• No results found

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase 2

N/A
N/A
Protected

Academic year: 2020

Share "Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase 2"

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

Cultured lung fibroblasts isolated from patients

with idiopathic pulmonary fibrosis have a

diminished capacity to synthesize

prostaglandin E2 and to express

cyclooxygenase-2.

J Wilborn, … , R M Strieter, M Peters-Golden

J Clin Invest.

1995;

95(4)

:1861-1868.

https://doi.org/10.1172/JCI117866

.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this

study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary

fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with

respect to their capacity for PGE2 synthesis and their expression and regulation of

cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating

immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in

F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity

and protein expression of the inducible form of COX, COX-2, and these responses were

inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate

increases in COX-2 protein expression or COX activity in response to these agonists. Under

conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P <

0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the

antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished

induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to

down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response

in IPF. Moreover, we believe that this represents the first description of a defect in COX-2

expression in association with a human disease.

Research Article

(2)

Cultured Lung

Fibroblasts

Isolated from

Patients with Idiopathic

Pulmonary

Fibrosis

Have

a

Diminished

Capacity

to

Synthesize Prostaglandin E2

and

to

Express

Cyclooxygenase-2

Jerome Wilbom,* Leslie J. Crofford,* Mane D. Burdick,* Steven L. Kunkel,§Robert M.Strieter,* and MarcPeters-Golden*

Divisions of *Pulmonary and Critical Care Medicine and

*Rheumatology,

Department of Internal Medicine, and §Departmentof Pathology, University of Michigan and Department ofVeteransAffairsMedical Centers,AnnArbor, Michigan48109

Abstract

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblastsisolated frompatientswithidiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgeryforlungcancer(F-nl)with respect to theircapacity

forPGE2 synthesis and their expression and regulation of

cyclooxygenase (COX) proteins. Basal COX activity,

as-sessed by quantitating immunoreactive PGE2 synthesized

from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. InF-nl, incubation with the agonists PMA, LPS,orIL-1increased COXactivityandprotein expression

of the inducible form ofCOX, COX-2,and theseresponses

were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in

COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximalinduction, COX

ac-tivityin F-IPFwassixfold less than thatin F-nl(P< 0.05).

Our data indicate that F-IPF haveastrikingdefect intheir

capacity to synthesize the antiinflammatory and

antifibro-genic molecule PGE2, apparently because of adiminished

induction of COX-2 protein. This reduction in the

endoge-nous capacity of F-IPFto down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, webelieve that thisrepresents

the firstdescription ofadefect in COX-2expression in

asso-ciation withahuman disease.(J. Clin. Invest. 1995.

95:1861-1868.) Key words: arachidonic acid *

Iipopolysaccharide-eicosanoids * interleukin-1l prostaglandin H synthase

Introduction

Idiopathic pulmonary fibrosis (LPF)1 is the most common of

the interstitiallung diseases,a groupof disorders characterized

AddresscorrespondencetoMarcPeters-Golden,M.D.,Division of

Pul-monaryandCritical CareMedicine, 3916 Taubman Center, University

ofMichigan Medical Center,AnnArbor, MI 48109-0360. Phone: 313-936-2612;FAX:313-764-4556.

Receivedfor publication 29 July 1994 andinrevisedform 24 Octo-ber 1994.

1.Abbreviations used inthispaper: COX, cyclooxygenase(PGH

syn-thase); EIA, enzyme-linked immunoassay;F-IPF,lung fibroblasts

iso-lated from patients with IPF; F-nl, normal lung fibroblasts;IPF,

idio-pathic pulmonary fibrosis; PLA2, phospholipase A2; RT,reverse

tran-scriptase.

TheJournal ofClinicalInvestigation, Inc. Volume95, April 1995, 1861-1868

by inflammatory injury and fibrosis of the lung parenchyma. Fibrosis is generally considered to be an irreversible finding and is the hallmark of IPF. This fibrotic component of IPF

is characterized by both a striking increase in the number of

fibroblastsand thedepositionof fibroblast-derivedextracellular

matrixproteins, especially collagen ( 1).Fibroblastproliferation and collagen synthesis are therefore of central importance in

IPF, and the modulation of these processes byeffector

mole-cules such ascytokines (2-4), growth factors(5, 6),and

eico-sanoids (7) is of great interest.

Amongthose factorsthatdownregulate fibroblastfunction, prostaglandin E2(PGE2)has been themostextensively studied. Thislipid mediator has been showntodecrease fibroblast prolif-eration (8, 9)andreducecollagen levels by inhibitingits synthe-sis (10, 11) and promoting its degradation (12). Since PGE2

isamajor eicosanoid product of fibroblasts ( 13 -15 ), it is plau-sible to speculate that this molecule might regulate fibroblast function in anautocrine fashion.

Theinitial steps in the metabolism of arachidonic acid (AA)

to PGE2 and otherprostaglandins (PGs) are catalyzed by the enzyme PGH synthase, orcyclooxygenase (COX) (16).Two

distinct isozymes of COX, COX-1 and COX-2, have been iden-tified. WhereasCOX-1 is constitutively expressedin many tis-sues(17), COX-2 has been shown to be inducibleby a variety of stimuli, such as PMA, IL-1, and LPS, in anumber of cell types ( 18-21). In Swiss 3T3 fibroblasts, for example, increases inCOXactivity in response toIL- I andPMAhave been shown

tocorrelate with increases in COX-2 protein (21).

We undertookthis study todetermine whether the eicosa-noid profile and the regulation of PG synthesis in fibroblasts isolated from patients with IPF (F-IPF) differed from those observed in normal human pulmonary fibroblasts (F-nl). Our dataindicate that, although F-nl andF-IPFhad identical eicosa-noidprofiles, F-IPF exhibited significantly less COX activity at baseline than F-nl. Moreover, when cells from the two popula-tionswerestimulated with IL-1, PMA, or LPS, F-nl responded

by increasingtheir PGE2 production overbaseline, but F-IPF

did not. In cells from both groups, these metabolic responses correlated withtheir capacitiestoup-regulate the expression of COX-2protein.

Methods

Patientpopulations.TheIPFstudy group consisted of nine previously untreatedpatientsfrom the University of Michigan Specialized Center of Research in Interstitial Lung Disease project. These patients displayed

radiographicand clinicalfindings consistent with IPF; pathologic con-firmation of[PFwasmadebyopenlungbiopsy. Therewerethree males and sixfemales,with amean±SEM age of55.2±14.9yr (range

28-69 yr).Allpatientswerecurrentnonsmokers. Thedegreesof

(3)

using publishedcriteria (22), and the mean inflammation and fibrosis scores from thethree specimens were calculated for each patient. The study group from whichF-ni were isolated consisted of eight patients undergoing pulmonary resection for bronchogenic carcinoma. There were five males and three females, with a mean age of54±15.1 yr (range 39-72 yr). One was a current smoker, five were former smokers (ceased at least 2 mo before surgery), and two were patients who never smoked. F-nl were isolated from areas of lung parenchyma that were consideredhistopathologically normal. The experimental protocol was approved by the University of Michigan Medical Center Institutional Review Board for Approval of Research Involving Human Subjects. The number of patients from whom cells were taken for each experiment isas indicated inthe text and figure legends.

Isolation and culture of pulmonary fibroblasts. Fibroblasts were isolated from open lung tissue biopsy specimens using methods pre-viously described (23). Briefly, lung tissue samples were isolated under sterile conditions, and1-mm3fragments from each of the three sampled segments werepooled and placed in DME (Gibco Laboratories, Grand Island, NY) with 10% LPS-free FCS (HyClone Laboratories, Inc., Lo-gan, UT). Fibroblasts proliferatingfromthese specimens were grown to80% confluence and serially passed in the same medium. At the fifth passage, fibroblasts wereseeded into96-wellplates (0.25 X 105 cells perwell) for metabolic studies, 60-mm dishes (4 x 105 per dish) for immunoblot analysis, and 175-mm flasks(IO'perflask) for RNA analy-sis.Confluent cells werewashed and rendered quiescent by culturing in serum- and LPS-free DME. They were then cultured for 1-72 h in the presence or absence of LPS from Escherichia coli (serotype 01ll:B4, Sigma ChemicalCo., St. Louis, MO) (100 ng/ml), human recombinantIL-l1, (Collaborative Biomedical Products,Bedford,MA)

(2.5 ng/ml),PMA(SigmaChemicalCo.)(50nM), and/or dexametha-sone(SigmaChemicalCo.)(1 MM)beforeharvesting. Cellswere cul-tured at370Cin ahumidifiedatmosphereof 5% CO2in air.

Separation of eicosanoids. Cellularlipidsof fibroblasts cultured in 96-wellplateswereprelabeled by including 1 MCi of[3H]AA (spact

76-100 Ci/mmol; Du Pont-New England Nuclear, Boston, MA) in the medium for 16 h. Unincorporated label wasremovedby washing

cellswith DME, and cells werethenincubated for 30 min with iono-phore A23187 (5MM).Radiolabeled free AA and its eicosanoid metab-oliteswereextracted from themedium,andpooled lipidextractsfrom four wells were subjected to reverse-phase HPLC as previously de-scribed(24). Radioactivitywasdeterminedon-lineusingaRadiomatic

Flo-One Beta detector(PackardInstrumentCo.,DownersGrove, IL).

DeterminationofPGE2accumulation. Aftera16-or72-h incubation in serum-free medium, cumulative PGE2 synthesis from endogenous

AA was determined byassaying supernatants with anenzyme-linked

immunoassay(EIA; Cayman Chemical,AnnArbor, MI)accordingto

the manufacturer's instructions.

DeterminationofCOX and PGE isomerase activities.PGE2

accumu-lation reflects the actions of bothphospholipase A2 (PLA2)andCOX. Tobypass PLA2andthereforeestimatemaximal COX metabolic

capac-ity,cellswereincubatedasalreadyoutlined for 1, 16,or72h, washed,

andincubated for 30 min in DMEcontainingexogenous AA(10 MM).

PGE2formation in the supernatantwasthenquantitated by EIA. Prelimi-nary experiments indicated that this dose of AA was saturating, as

indicated bymaximal PGE2synthesis (data not shown). Inasimilar

fashion,PGE isomeraseactivitywasdetermined aftera15-min incuba-tion with exogenous PGH2and subsequent quantitation ofPGE2. All assayswere performed induplicate, and activities were expressed as

PGE2 formed in nanograms permicrogramof cellularprotein. Protein concentrationwasdeterminedbyamicrotiterplatemodification(Pierce Biochemical, Rockford, IL) oftheBradford method (25) usingBSA

as astandard.

Preparation ofcrudecelllysates.F-nlorF-IPFwereharvested from 60-mm plates by scraping witharubberpoliceman in 250Ml of ice-coldhomogenizingbuffer(50mMpotassium phosphate,0.1 MNaCl,

2 mMEDTA, 1 mMDTT,0.5 mMPMSF,60

pg/ml

soybean trypsin

inhibitor, pH 7.1). The cell suspension was sonicated onice using a

model 250 sonifier (Branson Ultrasonics Corp., Danbury, CT) at a power level of1, 20% duty cycle for 1.5 min.

Immunoblot analysis. Aliquots ofF-nland F-IPF lysatescontaining 20Mgof total protein were combined 1:1 (vol/vol) with SDS sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 10%

/13-mercaptoeth-anol, pH 6.8) and immediately boiled for3min. They were then

sub-jectedtoSDS-PAGE on 10% acrylamide gels overlaid with a 5% stack-ing gel by the method of Laemmli (26). High molecular weight rainbow markers (Amersham Corp., Arlington Heights, IL) and COX-1 and COX-2 standards were run in parallel in each gel. TheCOX-l standard

(Oxford Biomedical Research, Inc., Oxford, MI) was purified from sheep seminal vesicle, and the COX-2 standard (generously provided by D. DeWitt, Michigan State University, East Lansing, MI) consisted of microsomes from cos cells transfected with the cDNA for murine COX-2(27). Proteinsweretransferredtonitrocellulose (Bio Rad Labo-ratories, Richmond, CA) using a Trans-Blot Cell (Hoefer Scientific Instruments, San Francisco, CA), and membranes were blocked for 1 h with 10% nonfat dry milk reconstituted with 0.1% Tween in Tris-buffered saline (50mM Tris-HCl, 150 mM NaCl,pH 7.5) (TBST).

The membranes werewashedfor 5 min in TBST and then incubated for 1 h with either anti-COX-1 antiserum(1:5,000 dilution)or anti-COX-2 antiserum (1:300 dilution).The former is arabbitpolyclonal antibodyraisedagainst sheepseminal vesicleCOX(28),and the latter isarabbit polyclonal antibodyraisedagainsta 17-amino acidpeptide

derived from the- murine COX-2 sequence that is notpresent in COX-1 (29).After another 15-min wash inTBST,the membranewas incu-batedfor 1 h with horseradishperoxidase-conjugatedgoat anti-rabbit

IgGsecondary antibody (1:10,000forCOX-l and1:5,000forCOX-2) followedbythree5-min washes in 0.3% TBST and three 5-min washes inTBST. Detectionwasaccomplished usingtheECL enhanced chemilu-minescence method(Amersham Corp.) accordingtothemanufacturer's directions. Multipleexposureswere obtained for eachmembrane,and thedensities of bandscorrespondingtoCOX-Iand COX-2were

quanti-tatedbyvideodensitometry ofappropriately exposed autoradiographs usingimage analysissoftware from Scion(Frederick, MD).To compare the steady-statebasal levels ofCOX-1 and COX-2 among all the F-nl and F-IPFpatients,allsampleswereloaded inarandom fashiononthe

samegelandprocessedinparallel;banddensities, expressedinarbitrary

densitometricunits,weredetermined. Toanalyzetheeffects ofagonists

on COX protein levels,different experimental samples from a given

patient wereloadedonthesamegelandprocessedinparallel,and the densities of bands from agonist-treated samples were expressed as a

percentage of that from the control(unstimulated) sample.

RNApreparationandanalysis. TotalRNA waspreparedfrom cul-tured fibroblastsbytheTri-Reagentmethod(MolecularResearch Cen-ter,Inc., Cincinnati, OH).ForPCRanalysisofRNA,cDNAwas

pre-paredbyreversetranscriptionof 1 Mgof each RNAsampleina50-Ml

reaction containing 50 mM Tris-HCl, pH 8.3, 40 mM KCl, 6 mM

MgCl2, 1 mM DTT, 0.4 mMdNTPs,2 MM random hexamerprimers

(Perkin Elmer-Cetus, Norwalk, CT),0.2U/MlRNaseinhibitor(Perkin Elmer-Cetus),and8U/Ml Moloney-murineleukemia virusreverse

tran-scriptase (RT)(LifeTechnologies,Inc., Bethesda, MD).Thereaction mixtures wereincubatedat roomtemperature for 10 min, at420C for 30 min, andat95TCfor 5 min. The cDNAswerethen dilutedto 100

Ml,

and thesame cDNAmixtureswereused in all PCRs. PCRsperformedin

a50-Mlreaction volumecontaining5 Mlof eachcDNA, 10 mM

Tris-HCl, pH8.3,50 mMKCl,25 mMMgCl2,50MMeach ofdATP,dCTP,

dGTP, and dTTP, 5 Ml of

[a-32P]dCTP

(3,000 Ci/mmol; Du

Pont-NewEngland Nuclear),and 0.05MlofTaqpolymerase(Perkin

Elmer-Cetus).Theprimersusedwere(a)humanCOX-1,sense5'-TGC CCA GCT CCTGGC CCG CCGCTf-3'andantisense 5 '-GTG CAT CAA CAC AGG CGC CTCTTC-3';(b)humanCOX-2,sense5'-TTCAAA TGA GAT TGTGGGAAAATTGCT-3'and antisense 5 '-AGA TCA TCTCTG CCTGAGTATCTT-3';and (c) G3PDH, sense5'-CCA

CCC ATG GCA AAT TCC ATG GCA-3' and antisense 5 '-TCT AGA CGG CAG GTC AGG TCC ACC-3'. All primer pairs amplified a

fragment that crossed an intron, thereby distinguishing cDNA from

(4)

18- Figure 1. PGE2

accumu-*T

F-ni 111 lation over 16 h inF-nl

5;

| F-IPF and F-IPF. BothF-niand

:0

X 12- F-IPF were grown to

80%confluence. They

N

W 6 werethenincubated for

0

a. 16hinserum-free

me-dium. Aliquots of the

su-0 pernatants wereusedto

F-nI F-IPF quantitate PGE2 byEIA. Results areexpressed in nanograms of PGE2 permicrogramof total cellproteinperwell.Each

barrepresents mean±SEM fromn =3. *P<0.05versuscorresponding

F-nl valueby unpaired t test.

Theconditions foramplification were as follows: COX-2, 950Cfor 2 min for 1 cycle, 950Cfor 1 min, 600C for 1 min,720Cfor 1 min for 35 cycles, and 720Cfor 7 min for 1 cycle; and COX-I and G3PDH,

95°C for 1 min,60°C for 1 min, and 72°C for 1 min for 25 cycles.

Bothcycle programswerepreceded by2 min at the stated denaturation temperature and followed by 7 minat72°C. Cyclecurvestudies between 25and 35cycles confirmed that, for the amounts of cDNAbeing

ampli-fied, the reactions had not reached the plateau of theamplificationcurve

for any primer pair. Negative controlsperformed withnoRNAadded to the reverse transcription reaction or no RTyielded nodetectable fragmentswith either primer pair.

Data analysis. Where applicable, data are expressed as mean±SEM. Paired or unpaired Student's t tests were used to analyze differences in enzymeactivity or steady-state COX protein levels. Linear correlation analysis was used to assess the correlation between PGE2 synthetic

capacity and fibrosis scores. Significancewasinferred from a P value

< 0.05.

Results

ConstitutivePGE2 accumulation in cultures of F-nl and F-IPF. To assess the capacity of F-nI and F-IPFto produce PGE2 in the absenceofanagonist, equal numbers of cellswerecultured

in serum- andLPS-free DMEM for 16 h, and PGE2 released intothemedium wasquantitated. F-IPFproduced6.5-fold less immunoreactive PGE2 per

1ug

of protein than did F-nl (P

< 0.05,n =3 for F-nI and F-IPF)(Fig. 1). The proteincontent

did not differsignificantly betweenF-nlandF-IPF(- 130

pg

ofcrude lysateprotein per60-mm dish at80% confluence for

both F-nl andF-IPF).

Eicosanoid profile in F-nl and F-IPF. PGE2 synthesis from endogenous AA reflects the sequential activities ofPLA2 and COX.Todetermine whether thedefect inF-IPFPGB2synthesis reflected adefect in PLA2-dependentAArelease, we assessed thereleaseof radiolabeled products from prelabeled cells

stimu-lated with A23187 for 30 min. Completely separating freeAA andall its metabolites by HPLC further permittedus to

deter-mine whether the two cell populations metabolized AA into

different eicosanoid products and, in particular, whether there was adifferential capacity forthese cells to synthesize

antiin-flammatory PGandproinflammatory leukotrienes.As shown in theexperiment depicted in Fig. 2, which is representativeof a totaloftwoindependent experiments, bothcell types produced a similar profile of eicosanoids upon stimulation, with PGE2

and free AA being the major products. Neithercell type

pro-ducedany detectableproducts of the 5-, 12-,or15-lipoxygenase

pathways. These data clearly establish thatF-IPF were capable

of releasing endogenous AA; however, the ratio of PGE2tofree

a-._

*t

._o

0

-0

0

400-PGE2

F-nl

300

-0--FIPF 8 712

0 20 40 60 E6 100 120

time

(min)

Figure2. Radiolabeled eicosanoidprofileofionophore-stimulatedF-nl and F-IPF. Cellularlipidsof fibroblastswereprelabeledwith[3H]AA

duringa 16-hculture in 96-wellplates. Unincorporatedlabelwas

re-moved, and cellswerewashed and then incubated for 30 min with 5

juMA23187.Radiolabeledproductsinpooledextractsfromfourwells

wereseparated by HPLC,andradioactivitywasdeterminedvia on-line detection. Peaks ofradioactivitywereidentifiedonthe basis of

comigra-tion with authentic standards. Similar resultswereobtained inaseparate

experimentusinganotherpairof F-nl and F-IPF.

AA was strikingly lower (- 18-fold in the example shown in Fig. 2) in F-IPF than in F-nl. This indicates that theprimary

limitation inPGE2 synthesis was inutilization of released AA

by the COXpathway of F-IPF.

COX activity in unstimulated F-nl and F-IPF. To focus

more directly onthisapparentdefect in COX metabolism, we

added exogenous AA in order to bypass PLA2, thereby

as-sessing maximal COX activity by quantitating PGE2 formation.

Becauseboth cellproliferation (30) andserum(31)can

influ-encePGsynthesis and COX activity,westudied cellsatvarious time points up to 72 h after the removal of serum to ensure

that anyresidual effects ofserum had abated. There were no

significant differences in COX activity over the 72-h culture period (i.e., at 1, 16, or72 h) for either fibroblast population (Fig. 3). Interestingly, COX activitywas 2.5-fold lower in F-IPF ascomparedwith F-nl (P<0.05)atalltimepoints studied (Fig. 3). All subsequent experiments wereperformedat 16 h.

ls-F-nl

2

Figure

3. COX

activity

F-c

IPF in F-nl and

F-IPF.

Sub-confluent F-nl and F-IPF

10 were cultured in

serum-free medium for1,16,or

cm 72h, washed,and then

*o

5-*1 * incubated with 10

ILM

AAfor 15 min.

Aliquots

of the cellsupernatants

0 1 16 7 h wereusedto

quantitate

PGE2 by ETA.Resultsare

expressed in nanograms ofPGE2permicrogramof total cellproteinper well. Each bar represents

mean±SEMfromn =4. *P< 0.05 versuscorrespondingF-nl value

(5)

A

COX-1

69

k-I

IPF IPF nl nI IPF

69kD-l*

B

200-100 eo e

.82()0-@2 F-nl

F-Il

-S

COX-2

std. IPF IPF nI ni

69

kD-

:Uj

c

COX-1 at

std. 0

01-CY

A_

0_

(

T

COX-1 IPF ni std.

Mil

au

F-nI

50- t

F-IPF*

t

40-

t

30-

20-1:

0**

I

I

I~~~~

cont LPS LPS/dex PMA PMA/dex IL-1 Figure 5. COXactivity in stimulatedF-nIand F-IPF. Cells were incu-bated for 16 h in the presence or absence(control;cont.) of 100ng/ mlLPS (n= 7for F-nl; n = 8 forF-LPF),50 nM PMA (n =5 for bothF-nIandF-IPF),or2.5 ng/mlIL-i,6(n= 3 for both nl and F-IPF) and 1

/iM

dexamethasone (dex). Cells were washed and then incubated with 10

MM

AAfor 15 min.Aliquots of the supernatants were used toquantitatePGE2 by EIA. Results areexpressedin nano-gramsofPGE2 permicrogramof total cellprotein per well. Each bar represents mean±SEM. *P<0.05versuscorresponding F-nivalueby unpairedttest;tP <0.05 versus unstimulated control valueby paired

ttest.

Figure 4. Immunoblot analysis of COX proteins in unstimulated F-ni

and F-IPF. Cells were culturedfor 16 h inserum-freemedium and harvestedfor immunoblotanalyses asdescribedin Methodsusing20

sgof crudelysate proteinper lane.(A)Autoradiograph depicting COX-1 expressionin cellsfromeightnlandeight IPF patients. Migration of

amolecularweight markerisindicatedbythearrows.COX-1 standard was run in theright lanes. (B) Densitometric analysis of COX-1 bands inF-ni andF-IPFfrom theaboveautoradiograph. Data areexpressed

asarbitrary densitometric units,andeachbar representsmean±SEM.

(C) Autoradiograph depicting COX-2 expressionin cellsfrom threenI

and three IPFpatients. Migration ofamolecularweightmarker is indi-catedbythe arrow. ACOX-1 standardwas runin theright lane,anda

COX-2 standard in theleft.

The formation of PGE2 from AA requires the actions of not

only COX, but also PGE isomerase. To exclude thepossibility

thatdifferences in PGE2 synthesis wereattributable to

differ-ences inPGE isomerase activitybetween thetwo cell

popula-tions, PGE2 formation from exogenously supplied PGH2 (5

ILsM)

wasdetermined. Both F-ni and F-IPFsynthesized equally large

amountsof PGE2 from exogenouslysupplied PGH2 (396±84.3

ng per jg ofproteinforF-nIand 473±67.8 ng per

qg

ofprotein

for F-IPF) (P > 0.05, n = 3). Moreover, this 40-fold (in

F-nl) to 100-fold (in F-IPF) greater capacity forproduction of PGE2 from exogenous PGH2 than from exogenousAAconfirms thatCOX itself is the rate-limiting step within this metabolic pathway. Our finding that differences between F-nl and F-IPF in PGE2 production from exogenous AA were attributable to

differences in theactivityof COX itselfwasthereforeexpected.

Immunoblotanalysis ofCOX proteins in unstimulated fi-broblasts. The expressionof COX-1 and COX-2proteins was

examined in F-nl and F-IPF cultured for 16 h in serum- and LPS-free medium. Crude lysates from both cell populations

weresubjectedtoimmunoblotanalysisusing polyclonal antisera

specific for

COX-l

and COX-2. As shown in Fig. 4A, most

of the cellsamples in both groups (n = 8 foreach) expressed COX-1 in the unstimulated state. Densitometric analysis

re-vealed that COX-1 expression in thetwopopulationswas simi-lar(Fig.4B). Thiswas an unexpected finding, given the 2.5-fold lower COX activity in unstimulated F-IPF as compared with unstimulated F-nl, and ledus toconsider that differences in COX-2 expression might be responsible for this difference in COX activity in the basal state. We found, however, very low levels of basal COX-2 expression in both groups. After prolonged exposure of autoradiographs, COX-2 was detectable insomesubjects,butnodifferenceswereapparentbetween the twopopulations (Fig.4 C).

COX activityinstimulatedfibroblasts. Inview of the reduc-tion in COXactivity inF-IPFin the basal state,we next

exam-ined COXactivityin stimulated cellsfrom thetwopopulations.

Previous studies have shown that variousagonists can induce COX-2 protein and that coincubation with the glucocorticoid

dexamethasonecanprevent this induction. Cellsweretherefore incubated for 16 h with medium alone or in the presence of LPS (100 ng/ml),PMA(50 nM),or

IL-1,8

(2.5 ng/ml), after which COXactivitywasassessedbyquantitating PGE2 formed from exogenous AA. As shown inFig. 5, F-nl increased their activity approximatelytwo- tothreefold in responseto stimula-tion with each of these threeagonists. Bycontrast,F-IPF exhib-itednoincrease in COXactivity with any of theagonists. Thus,

in the stimulatedstate the difference in maximal COXactivity

between F-nl and F-IPF was even more pronounced: F-IPF

exhibited sixfold less maximal COX activitythan didF-nl (P

< 0.05 for LPS, PMA, and IL-1 stimulation). As expected,

dexamethasonepreventedtheagonist-inducedincrease inCOX

activity in F-nl. SinceF-IPF exhibitedno induction of COX-2, it was not surprising that these cells were unaffected by dexamethasone.

Immunoblotanalysis of COX proteins instimulated

(6)

C

69 kD -_

F-nl

F-IPF

Figure6.Immunoblotanalysisof COXproteinsin stimulated F-nl and F-IPF.Cellswereincubated for 16 h in the absence(control; cont)or

pres-enceof 100ng/mlLPS,2.5ng/ml

IL-1,B,or 1

ttM

dexamethasone (dex).

Crudelysate protein (20,g)was

sub-jectedtoimmunoblotanalysis for COX-2proteinasdescribed in Meth-ods.(A) Representative

autoradio-graphwithmigrationof molecular

weightmarkersindicated bythe arrows.ACOX-2 standard was run in therightlane.(B) Densitometric

analysisofCOX-2 bands inF-nIand F-IPFincubated with medium alone

(control; cont),LPS(LPS),and LPS

plusdexamethasone(LPS/dex). Rel-ativeCOX-2 levels areexpressedas apercentageof control andrepresent mean±SEM fromseven nl andeight

69 kD IPF. *P < 0.05 versus F-nl by un-pairedttest;and tp < 0.05versus LPSby pairedttest.(C)

Representa-tiveautoradiograph showingCOX-2 induction inresponseto 2.5 ng/ml IL-1.

cells stimulated with LPS, PMA, and IL-1. A representative autoradiograph shown in Fig. 6 A reveals that steady-state COX-2 protein expression was increased by LPS in F-nl, and this inductionwaspreventedby coincubation with dexamethasone. Such induction of COX-2 proteinwasnotfound in F-IPF. Den-sitometric analysis of COX-2 protein expression (Fig. 6 B) indicates that in F-nl (n = 5), relative COX-2 expression

in-creased approximately twofold (P <0.05versuscorresponding controlvalue) inresponsetoLPS and decreased below baseline control levels (P < 0.05) when cells were coincubated with LPS plus dexamethasone. Bycontrast,COX-2wasnotinduced inF-IPF inresponsetoLPS(n=5).Moreover,LPS-stimulated

F-IPF expressed twofold less COX-2 than did LPS-stimulated F-nl. Inasimilar fashion, IL-1 induced COX-2 protein expres-sion substantially overthe control level inF-nl, butnot in F-IPF(Fig. 6 C). Similar induction of COX-2 in F-nl butnotin F-IPF was observed with PMA treatment (data not shown). Agonist stimulation hadno effecton COX-1 levels, and there was nosignificant difference in COX-1expression between the

two populations inthe stimulatedstate (datanotshown).

Expression of COX-2 mRNA inF-nland F-IPF. To deter-mine whether the defect responsible for the lack of COX-2 protein expression in F-IPF was pretranslational, we isolated total RNA from F-nl and F-IPF that had been stimulated with orwithout 100 ng/ml LPS. Fig. 7 demonstrates that cells from

each individual patient examined were representative of the

entiregroupwithrespect totheirCOX activity (top panel) and COX-2 protein expression (middle panel), in that F-nl (Fig.

7 A) but not F-IPF (Fig. 7 B) exhibited increases with LPS stimulation. RT-PCR analysiswasperformedontheseF-nl and F-IPF in the basal and LPS-stimulated states. COX-2 mRNA

in basal F-IPF was detectable only after 35 PCR cycles and

failed to increase afterincubation with LPS (Fig. 7 B, bottom panel), mirroring the results obtained for COX-2 protein ex-pression under thesametreatmentconditions (Fig. 7B, middle

panel). Abundant mRNA was detected in F-nl at 35 cycles (Fig. 7A, bottom panel); in fact, COX-2was detectable after only25cycles inF-nl. When normalizedtoG3PDH mRNA, a 2.8-fold increase in the COX-2 PCR fragmentwasobserved in

F-nltreated with LPS ascompared with control, whereas there was noincrease in COX-2 transcript after LPS treatmentin F-IPF. In sharpcontrast tothese striking differences in basal COX-2 mRNA expression, basal COX-I levelswere similar inF-nl and F-IPF (datanotshown).

Correlation between COX activity and fibrosis. To

deter-mine whether there was a correlation between COX activity andthe degree of pulmonary fibrosis, COX activities in both basal and LPS-stimulated fibroblasts from patients with IPF wereplotted againstmean fibrosisscores. As shown in Fig. 8, a statistically significant inverse relationship existed between thePGE2synthetic capacity of LPS-stimulated F-IPF and fibro-sisscores (r= 0.804). A similar correlation (r= 0.774)was noted for unstimulated F-IPFaswell (datanotshown).

Discussion

Fibroblasts have been studied extensively as targetcells in fi-broticdisorders, includingIPF.Lessis known about the possible role of fibroblastsas effector cells actively participating in the evolution of IPF. Because fibroblasts areknown to synthesize PGE2, and this PG has the ability to downregulate fibroblast proliferation and collagen synthesis, we hypothesized that F-IPF might exhibitadiminished capacity for PGE2 synthesisas compared withF-nl. This hypothesiswastestedinprimary

cul-tures of fibroblasts isolated from lung biopsy specimens from patients with IPF and from nonfibrotic control patients undergo-ing resection for bronchogenic carcinoma. Our study yielded several major findings. (a)F-IPFexhibited diminished capacity

to synthesize PGE2 from endogenous AA in both basal and ionophore-stimulated conditions. (b) Similar reductions in

A

LPS

92.5 kD P 69 kD-*

F-nl

LPFS COX-2

4-92.5 kO

4-69kD

F-IPF

U

U

El

B

200-B c( 2 150-x E

0 °

O-e

a

-CD

50-

O-cont. LPS LPS/dex

*

(7)

Figure7. COX activity and expression of COX-2 protein andmRNA in F-nl and F-IPF. Cells from a patient without (A) and with (B) IPF were incubated for 16 h in the absence (-) or presence ( + ) of100

ng/ml LPS.COX activity was assessed as described in Methods(-LPS, open bars; +LPS, hatched bars), and expressed in nanograms of PGE2 per microgram of total cell protein per well (top pan-els). Crudelysate protein (20

,g)

was subjected to immunoblotanalysis for COX-2 protein as described in Methods(middle panels), and RT-PCR (35 cy-cles)analysis of COX-2 mRNA expression was

per-formedasdescribed in Methods(bottompanels).

PGE2synthesis from exogenous AA (but not exogenous PGH2) suggest a defect at the level of the COX enzyme itself. (c) Unlike F-nl, F-IPFwereincapable of increasing COX activity after pretreatment with the agonists LPS, PMA, and IL-143. (d) This inability to augment COX metabolic activity correlated with a failure to increase steady-state levels of COX-2 protein and mRNA. (e) Finally, a significant inverse correlation was observed between the PGE2 synthetic capacity of F-IPF and semiquantitative fibrosis scores of lung tissue from which these

F-IPF were obtained.

Several methodological features ofourstudy deserve com-ment.First, cells from three separate lung segmentswerepooled

to obtain theF-IPF used. In the interstitial lung diseases, the degrees of pulmonary inflammation and fibrosis are well known

tobe heterogeneous in distribution (1). Although pooling cells

CL

w

a..

0) c

9-

3-0 5 10

fibrosisscore

15 20

Figure8.Correlationbetween COXactivity and fibrosisscore.Mean pathologicscoresfromthreebiopsy specimensareplotted'against COX activity (expressedasnanogramsofPGE2permicrogram ofprotein) in theLPS-stimulatedstatefor each ofseven lPFpatients. The relation-ship between PGE2 synthetic capacity and fibrosis is described by the equationy =9920.8 - 362.83x;r= 0.804.

from different regions minimizes the possibility of sampling errors, it also averages out cellular heterogeneity, thereby tend-ing to underestimate the magnitude of abnormalities in the most severely affected areas. Second, because alterations in eicosa-noidprofiles have been reported to accompany serial passage of fibroblasts(32), we chose to study cellsatthe fifth passage, reasoning that this was a sufficient passage number to ensure purity, but not so great that differences present in vivo might be lost. Nonetheless, the differences in maximal COX activity between F-nl and F-IPF thatweobserved havepersisted through the 12th passage (n = 2 for both cell populations; data not

shown). Third, because both the proliferativestateand the se-rum itself can influence PG synthesis and COX activity, we studiedquiescent cellsat various timepoints up to 72 h after removalof serum. The defects in COX activity in F-IPF were observedatalltimepoints.

Weinitially observed that constitutive PGE2 accumulation in F-IPFwas 6.5-foldlower than that in F-nI when cells were

studiedat16 h inculture. We therefore performed HPLC analy-sistodetermine whether there were any differences in the eico-sanoidprofilesbetween the twocell types that wouldaccount

for this finding. A previous report indicated that human skin fibroblasts failed to synthesize 5-lipoxygenase metabolites of

AA (33). However, since leukotrienes have the capacity to stimulate fibroblastproliferation and collagen synthesis (34), itwasneverthelessof interest to determine whetherthese metab-olites were synthesized by F-IPF. In fact, HPLC analysis de-tectednolipoxygenase products elaborated by fibroblasts from eitherpatientgroup. Rather, in both F-IPF and F-nl the major

productsobservedwerefreeAAandPGE2. Whatwas striking

was themarkedly reducedcapacityof F-IPFtometabolize the releasedAAtoPGE2,ascompared with F-nl. Although we did

notcompare thetwopopulations for theirquantitativecapacities

torelease AA, HPLC analysis of prelabeled cells clearly indi-cated that F-IPF were capable of releasing free fatty acid, thereby implicating adefect distal toPLA2. This does not

ex-cludeapossibleconcomitant reduction inAAlevels thatmight beavailable as substratefor COX (i.e., decreased deacylation

orincreasedreacylation)in F-IPFascomparedwith F-nl. This

possibilitymust beexploredin future studies.

0

0

0

(8)

Thecapacityfor PGH2 metabolismwasfargreater than the cells' capacity forPGH2formation, suggestingthat COX is the rate-limiting enzyme. Moreover, no differences in PGE iso-merase activity were detected between the two cell types in stimulated (datanot shown) orbasal states. But F-IPF did

ex-hibit 2.5-fold less COX activity than F-nl in the basal state.

Despite this, wedetected no differences in steady state levels

of COX-1 proteinand noappreciableCOX-2protein expression

in the basal state. Although the mechanism for reduced basal COXactivityin F-IPF has notbeen elucidated in this investiga-tion, several possibilities exist. First, COX-1 enzyme activity

in F-IPF may be less per molecule than that in F-nl,reflecting either intrinsicenzymemodificationsordifferences in modulat-ing factors. Second, the subcellular localization of COX-1 in F-IPF and F-nl might differ, resulting in decreased access of the enzyme in F-IPF to endogenous and exogenous substrate.

Third, differences in constitutive expression of COX-2 might

exist in the two populations, but may be below the limits of

sensitivity of the Western blotting procedure used. Using RT-PCR, wedidfind that whereasCOX-1 mRNA levels were simi-lar, COX-2 mRNA levels were indeed lowerin F-IPF than in

F-nl in the basal state. Identifying the mechanism responsible for this reduction in basal COX activity in F-IPF will require furtherinvestigation.

COX activity in F-nl wasincreased above baseline by the

addition of the agonists LPS, PMA, andIL-1p6 to the culture medium. Evidence that this increase inactivitywasdueto

COX-2induction wasprovided bythe observations that the increase in COX activity correlated with COX-2 protein and mRNA

expression, that there was no increase in COX- 1 expression

with agonist stimulation, and that the increases in activityand COX-2protein expressionwerebothprevented bycoincubation with dexamethasone. In contrast to F-nl, F-IPF wererefractory

to increases in COX activity orCOX-2 protein expression by

these same stimuli. Thatrefractoriness was observedfor three unrelated agonists stronglysuggests that the defect resides not atthe level ofareceptor orsignaling mechanism, but at a more

distal efferentstep in enzymesynthesis. RT-PCR resultssuggest a failure to increase steady-state mRNA levels upon agonist

stimulation. The molecular mechanism underlying this lackof

mRNAinducibility remains tobedetermined, but couldreflect

enhanced turnover rates forthe transcriptorreduced

transcrip-tion rates. This in turn mightresult from anacquired alteration

in theCOX-2promoter,fromaninabilitytoalternatively splice aprematuretranscript (35),orfrom alterations in theregulation

oractions oftranscription factors. It will beofgreatinterestin

future studies to determine whether other lung cells share this defect in COX-2 inducibility identifiedin F-IPF.

To ourknowledge, this is thefirst demonstrated association ofa lack of inducibility of COX-2 with a human disease. A great deal of attention has been focused onthe roleofCOX-2 ininflammation (36), and thedevelopment of selective inhibi-tors ofCOX-2 offersthe prospect ofantiinflammatory efficacy withlessgastrointestinal and renal toxicitythan current nonse-lective COX inhibitors (37). However, it is also recognized that certain PGs, including PGE2, can exertpotent inhibitory actions on leukocytes (38, 39), lymphocytes (40, 41), and

fibroblasts (8, 9, 11,42), whichparticipate inthe processes of

inflammatory injuryandrepair.Induction ofCOX-2byagonists

present in the inflammatory milieuof thelung might represent

animportant mechanism bywhichfibroblastscanincreasetheir

PGE2 synthetic capacity and therebylimit cellularproliferation,

collagen synthesis, andproductionofinflammatory mediators. A defect in this homeostatic process may promote or sustain

inflammation and fibrosis in the lung.In this regard,it is

intri-guing that, despitethe smallsample size,weobserveda

signifi-cantinverse correlation between thedegreeof fibrosis and COX

activity (PGE2 synthetic capacity) in fibroblasts isolated from IPFpatients. Of note, Boroketal. (43)foundsignificantlyless

PGE2 in lavage fluid obtained from patients with IPF than in normal subjects. These data therefore highlight the potential

detrimentalconsequences thatmightresult frompharmacologic inhibition ofCOX-2, at least during the reparative phases of

inflammatory injuryin thelung. Finally, ourobservations may havetherapeutic ramifications.AugmentationofPGE2levels in thelung viaaerosolizationofastablePGE2 analog (43)orvia

transfectionof the cDNAencodingCOX- 1orCOX-2(44)has thepotential to reduce fibrosis and ultimately to improve the

prognosis for this oftendevastating disease.

Acknowledgments

WethankBingTan, RobertMcNish, HollyEvanoff,and JanetHampton

for technical assistance, Drs. Mark Orringer and Richard Whyte for

providing lung tissue specimens, and Drs. William Smith and David DeWittfor kindly providing the COX-1 and COX-2 antisera,

respec-tively.

This work was supported by grants from the National Institutes of Health

(ROl-HL47391

and Specialized Center of Research

P50-HL46487). M. Peters-Golden is therecipientofaCareerInvestigator

Award fromthe American Lung Association. J. Wilborn is therecipient

ofaMinority InvestigatorResearch SupplementAward from the

Na-tionalHeart, Lung, and Blood Institute.

References

1.Panos, R.,and T.King.1991.Idiopathic pulmonaryfibrosis. In Immunologi-callyMediatedPulmonaryDiseases.J. P.LynchIII and R. A.DeRemee,editors. J. B.Lippincott, Philadelphia,PA. 1-39.

2.Jordana, M.,M. Newhouse,and J.Gauldie. 1987. Alveolar macrophage-andperipheralbloodmonocyte-derivedfactors modulateproliferationofprimary linesof humanlungfibroblasts. J. Leukocvte Biol. 42:51-60.

3.Elias, J.,B.Freundlich,J.Kern,and J. Rosenbloom. 1990.Cytokine

net-worksintheregulationof inflammation and fibrosis in thelung.Chest. 97:1439-1445.

4. Gauldie, J., M. Jordana, and G. Cox. 1994. Cytokines and pulmonary fibrosis. Cvtokines. 4:931-935.

5.Clark, J.,D.Madtes,and G.Raghu.1993.Effects ofplatelet-derived growth factor isoformonhumanlungfibroblastproliferationandcollagengene expres-sion. Exp. Lung Res. 19:327-344.

6.Fine, A.,and R.Goldstein. 1986. The effect oftransforming growth

factor-,6oncellproliferationandcollagenformationby lungfibroblasts. J. Biol. Chem. 262:3897-3902.

7. Elias,J., M.Rossman,R.Zurier, andR.Daniele. 1985. Humanalveolar macrophageinhibition oflungfibroblastgrowth:aprostaglandin-dependent

pro-cess.Am. Rev.Respir.Dis. 131:94-99.

8.Bitterman, P.,M.Wewers,S.Rennard,S.Adelberg,andR.Crystal. 1986. Modulation of alveolarmacrophage-drivenfibroblastproliferation byalternative macrophagemediators. J. Clin.Invest.77:700-708.

9.Elias,J. 1988. Tumor necrosis factor interacts with interleukin-l and inter-feronstoinhibit fibroblastproliferationviaprostaglandin-dependentand indepen-dent mechanisms.Am. Rev.Respir.Dis. 138:652-658.

10.Goldstein, R.,andP.Polgar.1982. Theeffect and interaction ofbradykinin andprostaglandinsonproteinandcollagen production by lungfibroblasts. J. Biol. Chem. 257:8630-8633.

11. Kom, J.,P.Halushka,andE.Leroy.1980. Mononuclear cell modulation of connective tissue function: suppression of fibroblastgrowthbystimulationof endogenousprostaglandin production. J. Clin. Invest. 65:543-554.

12.Baum, B.,S.Moss,R.Breul,and R.Crystal. 1980.Effect ofcyclicAMP

onthe intracellulardegradation ofnewlysynthesized collagen. J. Biol. Chem. 255:2843-2847.

(9)

radioimmunoas-say before andafter separation by high pressure liquid chromatography. Prosta-glandins Med.3:295-304.

14. Korn, J., D. Torres, and E. Downie. 1983. Fibroblast prostaglandinE2 synthesis. J. Clin.Invest. 71:1240-1246.

15. Shindo,N., T. Saito, and K. Murayama. 1988. Rapid quantification of 11 prostanoids bycombined capillary column gas chromatography and negative ion chemicalionization mass spectrometry: application to prostanoids released from normal humanembryonic lung fibroblasts W138 in a culture medium. Biomed. Environ. Mass.Spec. 15:25-32.

16. Smith, W., and L. Mamett. 1991. Prostaglandin endoperoxide synthase: structure andcatalysis. Biochem. Biophys. Acta. 1083:1-17.

17. O'Neill, G., and A. Ford-Hutchinson. 1993. Expression of mRNA for cyclooxygenase-l and cyclooxygenase-2 in human tissues. FEBS (Fed Eur. Bio-chem. Soc.) Lett. 330:156-160.

18.Fu, J.-Y., J. L. Masferrer, K. Seibert, A. Raz, and P. Needleman. 1990. The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in humanmonocytes. J. Biol. Chem. 265:16737-16740.

19. Jones, D., P. Carlton, T. McIntyre, G. Zimmerman, and S. Prescott. 1993. Molecular cloning of human prostaglandin endoperoxide synthase type 2 and demonstration of expression in response to cytokines. J. Biol. Chem. 268:9049-9054.

20.Masferrer,J., K.Siebert,B.Zweifel, and P. Needleman. 1992. Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme. Proc. Natl. Acad. Sci. USA.89:3917-3921.

21. Kujubu, D., S. Reddy, B. Fletcher, and H. Herschman. 1993. Expression of theproteinproduct of the prostaglandin synthase-2/TIS1O gene in mitogen-stimulated Swiss 3T3cells. J. Biol. Chem. 268:5425-5430.

22. Watters, L., T. King, M. Schwarz, J. Waldron, R. Stanford, and M. Reuben. 1986. A clinical, radiographic, andphysiologicscoring system for the longitudinal assessmentofpatients with idiopathic pulmonary fibrosis. Am. Rev. Respir. Dis. 133:97-103.

23.Rolfe, M., S. Kunkel, T. Standiford, S. Chensue, R. Allen, H. Evanoff, S. Phan, and R. Strieter. 1992.Pulmonaryfibroblastexpressionof interleukin-8: a modelfor alveolarmacrophage-derived cytokine networking.Am. J.Respir. Cell. Mol. Biol. 5:493-501.

24.Peters-Golden, M., R. W. McNish, R. Hyzy, C. Shelly, and G. B. Toews. 1990. Alterations in the pattern of arachidonate metabolism accompany rat macro-phage differentiation in the lung. J. Immunol. 144:263-270.

25.Bradford, M. 1976. Arapidandsensitive method for thequantitationof microgram quantities ofproteinutilizing the principleofprotein-dye binding. Anal.Biochem. 72:248-254.

26.Laemmli,U. 1970. Cleavage of structuralproteins duringtheassembly of the head ofbacteriophage T4. Nature (Lond.). 227:680-685.

27.Meade, E., W. Smith, and D. DeWitt. 1993. Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirinand other non-steroidalanti-inflammatory drugs.J.Biol. Chem. 268:6610-6614.

28.Smith, W., and T. Rollins. 1982. Characteristics of rabbit anti-PGH syn-thaseantibodies and use inimmunochemistry.MethodsEnzymol. 86:213-222.

29. O'Sullivan,M., E. Huggins, Jr., E. Meade, D. DeWitt, and C. McCall. 1992.Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macro-phages. Biochem.Biophys. Res. Commun. 187:1123-1127.

30. Hong, S., G. Patton, and D. Deykin. 1979. Arachidonic acid levels in cellularlipids determine the amount of prostaglandins synthesized during cell growth in tissueculture. Prostaglandins. 17:53-59.

31. O'Banion, M., H. Sadowski, V. Winn, and D. Young. 1991. A serum-andglucocorticoid-regulated4-kilobase mRNA encodes a cyclooxygenase-related protein. J. Biol. Chem. 206:23261-23267.

32. Polgar, P., and L. Taylor. 1980. Alterations in prostaglandin synthesis during senescence of human lung fibroblasts. Mech. Ageing Dev. 12:305-313.

33. Bernd, M., R. Ludwig, E. Zenzmaier, H. Gleispach, and H. Esterbauer. 1984.Characterization of lipoxygenase metabolites of arachidonic acid in cultured humanskin fibroblasts. Biochim. Biophys. Acta. 795:151-164.

34. Baud, L., J. Perez, M. Denis, and R. Ardaillou. 1987. Modulation of fibroblastproliferationbysulfidopeptide leukotrienes: effect of indomethacin. J. Immunol. 138:1190-1195.

35. Diaz, A., A. Reginato, and S. Jimenez. 1992. Alternative splicing of human prostaglandin G/Hsynthase mRNA and evidence of differential regulation of the resultingtranscripts by transforming growth factor ,, interleukin1I3,and tumor necrosis factor a. J. Biol. Chem. 267:10816-10822.

36. Vane, J. 1994. Towards a betteraspirin.Nature(Lond.).367:215-216. 37.Masferrer,J., B.Zweifel,P.Manning,S.Hauser,K.Leahy,W.Smith,P. Isakson, and K. Seibert. 1994. Selective inhibition of inducible cyclooxygenase-2in vivo isantiinflammatory andnonulcerogenic. Proc.Natl. Acad. Sci. USA. 91:3228-3236.

38.Ham,E.,D.Soderman,M.Zanetti,H.Dougherty,E.McCauley,and F. Kuehl, Jr. 1983. Inhibition by prostaglandins ofleukotriene B4 release from activatedneutrophils. Proc.NatI.Acad. Sci. USA. 80:4349-4353.

39.McLeish, K., G. Stelzer, and J. Wallace. 1987. Regulationof oxygen radical releasefrom murineperitonealmacrophages bypharmacologicdoses of PGE2. Free Radical Biol. Med. 3:15-20.

40.Oppenheimer-Marks, N.,A.Kavanaugh,and P.Lipsky. 1994. Inhibition of thetransendothelialmigrationof human Tlymphocytesby prostaglandinE2. J. Immunol. 152:5703-5713.

41.Betz, M., and B. Fox. 1991.ProstaglandinE2 inhibitsproductionof Thl lymphokines but not of Th2lymphokines. J. Immunol. 146:108-113.

42. Goldstein, R., and A. Fine. 1986. Fibrotic reactions in the lung: the activation of thelungfibroblast.Exp. LungRes. 11:245-261.

43. Borok, Z., A. Gillissen, R. Buhl, R. Hoyt, R. Hubbard, T. Ozaki, S. Rennard,and R.Crystal.1991.Augmentationof functionalprostaglandinE levels on therespiratoryepithelialsurfacebyaerosoladministration ofprostaglandinE. Am.Rev.Respir. Dis. 144:1080-1084.

44.Conary, J.,R.Parker,B.Christman,R.Faulks,G.King,B.Meryick,and K.Brigham. 1994. Protection of rabbitlungsfrom endotoxininjury byin vivo hyperexpression of the prostaglandin G/H synthase gene. J. Clin. Invest. 93:1834-1840.

References

Related documents

This two aspects of Ebo- la hemorrhagic fever can be related to fol- lowing concept mentioned in Ayurveda- If a disease is caused due to strong or many etio- logical factors

Although groundnut is self-pollinating and possesses limited variability, the statistically significant differences among the studied groundnut varieties in terms

At 3 weeks after chondrogenic induction, both Alcian Blue staining and quantitative analysis results showed that glycosaminoglycan formation was inhibited in HOXC8 si- lenced

A: Peak velocity of late filling wave; DIVPG: Diastolic intraventricular pressure gradient.; E: Peak velocity of early filling wave.; e ’ : Mitral annulus early peak velocity.;

The purpose of the study was to assess the usefulness of dynamic IVC-derived parameters (collapsibility index, distensibility index) in comparison to passive leg raising,

Further, several duck virome sequences had homologous with the insect viruses of the Poxviridae , Alphatetraviridae , Baculoviridae, Densovirinae, Iflaviridae and Dicistroviridae