A

- A* * *

- 

## Response of 

## Mouse Lung to 

## Carbon 

## Deposition During Injury and 

## Repair

Ian 

### Y.R. 

### Adamson and

### HeatherL. 

### Prieditis Department of Pathology, University of Manitoba, Winnipeg R3E OW3 Canada

Increased 

### respiratorydisease and.

### d'iyAWor-':

ras associated wi i offine p e a po a W j ined t.he 

### possibility that de i o 

### eenz inertparticles to

### previously 

### injured lun mayaccente

### pumonar d by

### investigating how the 

### lunghandles sMall 

### caron: particles delivered during acute 

### injury or during fibrotic repair. Mice received 2 .g carbon 

### by intratracheal instillation into," 

### lungs 

### already showing 

### acute injury, 

### 3 d after bleomycin (BL), or into 

### lungs ith 

### fibrosis, 4 weeb 

### after 

### IL. 

### .At3 s ater 

### BL, injury to the 

### tye I



## p` voola 

### .p...... resulted in

### high p els in lavae 'fluid. Intlingr carbon Itthstu idcd

*,a.ta 9.,w a 

### l}arge¢ increase in 

### inflam..mat*ry cdts 

## 'though m i r t inser?.,- 

### tiumn ada a 

### rtionwa 

### .,,,,w.up to 16 

### wees le H al..e: 

### mice

### wasequal to



### that fndaer l

### faoe In the mice that received 

### ca 

### n4w after

### bleomycin, fibrotic repar. 

### occurred, and the 

### epitheli 

### sprface as., restard 

### beorte pDart 

### caro,the> susqun *

, 

### tio: 

### cdmost

### paie 

### interstitiun, and 

### carbon reti 

### .t 16 

### weeks was not 

### differe frm th : the 

### carbon-onlygroup. 

## t.0.l0 

## pa,ticles.# nt 

### fibrotic 

### luing diadnot 

### indufce a4fion#It :fibroblast 

### growth or 

### colagenip The results 

### indiate 

### instili oparticulates tothealveoliata

### tim f t al 

### injuryresults inin to the 

### interiim. H , d of 

### purecarbo t injuredlupsp aloesn i.- ther stimulate an. on 

### fibtoi pr 

### cs, 

### although it alters the 

### patens.' ofp 

### .aricle deposition and retention in the

### :lu.i Ky 

### words: air pollution, 

### cabonpa 

### lare,0 bleomycin, 

### lung.injury, 

## phaC`YPOtosiA. XnvronHealchPerpectl3:7

Human exposure to various 

### inhaledpartic

### ulates may

### result in

### lung

### disease, whichhas been attributed to 

### the nature 

### and toxicity 

### of

### specificparticle types, 

### for

### example silicainduced 

### pulmonary fibrosis 

### (1-3). One 

### of the most common 

### inhaled air

### pollutants is 

### the carbon 

### particle, and 

### deposits are 

### fre

### quently seen upon 

### autopsy 

### of human 

### lungs, particularly 

### lungs of city dwellers 

### (1,4). It is 

### generally assumed that 

### carbon 

### itself is 

### inert, and any 

### associated 

### lung disease arises 

### from the 

### effects of co-inhaled toxic 

### substances, such as 

### mineral 

### dusts, metal ions, or

### acidicgases. Recent 

### epidemi

### ologic 

### evidence, 

### however, points to 

### the need for more 

### investigation into 

### the health 

### effects ofinhaled

### particulate matter ingeneral. 

### Studies now 

### linkexposure to 

### fine par

### ticles with 

### increased mortality, and there is 

### evidence 

### that diminished lung 

### function in persons 

### with 

### lung disease accompanies inhalation of particulate air 

### pollutants (5-8). Inarecent 

### study, the

### dailymortality rate in six U.S. 

### cities was 

### associated with 

### the level 

### of air 

### pollution, after correcting 

### for other factors such as 

### smoking (8). 

### The strongest 

### correlation with mortality was 

### found in 

### levels of fine respirable 

### particles rather thanwithaerosol acids orgases such
asnitrogen

### dioxide (8). Many 

### experimental studies have been 

### published to 

### describe the 

### deposition, clearance, 

### and 

### pathologic effects, if any, 

### of various particles 

### introduced into normal 

### lungs (9,10), 

### but not 

### much is 

### known 

### about 

### possible synergistic 

### effects of 

### depositingparticles, whether toxic or inert, into 

### abnormal lungs. Fine particulates (< 2.5 pm

### diameter) suchas soot can 

### reach the most 

### distal regions 

### of the 

### lung, and 

### although such particles are 

### usually considered inert, 

### there is 

### little 

### information on 

### their effects on

### previously injured 

### lungs. If 

### fine

### particles are

### deposited into

### alveoli during an acute injury 

### phase, damage at 

### the 

### air-blood barrier may increase 

### particle translocation to 

### the interstitium (11). On 

### the other 

### hand, it is 

### also 

### possible that the increased number 

### of inflammatory cells 

### recruited to 

### the 

### alveoli could enhance phagocytosis and 

### subsequent clearance. 

### Theremay

### alsobe

### differences in

### deposition and clearance if

### particles are 

### introduced into a 

### fibrotic lung in 

### which 

### function as 

### well as structure is 

### altered (12). In many 

### examples of 

### fibrosis, activation 

### of macro

### phages is 

### believed to be a 

### key event in 

### cytokine production 

### (13,14). How 

### these 

### activated 

### pulmonary 

### macrophages then 

### respond, even to an inert 

### particle, is not 

### known. During

### phagocytosis and attempt

### ed 

### clearance, there may 

### be synergistic 

### effects within 

### the 

### macrophage to increase 

### cytokine secretion so 

### that 

### phagocytosis of
an inert 

### particle may 

### become an extra 

### fibrogenicstimulus. We 

### studied these questions on

### howthe injured 

### lung responds to 

### deposition 

### offine 

### particulates using 

### intratracheal 

### (IT) instil

### lation of colloidal carbon as an inert 

### particle. Carbon was given to mice a 

### few 

### days after IT 

### bleomycin, which 

### induces acute injury to 

### the 

### alveolar 

### wall, and to mice 4 

### weeks 

### after 

### bleomycin when the 

### epithelial surface is 

### restored

### but fibrosishas 

### developed. Lung responses 

### such as injury, 

### inflammatory cell 

### numbers, particle retention, and the 

### production of fibrosis
were 

### compared overa subsequent 16-week 

### period. 

### Materials and 

### Methods Swiss mice 

### (25-g

### males) were 

### divided into 5 groups 

### of 40 

### which received intratracheal injections while 

### under mild nembutal anesthesia. Group 1 

### received 2 mg 

### ofa 

### carbon suspension 

### (carbon black in 

### hydrolyzed gelatin, 40-nm 

### diameter; Faber-Castell Corp., 

### Newark, NewJersey) in 0.1 ml water; group 2 received 0.15 units 

### ofbleomycin (Bristol-Myers Squibb Co., 

### Evansville, Indiana) 

### suspended in 

### 0.1 ml water; group 3 received the same 

### dose of

### bleomycin, then 3 

### days later received 2 

### mg carbon; group 4 

### was instilled with 

### bleomycin, then the same

### dose ofcarbon 4 weeks 

### later; and group 5 

### received 0.1 ml 

### distilledwater

### only. 

### Animalswere

### killed in groups 

### of 

### four by 

### barbiturate overdose at the 

### following times 

### after bleomycin or water 

### (groups 2 

### and 5) or 

### after carbon (groups 1, 3, 4): days 0, 

### 1 and 3, weeks 1, 2, 4, 8, 12, 

### and 16; mice 

### received 2 

### pCi/g tritiated 

### thymidine 1 

### hrbeforedeath. A 

### tracheotomywas 

### performed on 

### each animal, and the 

### lungs were 

### washed four times 

### with 1 ml saline. We 

### pooled the 

### bronchoalveolar 

### lavage (BAL) 

### fluid and determined the total 

### cell count 

### by hemocytometer. 

### The cell suspension was cen

### trifuged; a cytospin preparation was 

### made and stained

### bythe Giemsa

### method; differential counts 

### of

### polymorphonuclear leukocytes 

### (PMN) and 

### alveolar 

### macrophages (AM) were 

### made on 500 

### cells per 

### slide. We 

### determined 

### the total 

### number 

### of cells ofeach type 

### for each time 

### studied, andthe
mean ± SE 

### for four mice per group was 

### calculated. We 

### used the supernatant 

### ofthe 

### lavage fluid fromeachmouse to 

### determine total protein content as an 

### index of 

### lung injury 

### (15,16). After 

### lavage, the bronchus 

### leading to 

### the 

### right 

### lung was 

### clamped; this 

### lung was 

### removed, weighed, 

### and frozen for biochemical 

### analysis. The 

### left 

### lungwas 

### inflat

### edwith 0.5 ml 

### of 2% 

### buffered

### glutaraldehyde and 

### removed; most 

### ofthe tissue was 

### processed for 

### embedding in 

### glycol methacrylate. Sections 

### (0.75 

### jim thick) from three 

### randomblocks per

### animal were 

### prepared for 

### histologyand for 

### autoradiographywith 

### the use 

### of

### KodakNTB2 

### emulsion. We 

### determined the percentages 

### of 

### 3H-thymidine-labeled cells at 

### each time

### by

Address correspondence to I.Y.R. Adamson, Department ofPathology, UniversityofManitoba, 236-770 BannatyneAvenue, Winnipeg R3E 0W3 Canada. This research project was supported by a grant fromtheMedical Research Council ofCanada. Received 11 July 1994; accepted 26 October 1994.

Environmental Health 

### Perspectives 72- 

## eem Ie 

### wan *9m 

# 1--

counting 3000 alveolar 

### lung cells per animal. The 

### means and SEs were 

### calculated for each group 

### and 

### plotted as time 

### after carbon 

### instillation. Significant 

### differences
were 

### determined using ANOVA or Student's t-testas appropriate. The right lung 

### of each mouse was 

### homogenized in water, 

### and 

### biochemical assays were 

### performed on 

### duplicate sam

### ples. We 

### determined total protein, 

### and as an 

### index 

### of collagen content, we 

### determined 

### hydroxyproline (HYP) 

### levels after 

### hydrolysis with 

### hydrochloric acid 

### (16,1X). In addition, six extra mice per group were 

### killed at 

### the end 

### of the experiment 

### (16 weeks) to assess 

### the amount 

### of carbon 

### retained in 

### thewhole lung. 

### The lungs were 

### removed, 

### immediately chopped, and incu

### bated in 

### 40% potassium 

### hydroxide overnight in a 80°C 

### waterbath. When the tissuewas 

### completelydigested, the 

### solution

was 

### centrifuged at 1500 rpm 

### for 15 min, 

### and a 

### residue was 

### obtained. This was 

### washed twice in 

### distilledwater, 

### resuspended in water, 

### and 

### transferred to a 

### weighed tube. The residue was 

### dried to constant 

### weight. Means ± SEs were 

### calculated 

### for each group 

### of six, 

### and the 

### significance of any 

### difference from control and from car

### bon

### onlygroupswas

### determined. 

### Results Lung

### Morphology
Mice that 

### received onlywater 

### showed normal 

### lungs 

### by 

### light microscopy. 

### The 

### lungs of animals instilled with 

### only carbon 

### had increasedcellularity, 

### particularly in

### the first 2 

### weeks when most 

### carbon was 

### phagocytized in

### alveoli 

### byAM 

### and PMN, as previ

### ously described 

### (18,19). A 

### few 

### large AM 

### with carbon 

### persisted for several 

### weeks, and some 

### carbon was seen in 

### interstitial 

### macrophages, 

### particularly in

### peribronchiolarlocations. Lungs 

### examined 3 

### days after

### bleomycin showed diffuse injury to 

### the alveolar wall 

### and an 

### inflammatory response 

### ofAM 

### and PMN 

### (Fig. 

### IA). After these mice 

### received 

### carbon, additional 

### inflammatory cells were seen in 

### the alveoli 

### and, as 

### the experiment 

### progressed, it 

### appeared that more 

### carbon

was present in 

### the 

### pulmonary interstitium 

### and remained there as 

### fibrosis 

### developed (Fig. 

### 1B). Lungs 

### examined 4 

### weeks 

### after 

### bleomycin 

### only showed areas 

### of extensive 

### fibrosis (Fig. 2A). 

### When these 

### animals received 

### carbon, an 

### inflammatory response 

### with

### increasedAM

### and PMN in

### thealveoli

was seen a 

### few 

### days later, 

### particularly in 

### less 

### fibrotic areas 

### of

### lung. Most 

### ofthe car

### bon 

### appeared to 

### be 

### cleared 

### by 4-8 weeks when 

### the 

### inflammatory response was reduced, and little carbon was found in 

### fibrotic areas 

### of interstitium 

### (Fig. 2B). There were no 

### apparent 

### differences in the severity 

### offibrosis seen 

### morphologically in any 

### ofthe bleomycin groups at the end of 

### the experimental period. 

### Bronchoalveolar Lavage Values 

### obtained 

### for the numbers ofAM 

### and PMN and 

### for the protein level in 

### water-injected 

### controls were not 

### different from 

### noninjected (time 0) mice and so are not 

### shown at 

### each time to 

### simplify the 

### figures. 

### The number 

### ofAM 

### recovered after bleomycin alone 

### increased 

### fourfold in the 

### first week, 

### and, though it fell 

### somewhat, it
was still greater 

### than baseline over 

### the 16- week 

### period (Fig. 3). 

### After carbon only, AM 

### numbers peaked at 8-10 times normal, then 

### fell slowly. A 

### similar response in AM 

### numbers was seen in groups 

### that receivedbleomycin 3 

### days or 4weeks

### before carbon instillation (Fig. 3). A 

### similar patternwas seen

### when the PMN responsewas 

### quantitated. A 

### small increase in 

### lavaged cellswas 

### found soon

### after

### bleomycinalone, whereas the increasewas

### muchgreater

### after carbon injection (Fig. 

### 4). A 

### comparable PMN response in intensity and duration
was 

### found after instilling 

### carbon 3 

### days 

### afterbleomycin or 4 

### weeks later in 

### fibrotic lungs. To quantitate lung injury, 

### particularly any 

### permeability change at 

### the air-blood 

### barrier, protein 

### levels were 

### measured in BAL 

### fluid. Normal values were 

### low, at 

### around 90 

### jig/ml, and carbon instillation 

### caused a 

### sharp but transient increase 

### (Fig. 5). Asimilarpatternwas seen inmice with 

### fibrotic lungs due to 

### bleomycin administration 4 

### weeks before receiving 

### carbon.

Figure 1. (A) Mouse lung, 3 days after bleomycin, shows diffuse alveolar injury (arrows) with cell debris, red blood cells, and inflammatory cells in alveoli. (B) Lung section from mouse to which carbon was instilled 3 days afterbleomycin, killed 8weeks later. Many carbon-laden macrophages (arrows) are seen infibrous interstitium. x655.

Figure 2. (A) Mouse lung, 4weeks afterbleomycin only, shows extensive interstitial fibrosiswhile alveoli 

### (A) are mostly clear of cell debris and inflammatory cells. (B) Lung section from mouse to which carbon

was instilled 4weeks afterbleomycin and killed 8weeks later. Little carbon (arrows) is seen inthe fibrotic interstitium, while a fewcarbon-laden alveolarmacrophages are seen inthe airspace (A). x655.

Volume 103, Number 

### 1, January 1995 

### 73A

. e

-

0 2 4 6 10 12 14 16

Weeks after Carbon

Figure 3. The number of alveolar macrophages (AM) recovered by bronchoalveolar lavage (BAL) from the

four treatment groups up to 16 weeks after receiving carbon. There were no statistical differences

among the three groups that received carbon.

4 M arboIn|

- | |

-

Figure 4. The number of plvolyrmocrponuchargeukcye recovered by bronchoalveolar lavage (A)fo h

~~~~~~le

omycin-u fonly t t groups r we(eno 0s.01a) 

### fh~~~~~~~~~~~~~~A Blomci th days i Carbon

3500

_ _ _ _ _ _ 1 s

|| S

_ _ _ _~~~~~~~~~~~~~~~~~~~~~~~~~------ -emcnWeBeomcisdays}e+ Carbon_ 

### Figur 4_$enme 

### fplmrhncla ekcts(M )rcvre 

### ybocolelrlvg BA)Th 3da blo 

### yipls-crbnvalu wa infcnl ihrta hti te rusa a onl ( <0.1)

The highest levels ofalveolar protein were found in mice in the acute injuryphase up to 1 week after bleomycin instillation, when levels were 16-20 times normal. These values were obtained in BAL after bleomycin only and in mice that received carbon 3 days after bleomycin. In these

groups, protein levels did not return to normal for several weeks (Fig. 5). Lung Fibrosis
We examined autoradiographs of lung to determine if there was evidence of extended cell proliferation, particularly in the fibroblast population. In the first week after injecting carbon, the percentage of thymidine-labeled cells increased in all experimental groups (Fig. 6). This early increase appearedmainlydue to interstitial monocyte/macrophage labeling, though mice that received bleomycin also showed

some epithelial and endothelial cell labeling in the first 2 weeks. Labeling in the carbon-only groupdroppedsharply to control values by 2 weeks, while DNAsynthesis was higher inall bleomycin groups (Fig. 6). After 1 week, labeling in the various bleomycin groups was similar, and in each

case, thepredominant labeled cell type was the fibroblast. The similar fibrotic response in the three bleomycin groups was confirmed by the HYP measurements. Mice that received carbon only showed a small increase in HYP, not different from water 

### controls, andconsistentwithaging (Fig. 7). Animals ingroups 

### that receivedbleomycin, with or without carbon at different stages 

### of the 

### post-bleomycin response, 

### showed a rapid rise in tissue HYP beginning at 2 weeks. Thereafter, all bleomycin groups were equivalent, and HYP 

### levels were 

### all 

### significantly greater 

### than the control or 

### carbon-onlygroups (Fig. 7). 

### Lung

### Residue
An amorphous, nonparticulate residue weighing 0.2 + 0.05 mg was obtained 

### from control lungs, and a similar weight 

### of residue was 

### obtained from fibrotic lungs 

### after bleomycin alone (Fig. 8). Particle retention in mouse lung 16 weeks 

### after 

### instilling the 

### large dose of carbon 

### could be 

### quantitated; the residue was black

### and

### weighed significantlymore 

### than that from controls. A 

### similar 

### weight 

### of residue was measured from lungs that 

### received carbon only and carbon 4 

### weeks 

### after bleomycin. The 

### highest weight of residue was 

### recovered from lungs that received carbon during the acute injury 

### phase, 3 

### days 

### afterbleomycin. The 

### residue from this group 

### weighed 

### significantly
more 

### than 

### that obtained from all other groups (Fig. 8). 

### Discussion Baseline studies on 

### deposition, clearance, and 

### pathologic effects that determine 

### whether a 

### particle is toxic or 

### inert," are 

### usuallycarried out onnormal 

### animal 

### lungs after inhalation or 

### intratracheal instillation. However, in 

### human terms, exposure to 

### particulate air

### pollutants often occurs

### when the 

### lung is 

### compromised. It is

### possible that exposure to a 

### particle such as 

### carbon, which causes no 

### effects in 

### normal 

### lung, may 

### induce a 

### different reaction when 

### introduced to 

### lungs at 

### different stages 

### of
an injury-repair 

### cycle. In 

### general, particles deposited in 

### the 

### lung are

### mostlycleared

### by the 

### mucociliary system, 

### which can eliminate 

### freeparticles as 

### well as 

### those 

### engulfed 

### byAM. As 

### the dose increases, an 

### adaptive increase inAM occurs, 

### and at

### higher levels PMN 

### also migrate to 

### the alveoli to augment 

### phagocytosis and clearance 

### (18,19). These initial 

### responses occur 

### whether the 

### particle is 

### considered inert or toxic 

### (3) and
were seen here in 

### all 

### carbon-injected groups. There were 

### differences, however, in the 

### subsequent fate of

### particles, which

can

### be 

### related to the 

### integrity

### ofthe 

### alveolar

### epithelium. Little is 

### known

### about

### particlehandling by the 

### lungwhen there is 

### alveolar 

### epithelial cell injury. 

### The normal 

### epithelium is a barrier to 

### particle translocation, and it is 

### only crossed in 

### overload situations when 

### particles are 

### transported across type I cells

Environmental Health Perspectives 250

20o

x 150

100

74A

- **

- -e aa

- - 

### I

to 

### reach the interstitium, 

### where they are phagocytized by interstitial macrophages (18-20). Particles mayremain inthis location 

### for long periods, and in 

### the case 

### of carbon, no 

### fibrotic reaction ensues. In 

### the present studywe created a diffuse alveolar injury by instilling bleomycin 3 days 

### before giving a 

### large dose 

### of carbon. Because 

### transepithelial passage increases 

### with particle number 

### (18,20), we 

### used a 

### level near the maximum 

### deliverable in a 

### single dose to a mouse to 

### help detect any possibile changes in the 

### fibrotic response due to 

### particle exposure 

### of 

### injured lung. Intratracheal bleomycin induces type 1 

### epithelial necrosis (21) 

### and results in a 

### high level of leaked protein 

### that is measured in BAL 

### fluid. Although carbon 

### does not increase 

### this level 

### of damage, 

### the 

### epithelial injurypresent 

### for about 2 weeks 

### allows many 

### free particles to reach the interstitium forphagocytosis byinterstitial 

### macrophages. This was seen 

### morphologically, and later the increased translocation and retention 

### ofcarbonwas 

### confirmed by the increased 

### weight 

### of a 

### black lung residue recovered at 16 weeks. The increase in particle translocation to the interstitium 

### occurred despite a 

### brisk 

### inflammatory cell response to carbon that increased the number ofAM and PMN in 

### the alveoli. Most 

### ofthese cells would have recently arrived in the alveoli and so were likely immature andpoorphagocytes. The 

### observation that acute 

### inflammatory lung injury retards particle clearance has also 

### been reported in 

### calves with viral pneumonitis, in

### whichclearance 

### ofcobalt oxide

2000

I80

1600

lam 

### Ej 12W -M

600n UO 02

m

400

200

U la WeeksafterCarbon
Figure 5. Protein content of bronchoalveolar lavage (BAL) fluid. Values in the firstweek afterbleomycin alone orbleomycin 3days plus carbonwere significantlyhigherthanthe othergroups (p<0.01).

4, .0 3 

## .5||fi|lEEEEEEaa|~- Carbon
111

__ _ _ 

### -O~~~~~~~0- Bleomycin_

1111

-

_____",~~~~~~~~Beomycin(4wookss+Carbon_

M 3.0 1 ___Bleomycin(3days) +Carbon_

r .5

0.5

20 s~~~~~~~~~ek aferCabo .X 6ln_ s dfr c

0q 2- 4 B 10 12 14 1 

## aR~~~~~~~~ekafter_Carbon Fiue6ecetgso llln el ta noprae rtaedtyiie(3T tvrou ie.N sigifian difrne nlbln atr fe ekwrese ntetrebemcnijce rus was reducedandparticleswere sequestered in the interstitium 

### (S). The fact that particles deposited in the 

### alveoli 

### duringepithelial injurysubsequently results in an increased interstitial bur

### den constitutes a 

### potential hazard. In sev

### eral studies, a 

### clear link 

### has been established between interstitial macrophage activation 

### by particles such as 

### silica or 

### asbestos and the 

### subsequent development 

### ofpulmonary fibrosis (22,23). This also
occurs in other 

### examples of fibrosis, such
as 

### the 

### pulmonary response to 

### bleomycin alone, in which activated macrophages, 

### particularly those in the interstitium, have 

### been 

### identified as the prime source 

### of 

### cytokines that stimulate 

### fibroblast

### proliferation 

### and 

### collagen deposition (24). It was 

### thought possible that an extra 

### burden on these macrophages, even 

### by an inert parti

### cle, might 

### further stimulate an 

### activated 

### cell in a synergistic reaction. However 

### that didnot 

### happen in 

### these experiments using pure 

### carbon; 

### despite the extra 

### particulate load

### retainedup to 16 

### weeks, no 

### additional fibrotic response was seen or 

### measured 

### biochemically. Although we 

### used 

### particle instillation, our 

### results are in agreement 

### with an 

### earlier publication in 

### which 

### adding titanium

### dioxide as an 

### "inert" par

### ticle to 

### inhaled asbestos did not increase 

### fibrosis over 

### that induced 

### by asbestos 

### alone (25). A 

### different 

### pulmonary response was seenwhen carbonwas 

### instilled to the 

### lung 4 weeks 

### after 

### bleomycin. At this time, 

### epithelial repairwas 

### complete, and alveolar protein levels had returned to nearnormal,
even 

### though interstitial fibrosis had been produced. 

### Instilling carbon induces the usual 

### inflammatoryresponse, 

### thoughparti

### cles were 

### mostly 

### deposited in 

### the 

### less fibrotic areas 

### of 

### lung. This may 

### be the result of relative variations in 

### ventilation due to 

### the fibroticprocess. In one previous 

### study of inhaled 

### particle retention in 

### fibrotic 

### lungs of 

### hamsters, it was 

### noted that diseased areas 

### of 

### lung received fewer 

### particles (12). Greater

### deposition ofcarbon in 

### the more 

### functionallynormal regions 

### of lung, 

### together with the restoration 

### of the 

### epithelial barrier before at 

### the time 

### of 

### instillation, allows a more 

### regular pattern 

### of

### phagocytosis and

### clearance, whichwould 

### explain why the retained carbon at 16 

### weeks was 

### equal to that 

### ofthe carbon 

### only group 

### (Fig. 8). Despite the 

### likely presence 

### of activated 

### macrophages in 

### these 

### bleomycin-injected 

### lungs (24), few instilled 

### particles reached the interstitium, 

### and there
was no 

### additional fibroblast 

### stimulation as 

### judged by the 

### equal levels of HYP 

### after 

### bleomycin, withor

### without carbon. The results demonstrate that deposition 

### ofa 

### large number 

### of inert 

### particles in
a 

### single dose to the 

### lungduring a

### phase of

Volume 103, Number 1, 

### January 1995 

### 75A 

## -

# A*I

-

300 =211

20 thre bleomycingrup wer found

.

_ 0

- Boryi

10 -* Bloyi (4weks Cabo

300

>~~~~~~~~ek afte_-l-.rc

Mean -O 

### SEsonfrcroBnyadbemcn rus rm2wesowralbeomycin grus{ r the bl_

_ _ __igroupsc werfound.Cabo

_

- 2.0

,1.0

wes afercarbo 

### (C instBIlainp < 0.1 cm ._

_ Is X~ 5

p 0.0 C BL BL{3dasys) BL({4weeks) TIC .IC Figure 8. Weight of lung residues (mean + SE) 16 weeks after carbon (C) instillation. tp < 0.01 corn pared to all other groups; *p < 0.01 compared to bleomycin (BL) alone orwater control. 

### epithelial injury or 

### of 

### fibrotic repair 

### does not 

### exacerbate the 

### fibrotic response. However, it is 

### possible that repeated exposure 

### of injured lung to inert or to toxic 

### articulates found in urban environments mayproduce a 

### different outcome. In particular, exposure 

### of the lung at a time 

### of 

### epithelial injury is 

### potentiallymore serious 

### due to the likelihood 

### of increased 

### particle translocation and retention in the 

### pulmonaryinterstitium. REFERENCES

1. Parkes &JR. Occupational lung disorders. London:Butterworths, 1974. 2. Heppleston AG. Pulmonary toxicology of silica, coal andasbestos. Environ Health Perspect 55:111-127 (1984). 3. Bowden DH. Macrophages, dustandpulmonarydisease. Exp Lung Res 12:89-107 (1987). 4. Spencer H. Pathology of the lung, 4th ed. Oxford:Pergamon Press, 1985;413-510. 5. Utell MJ, Samet JM. Particulate air pollution and health: new evidence on an old problem. AmRevRespir Dis 147:1334-1335 (1993). 6. Schwartz J. Total suspendedparticulate matter and daily mortality in Cincinnati, Ohio. Environ Health Perspect 102:186-189 (1994). 7. Xu X, Wang L. Association of indoor and outdoorparticulate level with chronic respiratory illness. AmRevRespirDis 148:1516-1522 (1993). 8. Dockery DW, Pope CA, Xu X, Spengler JD, Ware JH, Fay ME, Ferris BG, Speizer FE. An association between air pollution and mortality insixU.S. cities. NEnglJ Med 329:1753-1759 (1993). 9. BrainJD, Valberg PA. Deposition ofaerosol in the respiratory tract. Am Rev Respir Dis 120:1325-1373 (1979). 10. Oberdorster G. Lung clearance of inhaled insoluble and soluble particles. J Aerosol Med 4:289-330 (1988). 11. Slauson DO, LayJC, Castleman WL, Neilsen NR. Acute inflammatory lung injury retards pulmonary particle clearance. Inflammation 13:185-199 (1989). 12. SweeneyTD, BrainJD, TrykaAF, GodleskiJJ. Retention ofinhaled particles in hamsters with pulmonary fibrosis. Am Rev Respir Dis 128:138-143 (1983). 13. Elias JA, Freundlich B, Kern JA, Rosenbloom J. Cytokine networks in the regulation of inflammation and fibrosis in the lung. Chest 97:1439-1445 (1990). 14. Schapira RM, Osornio-Vargas AR, BrodyAR. Inorganic particles induce secretion of a macrophage homoglogue of platelet-derived growth factor in a density and time dependent
manner in vitro. Exp Lung Res 117: 1011-1024 (1991). 15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol reagent. J Biol Chem 193:265-269 (1951). 16. Young L, Adamson IYR. Epithelial-fibroblast interactions in bleomycin-induced lung injury and repair. Environ Health Perspect 101:56-61 (1993). 17. WoessnerJF. Determination ofhydroxyproline in connective tissue. In: The methodology of connective tissue research. Oxford:Joynson and Bruvvers, 1976;227-233. 18. Adamson IYR, Bowden DH. Dose response of the pulmonary macrophagic system to various particulates and its relationship to transepithelial passage of free particles. Exp Lung Res 2:165-175 (1981). 19. Bowden DH, Adamson IYR. Pathways ofcellular eflux and particulate clearance after carbon instillation to the lung. J Pathol 143:117-125 (1984). 20. Morrow PE. Dust overloading of the lungs: update and appraisal. Toxicol Appi Pharmacol 113:7-12 (1992). 21. Adamson IYR. Drug-induced pulmonary fibrosis. Environ Health Perspect 55:25-36 (1984). 22. Adamson IYR, Letourneau HL, Bowden DH. Comparison of alveolar and interstitial macrophages in fibroblast stimulation after silica and long or short asbestos. Lab Invest 64: 339-344 (1991). 23. BrodyAR, BonnerJC, OverbyLH, BadgettA, KalterV, Kumar RK, Bennett RA. Interstitial pulmonary macrophages produce plateletderived growth factor that stimulates rat lung fibroblast proliferation in vitro. J Leuk Biol 51:640-648 (1992). 24. Phan SH, Kunkel SL. Lung cytokine production in bleomycin-induced pulmonary fibrosis. Exp LungRes 18:29-43 (1992). 25. Davis JMG, Jones AD, Miller BG. Experimental studies in rats on the effects ofasbestos inhalation coupled with the inhalation of titanium dioxide or quartz. Int J Exp Pathol 72: 501-525 (1991).

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