at an equivalent concentration.

Results

Fig. 1 illustratesaNorthernblotprobed withaporcine

TGFfl1

cDNA probe. Lane 1 was loaded with total RNAextracted fromahuman lung fibroblast culture stimulated withIL-Iain

vitro.Asingle2.5-kbtranscriptwasdetected characteristic of TGFf3,subtype 1 (17, 23). No signal could be detected in total RNA extractedfromnormal nasal tissues (lanes 2 and 3). How-ever, apositive signalatthe 2.5-kb sizewasidentified in total RNA from nasal polyp tissue (lane 4). In addition, thesame

2.5-kb signalwas seen in polyA' RNA fromasecondpolyp

specimen(lane S)aswellasfrom allergic rhinitis tissue (lane6).

Thus,wecouldvisualize

TGF#31

mRNA by Northern blot

anal-ysis in chronically inflamed tissues but not in normal nasal tissues.

To detect thepresence of TGF Il expressing cells in the

normalnasalmucosaandin nasal polyps, tissue sectionswere

subjectedtoin situhybridizationwith thesameTGFj I cDNA probeused in the Northern analysis. Fig. 2 shows representa-tive fields of nasal tissues. No posirepresenta-tive cells could beseenwith either the cDNAprobe(Fig.2A)or acontrol DNA ladder(Fig.

2B) in the normal nasalmucosa.In contrast, numerous cells with distinct clusters of silver grains indicating presence of

1

2

3

4

5 6

28

S

18S-"*

Figure 1. Northern blot analysis of RNA from nasal tissues with a porcineTGFjBl cDNAprobe. 10ggof total RNA was loaded to lanes

1, 2, 3, and 4, and polyA' RNA isolated from 200 fg of total RNA was loaded in lanes 5 and 6. An equal amount of RNA in lanes 1-4 was confirmed with the assessment of 28S and 18S ribosomal RNA stained with ethidium bromide after electrophoresis. RNA from cul-tured human lungfibroblasts stimulated with interleukin- 1awasused as apositive control (lane 1). Lanes 2 and 3wereloaded withRNA from normal nasal tissues. Lane 4 was loaded with RNA from a nasal polyptissue. Lanes 5and6wereloaded with RNAfrom another nasal polyp and anallergic rhinitis tissue, respectively. A single 2.5-kb sizetranscript, characteristic ofTGFfl1 was detected inlanes 1, 4,5, and 6, but not in lanes2and 3.

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TGF#3l

mRNAtranscripts were found in nasal polyp tissues hybridizedwith the TGF (31 cDNA probe (Fig. 2 C) but not with the DNA ladder (Fig. 2 D). Similar observations were made in a nasal tissue obtained froma patient withallergic rhinitis (data not shown). Binding ofthe

TGF#31

probe to these tissues was specific because pretreatment with chromotrope 2R,which has been shown to prevent nonspecific bindingof DNAtocells,specifically eosinophils (24),blockedbindingof the ladder butnotof the

TGF#

I probe.

Thedistribution of

TGF#1

expressingcells in nasalpolyp tissueswas reminiscent of theeosinophildistribution

charac-teristic of these tissues. Therefore, it was reasonable to ask whether thecellsexpressingthe

TGF#

I gene in thepolyp

tis-sueswereinfacteosinophils.Tothisend,weperformedinsitu

hybridizationin nasalpolyptissue sectionsstained,before

auto-radiography,withchromotrope2R,aspecific stain for

eosino-phils (21). AsFig. 3 shows most, ifnotall, positive cells for

TGF#31

mRNA,werealsostained with the chromotrope 2R.

Conversely, - 50% of the cells stained with thechromotrope 2R, werepositive within situhybridizationfor

TGF(3l

mRNA.

Sincethe presenceof

TGF#

1 mRNA in cells doesnot

neces-sarily indicatethatTGF31 proteinisbeingreleased in situ, we alsostained tissuesections with antibody raised against

TGF#

I

peptides. Thisantibodyreacts with active

TGFO

1 but not with inactive precursor molecule. As shown in Fig. 4 A,

anti-TGFfOl

minimally stained normal nasal mucosa. In contrast, strong

stainingwiththisantibodywasvisualizedin nasal polyptissue

(Fig. 4C),andslight staining was seen when these tissues were

stained with the control antibody (Fig. 4 B). In addition to

localization incells, substantial

TGF#

l stainingwas notedin

association withextracellularmatrixstructures(Fig.4D).

Discussion

Our studies demonstrate the presence of a characteristic

TGF#

1 signalby Northernblot analysis inRNAextractsfrom

nasal polyp and allergic rhinitis tissues but not normal nasal mucosa. We also demonstrate the presence of cells expressing the genefor

TGF#

1 only ininflamed tissues.Studies in which the tissuesectionswerepretreatedwithchromotrope 2R and

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Or,, 4*

.i.e.

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Figure 3.Autoradiogram ofasection froma nasal polyptissue.Exampleof sections whichwerepretreated with chromotrope 2R, hybridized with theTGFj 1 cDNAprobe, and counterstained withchromotrope 2R. Note apositivehybridization signal onlyonpositively counterstainedcells (eosinophils). (Magnification, 1,000).

ProductionofTransformingGrowth Factorf31byEosinophils 1665

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counterstained,afterhybridization, with chromotrope 2R

indi-catethat the TGFf Iprobe bound almost exclusivelyto

eosino-phils. Approximately 50% of the eosinophils infiltrating the

inflamed tissueswereshownto expresstheTGF31 gene. While

macrophages, fibroblasts, and endothelial cells have been

showntoproduce

TGF#

Iinvitro,wecouldnotdetect

TGF#

I geneexpressionincells other thaneosinophilsin the tissueswe

studied. Whether this is indeed the caseor,alternatively, the

levelofexpression ofthe

TGF#

1 genein these cells is belowthe

levelof detection of thetechniquecurrently used isuncertain.

Immunohistochemistry studies demonstrate that TGFf l protein is produced in situ in nasal polyp tissues. It is

interest-ing that in addition to localization in cells, a great deal of

TGFO31

appears tobeassociatedwith the extracellular matrix. Thispatternofdistribution issimilartothatrecently described by Broekelmann etal. in lung tissue from patientswith

idio-pathic pulmonary fibrosis, in which TGFJ3wasdistinctly

local-izedin macrophages by in situ hybridization while

immunohis-tochemical studies demonstrated marked localization ofthe

protein in the lung matrix (25). Our findings and those of

Limper are consistent with the concepts recently described by Nathan and Sporn regarding cytokine/matrix relation-ships (26).

Afeatureofboth nasalpolyposis and idiopathic pulmonary fibrosis is the presence ofvarying degrees of fibrosis in the stroma aswellas basement membrane thickening. It isthus

temptingto suggestthat TGF3 species might be directly

in-volvedin the development of these structural alterations.

How-ever,itmustbeemphasizedthat theabilityofTGFf3to stimu-late collagen deposition may be affected by the presence of othercytokines (27, 28). Hence, thepresenceofTGFf,while probablynecessary, may notbe sufficientandfurther studies need be carried out to fully describe the cytokine profile in thesetissues.Inadditiontoits role in theregulation ofextracel-lular matrixproteins production,TGF3hasabroadspectrum

ofinhibitory effectsonlymphocytes(5), macrophages (29),

neu-trophils(30), andmastcells(31). Thus,ourdatapose interest-ing questions in regardtotheroleofeosinophil-derived

TGF#Il

in theregulation oftheinflammatoryresponse.

Productionof cytokinesrepresents anovel andimportant

property of eosinophils. Inaddition to our dataon

TGFO

1,

Wong et al. have shown thateosinophils infiltrating colonic

malignant tissuesaswellasperipheral blood eosinophils from

patients with hypereosinophilicsyndrome produce TGFa (32).

Wehave recently had theopportunity toexamineperipheral

bloodeosinophils from apatient with hypereosinophilic

syn-drome and have shown that theyspecificallyhybridize withour

TGF3l

cDNA probe but not with the DNA ladder

(unpub-lished data). Furthermore,wehaveobtained evidencethat

eo-sinophilsin nasal polyp tissues alsoexpressthegenefor

granu-locyte macrophage-colony-stimulating factor (33). Therefore, theabilitytoreleasecytokinesrepresents a new waybywhich eosinophilsmay contribute to theregulation ofinflammatory

and immunologic responsesin airways tissuessuch as nasal

polyposis, allergic rhinitis and, by extension,asthma.

Acknowledgments

WeareindebtedtoDrs. D.Hitch and P.Lappfrom theENT Depart-ment,Chedoke-McMaster

Hospital,

whoprovideduswith tissues for

study.

This work wassupported by grants fromthe Medical Research Councilof Canada(MT 6447 and MA11015).

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