Modulation by T Cells of Pulmonary Inflammation and Fibrosis in an Experimental Model of Silicosis

12 Christman JW, Emerson RJ, Graham GB, Davis GS. Mineral dust and cell recovery from the bronchoalveolar lavage of healthy Vermont granite workers. Am Rev Respir Dis 1985; 132:393-99 13 Emerson RJ, Davis GS. Effect of alveolar lining material-coated silica on rat alveolar macrophages. Environ Health Perspect 1983; 51:81-84 14 Callis AH, Sohnle PG, Mandel GS, Wiessner J, Mandel NS. Kinetics of inflammatory and fibrotic pulmonary changes in a murine model of silicosis. J Lab Clin Med 1985; 105:547-53 15 Schuyler M, Gauner HR, Stankus RP, Kaimal V, Hoffman E, deSalvaggio J. Bronchoalveolar lavage in silicosis. Lung 1980; 157:95-102 16 Nathan CF, Murray HW, Cohn ZA. The macrophage as an effector cell. N Engl J Med 1980; 303:622-26 17 UnanueER. Cooperation between mononuclear phagocytes and lymphocytes in immunity. N Engl J Med 1980; 303:977-85

Modulation by T Cells of Pulmonary Inflammation and Fibrosis in an Experimental Model of Silicosis* Andrea H. Callis, Ph.D.; and David O. Lucas,

T

Ph.D.

h e p a t h o g e n e t i c m e c h a n i s m o f silicosis, a fibrotic p n e u m o c o n i o s i s , has b e e n p o s t u l a t e d to b e a n o n i m m u n o -

logic granulomatous r e s p o n s e involving t h e destruction o f alveolar m a c r o p h a g e s , t h e influx o f additional m a c r o p h a g e s , stimulation of collagen formation and hyalinization o f t h e collagen.

1

C u r r e n t r e s e a r c h in our laboratory explores t h e

h y p o t h e s i s that t h e i m m u n e system m o d u l a t e s silica-induced

18 Schmidt JA, Oliver CN, Lepe-ZunigaJL, Green I, Grey I. Silicastimulated monocytes release fibroblast proliferation factors identical to interleukin-1: a potential role for interleukin-1 in the pathogenesis of silicosis. J Clin Invest 1984; 73:1462-72

p u l m o n a r y d a m a g e . O n e o f o u r approaches has b e e n

19 Kampschmidt RF. The numerous postulated biological manifestations of interleukin-1. J Leukocyte Biol 1984; 36:341-55

c y t e s in m o d u l a t i n g silica-induced p u l m o n a r y inflammation

20 Dauber JH, Rossman MD, Pietra GG, Jimenez SA, Daniele RP Experimental silicosis: morphologic and biochemical abnormalities produced by intratracheal instillation of quartz into guinea pig lungs. Am J Pathol 1980; 101:595-607 21 Lugano E M , Dauber JH, Daniele RE Silica stimulation of chemotactic factor release by guinea pig alveolar macrophages. J Reticuloendothel Soc 1981; 30:381-90 22 Doll NJ, Stankus RP, Hughes J, Weill H, Gupta RC, Rodriguez M, et al. Immune complexes and autoantibodies in silicosis. J Allergy Clin Immunol 1981; 68:281-85 23 Calhoun WJ, Christman JW, Ershler W B , Graham GB, Davis GS. Elevated immunoglobulins in the lavage fluid of healthy granite workers. Thorax (in press) 24 Snyder GL. Interstitial pulmonary fibrosis—which cell is the culprit? Am Rev Respir Dis 1983; 128:535 25 MartinTR, Altman LC, Albert RK, Henderson UR. Leukotriene B4 production by the human alveolar macrophage: a potential mechanism for amplifying inflammation in the lung. Am Rev Respir Dis 1984; 129:106 26 Bateman E D , Emerson RJ, Cole PJ. A study of macrophagemediated initiation of fibrosis by asbestos and silica using a diffusion chamber technique. Br J Exp Pathol 1982; 63:414-25 27 Lugano E M , Dauber JH, Elias JA, Bashey RI, Jimenez SA, Daniele RP. The regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis. Am Rev Respir Dis 1984; 129:767-71

to

e x a m i n e t h e r e s p o n s e s o f Swiss n u d e , T - c e l l - d e f i c i e n t m i c e , and t h e c o n t r o l T-cell-sufficient

Swiss n u / +

m i c e in an

e x p e r i m e n t a l m o d e l of silicosis. T h u s , the role for T l y m p h o and fibrosis can b e assessed by m e a s u r i n g in t h e s e animals p a r a m e t e r s o f p u l m o n a r y inflammation and collagen deposition. METHODS The mice used in these studies were 8- to 12-week-old male or female Swiss (nu/ + ) and Swiss nude (nu/nu) purchased from Jackson Laboratories. Animals were exposed to silica by an intratracheal injection of 5 mg of silica crystals as previously described. 23

An increase in lung indices is a general measure of pulmonary inflammation measured as wet lung weight relative to body weight. Based on the following equation, an index of 1.0 is normal: wet lung wt/body wt silica-injected animal 4

wet lung wt/body wt saline-injected animal Collagen deposition is determined by total hydroxyproline per pair of lungs hydrolyzed in acid. Analysis of the lung lavage fluid provides a good measure of the cellular inflammatory response. The lungs are lavaged in situ with 1 ml of saline and the supernatant fluid is assessed for total protein and various enzymes indicative of inflammation; the cells in the lavage are enumerated under light microscopy. 5

6

RESULTS T h e i n f l u e n c e o f T l y m p h o c y t e s on silica-induced p u l m o nary cellular inflammation was m e a s u r e d by differences in

28 Heppleston AG, Stiles JA. Activity of a macrophage factor in collagen formation by silica. Nature (London) 1967; 214:521-22

total cell n u m b e r and t h e protein c o n c e n t r a t i o n of t h e lavage

29 Burrell R, Anderson M. The induction of fibrogenesis by silicatreated alveolar macrophages. Environ Res 1973; 6:389-94

animals at 1, 4 , a n d 12 w e e k s post-instillation o f 5 m g o f silica

30 Harrington JS, Ritchie M, King PC, Miller K. The in-vitro effects of silica-treated hamster macrophages on collagen production by hamster fibroblasts. J Pathol 1972; 109:21-37

t r a c t e d from t h e values for silica-injected animals to r e p r e -

31 Richards RJ, Wusteman RS. The effects of silica dust and alveolar macrophages on lung fibroblasts grown in-vitro. Life Sci 1974; 14:355-64 33 Kovacs EJ, Kelley J. Lymphokine regulation of macrophagederived growth factor secretion following pulmonary injury. Am J Pathol 1985; 121:261-68

n u m b e r s ( 2 3 . 8 ± 4 . 9 X 1 0 / cells/ml) and p r o t e i n

34 Ruoslahti E E , Engvall E, Hayman E G . Fibronectin: current concepts of its structure and functions. Coll Relat Res 1981; 1: 95-128 34 Ruoslahti E E , Engvall E, Hayman EG. Fibronectin: current concepts of its structure and functions. Coll Relat Res 1981; 1: 95-128

fluid. Swiss n u d e m i c e w e r e c o m p a r e d with t h e Swiss nu/ + crystals. Values for s a l i n e - i n j e c t e d c o u n t e r p a r t s w e r e subs e n t t h e i n c r e a s e i n d u c e d b y t h e i n j e c t i o n o f silica crystals. At o n e w e e k , t h e Swiss n u d e m i c e h a d substantially lower cell concentra-

tions (415.1 ± 1 7 2 . 8 (jig/ml) in t h e lavage fluid c o m p a r e d with t h e Swiss n o r m a l m i c e (cells, 5 0 . 8 ± 1 0 . 5 X 1 0 cells/ml; pro5

tein, 9 5 7 ± 3 0 8 . 0 (xg/ml). B y 4 and 12 w e e k s post-introduction o f t h e p a r t i c u l a t e , the differences w e r e not as m a r k e d , and

*From the Department of Microbiology, Arizona Health Science Center, Tucson. This research was supported by NIH grant HL33754. Reprint requests: Dr. Callis, Department of Anesthesiology, University of Arizona, Tucson 85724 C H E S T / 8 9 / 3 / M A R C H , 1 9 8 6 / Supplement

169S

the two groups o f animals a p p r o a c h e d e a c h o t h e r in values. T h e i n f l u e n c e o f T l y m p h o c y t e s on collagen deposition was d e m o n s t r a t e d b y levels o f hydroxyproline p e r lungs (above s a l i n e - i n j e c t e d counterparts). C o l l a g e n deposition in

the

Swiss m i c e rose m a r k e d l y b e t w e e n w e e k s l a n d 4 ( 7 7 . 7 ± 3 3 . 8 to 1 5 4 . 4 ± 2 7 . 3 (ig/lungs) and leveled off by w e e k 12. T h e r e was, however, a delay or suppression in collagen deposition in t h e n u d e mouse at t h e 4 - w e e k point ( 6 9 . 2 ± 15.1 u,g/lungs), T h i s delay in n u d e s was followed by a substantial i n c r e a s e in collagen deposition b y w e e k 12 (261.8 ± 9 0 . 6 (jug/lungs). L u n g indices rose in b o t h types o f m i c e at w e e k 1 ( b e t w e e n 2 . 1 and 2.2), b u t did not differ throughout t h e 12-week p e r i o d . Levels of angiotensin-converting

enzyme

(ACE)

were

m e a s u r e d in t h e lung lavage fluid o f b o t h silica- and saline7

i n j e c t e d n u d e and Swiss m i c e at all t h r e e intervals. A C E activity is e x p r e s s e d as u,mol/mg o f lung lavage protein. L e v e l s t h e n reflect local synthesis or r e l e a s e , not m e r e l y a n o n s p e c i f i c leakage o f plasma p r o t e i n s . S a l i n e - i n j e c t e d ani8

mals o f b o t h strains showed no d e t e c t a b l e A C E in t h e lavage fluid. An intratracheal i n j e c t i o n o f silica into t h e Swiss n u / + animals i n d u c e d levels o f A C E u p to 4 - 5 Limol/mg p r o t e i n . Swiss n u d e m i c e , on t h e o t h e r h a n d , had levels o f 1 5 . 2 ± 2 . 1 and 1 3 . 3 ± 4 . 6 u.mol/mg protein at w e e k s 1 and 4 , r e s p e c tively. A C E levels in Swiss n u d e m i c e r e t u r n e d to levels

3 Callis AH, Sohnle PG, Mandel GS, Mandel NS. The role of complement in experimental silicosis. Environ Res (in press) 4 Allen E M , Moore VL, Stevens JO. Strain variation in BCGinduced chronic pulmonary inflammation in mice; I. Basic model and possible genetic control by non H-2 genes. J Immunol 1977; 119:343-47 5 Woessner J E The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 1961; 93:440-47 6 Lowry OH, Rosebroiigh NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-75 7 Friedland J, Silverstein E . A sensitive fluorimetric assay for serum angiotensin-converting enzyme. Am J Clin Pathol 1976; 66:416-24 8 Krieger B, Schwartz J, Loomis W, Marsh J, Spragg R. Nonspecificity of elevated angiotensin-converting enzyme activity in bronchoalveolar lavage fluid from high permeability lung edema states. Am Rev Respir Dis 1984; 129:499-500 9 Hunninghake GW, Garrett KC, Richerson HB, Fantone J C , Ward PC, Rennard SI, et al. State of the Art: Pathogenesis of the granulomatous lung diseases. Am Rev Respir Dis 1984; 130:476-96 10 Nordman H, Hoskinen H, Froseth B. Increased activity of serum angiotensin converting enzyme in progressive silicosis. Chest 1984; 86:203-07

c o m p a r a b l e to those s e e n in t h e Swiss nu/ + strain b y w e e k 12. DISCUSSION D u r i n g t h e acute inflammatory stage at I w e e k post-silica instillation, T l y m p h o c y t e s , p e r h a p s T h e l p e r cells, i n f l u e n c e initial inflammatory cell influx o r replication as shown b y t h e

The Role of Migrating Inflammatory Cells in Proliferation of Lung Interstitium and Epithelium* Susan G. Shami D.Sc.;i Larry A. Martinez, B.S.; and Michael]. Evans, Ph.D.

d e c r e a s e d cellularity in t h e lavage fluid o f Swiss n u d e m i c e at 1 w e e k p o s t - i n j e c t i o n . Swiss n u d e m i c e also showed e v i d e n c e o f a d e l a y or suppression in t h e deposition o f l u n g collagen at t h e 4 - w e e k p o i n t — a trend that was r e v e r s e d over t h e next 8 w e e k s . W h e t h e r T cells directly i n f l u e n c e acute inflammation and later collagen deposition or m e d i a t e t h e s e events via i n f l u e n c e on o t h e r cells is not yet clear. Certainly, T cells m a y play a role in modulating inflammation b y e n h a n c i n g o r suppressing m a c r o p h a g e f u n c t i o n .

9

T cells also appear to

i n f l u e n c e t h e target o f early inflammatory d e s t r u c t i o n as shown b y A C E activity. T h e e n d o t h e l i a l cells in t h e capillary b e d o f t h e lung have a high c o n t e n t o f this e n z y m e . T h e r e fore, high levels o f A C E in the lavage fluid or s e r u m could indicate g r e a t e r p u l m o n a r y e n d o t h e l i a l c e l l injury in Swiss n u d e m i c e . Alternatively, since h u m a n alveolar m a c r o p h a g e s are also known to contain A C E ,

1 0

t h e h i g h e r levels of A C E in

t h e Swiss n u d e animals c o m p a r e d with Swiss n o r m a l m i c e may reflect g r e a t e r destruction o f alveolar m a c r o p h a g e s . T l y m p h o c y t e s appear to play a role in initial p a r t i c u l a t e i n d u c e d inflammatory c e l l influx o r replication. T h e s u b s e q u e n t s y n t h e s i s , degradation, and e v e n t u a l deposition o f silica-induced collagen also appears to b e m o d u l a t e d b y T cells or t h e inflammatory cells t h e y i n f l u e n c e . REFERENCES 1 Heppleston AG. The flbrogenic action of silica. Br Med Bull 1969; 25:282-87 2 Callis AH, Sohnle PG, Mandel GS, Wiessner J, Mandel NS. Kinetics of inflammatory and fibrotic changes in a mouse model of silicosis. J Lab Clin Med 1985; 105:547-53 170S

P

roliferation o f cells in t h e airway and alveolar e p i t h e l i u m o f t h e lung normally occurs at a very low rate. W h e n the

lung is i n j u r e d , this proliferative rate increases dramatically, and a repair r e s p o n s e e n s u e s ,

I n c r e a s e d proliferation in

4

airway and alveolar e p i t h e l i u m and alveolar interstitium has also b e e n o b s e r v e d w h e n t h e r e is no morphologic e v i d e n c e o f cellular injury.

The

involve inhalation

studies reporting such

proliferation

or intratracheal instillation o f various

particles and fibers in r o d e n t s .

2 7

A major question arising

from t h e s e studies c o n c e r n s the mechanism that causes c e l l proliferation in t h e a b s e n c e o f morphologic e v i d e n c e

of

c e l l u l a r injury to the tissue. Associated with particle and fiber exposure is an influx of inflammatory cells ( m o n o c y t e s and p o l y m o r p h o n u c l e a r l e u k o c y t e s [ P M N ] ) into the alveolar spaces o f t h e lung from t h e blood vasculature. W e hypothe s i z e d that the migration o f inflammatory cells through t h e alveolar e p i t h e l i u m during and after particle exposure is related to t h e cell proliferation observed in the a b s e n c e of m o r p h o l o g i c signs o f injury. T h i s study was d e s i g n e d to test t h e hypothesis that the migration o f inflammatory cells is related to cell proliferation. To a c c o m p l i s h this, normal m i c e and m i c e whose b o n e marrow h a d b e e n d e p l e t e d o f white blood cells ( W B C ) and, thus, *From the Inhalation Toxicology Research Institute, Lovelace Biomedical and Environmental Research Institute, Albuquerque. tCurrently at Pulmonary Division, Department o f Medicine, UCLA Center for Health Sciences, Los Angeles. Reprint requests; Dr. Shami, Pulmonary Division, Department of Medicine, UCtA Medical School, Los Angeles 90024 28th Annual Aspen Lung Conference