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1 Anlysis of ellulr nd Protein ontent of Bronho-Alveolr Lvge Fluid from Ptients with Idiopthi Pulmonry Fibrosis nd hroni Hypersensitivity Pneumonitis HERBERT Y. REYNOLDS, JAK D. FULMER, JOHN A. KAZMIEROWSKI, WILLIAM. ROBERTS, MIHAEL M. FRANK, nd RONALD G. RYSTAL From the Lbortory of linil Investigtion, Ntionl Instituite ofallergy nd Infetiotus Disese; the Pulmonry Brnh, Ntionl Hert nd Lung Institute; nd the Setion of Pthology, Ntiotnl Hert nd Ltung Instittute, Ntionl Instituites of Helthl, Bethlesd, Mlrylnd 214 A B S T R A T To evltute ellulr nd protein omponents in the lower respirtory trt of ptients with idiopthi pulmonry fibrosis (IPF) nd hroni hypersensitivity pneumonitis (HP), limited bronholveolr lvge ws done in 58 ptients (19 IPF, 7 HP, nd 32 ontrols). Anlysis of the ells nd protein in the lvge fluiids from ptients with IPF reveled n inflmmtory nd eosinophili response nd signifint elevtion of IgG in the lungs. With ortiosteroid therpy, inflmmtion diminished buit eosinophils remined. Lvge fluid from ptients with HP lso hd eosinophils nd elevted levels of IgG. However, in ontrst to IPF, lvge fluid from HP ptients ontined IgM, fewer inflmmtory ells, ndl strikingly inresed number (38-74%) of lymphoytes. Identifition of lvge lymphoytes in HP showed tht T lymphoytes were signifintly elevted nd B lymphoytes were deresed ompred to peripherl blood. These studies suiggest tht the lung in IPF nd HP my funtion s reltively independent immune orgn, nd tht nlysis of ells nd proteins in bronho-lveolr lvge fluid my be of dignosti, therpeuti, nd investigtive vlue in evluting ptients with fibroti lung disese. This pper ws presented in prt t the Amerin Federton for linil Reserh, Atlnti ity, N. J., 2 My An bstrt ppered in 1976 lin. Res. 24: 388. Requests for reprints my be ddressed to: Ronld G. rystl, Room 6D6, Building 1, Ntionl Institutes of Helth, Bethesd, Md Reeived for publition 14 June 1976 nd in revised form 29 September INTRODUTION The fibroti lutng diseses re heterogeneouis groupl) of hroni, usuilly ftl disorders, hrteried rdiogrphilly by pttern of interstitil infiltrtion nd physiologilly by the loss of lutng voluime nd derese in difftusing pity. Histologilly there is ellulr infiltrtion of lveolr sept nd n pprent inrese in prenhyml ollgen (1, 2). Althouigh some ptients witlh ptulmonry fibrosis n be grouped ording to etiology (e.g., otuptionl, environmentl, infetiouis, ttumor, or drtug) or by hrteristi lung histology (e.g. eosinophili grntulom), mny n be lssified only s hving "idiopthi pulmonry fibrosis" (IPF).' Althouigh the term 'IPF' implies tht the etiology nd pthogenesis of the disorder is unknown, tlhere re luies whih stuggest tht inflmmtory nd/or immtune-medited mehnisms re relted to the ontintued tivity of the disese (3). In ontrst to IPF, hroni hypersensitivity pneuimonitis (HP) is grouip of fibroti lutng diseses in whih the etiology is often known. With hroni inlhltion of ertin orgni ntigens, stuseptible individtuls develop grnulomtous interstitil disese whili often leds to signifint prenhyml fibrosis (4). Animl studies hve stuggested tht in hypersensitivity 'Abbrevitions used in this pper: HP, hroni hypersensitivity pneumonitis; 4, 6, hemolyti omplements; E, erythroyte; FEV,/FV, fored expirtory volume (1 s)/ fored vitl pity; IPF, idiopthi pulmonry fibrosis; Po2, men resting rteril blood oxygen tension; PMN, polymorphonuler leukoytes; SRB, sheep red blood ells. The Journl of linil Investigtion Volume 59 Jnury

2 pneumonitis, lol immune retions in the lungs, involving both humorl nd ellulr mehnisms, re intimtely relted to the pthogenesis nd tivity of the disese proess (5, 6). Our understnding of these diseses, however, is restrited by limittions in the tehnologies vilble for their study. Lung tissue is not essible for repeted studies nd rdiogrphil, physiologil, nd peripherl blood studies do not give true ssessment of the dynmi inflmmtory nd immune mehnisms operting in the lol environment of the lung. The present study ttempts to evlute these mehnisms through n nlysis of ellulr nd protein omponents in bronho-lveolr lvge fluid from representtive portion of the epithelil surfe of the lower respirtory trt in ptients with IPF nd HP. METHODS Study ptients. A totl of 26 ptients, 19 with IPF nd 7 with HP, were inluded in this study. All underwent extensive linil evlution inluding bronhosopy nd 2 hd open biopsies of the lung. The men ge of the 19 ptients with IPF ws yr;2 12 were men nd 7 were women. All ptients with IPF met the following riteri: n interstitil pttern on hest film, redued totl lung pity, redued lung ompline, redued single breth diffusing pity, resting hypoxemi whih worsened with exerise, nd norml fored expirtory volume (1 s)/fored vitl pity (FEV,/FV). None hd signifint environmentl exposure, symptoms suggestive of hypersensitivity lung disese, left ventriulr rdi filure, or history of hroni pulmonry infetions. 17 of the 19 ptients with IPF hd lung biopsies done within 2-11 mo before the bronho-lveolr lvge study. All biopsies showed septl fibrosis, septl ellulr infiltrtion, nd intr-lveolr ellulrity. None hd grnulom nd none hd refrtile prtiles under polried light mirosopy. Seven ptients urrently smoked igrettes. One ptient hd joint symptoms omptible with ollgen-vsulr disese but hd no supporting serologi evidene; one ptient hd positive rheumtoid ftor (titer 1:64) but no stigmt of joint disese; nd one hd positive ntinuler ntibodies nd ws believed to hve mixed ollgen-vsulr disese. Seven ptients were untreted t the time of bronholveolr lvge; the other twelve were reeiving prednisone (men dose mg dily). Peripherl blood vlues, inluding hemoglobin (14.±.5 g/1 ml), totl leukoytes (8,3+747 ells/mm3), nd perentge of lymphoytes on peripherl smer ( %) were within norml limits for ll 19 ptients. For untreted ptients with IPF, the men vitl pity ws 64.±5.% of predited norml vlues, funtionl residul pity ws % predited, totl lung pity ws % predited, but FEV,/FV ws norml. rbon monoxide diffusing pity ws 48.3±6.9%o predited. The men resting rteril blood oxygen tension (Po2) for this group ws mm Hg (on room ir) nd deresed to mm Hg with exerise. The ltter ws done with grded stedy-stte exerise protool, eh work level ws for 6 min with rteril blood gses determined during the lst 2 min. 2 All dt re presented s men+sem. 166 Reynolds, Fulmer, Kmierowski, Roberts, Frnk, nd rystl For treted ptients with IPF, these prmeters were similr. The men vitl pity ws % predited, funtionl residul pity ws 63.±3.6% predited, totl lung pity ws % predited, nd the FEV,/FV ws norml. The rbon monoxide difftising pity ws % predited, while the resting Po2 ws 73.3±4.2 mm Hg nd deresed to 53.7±2.1 mm Hg with exerise. Seven dditionl ptients were referred to us s hving IPF, but the episodi nture of the respirtory symptoms, serologil studies, or histopthology of the lung biopsy suggested they hd form of hypersensitivity pneumonitis. All of the ptients hd history of hroni intermittent respirtory nd systemi symptoms relted to their home or work environment. Four of the ptients hd ute episodes hrteried by hills nd fever ompnied by ough nd dyspne. The other three hd more insidious onset of ough nd progressive dyspne with no hills or fever. Four ptients noted relief of symptoms when removed from their home or work environment nd n exerbtion of symptoms with re-exposure to the pprent soure of ntigens. In four ptients, the hest X-ry bnormlities hve tended to persist even with removl from the pprent ntigeni soure. The men physiologi dt for these seven ptients were: vitl pity, 82.1±6.4% predited; funtionl residul pity, 7.2±5.6% predited; totl lung pity, 8.4±5.3% predited; FEV,/FV norml (in ll ptients), rbon monoxide diffusing pity, 66.1±6.4% predited. The men Po2 ws 76.1±6.4 mm Hg nd deresed to 68.2±4.8 mm Hg with exerise. Additionl linil nd lbortory informtion bout individul ptients with HP is given in Tble I. ontrol ptients. 32 ptients (2 men, 12 women) with undignosed lesions limited to the right upper lung underwent bronho-lveolr lvge in the lingul or left lower lobe s prt of the dignosti bronhosopy (7). 21 smoked igrettes nd 11 did not. None hd evidene by physil exmintion or hest X ry of disese in the left lung nd none were reeiving drugs. The ontrols were ge-mthed for the IPF ptients (nonsmokers 52.±3. yr; smokers 48.±2.4 yr). Bronhosopy nd bronho-lveolr lvge. Informed onsent ws obtined from ll ptients. Ptients were premedited intrmusulrly with tropine (.6 mg) nd diepm (1 mg); topil lidoine spry (2%) ws used to obtin lol nesthesi in the respirtory trt. 'Bronhosopies were performed trnsnslly with flexible fiberopti instrument (model BFT 5B-2, Olympus orportion of Ameri, New Hyde Prk, N. Y.). Bronhosopy ws done in the morning, pproximtely 3 h fter prednisone ws given to ptients reeiving this medition. The proedure ws done with onstnt rdi monitoring nd with n intrvenous infusion in ple. If the resting rteril P2 on room ir ws below 7 mm Hg, the bronhosopy ws done with supplementl oxygen given by nsl prongs or fe msk (with side port for the instrument). After routine exmintion of the mjor irwys, the bronhosope ws positioned in segment of the lingul (B4 or 5). A totl of 1 ml of sterile.9%o sline (Abbott Lbortories, North higo, Ill.) ws infused (3 ml twie nd then 4 ml) nd spirted by sution fter eh infusion. The lvge fluid ws immeditely strined through single lyer of gue to remove gross muus nd then entrifuged (5 g, 5 min) to seprte ellulr nd protein omponents. The supernte ws refully dented from the ell pellet for subsequent protein nlysis. ellulr nlysis. The ell pellet ws resuspended in modified Hnk's blned slt solution (without lium nd mgnesium ions) nd pelleted gin with low-speed entrifugtion. The ells were ssyed for totl number (model

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4 TABLE II Generl hrteristis of Lvge Fluid from Ptients with IPF nd from ontrols Reovery of Number infused onentrtion onentrtion Grotups of lvge Totl ells ell ptients fluiid reovered vibility of protein in lvge fluid* of lbumin in lvge fluid %7 x 1' % ing/nl' mng/ml ontrol, Nonsmokers ±6.3.63± ±3. 3.8±.8 1.7±.5 (3-8) (.1-1.1) ( ) (.5-3.) Smokers ± ± ± ±.3 1.±.1 (1-77) (.2-7.7) (.7-5.2) (.3-1.9) IPF, No therpyl 7 31.± ± ± ±.4 (15-65) (1.3-7.) ( ) (.3-2.8) On therpyt ± ± ± ±.4.9±.2 (2-74) (.3-3.6) (.7-4.3) (.3-2.) Nonsmokers ±.6 84.±5. 3.4±.6 1.±.2 (15-74) (.2-8.) (1.1-8.) (.3-2.6) Smokers ± ± ± ±.8 1.1±.4 (15-65) (.2-3.6) (.8-7.5) (.2-2.8) HP 7** ± ± ± ±.5 (3-66) (1.-3.7) (7.-26.) (.8-6.3) * Bronho-lveolr lvge ws onentrted to 1.-ml volume. 4 Inludes nonsmokers nd smokers. Men±SEM, rnge for the group observed given in prentheses. The differene in totl ells reovered in IPF (no therpy) nd ontrol (nonsmokers) ws signifintly different (Student's t test, P <.5). The onentrtion of protein nd lbumin in lvge fluid of ptients with HP ws signifintly greter thn tht in ll other groups (P <.5 ll ses). ** 1 lvges in seven ptients; repet lvges fter generl linil improvement, generlly showed derese in totl protein onentrtion (J. G mg/ml; R. B mg/ml; W mg/ml). 168 Reynolds, Fulmer, Kmierowski, Roberts, Frnk, nd rystl FN, oulter Eletronis, Hileh, Fl.), vibility (8) nd differentil ounts. The ltter were mde from ytoentrifuge smers stined with Wright-Giems. For ptients with HP, the reltive proportions of bone mrrow-derived (B) nd thymus-derived (T) lymphoytes were determined in lvge nd peripherl blood. Lymphoytes were seprted from the respirtory ell pellet or from heprinied blood with two-step proedure (9). First, mononuler ells were bnded by density grdient entrifugtion with Hypque-Fioll (Phrmi Fine hemils, In., Pistwy, N. J.) (1) nd subsequently wshed three times in Egle's minimum essentil medium (Grnd Islnd Biologil o., Grnd Islnd, N. Y.). Seondly, olumns of Sephdex G-1 (Phrmi Fine hemils, In.) were used to deplete the ell preprtions of monoytes or lveolr mrophges (11, 12). For respirtory ells, olumn of 1 ml Sephdex gel ws equilibrted with Egle's minimum essentil medium ontining 1% fetl lf serum, 17 ells were wshed into the olumn nd the nondherent ells were eluted with Egle's minimum essentil medium t room temperture. For blood ells the olumn ws mde with 3 ml of Sephdex nd 18 ells were used. This olumn method onsistently yielded 95-98% pure popultion of lymphoytes with vibility of t lest 95%. The net reovery of totl lymphoytes from the originl ell pellet ws 85.±4.%o. omprison of the originl ell pellet nd the postolumn purified lymphoytes showed no hnge in the reltive proportion of T nd B lymphoytes (12). The perentge of T lymphoytes were defined s those forming erythroyte (E) rosettes with sheep red blood ells (SRB) (13). The perentge of B lymphoytes ws defined s those forming rosettes with SRB (E)-rbbit IgM-nti-SRB ntibody (A)-mouse serum omplement () omplexes (EA rosettes) (14, 15). Detils of these regents nd proedures hve been desribed elsewhere (16). Protein nlysis. The supernte from the initil entrifugtion ws onentrted t 4 to finl volume of 1. ml with ombintion of positive pressure ultrfiltrtion (Diflo, UM-1 membrne, Amion orp., Sientifi Sys. Div., Lexington, Mss.) nd negtive pressure dilysis. Quntittive mesurements in this onentrted lvge inluded: totl protein by the Lowry method (17); lbumin nd immunoglobins G nd M by rdil immunodiffusion in gr (Behring Dignostis, Amerin Hoehst, Somerville, N. J.); IgE with rdioimmunobsorbent method (Phrmi Lbortories In.); nd hemolyti omplement (4 nd 6 titers) (18, 19).

5 Similr methods were tused to quintitte sertum protein vltues. When initil sttudies of lvge fluiid from ptients with IPF indited tht pproximntely 25% of the totl IgA ws monomiieri (7S) nd 75% ws polymeri (11S), it ws pprent tht rdil immtunodifftusion preipittioin, uising single IgA stndrd protein, woutld not turtely quintitte the two popultions of IgA present. To obvite this problem,.3 ml of the onenitrted lvge fluiid ws sedimented in 1-4% liner suirose density grdients (2); the 7S nd 11S IgA frtions were seletively pooled, dilyed to remove residtul sturose, nd quintitted using humn monomeri IgA ind seretory IgA stndrds, respetively (7). The 11S omponient ws identified s seretory IgA by sie, imiuinilioretivity with seretory omponent ntiserumiii nd the presene of J-hlini. Totl IgA ws ltulted from the stum of the monomiieri nd seretory vltues nd extrpolted to 1-nd volume. Speifi preipittinig ntibodies ginst vriety of ntigens' known to uise HP were ssyed by Oulhterlony rdil immuntiiiodiffuision in the lbortories of F. Wenel t the Mrshfield lini, Mrshfield, Wis.; similr ssys were lonie in outr lbortories uising ntigens obtined from Hollister-Stier Lbs, Spokne, Wsh. (lot 8971) nd Greer Lbs, Lenoir, N.. (lot K5B-8). Stitistil omnprisotns. Grouip dt re expressed ts men±tsem; imen vltues were ompred with Stuidenit's t test (two-tiled) nlysis. 1 8n 6_ D u -j4 )_ 11 LLJ L 32 -J LU -J 1 (D3 uj - 2_ A - S 8 * 4 Polymorphonuler Leukoytes Mrophges. I *o * X o r oo Eosinophils D Lymphoytes B RESULTS 61 ltge proedures were pelrlformed in 58 ptients (19 IPF, 7 HP, nid 32 ontrols). Lvge re(quiredl 5-1 mnin to omplete. It ws well tolerted by pttients nd dlid not ldd to the disomfort or morbidity of bronlhos opyt. Sinie prior studlies lhvxe demonstrted inl normiil subjets tht igrette smiioking n signifintly influiene thle reovery of respirtory ells (7, 21-23), resuilts fromii ptients with IPF re lso segregtedl s to smloking sttuis (Tble II). The perentge of lvge flutid reovered fromi ptients with IPF on no therpy tended to be less thln from other grouips, prtiiulrly the nonsmoking ontrols (P =.8) (Tble IL). Anilysis of ells ini IPF lvge. The totl number of ells reovered fromii lvge of ptients with tuntreted IPF ws signifintly greter thn from nonsmiiokin-g ontrols (P <.5) but not signifintly different tlhin from smioker ontrols (P >.1). Reovery of ells from IPF ptients on therpy ppered to be less tlhin f'romi those ulntreted, but this differene ws not signifint (P >.1). Smoking stttus did not influiene the totl nulmber of ells reovered friom ptients with IPF. The vibility of lvge fluiid ells ws similr between ll grouips (Tble II). Of ptients witlh IPF (nontreted nd t-eted), 73% 3Antigens used inluded: Miropolyspor feni, Thermotinomyes vulgris, T. viridis, T. shri, Aspergillus futinigtus, Aslp. sydotvi, Asp. gluus, Asp. niger, pigeon ser, pigeon droppings, ryptostro?n ortile, nd Atureobsidijumn pulltulnrs. 1 (5- S I1F IPF Non- Smoker on Therpy Smoker *& --O _ + so IPF IPF Non- Smoker on Therpy Smoker onb FIGURE 1 Perentge of ells identified in the differentil ount of lvge fluid is shown for ptients with uintreted IPF, ptients with treted IPF, nonsmoker ontrols, nd smoker ontrols. A horiontl line represents the men for the grouip. Nonsmokers () nd smokers () re designted. In nonsmoking ontrols, there were no PMNs fouind; in nonsmoking ontrols nd smoking ontrols no eosinophils were present. hd greter perentge of polymorplontuler leekoytes (PMN) in their lvge differentil ouniit thn did ontrols (Fig. IA). Althotugh there ws brod rnge whih overlpped, the mein perentge of' PM\Ns in treted IPF (14.+4.%) ws signifiintly lower (P <.5) thn tht in tuntreted IPF (33.+8.%). In the smoking ontrols, PMNs were infrequient (3.+.4%) nd in the nonsmoking ontrols tlhey were bsent. There ws no signifint differene in the perentge of PMNs in lvge from IPF nonsmiiokers nd smokers. The perentge of mrophges (Fig. IB) fouind in ontrol grouips nd for treted IPF ptients were not signifintly different. The lower perentge of mrophges in the uintreted IPF grotup (P <.5) ws Ltung Lvge in Pulmonry Fibrosis 169

6 < o *. IPF IPF Nornsmoker Smoker Thery ontrol IgG/ALBUMIN 8 *.o o *. * o * OSn ~~ 6. ~ 2 l :ge/albumin LAVAGE * S~~~~~~~~~~ S~~~~ ~~~~~~~~~~ 3 * so SERUM IPF IPF Nonsmoker Smoker On The on'rs FIGURE 2 A omprison of immunoglobulin levels in lvge fluid nd serum is shown for ptients with untreted IPF, IPF ptients under tretment, nonsmoker ontrols, nd smoker ontrols. The immunoglobulin dt re presented s rtio to the respetive lbumin level in the sme smple (Results). A horiontl line denotes the men for the group. Nonsmokers (), smokers () re designted. IgG/lbumin nd IgA/lbumin rtios re expressed s milligrm per milligrm. IgE/lbumin rtios re in nnogrm per milligrm; these levels re not vilble for the nonsmoker ontrols. probbly seondry to the reltive inrese in PMNs in these ptients. The perentge of eosinophils (Fig. I) in the IPF ell differentils ws signifintly higher (P <.1) thn for ontrol subjets in whom eosinophils -were not deteted. Eosinophils persisted in IPF despite nti-inflmmtory therpy. The number of eosinophils in the peripherl blood of these ptients ws not elevted exept for mildly inresed bsolute irulting eosinophil ounts in 3 of the 19 IPF ptients ( eosinophils/mm3 for the 3 ptients). The perentge of lymphoytes in IPF lvge fluid 17 Reynolds, Fulmer, Kmierowski, Roberts, Frnk, nd rystl (Fig. ID) ws neitlher inresed oml)pre(l witlh ontrols nor ltered ppreibly by therpy. Erythroytes in the respirtory ell pellets generlly weere less thn 3%. Protein nlysis in IPF. Quntittive nlysis of proteins reovered from the lower respirtory trt of humn subjets is diffiult, sine the bronho-lveolr bthing fluid n only be reovered by "wshing" it out. Two pprohes n be uised to obvite this problem. First, stndrdied lvge proeduire (1 ml infusion) ws used. Although vrible mouniit of lvge fluid ws reovered from individul ptients, the men voltumes reovered from thie diff'erenit ptient grotups were not signifintly different (Tble II), nd quintittive mesurements of' lvge pr-otei us n be expressed s uinits per milliliter of onienitrted lvge fluiid. For exmple, there were no signifint differenes between the totl protein onentrtions fouind in lvge fluiid of ll groups nilvyed (Tble II). Seondly, lvge protein vltues n be expresse(l inl reltion to ref'erene protein in the lvge. Albumin ws hosen s this referene stndrd (24) beuse it is not synthesied in the lutng nd its presene in bronho-lveolr fltuid reflets trnstuidtion f'romii the intrvsulr omprtment. In ddition, the reltive mouints of lbtumin in lvge nlyses were not signiifintly different in the vriouis ptient groups (Tble II). Likewise, lbtumin vltues in sertum were omprble for the vriouis grouips (dt not slhown). The rtio of speifi protein (milligrmiper milliliter) to lbtumin (milligrm per milliliter) in lvge fluiid ouild be ompred diretly to the rtio of' these proteins in serum. Thtus, the rtio of' speifi proteini to lbtumin in lvge should be the sme s tht in serim if proteins re present in the lower respirtory trt seondry to trnsudtion f'rom sertum. A htighi rtio of speifi protein to lbtumin in lvge ws tken s evidene of reltive inrese of tht protein in the lower respirtory trt ompred to serumiii. This seond method ws found to be mtuh more uiseftul, nd ll of the dt re presented in this mnner. The men onentrtions of eh protein (mouint per milliliter) in lvge n be lutlted from the protein to lbumin rtios by tusing the men lbunmin onentrtions for eh grouip (Tble II). Serum IgG to lbumin rtios were similr in ll ptients; the individul rtios were lwys less thln.65 (Fig. 2). In ontrol subjets, lvge IgG/lbumin rtios, likewise, were lwys less thn.65. In IPF ptients, however, lvge IgG/lbumin rtios were greter thn.65 in 63% (12 of 19) ptients. The men rtios for lvge fluid in uintreted IPF ( ) nd treted IPF ( ) were signifintly higher thn those for ontrols (P <.1 ll ompri-

7 sons). The elevted lvge IgG/lbumin levels in IPF were not dependent upon the smoking stttus of the ptient (Fig. 2). In generl, ptients on tretment for IPF hd lower rtios of lvge IgE/lbumin thn smoker ontrols (P <.25) despite onsiderble vrition in the ontrol vlues. 5% of the untreted IPF ptients lso hd lower lvge IgE/lbumin vltues thn ontrols, but the men for this group ws not signifintly lower thn ontrols (P >.1). Unfortuntely, dt were not vilble for nonsmoker ontrols, btut restults obtined in nonsmoking voltunteers demonstrte lvge IgE/lbumin vlues omprble to those fouind in the smoker ontrols (7). Sertum levels of IgE/lbumin were similr in ll groups. Lvge IgE/lbumin for some IPF ptients tended to be higher thn tht in seruim; for ontrols the lvge vlues were signifintly higher thn serum vlues (P <.1) (Fig. 2). One subjet (A. R.) in the untreted IPF group ws exltuded beuse of history of topi disese. Interestingly, his serum IgE level ws 3, ng/ml (IgE lbumin rtio of 1) while his lvge IgE ws 24 ng/ml (IgE/ lbumin rtio of 9). IgA/lbumin vlues in the serum of ll ptients were in nrrow rnge (rtio.5-.2 in ll ses) while the IgA/lbumin vlues in lvge fell brodly over 1-fold rnge (.2-2.). As expeted in ll grouips, lvge IgA/lbumin ws signifintly higher thn serum vlues, but there were no signifint differenes in lvge vlues between groups (Fig. 2). In ontrols, 9% of lvge IgA ws monomeri (7) while in IPF 25-3%/o of lvge IgA ws in the monomeri form. Interestingly, the use of IgA/lbumin rtios to nlye IPF serum vlues obsured the ft tht sertum IgA levels were inresed (>3.5 mg/ml) in 71% of the uintreted IPF group but only in 25% of the treted IPF ptients. With double-diffusion immunopreipittion nlysis of lvge fluid, IgM ws deteted in only 15% of speimens from IPF ptients (sensitivity of the IgM ssy ws.1 mg/ml). Likewise, this immtunoglobtulin ws found in pproximtely 5% of ontrol speimens (7). Serum IgM vlues were within the norml rnge for our lbortory ( mg/ml) for IPF ptients on no therpy ( mg/ml) nd for those on tretment ( mg/ml). Serum vlues for ontrol nonsmokers ( mg/ml) nd smokers ( mg/ml) were omprble. 4 hemolyti omplement titers in lvge, expressed s rtio to lbumin, were the sme for the IPF grouips nd for smoker ontrols (Fig. 3). However, men rtios in lvge fluid were signifintly lower (P <.1) thn orresponding serum rtios for the IPF group on therpy nd for smoker ontrols. Likewise, men lvge vltues of 6/lbumin were omprble in ll grouips, but were lso signifintly lower thn respetive seruim Fr LAVAGE SERUM 4/ALBUMIN ~~~~ I~~~~~~~ 8~~~~ ~ ~ ~~~~~ ~~~~~~~~~ o. : : IPF IPF Smoker on Therpy ontrols IPF IPF Smoker on Therpy oro FIGURE 3 A omprison of omplement levels in lvge fluid nd serum is shown for ptients with untreted IPF, ptients with IPF uinder tretment nd smoker ontrols. The men vlue for eh group is given by horiontl br. Nonsmokers (), smokers () re designted. The 4 dt re ltulted s 1-3 hemolyti titer per milligrm lbumin; the 6 dt re lulted s hemolyti titer per milligrm lbumin. rtios (P <.1). In ddition, men serumil 6/lbunmiin vltues in the IPF grouips were signifintly greter (P <.1) thn those of smoker onitrols. ell nd protein nlysis itn HP. The meni reovery of the lvge fluiid from ptients witlh HP (bouit 48%) ws generlly better thn in the IPF groups but the totl ell reovery ws omprble (Tble II). PMNs represented n verge of 8.5% of respirtory ells in HP (Fig. 4). (A higlher perentge ws noted for ptient R. K. nd for the seond nlysis in R. B. nd W..; both of these ltter ptients hd n episode of bronhitis 3-4 wk before the seond lvge study.) Eosinophils represented.5-2% of ll respirtory ells of ptients with HP wlhile pproximtely 29% of the lvge ells were lveolr mrophges with distintive fomy mroplhges onstituting bout 1% of the mrophge popuiltion. In striking ontrst to ptients with IPF nd to ontrols, lymphoytes ouinted for pproximtely 62% of the lvge ells in HP. Whien lvge lymphoytes were hrteried by sheep erythroyte (T ells) nd omplement (B ells) rosette tehlniquies (Fig. 4), the Lung Lvge in Pulmonry Fibrosis 171 8

8 LAVAGE BLOOD. A -J 6H,.(PO tlo m e D 6 u -J LA J /) -J -j Ul U- w w L 4h- 2F- 8 *A A LLJ LL 4 U- Z -J.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I 2w 2 (I) i w UJ i PMN EOS PAM LYMPH E EA E/EA LYMPH E EA E/EA FIGURE 4 Perentge of ells identified in the differentil ount of lvge fluid of seven ptients with hroni hypersensitivity pneumonitis. There were 1 lvges done in 7 ptients. A single symbol is used for eh ptient desribed in Tble I: J. G. first lvge (); J. G. lvge 7 mo lter (-); R. B. first lvge (]); R. B. lvge 11 mo lter (U); W.. first lvge (A); W.. lvge 11 mo lter (A); N. M. (1); E. W. ( ); R K. (); G. A. (A). (PMN, polymorphonuler leukoytes; EOS, eosinophils; PAM, pulmonry lveolr mrophges; LYMPH, lymphoytes; E, T lymphoytes; EA, B lymphoytes; E/EA, rtio of T to B lymphoytes.) A horiontl line represents men for the group. For the lvge dt, shded res represent the rnge of vlues found in lvge of ontrols (nonsmokers nd smokers) given in Fig. 1. In normls, no eosinophils re found in lvge nd the rnge for mrophges is 63-92%. For the blood dt, the shded res represent the ontrol vlues estblished for peripherl blood lymphoytes from young dults (n = 15). All of the dt in this figure is expressed s perentge of totl ells in differentil ount exept for the E/EA olumn (lvge nd blood) whih is presented s the rtio of perentge E to perentge EA. vst mjority of respirtory lymphoytes were identified s T lymphoytes (mnen 7±2.5%) nd only bout 6% ould be identified s B lymphoytes. The men rtio of E/EA (T/B ells) ws bout 14. Approximtely 24% of the respirtory lymphoytes were unidentified with our immune regents nd were onsidered null ells. In ontrst, vlues for periplherl blood leukoytes nd lymphoytes were withini the norml rnge in ll ptients with HP. Anlysis of the blood lymphoytes demonstrted tht 59% were T ells nd 21% were B ells, giving blood T to B men rtio of 3:1. The perentge of T lymphoytes in lvge ws signifintly greter (P <.25) thn in blood nd the perentge of B lymphoytes in lvge ws signifintly less (P <.5). The restulting T to B rtios in lvge ompred with those in blood were lso signifintly different (P <.5). The men totl protein onentrtion p)resent in the onentrted lvge fluiid from these ptienits ( mg/ml) ws signifintly higher thni tht found in IPF grouips or ontrols (P <.1) (Tble II). This ws not duie simply to more trnstudtion of sertum, sine the rtio of lbtumin to totl protein in HP (men.14±.2) ws muth less thn in IPF ( ) or ontrols (.44+.7) (P <.1 for ll omprisons). Repet lvges in three HP ptienits, done t times when there hd been generl linil improvement, yielded less totl protein (Tble II). Prt of the exess bronho-lveolr protein inl HP ws IgG. With the exeption of the seond nlysis of R. B., the rtios of lvge IgG/lbumin were.1.1 in ll ses of HP. These rtios were higlher thn those fotund in 75% of the IPF ptients nd higher thn ny ontrols (Fig. 5 vs. Fig. 2). In ontrst, the orresponding seruim rtios in HP (men.42+.3) were similr to IPF nd ontrols. IgG-rih frtions reovered from sturose grdient frtiontion of lvge 172 Reynolds, Fulmer, Kmierowski, Roberts, Frnk, nd rystl

9 nd serum of ptients J. G. nd R. B. ontined preipitting ntibody to thermophyli tinomyetes (M. feni) ntigens when ssyed by double-diffusion immunopreipittion in gr. IgM vs mesurble in ll lvge speimens in HP; its mere detetion mde it unusul in omprison with IPF (15% hd lvge IgM) or ontrols (5% hd lvge IgM). The IgM/lbumin levels in lvge vs. serum (Fig. 5) indited proportionlly greter mount in the bronholveolr fluid thn in serum. No preipitting ntibodies were deteted in lvge or serum IgM frtions reovered from surose grdients. IgE/lbumin levels in HP lvge were higher thn those found in serum. The lvge nd serum rtios, however, were norml ompred to the smoker ontrols (Fig. 5 vs. Fig. 2). Totl IgA/lbumin in HP lvge ws elevted ompred with serum but ws similr to ontrols. As found in IPF nd ontrols, 4/lbumin nd 6/lbuimin levels were lower in HP lvge fluid s ompred with serum. r IgG/Albumin - - LAVAGE I.4 _-.2 SERUM gm/albumin A A LAVAGE SERUM LAVAGE SERUM 3 _ /Albumin - _o t - L A f DISUSSION Limited lvge of the lower respirtory trt is simple extension of routine fiber-opti bronhosopy. While routine mesurements, suh s hest films nd pulmonry funtion studies, monitor generl lung ftuntion, nlysis of bronho-lveolr lvge fluid is diret ttempt to evlute speifi mehnisms whih my ontribute to the ontinued tivity of lung disese. Quntittion of ells nd proteins in lvge of ptients with IPF nd HP hs demonstrted tht the epithelil surfe of the lower respirtory trt is involved in inflmmtory nd immunologi retions tht would not be suspeted from nlysis of blood lone. In ddition, omprison of lvge ells nd immunoglobulins from IPF nd HP show number of similrities nd differenes whih my be importnt in understnding these disorders. Anlysis of the lvge fluid from ptients with IPF indites tht these ptients hve lol inflmmtory proess whih ws not seen in ontrols. How the presene of polymorphonuler leukoytes reltes to the fibroti proess is not known, buit these studies suggest tht when ptients with IPF re treted, this inflmmtory response tends to diminish. In ddition to the lol umultion of PMNs, the lower respirtory trt in IPF hs striking inrese in IgG ompred with serum IgG nd witl ontrol lvge IgG. Sine the IgG/lbumin levels in the lvge fluid of these ptients re signifintly higher thn their serum IgG/lbumin levels, it is unlikely tht the elevted IgG is seondry to trnsudtion restulting from the inflmmtory proess. It is probble tht in IPF there is lol pulmonry prodution of IgG (25, 26) LAVAGE SERUM LAVAGE SERUM LAVAGE SERUM FIGURE 5 Quntittion of proteins in bronho-lveolr lvge nd serum from ptients with hroni hypersensitivity pneumonitis. The symbols for the ptients re s in Fig. 4: J. G. first lvge (); J. G. lvge 7 mo lter (); R. B. first lvge (); R. B. lvge 11 mo lter (E); W.. first lvge (A); W.. lvge 11 mo lter (A); N. M. (1); E. W. (e); R. K. (); G. A. (A). The immunoglobulin dt (lvge nd serum) re presented s rtio to the respetive lbumin level in the sme smple: IgG/lbumin (milligrms per milligrm), IgE/lbumin (nnogrms per milligrm), IgM/lbumin (milligrms per milligrm), IgA/lbumin (milligrms per milligrm). 4 dt re presented s 1-3 hemolyti titer per milligrm lbumin; 6 dt re presented s hemolyti titer per milligrm lbumin. Shded res re the rnge of vlues for ontrol (nonsmokers nd smokers) given in Fig. 2 nd 3. For ontrol lvge IgM/lbumin is <.1 nd, IgA/lbumin is.3 to 1.8. For ontrol blood, 4/lbumin is nd/or the mehnisms of IgG tbolism or removl re fulty. It is thus possible tht prt of the pthogenesis of IPF is seondry to IgG ntibody being formed in the lung prenhym in response to s yet unidentified ntigen(s) resulting in lol ntibodyntigen omplexes. In HP, the lol umultion of IgG is even more impressive. This finding is onsistent with the onept tht the pthogenesis of hypersensitivity pneumonitis is ssoited with type III immune retion in lung (3). In ord with this onept, two of the HP ptients (J. G. nd R. B.) hd bronho-lveolr IgG preipitins ginst thermophyli tinomyetes ntigens. The lvge fluid from the HP ptients lso ontined signifint quntities of IgM (ompred to Lung Lvge in Pulmonry Fibrosis 173

10 serumtii) htirther emphsiing the possilble role of' lihumoorl imimuiniity in this diseise. Wlile the elevted levels of respirtory IgG in IPF ndl IgG ind IgM in HP suiggest lol type III imimutine reitionis, 1)oth IPF nd HP ptients lso lhd eosinoplhils )resent in their lvge flutid. This find(ing is interestinig in severl respets: () in generl, ptients xvith IPF ind( HP did not hive 1)lood eosinoplhili; (b) onitrols lhd no eosinioplhils in their lvge fluid; nd () therpy withi ortiosteroids didl not infltuene the lol eosinophil tumuiltioin. Althouigh these findin1igs suiggest th(wt loilied illergi retioin mlighit be oirrinig in the luinig prenhym of these Itients, the genierlly low levels of IgE/lbumin in lvge fluii(d f'romi ptienits withi IPF ind HP rguie gin<st the ltssi type tyipe I regin-medlited lting rettioni being iml)ortnt (3, 27-29). Tlhtus disese mehnisms in IPF nd HP re probbly not ssoittedl witlh ombined type I iud III immuiiitine retion;s fiound in disorder stuhl s lllergi bronhopulmiiointry sp)ergillosis (3). In both IPF ind HP, the totl IgA/lbumin levels in lvge were normiil. Howev%er, in both disorders, the reltive proportion of' monomileri IgA in the bronholveolr fluti l wts inresed oml)red withi ontrols. The reson for this fiidinig is; not ler lithouigih it is possible thlt the imioniomiieri IgA represenits trnstudttion froom the intrvsulr omprtment. Alterntively, inflmmiiiitioni enhlne(d proteolyti degrldtion of, lolly prodtued seretory IgA ouild explin this finding. Irrespetive of' the utise of' inresed mlonomneri IgA in the lower respirtory tret in IPF nd HP, neithler disorder ppers to be ssoitedl with lol lborm-ility of seretory IgA. There isi no diret evidene in these sttudies tlt either IPF of' HP re ssoited with lol omplement-miiedi ted retionis. Althouigh 4/lbu min levels in the bronho-lveolr fluiid tended to be lower thlnl serumiii (Figs. 3, 5), there ws n1o differene between IPF, HP, or onitrols. The lower vltues of omplemiient tivity in ll lvge nlyses my be more pprent tlhin biologilly signifint. Althouighi omplemiienit titers were promptly ssyed, its tivity is known to be lbile ind undoubtedly some tivity is lost in the lutng wshouit nd onenitrtinig piroeduires. The elevted levels of seruim 6 in IPF ind HP miy be duie to 6 ting s n tutte phlse retint in these pti ents. While the eosinophils, IgG, IgA, IgE, 4, ind 6 levels in the lvge fluiid of IPF n HP re quiite similr, nlysis of other elluilr nd protein ontents of the bronlho-lveolr fluiid in HP provides striking ontrst to thst in IPF ind ontrols. Lvge fluiid of these ptients ontined lveolr mrophges with fomiiy ytoplssm whih re onsidered to be hrteristi of the histology fouind in the hypersensitivity ltng disorders (4). Suih ells were not noted in IPF or 174 Reynolds, Fulmer, Kmierowski, Roberts, Frnk, nd rystl ontrols. In 1 lvge nlyses from 7 ptients witlh HP, the verge perentge of bronho-lveolr lymplhoytes ws 62% omnpred to bouit 1% in IPF (treted nd uintreted), 8% in smoker ontrols nl 18% in nonsmoker ontrols. Even thouigh ll ptients with HP hd elevted bronho-lveolr IgG nd IgM, nd two hld IgG preipitting ntibodies in the lvge fluiid ginst speifi fu-ngl ntigen, the exggerted bronho-lveolr lymiiplhoyte response in HP is not one of B lym)phoytes, s miglht be expeted, but rther one of T lymphoytes. This finding suiggests, s in experimiienitl niimnl models of hypersensitivity pnetimonitis (5, 6), thlt ell-medited mehnismns miy lso be imsportint in HP. Tlhuis, in hulmtlln hypersenisitivity disese, there is L)reliminry evidene of' lolied humniorl ind elluilr immuiwtnity in the pthogenesis of'the dlisordler. The onsequienes of'this T-ell tumutiltion in HP re uniiknowni, lthouiglh it is possible tht tivtion of these ells to liberte lol meditors my provide stimulus for grnulom formtion, eosiinophil u muititi oi nl/or ell injtury. Bsed on the highi rtio of T/B lymplhoytes foutnd in HP lvge fluid ompred to blood (Fig. 4), T ells ppered to be seqtiestered in ffeted pulmonry tisstue. This find(iniig ws even more striking when omlpred to the reent stuidies of' Dniele et l. (3) wlho noted tht in norml hui1mins, bronho-lveolr T/B rtios were the sml1e s in 1)lood. These lymiiplhovte restilts together witlh the immutnoglobuilin dt in both IPF ind HP stuggest tht the luing ni fuintion prtilly independent of systemi htumorl- nnd ellmedited immuniiie systemiis. Ltung biopsies were vilble fromii 17 of the IPF ptients und f'romn 3 of the HP ptients. In most, the biopsy ws done somiie montlhs before the lvge sttdly. Nevertheless, in 17 of' the 2 ptients in whomn biopsy ws perfori'med, the ells in the lveoli nd smll irwys were similr nd in smiie reltive proportions s fouind( in ell differentil ouints ol)tined from bronho-lveolr lvge. Thuts, ouir findinigs orroborte those of' Dvis et l. (31) who fouind ell ontent of lvge to be similr to lveolr ellulir onitent in ptients witlh interstitil pneui mionitis. It is doubtful tht nlysis of bronho-lveolr lvge fluiid n ever give s mu1iiih inforimtion bout 1)reihyml pthology in lutng disese s n n open luing biopsy speimen. However, wheres open lutng biopsy is uisuilly done only one to estblish dignosis, bronho-lveolr lvge does permit periodi ssessment of' the disese proess with little risk nd disomfort to the ptient. Thtus, nlysis of lvge fluiid from ptients with fibroti lutng disese dds noth-er dimension to evltutioni of these disorders. Lvge fluiid represents the epithelil milieui of the luing prenhym nd therefore gives fturther insight into lol phenomen using the lutng disese. It my prove uiseftul in following the effet of therpy Oil

11 these disorders. Most importntly, the differenes between the ellulr nlyses in HP ompred to IPF nd ontrols indite tht bronho-lveolr lvge otuld be uiseftul dignostilly in tegoriing ptients with ptulmonry fibrosis exen when speifi orgni ntigens nnot be redily identified. AKNOWLE DGM ENTS We thnk Dotors Norton Elson, Joel Moss, nd Edwrd Stool who did the lvge proedtures; Dr. Frit Weiuel of the Mrshfield lini for serologi stuidies; Dr. Anthony S. Fuii nd Dr. hrles H. Kirkptrik for their dvie; Ms. Thelmil Gither, Ms. Ptrii A. Johnson, nd Mr. Geoffrey Htudson for tehnil help; Dr. Dvid W. Alling for help with the sttistil nlysis; nd Mrs. Nny Wyne, Mrs. Joy Brhers, nd Mrs. Kristen ook for their editoril ssistne. REFERENES 1. Liebow, A. A., nd. B. rrington, The interstitil pneumonis. In Frontiers of Ptulmonry Rdiology. MI. Simon, E. J. Pothen, nd M. Lemy, editors. Grune & Strtton, In. New York Fulmer, J. D., nd R. G. rystl The biohemil bsis of pulmonry funtion. 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