Document Qg882zke7gmeBo67oaanyXQek

I JVMOoaUlruyRmNAeAn1nL, NLOiueFmbAebieEir,R4IOn, cS1.O9, 8PL8uMbliEsDheIrCsINE nfant, child, and >r.Thejournai i? immunoli .s the impar.. Vf.D. ! Syndrome d " nrciseilt...ani, ernie Lupus Rheumatoid lunology. ipetence in i Lung ClearaSncoeluoblfeInPhaartliecdleIsnsoluble and G. OBERDORSTER DepUarntimveernst6it0oy1foBEfiloRmpowhcyhoseoiscdtse,Ar,vEeSnncvuhiero,oonRlmooecfnhMteaseltedHric,eianNletYha1nS4dc6ieD4n2ecnetsisCtreyn, ter, Table of Contents Introduction Overview of Clearance Mechanisms Tracheobronchial Clearance Alveolar Clearance Insoluble Particles Soluble Particle s Concluding Remarks References Page 289 291 293 298 298 307 317 320 INTRODUCTION E f fic ie n t elim ination of inhaled soluble and insoluble p a rticu la te compounds deposited in the resp irato ry tra c t is necessary to keep it s mucosal surfaces clean and fu n c tio n a lly in ta c t. In addition, the c h a ra c te ris tic clearance processes and kin e tic s p revailin g in d iffe re n t regions of the resp ira to ry tra c t determine the retained dose of an inhaled substance in structures of the resp irato ry system. Thus, whether deposition and subsequent retention of inhaled p a rtic le s is considered in c lin ic a l situ atio n s involving diagnostic or therapeutic aerosols or in toxicology with occupational or environmental pollutant aerosols, knowledge of the clearance of these substances is crucial in understanding th e ir resp ira to ry tra c t dosimetry. Clearance mechanisms are not only d iffe re n t for d iffe re n t regions of the resp irato ry t r a c t , but also depend on physico-chemical c h a ra c te ris tic s of the deposited p a rticu la te m aterial.Several recent review a r t ic le s provide detailed descriptions and discussions of d iffe re n t processes governing the removal o f Inhaled solid and solute p a rtic le s a fte r deposition in the resp irato ry tra c t (Lauweryns and Baert, 1977; Morrow, 1977; Camner, 1980; Jones g i a l . , 1982; Effros and Mason, 1983; Jones, 1984; Pavia, 1984; Morrow and Yu, 1985; Schlesinger, 1985; Brain, 1985, 1986; Cuddihy and Yeh, 1988). Because of the multitude and complexity o f the diverse resp ira to ry tra c t clearance processes, the present a r t ic le w ill s e le c tiv e ly review aspects of soluble and insoluble p a rtic le removal from tracheobronchial and deep lung regions. In addition, the a r t ic le emphasizesc lin ic a l and toxico log ical im plications of these removal processes and considers some unresolved issues related to lung clearance. Key words: p a rtic le s ; tracheobronchial clearance; pulmonary clearance; s o lu b ilit y ; a lv e o la r macrophage; phagocytosis; p a rtic le overload; e p ith e lia l perm eab ility 289 Figure 1 is a schematic overview of clearance mechanisms, separated by those for insoluble and for soluble p a rtic le s . This scheme is sim plified in so far as i t assumes that insoluble p a rtic le s are not dissolved in the lung (no in vivo s o lu b ilit y ) and that inhaled soluble p a rtic le s w ill not remain in p a rtic u la te form a fte r deposition 1n the lung (high in vivo s o lu b ilit y ). One might predict that compounds of low water s o lu b ilit y are cleared from the lung more slowly than compounds of high water s o lu b ilit y . However, many "insolu b le" p a rtic le s show in vivo dissolution in the lung, very lik e ly w ithin the phagolysosomes of alve o la r macrophages due to the low pH (Lundborg si 41-, 1984). Thus, many solid p a rticle s are cleared from the lungs involving mechanisms for both insoluble p a rtic le s and solutes. Water s o lu b ilit y of an Fig. 1: Elimination of Inhaled Particles: Clearance Mechanisms Schematic representation of clearance mechanisms of Inhaled p a rtic le s deposited in the lung. "In so lu b le " and "so lub le" refers to in vivo s o lu b ilit y rather than s o lu b ilit y 1n water. Since most "In so lu b le " p a rtic le s undergo dissolution in the lung to some degree, the dissolved m aterial from " insoluble" p a rtic le s and from solid p a rtic le s with rapid 1q vivo d issolution w ill be cleared by pathways lis te d under soluble p a rtic le s . Dissolution of p a rtic le s of low in vivo s o lu b ilit y can become an important clearance mechanism a fte r retention of months and years in the a lve o la r and in t e r s t it i a l compartment. The trachea and lymph/blood c irc u la tio n are the fin a l pathways for elim ination out of the lung. Numbers 0-23 re fe r to respective generations in W eibel's lung model. 290 inhaled com lung. A f compounds m of rats rap less than < wi th a re (Oberdorste very low whalftime, w a l . , 1981) mechanical dissolved ; mechani sms low water : at a fast r rats (Oben the lung a nor in vi_y rate of a c Mercer as a funct p a rtic le me material ir pointed ou: for some needed bef p a rtic le s , quite d iff layer of t c e lls , or i ncreased possibly di The TcRadiologici i ncorporatf lung and r V compoun considered compounds (Class W) With this to th e ir pi The IC mechani cal general re following A (Morrow, 1 f n is the bn the cl correlated b, The ma region is by Diified ie lung lain in 1. One ie lung Dluble" i n the t a l -, /olving of an nOg* n -L3Qa u QO1. rst. cells) ling cells) josi ted rather >lution tid es red by low in ion of lea and } f the inhaled compound alone is not a good predictor of it s clearance behavior in the lung. A few examples on the pulmonary retention k in e tic s of heavy metal compounds may illu s t r a t e th is : Water soluble N iC l2 is cleared from the lungs of rats rap id ly (English fii 1, 1981) corresponding to a retention halftim e of less than one day, whereas water soluble CdCl2 is retained in the ra t lung with a retention halftim e of about two months due to chemical binding (Oberdorster fii 4J.., 1979a). As one might expect, CdO and NiO p a rtic le s of very low water s o lu b ilit y are retained in the rat lung with a long retention halftim e, which is also about two months (Oberdorster g i a l . , 1979a; English gt a l . , 1981). However, whereas NiO lung retention re fle c ts probably mainly mechanical lung clearance of the NiO p a rtic le s , the CdO p a rtic le s are rap id ly dissolved in the lung (Hadley fit a l . , 1980) and are then cleared by the same mechanisms and at the same slow rate as CdCl2 In contrast, ZnO p a rtic le s of low water s o lu b ilit y , which are also rap id ly dissolved in the lung, are cleared at a fast rate corresponding to a pulmonary retention halftim e of six hours in rats (Oberdorster fit a l . , 1979b). Thus, in vivo d issolution of a p a rtic le in the lung can be d is t in c t ly d iffe re n t from water s o lu b ilit y , but neither water nor in vivo s o lu b ilit y are unequivocal predictors of the pulmonary clearance rate of a compound. Mercer (1967) proposed a clearance model based on dissolution of p a rtic le s as a function of th e ir surface area. In his model, knowledge about the p a rtic le mass d is trib u tio n and the nonequilibrium s o lu b ilit y c o e ffic ie n t of the material in lung flu id is required to predict it s clearance from the lung. As pointed out by Morrow (1974), such predictions turned out to be quite accurate for some p a rtic u la te compounds, e .g ., UO2 . yet much more information is needed before th is s o lu b ilit y concept can be applied g enerally to inhaled p a rtic le s . In p a rtic u la r, dissolution of p a rtic le s in the lung is conceivably quite d iffe re n t depending on whether the p a rtic le s are in the liq u id surface layer of the epithelium , phagocytized 1n a lve o la r macrophages, in e p ith e lia l c e lls , or in the in te rstitiu rn . For example, Lundborg i a l. (1984) found increased disso lu tio n of p a rtic le s of metal oxides in a lv e o la r macrophages, possibly due to the low pH in th e ir phagolysosomes. The Task Group on Lung Dynamics of the Intern atio nal Commission of Radiological Protection (ICRP, 1966) proposed a lung clearance model which incorporates three classes of compounds with d iffe re n t s o lu b ilit ie s in the deep lung and respective retention halftimes of days, weeks, and years for D, W, and Y compounds. Both absorptive and mechanical clearance mechanisms were considered, and resultin g pulmonary retention halftim es for very soluble compounds (Class D) were given as 0.5 days, for intermediate soluble compounds (Class W) as 50 days and for highly insoluble compounds (Class Y) as 500 days. With this sim p lified clearance model, compounds could be c la s s ifie d with respect to th e ir pulmonary clearance, based so le ly on th e ir physico-chemical nature. The ICRP clearance model assumes that each clearance pathway (absorptive or mechanical) clears a p a rtic u la r compound at a constant rate. Thus, an ICRP general retention equation for the tracheobronchial or pulmonary compartment following a single inhalation exposure can be w ritten as: At - A 0 I f n e x p (- b pt ) n ( 1) (Morrow, 1977) where A^ and A0 is the amount retained at time t and time 0, f n is the fra ctio n of A0 cleared with each of n clearance pathways, and bn the clearance rate for each of n clearance pathways; bn is in versely correlated with the retention halftim e T-|/2: In 2 Tl/2 ( 2) OVERVIEW OF CLEARANCE MECHANISMS The main clearance pathway for insoluble p a rtic le s in the tracheobronchial region is the m ucociliary escalator consisting of the c ilia t e d epithelium moving a mucous layer on top of i t . This layer is composed of a sol phase of low v is c o s ity (hypophase) in which the c i l i a beat, and a blanket of an overlaying gel phase of high v is c o s ity (epiphase) which is thought to be moved by c i l i a r y motion towards the pharynx. Although i t is g enerally believed that this mucous blanket is continuous throughout the resp ira to ry t r a c t , contrasting observations in rats have been made which led to the suggestion that th is layer may be discontinuous under normal conditions and becomes more widespread only under conditions of bronchitis (Ira v a n i and van As, 1972; van As and Hebster, 1974; van As, 1980). In support of his observation of mucus is le ts in the conducting airways, van As (1980) reasoned that extremely high v e lo c itie s of tracheal mucus would be necessary to accommodate clearanceof a continuous mucous blanket o rig in atin g in the terminal bronchioles with th e ir large aggregated circumference. Obviously, such discontinuous mucous layer would make m ucociliary clearance of p a rtic le s deposited in the tracheobronchial rjegion less e ff ic ie n t . Other investig ato rs could not confirm the discontinuous mucous blanket, but did find a lack of the high v is c o s ity epiphase in the bronchioles (G il and Weibel, 1971; Lu ch tel, 1982) which would also reduce the e ffic ie n c y of m ucociliary movement in these generations. Clearance of p a rtic le s depositing in the tracheobronchial region also occurs through phagocytosis by airway macrophages which are eith e r a lv e o la r macrophages moving up with the m ucociliary escalator (Lehnert and Sanz-Rodriguez, 1988) or macrophages entering the airways v ia bronchial and bronchiolar mucosa (Morrow, 1974; Robertson, 1980). The importance of these macrophages for p a rtic u la te clearance from the conducting airways as well as th e ir orig in needs further in vestig atio n (B ra in , 1988; Gehr et a l ., 1988). Another clearance mechanism, which is less important in terms of the amount being cleared in the tracheobronchial region, is penetration of insoluble p a rtic le s of submicronic size through the epithelium (Gore and P a tric k , 1982), very lik e ly due to endocytosis by e p ith e lia l c e lls of the conducting airways (Sorokin and Brain, 1,974). In contrast to the resu lts with small submicronic p a rtic le s , no evidence was found that bigger p a rtic le s (7.9 pm) penetrated the bronchial epithelium (Velasquez and Morrow, 1984). F in a lly , cough can be a very e ff ic ie n t clearance mechanism in the conducting airways (L e ith , 1977; Khler at a l . , 1986), but is probably lim ited to the upper generations of the conducting airways (Mossberg, 1980). However, formation of a flow lim itin g segment (FLS) in the trachea or upper generation bronchi due to repeated frequent coughing maneuvers could a c tu a lly lead to a decrease in m ucociliary clearance (Smaldone a t a l . , 1979; Smaldone, 1986). This observation corroborates c lin ic a l experience in patients with chronic obstructive pulmonary disease and asthma who show regional defects in clearance associated with the location of FLS (Smaldone, 1986). Soluble p a rtic le s depositing in the tracheobronchial tree are mainly cleared by absorptive mechanisms consisting of tra n se p ith e lia l permeation via in te r c e llu la r pathways (tig h t In t e r c e llu la r jun ctions) or by a ctive and passive tra n s c e llu la r transport (B h a lla and Crocker, 1986). In addition, mechanical clearance along the m ucociliary escalator or cough can contribute to tracheobronchial clearance of inhaled solutes. Chemical reactions can also influence the rates of clearance of soluble substances from the tracheobronchial tre e , i . e . , reactions with and binding to c e llu la r and e x tra c e llu la r components. The most e ffe c tiv e clearance mechanism for insoluble p a rtic le s in the a lv e o la r region is phagocytosis by alve o la r macrophages (AM) (Fe rin a t a l-, 1965; Green, 1973; Morrow, 1973; Hocking and Golde, 1979; Robertson, 1980; Herscowitz, 1985; van Furth, 1985; Brain, 1986) and transport in turn to the m ucociliary escalator for removal towards the larynx. Some AM may penetrate back Into the In te rs titiu m (H o lt, 1980; Corry a t a l . , 1984), from where the p a rtic le laden AM may reach the regional lymphnodes (Harmsen a t a l . , 1985) although this 1s disputed (Adamson and Bowden, 1978; Lehnert a t a l . , 1986). AM - p a rtic le encounters in the deep lung could be f a c ilita t e d through the a ctiva tio n of serum components by deposited p a rtic le s which then act as chemoattractants for AM (W arheit, a t a l- , 1985, 1988). Likewise, chemotropism may be responsible for migration of AM towards the m ucociliary escalator, althou Baert, epithe cleara . fibers Baert, nonfib across remova mechan surfac respir Brain, study and br Tr subste the r transp rate hydrop 1972a, for s Schani depenc affed volumf epithe later solute molect C clean; reach; inter (Fig. endoc. endot lymph fiber Sebas W w ill lymph clear resul oncot lymph and B inter Indee more more via 1 Depem inter p depos 292 although such chemotactic movement has not been demonstrated (Lauweryns and Baert, 1977). Endocytosis by type I - and to a lesser degree by type I I e p ith e lia l c e lls and subsequent exocytosis into the in te rstitiu m is another clearance pathway in the deep lung by which deposited smaller p a rtic le s and fib ers can reach in t e r s t it ia l site s (Greenberg s i a l . , 1972; Lauweryns and . Baert, 1974; Sorokin and Brain, 1974; Brody, 1979; 1981), whereas larger nonfibrous p a rtic le s (>9 pm) are probably less lik e ly to be translocated across the epithelium (Snipes and Clem, 1981; Snipes s i a l . , 1984). For the removal of free p a rtic le s from resp irato ry bronchioles and a lve o la r ducts a mechanical mechanism caused by a drag or flu id flux supported by continuous surfactant production, cranial movement of the m ucociliary layer and the tid a l resp irato ry movement of the lung was suggested (M acklin, 1955; C asarett, I960; Brain, 1970; Robertson, 1980) but never proven experim entally. The re s u lt of a study by Faridy (1976) showing cranial movement of surfactant into bronchioli and bronchi during v e n tila tio n of rat lungs support this suggestion. Transepithelial transport is the major mechanism for clearance of soluble substances from the alve o la r region. In t e r c e llu la r d iffu sio n al transport in the region of tig h t junctions as well as a ctive and passive tra n s c e l1ular transport is involved (Lauweryns and Baert, 1977). Major determinants for the rate at which these absorptive processes occur are the 1ipophi1i c i t y and h yd ro p h ilicity of the solutes and th e ir molecular size (Enna and Schanker, 1972a,b). Saturable, c a rrie r type transport processes have also been described for some anionic compounds including the amino acid glycoleucine (L in and Schanker, 1981), and for inorganic cations such as Cd a calcium channel dependent uptake into c e lls can exist (Hinkle s i a l - , 1987). Other factors a ffe ctin g pulmonary clearance rates of hydrophilic substances Include lung volume, e p ith e lia l surface area and d is trib u tio n of the substance in the e p ith e lia l surfactant layer (E ffro s and Mason, 1983) which w ill be discussed la te r . Endocytosis by AM and Type I e p ith e lia l c e lls can also contribute to solute clearance from the a lveo lar space of the lung, in p a rtic u la r for large molecular weight hydrophilic compounds (B h a lla and Crocker, 1987). Clearance mechanisms 1n the alve o la r space provide an e ffe c tiv e means to cleanse the a lve o la r epithelium of insoluble p a rtic le s . Only a small fra ctio n reaches the in te rstitiu m under normal conditions. Once they have reached the in te rstitiu m , several mechanisms can contribute to th e ir further removal (F ig . 1). These include phagocytosis by I n t e r s t it ia l macrophages (IM ), endocytosis and luminal exocytosis of small (< 0.1 pm) p a rtic le s by endothelial c e lls , directed fluid flux via cle fts of in te rce llu la r junctions of lymph c a p illa r ie s , and d ire c t mechanical movement (fo r example of mineral fib e rs ) through the in te rstitiu m (Morrow, 1972, Lauweryns and Baert, 1977; Sebastien s i a l - . 1979; Leak, 1980). Water-soluble substances that reach the in te rs titiu m , on the other hand, w ill be cleared by diffu sio n into blood c a p illa r ie s and to a lesser degree into lymph c a p illa r ie s depending on th e ir molecular size. The major determinants of clearance of hydrophilic solutes into c a p illa r ie s are the S ta rlin g forces which re s u lt from the balance between in travascu lar and in t e r s t it i a l hydrostatic and oncotic pressures (S ta r lin g , 1896). Since the In t e r c e llu la r junctions of the lymph c a p illa r ie s are less tig h t than those of the blood c a p illa r ie s (Lauweryns and Baert, 1969) one would predict that larger sized hydrophilic solutes in the in t e r s t it ia l space - lik e proteins - are cleared p re fe re n tia lly into lymphatics. Indeed, Meyer s i a l. (1969) calculated lymphatic uptake to be almost 40 times more e ffe c tiv e than absorption v ia blood c a p illa r ie s ; however, q u a n tita tiv e ly more of such solutes were found to be cleared v ia the blood c irc u la tio n than via lymph due to the high blood flow to lymph flow ra tio (Meyer s i s i . , 1977). Depending on the chemical nature of solutes, binding to e p ith e lia l or in t e r s t it i a l c e ll structures can occur and delay th e ir clearance. TRACHEOBRONCHIAL CLEARANCE Figure 2 shows a hypothetical retention curve of inhaled insoluble p a rtic le s deposited in the lower resp irato ry tra c t, 1.e ., from trachea to a lv e o li. Two FIGURE 2 Hypothetical Retention of P a rtic le s in the Lower Respiratory Tract I t is assumed in th is case that 80% of the deposited p a rtic le mass clears rap id ly from the tracheobronchial tree (retentio n halftim e 0.3 days) and 20% more slowly from the a lve o la r region (retentio n halftim e 50 days). Alveolar deposition is assumed to be represented by the intercep t of the slow clearance phase with the R-axis. Both tracheobronchial and a lve o la r retention could probably be described more accurately by increasing the numbers of exponential terms in the retention equation. d is tin c t phases can be distinguished, a rapid phase which in this case clears 80% of the deposited m aterial with a retention halftim e of 0.3 days and a slow phase with a retention halftim e of 50 days for 20% of the deposit. The r e la tiv e amount being cleared in eith e r phase depends on the amount deposited in the tracheobronchial and in the pulmonary region, which in turn is a function of the c h a ra c te ris tic s of the p a rtic le and of breathing (Heyder, 1982). The rapid phase of clearance is u su ally regarded as re fle c tin g tracheobronchial clearance, which is presumed to be e s s e n tia lly complete 24 hours a fte r the exposure. As pointed out by Morrow and Yu (1985), a better f i t could be achieved by a three exponential term, and additional exponents In the general equation ( 1) would improve the f i t s t i l l more. Therefore, the numbers of exponents one could use is a rb itra ry since there appears to be no physico-chemical or physiologic basis for selectin g a certain number. In this context. Morrow and Yu (1985) discussed also a time dependent tracheobronchial retention fo r which a power function could be applied to describe the retention k in e tic s . The use of exponential terms, however, is very convenient and appealing since they allow clearance k in e tics to be expressed in terms of clearance rates and retention halftim es which are e a s ily understandable. Describing tracheobronchial clearance by several clearance rates may have a physiological basis, since c ilia r y beat frequency increases from terminal bronchioles to trachea resultin g in increasing v e lo c itie s of the mucus (Ira va n i and van As, 1972). Indeed, Hilkey i 1. (1980) estimated from studies in humans that bronchial clearance of inhaled 7.9 pm p a rtic le s was fa s te s t in the central zone, corresponding approximately to generations 1-6 , and slowest for the peripheral zone, approximately generations 14-16. Respective retention 294 hal ftimes experimer clearance proposed experimer di chotomc lin es the vivo con bronchio when the velocity tracheal noni nvas v e lo c iti' parti cle 1 .. 198 Yu and d e e independ and inte veloci ti to gener a veloci Using tl" predicti Both 14-16) t V e lo c iti human c (1979). mucoci1i region of mucu and di 1 halftim es were 1.97 hrs, and 2.62 hrs. The intermediate zone in their, experiment showed a s lig h t ly shorter retention halftim e (1.70 h rs ). Predicted clearance of Inhaled 7.9 pm p a rtic le s based on a tri-zonal clearance model proposed by the same group(Lee at a l - . 1979) agreed reasonably well with th e ir . experimental findings. Their mathematical model is based on W eibel's symmetric dichotomous lung model, a d d itio n a lly assuming that the mucous blanket which lin es the airways is uniformly thick throughout the e n tire lung. Simulating in vivo conditions, transport rates of mucus decrease from trachea to terminal bronchioles In th e ir model and gave the best agreement with experimental data when the v e lo c ity of mucusin the trachea was set at 5.5 mm/min, resultin g in a v e lo c ity in the terminal bronchioles of 4.6 pm/m1n. The applied v e lo c ity for tracheal mucus 1s well within the range of rates measured experimentally with noninvasive radioaerosol boli techniques (Yeates at a l - . 1975). Transport v e lo c itie s of tracheal mucus measured a fte r in tratrach eal in s t illa t io n of test p a rtic le s 1n dogs were about 10 mm/min and in rats about 2 mm/min (F e lic e t t i a l - . 1981). Yu at a l. (1986) proposed a multicompartmental tracheobronchial deposition and clearance model by considering each airway generation as equivalent to an independent m ucociliary escalator with it s own length and transport v e lo c ity and in te ractin g with the other escalators as a linked serie s. The resultin g v e lo c itie s of mucus are sim ila r to the model by Lee at a l . (1979) for trachea to generation 5, but are fa s te r for the rest of the tracheobronchial tree with a v e lo c ity o f the mucus in the terminal bronchioles of 30 pm/min (F ig . 3). Using th e ir model, Yu at al- (1986) could improve the agreement between model prediction and the experimental resu lt by Hilkey at al- (1980). Both models predict the longest retention halftimes in zone I I I (generations 14-16) to be between 2 and 8 hours, apparently in agreement with the prevailing s % r FIGURE 3 Mucociliary Escalator .1t s iW e le )f d nS ?e :d ;e 5is) er Df 3W on V e lo c itie s of mucus in generations 0 (trachea) to 16 (term inal bronchioles) of human conducting airways were modeled by Yu a t al- (1986) and Lee a t al(1979). Both models simulate reasonably well experimental results of m ucociliary escalato r mediated p a rtic le clearance from the tracheobronchial region (modified a fte r Yu at a l - . 1986). V0 and d0 re fe r to the v e lo c ity of mucus and the diameter, re s p e ctive ly , of generation 0 (tra c h e a ), and Vi and di to v e lo c ity and diameter of the i-th generation. 295 v}ew that tracheobronchially deposited p a rtic le s are e s s e n tia lly a ll cleared within 24 hours. However, recent results by Stahlhofen e i 1- (1986) seem to suggest that a substantial portion of tracheobronchially-deposited p a rtic le s may remain in the resp irato ry tra c t of humans beyond 24 hours: When a bolus of uniform 2.9 pm p a rtic le s was inhaled with a breathholding maneuver to a volumetric depth in the lung of ~50 cm3,i . e . , corresponding to the upper generations of the resp irato ry tra c t, a s ig n ific a n t retention of 401 of the deposited p a rtic le s was observed beyond 24 hours (F ig . 4 ). This can be explained by e ith e r a much longer retention of p a rtic le s in c ilia t e d structures of the tracheobronchial tree or else by penetration of 401 of the p a rtic le s into nonciliated airways due to mechanisms of flow-dynamics and d istrib u tio n not yet understood (e .g ., axial core flow, nonuniform asymmetric f i l l i n g ) - in which case postulation of extended tracheobronchial retention is not required. Although prolonged tracheobronchial retention of some p a rtic le s could be explained by factors such as endocytosis of p a rtic le s by e p ith e lia l c e lls , a discontinuous layer of the mucous blanket forming s ite s of in e ffic ie n t clearance, or lack of a mucous epiphase in the bronchioles, the magnitude of the p a rtic le s with prolonged retention (>401 of the deposited mass) is surprising and cannot e a s ily be explained by such mechanisms. Retention due to p a rtic le phagocytosis by airway macrophages which adhere to e p ith e lia l c e lls underneath the mucous layer could possibly be a more lik e ly explanation since the d is trib u tio n of these c e lls along the airways with regard to surface area FIGURE 4 Tracheobronchial Retention of P a rtic le s 2 0 0 cm3 -- 7 0 cm3 50 cm3 Retention of 2.9 pm p a rtic le s in human lower resp ira to ry tra c t a fte r th e ir in h alatio n as boli to volumetric depth of 50, 70 and 200 cm3 (Stahlhofen t a l . , 1986). S ig n ific a n t long-term retention beyond 24 hours occurred even a fte r in halation to volumetric depth of only 50 cm3, presumably reaching the upper airway generations only. The retention of the rad ioactive tracer p a rtic le s was measured by noninvasive external gamma-counting (courtesy of Or. Stah lho fen). Vp is the depth of the aerosol pulse between larynx and the front of the aerosol bolus at end expiration, given in cm3. 296 densi t Sanz-F the a; norma1, (bacte detox1 t aj parti c They i ni t i ; earner; norma littlperip: prolo requi parti trach (1988 an i r asses more regio S to pi deduc funct site size, phys i propt mucoc mucoc Cougi Phys Inha (I S02 03 (1 h2s o . Cig. Phar; (Mos 1984 For density is sim ila r to that of the alve o la r compartment (Lehnert and Sanz-Rodriguez, 1988). I f such macrophage mediated p a rtic u la te retention in the airways can be demonstrated these c e lls then may appear as in h ib ito rs of a normally fa st bronchial clearance. However, phagocytosis of p a rtic le s (b a c te ria , antigens, e tc .) by airway macrophages serves at the same time as a detoxifying and protective mechanism, as pointed out by Brain (1988). Smaldone gt a l. (1988) also reported s ig n ific a n t retention of ra d io a c tiv e ly tagged p a rtic le s presumably in central airways of humans at 24 hours post inhalation . They believe that the p a rtic le s had fa ile d to c le a r from central site s of i n i t i a l deposition since the 24 hour retention pattern observed with a gamma camera correlated with the i n it i a l central deposition pattern. In addition, normal persons who deposited i n i t i a l l y more in the periphery of the lung showed l i t t l e retention in central airways at 24 hours so that movement of p e rip h e ra lly deposited material into central regions cannot account for a prolonged central retention (Smaldone t a l . , 1988). Further studies are required to examine the i n i t i a l location and disposition of the deposited p a rtic le s within the tracheobronchial tree to resolve the controversy of tracheobronchial retention beyond 24 hours. In a recent e d ito r ia l, Foster (1988) dealt in more d e ta il with the question of the 24 hour lung retention as an index of alve o la r deposition. He concluded also that a more accurate assessment of aerosol deposition within central bronchi is needed to determine more p recise ly the effectiveness of m ucociliary function within sp e c ific lung regions. Several investigato rs have studied a lte ra tio n s of m ucociliary clearance due to physiologic, pharmacologic or toxicologic stim u li. In general, as can be deduced from the preceding discussion, the rate of m ucociliary clearance is a function of c i l i a r y a c t iv it y , the v is c o e la s tic properties of mucus and of the s ite of deposition of the inhaled p a rtic le s - which in turn depends on p a rtic le size, breathing parameters, and airway geometry. Changes in biochemical and physical properties of airway secretions and in vagal tone can a ffe c t the properties and thickness of mucus as well as the c i l i a r y a c t iv it y and m ucociliary transport (P a v ia , 1984). Table 1 summarizes factors known to a lte r mucociliary clearance. TABLE 1 Factors Influencing Mucociliary Clearance Increase Decrease Cough Physical exercise Inhalation of inocuous p a rticle s (high concentration, short duration) S02 O3 (man) H^SCLjdow concentration) Cig. smoking ( i n i t i a l l y ) Pharmaceuticals: beta-adrenerg1cs cholinergics aminophyl1in ami loride m ucolytics(?) hi stamine Sleep Age Inhaled gases and p a rtic le s : 02 ;03(r a t) H^S04;(h igh concentration) cig . smoke (chronic) Diseases: bronchitis bronchiectasis asthma bronch. carcinoma cystic fibrosis (?) chronic flow lim itation primary dyskinesia syndrome Pharmaceuticals: anticholinergics (? ) anesthetics aspirin (Mossberg, 1980; Kenoyer i l a i- . 1981; Matthys i L . , 1983; Schlesinger i al.., 1984; Pavia, 1984; Khler i a l . , 1986; Smaldone, 1986; Foster i a l . , 1987). For more detailed information, see Pavia, 1984. 297 ALVEOLAR CLEARANCE Insoluble Particles As previously discussed (F ig . 1), the most e ff ic ie n t and prominent a lveo lar clearance mechanism for insoluble p a rtic le s involves phagocytosis by a lveo lar macrophages (AM). AM are large mononuclear c e lls , representing more than 95-981 of the normal free c e ll population in the a lve o la r area. They o rig in ate from bone marrow precursor c e lls and are carried as monocytes to the lungs where they can mature and m ultiply in the lung in te rstitiu m (P in k e tt fii a l- , 1966; Brain et a l . , il977; Blusse van Oud Alblas and van Furth, 1979; Johnson fii a l- . 1980). AM cah also m ultiply in the alve o la r space (Blusse van Oud Alblas i a l . , 1983; Evans fit a l . , 1986). Depending on the p a rtic le load in the a lv e o li a fte r in halation exposure, there can be an immediate influx of monocytes from blood into the a lv e o li followed by a delayed in flu x of macrophages from in t e r s t it ia l spaces (Adamson and Bowden, 1981). Endocytosis of p a rtic le s by AM involves the recognition of the p a rtic le , adhesion and a ctive phagocytosis (Ueda fit a l. , 1981; Ito fii a l . , 1981; Lehnert and Tech, 1985). A directed movement of AM to the s ite of deposition of p a rtic le s and th e ir subsequent phagocytosis by AM can be f a c ilit a t e d by chemotactic movement, probably involving a c tiv a tio n of the complement cascade in serum by p a rtic le s (Warheit i s i . , 1985, 1988). Although there is no evidence that this e a rly chem otactically f a c ilita t e d AM-particle encounter influences the long term AM mediated clearance of p a rtic le s i t may prevent free p a rtic le s from entering the in te rstitiu m via endocytosis by e p ith e lia l c e lls . Phagocytosis of p a rtic le s leads to metabolic a ctiva tio n of AM (re sp ira to ry burst) which in turn release a battery of mediators of d iffe re n t biological a c t iv it ie s including oxygen m etabolites, neutrophil chemotactic facto rs, lysosomal hydrolases, other proteinases, prostaglandins, plasminogen activa to rs and fib ro b la st growth factors (Bitterman fii a l . , 1982; Nathan, 1987). The rate of phagocytosis seems to depend on p a rtic le size, with an optimum size around 1.5-3 urn and a slower rate of phagocytosis occurring for smaller and for larger p a rtic le s (Holma, 1967; Hahn fii a l . . 1977). AM which have phagocytized p a rtic le s w ill normally reach the m ucociliary e scalato r, whether by random or by directed movement is not known. In vestig atio n of clearance mechanisms of the tra n sitio n a l zone, i. e . the in teractio n of a lve o la r and m ucociliary transport mechanisms, need c e rta in ly more atten tion . The old concept that AM can migrate back into the in te rstitiu m a fte r phagocytosis of p a rtic le s (Cummins and Sladden, 1930; Cole, 1944) was disputed by Lauweryns and Baert (1977) in th e ir comprehensive review of a lve o la r clearance mechanisms and by Lehnert fii al- (1986); however, this concept was recen tly re v ita liz e d by Harmsen si a l. (1985) who found in dogs that a fte r intrabronchial in s t illa t io n of AM containing eith e r green or red fluorescent microspheres that individual macrophages in regional lymphnodes contained almost only p a rtic le s of one fluorescent color, ra re ly both. Thus, the p o s s ib ility of a lve o la r clearance of p a rtic le s within AM to regional lymphnodes should not be excluded but needs additional confirmation. Several authors suggested that AM which have entered the in te rs titiu m re-enter the airways at the level of the c ilia t e d airways (Green, 1973; Tucker et a l . , 1973, K ilburn, 1974; Holt, 1982). However, unequivocal proof of such migratory behavior of p a rtic le laden macrophages is not a v a ila b le . Free p a rtic le s entering the in te rstitiu m can be carried to regional lymphnodes, entering the lymphatic c a p illa r ie s through th e ir in te r c e llu la r c le ft s or a fte r phagocytosis by the lymphatic endothelium (Lauweryns and Baert, 1977; Leak, 1980). There appears to be a lim ita tio n in diameter for p a rtic le s which can be moved along those channels before they accumulate in the lymphnodes. For example, studies with 3-15 pm p a rtic le s in rats and dogs in d icate that 3 and 7 pm p a rtic le s in s t ille d into the lungs can be translocated to regional lymphnodes, but not 9-15 pm p a rtic le s (Snipes and Clem, 1981; Snipes i a l . , 1984). I t remains to be studied whether the lim itin g facto r for transport of larger sized p a rtic le s to lymphnodes lie s in the tra n se p ith e lia l tran sfer or in the translocation within the lymphatic 298 channels. early, studi (Ludwig, 19 penetrate t high degree Fibrous par the postno (Oberdorste(Godwin and lymphnodes burden is h subsequentl; Ferin ( on it s remc high inhale in a high translocati lymphnodes. burdens of completely 1986; Wolff Measured p insoluble , d if f e r cons halftimes r hundred dav a l . , 1983; clearance c can better 1 co o o TVJ c -o OL 0 Mechanical p a rtic le s pulmonary 1983, 1985; was estimt were adjust channels. Penetration of p a rtic le s through lymphnodes was reported in the e a rly studies of Drinker fit a l. (1934), and from th is and other studies (Ludwig, 1971) i t appears that p a rtic le s above 10 pm in diameter hardly penetrate the nodes. Even smaller p a rtic le s of a few pm are retained to a high degree in the lymphnodes (Morrow, 1972), but some w ill penetrate them. Fibrous p a rtic le s were also found to penetrate the lymphnodes and to appear in the postnodal lymph c irc u la tio n , thus reaching the blood circ u la tio n (Oberdorster i a l . , 1988) and leading to a widespread systemic d istrib u tio n (Godwin and Ja g a tic , 1970). Although the fractio n of p a rtic le s penetrating the lymphnodes is minor, i t may become more important when a lung p a rticu la te burden is highly increased under conditions of high p a rtic u la te exposures which subsequently increase p a rticu la te burden of the lymphnodes. Ferin (1972) investigated the e ffe c t of the absolute lung burden of a dust on it s removal rate. He found in th is and other studies (F e rin , 1977), that high inhaled and in s t ille d concentrations of TiO2 p a rtic le s in rats resultin g in a high p a rticu la te burden of several mg/g lung led to a highly increased translocation of those supposedly innocuous p a rtic le s to the thoracic lymphnodes. In addition, several Investig ators reported that at such high lung burdens of p a rtic le s , alve o la r clearance is severely retarded or can even cease completely (Fe rin and Feld stein , 1978; Muhle fit a l . , 1987; Bellmann fit 1 ., 1986; Wolff fit a l . , 1987). This aspect w ill be discussed in more d e ta il la te r. Measured pulmonary clearance rates due to mechanical clearance of highly insoluble p a rtic le s (1-4 pm radiolabeled fused alum ino-silicate p a rtic le s ) d if f e r considerably in d iffe re n t animal species (F ig . 5). Respective retention halftim es range from about 50-100 days in rats, mice and hamsters to several hundred days in dogs, guinea pigs, and man (B a ile y fit a l . , 1985a,b; Snipes fit a l . . 1983; Snipes and McClellan, 1985). I t appears that mechanical lung clearance cannot always be described by monoexponential clearance k in e tic s , but can better be expressed as a multi phasic process. This is best illu s tr a te d by FIGURE 5 Lung Clearance of Highly Insoluble P a rtic le s Mechanical lung clearance of respirable radiolabeled fused alum ino-silicate p a rtic le s - excluding disso lu tio n - was estimated in d iffe re n t species by pulmonary retention measurements over several hundred days (Snipes fit a l . , 1983, 1985; B a ile y fii 1-, 1985a,b). Low i_n vivo disso lu tio n of the p a rticle s was estimated from urinary excretion data of the la b e l, and retention curves were adjusted for th is in vivo d isso lu tio n. resu lts of Snipes fit fii. (1983) in rats (F ig . 5) which shows estimated mechanical clearance of inhaled 134cs-label ed fused alu m in osilicate p a rtic le s from retention measurements of up to 850 days. About 5% of the i n i t i a l lung burden of a rat was estimated to be cleared mechanically with a long-term retention halftime of about 700 days, whereas the retention halftim e for most of the pulmonary p a rtic u la te burden changed with time from about 35 days during the f i r s t week a fte r exposure to about 120 days a fte r 6 months and to over 450 days a fte r more than one year. A s im ila r prolongation in retention halftimes in rats with time was reported by B a ile y s i 1. (1985a), with halftimes of 32 days in the early phase of clearance and of 173 days a fte r one year. Reported monophasic retention halftimes in rats of about 50-70 days for tracer p a rtic le s in other studies (F e rin , 1978; Oberdorster fii f i i. , 1984; Lehnert and Morrow, 1985; Bellmann fit al.., 1986) might be due to the fa ct that clearance was followed only for 120 days or less by those in vestig ato rs. As can be seen from Fig. 5, a constant clearance rate (monoexponential) with a corresponding retention halftim e of about 60 days w ill very well describe the pulmonary retention c h a ra c te ris tic s of insoluble p a rtic le s in the rat up to day 120. Multiphasic a lve o la r clearance curves, although with d iffe re n t parameters and rates changing not as much as in the r a t, were also found to f i t observed data in dogs, guinea pigs, hamsters and mice (Snipes fit f ii. , 1983; B a ile y fit al.., 1985a; Kreyllng fit f ii. , 1986, 1988). Likewise, B a ile y fii 1. (1985b) estimated from th e ir studies in humans pulmonary mechanical clearance rates which changed with time (Fig. 6), s ta rtin g at about 4 x 10" 3 per day (equivalent to a retention halftim e of about 170 days) and decreasing to a constant rate of about 1 x 10-3 per day (about 700 day retention halftim e) at 300 days post inhalation . Since dissolution of the highly insoluble p a rtic le s used in th e ir study and subsequent absorption of the dissolved material was estimated to occur at rates approaching the mechanical long-term clearance rates, this was taken into account in those studies by measurement of in v itr o s o lu b ilit y and in vivo urinary excretion rates^ In addition, an estimated clearance due to transport into regional lymph nodes was considered in the calcu lations of B a ile y fii 1. (1985). I t appears that several hundred days a fte r Inhalation of even highly insoluble p articles dissolution becomes a major clearance mechanism in the lung (Kreyling g i a l. , 1988). However, lack of knowledge of exact in vivo d issolution rates and the p o s s ib ilit y of chemical reactions of the dissolved labels in the lung are factors of uncertainty in the estimates of mechanical clearance rates. Bohning fii fii. (1982) could describe e ffe c tiv e clearance rates (including mechanical clearance and disso lu tio n ) of Sr-85-labeled polystyrene p a rtic le s in normal humans by two exponential phases, with 27X of the p a rtic le s being cleared with a corresponding retention halftim e of 30 days and the rest with a halftime of 296 days. In th e ir opinion, this suggests the existence of two d iffe re n t mechanisms of AM-mediated clearance, which could also be consistent with the data of B a ile y i fii. (1985b). Based on the results of Baile y fii 1. (1985b) and s im ila r results in humans with labeled teflon p a rtic le s by Phi 1ipson fii fii. (1985), Cuddihy and Yeh (1988) proposed a mathematical model which makes use of variab le clearance rates that are expressed 1n terms of the changing fraction s of the remaining lung tissue burdens cleared per day. They suggested that the mechanical clearance rates for insoluble p a r title s in man, M (t), vary with time according to: M (t) - 0.005e--02t + o .001 (3) (Cuddihy and Yeh, 1988). As one can see, at la te time points a fte r inhalation (>200 days) the clearance rate approaches a constant corresponding to a retention halftim e of about 700 days (see also Fig. 6). A comparison of retention data of d iffe re n t p a rtic u la te m aterials (B a ile y et a l . , 1985b) and the studies described in the preceding paragraph support the view that in a given animalspecies AM mediated clearance changes with time and is independent of the p a rticu la te compound as long as the compound exhibits very low in vivo s o lu b ilit y and c y to to x ic ity . Results of some other studies do not seem to f i t thisconcept. For example, studies in ra ts , dogs and humans with inhaled m etallic iron, Fe203, Fe304, UO2 orsoot p a rtic le s showed a 300 o .a 4x1 0 Vo co o 3x 10 r0a. c 1 2x1 C oo 'Joo0cZ 1 x1 C 2 o co s 3 CL Fractions c d a ily from correction 1985b). A particles 1 particles b estimated d' by the auth both p a rtic particles i as 6.9 x I urn p a rtic le be 3.5 x Radiologi ca 1 - 2 x 10- pulmonary r and Arnett, a l . , 1979; ' di ssolution of the obse retention particles s with low l affected b; however, nc mediated c to x ic ity at common mech The cl inhalation cal from ?n of Ftime Dnary week more time jarly -ition .dies al., s or ance days uble and a in ;85a; from wi th tion TO"3 ince uent hi ng hose tion "h c ies al.. the :tors di ng s in :d le of :rent data .mans l) : are dens uble ;tion tion y t vi ew d is not wi th :a FIGURE 6 Alveo lar Mechanical Clearance Rates v Days after Exposure Fractions of 1 and 4 pm labeled fused alum ino-silicate p a rtic le s cleared d a ily from the pulmonary region of the human lung were estimated a fte r correction fo r disso lu tio n and transport to lymph nodes (a f te r B a ile y s i a l . , 1985b). A s lig h t ly slower mechanical clearance rate estimated for the 1 pm p a rtic le s up to day 100 and a s lig h t ly slower clearance rate for 4 pm p a rtic le s beyond day 200 was found; y e t, in view of the uncertainties in the estimated disso lu tio n rates, these differences were not regarded as s ig n ific a n t by the authors (B a ile y a l a l- , 1985b). Thus, an averaged clearance rate for both p a rtic le s is shown in this fig u re. Fractional d isso lu tio n rates for these p a rtic le s in the human lung were estimated from urinary excretion of the label as 6.9 x 10-4 per day for 1 pm p a rtic le s and as 2.2 x 10-4 per day for 4 pm p a rtic le s ; fra ctio n a l mechanical transport to lymph nodes was assumed to be 3.5 x 10-4 per day as proposed by the Intern atio nal Commission on Radiological Protection (ICRP, 1966), which is in the same range as values of 1 - 2 x 10~4 per day estimated for th is translocation pathway from dog studies. pulmonary retention halftim e in a ll three species of about 60-100 days (A lb ert and A rnett, 1955; LaBelle and Brieg er, 1961; Gibb and Morrow, 1962; Cohen t 4l . , 1979; Chan e i a l- , 1984). However, i t is not known to what extent p a rtic le dissolution contributed to these retention halftim es; in addition, the length of the observation period may not have been long enough to determine a longer retention halftim e as discussed above. Other studies with inhaled UO2 p a rtic le s showed a high co rrelatio n of pulmonary clearance rates in rats even with low UO2 lung burden (Downs a t a l- , 1967). Whether AM function was affected by the low UO2 lung burdens (45-410 pg/g lung) is not known, however, no pathological e ffects in the lungs were observed. Possib ly, AM mediated clearance is s p e c ific for some p a rtic u la te compounds of very low to x ic ity and of low In vivo s o lu b ilit y thus making i t d if f ic u lt to apply a common mechanical pulmonary clearance rate to a ll p a rtic le s of th is kind. The clearance k in e tic s discussed above were observed a fte r a single in halation exposure to p a rtic le s of low to x ic ity (b io lo g ic a lly " in e r t " ) leading 3 to a low p a rticu la te lung burden of a few micrograms per gram lung tissue. However, for b io lo g ic a lly a ctiv e (o r toxic) p a rtic le s - e .g ., s ilic a - lung clearance could be quite d iffe re n t. A d d itio n a lly, e ffe c ts of such toxic p a rtic le s on AM could lead to an impairment of lung clearance of other "in e rt" p a r t ic le s , as demonstrated by the prolongation of Fe203 p a rtic le ( " in e r t " ) clearance a fte r long-term exposure to low concentrations of NiO and CdO (to x ic ) aerosols was observed (Oberdorster and Hochrainer, 1980a,b). As mentioned before, exposure even to so called " in e r t " or "nuisance" parft ic le s at high concentrations resultin g in lung burdens in the mg/g lung t it>ssue range can lead to s ig n ific a n t retardation or even to sta sis of p a rtic u la te lung clearance. For example, i t was reported that the elim ination of p a rtic le s from the a lve o la r compartment was decreased in rats exposed ch ro n ica lly to high concentrations of TiC>2 p a rtic le s (Bellmann et a l . , 1986; Muhle e i al.., 1988). The in a b ilit y of AM to elim inate the deposited p a rtic le s resulted in a much higher lung burden of inhaled T1O2 p a rtic le s than predicted from retention data derived from lower le ve ls of exposure. Concurrently, re d is trib u tio n of p a rtic le s in to In te rs titiu m and lymphatic tissue was also found, confirming e a r lie r resu lts by Ferin (1977) that a high p a rtic u la te lung bufden w ill lead to an increased accumulation of p a rtic le s in regional lymph nodes. As evidenced by many long-term studies in rats in which d iffe re n t dust m aterials were used, e .g ., TiO2 . coal dust, carbon p a rtic le s , f l y ash, PVC p a rtic le s , diesel p a rtic le s , photocopier toner p a rtic le s . SiO2 and asbestos (Le Bouffant, 1971; Ferin, 1972; Davis et a l . . 1978; Bolton a l . , 1983; Hehner a l . , 1983; Chan t a l- , 1984; Bellmann a l- , 1986; H o lff a l- , 1987; Lee t a l- , 1987; Muhle a t a l- . 1987, 1988), the prolongation of dust retention in thp lungs of laboratory animals exposed for many months to high dust exposure le ve ls appears to be a nonspecific phenomenon seen with many d iffe re n t kinds of p a ftic le s including so-called b io lo g ic a lly " in e r t " dusts. Impaired p a rtic le clearance was t y p ic a lly accompanied by an inflammatory response in the resp ira to ry tra c t, macrophage recruitment and e p ith e lia l c e ll p ro life ra tio n . From such observations of nonspecific in h ib itio n of pulmonary clearance mechanisms the concept of the "overloading" of the pulmonary clearance has emerged, i . e . , the accumulation and persistence of excessive amounts of a non-biodegradable p a rtic u la te material in the lung retards lung clearance and e ven tu ally induces lung in ju ry . This includes - in addition to Inflammatory reactions and macrophage aggregations - the development of f lb r o tic changes and even lung tumors (Campbell, 1937; Martin a t a l . , 1977; McClellan i 4l . , 1986; Heinrich fil a l . , 1986; V o s ta l, 1986; Lee e i a l . , 1985). This concept of overload implies that b io lo g ic a lly " in e rt" p a rtic le s do not e x is t, but that any p a rtic le at s u f fic ie n tly high lung burdens w ill eventually cause lung damage. Underlying biochemical mechanisms of damage include the release of mediators from activated AM and chem otactically recruited neutrophils affectin g tissue functions (e .g ., released oxygen metabolites a ffe ctin g a lve o la r membrane in te g r ity ; Welsh a t a l- , 1986; P it t a t aL-, 1987). I t appears, that a threshold concentration of the dust in the lungs exists above which the e ffec ts of "overloading" occurs, and th is concentration seems to be above 1 mg of dust per gram lung tissue (Bolton a t a l- , 1983; Morrow and Mermelstein, 1988), possibly as high as 2 or 3 mg/g. Although high dust loads of "in e r t " p a rtic le s in the a lv e o la r space lead to adaptive responses including influx of blood-borne macrophages and neutrophils (e a rly phase) and of p ro life ra tin g in t e r s t it ia l macrophages (la t e r phase) into the a lv e o li (Adamson and Bowden, 1981), physical "overloading" of the individual macrophages may eventually occur when they are no longer able to cope with the high dust burden, and the dust clearance function subsides (Bowden, 1987). Lauweryns and Baert (1977) described this as AM reaching an exhaustion stage dependent on the maximal capacity of the plasmalemma, and they report a value of 50% of the c e ll membrane to be capable of incorporating ingested p a rtic le s (Chapman-Andresen, 1963, cited in Lauweryns and Baert, 1977). Recently, Morrow (1988) hypothesized that i t is the volume of the phagocytized p a rtic le s per AM rather than the mass burden per gram lung which in h ib its AM function under conditions of "overload". According to this hypothesis, a maximum phagocytized volume would in cap acitate AM movement. 302 Morrow (196 d etailed di vol ume i s functional 1 recrultment of a rat , that phagoc could 1eac phagocytize overloading retention 1 at low par was determ particles period of retention retention particles p a rtic le s v. recent pilc particles v that AM fi highly immc hardly cap Rheologic c for AM func .1 0 , ao -wco a a> a: The p a rtic (50 pg 0 p a rtic le s subsequent of the ret in the lun translocat lymphatic Morrow (1988) described this as the "volumetric lim it" of the AM in his detailed discussion on dust overloading mechanisms. He suggested that this volume is about 60% of the normal macrophage volume which leaves the AM fu n ctio n a lly impaired, a value which he estimated from published data on AM recruitm ent, lung dust burdens and dust clearance. Considering a normal volume of a ra t AM of about 1000 pm3 (le h n e rt and Morrow, 1984), th is would mean that phagocytosis of 74 p a rtic le s with a diameter of 2.5 pm by a single AM cpuld lead to it s volumetric overloading. A lte rn a tiv e ly , one single phagocytized p a rtic le of 10.5 pm diameter would also re s u lt in volumetric overloading. Indeed, Snipes and Clem (1981) found in rats that the pulmonary rtention halftim es (T 1/2) increased s ig n ific a n tly for large p a rtic le s even at low p a rticu la te lung burdens (F ig . 7): Whereas a normal T1/2 of 70 days was determined for 3 pm p a rtic le s , i t increased to 580 days for 9 pm p a rtic le s and was not measurable for 15 pmp a rtic le s over the observation period of 106 days. S im ila r results were obtained in dogs where the normal retention halftim e of 820 days for 3 pm p a rtic le s increased to very long retention halftimes not measurable over a 120 day period for 7 and 13 pm p a rtic le s (Snipes t a l- , 1984). No proof was provided that the bigger p a rtic le s were a c tu a lly phagocytized by AM. However, we could demonstrate in a recent p ilo t experiment in rats (unpublished re s u lts ) that 10.3 pm polystyrene p a rtic le s were re a d ily phagocytized by AM (F ig . 8). I t is e a s ily conceivable ttiat AM f ille d with a phagocytized p a rtic le volume as shown in Figure 8 wi 11 be highly immobile: the normally ru ffle d surface is stretched smoothly and seems hardly capable of forming pseudopodia or of engulfing additional p a rtic le s . Rheologic c e ll properties such as cytoplasmic v is c o s ity and e la s t ic it y important for AM function (Valberg and Feldman, 1987) might also be disturbed resultin g in FIGURE 7 Lung Clearance of Monodispersed Polystyrene P a rtic le s of D iffe ren t Sizes in Rats (Snipes and Clem, 1981) 1 f r y Days after Instillation The p a rtic le s had been in s t ille d 1n tra tra c h e a lly as a mixture of a ll three sizes (50 pg of 14'Ce labeled 3 pm p a rtic le s ; 5 pg of 3Sr labeled 9 pm p a rtic le s and 2 pg of 4^Sc labeled 15 pm p a rtic le s ) on day 0, and subsequent pulmonary retention was followed for up to 106 days for determination of the retention halftim es. The label of the 3pm p a rtic le s only was found in the lung associated lymphnodes, indicating a size lim ita tio n for p a rtic la te translocation eith e r across the a lve o la r epithelium or within pulmonary lymphatic vessels. 303 FIGURE 8 A lveolar Macrophage in Broncho-Alveolar Lavage of a Rat with Phagocytized 10.3 pm Polystyrene P a r tic le Partie The monodispersed 10.3 ym p a rtic le s had been in s t ille d in tra tra c h e a lly 24 hr p rio r to the broncho-alveolar lavage. Almost a ll of them were found to be phagocytized by AM. C ells were fixed in 21 glutaraldehyde in 0.1 m phosphate buffer and prepared for SEM. a disruption of the cytoskeleton. Possib ly, In less extreme cases as that shown in Fig. 8 - i . e . , with many small p a rtic le s instead of one big p a rtic le - disturbances of AM f lu id it y may not be as severe. ' So fa r , the phenomenon of chronic p a rtic le overload has only been reported in experiments with rats in which high a ir concentrations (>5 mg/m^) of in ert p a rtic le s inhaled over weeks and months resulted in a s ig n ific a n t reduction of AM mediated p a rtic u la te clearance and eventually f ib r o tic lung disease. Many questions remain unanswered at this point, e .g ., is the loss in AM function re v e rsib le ? Does the i n i t i a l adaptive increase in AM numbers seen in acute overload situ atio ns (Adamson and Bowden, 1978) p ersist in chronic overload conditions? What is the significan ce of the dust deposition rate vs. the total lung dust burden? Is there a common pathogenetic mechanism of chronic in t e r s t it i a l lung disease caused by overloading with low to x ic ity dust and by lower lung loads of cytotoxic dust? and f in a lly : what are the im plications for human exposure to high dust le ve ls? Answers to such questions w ill not only shed lig h t on mechanisms involved in overload situ atio ns but are also important for the understanding of basic clearance mechanisms and the pathogenesis of lung diseases. For example, Rom at a l. (1987) and Bowden (1987) discuss the central role of activated AM in the pathogenesis of pneumoconioses a fte r chronic exposure to high concentrations of inorganic dusts since they found under such exposure conditions an increased release from AM of AM derived growth factor and fib ro n ectin which are important for fib ro b la st a ttra c tio n and re p lic a tio n . Predicted k in e tic s of dust retention in the lungs during chronic high exposure conditions are shown in Figure 9 fo r a ra t lung based on the experimental findings ofs e v e ra l. groups (Chan at 41-, 1984; Bellmann e i a l . , 1986; W olff a t 1-. 1987; Muhle a t a l . , 1988). At low to moderate exposure 304 The k in e tic and high e results of Muhle e t al burden sin P a rtic u la te particles ii At low expo a steady si rates accc p a rtic le vc k - alveola volume (Vc, clearance b At the maxi predicted t< become equi' concentrate lung or AM l is equivalei the ra t. H( burdens hav be impairec ("vo lu m etrif P a r t ic le ac but may eve p a rtic le s , clearance parti culate A1though p a rtic le s hi rats as ca: large parti hr be that : 1e - )rted nert )n of Many :tion .cute load otal -onic d by for only tant 1ung trai onic such .ctor n. high the il- . sure The k in e tics of p a rtic le accumulation in the lungs of rats during chronic low and high exposure concentrations of "nuisance" p a rtic le s are derived from results of Chan si i- (1984), Bellmann si i- (1986), Wolff et al_. (1987) and Muhle i a i. (1988). The p a rtic le burden is expressed as volumetric p a rtic le burden since th is seems to correlate with AM m obility (Morrow, 1988). P a rtic u la te burden in whole lung compartment re fle c ts p a rtic le s in AM, free p a rtic le s in the a lve o la r epithelium and p a rtic le s in the pulmonary lymph nodes. At low exposure concentrations, the p a rticu la te burdens in the lung and AM reach a steady s ta te , which can be predicted by the d a ily deposition and clearance rates according to: - V o /k[l-(ex p - kt)], where = accumulated p a rtic le volume at time t ; Vq d a ily deposited volumetric p a rtic le burden; k a lve o la r clearance rate. At high exposure concentrations, when a c r it i c a l volume (Vc r i t ) of phagocytized p a rtic le s is reached, AM mediated p a rtic le clearance begins to slow and accumulation of p a rtic le s in the lung increases. At the maximum phagocytized volume (Vmax), AM mediated p a rtic le clearance is predicted to subside and the rate of accumulation of p a rtic le s in the lung may become equivalent to th e ir deposition rate. concentrations of p a rtic le s , the lung burden - here expressed as volumetric lung or AM burden - w ill reach an equilibrium when the pulmonary clearance rate is equivalent to the pulmonary deposition rate , i . e . , a fte r about 12 months in the ra t. However, during chronic high exposure concentrations when AM p a rtic le burdens have reached a c r it i c a l value, normal AM mediated clearance begins to be impaired and eventually may cease when a maximum phagocytized volume ("volum etric lim it " ) of p a rtic le s is reached in the AM (Morrow, 1988). P a r t ic le accumulation in the lung then does not follow the predicted pattern but may even tu ally become lin e a r re fle c tin g the deposition rate of the inhaled p a rtic le s . Muhle i L . (1988) determined e m p irically that the pulmonary clearance rate of p a rtic le s decreased lin e a r ly with the logarithm of p a rticu la te mass in the lung once a c r it i c a l mass burden had been reached. Although the existence of such overload related impaired lung clearance of p a rtic le s has not been demonstrated in humans i t is probably not re s tric te d to rats as can be deduced from the previously mentioned retention studies with large p a rtic le s in dogs of Snipes si 4l- (1984). Furthermore, Muhle (1988) 305 observed in hamsters an impairment of p a rtic le clearance a fte r exposure to high concentrations of photocopier-toner p articles. I t is conceivable, therefore, that humans occupationally exposed to high dust concentrations on a chronic basis might show signs of "overload" related Impaired p a rtic le clearance and lung damage (Bowden, 1987). For example, i t is well known from studies in coal miners that exposure to excessive amounts of respirable coal mine dust w ill lead to an increased ris k of progressive massive fib ro s is , chronic bronchitis or emphysema (Ruckley i al.., 1984; M ille r and Jacobsen, 1985; Rom e i a l . , 1987). In e a r lie r studies, Nagelschmidt (1965) reported that in ,coa 1 workers extremely high amounts of lung dust of 9 g to over 60 g correlated with the s e ve rity of pulmonary fib ro s is . However, they pointed out in addition that the rank of the coal is of importance for it s flbrogenic p o te n tia l, I t i s in trig u in g to view th is response to chronically-inhaled high levels of coal dust as a consequence of dust overloading of AM mediated clearance in humans analogous to results of rat studies. Indeed, Freedman and Robinson (1988) found in coal workers a decreased clearance of the inhaled coal dust as one iindicator of an e ffe c t of chronic exposure. The demonstrated importance of coal rank for the fibrogenic response may in dicate that c y to to x ic ity of the d iffe re n t coal dusts could also play a role. In contrast to the coal dust, much lower amounts of lung dusts of 2-6 g in fib r o tlc coal workers' lungs were found when 30-501 of the retained dust consisted of the more cytotoxic quartz p a rtic le s (Nagelschmidt, 1965). In view of the results of the animal experiments i t is therefore prudent not to exclude the p o s s ib ility of dust overload e ffec ts in humans. Considering a recommended threshold lim it value (TLV) in the U.S. for occupational exposure to so-called "nuisance dust" of 10 mg/m3 (ACGIH, 1987), a chronic eight hour d a ily exposure to th is permissible level could lead to dust accumulation of several mg dust per gram lung. 1 This should a le rt us to an important issue in occupational health which c a lls for further attention and re-evaluation of -is present nuisance dust standards by health au th o ritie s. Figure 10 depicts a compartmental scheme of p a rtic le translocation in the peripheral lung region under conditions of normal low exposure concentrations and high exposure concentrations (overload). Differences in translocation pathways, r e la tiv e translocated amounts and translocation rates between "normal" ahd "overload" are indicated as well as gaps in our knowledge for such tran slo catio ns. The scheme is sim p lified 1n several respects: P a rtic le s in in te rstitiu m and lymph nodes are placed into the same compartment as representing a common pool of p a rtic le s which have crossed the a lveo lar membrane. Obviously, biological consequences are quite d iffe re n t whether p a rtic le s are in the in te rstitiu m or in lymph nodes. I t is assumed, that released p a rtic le s from dying AM (k 2> w ill be taken up by other AM under normal conditions (k ]) - a repeatedly described concept (Heppleston, 1963; Heppleston and Young, 1973; Lehnert t a l . , 1986) which is supported by recent studies of Lehnert t a l. (1988) - whereas they remain mostly in the free p a rtic le pool under conditions of overload. Based on the suggested immobility of overloaded AM i t is furth er assumed that overloaded AM do not tran sfer into the in te rstitiu m (k4 ) but that the reported in t e r s t it ia l dust accumulation occurs v ia the free p a rtic le pool (k 3) (Lehnert et a l . , 1986; Bowden, 19B7). C le a rly , more studies are needed to elucidate the mechanism of in t e r s t it ia l dust accumulation in overload situ a tio n s. The scheme also sim p lifie s by Assuming (1) these are respirable p a rtic le s (mass median aerodynamic diameter - 3.5 pm with geometric standard deviation of 1.5 ); (2) a (co n se rva tive ly low) pulmonary deposition of 15%; (3) an inhaled volume of 10 m3 per day; (4) a pulmonary retention halftim e of 500 days; and (5) a human lung weight of 950 g ), the accumulated dust concentration in the lung would reach a steady state of 11 mg dust per gram lung, a level which is known from rat studies to cause lung damage due to overload as discussed in the preceding paragraphs. Even the legal OSHA standard in the U.S. of 5 mg/m3 resp irab le p a rtic le s could lead to lung dust accumulation of over 5 mg/g which Is s t i l l an excessive le v e l. showi ng p a rtic le : represent under. no movement overload acute hie Bowden, become e dying. (1988) di esel In (1986) function pulmonar concentr (1984) a i ntroduc very lor (Bolton a small prolonge sequestr plaques" which p< Baert, 1 Most clearanc mechani s transfer dying A quanti fi i ntroduc This moc normal ; a fte r e: as well was rec which c saturabI by Bioz possi b i' wi thi n specific particle adjustat kinetic: attempt: mechani: normal conseque Sol e The number i exchang macromo additioi airborne the pre h I\jn ore, oni c and coal will iti s a].., kers the the is coal ns 988) one ; of the ust, were artz not ng a sure hour 1 of ssue n of be L. mal " such s in as ol ar ther that nder 963; cent free 1ity into tion 87). ti al by eter showing the same designation for slow clearance processes of AM containing p a rtic le s ( kg) for normal and overload conditions; y e t, th is pathway represents more than one clearance mechanism which are very lik e ly d iffe re n t under normal and overload conditions, i . e . , a ctive movement of AM and passive movement due to flu id fluxes. An adaptive increase in AM numbers under chronic ` overload conditions - as suggested in Fig. 10 - may reasonably be assumed from acute high p a rtic le load studies (B ra in , 1971; Adamson and Bowden, 1978, 1980; Bowden, 1987) unless the in t e r s t it i a l macrophage pool and/or blood monocytes become exhausted and cannot replenish AM at the same rate at which they are dying. This seems, however, u n lik e ly since Strom (1984) and Henderson t a l. (1988) found that a fte r two years of exposure of rats to high concentrations of diesel exhaust the numbers of lavagable AM were doubled compared to controls. In an attempt to model overload related clearance phenomena, Yu and Morrow (1986) created a nonlinear mathematical model in which the clearance rate is a function of mass burden in the lung and which could describe increasing pulmonary p a rtic le accumulation observed in ra t studies with high exposure concentrations. Likewise, the nonlinear sequestration model by Chan et 1. (1984) and Strom and Chan (1988) provides a description of overload e ffects by introducing a sequestration compartment - represented by aggregated AM - with a very long retention halftim e. Introduction of a "sequestration" compartment (Bolton fii a l . , 1983) could accommodate both a prolonged long-term retention of a small fra ctio n of p a rtic le s observed under normal conditions (F ig . 5) and the prolonged retention under overload conditions. The anatomical co rrela te of the sequestration compartment in overload situations might be the so-called "dust plaques" (Gross and Hatch, 1962) which could be considered as a rese rvo ir from which p a rtic le s are liberated into the alve o la r in te rstitiu m (Lauweryns and Baert, 1977). Most rece n tly, Stber (1988) proposed a multicompartmental a lveo lar clearance model for insoluble p a rtic le s which takes into account physiologic mechanistic features such as AM life tim e , decreasing macrophage mediated tra n sfer rates with increasing lung dust burden, and p a rtic le release from dying AM with subsequent re-phagocytosis from the e p ith e lia l surface. The q u an tifica tio n of th is la t t e r tran sfer rate with a ll it s consequences was f i r s t introduced in to a model by Stber (1988) who termed i t "reverse tran sfer ra te ." This model may give reasonable predictions of both p a rtic le accumulation during normal and excessive loading and subsequent clearance or ir re v e rs ib le retention a fte r exposure cessation. A d iffe re n t model incorporating p a rtic le deposition as well as the d iffe re n t aspects of p a rtic le clearance discussed in th is section was recen tly described by Smith (1985). This model features clearance rates which change with dust load due to a b io logical feedback system; i t assumes saturable Michaelis-Menten kin e tic s for uptake of p a rtic le s in to AM as described by Biozzi a l. (1953) and takes into account disso lu tio n of p a rtic le s . The p o s s ib ilit y of clearance of free p a rtic le s as well as p a rtic le sequestration within the in t e r s t it i a l compartment are also included. Although th is model was s p e c ific a lly developed for describing the clearance k in e tic s of quartz p a rtic le s and the related development of f ib ro s is , it s parameters are e a s ily adjustable to new data as they become a v a ila b le to model pulmonary clearance k in e tics of p a rtic le s of low c y to to x ic ity . However, these useful modeling attempts should not d is tr a c t from the necessity that more k in e tic and mechanistic experimental studies and data on AM behavior and function under normal and overload conditions are needed to understand the pathogenetic consequences. Soluble Particles (Solutes) The a lv e o la r- c a p illa ry membrane is extremely well endowed to f u l f i l l a number of d iffe re n t physiological tasks. Besides it s function of e f f ic ie n t ly exchanging oxygen and carbon dioxide, i t also provides transport of macromolecules in both d irectio n s. Two major physiological functions, in addition, are the formation of a b a rrie r against the penetration of harmful airborne p a rtic u la te matter a fte r deposition in the a lv e o la r compartment and the prevention of leakage of solutes from c a p illa r ie s into a lv e o la r spaces. 307 Fig. 10: Translocation of highly insoluble particulate material of low cytotoxicity in the distal lung during chronic exposure to low (normal) and very high (overload) particle concentrations in the air. k7 f j ko escalator Low particulate exposure Particulate burden in lung reaches steady state and is nearly equivalent to AM particulate burden (normal) High particulate exposure Particulate burden in AM reaches steady state; particle build up in lung continues with exposure (overload) free particle pool particles in resident and newly arrived AM particles in interstitium and lymph nodes The scheme is complicated by the appearance of numerous PMN in overload conditions which also phagocytize particles yet have a much shorter life span in the alveolar space than AM. In addition, release of mediators from activated AM and PMN affects function of these cells and of the epithelial barrier Dissolution of particles can also play a role, and the dissolution rate depends on the particulate material and on whether it is located in AM, on the epithelium or in the interstitium. Flux of Particulate Mass Fractional Transfer Rate in Translocation Pathway Mechanism of Translocation Normal Overload Overload Compared to Normal Input kg deposition of inhaled particles low high no change unless change in particles yet have a much shorter life span in the alv \r space than AM. In addition, release of mediators from activated AM and PMN affects functi of these cells and of the epithelial barrier. Dissolution of particles can also play a role, and the dissolution rate depends on the particulate material and on whether it is located in AM. on the epithelium or in the interstitium Translocation Pathway Flux of Particulate Mass Mechanism of Translocation Fractional Transfer Rate in Normal Overload Overload Compared to Normal Input k0 deposition of inhaled particles on alveolar epithelium k1 phagocytosis of free particles by resident AM low low high high no change unless change in lung structure or respiration lower k2 release of particles from dying AM into free particle pool low high higher Intrapulmonary k3 translocation transepithelial transport of free particles into interstitium and lymph nodes very low high no change (?) k4 migration of A M containing particles into interstitium and k lymph nodes low none (?) lower k5 fast clearance of mobile AM via mucociliary escalator high none lower Output X k6 slow clearance of AM via mucociliary escalator low low lower k7 removal of free particles via very low low (?) mucociliary escalator no change (?) k8 postnodal clearance of k particles from lymph nodes low low (?) lower (?) Thus * the in te g r ity of the a lv e o la r- e p ith e lia l b a rrie r is important for several physiological functions. Inhaled aerosolized soluble compounds which are deposited in the a lveo lar region are mainly cleared into the in te rs titiu m via tra n s e p ith e lia l transport (F ig . 1). This transport and the resultin g pulmonary retention of these compounds are influenced by th e ir water or lip id s o lu b ilit y , th e ir molecular size and by chemical reactions (e .g ., binding to proteins, c e ll structures) in the lung. Absorption studies with non-reacting lip id - so lu b le and water soluble solutes showed that they are absorbed from a lv e o li by d iffu sio n across a lipoid-pore membrane (T aylor and Gaar, 1970; Enna and Schanker, 1972). Lipid soluble substances of diverse chemical structure and degree of ionization penetrate the a lve o la r epithelium at rates which increase roughly with th e ir lip id water p a rtitio n c o e ffic ie n t, passing e a s ily through the b ilip id cell-membrane rather than the in te r c e llu la r junctions (Jones t al.., 1982). Since th is is a very rapid process the clearance rate of lip o p h ilic compounds from the a lv e o la r space is lim ited mainly by blood perfusion which needs to be s u f f ic ie n t ly high to maintain a concentration gradient. However, pulmonary retention of lip o p h ilic compounds, and possibly h ydroph ilic compounds, can markedly be increased by adsorption onto p a rtic le s . For example, adsorption of benzo-a-pyrene (BaP) onto inhaled diesel p a rtic le s increased the pulmonary retention of BaP one day a fte r inhalation from about 11, when BaP was inhaled alone, to about 501 when i t was adsorbed onto inhaled diesel p a rtic le s (Bond i a l . , 1986) (F ig . 11). Since this longer retention means a prolonged exposure of lung target c e lls to BaP, i t may conceivably augment the adverse e ffe c t of th is carcinogen as suggested by S a f f io t t i et a l. (1968). In general, the prolongation of pulmonary retention of the solute FIGURE 11 E ffe c t of P a rtic le s on Lung Retention Pulmonary retention of a lip o p h ilic organic compound (Benzo-a-pyrene, BaP) is a lte re d depending on whether i t 1s inhaled alone or adsorbed onto inhaled diesel p a rtic le s (a f te r Bond i 1 . , 1986). Although the I n i t i a l dose rate of BaP delivered to target c e lls on day one 1s much higher when the compound is inhaled alone, adsorption onto the p a rtic le s resu lts 1n a higher dose delivered d a ily to target c e lls a fte r day one. Since diesel p a rtic le s are phagocytized by AM the prolonged retention re fle c ts e ith e r BaP s t i l l on the p a rtic le s or retention of BaP a fte r desorption from the p a rtic le s in AM. 310 adsorbed the part ioteracti ' 1mpT1c-ati smoke i physico-c enhance example, nasophan and react In c lim ited "effect! \ e p ith e li; lower me normal a Gaar, 19 Nelson, derived nm and tl assumes by inhal endothe! the regi 1976; Ch molecula rate 1i m and an i exists ( via inti pinocyto' particulc i ntercel transcel tracheob: 1986, 19! Ref 1c to descr barri e r, and a re permeable the corr pulmonar; shows, t hydrophi' than the the pulmi the alve contrast hydrophi impermeat workers : protein lung (Ac protein average transpor Basec noninvas inhaled \ measuremc 99mTc_ i at ra! 1ar ort ese lar in ble a pid 1on ei r pid 2). nds be bly es. 1es iout il ed :ion ably aj_. ute ) is al ed e of d is ered sized s or adsorbed onto p a rtic le s depends on the physico-chemical nature of both the p a rtic le s and the adsorbed compound (Bond e i a l . , 1986). Such physical in teractio ns and resultin g physiological consequences may have p ractical im plications for e ffects of compounds adsorbed onto p a rtic le s in cig arette smoke and in the urban and occupational environment. Likewise, ' physico-chemical reactions of inhaled gaseous compounds with p a rtic le s could enhance the e ffects of the gaseous substance due to a c a r rie r e ffe c t. For example. Inhaled SO2 . which is normally e ff e c tiv e ly absorbed in the nasopharyngeal region, can penetrate the upper airways when i t is absorbed into and reacts with a wet aerosol (M cJilto n a l . , 1976). In contrast to lip o p h ilic solutes, clearance of hydrophilic solutes is lim ited by th e ir diffu sio n rate. Several in vestig ato rs have determined "e ffe c tiv e " pore sizes for water soluble substances in both the a lveo lar e p ith e lia l and the c a p illa r y endothelial layer by using macromolecules and lower molecular weight solutes given in tra tra c h e a lly or intravenously under normal and altered physiological conditions (Taylor t a l . , 1965; Taylor and Gaar, 1970; Enna and Schanker, 1972; Schneeberger, 1976; Jones e i a l . , 1978; Nelson, 1978; Gardiner, 1978; Egan, 1980). From resu lts of such studies i t was derived that pore sizes of the a lveo lar epithelium are in the range of 0 . 6-1.5 nm and those of the endothelium in the range of 2-13 nm. The concept of pores assumes that hydrophilic solutes of low molecular weight deposited in the lung by inhalation w ill diffu se through the alve o la r epithelium and c a p illa ry endothelium via these pores, whose anatomical co rrelate is thought to lie in the region of the In t e r c e llu la r tig h t junctions '(Schneeberger and Karnovsky, 1976; Chopra L-, 1979; Jones t a l . , 1982; Effros and Mason, 1983). The molecular size of the hydrophilic solutes deposited in the lung represents the rate lim itin g factor for d iffu sio n al clearance of those solutes from the lung, and an inverse relation sh ip between clearance rate and solute molecular weight exists (E ffro s and Mason, 1983). In addition to these d iffu sio n a l processes v ia in te r c e llu la r tig h t jun ctions, tra n s c e llu la r transport of solutes by pinocytotlc a c t iv it y 1n e p ith e lia l c e lls occurs (Chinard, 1980). This is p a rtic u la rly important for larg er solute molecules, such as proteins, for which in te r c e llu la r junctions are highly impermeable. An increased tran sfer by this tra n s c e llu la r route was reported following e p ith e lia l damage in the tracheobronchial and alve o la r region a fte r O3 exposure (B h a lla and Crocker, 1986, 1987). R eflectio n c o e ffic ie n ts as a measure of osmotic effectiveness can be used to describe hydrophilic solute perm eability across the alveolar-endothelial b a rrie r, i . e . , a re fle c tio n c o e ffic ie n t of 0 means a fre e ly permeable membrane and a re fle c tio n c o e ffic ie n t of 1 represents an ideal semipermeable membrane, permeable for the solvent, but not the solute. Figure 12 shows schem atically the co rrela tio n between re fle c tio n c o e ffic ie n ts for a lv e o la r epithelium and pulmonary c a p illa r y endothelium for d iffe re n t molecular solute r a d ii. I t shows, that the perm eability properties o f the a lv e o la r c a p illa r y membrane to hydrophilic solutes 1s determined p rim arily by the a lve o la r epithelium rather than the c a p illa r y endothelium. Corresponding perm eability c o e ffic ie n ts for the pulmonary c a p illa r y endothelium are approximately 10 times greater than for the a lve o la r epithelium (Staub, 1974). Thus, the a lv e o la r epithelium , in contrast to the c a p illa r y endothelium, is only s lig h t ly permeable to hydrophilic solutes with molecular weights above 15000 daltons and e s s e n tia lly impermeable to molecules above 40,000 daltons (Jones a l . , 1982). Several workers suggested that the normal alve o la r b a rrie r Is completely impermeable to protein (Egan a t a l- . 1977; Nelson a t a l - 1978; Egan, 1982) lik e in the fetal lung (Adams, 1966). P h y s io lo g ic a lly , however, a f in it e perm eability for protein 1s present, and the results of several studies in dogs showed an average clearance rate for protein of I I per hour (Staub, 1983). Pin ocytotlc transport may contribute to th is rate, as mentioned above. Based on these physiological p rin cip les of e p ith e lia l perm eability, a noninvaslve method of determining the in te g r ity of the a lv e o la r epithelium with Inhaled hydrophilic solutes has been developed. The technique makes use of the measurement of the pulmonary clearance rate of a small molecular weight tra c e r, 99mTC-iabeled diethylenetriam inepentaacetic acid (DTPA), a stable chelator 311 FIGURE 12 Reflectio n C o efficien ts of Alveolar Epithelium and Pulmonary C a p illa ry Endothelium for Hydrophilic Solutes S 5` c The molecular radius for NaCl2 is -0.23 nm, for albumin -3.6 nm (modified a fte r Jones s i a l . , 1982). The re fle c tio n c o e ffic ie n t is the ra tio of the actual osmotic pressure and the ideal osmotic pressure for a given concentration of the solute: A re fle c tio n c o e ffic ie n t of 1 represents an ideal semipermeable membrane, and of 0 a fre e ly permeable membrane. The a lve o la r retention halftim es, Tl/2, shown for the solute diethylenetriam inepentaacetic acid (DTPA) with a molecular radius of 0.57 nm, in dicate a value of about 60 min for the normal a lve o la r epithelium which w ill decrease s u b s ta n tia lly for the damaged a lve o la r epithelium , shown here as a hypothetical dotted lin e . In theory, the a lve o l r epithelium could be damaged to a degree that solute clearance becomes lim ited by the endothelial b a rrie r of the pulmonary c a p illa r ie s , provided, the c a p illa r y endothelium remains unaltered in such situ a tio n s. (Chopra i a l . , 1979; Rinderknecht si a l . , 1980; Jones i a l . , 1982; Coates and O'Brodovich, 1986). As pointed out by Effros and Mason (1983), i t is necessary that solutes used in th is method, lik e DTPA, do not react or bind to sp e c ific site s or cross a c tiv e ly the e p ith e lia l membrane. This tra ce r is inhaled as an aerosol of submicronic p a rtic le s so that they are deposited p rim arily in the a lve o la r region of the lung. Upon deposition of the solute d roplets, they merge With the flu id lin in g the a lve o la r surface, a process which is possibly influenced by surface tension. The molecular size of DTPA of 492 daltons corresponds to a molecular radius of 0.57 nm (Jones i 41-, 1982) and thus DTPA can e a s ily penetrate the endothelial layer (F ig . 12). However, i f unaltered, the tig h t a lve o la r epithelium represents a b a rrie r to DTPA d iffu sio n so that it s clearance occurs at a rate corresponding to a halftim e of about 60-85 minutes' in humans (Mason 4].., 1985; Dusser i a l . , 1984; Rees i a l . , 1985; Marks si a l - . 1985; Langford i a l . , 1986; U te ll a t a l - . 1985). This clearance rate can be determined noninvasively in vivo by gamma camera imaging of the aerosolized 99fir|-c_DTPA deposited in the lung, representing g enerally f i r s t 312 order clear in the bloc indicated ii the r e fle c t 1ung's become been demons origins, i diseases, : (Chopra et 1982; Dussc Kehrl et a l Alveola when no lur volumes (M1986). The alve o la r me deposi t i on Effros and soluble par Ct where ct - cc C0 i n P pe S = ep V - vc I f the rat changes in i ndependent increase i a lv e o la r ep Wool man et Figure 99mTc_ ia b e l different ' min therea (Oberdorste increased ' lung cleara Two minute at normal 1 change subs clearance v. and cleara Fig. 13). a lv e o la r si an increas However, re addi tio n a l1 could poss (E ffro s and Such j from result DTPA diffus lung volume in fl uence aerosol - i spreading diffusion ( order clearance kin e tics since there is i n i t i a l l y l i t t l e build up of a c t iv it y in the blood compartment due to rapid elim ination of DTPA v ia the kidney. As indicated in Figure 12, DTPA retention halftimes w ill become much shorter when the re fle c tio n c o e ffic ie n t of the a lveo lar membrane decreases, i . e . , when the lungs become more "leaky" due to damaged a lv e o la r epithelium. Such damage has been demonstrated in acute and chronic lung in ju rie s of d iffe re n t pathogenetic o rig in s, including inflammatory reactions, edema, in t e r s t it i a l pulmonary diseases, pulmonary emboli, asthma, cig arette smoking, and ozone exposure (Chopra t a l . , 1979; Rinderknecht a i a l- , 1980; Mason, 1985; Dolovich si a l- , 1982; Dusser t a l . , 1984; Mason e_ a l- , 1985; Buxton-Thomas i a l- , 1986; Kehrl et a l . , 1987). A lveolar clearance of DTPA can also increase under physiological conditions when no lung in ju ry is present, e .g ., during exercise and at increased lung volumes (Meignan et a l - , 1986; O'Brodovich i a l - . 1986; Hoolman t a l - , 1986). Thus, DTPA clearance does not only r e f le c t the perm eability of the alve o la r membrane since those and other factors Including s ite s of aerosol deposition and surface area can s ig n ific a n tly influence DTPA clearance rates. Effros and Mason (1983) described the clearance k in e tic s of inhaled water soluble p a rtic le s in analogy to CO d iffusion studies by the relatio n sh ip Ct - C0*e-<PS/V)t (4) where ct - concentration in lung at time t (pg/g) C0 - i n i t i a l concentration in lung (pg/g) P - perm eability of the alve o la r epithelium (cm/sec) S - e p ith e lia l surface area (cm2) V - volume of solute d istrib u tio n (cm3; e p ith e lia l lin in g flu id volume) I f the ra tio S/V remains constant, the changes vn solute clearance r e fle c t changes in perm eability of the membrane only and clea'rance rates are r e la t iv e ly independent of changes in surface area (E ffro s and Mason, 1983). However, an Increase in surface area w ill also increase solute clearance across the a lve o la r epithelium as seen in studies with increased lung volumes (Egan, 1982; Woolman a l . , 1986; Rizk t al-, 1984; Marks 1 a l . , 1985). Figure 13 shows resu lts of an experiment in our laboratory in which 99mfc-labeled aerosols were delivered for two minutes to anesthetized dogs at d iffe re n t lung volumes, and lung clearance of the solute was followed for 30 min th e re afte r at e ith e r spontaneous breathing or at increased lung volume (Oberdorster a t a l - , 1985): When the solute aerosol was delivered at an increased lung volume (t id a l volume 55 ml/kg body weight, b in Fig. 13) solute lung clearance was increased s ig n ific a n tly over control le v e ls (a in Fig. 13). Two minute v e n tila tio n at th is same high lung volume p rior to aerosol d e liv e ry at normal lung volume (t id a l volume 13 ml/kg body weight, c in Fig. 13) did not change subsequent solute clearance. The most dramatic increase in lung solute clearance was seen when the solute aerosol was delivered at normal lung volume and clearance was measured under conditions of increased lung volume (d in Fig. 13). In th is la t t e r case, the ra tio S/V in Eq. (4) increased since the a lve o la r surface area, but not the volume of d is trib u tio n increased, and thus an Increased surface area may p a rtly explain the increased clearance rate. However, resu lts from flu id in s t ille d lungs suggest, that lung hyp erinflation a d d itio n a lly increases the perm eability of the membrane, P in Eq. (4 ), which could possibly be due to stretching of the in te r e p ith e l1al tig h t junctions (E ffro s and Mason, 1983). Such junctional stretching during lung h yp erin flatio n Is also suggested from results of studies in rats with l . v . administered DTPA where the amount of DTPA d iffu sin g into the a lve o la r space was found to be increased with increased lung volumes (F io r ic a si a l . , 1988). In addition, high lung volumes might influence the d iffu sio n c h a ra c te ris tic s of the solute - when delivered as aerosol - into the surface lin in g f lu id , I . e . , i t may increase the d iffu sio n al spreading and thus increase the surface area a v a ila b le for tra n se p ith e lia l d iffu sio n (O'Brodovich and Coates, 1987). Spreading on a f la t surface occurs FIGURE 13 Lung Volume and Solute Clearance abed A lveo lar clearance rates in dogs of 99mTc-DTPA administered for 2 min as an aerosol show the influence of lung volume (Oberdorster i a l - . 1986b). a: DTPA aerosol d e liv e ry at normal lung volume, clearance measurement at normal lung volume, b: DTPA aerosol d e liv e ry at high lung volume, clearance measurement at normal lung volume (s ig n ific a n tly d iffe re n t from a, p < 0.05). c: Two minute v e n tila tio n at high lung volume p rior to DTPA aerosol d e liv e ry at normal lung volume and clearance measurement at normal lung volume. d: DTPA aerosol d e liv e ry at normal lung volume and clearance measurement at high lung volume (s ig n if ic a n t ly d iffe re n t from a, p < 0 .02). u su ally ra p id ly , w ithin a few seconds, and thus should g re a tly exceed the lung clearance rate of the solute across the alve o la r epithelium (E ffro s and Mason, 1983). However, the hydrophobic surface of the a lve o la r lin in g flu id could conceivably slow down the d iffu sio n of hydrophilic solutes into the lin in g flu id (O'Brodovich and Coates, 1987), a facto r that needs further in ve s tig a tio n . I t is in te restin g to speculate that d e liv e ry of the solute aerosol at high lung volumes in our study described above (b in Fig. 13) might have led to deposition of the p a rtic le s in a lv e o li with less lin in g flu id (lower volume o f d is trib u tio n , V in Eq. (4) since they are not in fla te d during normal v e n tila tio n , thus explaining the observed fa s te r lung clearance rate. Although applying th is technique of noninvasively measuring an index of a lv e o la r perm eability with " mTc-DTPA aerosols c l i n i c a l l y Is very appealing, several unresolved questions require further basic research for interpreting re s u lts from th is method before i t should be used ro u tin ely. O'Brodovich and Coates (1987) also emphasized in a recent review that th is method remains an experimental in v e s tig a tiv e tool that is not yet ready for widespread c lin ic a l ap p lica tio n . For example, c la r if ic a t io n is needed on questions related to the sig n ifica n ce of lung surfactant; the s t a b ilit y of the ^Tc-D TPA complex (Nolop i a l - . 1986; Haldman a i . , 1987; Dolovich a a i - . 1987); unexpected decreases in clearance rates in the beginning phases of an inflammatory reaction 1n the lung (Oberdorster a i a l - . 1986a; Pla tn e r and Morrow, 1988); the Importance of DTPA build-up in the blood compartment during accelerated lung clearance (Jones i a l - . 1982; Langford i a l - . 1986); the re p ro d u cib ility w ithin and between laboratories and standardization of the technique (O'Brodovich and Coates, 1987). 314 I marke voi UtT mease large such of d stab i pharr (d iff phys; \ has 1 with Oberc half! stud: absoi and inha spec' the sol u to t betw Spec sole mous Resp the 11 pc (aft .... ^ 1U^ -- . .V.-.--.r. ..u- I t is conceivable that the DTPA molecule is too small, representing a marker which is too s en sitive towards small physiological changes in lung volume thus making i t d if f ic u lt to use i t as a routine diagnostic tool for measuring an in dicato r of alve o la r e p ith e lia l perm eability. A solute tra ce r of larger molecular weight might be better suited for th is purpose. The choice of such tracer depends on how it s clearance is influenced by factors such as: s ite of deposition 1n resp irato ry tra c t; hydrophi1i c i t y , 1ipophi1i c i t y ; chemical s t a b ilit y and reactions (binding, receptors); lung disorders and administered pharmaceuticals (see Jones 1984 and Newhouse t al.., 1987); re c irc u la tio n (d iffu sio n gradient, e .g ., influenced by renal e lim in a tio n ); lung volume; physical exercise. With regard to pulmonary solute clearance in d iffe re n t animal species i t has been found that clearance of inhaled DTPA in dogs is fa s te r than in humans, with pulmonary retention halftimes of about 30 minutes (Rizk et al.., 1984; Oberdorster s i a l . , 1986b). On the other hand, respective DTPA retention halftimes were longer in rabbits (T j /2 - 127 min, Je ff e r ie s e i a l - , 1984). A study in d iffe re n t species with a range of inhaled solutes showed that absorption of hydrophilic solutes was fastest 1n the mouse, followed by the rat and was slowest in the rabbit (Schanker s i a l - . 1986). In contrast, a given inhaled lip o p h ilic solute was absorbed at about the same rate in a ll three species (F ig . 14). Thus, lung clearance of hydrophilic solutes depends on both the animal species and the solute molecular weight whereas for lip o p h ilic solutes i t seems to depend only on th e irlipid/w ater p a rtitio n c o e ffic ie n ts and to be independent of animal species. Since the ra tio of clearance rates between species was found to be constant for hydrophilic solutes, re fle c tin g FIGURE 14 Lung Clearance of Inhaled Solutes in D ifferen t Species Minutes after Inhalation Species dependency was observed fo r h yd rop h ilic, but not for lip o p h ilic solutes. Ratios of clearance rates o f d iffe re n t hydrophilic compounds for m ouse:ratrrabbit were remarkably constant with approximately 0.4 :1:2.5 . Respective clearance rates for d iffe re n t hydrophilic compounds increase with the molecular size of the compound. In con trast, clearance rates for 11pophl11c compounds increases with th e ir lipid/w ater p a rtitio n c o e ffic ie n t (a fte r Schanker s i a l . . 1986). 315 the speclies sp e cific porosity of the epithelium, i t may be possible to predict the rate of pulmonary absorption of a given drug in one species from data obtained for the same drug in another species (Schanker a l . , 1986). A leaky a lve o la r epithelium in diseased lungs leading to increases of the clearance of hydrophilic solutes from the a lveo lar space implies that lung retention of soluble compounds given as aerosols is decreased compared to a healthy lung, e .g ., a therapeutical aerosol could have a s ig n ific a n tly shorter residence time - and thus is less e ffe c tiv e - at a target s ite in the lung than would be expected from it s kin e tics under healthy conditions. Encapsulation of such therapeutic agents in p a rticle s with membranes of low in vivo s o lu b ility could increase th e ir pulmonary retention. Obviously, such m odifications depend also on whether a systemic d istrib u tio n of an inhaled therapeutic agent or a more lo calized e ffe c t in the lung is desired (Lourenco and Cotromanes, 1982). Chemical reactions influencing pulmonary retention of solutes (F ig . 1, Table 2) are of p a rtic u la r importance Tor inhalation toxicology. Many water soluble inhaled substances w ill not be cleared from the a lv e o la r space simply by d iffu sio n a l processes but may react with lung flu id s or c e lls , e .g ., being hydrolyzed to less soluble compounds or bound to proteins. Resulting clearance rates are unpredictable and can also d if fe r quite s ig n ific a n tly among animal species. Conversely, substances with low water s o lu b ility can be cleared from the lungs quite rap id ly because th e ir i_n vivo dissolution in the lung is very rapid. As mentioned in the Introduction, examples both for a fa st lung clearance of water insoluble compounds(high In vivo s o lu b ilit y ) and of low lung clearance of water soluble compounds (chemical binding) are found a fte r in halation o f heavy metals. For instance, inhaled aerosols of ZnO and Pb (OH) 2 (low water s o lu b ilit y ) and other lead compounds were found to have a monoexponential retention halftime of a few hours only (Bianco & a l- , 1974; Oberdorster e l a l . , 1979; Morrow a l a l- . 1980). Inhaled aerosols of CdCl2 (water so lu b le ), on the other hand, showed a much longer pulmonary retention than predicted from th e ir water s o lu b ilit y and molecular size. Moreover, pulmonary retention of CdCl2 a fte r a single exposure is s ig n ific a n tly d iffe re n t between ra ts , dogs, and monkeys (F ig . 15, Oberdorster and Cox, 1988): rats exhibited a monoexponential clearance pattern corresponding to a halftime of 85 days, in dogs 701 of the deposited CdCl2 as cleared with a retention halftim e of 153 days and in monkeys a monoexponential clearance with a retention halftim e of 818 days was found over an observation period of 650 days. Although th is species dependent pulmonary clearance o f water soluble Cd-compounds appears to be very sim ila r to the pulmonary clearance c h a ra c te ris tic s of highly insoluble p a rtic le s (F ig . 5) - which is related to AM function - pulmonary clearance of cadmium is not mediated by AM. This was demonstrated in rats when during long-term inhalation of CdO aerosols the a lv e o la r clearance of Fe203 te s t p a rticle s was s ig n ific a n tly retarded, but not that of a CdCl2 test aerosol (Oberdorster and Hochrainer, 1980). Water insoluble CdO p a rtic le s are cleared with the same rates as CdCl2 . due to a rapid i n i t i a l in vivo dissolution of those p a rtic le s , probably in the phagolysosomes of AM (Hadley 1 ., 1980; Lundborg i 1 ., 1984). Following this 'apid in vivo dissolution of CdO, subsequent elim ination occurs at the slow clearance rate of soluble cadmium compounds. This slow clearance may in volve binding to serum and c e llu la r proteins, most lik e ly including m etallothionein (Oberdorster, 1986) which is induced in the lung due to cadmium exposure ;(Post a l . , 1982). The measured pulmonaryretention halftimes of soluble p a rticle s are undoubtedly the re s u lt of several clearance processes whose r e la tiv e contributions change depending on the physico-chemical properties o f a sp e cific p a rtic u la te matter. These processes - whether reactions with s p e c ific ligands, d iffu sio n rates across membranes, disso lu tio n rates in AM, or mechanical transport rates - are of d iffe re n t magnitude in d iffe re n t mammalian species and thus make i t d if f ic u lt or even impossible to extrapolate d ir e c t ly results of pulmonary retention measurements of a s p e c ific compound in animals to humans. 316 1 Par- -1.0 J 0.5 oo "0O) `o Qd 0.2 0.1 C Retention meas Pulmonary cadi monoexponentia days, respecti constant fitte Basic d iff p a rtic le s from p articles is results of rec be substantial which phagocy otherwise fast p a rticle s are rapid rate wit on U p o p h iU c i to tissue and can Increase retention of a than in the ; 1986b), althou alveolar epith vivo is possil liquid layer 1 Alveolar c and transport p a rticle s are by a lv e o la r m pathway as f r p a rtic u la te cc dissolution ra can involve di ... . -* FIGURE 15 Pulmonary Retention of Inhaled Hater Soluble CdCl2 P a rtic le s in D ifferen t Species (Oberdorster and Cox, 1988) Retention measurements were started one day a fte r exposure to CdCl2 aerosol. Pulmonary cadmium retention 1n rat and monkey could be described by a monoexponential term with bio log ical retention halftim es of 85 days and 835 days, re s p e ctive ly ; in dogs, a b io logical retention halftim e of 153 days plus a constant f itt e d the data best. CONCLUDING REMARKS Basic differences exist 1n the clearance of Insoluble and soluble inhaled p a rtic le s from the resp irato ry tra c t. Conducting airway clearance of insoluble p a rtic le s is fa s t and maybe e s s e n tia lly complete within one day, although resu lts of recent studies in d icate that retention in the conducting airways may be su b s ta n tia lly longer than g enerally presumed. Possib ly, airway macrophages which phagocytlce p a rtic le s in th is region may act as "in h ib ito rs " of an otherwise fast bronchial clearance of insoluble p a rtic le s . Inhaled soluble p a rtic le s are cleared from both the conducting and the a lv e o la r zone at a very rapid rate with corresponding retention halftimes of minutes or hours, depending on lip o p h illc it y , h y d ro p h ilic ity , and molecular size. However, binding with and to tissu e and c e llu la r components and adsorption onto highly Insoluble p a rtic le s can increase retention halftim es s ig n ific a n tly to days and months. In vivo retention of an inhaled hydrophilic solute is longer in the conducting airways than in the a lve o la r region (Brown and Schanker, 1983; Oberdorster e a ! . , 1986b), although pore equivalents seem to be of larger size in bronchial than a lve o la r epithelium (Gatzy and Boucher, 1982). This longer retention seen i_n vivo is possibly due to differences in the thickness and composition of the liq u id layer lin in g the e p ith e lia l surfaces-of a lv e o li and conducting airways. A lveo lar clearance mechanisms for solute p a rtic le s involve mainly d iffusion and transport v ia in te r c e llu la r and tra n s c e llu la r pathways, whereas insoluble p a rtic le s are transported up the m ucociliary escalator mostly a fte r phagocytosis by alve o la r macrophages, and a minor fra ctio n may leave the lung v ia this pathway as free p a rtic le s . E ffe c tiv e pulmonary clearance rates of Inhaled p a rtic u la te compounds are determined by th e ir mechanical clearance ra te , th e ir dissolution rate and the clearance rate of the dissolved compound. The la tte r can involve d iffu s io n , a ctive transport or chemical reactions in the lung which 317 .mi .... i**. .. . ... --- '__ _ - lik e the other factors - can be d iffe re n t in d iffe re n t animal species. I t is therefore d if f ic u lt to determine or to predict mechanical clearance rates of p a rtic le s with unknown in vivo s o lu b ilit y and r e a c t iv it y from In vivo retention measurements in a given animal species. Estimated mechanical pulmonary clearance rates of solid p a rtic le s showed a species dependency and became slower with time. However, some uncertainties remain with regard to the accuracy of th4 estimates of in vivo dissolution rates and transport rates of p a rtic le s into lymph nodes which need to be known for calcu latin g mechanical clearance rates. Although the mechanical clearance rate of insoluble p a rtic le s Is very slow fo r the a lve o la r compartment compared to the tracheobronchial region, i t is s t i l l very e ff ic ie n t in handling p a rtic u la te burdens resu ltin g from inhalation of typ ical dust concentrations in ambient a ir . However, studies in rodents showed that under extremely dusty conditions lastin g for an extended period of time AM mediated clearance becomes in creasing ly impaired resu ltin g in the development of chronic lung disease. Transep ithelial transport of solid p a rtic le s across the a lve o la r epithelium occurs normally at a low rate only, but can increase su b s ta n tia lly under conditions of high p a rtic u la te lung burdens. I t appears that whenever AM-mediated p a rtic le clearance is impaired, an increased penetration of p a rtic le s in to the in te rs titiu m takes place and an increased build-up of p a rticu la te burden in the regional lymph nodes occurs (e ,g ., Ferin and Feld stein , 1978). Conceivably, AM function may be affected s y n e rg is tic a lly by a combination of high exposure concentrations and c y to to x ic ity of the inhaled compounds, e .g ., in halation of high concentrations of quartz and asbestos (F e rin , 1972; Ferin and Leach, 1976) or of diesel exhaust with it s many chemicals of known and unknown t o x ic ity in both the gas and p a rtic u la te phase (Chan 1 ., 1984; W olff e i a h , 1987). Linder such conditions, AM clearance function should be affected at lower p a rtic u la te lung burdens than at those with "nuisance" p a rtic le s of low to x ic ity lik e TiO2 - The resu lts of Wolff i al.. (1987) seem to support th is view since exposure of rats for 18 months to low concentrations of diesel exhaust resu ltin g in a lung burden of 320 pg soot/lung - below a concentration of p a rtic le s of low to x ic ity in the lung known to cause impaired lung clearance in rats - led to an accelerated accumulation of soot upon furth er exposure. Diseases of the lung can a ffe c t clearance of soluble and insoluble p a rtic le s d iffe re n tly : elim ination of inhaled insoluble p a rticle s is retarded, whereas elim ination of inhaled solute p a rtic le s is accelerated. This is demonstrated in Figure 16 in which the e ffe c t of smoking on a lv e o la r lung clearance of highly insoluble and highly soluble p a rtic le s is shown. Long-term lung clearance of trace amounts of magnetite te s t p a rtic le s was s ig n ific a n tly decreased in smokers (Cohen i a l . , 1979), whereas 99mTc-DTPA solute lung clearance in smokers was s ig n ific a n tly increased (Jones a l . , 1980). In addition to showing the q u a lita tiv e differences in the e ffe c t of smoking on a lv e o la r lung clearance, differences in the rate of clearance between the two p a rtic le types become apparent from the time scale, months for the solid p a rtic le s and minutes for the liq u id p a rtic le s (F ig . 16). Obviously, such differences are re a d ily explained by the d iffe re n t underlying clearance mechanisms for these two types of inhaled p a rtic le s . However, in both cases AM probably play a central role in the mechanisms of altered a lv e o la r clearance in lungs of smokers and nonsmokers: S o lid p a rtic le clearance decreased in smokers due to impaired AM function (Green, 1985), and solute clearance may have increased due to e ffects on the a lve o la r e p ith e lia l b a rrie r of mediators released from activated AM (Jones i a l . , 1983). As already mentioned, the im plications for diseased lungs - in this case, smokers' lungs - are that retention of water soluble therapeutic aerosols in the deep lung may be less than anticipated and that - in contrast - smokers accumulate s ig n ific a n tly more of deposited nuisance dust or other solid p a rtic le s in the a lv e o la r region than nonsmokers. The la t te r was corroborated by findings of an increased retention of p a rtic u la te m aterial in the lungs of smokers (Churg and Wiggs, 1987). However, i t is possible that th is greater retention and resu ltin g dust accumulation in lungs of smokers could somewhat be o ffs e t by a lower pulmonary deposition due to a greater central deposition in th e ir conducting airways i f chronic bronchitis is present. 318 100 80- co I 60 4> QL ctot) 40- 4> CL 20 0-C In lung p a rtic le 1985); p of othe F6304 t nine no conclude (Cohen g simi la r smokers solute ( alveolar of this smokers and 20 nonsmoke i 41-. Know lung is occupati dosimetr humans determi r bases 0 particul importar t is of tion nary ower ' of into s. slow is :ion ?nts i of the ol id iiy . 1ung 'ed, i an :urs i of 9-. and lown , )wer :ity iew '."'t . ii bl e ed, is ung erm tly ung In on two lid uch nee AM in ers ave ors the hat ess ore han ion 7). ust ary if FIGURE 16 Smoking and Lung Clearance Smoking and Lung Clearance of Particles Solid Particles (Fe3 0 4 ) Liquid Particles ( " mTc--DTPA) In lung In ju ry caused by smoking, pulmonary clearance of highly Insoluble test p a rtic le s (FegOi*) is retarded probably due to decreased AM function (Green, 1985); pred ictab ly, smokers w ill accumulate in addition to smoke p a rtic le s more of other inhaled p a rtic le s in th e ir lungs than nonsmokers. Retention of Fe3<)4 te s t p a rtic le s was measured noninvasively by magnetopneumography in nine nonsmoking and three heavy smoking male subjects, and the authors concluded that smoking was the cause of retardation of te s t p a rtic le clearance (Cohen 41-, 1979). Bohning t a_L. (1982) came to the same conclusion in a sim ila r study a fte r inhalation of radiolabeled polystyrene te s t p a rtic le s by smokers and nonsmokers. In contrast, pulmonary clearance of the hydrophilic solute DTPA inhaled as an aerosol is increased in smokers due to a more leaky alve o la r epithelium. The DTPA study (Jones et a l . , 1980) was the f i r s t study of th is kind measuring lung clearance of ^ 'mTc-DTPA with a gamma camera in smokers and nonsmokers. Mean retention halftim es were 59 m1n for nonsmokers and 20 m1n for smokers. The resu lt of th is study in ten smokers and ten nonsmokers was confirmed la te r in other studies (Dolovich i a l . , 1982; Mason fii 41-, 1983; Mason, 1985). Knowledge of lung clearance processes of inhaled p a rtic le s in the healthy lung is essential for the understanding of e ffec ts of Inhaled environmental, occupational and c lin ic a l aerosols. I t is also a necessary presupposition for dosimetric extrapolation modeling of resu lts from animal inhalation studies to humans since retained doses of substances at pulmonary target s ite s are larg ely determined by th e ir clearance behavior. Unraveling of c e llu la r and molecular bases of lung clearance mechanisms requires considerable additional work. In p a rtic u la r, the mechanisms of clearance behavior in lung diseases are an important issue which needs further attention . ACKNOWLEDGEMENTS The author g ra te fu lly acknowledges the helpful review and valuable suggestions by Dr. P.E. Morrow, Dr. J . Ferin, Dr. M .J. U t e ll, and Dr. S.C. Soderholm. Special thanks goes to P.A. Morse and N.L. Corson for helping to prepare the figures of th is a r t ic le . 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A r tic le received: March 8, 1988 Reviewed by: W.M. Foster Address rep rin t requests to: Or. Gunter Oberdorster Department of Biophysics Environmental Health Sciences Center U niversity of Rochester School of Medicine and D entistry 601 Elmwood Avenue Rochester, NY 14642 330 l JVMOoaUlruyRmNAeAnlnrLNLOiueFri ctsbrrdtrtcdttpcoiceealheoieloyeoeoelxmnmesdcaanttpspacohtyart,auaet,rreaIpieyiniglenarbnodlibaoosena(rtcynoterppdonnseite,fiohuoomtttnchiLiii:(it; aoinnbhdsaelrhTevedyatnomiect baracarneaedytdiiooononanoetdthtoiefIftner organKeellye v r