Mast cell tryptase is a mitogen for cultured fibroblasts

(1)

Mast cell tryptase is a mitogen for cultured

fibroblasts.

S J Ruoss, … , T Hartmann, G H Caughey

J Clin Invest.

1991;

88(2)

:493-499.

https://doi.org/10.1172/JCI115330

.

Mast cells appear to promote fibroblast proliferation, presumably through secretion of growth

factors, although the molecular mechanisms underlying this mitogenic potential have not

been explained fully by known mast cell-derived mediators. We report here that tryptase, a

trypsin-like serine proteinase of mast cell secretory granules, is a potent mitogen for

fibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulate thymidine

incorporation in Chinese hamster lung and Rat-1 fibroblasts and increase cell density in

both subconfluent and confluent cultures of these cell lines. Tryptase-induced cell

proliferation appears proteinase-specific, as this response is not mimicked by pancreatic

trypsin or mast cell chymase. In addition, low levels of tryptase markedly potentiate DNA

synthesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin.

Inhibitors of catalytic activity decrease the mitogenic capacity of tryptase, suggesting,

though not proving, the participation of the catalytic site in cell activation by tryptase.

Differences in Ca++ mobilization and sensitivity to pertussis toxin suggest that tryptase and

thrombin activate distinct signal transduction pathways in fibroblasts. These data implicate

mast cell tryptase as a potent, previously unrecognized fibroblast growth factor, and may

provide a molecular link between mast cell activation and fibrosis.

Research Article

(2)

Mast Cell Tryptase Is

a

Mitogen for Cultured Fibroblasts

Stephen J.Ruoss,*Thomas Hartmann,* and George H.

Caughey*"

*Cardiovascular Research Institute and tDepartment ofMedicine, UniversityofCalifornia, San Francisco, CA 94143

Abstract

Mast cellsappeartopromotefibroblast proliferation,

presum-ably through secretion of growth factors, although the molecu-lar mechanisms underlying this mitogenic potential have not

beenexplained fully by knownmastcell-derived mediators. We

report herethat tryptase, a trypsin-like serine proteinase of

mastcellsecretorygranules, isapotentmitogen forfibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulatethymidine incorporationinChinese hamsterlung and Rat-i fibroblasts and increase cell density in both subconfluent and confluent cultures of these cell lines.Tryptase-induced cell proliferation appears proteinase-specific, as this response is

notmimicked by pancreatic trypsinor mastcellchymase. In addition, low levels oftryptasemarkedly potentiateDNA

syn-thesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin. Inhibitors of catalytic activity

de-creasethemitogenic capacity oftryptase, suggesting, though

notproving, the participation of the catalytic site in cell activa-tionbytryptase.Differencesin

Ca"

mobilizationand sensitiv-itytopertussis toxinsuggestthattryptaseandthrombin

acti-vatedistinct signal transduction pathways in fibroblasts. These dataimplicatemastcelltryptaseasapotent,previously

unrec-ognizedfibroblastgrowth factor,andmayprovideamolecular

link betweenmastcellactivation and fibrosis. (J. Clin. Invest. 1991. 88:493499.) Key words: proteinase - proliferation

growth factor * signal transduction-chymase

Introduction

Severallines ofevidencesuggestparticipation ofmastcells in fibroblast proliferation. Increased numbers ofmast cells are

foundincloseproximitytoproliferating fibroblasts inhealing wounds and infibrotic diseases of the lung and skin in humans (1-4). These observations have receivedsupportfromanimal models oflung fibrosis induced by ionizing radiation, bleomy-cin,orasbestos, which leadto mastcellhyperplasia early in the development of fibrosis (5-7). In the hypersensitivity

pneu-monitis model oflung fibrosis in mast cell-deficient W/W"

mice, mast cells arerequired to produce the fullpathologic

response(8), suggestingacentral role formastcells in the

fibro-blast proliferation. Furthermore, morphologic studies of the lung parenchyma in interstitial fibrosis (1), fibrotic airway subepithelium in chronic asthma (9), and lesional skin in

Address correspondence and reprint requests to Dr. George H. Caughey,CardiovascularResearch Institute, University ofCalifornia, SanFrancisco,CA 94143-091 1.

Receivedfor publication 22 June 90 and in revised form 25 March 91

scleroderma(10)provide evidenceofmastcelldegranulation, suggesting that granuleconstituents mayparticipatein the pro-liferativeprocess.This hypothesishas been examined in vitro in rodent experiments, which demonstrate cell proliferation aftermastcelldegranulation (1 1).Activation ofmastcells

re-sults inextracellularrelease ofhistamine,the serineproteinases tryptaseandchymase,and other mediators(12-15). Although histaminehas somegrowth-promoting potential (16),its activ-ity accounts onlypartiallyfor themitogenic potentialofmast

cell granule contents(17, 18),

implying

theexistence of addi-tionalmastcell-derivedgrowthfactors.

Extensive work has established mitogenic activity for thrombinand otherproteinasesinvariouscell typesincluding fibroblasts (19-21).We haveexploredthepossiblerole formast

cell proteinases in fibroblast

proliferation

by examining the mitogenic activity ofpurified dogtryptaseandchymase in cul-turedfibroblasts.

Methods

Materials. Benzoyl-val-gly-arg-p-nitroanilide (VGR-pNA),' D-Phe-L-pipecolyl-Arg-p-NA, succinyl-phe-pro-phe-p-nitroanilide (FPF-pNA), amiloride, bovine heparin, leupeptin, diisopropylfluorophosphate (DFP), epidermal growth factor (EGF), bovine insulin, and human a-thrombin (3,700 NIH U/ml) were obtained from Sigma Chemical Co.(St.Louis, MO). Basic fibroblast growth factor (bFGF)waskindly

provided by Dr. D. Gospodarowicz (University ofCalifornia, San Fran-cisco). Pertussis toxinwasfrom ListBiologicalLaboratories (Camp-bell,CA).Fura-2-acetoxymethylester(AM)wasobtainedfrom Molecu-larProbes, Inc. (Eugene,OR).(Methyl-3H)-thymidinedeoxyriboseand

['4C]benzoicacidwerefrom ICN Biomedicals(Irvine,CA), and

myo-(2-3H)-inositol was obtained from Amersham Corp. (Arlington Heights, IL). Mast cell tryptase (22) and chymase (23)werepurified

from dog mastocytoma cells.

Cell culture. Chinese hamster lung (CHL) fibroblasts (clone CCL39),anestablisheddiploid cell line,wereobtainedfrom the Ameri-canType Culture Collection(Rockville,MD).Rat-l fibroblastswere obtained from the laboratory of Dr. H. Bourne(Universityof Califor-nia, SanFrancisco). All cells were grown in DMEM containing 4.5 g/liter glucose,andsupplemented with 10% FCS, 50 U/ml penicillin, 50 gg/ml streptomycin, and 25 mM Na+ bicarbonate, at 37°Cin 5%CO2.

Mastcell proteinasecatalyticassays. Dog mast celltryptase and chymase wereassayed using the specific chromogenic peptide sub-stratesVGR-pNAand FPF-pNA,respectively,aspreviouslydescribed (22, 23). Briefly, purifiedenzyme solutionswereincubated with the respective p-nitroanilide substratesat37°C, withmeasurementof ab-sorbanceat394nmperformed usingan845 IA Diode Array

Spectro-photometer (Hewlett-Packard, Palo Alto, CA). Established specific ac-tivities for theseproteinaseswereusedtodetermine molar concentra-tions(22, 23), usingthetetrameric molecular weight (140,000 D) for tryptaseandmonomeric molecularweight for chymase.

1.Abbreviations used in this paper: bFGF, basic fibroblast growth fac-tor;CHL, Chinese hamster lung; DFP, diisoprophylfluorophosphate; EGF, epidermal growth factor; fura-2-AM, fura-2-acetoxymethylester; FPF-pNA,succinyl-phe-pro-phe-p-nitroanilide; VGR-pNA, benzoyl-val-gly-arg-pNA.

J.Clin. invest.

0021-9738/91/08/0493/07 $2.00

(3)

Measurement of DNA synthesis. CHL cells were grown to con-fluence in 24-well plates and rendered quiescent by 24 h incubation in serum-free DMEM. After serum deprivation, cells were incubated for 24 hwithagonists in DMEM/Ham's F-12 medium (1:1) in the pres-ence of[3H]thymidine(0.5 uCi/ml). Growth factors were added simul-taneously in experiments where multiple mediators were applied. Puri-fied tryptase or chymase, when added, were applied to cells in 270 mM

NaCl,10 mM Bis-Tris (pH 6.1) buffer. When present, bovine heparin was added in a 2.5:1 weight excess over tryptase. Controls included buffer andheparinalone.After the 24-h incubation period with [3H]-thymidine,trichloroaceticacid-precipitablematerial was solubilized in 0.3 NNaOH, and the incorporated radioactivity was assessed by liquid

scintillationcounting. Thymidine incorporation assays for Rat-l cells followed the same protocol as above, with the exception that

[3H]-thymidine (0.55Ci/ml) was added to the culture medium for the final 6 hof the total 21 h of incubation with mitogens.

Pertussis toxin sensitivity assays. Assay of pertussis toxin-induced inhibition of DNA synthesis was performed as previously described (24). Briefly, pertussis toxin was added to quiescent CHL fibroblasts, at theconcentrations indicated in Results, 4 h before stimulation and was present throughout the 24-h incubation with growth factors.

[3H]-Thymidineincorporation was assessed as described above.

Cellproliferationassays. Stimulation of cells at low density and at highdensitywas used to establish the characteristics of cell prolifera-tion in response to tryptase.Inthe lowdensity experiments,CHL cells were plated at 2,500cells/cm2inmulti-well platescontainingDMEM with 10% FCS. After 24 h, cells were washed three times with serum-freeDMEMandcovered with DMEM/Ham'sF-12medium (1:1), sup-plemented with transferrin (5 tg/ml)andinsulin(10ug/ml). Growth

factors,aslisted inResults,wereaddedtocells inserum-freemedium atthe time of mediumchange, and every second daysubsequentlywith

accompanyingmedium change. Ondaysof mediumchange,cellswere

suspendedwithtrypsinandcountedusingahemocytometer; duplicate

wells were counted for eachcondition.

Inthehigh density experiments, CHL cells were grownto con-fluence in 10% FCS thendeprivedofserumfor 24 h in DMEM.

Quies-cent cellswerethen stimulated withtryptase(2 nM), thrombin(10

nM),or 10%FCS. Aftera36-h incubation withgrowth factors, cell

densitywasdeterminedby countingasabove.

Studies with tryptase inhibitors. Toexaminethe roleof tryptase

catalytic activityin the observed mitogenicresponses, theeffectsof activesite inhibition byleupeptinand DFPwereexamined.

(a) Leupeptin studies. Thereversible, arginal-classinhibitor

leu-peptinwasaddedtoquiescentCHL cellsconcomitantwith tryptaseor othergrowth factors,asindicatedbelow;thefinalleupeptinandgrowth

factor concentrationswere asgivenin Results. Inhibition ofmitogenic activitywasassessedby

[3H]thymidine

incorporation.

(b) DFPstudies. Tryptase(3 ,uM)andthrombin(1MM)were incu-batedindependentlywith DFP for 21 hat4°C beforeexposuretocells. Molar ratiosofDFP toenzymerangedfrom 3X10'to104for tryptase, andfrom10-2to104for thrombin. The 21-hpreincubationswere neces-sarytoallowhydrolysisofexcessunincorporated DFP,which is cyto-toxic. All incubationswere

performed.

in 270 mMNaCl,10mMHepes

(pH 6.1) buffer. Control DFPsolutions,inconcentrations correspond-ingtothose used in theproteinase coincubations,alsowereincubated at4°Cfor 21 h.CompleteDFPhydrolysisafter 21 h of incubationwas

confirmedbytheinabilityof the incubation solutionstoinhibit the

amidolytic activityof fresh tryptaseorthrombin.Amidolytic activity

wasassessed with chromogenic substratesasdescribed below.Aliquots ofproteinasesin the absence ofDFP wereincubated underidentical conditions toassurepreservation ofproteinase activity duringthese incubations.Aliquotsof theproteinase preparationsabovewereadded toquiescentCHL cellstofinalconcentrationsof 7.5 nM(tryptase)or

10 nM (thrombin).

[3H]Thymidine

incorporation wasmeasured as described above.Catalytic activityof tryptase andthrombinwas

mea-suredimmediatelyafter the 21 h incubationat4°C. Tryptasewas

as-sayed using VGR-pNA (22),andthrombincatalytic activitywas

as-sessedusingthe methodofWittingetal.(25),with

D-Phe-L-pipecolyl-Arg-p-NA(200

AM).

Determinationofintracellular pHchange.Intracellular pHchange

wasdetermined from theequilibriumdistribution of

['4C]benzoic

acid asdescribed(26).Briefly,quiescentCHL fibroblasts in 12-wellplates

wereequilibratedfor 1 h in bicarbonate-freeHepes-bufferedDMEM (pH7.4),at37-C.

['4C]Benzoic

acid(1

,Ci/ml)

waspresent for the final 30 min ofequilibration.Cellswerethen stimulated for 10 min with tryptaseorthrombinatconcentrations of 7.5 and 1nM,respectively.

When present, amiloride(1mM)wasadded 5 minbeforeaddition of

growthfactors. Stimulation of cellswasstopped by aspirationof exter-nalmedium andrapid washingfour times with ice-cold unlabeled

me-dium. Retained

[14C]benzoic

acidwasmeasuredbyliquidscintillation

counting. Changes in intracellularpH werecalculatedaspreviously described(27).

Measurement ofintracellular calcium. Free cytosolic

Ca"+

was

measured in CHL cellsusingthe fluorescent indicator fura-2. CHL cells growntoconfluenceonglasscoverslipswereloaded with fura-2-AM(1

MM)

in Hank'sbuffer with 20mMHepes (pH 7.4),for 15 minat

370C.

Cellswerethen washed twice withdye-freebuffer and mounted inacuvettewith continuousstirring. Tryptase (7.5 nM)and thrombin

(1 nM)wereaddedtothecuvettewhilerecording.Fluorescencewas

recorded inafluorimeter(SLM8000; SLM Instruments, Inc., Urbana,

IL), changingexcitation wavelengths every second between 340 and 380nm. Emittedlightabove495nmwasrecorded. Backgroundwas

determined afterpermeabilizingthe cells with mellitin(10

Mg/ml)

and addition of 1 mMMnCl2.Theratiooflightintensities upon excitation at340 and380nmwasusedtocalculatefreecytosolic Ca"

concentra-tion,assumingaKdof 224nMfor theCa++-fura-2complex (28).

Measurementof inositol phosphates. Formation of inositol phos-phateswasassayedaspreviouslydescribed(29).Confluent CHL fibro-blasts in 12-wellplateswereincubated for 24 h in serum-free DMEM

containing[3H]inositol(2

gCi/ml).

Afterequilibrationin

Hepes-buf-fered DMEM(pH 7.4)for 30 min, 10mMLiClwasaddedtoincrease assay sensitivity through inhibition ofinositol-l-phosphatase (30). LiCl-treatedcellswereexposedtotryptase(7.5 nM)orthrombin(1 nM)for10min. Cellswerethenextracted with 10 mM formicacid,and totalradiolabeled inositolphosphateswereassessedby anion-exchange chromatographyandliquidscintillationcounting (29).

Results

Tryptase inducedthe

incorporation

of

[3H]thymidine

in

con-fluent

quiescent

CHLcells upto22-foldabovecontrollevels. This

corresponded

to65% of theresponseto 10% FCS

(Fig.

1

a). Tryptase

also initiatedDNA

synthesis

in confluent

serum-deprived

Rat-l fibroblasts. The

thymidine incorporation

re-sponsetotryptaseobserved inRat-I wasgreater than that ob-servedin CHL

cells,

with theresponseofRat- I cellsto15 nM tryptase

equaling

theresponseto 10% FCS

(Fig.

1

b).

The half-maximalresponse tryptase

concentration

was- _{4 nM}_{for both}

cell lines. In contrast to tryptase, the

thymidine uptake

ob-served inRat- 1 cells inresponseto

thrombin,

trypsin,

or

chy-mase (a

chymotrypsin-like

mast cell serine

proteinase)

was

minimal

(Fig.

1

b). Higher

concentrations of

trypsin

or

chy-maseresulted in cell

rounding

anddetachment from the

plates,

whereas lower concentrations ofthese

proteinases produced

thymidine

uptake

that

only

approached

control levels

_(Fig.

1

b).

Thus,

tryptase-induced

DNA

_synthesis

appeared

to be a

specific

response.Similar effects for

chymase

and

_trypsin

were

observed in

quiescent

CHL cells

(data

not

shown).

These

re-sults suggest that

proteolysis

perseisnotsufficienttoinduce DNA

synthesis

in the fibroblast cell lines

investigated.

Toconfirm thatcell

growth

accompanies

DNA

synthesis

aftertryptase

stimulation,

cell numbersweredetermined

after

CHL cellexposuretotryptase undertwodifferent cell

_density

conditions.

First,

CHL cells wereseeded in 10% FCS atlow

(4)

S

co

cn

Co CO)

0

ae4C 21

E 0

aI 100

Co

0QLT 80

Un

o 60

0-, 40

CL2

.S 20

E

>1

E In

[tryptase],nM

(b)

I. _afl

control chymase trypsin thrombin tryptase

Figure1. Concentra-tion-dependent reinitia-tionof DNAsynthesis

infibroblastsby dog tryptase. (a)Quiescent, confluentCHL fibro-blasts were incubated

for24 hwithpurified dog tryptase, from 0.25 to22.5nM,in the

pres-enceof[3H]thymidine. (b) Serum-deprived

confluent Rat-I fibro-blasts were incubated for 18 hwith chymase (3 nM), trypsin(100

nM), tryptase (15 nM), andthrombin (10 nM). Results ofincorporation of[3Hjthymidineare normalized to the

re-sponseto 10% FCS. Mean±SEof5to20 in-dependent determina-tions are shown.

maintainedinthepresenceof insulinandtransferrinalone did notproliferate (Fig. 2). In contrast, cell numberincreased 8-fold after 6 d in thepresenceof2 nM tryptase, witha subse-quentplateau in cell

density (Fig.

2). Inthepresence of1 nM thrombin, CHL cell number increased 20-fold in thesame pe-riod(Fig. 2). CHL cells didnotreach

confluence

underthese

conditions,

but

confluence

wasachieved by 6 d with incuba-tion in10%FCSor 10 nMthrombin. Higher concentrations of tryptasedidnotincrease growthrate orfinalcelldensity.The mechanism foracessation of tryptase-induced cell growthat subconfluence remains unknown.

To examine induction ofcell proliferation by tryptaseat high cell density, confluent serum-deprived CHL cells were stimulated with7.5 nM tryptase,10nMthrombinor10% FCS. Cellcountsassessed 36hafter stimulation demonstrated that tryptase was

equivalent

tothrombinor10% FCS in

promoting

growth (Table I).Ineachcondition cell number increasedover controls by - 60%. Similar results were obtained for

con-fluent,

serum-deprived Rat- 1 cells, where cell number in-creased by 39.7 and 42.4% aftertryptase and FCSexposure,

50000 Figure 2. Effect of

tryp-/0M

tase on cellproliferation

in low density CHL

fi-CQ

300001

/broblasts.

CHL cells

_ _/ _were_seeded_at_2,500

8

₂₀₀₀₀

_cells/cm2

_{in medium}

containing 10% FCS.

10000- After24h,cellswere

F, P washedtl and covered

0 2 4 6 with

serum-free

me-incubationtime (days) diumsupplemented with transferrin and in-sulin. Fresh medium supplemented with transferrin and insulin was addedtocells alone (opentriangles),orwith 2 nM tryptase(closed squares)or I nMthrombin (opensquares),everysecond day. Cells were countedin a Neubauer chamber, triplicate wells were counted for each condition. Data represent mean±SD.

Table I. CellProliferation inConfluentFibroblasts after Mitogen Exposure

Cells(Xl0'/cm2)*

Stimulus CHL Rat-I

Control 2.43±0.15 4.03±0.38

7.5 nM Tryptase 3.80±0.31* 5.63±0.18*

10 nM Thrombin 3.90±0.45* 3.81±0.25

10%FCS 3.98±0.40* 5.74±0.29*

Cell counts 36 h after exposure tomitogens.

*_Mean±SEM_{for triplicate determinations.} *_A_{significant difference}

from control(P<0.05) byStudent t test.

respectively(TableI). Thrombin did not significantly increase Rat-I cell number overbaseline,in agreement with the failure ofthrombintostimulatethymidine incorporationinthese cells (Fig. 1 b).These resultsindicatethat tryptase notonly stimu-lates S-phase entry in fibroblasts, but also promotescompletion ofthe cellcycle, resultingin cell division.

To examine the interaction between tryptase and other growth factors, CHL fibroblasts wereincubatedwith tryptase in combination with EGF, bFGF, insulin, orthrombin. Thresh-old response concentrations of tryptase (0.75 nM) in combina-tion with eitherinsulin, EGF,orbFGF elicited increasing syn-ergisticresponses(Fig. 3,a-c). Synergy was produced without

anapparent shift in theconcentrationproducingthe half-max-imal response for all three growth factors. In contrast, the [3H]-thymidine incorporationresponse toconcomitant low

concen-tration tryptaseplusthrombin was additive rather than syner-gistic (Fig. 3 d).

Tryptaseisfound in mast cell preparationsnoncovalently linked to, and stabilized by, heparin, a constituent of mast cell secretory granules (31). Heparin is known to modulate the ef-fects of a number of mitogens, including members ofthe hepa-rin-bindinggrowth factor family, and may also have indepen-dent growth regulatory function (32). The

[3H]thymidine

up-take response of CHL cells to 7.5 nM tryptase was not altered by the addition of heparin when added to tryptase solutions in aweight ratio to tryptase of 2.5:1. Heparin alone at an equiva-lentconcentration hadnoeffectonthymidine incorporation (datanotshown).

Thepossibilitythatproteinasesexerttheirmitogeniceffects through a mechanism that is dependent upon their catalytic activitycontinues to be an issue of great interest(21,33). To determine whether preserved catalytic activity of tryptase is required for the mitogenic effect, we studied the effect of pro-teinasecatalyticinhibition on DNAsynthesis usingleupeptin andDFP,twoestablishedtryptase inhibitors(22).

(5)

40-(a)

30-

20-Xo

-10- W6

0 r-_I"

0 0.01 0.1 1.0 10 100

[insulin],pg/mi

40-(b)

30-20

10 * I

I

0 - . .I#

0 0.1 1.0 10 100

[EGF],ng/ml

inn.

80

60-40'

20'

(C)

0 1 3 10 30 100

[FGFJ, ng/ml

0.001 0.01 0.1

[thrombin],nM

J Figure4.Effect of

leu-100 I peptinonthe CHL

fi-W 80]~ZzJJ.broblastthymidine

up-80 \ _{take response to tryptase}

3_ 60 -₆₀

oT'(7.5

nM;closed

sym-bols)and EGF (100 ng/

E

c 40-

ml;

_open

symbols).

Cells

2 were incubated for 24 h

in the presence or

ab-o0

-- sence of the

leupeptin

0 0.1 10 1000o concentrations shown,

[leupeptin],

_jiM

with tryptase or EGF.

[3H]Thymidine

incor-poration results were normalizedtoresultsobtained in the absence ofleupeptin.Data represent mean±SD fromtriplicatedeterminations.

Figure 3.Reinitiation of

DNAsynthesis in CHL

fibroblastsby tryptase in combination with insu-lin, EGF, bFGF, and thrombin. Results rep-resent 24-h

[3H]-thymidineuptake re-sponsesfor insulin(a),

EGF (b), bFGF (c), and thrombin (d), alone (opensymbols), andin

combinationwith 0.75 nMpurifiedtryptase

(closed symbols). Ra-diolabelincorporation

dataarenormalizedto the responseto10%

fe-talbovineserum.Data aremean±SD froma

representative

experi-ments done in duplicate.

reversible inhibitor and couldpotentially affect other cellular proteins while in solution during the24-hstimulationperiod. Asanalternative toleupeptin, weusedDFP,an irrevers-ible, covalent serine proteinase inhibitor, whichcancompletely

inhibit thecatalytic activity oftryptase(22). When freein solu-tion, this inhibitor is cytotoxic. Taking advantage of the insta-bilityof free DFP inaqueoussolution,weincubated protein-ases(bothtryptase andthrombin) withDFP, and continued

theincubations for 21 hat4°Cto allow for thehydrolysisof

unincorporated DFP. Complete hydrolysis ofDFP was

con-firmedbyalack of inhibition ofproteinase amidolytic activity

by the incubatedDFPsolutions; amidolytic activity oftryptase

and thrombininthe absence ofDFPdidnotdiminishduring incubationat4°C (datanotshown).

While thecatalytic activity oftryptasewasinhibited 98.7%

by DFP,thereinitiation ofDNAsynthesiswasreducedby only

60.9%(Table II). For thrombin, catalytic activitywasinhibited

greaterthan 99.2%, while the mitogenicresponseof CHL cells

toDFP-thrombin wasinhibitedby 85.2%,in agreement with

published data (34) (Table II). These data, togetherwith the

leupeptin inhibition results,suggest thatexpressionof the full

mitogenic activity oftryptase isdependent uponan

uninhib-ited catalytic site,although acatalyticsite-independent compo-nent mayexist.Itremainsto be proven that aproteolyticevent is a necessary component of tryptase-induced mitogenesis.

Notwithstandingthefinding that tryptaseisamitogenfor Rat- I cellswhile thrombinisnot,thesimilarities in the struc-ture andcatalytic activity of these two mitogenssuggest the possibility ofasharedmechanismof cell activation. To investi-gate this possibility, we compared the participation ofboth mitogens in known signal transduction pathways and re-sponses.

Activationoftheamiloride-sensitive

Na+/H'

antiporter isa virtually universalresponseof fibroblaststomitogens, includ-ing thrombin, EGF, bFGF,andplatelet-derived growth factor (27, 35, 36).Measuredinbicarbonate-free conditions, stimula-tion of

Na+/H'

exchange results in a transient intracellular alkalinization. Tryptase, likethrombin, increased intracellular pHtransiently inCHLfibroblasts,aneffect thatwasabolished by amiloridepretreatmentofthecells

(Fig.

5).

Significant

alka-linization was evident by 5 min, wasmaximal 10 min after addition oftryptase,anddecreased overthenext 20min(data notshown).

Important earlycellularresponses togrowth factors include stimulation ofphospholipase C (resultingininositolphosphate

formation),

mobilization of

cytosolic

Ca",

and activation of

GTP-binding proteins

(35, 37,

38).

These responsesareknown to occurafter fibroblastexposure tothrombin.

Therefore,

we comparedtheeffects oftryptaseandthrombinonformation of inositol phosphates,onchangesincytosolic Ca++ concentra-tion, and onreinitiation ofDNAsynthesisinthe presence of pertussis toxin. The concentrations of tryptase and thrombin

Table II.

Diisopropylfluorophosphate

Inhibitionof Tryptaseand

Thrombin:DNASynthesisandCatalyticActivity Responses

Tryptase Thrombin$ Inhibition ofDNAsynthesis 60.9±2.7% 85.2±0.9% Inhibitionofcatalytic activity11 98.7% 99.2%

*_{Tryptase incubated at 3}

MM;

_molar_{ratio of}_DFP_to_tryptase_equaled

300:1. t Thrombin incubatedat 1,uM;molarratioof DFPto throm-binequaled 100:1.I_{Assayed by}_radiolabeled_{thymidine uptake (see}

Methods); compared with enzyme incubated 21 hat4°C,in the ab-senceof DFP; mean±SE fortriplicatedeterminations. 11 Assayed by cleavageofchromogenicsubstrate(see Methods);comparedwith enzyme incubated 21hat4°C,in the absenceof DFP.

s

n

a,

CI,

C.)

.c :2

0

a,

CD 80

60

40

20

(d)

K-

_i

(6)

0.3

0.2

0. _0.1

0.0

-0.1 - control tryptase thrombin

Figure 5. Effect of

tryp-taseandthrombin on intracellular pH. The figure shows the results of10-min incubation of CHL cells with tryp-tase (7.5 nM) or throm-bin (I nM), in the pres-ence(closed bars) or

ab-sence(openbars) of I mMamiloride pretreat-ment. Measurement of intracellular['4C]benzoicacid and calculation of changes in intracel-lular pH wereperformed as described in Methods. Data are mean±SD fortriplicatedeterminations.

usedin the following experimentswerechosen becausethey result insimilar

[3H]thymidine

uptakeresponses(seeFigs. 1 a and 3d).

The total inositol phosphates generated afterexposureof [3H]inositol-loaded CHL cellsto proteinases wasassessed by anion-exchange chromatography,aspreviously described (29). Noincrease ininositol phosphate formationwasdetectedafter tryptase

stimulation,

consistentwithalackof

phospholipase

C activation(Fig. 6 a).In contrast, thrombinproducedamarked increase in inositol phosphates,aspreviously reported (39).

Animportant cellularresponse to

phospholipase

C activa-tion is the mobilizaactiva-tion of intracellular

Ca",

mediated through

inositol-1,4,5-trisphosphate (37).

Alternatively,

cyto-solic Ca++ concentrationcanbe elevatedthrough influx of ex-tracellularCa++(36, 40).Todetermine whethertryptase alters Ca++ homeostasis through a

phospholipase

C-independent

pathway, weexamined the effect oftryptase on intracellular Ca++concentration,using the fluorescent

Ca++-indicator

fura-2 (28). No increase in

cytosolic

Ca++ concentration was ob-servedafter addition of7.5 nM tryptase toCHL cells

(Fig.

6

b);

2000 _(a)(

1500

1000

LiCo tryptase thrombin

(b)

tryptase thrombin

Figure 6. Effect of tryp-tase oninositol phos-phategeneration (a)and cytoplasmic Ca++ con-centration (b)in CHL cells.(a)Inositol

phos-phatesgeneratedafter

1O

minstimulationwith tryptase(7.5nM)or thrombin(1 nM)in the presenceof 10 mM

LiCl.Results represent mean cpm/well(±SD)

forthree determina-tions. (b)Freecytosolic Ca+ concentration measured inCHL cells

usingthefluorescent

indicator fura-2. Tryp-tase(7.5 nM) and thrombin

(1

nM) were addedsequentially

dur-ingcontinuous

record-ing. Thetracingshown is representative of three

independent

measure-ments.

however, a prominent

Ca"+

mobilization response was ob-served with 1 nMthrombin, as previously reported (41). These resultsindicate that cytosolic

Ca"+

is not used as a second mes-sengerinsignal transduction by tryptase.

Pertussis toxin-sensitive GTP-binding proteins have been shownto mediate themitogenicsignalof certain growth fac-tors,including thrombin (24, 38). Tryptase-induced stimula-tion of DNA synthesis in CHL fibroblastswas unaffectedby pertussis toxin over a concentration range of 1-10 ng/ml, as

was the response to EGF (Fig. 7), a mitogen thought to act through a pertussis toxin-insensitive pathway in fibroblasts (42). Pertussis toxin exposure produced significant inhibition of the thrombin-mediated mitogenic response (Fig. 7), in agreement withChambardetal.

(24).

Discussion

These data establish tryptase as a potent mitogen for fibroblasts invitro. Tryptase induces the synthesis of DNA in fibroblasts with concomitant cell proliferation. Prompt activation offibro-blasts occurs upon tryptase exposure, as evidenced by

Na+/H'

antiporter activation within 5 min oftryptase addition. Even at threshold concentrations, tryptase markedly potentiates the mitogenicresponseoffibroblaststobFGF, EGF,and insulin. Themarkedreduction oftryptase-inducedmitogenesisby pro-teinase inhibitors suggests that an intact, unoccupied catalytic site is

required

toexhibitthefull response. Intheserespects, tryptaseresemblesthrombin, aserineproteinase with similar catalytic features (22, 43). However, although both enzymes stimulate DNAsynthesis and cell proliferation in fibroblasts, and both activate theamiloride-sensitive Na+/H+antiporter, the two enzymes appear to activate different signaling path-ways. Theactivation offibroblasts bytryptaseinvolves neither thebreakdown ofphosphatidylinositol phosphatesnor an in-crease incytosolic

Ca",

both of which occur upon stimulation with thrombin. Furthermore, unlike thrombin,tryptase does not appear toactivate cells througha pathwayinvolving per-tussis toxin-sensitive G-proteins. Althoughtryptaseinducesan eightfold increase in CHL cell number over a 6-daygrowth period, confluence is not achieved. The mechanism for this limit inproliferation isnotknown.

The specific signal transduction mechanisms responsible forthe

mitogenic

effect oftryptaseremain unresolved. How-ever, two lines of evidence reinforce thespecificityof

tryptase-CnD

@Figure7.Pertussis toxin

E;!^5 sensitivityofthymidine

T

J

Tincorporation

in

re-100,

loo-sponse tothrombin(I

nM;

striped bars),

tryp-OL

75-

vxtase

_(7.5_nM;_black

bars),and EGF(100 ng/ml;graybars)in CHLfibroblasts.

Per-tussistoxin wasadded

E

&_Wto

R _

cells,atthe

concen-o-_ _trationsg | _

_s

_indicated,_{4 h}

0 1 10 before stimulation and

[pertussistoxin],ng/ml was present throughout

the 24-hincubation with growth factors.[3H]Thymidineincorporationresultswere nor-malized to 100% for each agonist in absence ofpertusistoxin and represent mean±SD fortriplicatedeterminations.

E

3L

400-300

._ C)

E~

iS

8

200-0

100

0 100 200 30(

(7)

induced mitogenesis. First, two additional serine proteinases, trypsinand mast cellchymase,werealsotestedfor their ability to induce DNA synthesis in fibroblasts. Trypsin has known mitogenic capacity in fibroblasts, particularly chick embryo fibroblasts, which appearexquisitely responsiveto a numberof proteinases (21, 44, 45). Chymase, achymotrypsin-like mast cell granule proteinase, has been implicated in extracellular matrix degradation andarchitectural alteration in fibroblasts aftermastcelldegranulation(46). InRat-l cells(Fig. 1 b)and CHL fibroblasts(datanotshown), neithertrypsinnorchymase increased

[3H]thymidine

uptake, whereas tryptase produced strikingresponses atconcentrations similarto, orlower than, those oftrypsinandchymase.Second,amarkeddifferencewas observedinthe[3H]thymidine incorporationresponsesof Rat-1 cells to tryptase and thrombin. While both tryptase and thrombin stimulatedDNAsynthesis in CHL cells, only tryp-taseproducedaresponseinRat- 1 cells.

Themechanismsunderlying mitogenic activity of protein-ases, such as

thrombin,

remain unclear despite considerable workin thisfield.Theparticipation of proteolyticeventsin the activation of cells by proteinases has been proposed, though notconclusively established (21).As reportedherein, inhibitors of catalytic

activity

(leupeptin and DFP)producesubstantial reductions in themitogenicresponseoffibroblaststo tryptase (Fig.4,and Table II).

Theobservationthatleupeptinand DFPdiminishthe mi-togenic

activity

oftryptaseisconsistent with anumberof hy-potheses. One isthat aspecificproteolyticeventisrequired for theinitiation of

signal

transduction leadingto DNA

synthesis

andcell

proliferation.

Asecond is that

binding

tothetryptase

catalytic

site is

required,

but that the

binding

protein (possibly

acellsurface receptor)acts as a

pseudosubstrate,

not

subject

to

cleavage.Inthelattercase,the

proteinase

would inducea criti-calconformational change in the transduction

protein

in the absence ofa

proteolytic

event,thereby

initiating

cellactivation. Athirdpossibility isa

binding

interaction withacellsurface molecule involvingapartoftryptasedistinct fromtheactive site.

Clearly, thesehypothesesneed not bemutually exclusive. Activation bytryptasemay

require

both

catalytic

and noncata-lyticevents.Itis

interesting

that thedegree of

mitogenic

inhibi-tion produced

by

leupeptin

and DFP differs.

Leupeptin,

a

tri-peptide

inhibitor, produces

virtually complete mitogenic

and

catalytic

inhibition,

whereas DFP

produces

a lesser degree of

mitogenic

inhibition of 60%, eventhough

catalytic activity

is inhibitedalmostcompletely. Giventhesizedifference between thesetwo

inhibitors,

thedifferencein

magnitude

of

mitogenic

inhibition couldbeexplainedby thepresenceofa

binding

epi-tope ontryptasethat extendsbeyond the immediate substrate

binding pocket.

This

binding region, although

adjacent

tothe catalytic

site,

may notdependon a

catalytic

eventforcell acti-vation. Evidence exists for thrombin thatacatalytic site-inde-pendent

epitope

mayparticipate in production ofa

mitogenic

signal

insometargetcells(47).In

addition,

a recent report

(33)

suggestsdissociation of

catalytic activity

and

mitogenic

effect for thrombin in smooth muscle cells, although, aswith the tryptaseresults presented here, the study reveals only

partial

inhibition of mitogenic

activity

byproteinase inhibitors. This wouldsupport amixedcatalytic site-dependent and -indepen-dentfunction of proteinasesin

mitogenic

activation. Thelack ofany

significant

effect of

trypsin

orchymaseon DNA synthe-sis in CHLand Rat- Icellssupports theconclusion that

nonspe-cific cell surface proteolytic events are not sufficient to induce S-phase entry in these fibroblasts.

Our in vitro results provide strong support for the hypothe-sis that mast cell activation can lead to fibroblast proliferation invivo, and for thepossibilitythat tryptaseplaysanimportant role as mediator of this response. This hypothesis is also sup-ported byexisting knowledgeconcerningtheactivity, concen-tration, and tissue distribution of this proteinase. Tryptase is released from mast cells as a heparin-associated tetramer of catalytically active subunits, and is unusual among serine pro-teinases inbeing active in serum and highly resistant to inacti-vation bycirculating inhibitors (48, 49). Furthermore, the con-centrations of tryptase sufficient in ourexperimentsto stimu-latefibroblast growth directly or in synergy with other growth factors are predicted to be achievedreadilyinthe microenvir-onment of the degranulatingmast cell (50, 51). Finally, the widedistributionoftryptase-containingmastcellsin humans suggests thattryptase may play a part in thedevelopmentof fibrotic disorders affectingavarietyoftissues,including lung, skin, andgut.

Acknowledgments

The authorsthankDr. K.Seuwen forassistance ininositol phosphate formation assays.

Dr. Ruossis aCystic FibrosisFoundation Fellow. Dr. Hartmann is supported by the DeutscheForschungsgemeinschaft.Dr.Caugheyisa recipient ofNational Institutes of HealthClinicalInvestigatorAward HL-07136 andofanRJR-Nabisco Research Scholar Award in Pulmo-nary.

References

1.Kawanami, O.,V. J.Ferrans, J.D.Fulmer, andR.G.Crystal. 1979.

Ultra-structureofpulmonarymastcells inpatientswithfibroticlungdisorders.Lab.

Invest.40:717-734.

2. Trabucchi, E.,E.Radaelli,M.Marazzi,D.Foschi,M.Musazzi,A.M. Veronesi, and W.Montorsi. 1988. The roleof mast cells in woundhealing. Int. J. Tissue React. 10:367-372.

3. LeRoy, E.C., E. A.Smith,M. B.Kahaleh, M.Trojanowska,and R.M.

Silver. 1989.Astrategy fordeterminingthepathogenesis of systemicsclerosis: is

transforming growthfactor,5the answer?Arthritis Rheum.32:817-825.

4.Claman,H. N. 1989. On scleroderma:mastcells, endothelialcells and

fibroblasts.JAMA(J.Am.Med. Assoc.)262:1206-1209.

5.Watanabe,S., K. Watanabe, T.Ohishi,M.Aiba,andKKageyama. 1974. Mast cells in theratalveolar septaundergoingfibrosis afterionizingradiation. Lab.Invest.31:555-567.

6.Goto, T., D.Befus,R.Low,andJ.Bienenstock.1984. Mast cell heterogene-ityandhyperplasiainbleomycin-induced pulmonaryfibrosis inrats. Am.Rev. Respir. Dis. 130:797-802.

7. Wagner, M. M., R. E. Edwards,C. B.Moncrieff, and J. C. Wagner. 1984.

Mastcells andinhalation of asbestosin rats.Thorax. 39:539-544.

8.Takiazawa, H.,K.Ohta,KHirai,Y.Misaki,T.Horiuchi,N.Kobayashi,J.

Shiga,and T.Miyamoto.1989. Mast cellsareimportantin thedevelopmentof hypersensitivity pneumonitis.Astudy with mast-cell-deficient mice.J.Immunol. 143:1982-1988.

9.Beasley, R., W. R.Roche,J. A.Roberts,and S.T.Holgate. 1989. Cellular

eventsin thebronchi in mild asthma andafter bronchialprovocation.Am. Rev. Respir. Dis. 139:806-817.

10.Claman,H.N., R. C.Giorno,and J. R. Seibold. 1990. Mast cell

degranu-lation in scleroderma(SS). Clin.Res. 38:349A.

11. Norrby, K,L.Enerback,and L. Franzen. 1976. Mast cell activationand

tissueproliferation. CellTissue Res. 170:289-303.

12.Schwartz,L. B. 1987.Mediators ofhumanmastcells and humanmastcell subsets(publishederratumappears in 1987.Ann.Allergy.59:316).Ann.Allergy.

58:226-35.

13. Schwartz, L.B., R.A.Lewis,D.Seldin,and K. F.Austen. 1981.Acid hydrolases and tryptase fromsecretory granulesofdispersedhumanlungmast

cells. J.Immunol. 126:1290-1294.

(8)

G. Dong,R. M.Lavker,D.Proud, and G. S.Lazarus. 1986.Identification of a chymotrypsin-like proteinase in human mast cells. J. Immunol. 137:962-970.

15.Caughey,G. H., S. C. Lazarus, N. F. Viro, W. M. Gold, and J. A. Nadel. 1988. Tryptase and chymase: comparison of extraction and release in two dog mastocytoma lines. Immunology. 63:339-344.

16. Jordana, M., A. D.Befus, M. T. Newhouse, J. Bienenstock, and J. Gaul-die. 1988. Effectsofhistamine on proliferation of normal adult lung fibroblasts. Thorax. 43:552-558.

17. Franzen, L., and K. Norrby. 1980. Local mitogenic effect of tissue mast cellsecretion.Cell Tissue Kinet. 13:635-642.

18. Marks, R. M., W. R. Roche, M. Czerniecki, R. Penny, and D. S. Nelson. 1986. Mast cell granules causeproliferation of human microvascular endothelial cells.Lab.Invest.55:289-294.

19. Chen, L. B., and J. M. Buchanan. 1975.Mitogenic activity of blood components. I. Thrombin andprothrombin. Proc. Natl.Acad.Sci. USA.

72:131-135.

20. Carney, D. H., and D. D.Cunningham. 1978. Cell surface action of thrombin issufficient to initiate division of chick cells. Cell. 14:811-823.

21.Scher, W. 1987.Biology of disease: the role of extracellular proteases in cellproliferation and differentiation. Lab. Invest. 57:607-633.

22. Caughey, G. H., N. F.Viro, J. Ramachandran, S. C. Lazarus, D. B.

Borson, and J.A.Nadel. 1987.Dog mastocytoma tryptase:affinitypurification,

characterization and amino-terminal sequence. Arch. Biochem. Biophys. 258:555-563.

23.Caughey, G. H., N. F.Viro,S. C.Lazarus,and J.A.Nadel. 1988. Purifica-tion andcharacterization ofdog mastocytomachymase: identification ofan

octa-peptideconserved inchymotryptic leukocyte proteinases. Biochim. Biophys.

Acta.952:142-149.

24.Chambard, J.C.,S.Paris,G.L'Allemain,and J.Pouyssegur. 1987. Two growthfactorsignallingpathways in fibroblastsdistinguished by pertussistoxin. Nature(Lond.). 326:800-803.

25.Witting, J. I., C.Pouliott,J. L.Catalfamo,J.Fareed,andJ. W. FentonII. 1988.Thrombin inhibition withdipeptidylargininals.Thromb. Res. 50:461-468. 26. Hartmann, T., K. Seuwen, M.F.Roussel,C. J.Sherr,and J.Pouyssegur.

1990.Functional expressionof the human receptorfor colonystimulatingfactor 1 (CSF- 1) in hamster fibroblasts: CSF-1 stimulatesNa+/H+exchangeand DNA

synthesisin the absence ofphospholipidbreakdown.GrowthFactors.2:289-300. 27.L'Allemain, G.,S.Paris,and J.Pouyssegur. 1984. Growth factor action andintracellular pHregulationinfibroblasts. Evidence foramajorroleof the Na+/H+antiport. J.Biol. Chem.5809-5815.

28.Grynkiewicz,G., M.Poenie,and R. Y.Tsien.1985.A newgenerationof Ca++ indicators withgreatlyimprovedfluorescenceproperties.J. Biol. Chem.

260:3440-3450.

29.Seuwen, K., I.Magnaldo, and J. Pouyssegur. 1988. Serotonin stimulates DNAsynthesisinfibroblastsacting through 5-HTlBreceptorscoupledto a Gi-protein. Nature(Lond.).335:254-256.

30.Hallcher, L. M., and W. R. Sherman. 1980. Theeffects of lithiumand other agentsontheactivity ofmyo-inositol-l-phosphatasefrom bovine brain. J.

Biol. Chem.255:10896-10901.

31.Schwartz, L. B., andT. R.Bradford. 1986.Regulation oftryptase from humanlungmastcellsby heparin. Stabilization oftheactivetetramer.J.Biol. Chem.261:7372-7379.

32. Ruoslahti, E. 1989. Proteoglycans in cellregulation. J. Biol. Chem.

264:13369-13372.

33. Bar-Shavit, R., M. Benezra, A. Eldorf, J. W. Fenton II, G. D. Wilner, and I. Vlodavsky. 1990. Thrombin immobilized to extracellular matrix is a potent mitogenfor vascular smooth muscle cells: nonenzymatic mode of action. Cell

Regulation. 1:453-463.

34.Glenn, K. C., D. H.Carney, J. W. I. Fenton, and D. D. Cunningham. 1980. Thrombin active site regions required for fibroblast receptor binding and initiation of cell division. J. Biol. Chem. 6609-6616.

35. Rozengurt, E. 1986. Early signals in the mitogenic response. Science

(Wash.DC). 234:161-166.

36. Moolenaar, W. H. 1986. Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol. 48:363-376.

37.Berridge, M. J. 1984. Inositol trisphosphate and diacylglycerol as second messengers.Biochem. J. 220:345-360.

38. Murayama, T., and M. Ui. 1987. Possible involvement ofa GTP-binding

protein,the substrateof islet-activating protein, in receptor-mediated signaling responsible for cell proliferation.J. Biol. Chem. 262:12463-12467.

39. Raben, D. M., and D. D. Cunningham. 1985. Effects of EGF and throm-bin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF. J Cell. Physiol. 125:582-590.

40.Pandiella,A., A.Malgaroli, J. Meldolesi, and L. M. Vicentini. 1987. EGF raises cytoslic Ca++ in A431 and Swiss 3T3 cells by a dual mechanism. Exp. Cell Res. 170:175-185.

41.Magnaldo, I., G. L'Allemain, J. C. Chambard, M. Moenner, D. Barritault, and J.Pouyssegur. 1986. Themitogenic signalling pathway of fibroblast growth factor isnotmediatedthroughpolyphosphoinositide hydrolysis and protein ki-nase Cactivation in hamster fibroblasts.J. Biol. Chem. 261:16916-16922.

42. L'Allemain, G., K. Seuwen, T. Velu, and J. Pouyssegur. 1989. Signal transduction in hamsterfibroblasts overexpressing the human EGF receptor. Growth Factors. 1:311-321.

43.Hemker, H. C. 1983. Substrates. In Handbook of Synthetic Substrates for theCoagulation and Fibrinolytic System. Matinus Nijhoff, Boston. 53-93.

44. Burger, M. M. 1970. Proteolytic enzymes initiating cell division and escape from contactinhibitionof growth. Nature(Lond.).227:170-17 1.

45.Zetter, B. R.,L. B.Chen, and J.M.Buchanan.1976.Effects of protease

treatment ongrowth, morphology,adhesion,and cellsurfaceproteinsof second-ary chickembryofibroblasts.Cell.7:407-412.

46.Ginsburg,H.,M.Amira,J.Padawer, and S. Davidson. 1989. Structural alterations in fibroblast monolayers caused by mast cell degranulation. J.

Peri-odontalRes.24:41-4.

47.Bar-Shavit,R.,A. J.Kahn, K. G. Mann, and G. D. Wilner. 1986.

Identifi-cationofathrombin sequence with growth factoractivityonmacrophages. Proc.

Natl. Acad.Sci. USA. 83:976-980.

48.Smith,T.J., M. W. Hougland, and D.A.Johnson. 1984. Humanlung

tryptase.Purificationandcharacterization.J. Biol. Chem. 259:11046-11051.

49.Alter, S. C., J. A. Kramps, A.Janoff, and L. B. Schwartz. 1990.

Interac-tions of human mast cell tryptase withbiological protease inhibitors. Arch.

Bio-chem.Biophys.276:26-31.

50.Schwartz,L.B.,A.-M. A. Irani,K. Roller,M.C.Castells, and N.M.

Schechter.1987.Quantitation of histamine,tryptase, andchymase indispersed

human T and TC mast cells.J. Immunol.138:2611-2615.