Document RjZwzbVN53YqrkeLJdkRRY2v8

FUNDAMENTAL AND APPLIED TOXICOLOGY 10, 369-384 (1988) ISSUES Possible Mechanisms to Explain Dust Overloading of the Lungs P. E. Morrow Environmental Health Sciences Center and Department o f Biophysics. University o f Rochester. School o fMedicine and Dentistry. Rochester. New York 14642 Received July 9. 1987; accepted November 10. 1987 Possible Mechanisms to Explain Dust Overloading of the Lungs. M O RROW , P. E. (1988). Fundam. Appl. Toxicol. 10, 369-384. This paper briefly reviews the available evidence on dust overloading of the lungs, a condition which has come to the forefront in many recently reported chronic inhalation studies. A general hypothesis is developed that dust overloading, which is typified by a progressive reduction of particle clearance from the deep lung, reflects a breakdown in alveolar macrophage (AM)-mediated dust removal due to the loss of AM mobility. The inabil ity of the dust-laden AMs to translocate to the mucociliary escalator is correlated to an average composite particle volume per alveolar macrophage in the lung. When this particulate volume exceeds approximately 60 iim'/AM. on the basis of a uniform distribution of particles over the AM pool size ( ~ 2.5 X IO' cells) in the Fischer 344 rat, the overload effect appears to be initiated. When the distributed particulate volume exceeds --600 ^m3per cell, the evidence suggests that AM-mediated particle clearance virtually ceases and agglomerated particle-laden macrophages remain in the alveolar region. This paper considers possible mechanisms why these particle laden cells are immobilized, viz., one is based on excessive particle-cell, cell-cell chemotactic interactions, and migratory inhibition factors; the other considers the volumetric increase by phagocytized particles, per se. as leading to an inability of the AM to spread and migrate proba bly through a competitive requirement for surface membrane and cytoskeleton in both endocytoticand migratory functions. 1988 Society of Toxicology BACKGROUND OF DUST OVERLOAD CONCEPT A substantial number of experimental obser vations (Adamson and Bowden, 1980, 1981; Bolton el a i, 1983; Bowden and Adamson, 1984; Bowden, 1987; Chan et a i, 1984; Davis et al., 1978; Ferin, 1972, 1977; Ferin and Feldstein, 1978; Green et at., 1983; KJosterkotter and Buneman, 1961; KJosterkotter and Gono, 1971; Le Bouffant, 1971; Lee el di, 1983; Matsuno el a i, 1986; Middleton et di, 1979; Muhle et a i, 1987a,b; Shami el a i, 1984; Wehner el a i, 1983, White and Bhag wan, 1981; Wolff et a i, 1985, 1987) has led to the general concept of dust overloading of the lungs. The essence of this experimental finding is bipartite: (a) long-term exposure to relatively high dust concentrations leads to excessive pulmonary dust burdens whereby the pulmonary clearance of persistently re tained particles by alveolar macrophages be comes progressively reduced until it essen tially ceases: at this time, lung dust burdens increase linearly at a build-up rate approxi mating the rate of dust deposition; (b) as ex cessive lung burdens develop, a number of al terations appear in both the disposition of re tained particles and their pattern of induced responses and toxic actions within the lungs. In both regards, interest is mainly with in haled particles whose retention half-times in 369 0272-0590/88 S3.00 Copyright 1988 by the Society of Toxicology All rights of reproduction in any form reserved. 370 P. E. MORROW the lungs are measured in weeks, months, or years, and whose toxicity depends upon chronicity. Thus, breakdown of macrophage-medi ated dust clearance creates an artifactual lung condition typified by a sequence of dysfunc tional and pathologic changes. While the temporal development of these nonspecific changes may differ with different dusts and the degree of dust overloading, most materi als appear to initiate a quantitatively similar pattern of changes, e.g., widespread accumu lations of dust-laden macrophages within al veoli; enhanced appearance of lymphoid-as sociated and interstitial dust deposits; and persistent inflammatory changes with in creased epithelial permeability, associated with neutrophil infiltration and activation. These conditions usually progress during chronic exposures until there is a develop ment of alveolitis and granulomatous lung disease, such as fibrosis (Adamson and Bow den, 1981; Bowden, 1987; Hunninghake et al., 1984; Gross, 1967; Pepelko et al., 1980; Reiser and Last, 1986). The toxicological implications of lung overloading should be obvious. Even many dusts which have generally been recognized as innocuous and considered to fall within the Nuisance Dust classification (ACG1H, 1981), have, under conditions of dust over loading, been shown to produce a diminution of dust clearance and a variety of unexpected toxicological endpoints including tumorigenesis(Brand, 1986; Holland et al., 1986; L e e # al., 1985). Dusts with established toxicities, such as silica, in overload circumstances, have been reported as producing a relatively high incidence of lung cancer: a pathological endpoint seldom seen in man or experimen tal animals under lesser exposure conditions and lower lung burdens of silica (Groth et al.. 1986). Several contemporary papers have pointed out the confounding nature of dust overloading in interpreting the outcome of recent chronic inhalation toxicity studies for diesel particles (McClellan, 1986; Vostal, 1986). It should be emphasized that lung over loading per se does not depend upon the in herent chronic toxicity of the investigated material. Whether the material is potentially tumorigenic, fibrogenic, or completely be nign, dust overloading superimposes its ac tions of modifying both the dosimetry and the toxicological effects of the test material. In chronic studies, the incorporation of a surrogate particle, e.g., 59Fe20 3, which is known to be cleared from the lungs by alveo lar macrophages (AMs) (Lehnert and Mor row, 1985b) has been shown to track the change in retention of the test dust. For this application the animal is exposed to micro gram quantities of the surrogate aerosol and its retention is measured independently but concurrently with the test dust. As the lung burden of the test dust becomes excessive and its retention time is prolonged, a concurrent prolongation of the surrogate particle reten tion has been observed. This circumstance adds credence to the involvement of the AM in the overload process (Morrow, 1986; Muhle et al., 1987a,b; Wolif et al., 1985, 1987). Several important aspects of dust overload ing are still poorly understood. One obvious need is for a better quantitative definition of the overload condition. While the level of dust burden causing overloading appears to be greater than 1-2 mg of a relatively persis tently retained dust in the lungs of a Fischer 344 rat, for example (Chan e la i. 1984; Wolff et al.. 1985, 1987; Muhle et al., 1987a,b), such a dust burden does not produce an allor-none type of overload response. Rather, the responses and effects produced become more severe as the lung burden exceeds this general level. Chronicity also appears to be a significant factor in its development. Another important informational defi ciency pertains to the general appropriateness of the overload phenomena to all persistent dusts and to other species besides the rat. Since the impact of overloading appears to increase with the absolute dust burden in the lungs, it is important to understand to what overhe in igated ntially ly be lts acy and rial, n of a ich is alveoMork the >r this nicro mi and ly but ; lung /e and arrent reten>tance e AM 1986; rloadivious ion of vel of ars to >ersisischer Wolff 7a,b), m allather, come s this ) be a defieness istent e rat. irs to n the what DUST OVERLOAD MECHANISMS 371 extent this condition of impaired macro known lung burdens associated with dust phage clearance is reversible. Intuitively, one overload in order to develop the primary hy would expect recovery to be faster at lung lev pothesis, viz., that a progressive increase in els which did not cause a cessation of dust the cumulative size of particle volume phago- clearance. cytized leads to AM immobilization. Esti In any case, dust overloading threatens to mates of the boundary conditions for particle compromise many expensive, long-term overloading are described. Third, possible : studies unless there is some reasonable strat bases for the unusual prolongation and ulti egy to limit inhalation exposures in ways mate cessation of AM-mediated particle analogous to those now applied to parenteral clearance are examined in the context o f the and oral administrations involved in other loss of translocation mobility of the " over chronic toxicity investigations (National loaded AM." Toxicology Program, 1984; Munro, 1977). Suggestions toward this goal were described in two recent publications (Morrow, 1986; Morrow and Mermelstein, 1987). KINETIC DESCRIPTION OF OVERLOAD CONCEPT Despite informational deficiencies and the Kinetically, the clearance of dust from the lack of any systematic study of dust overload alveolar or pulmonary region o f the lungs has ing, there appears to be a plausible mechanis usually been treated as a first-order process tic basis for the overload phenomenon. This (Morrow, 1977). This widely applied model paper develops mechanistic hypotheses is not based on specific clearance mecha largely from studies which have attempted to nisms although it is evident that several quantify the size and capacity of the pulmo known clearance processes are, or closely re nary macrophage pool and from studies semble, single first-order processes. Rather, | which have described the kinetics and re the model has been used mainly because it f sponses of AMs during quantitatively de provides a kinetically suitable description of scribed particle exposures (Lehnert and Mor pulmonary clearance and a relatively simple row, 1985b). dosimetric approach (Morrow, 1973). In depicting a single-compartment, first STATEMENT OF HYPOTHESIS order clearance model (Fig. 1A) z/ is the rate of pulmonary dust deposition and k is the j The principal overload hypothesis to be de fractional clearance rate of the lung compart : veloped in this paper proposes that the condi- ment removal and L is the dust content of | tion of dust overloading in the lungs is caused the lung compartment. One can see that the | and perpetuated by a loss in the mobility of build-up rate of L (L) depends upon the rela the alveolar macrophage. Moreover, this loss tionship L = d - kL mg day" 1, where d is j of mobility that impedes macrophage trans expressed as milligrams per day. L is ex I location from the lungs is produced by the pressed in milligrams and k equals In 2/t[ phagocytosis of excessive amounts of parti where t{ is the retention half time in days; ) des, a condition which can be expressed as therefore, k is expressed as fraction per day 1 the cumulative particle volume per alveolar (day-1). When d approximates the value of macrophage. the product kL, and L approaches zero (Fig. j The overall mechanistic hypothesis re 1B), the increasing similarity of absolute de quires a step-wise development. First, parti position and absolute clearance rates leads as cle build-up and clearance kinetics and how ymptotically to the steady-state lung burden, 1 these relate to chronic inhalation exposure Lm. The dust build-up rate before and at the conditions will be examined. Second, we will steady-state condition is accordingly ex review AM-mediated clearance and apply pected to resemble that depicted in Fig. 2. 372 P. E. MORROW A. Build-Up Phase d Lk (mg day"') (mg) (day'1) d > kL L = d - kL B. Steady-Slate Phase d L,, (mg) (mg day'1) (day' 1) d = K*.L,, L- 0 Fig . 1. Build-up and steady-state kinetics of single compartment lung model employing first-order dust clearance during a chronic dust exposure. (A) The kinetic relationships between dust deposition (d) and clearance (k) are depicted during the build-up phage of dust in the lungs. The build-up rate is described by L. (B) When the dust burden in the lungs achieves a certain level, the amount of dust removed by first-order clearance (k^) be comes equal to the rate dust deposition (d ). This heralds the achievement of a steady-state lung burden (L,,). This type of build-up curve was nicely demonstrated in the rat, dog, and monkey during chronic studies by Leach et al. (1970, 1973) and kinetically inferred by the analyses of coal miner lungs reported by Stber et al. (1967). It is conventional to accept five retention half-times (t\) as the approximate time re quired to produce La . In reality five t{ consti tutes only 97% of which, in theory, equals d{t\/In 2). Keeping the amounts of dust ex pressed in milligrams and time expressed in days, the predicted value of Lx in milligrams is approximately equal to 1.44 d t{ or can be determined from the ratio: d/k. The deposition rate d (mg day" ') is obvi ously determined by the exposure concentra tion (mg m"3), the respirable dust deposition fraction, and the ventilation of the exposed subject (m3 exposure day" 1). For illustrative purposes, we can assume a Fischer 344 rat, during a 8-hr exposure day, ventilates ( 180 ml min" 1 X 480 min/106 ml m"3) or 0.0864 m3day" '. Applying a 0.10 rat respirable deposition fraction, the rate of dust deposition (d) will equal the product of the amount of air breathed by the rat times the exposure con centration (C) times this deposition fraction, i.e., 0.0864 m3 day" 1 X (C) mg m3 X 0.10. This product, 8.64 X 10"3 (C) mg day"1, equals the value of d. Because chronic dust exposures are usually limited to 5 days a week, we can " adjust" the value of d by so that we can treat the rate of dust deposition as though it pertained to continuous daily ex posures. Thus, the adjusted d is equal to 6.17 X 10"3(C) mg day" '. The one remaining factor which deter mines the rate of achieving La and its abso lute value is the pulmonary retention half time for the dust. In order to examine the im pact of different exposure concentrations on L ,,, we will assume a 60-day retention half time, which means that the clearance rate co efficient, k = In 2/60 days = 0.0116 day"1, pertains to the dust. In the first example, we will consider (C) to be equal to 2 mg m "3. This signifies that the rate of dust deposition, d, equals 6.17 X 10"3 m3 day" 1 X 2 mg m"3 or 0.01234 mg day" 1. Fig . 2. Build-up rate of lung dust content during clear ance inhalation exposure. In this depiction the level of lung dust build-up is expressed as a fraction of the steadystate lung burden (L,,). Time is expressed in terms of the retention half-time of the test dust determined from the clearance rate coefficient (k). that is, /) = In 2/k. 0.0864 osition d) will of air re conaction, < 0 . 10. d a y '1, ic dust days a by i so osition aily exto 6.17 deter:s abso>n halfthe im ions on >n half rate co i d'W-1, r(C') to that the X 10" 3 l day" 1. ring dear ie level of hesteadyrms of the I from the k. DUST OVERLOAD MECHANISMS 373 TABLE 1 Interrelationships of Respirable Exposure Concentrations and Dust Retention in Achieving Steady-State Lung Burdens" Exposure concentration (C) (mg nT3) Deposition fraction Deposition rate (d ) (mg day'1) Retention half-time (days) Clearance rate(k) (day-1) Steady-state lung burden(L,,) (mg) Time to achieve L ,, (days) Case 1 Case 2 Case 3 Case 4 2 2 20 2 0.10 0.01234 60 0.0116 0.10 0.01234 120 0.0058 0.01 0.01234 60 0.0116 0.10 0.1243 60 0.0! 16 1.06 2.12 1.06 10.6 300 600 300 300 * In all examples, the rat is assumed to breathe 0.0864 m' per exposure day and all exposures are adjusted so as to resemble continu ous weekly exposures, as described in the text. From the lung model (Fig. 1B), we can see more than predicted by the first-order kinetic that under steady-state conditions, that is, model. Instead of reaching 97% of the pre when d is equal to 0.01234 mg day" 1, the dicted Lss (10.3 mg) in 300 days, the linear product of fcLa will also equal this value, build-up model indicates that this will hap therefore, L * = 0.01234 mg day~'/0.0116 pen in 83.5 days (10.3 m g/0.1234 mg day" 1) day" 1 = 1.06 mg. With the assumptions while at 300 days, 37 mg is the predicted made, the first-order clearance model pre value for the lung dust burden, L. dicts that in 300 days (5 /^) we will effectively The foregoing development and these sim achieve L actually 97% of ,, or 1.03 mg. plified estimations are predicated on the as If we had chosen a longer retention half- sumption that clearance mechanisms other I tiipe for the dust, say 120 days, the value of k than macrophage-mediated particle removal | would be one-half as large, i.e., 0.0058 d a y '1. are relatively unimportant over the chronic All other assumptions unchanged, the value exposure time. For instance, estimations of of would be twice as large, 2.12 mg, and it would be effectively reached in 600 days. This and several other changes in assumptions are , given in Table 1. Of the examples presented j in Table 1, let us focus on case 4 where the exposure concentration is taken as 20 mg m"3 I and the predicted is 10.6 mg. Available ev idence on dust overloading suggests the fore ; going method of predicting L x would not ap ! ply since more than 2 mg of dust would be j deposited in the rat's lungs within the first 30 days of exposure. In Fig. 3, we see the build ) up rate and L value associated with the case 4assumption (Table 1). Additionally, a linear build-up rate, i.e., a constant daily increase in lung dust content associated with an insig Fig. 3. Case 4 build-up kinetics. In this graphic depic nificant daily reduction in dust clearance, is tion, both a first-order build-up rate and a linear build shown having a value o f0.1234 mg day" 1, the up rate are given. Inasmuch as dust overloading would I value of d. This latter depiction, in turn, indi cates that at 40.5 days, for example, there occur with either build-up rate assumption in less than 30 days of exposure, the linear build-up will be closer to reality throughout the subsequent exposure time, pro j would be 5 mg o f dust in the lungs, slightly vided all assumptions remain constant. i 374 P. E. MORROW clearance by dissolution have often yielded relatively long half-times (Mercer, 1967). Such a removal process could account for a ~10% reduction in the lung burden achieved. This would lead to a more-or-less linear build-up rate intermediate to the two curves depicted and a somewhat smaller value for L. To have avoided a condition of excessive lung levels occurring in the foregoing exam ple (case 4), either the exposure concentra tion or the respirable deposition fraction would have had to be reduced by about a fac tor 10 (e.g., case 3 in Table 1), thereby, a pre dicted La of 1.1 mg would be expected and the build-up of dust in the lungs would follow the simple first-order kinetics initially de picted (Fig. 2). Kinetic descriptions of dust clearance us ing a single compartment and single or multi ple first-order clearance functions have proved both versatile and useful, but they uniformly depend upon the proposition that the value of k does not vary with the size of L. This has led to an alternate approach, for example, represented by Vincent et al. (1985) and by Strom and Chan (1984). Both models envision a nonlinear sequestration compart ment which has a very small clearance coefficient (long retention half-time). For example, in Strom and Chan's model, the sequestration compartment becomes more dominant as the mass of particles in the mac rophage compartment increases. This leads to a redistribution of particles to the seques tration and lymphatic compartments. Kinetically, these nonlinear models with se questration compartments are capable of de scribing overload effects by the use of a complex set of differential equations. The power of these models is somewhat offset by their inherent complexity and the recogni tion of profoundly abnormal events in the lungs induced by excessive dust burdens, in cluding a blockage of macrophage migration and a virtual cessation of AM-mediated dust clearance. Evolution of an abnormal or pathologic compartment does not befit the usual compartmental concept. Another version of this approach, is that of Yu and Morrow (1987) which makes use of a simpler nonlinear model, i.e., where the value of k is a function of the magnitude of L. This model has the capability of describing kinetically the prolongation of pulmonary dust retention both as a normal consequence of chronic exposures and as an overload phe nomenon without invoking a hypothetical compartment. In this case, " overloading" is an extension of the normal prolongation effect of an increasing lung burden. Linked to this decrease in pulmonary dust clearance with increasing lung burden is a concomitant increase in translocation of dust to the lym phatic drainage and lymphoid tissue (Adam son and Bowden, 1981, 1982; Ferin, 1972; Ferin and Feldstein, 1978; Strom and Garg, 1985; Yu and Morrow, 1987) which also oc curs in the condition of dust overloading. The solubility clearance model proposed by Mercer (1967) is another example of a nonlinear model where the decreased rate of dissolution (clearance) and the increased rate of lymph nodal uptake were both linked to increasing pulmonary dust levels. Mercer's model is focused on clearance by a non-equi librium dissolution process, whereas the im plications of the dust overload concept focus on the capacity of the alveolar macrophage system and its progressive incapacitation, a functional concept readily applied to the nonlinear models of Strom and Chan (1984), Vincent et al. (1985), and Yu and Morrow (1987). THE AM SYSTEM AND DUST OVERLOADING Studies have shown that the size of the AM population of the rat lungs is altered by the alveolar deposition of dust particles (Brain, 1971; Bowden, 1987; Lehnert et al.. 1985c). In a study designed to examine the effects of microgram (27-44 ng) lung burdens of an in- fit the that of se of a re the ude of ribing onary luence d phehetical ing" is gation Jnked arance mitant e lymAdarn. 1972; 1Garg, ilso ocngoposed le of a r >f .eo iate iked to lercer's n-equithe im>t focus ophage ition, a to the (1984), dorrow ST the AM I by the (Brain. 1985c). ffects of >f an in- DUST OVERLOAD MECHANISMS 375 nocuous dust on AM response in SPF Long- cells, we can convert 1 X 10-3 cm3to pm3 by Evans rats, Lehnert and Morrow (1985a,b), the factor 1 X 1012 pm3/cm 3; thus, 1.5 mg of using 59Fe oxide aerosol (1.6 0.1 pm unit density dust has a composite volume of MMAD) and multiple bronchoalveolar la 1.5 X 109pm3. Allocating this composite par vages, estimated the AM pool size as 2.14 ticle volume to ~ 2 .5 X 107 AM pool indi x 107 cells with a daily output from the rat cates an average particle volume of 60 pm3/ lungs of --2.8 X 105AM. This latter estimate AM under the initial conditions of excessive agrees reasonably well with that made by lung burdens whereby evidence of dust over Masse el al. (1977) of 7.5 X 105 AM per day loading begins to appear. and the AM pool size estimation of Crapo el Expressing lung burdens volumetrically in al. (1980) of ~ 3 X 107 cells, both investiga dicates th atT i0 2, for example, with a p = 4.3, tors using different strains of rats. requires four times the particulate mass bur The AM study of Lehnert and Morrow den to bring about the same volumetric load (1985b) provided evidence by lavagate analy ing of the macrophage pool, i.e., 4-8 mg ses that the pulmonary clearance of Fe20 3 T i0 2/g lung instead of 1-2 mg dust/g lung. particles is >90% pulmonary macrophage- This fits the experimental findings with T i0 2 mediated. On the basis that pulmonary reten of Lee el al. (1985). In retrospect, our initial tion half-times reported for Fe2C>3 by several description of overloading being associated ' | groups (Gibb and Morrow, 1962; Lehnert and Morrow, 1985b; Morrow el a l, 1964; Muhle el a l, 1987a) range between 53 and 65 days and average around 60 days, and the assumption that Fe2Oi clearance is com pletely due to AM removal, then a clearance coefficient, k, of In 2/60 days = 0.0116 day-1 should apply to the fraction of the macro phage pool translocated daily. Averaging the with 1-2 mg dust/g of lung arose from studies of coal, soot, diesel, and copolymer particles, all essentially unit density materials. To avoid the dust density issue, overloading can be expressed as starting at a lung burden of about 1-2 x 109 pm3 particulate volume/g lung (60 pm 3/AM X 2.5 X 107 AM/lung) in the rat lung. estimates cited regarding AM pool size to 2.5 The median volumetric size of a rat AM is x 107 cells, then the product, 2.5 x 107 cells approximately 1000 pm3 (Dethloff el a l, (0.0116 day-1), indicates 2.9 X 105 cells are 1987; Lehnert and Morrow, 1984; Strom, translocated day-1, a value similar to those 1984); therefore, this 60 pm3 particulate vol | cited earlier and determined on independent ume can be thought of as producing a 6% in bases. crease in the average AM volume. Since a 1 |i If we accept a provisional value of 1 mg pm spherical particle (p = 1.0) is 5 X 10-13 | dust/g of lung tissue as the lowest level pro cm3or 0.5 pm3, 60 pm3is represented by 120 ducing the overload condition then in the such particles. By analogy, for 3-pm particles, I adult Fischer 344 rat of ~ 1.5 g lungs, a 1.5 60 pm 3is represented by 4 or 5 such particles. mg burden is, in principle, relegated to an This and other volumetric computations in AM pool consisting of ~ 2 .5 X 107cells. This this paper neglect the complexity of void i is equivalent to ~ 6 X 10-8 mg dust/AM. spaces associated with close packing of ) Since it is logical to assume that the density spheres: these could, under some conditions, i of the particle is irrelevant to phagocytosis increase the composite particle volume of * (Morrow and Mermelstein, 1987), we can re uniform spheres by about one-third or more express the mass of the lung dust in terms of (from 1 - 4tt/ 18 to 1 - 4tt/ 24) (Stober, 1972). an equivalent volume with a density (p) equal Another neglected complexity pertains to the to 1.0, thereby, 1 mg of dust is volumetrically range of AM cellular volumes. As Strom | equivalent to 1 X 10-3cm3. To work with vol- (1984) reported, AM volumes, even under j umetric units more suitable for particles and control circumstances, are highly skewed and i 376 P. E. MORROW include some cells with volumes greater than The composite volume for 8 X 109uniform 4000 /zm3volume. particles of 1.0-Mm-diameter is 4 x 10%m3. In a recent review by Bowden (1987), sev From this calculation and the estimated AM eral collaborative studies with Adamson (Ad pool size, (4 X 109 ^m3/8 X 107 AM), we ob amson and Bowden, 1980, 1981, 1982; Bow tain an estimate of 50 #zm3/AM when the AM den and Adamson, 1984) are described which recruitment system is characterized by Bow analyzed the AM response in mice given vari den as " saturated" (Bowden, 1987). The sim ous particle sizes and burdens. Recruitment ilarity of this composite particle volume per of AMs was found to be related to the size macrophage to the 60 zm3/AM value ob and number of the administered particles. At tained on the basis of rat studies is apparent. constant lung loading, 0.03-^m carbon parti Bowden stressed that this " saturation" cles induced a three-fold greater increase in level of innocuous particles was associated the lavageable AM population than uniform with a general pulmonary inflammatory re latex particles of 1.0-^m diameter, with 0.1- sponse, an increased likelihood of free parti /im latex particles being intermediate in their cles in the interstitium, and increased local AM recruitment. ization of particles in peribronchial and peri Adamson and Bowden (1981) reported vascular connective tissue (Adamson and r>i '. Uli ` that in the mouse lung, approximately 1010 particles of 0.03 tin were needed to stimulate division of interstitial cells and produce a concomitant increase (up to a factor 10) in lavageable cells (AM) over a control rate of ~ 2 .6 X 105AMs per day in unexposed mice. If substantially larger numbers of particles were instilled in the mouse lung, for example, 8 X 1013carbon particles of 0.03-/zm size or 8 X 109 latex particles of 1.0-jzm diameter, no Bowden, 1980, 1981, 1982; Bowden and Ad amson, 1984). These responses are similar to those seen with particle overloading and ap pear to support the general hypothesis that the progressive prolongation of particulate re tention derives from a combination of events which includes the apparent inability of the AM system to adapt to excessive dust bur dens. The mean 50-60 tim}/AM volume change obtained by the foregoing calculations infers further elevation in lavageable AMs occurred each AM phagocytizes the same volume signifying a " saturation" of AM recruitment. (number) of particles. This is clearly not the The differences seen in macrophage recruit case as Kavet et al. (1978) and others have ment between the largest (1.0 zm) and small shown (Lehnert and Morrow, 1984; Lehnert est (0.03 (im) particle investigated was about etal. ( 1985d). Frequency distributions of par- a factor 3, with the maximal effect produced ticles/AM vs percentages of the AM popula within the first postinstillation week. tions have been found to be skewed and For purposes of this discussion, data for the sometimes Poisson-like (Kavet el al., 1978). l-/zm particle will be used since this particle At early times after AMs are exposed to uni size is typical of dusts used in inhalation stud form latex particles in either in vitro or in vivo ies and is close to the particle size used by systems (the latter being assessed by lavaged Lehnert and Morrow (1985a,b) for AM ki AMs), there is clearly a broad distribution of netics. If we accept 8 X 105AM as represent particle numbers per AM. For example, ing an average, sustained daily translocation Lehnert et al. (1985d) intratracheally admin rate from the dust-exposed mouse lung (Ad istered 4 X 108 uniform latex panicles of 1.9- amson and Bowden, 1981) and assume that Mm-diameter to each of five Sprague-Dawley the lavageability of mouse AMs is compara rats. After 24 hr, lavaged AMs (pooled data) ble to that determined for the rat, then the demonstrated that --99% of the particles activated AM pool size for the mouse would were cell-associated. Moreover, at this time a be about 8 X 107cells. mean of ~ 35% of the lavaged AMs contained DUST OVERLOAD MECHANISMS 377 no particles; 18% contained 1-5 particles/ AM; 12% contained 6-10 particles/AM; 6% contained 11-15 particles/AM; and 3% con tained 16-20 particles/AM. A substantial fraction, ~26% , of the lavaged AMs con tained more than 20 particles. Subsequently, Lehnert reexamined this portion of the fre quency distribution (1987). The extended grouped data were as follows: approximately 5% of the AMs contained 21-30 particles/ cell; ~5% contained 31-40 particles/AM; ~5% contained 41-50 particles/AM; ~3% contained 51-60 particles/AM; ~3% con tained 61-70 particles/AM; ~2% contained 71-80 particles/AM; and ~1% contained 81-90 particles/AM. The remaining catego ries 91-100, 101-110, 111-120, 121-130, and > 130 particles/AM were associated with mean percentages of 0.8, 0.2, 0.2, 0.2, and 0.1, respectively. A rough estimate based upon these original and extended data indicates that the average number of 1.9-jzm particles, each with a 3.6 zm3 volume, per AM was of the order of 15. Furthermore, this distribution signifies that ~73% of the AM had up to 60 zm3 of 1.9zm-diameter particles; 37% of the AM were distributed within the overload range, i.e., contained between 60 and 500 >im3/AM. Clearly, these highly skewed distributional data are not Poisson-like. Instead of visualizing the overload process as affecting all AMs uniformly, it is probably more realistic to envision an increasing frac tion of the AM population reaching some composite - particulate - volume limitation which results in a reduced ability to clear par ticles. With a chronic particle exposure, the increase in lung dust burden results in a pro gressive increase in the immobilized AM population until ultimately all cells reach this volumetric limitation and AM-mediated clearances ceases. There is only limited evidence on the mag nitude of the lung burden bringing about a virtual cessation of AM-mediated clearance. Such data indicate that a 10-fold greater dust burden is required than is present when over loading begins to bring about a significant prolongation of particulate clearance (Chan et al., 1984; Muhle et al., 1987b). Expressed in average terms, the volumetric limitation would constitute at least 600 *zm3/AM. For 1.9-izm-diameter uniform microspheres, this would be equivalent to --170 particles per cell, again neglecting the void-space volume which becomes more and more important with increasing numbers of phagocytized par ticles. For this specific case, adding the voidspace volume to the composite volume of 170 uniform 1.9-jzm particles/AM might ex ceed 780 izm3/AM, or conversely, only 150 such particles would be needed to exceed the 600 /zm3/AM volumetric limitation if close packing occurred. With this restatement of dust overloading, it is interesting to consider the study of Snipes and Clem (1981) with 3-, 9-, and 15-jzm di ameter polystyrene particles simultaneously administered intratracheally into the lungs of Fischer 344 rats. Polystyrene particles of each size were tagged with a different radionuclide so external counting was useful for determin ing the respective retention half-times. Fifty micrograms ( ~ 1.7 X 106 particles) of 3-/zm microspheres was found to be cleared biphasically with the slow (alveolar) half-time slightly longer than that for iron oxide, 69 vs 60 days, respectively. When 5 zg (--2 X 105 particles) of 9-jzm polystyrene microspheres were administered, the slow clearance phase had a 580-day half-time. Each of these 9-nmdiameter particles has a 382 zm3volume. By virtue of the low (0.01) ratio of particles to AM cells (2 X 105:2 X 107), only a fraction of the AM cells could phagocytize either I or 2 of these particles, but either event would be expected to lead to a prolongation of reten tion and this was found. For the 15-*zm-diameter polystyrene mi crospheres, except for an initial rapid clear ance of ~ 14% of the instilled particles proba bly due to entrapment of particles in small airways, the alveolar clearance phase was im measurable. Each of these 15-jzm particles has a 1.77 X 103 /zm3 volume which is larger 378 P. E. MORROW than the volume of the average AM and greatly exceeds the estimated overload limit for any cell, creating a virtual " all or none" situation as far as AM migration is con cerned; hence, no AM-mediated clearance should occur irrespective of the postexposure time, and this was found. The attainment of the condition of over loading which is termed the " volumetric limit" implies a loss of AM mobility, but not necessarily a concomitant loss or reduction of phagocytic function. Histological data do not reveal what the status of the dust-laden AMs may be in this regard. During the develop ment of the overload condition, the ability of the pulmonary macrophage to phagocytose particles which are not acutely cytotoxic does not appear to be impaired, since alveoli are regularly seen with particle-laden macro phages. Since these accumulations of par ticle-laden cells persist, it is logical to assume that normal AM cytolysis occurs with prompt reengulfment of released particles by new AMs. This condition is apparently not remedied with time if the dust exposure con tinues. Whether the condition reverses, how fast and to what extent, after dust-loading ceases has not been determined. Since the overload condition is sustained during chronic inhalation exposures of many months' duration, one can postulate that the inability of the dust-laden macrophage to translocate from the lungs is the critical fea ture of the condition. That the mobility of the pulmonary macrophage must be adversely affected by the excessive dust loading is clearly inferred, but whether this is a direct or indirect effect on the AM cannot be deduced from available data. FACTORS AFFECTING THE MOBILITY OF THE AM TO TRANSLOCATE FROM THE LUNGS Although much investigative effort has been directed at phagocytic mechanisms, particularly at the role of chemotactic factors and opsonins, AM activation, AM phago cytic receptors, nonspecific phagocytosis, and various cell-cell interactions (Silverstein, Steinman, and Cohn, 1977; Brain, 1986; Jones, 1984; Musson and Henson, 1984) rel atively less fundamental information has been acquired on macrophage mobility per se, and very little is known about AM mobil ity in the context of translocation from the lungs. At present, two general concepts prevail for AM translocation from the alveoli to the bronchial tree: this appears to be the most widely accepted and dominant translocation pathway for AMs. First, there is the random walk concept that implies a stochastic basis for a cell to encounter the active translocation region of mucociliary transport, commenc ing at the level of the terminal bronchioles. The second postulated concept is that of di rected migration through the transitional air ways to the same " mucociliary escalator." In the latter regard, the AM migration is pre sumed to be due to either (a) the directed mo tion of the alveolar fluid lining layer on which the AMs are passively transported or (b) a chemoattraci mt gradient which directly po larizes and facilitates AM migration along the alveolar epithelial surface. The first of the concepts of directed migra tion probably originated with the view of a continuous capillary effusate into the alveoli together with Macklin's description of " sumps" (Macklin, 1955). This concept was furthered by the intuitive model of a respira tory-driven " ebb and flow" for the superficial alveolar fluid lining layer (Gross, 1953; Kilburn, 1974). Unfortunately, the best avail able evidence refutes the existence of a nor mal capillary effusate on the alveolar surface and supports the viewpoint that the alveolar surfactant-hypophase layer is generally less than 0 .1(im thick on the alveolar surface and discontinuous (Gil, 1985). Lung surfactant cyclically forms multilayers and then re spreads, in situ, during breathing (dynamic compression and relaxation) and a continu ous alveolar lining layer is not required for DUST OVERLOAD MECHANISMS 379 appropriate reduction of surface tension (Notter and Morrow, 1975; Notter and Finkelstein, 1984). Of these several concepts, therefore, di rected migration by chemotactic factorfs) seems to be the least contentious. Kinetically directed and random migration by AMs would both resemble first-order processes, i.e., a constant fraction of the AM pool would translocate per unit time. However, the exis tence of powerful and multifarous chemoat tractants seems to provide a basis not only for chemotactic particle-cell interactions leading to phagocytosis, but also for modifications of cell-cell interactions and of AM mobility. Consequently, alterations in cell mobility ac companying particle overloading can be hy pothesized as based on excessive elaboration of these factors. Among the factors which have been dem onstrated to induce inflammation and in crease epithelial permeability, affect cell ad herence, enhance the formation of AM clus ters and granuloma, and inhibit AM migratory activity are certain lymphokines, e.g., macrophage migratory inhibition factor (MIF), and several glycoproteins elaborated by AMs and other cells, such as fibronectin and colony stimulating factors (CSFs) (Bur gess et al., 1977; Dauber and Daniele, 1980; Fowles et a i, 1973; Hunninghake et al., 1984; Kradin et al., 1986; Martin tal., 1984; Metcalf, 1985; Reiser and Last, 1986; Rocklin, 1974; Warr and Martin, 1973). In the latter group, M-CSF, for example, has im portant actions on AM adherence and self agglutination and on the proliferation and ac tivation of AMs and neutrophils. Both AMs and neutrophils produce prosta glandins and activated AMs affect phospho lipid production of many molecules with sup pressor feedback actions on the AM, leading Metcalf(1985) to suggest an internal self-reg ulation whereby, inter alia, the level of ma ture AMs controls new AM recruitment. Re iser and Last (1986) used this same informa tion, including some data on lymphokines, e.g., MIF, to propose a basis for increased cell matrix reactions and granuloma formation. Collectively, the actions of these chemoat tractants and activators and modulators of AM function fit the phnomnologie infor mation on particle-laden, aggregated, immo bilized AMs observed during particle over loading. Nevertheless, the evidence that these factors, mainly studied in in vitro systems, ex plain the progressive cessation of AM-medi ated particle translocation from the lungs is entirely circumstantial. It is conceivable that the foregoing factors come into play only in relation to the " satura tion" effect on AM recruitment described by Bowden ( 1987) and in terms of the AM agglu tination and granuloma formation. Under these conditions, the AMs may be physically unable to spread and migrate, notwithstand ing the possible persistence of a normal or en hanced chemoattractant gradient for directed AM translocation. It is likely that control of the AM translocation process is complex and that all of these speculations regarding AM mobility vary greatly in their relevance. It seems equally plausible to imagine that the failure of the engorged, particle-laden macrophages to possess normal motility may be due to the volumetric distortion induced in these cells with a concomitant reduction in available cell surface for spreading and to phagolysosomal interactions with the cytoskeletal system (Trotter, 1981; Cain and Kraus, 1981). In other words, a completely mechanical basis for AM immobilization seems possible based on the dual requirement of cell surface membrane and cytoskeleton for both migration and endocytosis (Aggler and Werb. 1982). In Fig. 4, we see an example of what might be involved. A fully spread macrophage which may be typical of a migrating cell is seen. On the basis of the field magnification, we can estimate this cell's surface area as --1250 Mm2, which is about 2.6 times more surface membrane than a " smooth" AM with a 1000 tm3 volume would possess (1250 ^m2/483 ^m2). The spherical cell volume of 380 P. E. MORROW FIG. 4. Alveolar macrophage spreading. These rat alveolar macrophages in this photomicrograph were taken from a cell-particle (sheep erythrocytes) suspension and allowed to adhere to a substrate before fixation and viewing by scanning electron microscopy (SEM). In this selected field, at least one fully spread AM appears almost devoid o f particles. The foreground AM, juxtaposed by two other macrophages, ap pears to have spread less and the surface of its spherical segment is smooth except for many proturbances caused by large numbers of phagocytized erythrocytes. (Photomicrograph provided by Dr. B. Lehnert.) 1000 /zm3 assigned the average AM is based on a displaced volume measurement which is relatively insensitive to the presence of a nor mally ruffled surface membrane. Hence, we can hypothesize that this " stored" surface membrane is available for phagocytosis and especially for migratory spreading in a some what competitive fashion. The AM in the foreground of Fig. 4 is engorged with many sheep red blood cells (>80), almost devoid of ruffles, and appears to have a limited ability to spread. Although SEM images of fixed cells are not dimensionally reliable, this micro graph possibly demonstrates features which may affect the ability of the cell to migrate from the alveolar spaces to the mucociliary region. Perhaps even more compelling than the surface membrane competition, is that docu mented for endocytosis, cell adhesion, spreading, and locomotion of the AM in rela tion to its cytoskeletal system (Aggler and Werb, 1982). Whatever the mechanistic basis for the in hibition of the AM to translocate from the al veolar region, one can deduce that the volu metric " limitation" of 600 Mm3 or there abouts can result from the phagocytosis of many small particles or of a single large parti cle such as a 15-/xm diameter latex sphere. In other words both the induction of dust over loading and the ultimate breakdown of AM mediated particle clearance should be consid ered both as a particle size effect such as that demonstrated by Snipes and Clem (1981) and in terms of a composite volumeof phago cytized particles discussed here in relation to excessive particulate burdens. I DUST OVERLOAD MECHANISMS 381 SUMMARY AND CONCLUSION A general mechanistic hypothesis is pre sented on how and why excessive particle loading brings about a debilitation of AM mediated particle clearance. The general hy pothesis contains several secondary hypothe ses; specifically, that particle overloading oc curs when a certain cumulative or composite particulate volume is reached in the alveolar macrophage, and that this, in turn, results, di rectly or indirectly, in the loss of AM mobility and AM-mediated particle transport. The ca pability of the lung to clear particles, even of low inherent toxicity, is an important defense mechanism. The longer insoluble particles reside in the lung, the more opportunities ex ist for adverse developments. Accumulation of sufficient number of persistently retained particles may lead to a variety of adverse effects including, pneumoconiosis, hypersen sitivity pneumonitis, and tumorigenesis. While it is clear that retardation of particle clearance represents a departure from normal behavior, it is unclear whether lung overload ing or in the extreme, cessation of particle clearance by itself represents a toxic endpoint or whether it merely leaves the lung more sus ceptible to infection and other sources of in jury. While this general picture is derived from limited experimental data, it appears to provide coherence to an assortment of highly germane, but seemingly isolated experimen tal results. In any case, the main features of the hypothesized mechanistic basis for parti cle overload proposed are subject to experi mental refutation or confirmation and, at least, should stimulate the development of additional evidence or alternative explana tions for our present, all-too-limited under standing of the phenomena of dust over loading. ACKNOWLEDGMENTS The valuable suggestions and criticisms provided by mycolleagues, Drs. G. Oberdorster, R. 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