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Cellular and Molecular Mechanisms of Asbestosis
Cellular and Molecular Mechanisms of Asbestosis*
T a b l e 1 — A m o u n t s of Cell Layer-Associated Collagen in Normal Rat Lung Fibroblasts (RL-82) Exposed to Crocidolite Asbestos (N=4 per group)
B. T. Mossman, Ph.D., R. Gilbert, B.S., F.C.C.P;J. Doherty, B.S.; M. A. Shatos, Ph.D.; J. Marsh, M.S.; and K. Cutroneo, Ph.D.
O
ccupational and e x p e r i m e n t a l e x p o s u r e to asbestos is associated with t h e d e v e l o p m e n t o f p e r i b r o n c h i o l a r
and interstitial p u l m o n a r y
fibrosis.
Like other asbestos-
1
associated diseases o f t h e respiratory tract (ie, m e s o t h e l i o m a and b r o n c h o g e n i c carcinoma), asbestosis appears to involve alterations in differentiation and proliferation o f affected c e l l types.
Whether
t h e disease process
i n c r e a s e in replication o f
fibroblasts
e n h a n c e d ability o f individual
involves e i t h e r in
fibroblasts
an
t h e l u n g o r an to p r o d u c e m o r e
collagen o r both is u n c e r t a i n . To address this q u e s t i o n , w e u s e d a c o m b i n a t i o n o f in vitro
and in vivo
approaches.
University o f Vermont) was m a i n t a i n e d
in
Minimal Essential Medium ( M E M , G I B C O ) containing 1 0 % fetal c a l f s e r u m and e x p o s e d to crocidolite asbestos ( U I C C r e f e r e n c e sample) at various c o n c e n t r a t i o n s in m e d i u m (ie, 1, 2 . 5 , and 5 y,g/em dish) w h e n cells r e a c h e d approximately 2
8 0 % confluency. At 2 4 - h o u r intervals, 1 0 ~ M a s c o r b a t e was 4
a d d e d to all plates. C e l l s t h e n w e r e p u l s e d with H - p r o l i n e 3
(10 u,Ci/mI m e d i u m ) for 2 hours p r i o r to assays.
After
homogenization o f cells and b o i l i n g for 10 m i n u t e s to inactivate proteases, cell s a m p l e s w e r e assayed in t h e p r e s e n c e and a b s e n c e o f purified bacterial collagenase (Advance Biofactures C o r p ; —40 U/assay t u b e ) to d e t e r m i n e t h e ratio o f newly synthesized collagen to n o n c o l l a g e n p r o t e i n . At e a c h 2
p e r i o d , H - t h y m i d i n e , o r H T (1 u,Ci/ml m e d i u m ) was added 3
to additional plates for 1 h o u r b e f o r e preparation for autoradiography.
3
Control (nonasbestos exposed) cells
were
t r e a t e d identically. Results show an i n c r e a s e in total c e l l layer-associated collagen at 4 8 and 7 2 hours after addition o f asbestos to R L - 8 2 cells (Table 1). I n c r e a s e d replication o f fibroblasts 3
48 hr
No asbestos Asbestos
8.52+1.13
18.38 ± 0 . 9 4
23.66±1.34
1.0 M.g/cm 2.5 jig/cm
6.39 ± 0 . 9 0 5.7 ±1.1.5
25.59±1.89*
5.0 u,g/cm
8.95
24.05 ± 2 . 0 6 t
21.92 ± 1 . 5 8 19.55 + 2.88 37.69+1.66*
z
2
2
72 hr
24.75 ± 2 . 1 8 *
* p < . 0 2 (Student's (test adjusting for multiple comparisons between groups). tp<.05. tp-c.OOl. collagen reflects a partitioning o f procollagen m R N A s into fibroblasts
to b l e o m y c i n ,
5
INHALATION E X P E R I M E N T S
lung ( R L - 8 2 o b t a i n e d from D r M a r l e n e Absher, D e p a r t m e n t
3
24 hr
VITRO
A n o r m a l fibroblast c e l l line derived from F i s c h e r 3 4 4 rat of M e d i c i n e ,
Group
p o l y s o m e s , a p h e n o m e n o n o c c u r r i n g after exposure o f lung
EXPERIMENTS USING LUNG FIBROBLASTS E X P O S E D TO A S B E S T O S IN
Total Collagen (cpm/ng DNA)
as d e t e r m i n e d b y n u m b e r s o f cells incorporating
H T did not o c c u r u n d e r t h e s e c i r c u m s t a n c e s (data not
shown).
T h e work d e s c r i b e d above indicates increased synthesis o f procollagen b y individual cells after exposure o f rat lung fibroblasts
to a s b e s t o s . To d e t e r m i n e w h e t h e r
proliferation o f
fibroblasts
abnormal
in t h e lung also occurs
after
inhalation o f asbestos, 6 - 8 - w e e k - o l d male F i s c h e r 3 4 4 rats (n = 3-5/group) w e r e exposed to crocidolite asbestos for 3 0 days (5 hr/day, 5 days/week) using a modified T i m b r e l l generator. Twenty-four hours before removal o f t h e lungs for vascular perfusion, the rats w e r e i n j e c t e d with H T (2 u,Ci/g). 3
After t h e lungs w e r e e m b e d d e d in paraffin, alternative 3-m tissue sections w e r e p r e p a r e d for histology and stained w i t h M a s s o n s t r i c h r o m e for d e t e c t i o n o f collagen.
Additional
slides w e r e p r o c e s s e d for i m m u n o c h e m i s t r y using an antib o d y to c o p p e r - z i n c superoxide dismutase ( S O D ) d e v e l o p e d in this laboratory. This allowed us to differentiate m a c r o 8
p h a g e s ( S O D - p o s i t i v e ) from
fibroblasts
( S O D - n e g a t i v e ) in
t h e i n t e r s t i t i u m o f the lung. After dipping and d e v e l o p m e n t for autoradiography, slides w e r e counterstained with h e m a toxylin b e f o r e c o u n t i n g b y light microscopy using 1,000 X magnification. E i g h t to 12 fields on each slide w e r e evaluated to d e t e r m i n e t h e p e r c e n t a g e o f l a b e l e d bronchiolar epithelial, alveolar epithelial and interstitial cells ( S O D - n e g a tive). I n c o n t r a s t to those o f control s u b j e c t s , t h e lungs of a s b e s t o s - e x p o s e d animals exhibited fibrotic changes as deter-
To d e t e r m i n e w h e t h e r t h e e n h a n c e d synthesis o f total
m i n e d b y i n c r e a s e d deposition o f trichrome-positive m a t e -
cellular collagen p e r n a n o g r a m o f D N A r e f l e c t e d an inc r e a s e d accumulation o f collagen t y p e I m R N A s in
fibroblasts
exposed to a s b e s t o s , t h r e e r e c o m b i n a n t plasmids containing t y p e I collagen-specific s e q u e n c e s (po^Ri, p a ^ , p o t j R J w e r e
T a b l e 2 — Q u a n t i t a t i o n of Numbers of Cells Incorporating "H-Thymidine in Fischer 344 Rat Lungs Exposed to Asbestos for 30 Days (N=8-12 fields/slide)
o b t a i n e d from C . G e n o v e s e , P h . D . ( D e p a r t m e n t o f P e d i Labeling Index, %
atrics, University of C o n n e c t i c u t ) . Q u i c k - b l o t analysis c o n 4
sistently failed to show a d i f f e r e n c e b e t w e e n t h e a m o u n t s o f procollagen t y p e I m R N A s in control and a s b e s t o s - e x p o s e d c e l l s . S u b c e l l u l a r fractionation studies now are in progress to determine
whether
asbestos-induced
synthesis
of pro-
*From the Departments of Pathology and Biochemistry, University of Vermont, Burlington. Supported by Pulmonary SCOR grant P50HL14212 from the National Heart Blood and Lung Institute. Dr. Shatos is a Parker B. Francis fellow of the Puritan-Bennett Foundation.
160S
Bronchiolar epithelial Alveolar epithelial Interstitial (SOD - ) *
Control
Asbestos-exposed
.78 ± , 1 7 .8 ± . 2 1.13±.2
4.13±.87t 3.07±.46t
1,26±.25
*As detected with an antibody to copper-zinc SOD prepared in this laboratory (Mossman et al, personal communication). t p < , 0 0 1 (Students ( test adjusting for multiple comparisons between groups). 2 8 t h Annual A s p e n Lung Conference
rial originating at t h e alveolar d u c t .
7
Autoradiography r e -
s t u d i e d t h e effect o f t h e e x p o s u r e in vitro
of mouse resident
vealed that n u m b e r s o f l a b e l e d alveolar epithelial cells and
alveolar ( A M 0 ) and p e r i t o n e a l m a c r o p h a g e s ( P M 0 ) to n o n -
interstitial
toxic c o n c e n t r a t i o n s o f asbestos on t h e i r suppressive capacity
fibroblasts
w e r e i n c r e a s e d significantly in as-
b e s t o s - e x p o s e d lungs (Table 2).
on t h e m i t o g e n - i n d u c e d proliferation o f s y n g e n e i c spleen
In contrast, t h e labeling indices o f b r o n c h i o l a r epithelial
l y m p h o c y t e s , on t h e i r ability to r e l e a s e a r a c h i d o n i c acid
cells in b o t h control and a s b e s t o s - e x p o s e d lungs w e r e c o m p a -
metabolites
rable.
z y m o s a n and on t h e s p o n t a n e o u s r e l e a s e o f i n t e r l e u k i n 1
and
superoxide
anion
(07)
in r e s p o n s e
to
(11-1). P M 0 purified b y a d h e r e n c e and A M 0 o b t a i n e d b y CONCLUSIONS
b r o n c h o a l v e o l a r lavage from 8-16-week-old C 3 H / H e N m i c e
O u r work suggests that at least two m e c h a n i s m s r e s u l t in
w e r e c u l t i v a t e d in flat-bottom wells in t h e p r e s e n c e o f c o n t r o l
increased
m e d i u m , latex b e a d s , or U I C C asbestos a m o s i t e , 8 0 u-g/ml,
synthesis o f total cellular procollagen b y individual fibro-
for 21 h o u r s . T h e m e d i u m was t h e n carefully aspirated and
asbestosis.
On
one hand,
asbestos
stimulates
blasts. This p h e n o m e n o n does not r e f l e c t an i n c r e a s e in
t h e M 0 f u r t h e r p r o c e s s e d . S u p p r e s s i v e capacity was evalu-
steady-state levels o f procollagen t y p e 1 m R N A s . I n addition,
a t e d as previously d e s c r i b e d
interstitial fibroblasts have an e n h a n c e d replicative potential
cells s t i m u l a t e d with an o p t i m a l d o s e o f C o n - A and evaluat-
in t h e lung after inhalation o f asbestos b y rats.
ing t h e incorporation o f tritiated t h y m i d i n e after 7 2 hours o f
5
by adding s y n g e n e i c spleen
c u l t u r e . T h e r e l e a s e o f Ol was m e a s u r e d b y f e r r i c y t o c h r o m e c r e d u c t i o n after stimulation with o p s o n i z e d zymosan. Pros-
REFERENCES 1 Craighead J E , Mossman BT. The pathogenesis of asbestosassociated diseases. N Engl J Med 1982; 306:1446-55 2 Newman RA, Cutroneo KR. Glucocorticoids selectively decrease the synthesis of hydroxylated collagen peptides. Mol Pharmacol 1978; 14:185-98 3 Mossman BT. Autoradiography for determination of DNA synthesis in the hamster bladder epithelium. TCA Manual 1977; 3:663-65 4 Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat a and ot collagen mRNAs and their use in studying the regulation of type I collagen synthesis by 1,25dihydroxywtamin D. Biochemistry 1984; 23:6210-16 t
2
taglandin E
2
( P G E ) and F , ( P G F . J , and l e u k o t r i e n e C 2
2 a
4
( L T C ) w e r e evaluated by radioimmunoassay after stimula4
tion w i t h zymosan 2 0 0 |i.g/ml in m e d i u m without s e r u m . I l - l was evaluated b y t h e C 3 H / H e J
thymocyte
proliferation
assay. T h e p r e s e n c e o f i n t e r l e u k i n - 2 (11-2) activity in t h e s u p e r n a t a n t s was assayed b y t h e ability to support
the
proliferation o f t h e C T L L c e l l l i n e . I n a g r e e m e n t
with
previous e x p e r i m e n t s " t h e e x p o s u r e o f A M 0 and P M 0 in
vitro
to a m o s i t e c a u s e d a d o s e - d e p e n d e n t d e c r e a s e in t h e i r suppressive capacity, w h e r e a s phagocytosis o f latex had n o effect. T h i s p h e n o m e n o n was not d u e to loss o f c e l l viability as
5 Sterling KM, Harris MJ, Mitchell J, Cutroneo KR. Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs: reversal of the bleomycin effect by dexamethasone. J Biol Chem 1983; 258:14438-44
j u d g e d b y L D H r e l e a s e and trypan b l u e exclusion, and was
6 Mossman BT, Marsh JP, Shatos MA. Alteration of superoxide dismutase (SOD) activity in tracheal epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants. Lab Invest 1986 (in press)
different effects on t h e z y m o s a n - i n d u c e d r e l e a s e o f P G E
7 Mossman BT, Shatos MA, Marsh JP, Doherty JM, Craighead J E , Hemenway D, et al. Rapid development of asbestosis in rats after inhalation of crocidolite. Am Rev Respir Dis 1984; 129:A148
p a r a l l e l e d b y a m a r k e d r e d u c t i o n o f t h e ability to p r o d u c e Oz. A s b e s t o s e x p o s u r e c a u s e d a small i n c r e a s e o f spontaneous P G E production from b o t h M 0 populations; however, it had 2
PGF
2
and
from A M 0 and P M 0 . I n fact, w h i l e P M 0 showed a
2 t t
r e d u c t i o n o f t h e r e l e a s e o f t h e s e m e t a b o l i t e s after e x p o s u r e to asbestos c o m p a r e d with u n t r e a t e d cells, A M 0 showed a m a r k e d i n c r e a s e . T h e r e l e a s e o f L T C , however, was d e 4
c r e a s e d in b o t h M 0 populations. L a t e x b e a d s had m i n i m a l effects on t h e s e activities. I n addition, a m o s i t e b u t not latex b e a d s i n d u c e d t h e r e l e a s e o f I l - l in c u l t u r e s of A M 0 and P M 0 .
Asbestos-induced Modulation of Release of Regulatory Molecules from Alveolar and Peritoneal Macrophages*
I l - l activity was p r e s e n t in t h e s u p e r n a t a n t s during t h e first 2 4 hours o f c u l t u r e , and t h e r e l e a s e c o n t i n u e d during f u r t h e r incubation, e v e n in s e r u m - f r e e m e d i u m . N o 11-2 activity or dialyzable inhibitors w e r e found in t h e s u p e r n a t a n t s . T h e s e data, s u m m a r i z e d in F i g u r e 1, confirm previous
P. Sestini, M.D.;A. Tagliabue, Ph.D.; M. Bartalini; and D. Boraschi, Ph.D.
EFFECTS OF AMOSITE ON RESIDENT MACROPHAGES PM0
A s b e s t o s inhalation causes several i m m u n o l o g i c a b n o r m a l i ties in exposed individuals and e x p e r i m e n t a l a n i m a l s ,
and it has b e e n s u g g e s t e d that t h e s e p h e n o m e n a have a role in t h e induction o f p u l m o n a r y fibrosis and c o u l d be r e l a t e d to alterations o f m a c r o p h a g e - m e d i a t e d regulatory a c t i v i t i e s . To 4
investigate w h e t h e r asbestos could affect t h e r e l e a s e
AM0
1 1
of
i m m u n o r e g u l a t o r y m o l e c u l e s from m a c r o p h a g e s , w e have
SUPPRESSION °2 PGE
PGF * 2
LTC *From Institute of Respiratory Diseases, University of Siena, and Sclavo Research Center, Siena, Italy. Reprint requests: Dr. Sestini, Institute of Respiratory Diseases, via Tufi 1, 53100 Siena, Italy
2
4
IL-1 FIGURE 1
C H E S T / 8 9 I 3 / M A R C H , 1986 / Supplement
161S
T a b l e 1 — A m o u n t s of Cell Layer-Associated Collagen in Normal Rat Lung Fibroblasts (RL-82) Exposed to Crocidolite Asbestos (N=4 per group)
B. T. Mossman, Ph.D., R. Gilbert, B.S., F.C.C.P;J. Doherty, B.S.; M. A. Shatos, Ph.D.; J. Marsh, M.S.; and K. Cutroneo, Ph.D.
O
ccupational and e x p e r i m e n t a l e x p o s u r e to asbestos is associated with t h e d e v e l o p m e n t o f p e r i b r o n c h i o l a r
and interstitial p u l m o n a r y
fibrosis.
Like other asbestos-
1
associated diseases o f t h e respiratory tract (ie, m e s o t h e l i o m a and b r o n c h o g e n i c carcinoma), asbestosis appears to involve alterations in differentiation and proliferation o f affected c e l l types.
Whether
t h e disease process
i n c r e a s e in replication o f
fibroblasts
e n h a n c e d ability o f individual
involves e i t h e r in
fibroblasts
an
t h e l u n g o r an to p r o d u c e m o r e
collagen o r both is u n c e r t a i n . To address this q u e s t i o n , w e u s e d a c o m b i n a t i o n o f in vitro
and in vivo
approaches.
University o f Vermont) was m a i n t a i n e d
in
Minimal Essential Medium ( M E M , G I B C O ) containing 1 0 % fetal c a l f s e r u m and e x p o s e d to crocidolite asbestos ( U I C C r e f e r e n c e sample) at various c o n c e n t r a t i o n s in m e d i u m (ie, 1, 2 . 5 , and 5 y,g/em dish) w h e n cells r e a c h e d approximately 2
8 0 % confluency. At 2 4 - h o u r intervals, 1 0 ~ M a s c o r b a t e was 4
a d d e d to all plates. C e l l s t h e n w e r e p u l s e d with H - p r o l i n e 3
(10 u,Ci/mI m e d i u m ) for 2 hours p r i o r to assays.
After
homogenization o f cells and b o i l i n g for 10 m i n u t e s to inactivate proteases, cell s a m p l e s w e r e assayed in t h e p r e s e n c e and a b s e n c e o f purified bacterial collagenase (Advance Biofactures C o r p ; —40 U/assay t u b e ) to d e t e r m i n e t h e ratio o f newly synthesized collagen to n o n c o l l a g e n p r o t e i n . At e a c h 2
p e r i o d , H - t h y m i d i n e , o r H T (1 u,Ci/ml m e d i u m ) was added 3
to additional plates for 1 h o u r b e f o r e preparation for autoradiography.
3
Control (nonasbestos exposed) cells
were
t r e a t e d identically. Results show an i n c r e a s e in total c e l l layer-associated collagen at 4 8 and 7 2 hours after addition o f asbestos to R L - 8 2 cells (Table 1). I n c r e a s e d replication o f fibroblasts 3
48 hr
No asbestos Asbestos
8.52+1.13
18.38 ± 0 . 9 4
23.66±1.34
1.0 M.g/cm 2.5 jig/cm
6.39 ± 0 . 9 0 5.7 ±1.1.5
25.59±1.89*
5.0 u,g/cm
8.95
24.05 ± 2 . 0 6 t
21.92 ± 1 . 5 8 19.55 + 2.88 37.69+1.66*
z
2
2
72 hr
24.75 ± 2 . 1 8 *
* p < . 0 2 (Student's (test adjusting for multiple comparisons between groups). tp<.05. tp-c.OOl. collagen reflects a partitioning o f procollagen m R N A s into fibroblasts
to b l e o m y c i n ,
5
INHALATION E X P E R I M E N T S
lung ( R L - 8 2 o b t a i n e d from D r M a r l e n e Absher, D e p a r t m e n t
3
24 hr
VITRO
A n o r m a l fibroblast c e l l line derived from F i s c h e r 3 4 4 rat of M e d i c i n e ,
Group
p o l y s o m e s , a p h e n o m e n o n o c c u r r i n g after exposure o f lung
EXPERIMENTS USING LUNG FIBROBLASTS E X P O S E D TO A S B E S T O S IN
Total Collagen (cpm/ng DNA)
as d e t e r m i n e d b y n u m b e r s o f cells incorporating
H T did not o c c u r u n d e r t h e s e c i r c u m s t a n c e s (data not
shown).
T h e work d e s c r i b e d above indicates increased synthesis o f procollagen b y individual cells after exposure o f rat lung fibroblasts
to a s b e s t o s . To d e t e r m i n e w h e t h e r
proliferation o f
fibroblasts
abnormal
in t h e lung also occurs
after
inhalation o f asbestos, 6 - 8 - w e e k - o l d male F i s c h e r 3 4 4 rats (n = 3-5/group) w e r e exposed to crocidolite asbestos for 3 0 days (5 hr/day, 5 days/week) using a modified T i m b r e l l generator. Twenty-four hours before removal o f t h e lungs for vascular perfusion, the rats w e r e i n j e c t e d with H T (2 u,Ci/g). 3
After t h e lungs w e r e e m b e d d e d in paraffin, alternative 3-m tissue sections w e r e p r e p a r e d for histology and stained w i t h M a s s o n s t r i c h r o m e for d e t e c t i o n o f collagen.
Additional
slides w e r e p r o c e s s e d for i m m u n o c h e m i s t r y using an antib o d y to c o p p e r - z i n c superoxide dismutase ( S O D ) d e v e l o p e d in this laboratory. This allowed us to differentiate m a c r o 8
p h a g e s ( S O D - p o s i t i v e ) from
fibroblasts
( S O D - n e g a t i v e ) in
t h e i n t e r s t i t i u m o f the lung. After dipping and d e v e l o p m e n t for autoradiography, slides w e r e counterstained with h e m a toxylin b e f o r e c o u n t i n g b y light microscopy using 1,000 X magnification. E i g h t to 12 fields on each slide w e r e evaluated to d e t e r m i n e t h e p e r c e n t a g e o f l a b e l e d bronchiolar epithelial, alveolar epithelial and interstitial cells ( S O D - n e g a tive). I n c o n t r a s t to those o f control s u b j e c t s , t h e lungs of a s b e s t o s - e x p o s e d animals exhibited fibrotic changes as deter-
To d e t e r m i n e w h e t h e r t h e e n h a n c e d synthesis o f total
m i n e d b y i n c r e a s e d deposition o f trichrome-positive m a t e -
cellular collagen p e r n a n o g r a m o f D N A r e f l e c t e d an inc r e a s e d accumulation o f collagen t y p e I m R N A s in
fibroblasts
exposed to a s b e s t o s , t h r e e r e c o m b i n a n t plasmids containing t y p e I collagen-specific s e q u e n c e s (po^Ri, p a ^ , p o t j R J w e r e
T a b l e 2 — Q u a n t i t a t i o n of Numbers of Cells Incorporating "H-Thymidine in Fischer 344 Rat Lungs Exposed to Asbestos for 30 Days (N=8-12 fields/slide)
o b t a i n e d from C . G e n o v e s e , P h . D . ( D e p a r t m e n t o f P e d i Labeling Index, %
atrics, University of C o n n e c t i c u t ) . Q u i c k - b l o t analysis c o n 4
sistently failed to show a d i f f e r e n c e b e t w e e n t h e a m o u n t s o f procollagen t y p e I m R N A s in control and a s b e s t o s - e x p o s e d c e l l s . S u b c e l l u l a r fractionation studies now are in progress to determine
whether
asbestos-induced
synthesis
of pro-
*From the Departments of Pathology and Biochemistry, University of Vermont, Burlington. Supported by Pulmonary SCOR grant P50HL14212 from the National Heart Blood and Lung Institute. Dr. Shatos is a Parker B. Francis fellow of the Puritan-Bennett Foundation.
160S
Bronchiolar epithelial Alveolar epithelial Interstitial (SOD - ) *
Control
Asbestos-exposed
.78 ± , 1 7 .8 ± . 2 1.13±.2
4.13±.87t 3.07±.46t
1,26±.25
*As detected with an antibody to copper-zinc SOD prepared in this laboratory (Mossman et al, personal communication). t p < , 0 0 1 (Students ( test adjusting for multiple comparisons between groups). 2 8 t h Annual A s p e n Lung Conference
rial originating at t h e alveolar d u c t .
7
Autoradiography r e -
s t u d i e d t h e effect o f t h e e x p o s u r e in vitro
of mouse resident
vealed that n u m b e r s o f l a b e l e d alveolar epithelial cells and
alveolar ( A M 0 ) and p e r i t o n e a l m a c r o p h a g e s ( P M 0 ) to n o n -
interstitial
toxic c o n c e n t r a t i o n s o f asbestos on t h e i r suppressive capacity
fibroblasts
w e r e i n c r e a s e d significantly in as-
b e s t o s - e x p o s e d lungs (Table 2).
on t h e m i t o g e n - i n d u c e d proliferation o f s y n g e n e i c spleen
In contrast, t h e labeling indices o f b r o n c h i o l a r epithelial
l y m p h o c y t e s , on t h e i r ability to r e l e a s e a r a c h i d o n i c acid
cells in b o t h control and a s b e s t o s - e x p o s e d lungs w e r e c o m p a -
metabolites
rable.
z y m o s a n and on t h e s p o n t a n e o u s r e l e a s e o f i n t e r l e u k i n 1
and
superoxide
anion
(07)
in r e s p o n s e
to
(11-1). P M 0 purified b y a d h e r e n c e and A M 0 o b t a i n e d b y CONCLUSIONS
b r o n c h o a l v e o l a r lavage from 8-16-week-old C 3 H / H e N m i c e
O u r work suggests that at least two m e c h a n i s m s r e s u l t in
w e r e c u l t i v a t e d in flat-bottom wells in t h e p r e s e n c e o f c o n t r o l
increased
m e d i u m , latex b e a d s , or U I C C asbestos a m o s i t e , 8 0 u-g/ml,
synthesis o f total cellular procollagen b y individual fibro-
for 21 h o u r s . T h e m e d i u m was t h e n carefully aspirated and
asbestosis.
On
one hand,
asbestos
stimulates
blasts. This p h e n o m e n o n does not r e f l e c t an i n c r e a s e in
t h e M 0 f u r t h e r p r o c e s s e d . S u p p r e s s i v e capacity was evalu-
steady-state levels o f procollagen t y p e 1 m R N A s . I n addition,
a t e d as previously d e s c r i b e d
interstitial fibroblasts have an e n h a n c e d replicative potential
cells s t i m u l a t e d with an o p t i m a l d o s e o f C o n - A and evaluat-
in t h e lung after inhalation o f asbestos b y rats.
ing t h e incorporation o f tritiated t h y m i d i n e after 7 2 hours o f
5
by adding s y n g e n e i c spleen
c u l t u r e . T h e r e l e a s e o f Ol was m e a s u r e d b y f e r r i c y t o c h r o m e c r e d u c t i o n after stimulation with o p s o n i z e d zymosan. Pros-
REFERENCES 1 Craighead J E , Mossman BT. The pathogenesis of asbestosassociated diseases. N Engl J Med 1982; 306:1446-55 2 Newman RA, Cutroneo KR. Glucocorticoids selectively decrease the synthesis of hydroxylated collagen peptides. Mol Pharmacol 1978; 14:185-98 3 Mossman BT. Autoradiography for determination of DNA synthesis in the hamster bladder epithelium. TCA Manual 1977; 3:663-65 4 Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat a and ot collagen mRNAs and their use in studying the regulation of type I collagen synthesis by 1,25dihydroxywtamin D. Biochemistry 1984; 23:6210-16 t
2
taglandin E
2
( P G E ) and F , ( P G F . J , and l e u k o t r i e n e C 2
2 a
4
( L T C ) w e r e evaluated by radioimmunoassay after stimula4
tion w i t h zymosan 2 0 0 |i.g/ml in m e d i u m without s e r u m . I l - l was evaluated b y t h e C 3 H / H e J
thymocyte
proliferation
assay. T h e p r e s e n c e o f i n t e r l e u k i n - 2 (11-2) activity in t h e s u p e r n a t a n t s was assayed b y t h e ability to support
the
proliferation o f t h e C T L L c e l l l i n e . I n a g r e e m e n t
with
previous e x p e r i m e n t s " t h e e x p o s u r e o f A M 0 and P M 0 in
vitro
to a m o s i t e c a u s e d a d o s e - d e p e n d e n t d e c r e a s e in t h e i r suppressive capacity, w h e r e a s phagocytosis o f latex had n o effect. T h i s p h e n o m e n o n was not d u e to loss o f c e l l viability as
5 Sterling KM, Harris MJ, Mitchell J, Cutroneo KR. Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs: reversal of the bleomycin effect by dexamethasone. J Biol Chem 1983; 258:14438-44
j u d g e d b y L D H r e l e a s e and trypan b l u e exclusion, and was
6 Mossman BT, Marsh JP, Shatos MA. Alteration of superoxide dismutase (SOD) activity in tracheal epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants. Lab Invest 1986 (in press)
different effects on t h e z y m o s a n - i n d u c e d r e l e a s e o f P G E
7 Mossman BT, Shatos MA, Marsh JP, Doherty JM, Craighead J E , Hemenway D, et al. Rapid development of asbestosis in rats after inhalation of crocidolite. Am Rev Respir Dis 1984; 129:A148
p a r a l l e l e d b y a m a r k e d r e d u c t i o n o f t h e ability to p r o d u c e Oz. A s b e s t o s e x p o s u r e c a u s e d a small i n c r e a s e o f spontaneous P G E production from b o t h M 0 populations; however, it had 2
PGF
2
and
from A M 0 and P M 0 . I n fact, w h i l e P M 0 showed a
2 t t
r e d u c t i o n o f t h e r e l e a s e o f t h e s e m e t a b o l i t e s after e x p o s u r e to asbestos c o m p a r e d with u n t r e a t e d cells, A M 0 showed a m a r k e d i n c r e a s e . T h e r e l e a s e o f L T C , however, was d e 4
c r e a s e d in b o t h M 0 populations. L a t e x b e a d s had m i n i m a l effects on t h e s e activities. I n addition, a m o s i t e b u t not latex b e a d s i n d u c e d t h e r e l e a s e o f I l - l in c u l t u r e s of A M 0 and P M 0 .
Asbestos-induced Modulation of Release of Regulatory Molecules from Alveolar and Peritoneal Macrophages*
I l - l activity was p r e s e n t in t h e s u p e r n a t a n t s during t h e first 2 4 hours o f c u l t u r e , and t h e r e l e a s e c o n t i n u e d during f u r t h e r incubation, e v e n in s e r u m - f r e e m e d i u m . N o 11-2 activity or dialyzable inhibitors w e r e found in t h e s u p e r n a t a n t s . T h e s e data, s u m m a r i z e d in F i g u r e 1, confirm previous
P. Sestini, M.D.;A. Tagliabue, Ph.D.; M. Bartalini; and D. Boraschi, Ph.D.
EFFECTS OF AMOSITE ON RESIDENT MACROPHAGES PM0
A s b e s t o s inhalation causes several i m m u n o l o g i c a b n o r m a l i ties in exposed individuals and e x p e r i m e n t a l a n i m a l s ,
and it has b e e n s u g g e s t e d that t h e s e p h e n o m e n a have a role in t h e induction o f p u l m o n a r y fibrosis and c o u l d be r e l a t e d to alterations o f m a c r o p h a g e - m e d i a t e d regulatory a c t i v i t i e s . To 4
investigate w h e t h e r asbestos could affect t h e r e l e a s e
AM0
1 1
of
i m m u n o r e g u l a t o r y m o l e c u l e s from m a c r o p h a g e s , w e have
SUPPRESSION °2 PGE
PGF * 2
LTC *From Institute of Respiratory Diseases, University of Siena, and Sclavo Research Center, Siena, Italy. Reprint requests: Dr. Sestini, Institute of Respiratory Diseases, via Tufi 1, 53100 Siena, Italy
2
4
IL-1 FIGURE 1
C H E S T / 8 9 I 3 / M A R C H , 1986 / Supplement
161S