Monocyte Procollagenase and Tissue Inhibitor of Metalloproteinases

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY 1987 hy The Americn Society for Biochemistry nd Moleculr Biology, nc. Vol. 262, No. 33, ssue of November 25, pp Printed in U.S.A. Monocyte Procollgense nd Tissue nhibitor of Metlloproteinses DENTFCATON, CHARACTERZATON, AND REGULATON OF SECRETON* (Received for publiction, June 11,1987) Edwrd J. Cmpbell, J. Dvis Cury, Cthy J. Lzrus, nd Howrd G. elgusl From the Respirtory nd Criticl Cre nd Dermtolopy Divisions, Deprtment of Medicine, Jewish Hospitl t shington University Medicl Center, St. Louis, Missouri 6311 Alveolr mcrophges hve been shown to secrete procollgense nd the tissue inhibitor of metlloproteinses (TMP), which re similr or identicl to the corresponding proteins of humn skin fibroblsts. Lit tle is known, however, bout the collgenolytic ctivity of norml humn monocytes. e hve pplied immunologic, biochemicl, nd moleculr biologic tools to exmine the collgenolytic profile of freshly isolted peripherl blood monocytes. Our studies indicte tht: collgenseinhibitory glycoprotein (the ltter lso known s tissue inhibitor of metlloproteinses (TMP)). Results to 1) monocytes re cpble of producing both procollgense nd TMP tht re identicl to the corresponddte hve indicted tht these proteins re immunologiclly ing products of skin fibroblsts, lveolr mcrophges, nd functionlly identicl to nlogous the products of humn nd U937 cells; 2) unstimulted monocytes in vitro skin fibroblsts. Anlyticl tools developed for the fibroblst secrete high levels of TMP, but little or no procoll proteins hve thus been pplicble to the study of collgenogense; 3) n s yet unidentified component(s) of serum lysis by mcrophges nd differentited U937 cells. re required for in vitro production of TMP (but not The proteinse profile of humn mononucler phgocytes procollgense) by monocytes; 4) even when stimu chnges during cellulr mturtion (1, 4, 12, 1416). Studies lted, monocytes secrete much smller quntities of of the U937 (12, 13) nd HL6 (16) continuous cell lines procollgense in comprison with mcrophges; nd hve shown tht the expression of collgense nd TMP is 5) regultion of the secretion of procollgense nd mrkedly ltered following differentition induced by phorbol TMP by monocytes exhibits high degree of individ ester compounds. Although some studies of humn monocyteul vribility, but is nevertheless subject to clerly derived collgense ctivity hve been reported (17, 18), biodifferent control mechnisms thn our previous find chemicl nd functionl chrcteriztion of the collgense ings would indicte for lveolr mcrophges. Mono of norml humn monocytes hs not been chieved, nd the cytes thus express mcrophgelike, rther thn only described collgenseinhibitory product of monocytes neutrophillike, profile of proteins cpble of medit hs been 2mcroglobulin (19). Creful chrcteriztion of ing collgen turnover, the regultion of which is dis the collgenolytic ctivity of monocytes is importnt becuse: tinct from tht of more differentited lveolr mcro 1) studies with the humn cell lines U937 (12) nd HL6 phges. Further study of monocyte nd mcrophge (16) hve suggested tht the cpcity to produce collgense collgenolytic ctivities my provide insights into both nd TMP my differ between immture nd mture monothe cell biology of mononucler phgocyte mturtion nucler phgocytes (the ltter typified by lveolr mcrond the mechnisms by which such cells medite the turnover of interstitil collgens. phges (3)); 2) monocytes nd monocytelike U937 cells hve been shown to contin neutrophillike serine proteinses which differ from enzymes produced by lveolr mcrophges (1, 4, 14, 15, 2); nd 3) neutrophils hve been shown to Mononucler phgocytes re developmentlly nd func possess n interstitil collgense which differs in both its tionlly complex cells tht subserve vriety of immunoreg immunologic nd ctlytic properties from the collgense ultory, phgocytic, nd secretory functions. A growing body secreted by lveolr mcrophges (2124). e now report of dt suggests tht humn mononucler phgocytes cn immunologic, ctlytic, nd moleculr biologic studies of colmodulte nd directly prticipte in the degrdtion nd lgense nd TMP of norml monocytes, long with prelimremodeling of extrcellulr mtrix (17). inry dt concerning the regultion of secretion of these The interstitil collgens (types 1111) re mjor structurl proteins. These results provide insights into chnges which proteins of the extrcellulr mtrix nd provide importnt mononucler phgocytes undergo during mturtion nd lso determinnts of the rchitecture nd functions of vriety of hve implictions for the role of monocytes in collgen turntissues. The triple helicl structure of these mcromolecules over in vivo. renders them resistnt to proteolytic degrdtion except by * This work ws supported in prt by United Sttes Public Helth Service Grnts HL3341, AM3585, nd HL7317 nd by the Council for Tobcco Reserch, U. S. A., nc. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. $ Recipient of Reserch Cncer Development Awrd AM specific collgenses (8). Such proteinses ctlyze both the initil nd the rtelimiting step of interstitil collgen degrdtion (811). Recent studies hve begun to clrify the direct role of humn mononucler phgocytes in collgen degrdtion. Humn lveolr mcrophges (3) nd phorboldifferentited U 937 cells (12, 13) secrete in vitro both collgense nd EXPERMENTAL PROCEDURES MterilsConcnvlin A (ConA), dimethyl sulfoxide, phorbol myristte cette (PMA), nd bcteril lipopolyscchride (LPS) The bbrevitions used re: TMP, tissue inhibitor of metlloproteinses; ConA, concnvlin A; PMA,phorbol myristte cette; LPS, bcteril lipopolyscchride; ELSA, enzymelinked immunosorbent ssys.

2 Monocyte were obtined from Sigm. Tris bse, phenylmethnesulfonyl fluoride, EDTA, soyben trypsin inhibitor, nd bovine pncretic trypsin were lso purchsed from Sigm. Phosphocellulose (P11) nd crboxymethylcellulose (CM52) mtrices were obtined from htmn. Monocytes nd Cell LinesMonocytes were seprted from humn peripherl blood by FicollHypque centrifugtion (251, followed by elutrition centrifugtion of the mononucler cell lyer (26). Mononucler cells, suspended in elutrition buffer (.1% humn serum lbumin,.15 M NC1,2.7 mm KCl, 2.3 mm phosphte, 6 mm glucose, 1 mm EDTA, ph 7.4), were pumped t flow rte of 8 ml/min into the elutritor rotor (JEGB rotor with stndrd chmber mounted in Beckmn 5221 centrifuge) t constnt 247 rpm. Lymphocytes, residul pltelets, nd erythrocytes were eluted from the chmber by incresing the flow rte to 16.5 ml/min. Monocytes were then eluted t flow rte of 22 ml/min. Differentil counting of rightstined cytocentrifuge preprtions (Shndon Southern nstruments nc., Sewickley, PA) reveled >9% monocytes; the reminder of the cells were lymphocytes. Neither polymorphonucler neutrophils nor pltelets were observed in the monocyte preprtions. Elutrited monocytes were wshed once with Hnks' blnced slt solution (The Tissue Culture Support Center, shington University Medicl Center), resuspended in 1:l (v/v) Hm's Fl2/Dulbecco's modified egle's medium, nd then plted in Linbro 6well cluster pltes (Flow Lbortories, nc., McLen, VA). After llowing the cells to dhere for 1 h t 37 "C, the medium ws replced with fresh medium contining the dditions s specified. At intervls, the conditioned medi were spirted, djusted to.5 M Tris, ph 7.5, supplemented to contin.1 M CCls, nd stored t 75 "C for lter nlysis. U937 cells were obtined from Dr. Hillel Koren, (Environmentl Protection Agency, University of North Crolin, Chpel Hill, NC) nd were cultured in RPM 164 medium supplemented with 1% fetl bovine serum nd 2 mm Lglutmine s previously described (4). HL6 cells were obtined from E. Humbermn (Argonne Ntionl Lbortory) nd cultured in scove's modified Dulbecco's medium contining trnsferrin (5 mg/ml) nd Lglutmine (2 mm) s previously described (16). U937 nd HL6 cells were differentited by exposure to lo* M PMA for 24 b to induce the synthesis of procollgense nd TMP (12.16) prior to hrvesting totl RNA. CollgensGuine pig type collgen ws prepred s described by Gross (27). Humn plcent type 111 collgen ws kindly donted by Dr. Robert Burgeson (Shriner's Hospitl, Portlnd, OR). Collgense nd TMPHumn skin fibroblst procollgense ws purified to homogeneity from serumcontining conditioned medi by sequentil mmonium sulfte precipittion, crboxymethylcellulose chromtogrphy, nd Ultrogel AcA34 chromtogrphy s de scribed previously by Stricklin et l. (28). TMP ws purified to homogeneity from the sme humn fibroblstconditioned medi using previously estblished methodology (29). mmunossyscollgense nd TMP were quntified by enzymelinked immunosorbent ssys (ELSAs) (3,31) using polyclonl ntibodies to collgense nd TMP purified from humn skin fibroblst cultures. The ELSAs mesure the totl mount of collgense or TMP present, including both free nd complexed proteins. Furthermore, the collgense ELSA does not recognize the collgense of polymorphonucler neutrophils (3). Double immunodiffusion ws performed in 1.% ongr (32). Prtil Purifiction of Monocyte ProcollgenselKinse Studies of Monocyte Collgense ActiuityTo purify prtilly monocyte collgense for determintion of kinetic constnts, 3 ml of ConAstimulted, 2% serumcontining conditioned medi were dilyzed ginst.1 M Tris, ph 7.5, contining.1 mm CCl2 nd then subjected to ionexchnge chromtogrphy using crboxymethylcellulose (CM52) mtrix. The dilyzed medi were pplied to 1.6 X 2.cm column nd eluted with.5 M NC1. The elute represented highly purified procollgense, since lmost ll serum nd medi proteins, including TMP nd smcroglobulin, were not retined by the mtrix t ph 7.5 (28, 29). The eluted mteril, following dilysis ginst.5 M Tris, ph 7.5, contining.1 M CCls, nd subsequent 1fold concentrtion by membrne centrifugtion (Amicon Centricon1 membrne) ws utilized for both the kinetic mesurements described below nd double immunodiffusion studies. Vlues for K,,, nd Kct were determined on monomeric type nd 11 collgens s described previously (lo), except tht the mount of collgense present in the concentrted, prtilly purified mteril ws mesured by ELSA (3) in order to permit clcultion of Kct. Procollgense ws ctivted by exposure to trypsin t room temperture for 1 min nd then dding 1fold molr excess of soyben Secretion of Procollgense nd TMP trypsin inhibitor. A trypsin titrtion ws performed to ensure mximl ctivtion. To determine K,,, nd kct vlues, vrying mounts (4 2 pg) of ech ntive collgen were diluted in buffer (.5 M Tris,.1 M CCls,.25 M NCl, ph 7.5) nd then incubted with prtilly purified monocyte collgense t 25 "C. The TCA digestion products were quntified by densitometric scnning of stined polycrylmide slb gels (O), nd LineweverBurk plots were then constructed. Prtil Purifiction of Monocyte TMP/Stoichiometry of nhibi tionfor studies of the stoichiometry of TMP ctivity, monocytes were cultured in the presence of 5% fetl bovine serum, nd the conditioned medi were collected fter 48 h. Fifty ml of conditioned medi were pplied to 1.6 X 5.cm phosphocellulose column (P 11) equilibrted in.5 M Tris, ph 7.5, nd eluted with.5 M mmonium sulfte. Under such conditions, smcroglobulin is not retined in the column, wheres both procollgense nd TMP bind nd re then eluted (29). The eluted pek ws dilyzed ginst 2 mm Tris, ph 7.5, contining.1 mm CCl, nd then lyophilized with reconstitution in wter to chieve 5fold concentrtion. This prtilly purified TMP ws employed in double immunodiffusion nlysis. The sme preprtion ws lso used to study TMP stoichiometry becuse ny collgense present ws in the zymogen form, which does not interct with the inhibitor molecule (33). The molr stoichiometry of inhibition ws determined by incubting pure humn skin fibroblst collgense with incresing quntities of either monocyteconditioned medi processed s described bove or pure humn fibroblst TMP. The TMP concentrtion in the prtilly purified mteril ws determined by ELSA (31). Collgen degrdtion ws quntified by solubiliztion t 37 "C of guine pig skin type collgen fibrils biosyntheticlly lbeled with [4C]glycine(34). RNA soltion nd Northern Blot AnlysisTotl cytoplsmic RNA ws isolted by gunidinium isothiocynte extrction ccording to estblished procedures (35). The totl RNA ws then frctionted on 1.2% grose gel contining 2.2 M formldehyde (36), trnsferred to nitrocellulose filters, nd then hybridized to either nicktrnslted humn fibroblst collgense (37) or TMP (38) cdna probe using hybridiztion conditions for ech cdna s re ported previously (37, 39). Approximtely 1 pg of totl RNA were pplied to ech slot of the grose gel. Autordiogrphy ws performed using Kodk XAR5 XOmt AR film developed t 7 "C (4). Regultion of Secretion of Monocyte Procollgense nd TMP To evlute procollgense nd TMP secretion by monocytes in response to externl influences, we quntified procollgense nd TMP in medi conditioned by monocyte cultures, both spontneously nd in response to selected puttive regultory gents. To evlute bsl secretion, conditioned medi from monolyer cultures in the presence of 2% utologous humn serum were collected t intervls up to 15 dys fter the cultures were estblished. The secretory responses to ConA (2 pglml), PMA (1 ng/ml), nd LPS (1 pg/ml) were evluted by ssy of culture medi conditioned for 48 h by newly isolted monocytes. Dimethyl sulfoxide ws used s solvent for PMA, nd low concentrtions (O.OOl%, v/v) were present in PMAtreted cultures. Prllel control cultures were thus exposed to.1% dimethyl sulfoxide s solvent control. RESULTS Monocyte Culture Conditionsn preliminry studies, we were unble to mintin monolyer cultures of monocytes for longer thn 4896 h in the bsence of humn serum; 2% utologous serum ppered optiml for longterm monocyte culture, s described by others (1). Since humn serum contins low concentrtions of TMP (31, 41), studies involving double immunodiffusion nlysis nd ssessment of the functionl ctivity of monocyte TMP were performed using medi supplemented with 5% fetl bovine serum nd were mintined for no longer thn 48 h. All other monolyers were mintined in the presence of 2% utologous humn serum. here required, the quntity of TMP secreted by monocytes ws corrected for the content of TMP in the dded humn serum, s determined by ELSA ( Fg/ml of serumcontining culture medium). Procollgense nd TMP Secretion by MonocytesELSAs for fibroblst/mcrophge collgense nd TMP were performed on medi conditioned by monolyer cultures of monocytes in the presence of 2% utologous humn serum nd

3 15864 Monocyte Secretion of Procollgense nd TMP hrvested fter 48h. These medi contined immunorective secreted products which were detected by both ssys (dt not shown). The reminderof the studies to be described were performed to chrcterize further these products both structurlly nd functionlly. Double mmunodiffusion AnlysisFig. 1 shows Ouchterlony ssys compring monocytederived procollgense nd TMP with the respective humn skin fibroblst products using polyclonl ntibodies rised to the purified fibroblst proteins. Precipitin lines of complete identity were observed, indicting tht monocyteconditioned medi contined proteins immunologiclly identicl to fibroblst procollgense (left) nd TMP (right). Ctlytic Actiuity of Monocyte CollgenseConcentrted, prtilly purified monocyteconditioned medi, prepred s described under "Experimentl Procedures," were essentilly devoid of collgense ctivity unless ctivted with trypsin (not shown). These dt suggest the secretionof collgense zymogen by monocytes, consistent with the mnner of its relese from other cell types (3, 28). Monocyte collgense cleved monomeric type nd 11 collgens in mnner Nm l/[substrote.pm] chrcteristic of mmmlin collgenses; t 25 "C, only FG.2. LineweverBurk plots of humn monocyte collsingle 3%4:1%4clevge ws observed, producing typicl %length gense ctivity ginst monomeric collgen types nd 111. TCA nd %length TC" frgments (not shown).fig. 2 shows LineweverBurk double reciprocl plots were generted s detiled fully under "Experimentl Procedures." Vlues for k,were obtined LineweverBurkdoublereciprocl plots of thectlytic clevge of monomeric collgen types nd 11 by trypsin using n ELSA (3) to quntify collgense concentrtion. Note the ctivted monocyte collgense. Similr vlues for the K, of linerity of the plots nd the mrkeddifferences between type nd 111 clevge. Comprison of these kinetic constnts with the correthe two substrtes were obtined, but the ctlytic rte for sponding vlues for humn mcrophge nd skin fibroblst collgentype 11 collgen exceeded tht ginst type collgen by ses ppers in Tble. pproximtely 16fold. heres pure monocyte collgense could not be obtined due to very the smll mountsof enzyme TABLE secreted, it ws possible to determine vlues for kctby quncomprison of collgen type specificity of humn monocyte, tifying the mountof enzyme present in the prtilly purified mcmvhpe. nd fibroblust colleenses mteril by ELSA. However, the kinetic prmetersfor type Collgen Enzyme source K, L 1 collgen could not be ccurtely mesured becuse of the type very slow turnover of this substrte nd the low concentrpm h" tions of enzyme vilble. This mrked type 11 collgen Monocyte" selectivity ws lso observed using concentrted, serumfree, 1 ND' ND ConAstimultedconditioned medi not subjected tony chromtogrphic purifiction (dt not shown). Furthermore, Mcrophge' collgense ctivity ws completely bolishedby 1,lOphenn throline or EDTA, wheres phenylmethnesulfonyl fluoride nd soyben trypsininhibitor were without effect (not shown). Skin fibroblstd Tble shows comprtive vlues of K, nd k., for mono1 1.o cyte, lveolr mcrophge, nd skin fibroblst collgenses. All show similr K, vlues of pproximtely 12 p~ for the 'Monocyte dt were derived s described for Fig. 2. nhibitor 'ND, not done. Ctlytic ctivity of vilble mounts of enzyme were too low to quntify ccurtely. Mcrophge dt for collgen types 1 nd 111 re from elgus et l. (3) nd represent reltive rtes of clevge, with the rte ginst type 1 collgen rbitrrily defined s 1..Vlues for guine pig type collgen hve not been previously published. Fibroblst dt refrom elgus et l. (1). vrious collgen types tested. Moreover, ll disply similr specificity for the ctlytic clevge of susceptible monomeric collgens; i.e. type 11 >>type >> type 11. By contrst, humn neutrophil collgense degrdes monomeric type collgen 15fold more rpidly thn type 11 (23, 24). Molr Stoichiometry of nhibition by Monocyte TMPAs FG.1. Double immunodiffusion nlysis of procollgense shown in Fig. 3, TMP fromprtilly purifiedmonocytend TMP secreted by monocytes. The centerwelb contined conditioned medi (see "Experimentl Procedures"), quntirbbit polyclonl ntibody ginst humn skin fibroblst procollgen fied by ELSAforhumn fibroblst TMP, exhibited n se (left) or TMP (right). Mo, concentrted monocyteconditioned pproximtely 1:l molr stoichiometrywith respect to its medi,prtillypurified s described under "Experimentl Procedures;" F, pure humn skin fibroblstprocollgense (left) nd TMP bility to inhibit the collgenolytic ctivity of exogenously (right). Note precipitinlines of complete identity in bothpnels. ddedpureskin fibroblst collgense. Therefore,humn

4 Monocyte Secretion of Procollgenuse nd TMP c> differentited withphorbol esters (16), did not contin hybridizble mrna (not shown). Northern blotting of totl cytoplsmic RNA from monoz cytes reveled n mrna of.9 kilobses which hybridized to 8 cdna probe for humn fibroblst TMP (Fig. 4, right). A w T M P mrna of identicl size ws identified from humn j 6skin fibroblsts, lveolr mcrophges, nd phorboldifferen u tited cell lines (U937 nd HL6). Thus, Northern blotting provided further evidence for the identity of procollgense nd TMP derived from humn monocytes, skin fibroblsts,lveolrmcrophges, U937 cells, nd (for TMP) HL6cells. Spontneous Secretion of Procollgenuse nd TMP by MonMOLES NHETOR / MOLE COLLAGENASE ocytesunstimulted monocytes (Fig. 5) cultured in the presfg.3. Molr stoichiometry of inhibition produced by hu ence of 2% utologous serum secreted miniml or undetectmn monocyte TMP. Dt indicte the inhibitory cpcity, ginst ble mounts of procollgense, when followed for up to 15 pure humn skin fibroblst collgense, of TMP purified from hu dys in uitro. n mrked contrst, lrge quntities of TMP mn skin fibroblsts ()nd prtilly purified monocyte TMP (). (pproximtely.2 pg/16 cells/24 h) were secreted into such The concentrtion of TMP in prtilly purified monocyte condiin n pproxitionedmedi ws determined by ELSA. Note tht TMPfrom both medi, nd TMP continued to ccumulte sources exhibits n pproximtely 1:l molr stoichiometry of inhibi mtely liner mnner for t lest 15 dys (Fig. 5). tion. Regultion of the Productionof Procollgenuse nd TMPn the first 48 h of in uitro culture, low to undetectble spontneous secretionof procollgense ws observed (Fig. 6, left). Although there ws considerble individul vribility, the group s whole showed no enhncementof procollgensesecretionin response to PMA, the dimethyl sulfoxide 28s solvent control (not shown), or LPS. There ws tendency for procollgense secretion to be stimulted by ConA, especilly in the bsence of serum (in five out of six subjects, 285 procollgense incresed in response to ConA). Procollgense secretion by one subject's monocytes showed high bsl 2.5kb levels nd decresedslightly with Conk this individul's 18s 18s results tended to obscure n otherwise 2fold stimultion in men procollgense secretion by the group in response to >O.Pkb ConA. n mrked contrst to ourprevious observtions with humn lveolr mcrophge procollgense secretion (3, 42), LPS filed to produce ny enhncement in the rte of procollgense relese by monocytes. hen cultured in the presence of 2% utologous serum, monocytes secreted substntil quntities of TMP (Fig. 6, right). Agin in contrst toprevious observtions with lveo loo[ FG.4. Northern blot of procollgense nd TMP mrna isolted from vrious humn cells. Totl RNA ws hrvested from lveolr mcrophges stimulted with LPS (for procollgense) or PMA (for TMP), skin fibroblsts, monocytes (stimulted with ConAforprocollgense), PMAdifferentitedHL6 cells, nd PMAdifferentited U937cells s described under "Experimentl Procedures." Northern hybridiztionws thenperformedusing cdna probe for humn skin fibroblst procollgense (left (37)) or TMP (right (38)).As shown in the left pnel, 2.5 kilobse (kb) procollgense mrna species ws identified from thefour cell types. No hybridizble procollgense mrna ws found in the HL6cells right pnel showstht.9kilobse TMP mrna (not shown). The species from the five cell types hybridized with the cdnaprobe. 2 z 2.5 / 1 V monocytederived nd fibroblst TMP pper to be function lly equivlent. DAYS Comprison of Procollgenuse nd TMP mrna from MonFG. 5. Bsl levels of collgense nd TMP secretion by ocytes nd Other Humn CellsNorthern blotting of totl humn monocytes. Unstimulted monocytes (2 X 16/ml) were held cytoplsmic RNA isolted from ConAstimulted monocytes in monolyer culture in the presence of 2% utologous serum for reveled 2.5kilobse mrna which hybridized with cdna vrious periods of time. Dt indicte the cumultive secretion of the probe to humn skin fibroblstprocollgense (Fig. 4, left). A respective immunorective proteins t the times shown nd re the procollgense messge of identicl size ws lso identified results of duplicte ELSAs. Medi were chnged every 5 dys. Ech type of symbol represents dtfrom monocytes derived from differfrom humn skinfibroblsts, lveolr mcrophges,nd phor ent subject., procollgennse;, TMP. Note tht the sponboldifferentited U937 cells (Fig. 4, left). HL6 cells, which tneously secretedcollgenolytic profilepredominntly orexclusively hve been shown not to synthesize collgense even when fvors collgense inhibition.

5 15866 Monocyte Secretion of Procollgense n MP.175L O(.17) T O.6 r v) z V D j.5 8 s2 Serum Control e B O *: A uc V" PMA LPS,.4 V L" D 4 % 8 Serum Control Con A FG. 6.' Effect of vrious gents on the production of monocyte procollgense nd TMP. mmunorective procollgense (left) nd TMP (right) secretion by monocytes ws quntified during the initil 48h period of in vitro monolyer culture. Ech symbol represents dt from monocytes derived from different subject; results re mens of duplicte ELSAs. Low to undetectble (detection limit =.5 pg/16 cells) spontneous secretion of procollgense ws observed. There ws tendency for procollgense secretion to be enhnced by ConA; there ws no stimultory effect of LPS or of PMA. There ws no mesurble secretion of TMP in the bsence of serum (detection limit =.25 pg/16 cells). n the presence of 2% utologous serum, monocytes from ll subjects spontneously relesed substntil mounts of TMP. None of the gents tested ugmented the secretion of TMP; secretion ws reduced by PMA nd LPS. Concentrtions were s follows: ConA, 2 pg/ml; PMA, 1 ng/ml; LPS, 1 pg/ml. lr mcrophges (3), none of the gents tested significntly enhnced this spontneous rte of TMP production; in fct, PMA nd LPS reduced TMP secretion. An unexpected finding ws tht there ws no mesurble secretion of TMP by monocytes in the bsence of dded serum. This ws not explicble by cell injury since procollgense secretory rtes were not similrly ffected (Fig. 6, left). The component(s) of serum which permits TMP production by monocytes hs not yet been identified. DSCUSSON The work described in this report hs demonstrted the bility of norml humn peripherl blood monocytes to secrete both n interstitil procollgense nd TMP. mmunologic, biochemicl, nd moleculr biologic nlyses ll would indicte tht these proteins re identicl to the corresponding proteins of skin fibroblsts (28, 29), lveolr mc rophges (3), U937 cells (12), nd HL6 cells (the ltter for TMP only (16)). The studies of procollgense nd TMP mrna reported here further strengthen the rgument tht the corresponding products of these cells re, in fct, identicl. Neutrophillike collgense ws not produced t significnt levels by humn monocytes, s demonstrted by: 1) the comprtive lck of monocyte collgenolytic ctivity ginst type reltive to type 11 monomeric collgens in both prtilly purified (Fig. 2 nd Tble ) nd freshly hrvested conditioned medium smples; 2) the extrcellulr locliztion of virtully ll collgense ctivity (not shown); nd 3) the bsence of demonstrble immunorectivity of both monocyte superntnts nd extrcts with monoclonl ntibody directed ginst neutrophil collgense.* Previous studies of collgenolytic ctivity of norml humn peripherl blood monocytes hve been few (17, 18, 43) nd hve not chrcterized the collgenolytic enzyme. The work of Louie et l. (17) demonstrted tht dherent humn pe ripherl blood mononucler cells nd elutrited monocytes secreted metlloproteinse collgense ctive ginst soluble K. Hsty, personl communiction. type collgen. Secretion of collgenolytic ctivity ws incresed by ConA nd diminished by cycloheximide. This work indictes tht monocyte collgenolytic ctivity cn be ttributed to collgense of the fibroblst/mcrophge type. Furthermore, the only collgenseinhibitory product of monocytes reported previously hs been 2mcroglobulin (19), which is secreted in trce mounts. n contrst, our studies demonstrte tht monocytes hve the cpcity to secrete lrge mounts of TMP (Fig. 5), levels tht cn mrkedly influence the collgenolytic potentil of such cells. Since polymorphonucler neutrophils hve been shown to possess metlloproteinse collgense which is distinct from the fibroblst mcrophge collgense (2224, 44); nd since monocytes nd undifferentited U937 monocytelike cells hve serine proteinses (1, 4, 14, 15, 2) tht re similr or identicl to the elstse nd cthepsin G of neutrophils, it ws of interest to lern tht monocyte collgense nd TMP were identicl to the nlogous products of lveolr mcrophges (3) nd different from proteins contined in neutrophils. Although monocytes shred the sme enzyme nd inhibitor with lveolr mcrophges, both quntittive nd qulittive spects of the spontneous nd stimulted secretion of these proteins differed between the two cell types, s shown when the present dt re compred to our previous work with lveolr mcrophges (3). The differences were: 1) monocytes spontneously secreted lrge quntities of TMP nd little or no procollgense, mrkedly fvoring collgense inhibition rther thn collgenolytic ctivity, wheres mcrophges spontneously secreted comprtively higher levels of procollgense nd somewht lower levels of TMP; 2) monocyte secretion of TMP demonstrted n bsolute requirement for one or more syet unidentified components of serum in uitro, wheres mcrophges secreted TMP in the bsence of serum; 3) monocyte secretion of TMP ws fixed t high levels nd ws unresponsive to the regultory gents tested, wheres mcrophge TMP ws subject to upregul tion by PMA nd LPS; 4) monocyte secretion of procollgense ws totlly unresponsive to the stimultory effects of LPS even though this stimulus is known to upregulte mrkedly

6 Monocyte other monocyte secretory products (45, 46), wheres mcrophge procollgense ws consistently nd substntilly upregulted by LPS; nd 5) even following monocyte stimultion with ConA, levels of secreted procollgense were much smller (51%) thn the quntities relesed by mcrophges. Mononucler phgocytes re known to chnge their complement of proteolytic enzymes during differentition (1, 4, 14, 15, 2, 47). The work described in this report identifies differences between humn monocytes nd mcrophges both in bsl (spontneous) levels of procollgense nd TMP secretion nd in the response of such cells to regultory stimuli; nevertheless, both monocytes nd mcrophges re Secretion of Procollgense nd TMP nd Teitelbum, S. L. (1985) J. Clin. nvest. 76, cpble of producing the sme proteins. n contrst, the serine 4. Senior, R. M., Cmpbell, E. J., Lndis, J. A., Cox, F. R., Kuhn, proteinses elstse nd cthepsin G hve been shown to be C., nd Koren, H. S. (1982) J. Clin. nvest. 69, present within monocytes (4, 14, 15, 2) but re gretly 5. Chpmn, H. A., Jr., nd Stone,. L. (1984) J. Clin. nuest. 74, reduced or bsent from monocytederived mcrophges nd 6. Golds, E. E., Snter, V., Killckey, J., nd Roughley, P. J. (1983) differentited U937 cells (1,12). Bsed upon the present nd J. Rheumtol. 1, previous work, it ppers tht n orderly progression is iden 7. Dyer, J.M., Brerd, J., Chess, L., nd Krne, S. M. (1979) J. tifible during humn mononucler phgocytic differenti Clin. nuest. 64, tion, in which these cells develop the cpcity to secrete TMP 8. Gross, J., nd Ngi, Y. (1965) Proc. Ntl. Acd. Sci. U. S. A. 54, prior to procollgense nd eventully re cpble of regult ing both proteins. Such n hypothesis is supported by the 9. elgus, H. G., Jeffrey, J. J., Stricklin, G. P., Roswit,. T., nd Eisen, A. Z. (198) J. Biol. Chem. 255, following observtions. 1) Phorboldifferentited HL6 cells 1. elgus, H. G., Jeffrey, J. J., nd Eisen, A. Z. (1981) J. Biol. re cpble of producing lrge quntities of TMP but fil to Chem. 256, synthesize ny procollgense whtsoever (16). 2) U937 cells 11. elgus, H. G., Jeffrey, J. J., Stricklin, G. P., nd Eisen, A. Z. in the bsl stte secrete mesurble (but smll) mounts of (1982) J. Biol. Chem. 257, TMP nd do not relese ny collgense; wheres following 12. elgus, H. G., Connolly, N. L., nd Senior, R. M. (1986) J. Clin. differentition by phorbol esters, such cells produce lrge nuest. 77, Hersh, C. L., Yeh, R. K., Cllwy, J. E., Grci, J. A., nd quntities of both procollgense nd TMP (12). 3) Our GilmoreHerbert, M. G. (1986) Biochemistry 25, Senior, R. M., nd Cmpbell, E. J. (1984) J. mmunol. 132, present dt indicte high constitutive level of TMP production by newly isolted peripherl blood monocytes (lbeit only under the influence of n syet undefined permissive serum component or components), but monocytes hve pprently little cpcity to secrete procollgense even with ConA stimultion. 4) Alveolr mcrophges hve the cpbility to secrete both collgense nd TMP in the presence or bsence of serum nd lso to regulte the secretion of both proteins in response to gents in the pericellulr milieu (3). Monocytes obtined from different volunteers ppered to exhibit mrked individul vribility in their collgenolytic profile. This vribility ws noted in bsl levels of TMP production (Fig. 5) nd, even more strikingly, in the response of procollgense secretion to regultory gents (Fig. 6, left). These results re too preliminry to support conclusions firm bout individul vribility; however, it is possible tht the collgenolytic profile of n individul subject s mononucler phgocytes during inflmmtion or tissue injury my be n importnt determinnt of the extent of collgen ccumultion or interstitil collgen degrdtion (nd thus n importnt determinnt of the outcome of chronic inflmmtory disese process). t will be of considerble interest to compre secretion of these two proteins in monocytes nd lveolr mcrophges obtined from the sme donor to determine whether there re donorspecific ptterns of procollgense nd TMP secretion. t is lso noteworthy tht other monocytederived proteinses re cpble of degrding collgen types 11 (48, Acknowledgmentse thnk M. A. Cmpbell, D. K. Kobyshi, C. Fliszr, nd C. Drgovich for their excellent technicl ssistnce. e lso thnk G. L. Goldberg (shington University School of Medicine) for use of collgense cdna probe nd J. J. Jeffrey nd R. M. 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