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The effect of phospholipid-containing surfactant from nickel exposed rabbits on pulmonary macrophages in vitro
Toxicology 21 (1981)169--178 © Elsevier/North-Holland Scientific Publishers Ltd.
THE E F F E C T OF PHOSPHOLIPID-CONTAINING SURFACTANT FROM NICKEL EXPOSED RABBITS ON PULMONARY MACROPHAGES IN VITRO
ANNA
WIERNIK, CONNIE JARSTRAND
and A N N E
JOHANSSON
a
Department o f Clinical Bacteriology at Roslagstull Hospital, B o x 5651, S-114 89 Stockholm and aDepartments o f Environmental Hygiene, Karolinska Institute and Section o f Inhalation Toxicology, Department o f Toxicology, National Institute o f Environmental Medicine, B o x 60 208, S-104 01 Stockholm and athe Wenner-Gren Institute, University o f Stockholm, S-113 45 Stockholm (Sweden)
(Received January 13th, 1981) (Revision received May 25th, 1981) (Accepted June 4th, 1981)
SUMMARY Alveolar macrophages from 9 normal rabbits were incubated in vitro for 3 h with and w i t h o u t phospholipid-containing surfactant from nickeltreated ones. The macrophages treated with surfactant showed morphological and functional criteria of increased activity. The cell surface had m a n y protrusions and the cytoplasma contained several lamellated structures. The oxidative metabolism, measured by the nitroblue tetrazolium (NBT)-test, at rest and after E. c o l i stimulation was increased, as was the a t t a c h m e n t and ingestion of yeast particles. The NBT-values were a b o u t the same as corresponding values of macrophages lavaged from the lungs of nickel-treated rabbits. Macrophages incubated with surfactant from untreated animals, had NBT values and phagocytic activity similar to cells incubated w i t h o u t surfactant. As this substance was administered in excess, the difference in macrophage response would probably be due to a qualitative alteration of the surfactant after nickel exposure.
INTRODUCTION The h u m a n disease pulmonary alveolar proteinosis described in 1958 by Rosen et al. [1] is characterized by large amounts of phospholipid- and protein-rich material accumulated in the alveoli [2,3]. The alveolar macroAbbreviations: FITC, fluorescein isothiocyanate; NBT, nitroblue tetrazoli~m; OD, optical density.
169
phages have been shown to be loaded with this material, and to have an impaired antibacterial function [4,5]. Also in rabbits exposed to metallic nickel dust (1 pg/m 3) we have found alveoli filled with an amorphous substance which was rich in phospholipids and consisted of lamellated structures [6, Johansson et al., unpublished]. The a m o u n t of phospholipids increased with the duration of the nickel exposure [ 7]. In addition, after exposure for 1 m o n t h there was an increased fractional volume of alveolar type II cells [6]. Increased size variation, phagocytic capacity and metabolic activity of alveolar macrophages were found as well as ultrastructural alterations [8,9]. After 3 and 6 months of nickel exposure the oxidative metabolism was high, b u t contrary to the controls and to the 1 m o n t h experiment the metabolism could n o t be further increased u p o n stimulation with bacteria. The phagocytic capacity was decreased [10] and morphologically the cells had a s m o o t h surface and the cytoplasm was engorged with lamellar bodies [11]. These changes suggested a decreased activity of the alveolar macrophages and an impaired capacity to metabolize the large amounts of surfactant produced. The aim of the present study was to investigate if the phospholipidcontaining surfactant which constitutes an outstanding feature in the pathological picture is responsible for the changes in function and morphology of the macrophages in rabbits exposed to nickel dust. Therefore we studied macrophages from healthy unexposed rabbits after incubation for a short time with surfactant from nickel exposed rabbits. MATERIALS AND METHODS
Exposure to nickel solution Nine rabbits were anaesthesized with Mebumal and intubated whereafter 1 ml of nickel suspension, 0.1 g metallic nickel/ml isotonic saline, was instilled into the trachea. The procedure was repeated after 2 weeks. After another 2 weeks the animals were sacrificed with an overdose of Mebumal. Collection o f macrophages and estimation o f their size The alveolar macrophages were collected according to a modification [12] of the m e t h o d of Myrvik [13]. The viability of the macrophages was tested by staining with eosin-Y. A smear of the cells from the lung wash was airdried, fixed immediately in methanol and stained with a toluidine blue solution. The size distribution was determined by measuring the diameters of 100--200 cells from each rabbit in a Lanameter (Reichert, Austria). Collection o f the surfactant The material from the lung wash was centrifuged at 200 g for 15 min. A milky precipitate located above the cell b u t t o n at the b o t t o m of the tube could be collected and was considered to b e t h e surfactant [ 8]. Incubation o f macrophages with the surfactant Half a milliliter of a macrophage suspension (30 X 170
10 6
cells/ml culture
medium which consisted of Parker 199 with 10% fetal bovine serum and 2 mM glutamine) from each of the unexposed rabbits was transferred to 2 plastic test tubes. 0.2 ml of the surfactant from an exposed or an unexposed rabbit was added to 1 of the tubes and the volumes in both tubes were adjusted to 1 ml with culture medium. In each experiment surfactant from different animals was used. The tubes were incubated in a water bath with a rocking device at 37°C for 3 h. The cells were then washed twice by centrifugation in Hank's Tris solution and resuspended in Eagle's medium. Thereafter the size, the morphology and the function of the cells were investigated.
Nitroblue tetrazolium (NBT) test Half a milliliter of macrophage suspension (6 × 106 cells/ml Eagle's medium) and 0.3 ml autologous serum were transferred to each of 2 plastic tubes. Thereafter, 0.5 ml NBT-solution (0.1 g NBT in 100 ml of Hank's solution) was added to 1 of the tubes while to the other was added 0.45 ml NBT-solution together with 0.05 ml of a saline suspension of heat-killed E. coli containing a b o u t 10 ~° bacteria/ml. The tubes were incubated for 30 min under continuous agitation in a 37°C water-bath. The reaction was then interrupted by the addition of 1 ml 0.5% HC1. After centrifugation the formazan produced was extracted with 1 ml dimethylsulfoxide and the optical density (OD) of the solution determined by s p e c t r o p h o t o m e t r y at 572 nm. Attachment and ingestion o f fluorescein labelled yeast particles A modification of the m e t h o d described by Hed [14] was used. Macrophage suspension (106 cells/ml Eagle's medium) was placed on glass slides (C1 301 Novakemi) in triplicates and the cells were allowed to adhere for 30 min at 37°C. Then the glass slides were rinsed with Ringerdex solution (Pharmacia, Sweden) at 37°C. Thereafter 0.1 ml of a suspension of yeast particles (Sac° charomyces cerevisiae 2.5 × 107 particles/ml) labelled with fluorescein isothiocyanate (FITC) and opsonised in pooled rabbit serum was added to each glass slide and the preparations were incubated for 30 min at 37°C. The reaction was interrupted by placing the slides in Ringerdex solution at 4°C. Crystal violet solution was applied for 10 s. The ingested particles were easily distinguishable by their fluorescence while the attached particles were coloured dark by crystal violet. The numbers of attached and ingested cells/ phagocytic cell were determined with a fluorescence microscope (Reichert, Austria). Electron microscopy The macrophages were fixed in 2.5% buffer (pH 7.2) postfixed in 1% OsO4 graded alcohol and e m b e d d e d in Epon LKB Ultramicrotome III and examined
glutaraldehyde in 0.1 M cacodylate in the same buffer, dehydrated in 812. Thin sections were cut on an in a Jeol 100S electron microscope.
Statistical methods Three-way analysis of variance was used with the following factors: 171
(1) rabbits; (2) addition -- no addition of surfactant; (3) with -- w i t h o u t
E. coli stimulation or ingested - - a t t a c h e d particles [15]. R ES ULTS
Morphology Many macrophages washed o u t of the lungs from the nickel-treated rabbits had a surface rich in long protrusions and microvilli (Fig. 1). The cytoplasm contained many lamellar bodies and lipid droplets. The cells also had polymorphic nuclei and well developed nucleoli. The Golgi zones were p r o n o u n c e d as was the granular endoplasmic reticulum. Many cells tended to accumulate in clusters and several cells with 2 or more nuclei, so-called "giant cells", were seen. However, 3 of the animals showed less response than the others and many macrophages in all rabbits appeared normal. When macrophages from unexposed rabbits were treated with surfactant from nickel-treated ones, the preparations contained, outside the cells, accumulations of lameUated material, cell debris and dead cells. These dead cells probably came from the surfactant fraction because they contained large amounts of lamellated material. Macrophages from the unexposed
b Fig. 1. Alveolar macrophage from a nickel-treated rabbit. The cytoplasm contains numerous lamellated bodies (LB) of different density. The surface is rich in microvilli and protrusions (x 6800).
172
rabbits contained lameUar bodies, although n o t as frequently as the macrophages from the nickel-treated rabbits (Fig. 2). The debris and lamellated material were phagocytized by the macrophages (Fig. 3). There were several cells accumulating around the surfactant material and the cells had protrusions and broad zones in close apposition. The surfactant was seen as lamellated structures, lamellae arranged in concentrical whorls, intermingled with a lattice-like net (Fig. 3). Similar but less pronounced reactions were seen in the preparations consisting of surfactant from unexposed rabbits and macrophages from the same kind of animals. The surfactant did n o t contain much debris and lamellated structures and fewer cells in the preparations contained lamellar bodies and phagosomes. In the macrophages exposed to surfactant t w o main types of lamellar bodies were present. One t y p e was characterized by electron dense, concentrically arranged whorls. The other t y p e was very electron lucent and had few lamellae (Fig. 4). The control macrophages incubated without surfactant appeared mostly normal as compared to our previous studies of alveolar macrophages [8,11]. Some cells contained occasional lamellar bodies and had many lysosomal structures {Fig. 5}.
Fig. 2. A l v e o l a r m a c r o p h a g e f r o m a n u n t r e a t e d r a b b i t , i n c u b a t e d for 3 h w i t h s u r f a c t a n t f r o m a n i c k e l - t r e a t e d one. T h e cell c o n t a i n s l a m e l l a t e d b o d i e s (LB) a n d t h e surface has p r o t r u s i o n s . Debris sticks to t h e cell surface ( a r r o w ) (× 8 5 0 0 ) .
173
Fig. 3. Macrophage from an untreated rabbit is sticking to a cluster of surfactant material, from a nickel-treated one, with protrusions from cell surface. Surfactant material is seen in a cleft between 2 protrusions (arrow) (x 20 400).
Size In 2 out of 3 animals examined the mean of the diameter and the mean of the variances of the diameter of the macrophages were larger, 10% and 35%, respectively, after incubation with surfactant from nickel-exposed animals than after incubation w i t h o u t this substance or with surfactant from unexposed rabbits.
Fig. 4. Fusion of 2 lamellated structures (arrow) of different density (× 20 400).
174
5 Fig. 5. Alveolar macrophage, from an untreated rabbit, incubated without surfactant. Several lysosomal structures are seen in the cytoplasm (x 8500).
Function The NBT-reduction at rest and with E. coli stimulation of the macrophages from 9 unexposed rabbits was significantly higher after preincubation for 3 h with surfactant from exposed rabbits than after incubation without this substance for the same time (P < 0.01). Incubation of macrophages from 3 o u t of the 9 control rabbits with surfactant from unexposed rabbits did n o t give a change of any importance in the NBT-values (Table I). Macrophages from the 9 nickel-exposed rabbits showed, without any incubation, NBT-values of the same magnitude as the corresponding cells of unexposed rabbits after incubation with the surfactant from nickeltreated animals (Table I). The attachment as well as ingestion of particles by macrophages from 7 untreated animals was significantly higher after preincubation with the surfactant from exposed rabbits for 3 h (P < 0.01) (Table II) than after incubation w i t h o u t this substance for the same time. Surfactant from unexposed rabbits had no influence on phagocytosis.
175
TABLE I NBT-REDUCTION OF MACROPHAGES FROM UNEXPOSED RABBITS AFTER 3 h I N C U B A T I O N IN V I T R O W I T H O U T A N D W I T H S U R F A C T A N T FROM EXPOSED AND UNEXPOSED RABBITS Values represent amount of formazan produced by 3 × 104 macrophages during 0.5 h, expressed as optical densities. Mean values for the NBT-reduction of the macrophages from the nickel-intubatedrabbits were at rest 0.27 and with E.coli stimulation 0.55. No surfactant
Surfactant from exposed rabbits
Surfactant from unexposed rabbits A t rest
E. coli stimulated
0.25 0.19 0.19 0.21±0.03
0.38 0.35 0.26 0.33±0.06
A t rest
E. coli stimulated
A t rest
E. coli stimulated
0.20 0.19 0.14 0.24 0.27 0.20 0.17 0.24 0.12 0 . 2 0 ± 0.05 a
0.32 0.35 0.33 0.33 0.33 0.25 0.24 0.37 0.25 0.31 ± 0 . 0 5
0.27 0.25 0.21 0.49 0.47 0.31 0.23, 0.45 0.20 0.32 ± 0.12
0.30 0.35 0.53 0.62 0.52 0.40 0.25 0.61 0.38 0 . 4 4 ± 0.13
a M e a n ± S.D.
T A B L E II NUMBER OF INGESTED AND ATTACHED YEAST PARTICLES/MACROPHAGE F R O M U N E X P O S E D R A B B I T S A F T E R 3 h I N C U B A T I O N IN V I T R O W I T H O U T A N D WITH SURFACTANT FROM EXPOSED AND UNEXPOSED RABBITS No surfactant
Surfactant from exposed rabbits
Surfactant from unexposed rabbits
Ingest particles/ macrophage
Attached particles/ macrophage
Ingested particles/ macrophage
Attached particles/ macrophage
Ingested particles/ macrophage
Attached particles/ macrophage
1.3 0.8 0.6 1.2 1.9 1.0 1.0 1.1±0.4 a
1.0 2.9 2.3 2.2 1.5 1.0 0.7 1.7±0.8
0.9 0.9 0.9 1.9 2.3 1.9 1.8 1.5±0.6
2.8 3.3 3.9 2.2 1.7 1.2 0.9 2.3±1.1
0.5 1.5 1.9 1.0 1.1 1.2±0.5
2.1 1.7 1.6 1.0 1.0 1.5±0.5
a M e a n + S.D.
176
DISCUSSION
The exposure to nickel dust was in this study simplified and consequently different from that previously used. However, the macrophages washed o u t from the nickel-intubated rabbits showed the same type of changes that was typical for such cells from rabbits after inhalation of nickel dust for I month. These cells had increased size variation and phagocytic capacity. Ultrastructurally, the cytoplasm contained numerous lamellar structures and the surface appeared activated in the sense that it was rich in microviUi and protrusions. These macrophages also had enhanced metabolic activity as measured by the NBT-reduction. Several publications have established that an elevated NBT-reduction can be a sign of activation in mononuclear phagocytes [ 16,17]. In the lungs of the intubated rabbits, however, a variation between them in the response to the nickel was observed and many macrophages looked apparently normal. This is most certainly due to the uneven distribution of the nickel caused by the intubation procedure. In this study macrophages from normal unexposed rabbits were incubated for 3 h with surfactant from nickel-intubated ones. After this time morphologic changes were seen that were less pronounced b u t of the same kind as noted in the nickel-exposed rabbits. The 2 types of lamellar bodies most likely represent surfactant in different stages of decomposition. Also the functional changes of the macrophages after incubation with the surfactant for 3 h were the same as in the nickel-exposed rabbits e.g. there was an increased NBT-reduction and an increased capacity to engulf particles. Although both surfactants compared to the in vivo situation were administered in excess, the one from unexposed rabbits did not give any morphological or functional changes. This indicates that the change of the surfactant by nickel exposure is qualitative rather than quantitative. The 2 surfactants used in this study were only compared by volume and n o t by biochemical analysis. However, previous studies of surfactant from rabbits exposed for 1 m o n t h showed increased amounts of phospholipid, especially phosphatidylcholine and phosphatidylinositol [18]. It is probable that the surfactants from the exposed animals used in this study were changed in the same way. Further, various works have shown that different lipids and fatty acids can be incorporated in the cell membrane and cause functional changes of the cell [19,20]. An activation of macrophages by lipids has also been demonstrated [21]. Therefore it is probable that the morphological and functional changes of the macrophages recorded in this and previous studies, is due to the phospholipids of the surfactant. The substance might be both engulfed by the macrophages and incorporated in the cell membrane. ACKNOWLEDGEMENTS
We are grateful to Marina Nyborg, Margot Lundborg and Karin WidskiSldOlsson for skilful technical assistance.
177
REFERENCES 1 2 3 4 5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21
S.H. Rosen, B. Castleman and A.A. Liebow, N. Engl. J. Meal., 258 (1958) 1123. J.E. Hawkins, E.V. Savard and J. Ramirez-Rivera, Am. J. Clin. Pathol., 48 (1967) 14. J. Ramirez-Rivera and W.R. Harlan, Am. J. Med., 45 (1968) 502. D.W. Golde, M. Territo, Th.N. Finley and M.J. Cline, Ann. Intern. Med., 85 (1976) 304. J.O. Harris, Chest, 76 (1979) 156. A. Johansson and P. Camner, Environ. Res., 22 {1980) 510. M. Casarett-Bruce, P. Camner and T. Curstedt, In "Proceedings of the 19th Annual Hanford Biology Symposium" October 21--24, 1979, Richland, Washington, U.S.A., 1980. P. Camner, A. Johansson and M. Lundborg, Environ. Res., 16 (1978) 226. C. Jarstrand, M. Lundborg, P. Camner and A. Wiernik, Toxicology, 11 (1978) 353. A. Wierrdk, P. Camner, C. Jarstrand and A. Johansson, Acta Soc. Med. Suecanae, 88 (1979) 452. A. Johansson, P. Camner, C. Jarstrand and A. Wiernik, Environ. Res. (1980) in press. M. Lundborg and B. Holma, Sabouraudia, 10 (1972) 152. Q.N. Myrvik, E.S. Leake and B. Faris, J. Immunol., 86 (1961) 128. J. Hed, FEMS Microbiol. Lett., 1 (1977) 357. K.A. Brownlee, Statistical Theory and Methodology in Science and Engineering, John Wiley and Sons, New York, 1960. P. Alf61dy and E.-M. Lemmel, Clin. Immunol. Immunopathol., 12 (1979) 263. G.G. Kreuger, B.E. Ogden and W.L. Weston, Clin. Exp. Immunol., 23 (1976) 517. M. Bruce, P. Camner, T. Curstedt, M. Hagman and A. Johansson, Acta Soc. Med. Suecanae, 87 (1978) 461. A.J. Schroit and R. GaUily, Immunology, 36 (1979) 199. M. Dianzani, M.W. TorrieUi, R.A. Canuto, R. Garcea and F. Feo, J. Pathol., 188 (1976) 193. N.R. Di Luzio, Lipid Res., 10 (1972) 43.
178
THE E F F E C T OF PHOSPHOLIPID-CONTAINING SURFACTANT FROM NICKEL EXPOSED RABBITS ON PULMONARY MACROPHAGES IN VITRO
ANNA
WIERNIK, CONNIE JARSTRAND
and A N N E
JOHANSSON
a
Department o f Clinical Bacteriology at Roslagstull Hospital, B o x 5651, S-114 89 Stockholm and aDepartments o f Environmental Hygiene, Karolinska Institute and Section o f Inhalation Toxicology, Department o f Toxicology, National Institute o f Environmental Medicine, B o x 60 208, S-104 01 Stockholm and athe Wenner-Gren Institute, University o f Stockholm, S-113 45 Stockholm (Sweden)
(Received January 13th, 1981) (Revision received May 25th, 1981) (Accepted June 4th, 1981)
SUMMARY Alveolar macrophages from 9 normal rabbits were incubated in vitro for 3 h with and w i t h o u t phospholipid-containing surfactant from nickeltreated ones. The macrophages treated with surfactant showed morphological and functional criteria of increased activity. The cell surface had m a n y protrusions and the cytoplasma contained several lamellated structures. The oxidative metabolism, measured by the nitroblue tetrazolium (NBT)-test, at rest and after E. c o l i stimulation was increased, as was the a t t a c h m e n t and ingestion of yeast particles. The NBT-values were a b o u t the same as corresponding values of macrophages lavaged from the lungs of nickel-treated rabbits. Macrophages incubated with surfactant from untreated animals, had NBT values and phagocytic activity similar to cells incubated w i t h o u t surfactant. As this substance was administered in excess, the difference in macrophage response would probably be due to a qualitative alteration of the surfactant after nickel exposure.
INTRODUCTION The h u m a n disease pulmonary alveolar proteinosis described in 1958 by Rosen et al. [1] is characterized by large amounts of phospholipid- and protein-rich material accumulated in the alveoli [2,3]. The alveolar macroAbbreviations: FITC, fluorescein isothiocyanate; NBT, nitroblue tetrazoli~m; OD, optical density.
169
phages have been shown to be loaded with this material, and to have an impaired antibacterial function [4,5]. Also in rabbits exposed to metallic nickel dust (1 pg/m 3) we have found alveoli filled with an amorphous substance which was rich in phospholipids and consisted of lamellated structures [6, Johansson et al., unpublished]. The a m o u n t of phospholipids increased with the duration of the nickel exposure [ 7]. In addition, after exposure for 1 m o n t h there was an increased fractional volume of alveolar type II cells [6]. Increased size variation, phagocytic capacity and metabolic activity of alveolar macrophages were found as well as ultrastructural alterations [8,9]. After 3 and 6 months of nickel exposure the oxidative metabolism was high, b u t contrary to the controls and to the 1 m o n t h experiment the metabolism could n o t be further increased u p o n stimulation with bacteria. The phagocytic capacity was decreased [10] and morphologically the cells had a s m o o t h surface and the cytoplasm was engorged with lamellar bodies [11]. These changes suggested a decreased activity of the alveolar macrophages and an impaired capacity to metabolize the large amounts of surfactant produced. The aim of the present study was to investigate if the phospholipidcontaining surfactant which constitutes an outstanding feature in the pathological picture is responsible for the changes in function and morphology of the macrophages in rabbits exposed to nickel dust. Therefore we studied macrophages from healthy unexposed rabbits after incubation for a short time with surfactant from nickel exposed rabbits. MATERIALS AND METHODS
Exposure to nickel solution Nine rabbits were anaesthesized with Mebumal and intubated whereafter 1 ml of nickel suspension, 0.1 g metallic nickel/ml isotonic saline, was instilled into the trachea. The procedure was repeated after 2 weeks. After another 2 weeks the animals were sacrificed with an overdose of Mebumal. Collection o f macrophages and estimation o f their size The alveolar macrophages were collected according to a modification [12] of the m e t h o d of Myrvik [13]. The viability of the macrophages was tested by staining with eosin-Y. A smear of the cells from the lung wash was airdried, fixed immediately in methanol and stained with a toluidine blue solution. The size distribution was determined by measuring the diameters of 100--200 cells from each rabbit in a Lanameter (Reichert, Austria). Collection o f the surfactant The material from the lung wash was centrifuged at 200 g for 15 min. A milky precipitate located above the cell b u t t o n at the b o t t o m of the tube could be collected and was considered to b e t h e surfactant [ 8]. Incubation o f macrophages with the surfactant Half a milliliter of a macrophage suspension (30 X 170
10 6
cells/ml culture
medium which consisted of Parker 199 with 10% fetal bovine serum and 2 mM glutamine) from each of the unexposed rabbits was transferred to 2 plastic test tubes. 0.2 ml of the surfactant from an exposed or an unexposed rabbit was added to 1 of the tubes and the volumes in both tubes were adjusted to 1 ml with culture medium. In each experiment surfactant from different animals was used. The tubes were incubated in a water bath with a rocking device at 37°C for 3 h. The cells were then washed twice by centrifugation in Hank's Tris solution and resuspended in Eagle's medium. Thereafter the size, the morphology and the function of the cells were investigated.
Nitroblue tetrazolium (NBT) test Half a milliliter of macrophage suspension (6 × 106 cells/ml Eagle's medium) and 0.3 ml autologous serum were transferred to each of 2 plastic tubes. Thereafter, 0.5 ml NBT-solution (0.1 g NBT in 100 ml of Hank's solution) was added to 1 of the tubes while to the other was added 0.45 ml NBT-solution together with 0.05 ml of a saline suspension of heat-killed E. coli containing a b o u t 10 ~° bacteria/ml. The tubes were incubated for 30 min under continuous agitation in a 37°C water-bath. The reaction was then interrupted by the addition of 1 ml 0.5% HC1. After centrifugation the formazan produced was extracted with 1 ml dimethylsulfoxide and the optical density (OD) of the solution determined by s p e c t r o p h o t o m e t r y at 572 nm. Attachment and ingestion o f fluorescein labelled yeast particles A modification of the m e t h o d described by Hed [14] was used. Macrophage suspension (106 cells/ml Eagle's medium) was placed on glass slides (C1 301 Novakemi) in triplicates and the cells were allowed to adhere for 30 min at 37°C. Then the glass slides were rinsed with Ringerdex solution (Pharmacia, Sweden) at 37°C. Thereafter 0.1 ml of a suspension of yeast particles (Sac° charomyces cerevisiae 2.5 × 107 particles/ml) labelled with fluorescein isothiocyanate (FITC) and opsonised in pooled rabbit serum was added to each glass slide and the preparations were incubated for 30 min at 37°C. The reaction was interrupted by placing the slides in Ringerdex solution at 4°C. Crystal violet solution was applied for 10 s. The ingested particles were easily distinguishable by their fluorescence while the attached particles were coloured dark by crystal violet. The numbers of attached and ingested cells/ phagocytic cell were determined with a fluorescence microscope (Reichert, Austria). Electron microscopy The macrophages were fixed in 2.5% buffer (pH 7.2) postfixed in 1% OsO4 graded alcohol and e m b e d d e d in Epon LKB Ultramicrotome III and examined
glutaraldehyde in 0.1 M cacodylate in the same buffer, dehydrated in 812. Thin sections were cut on an in a Jeol 100S electron microscope.
Statistical methods Three-way analysis of variance was used with the following factors: 171
(1) rabbits; (2) addition -- no addition of surfactant; (3) with -- w i t h o u t
E. coli stimulation or ingested - - a t t a c h e d particles [15]. R ES ULTS
Morphology Many macrophages washed o u t of the lungs from the nickel-treated rabbits had a surface rich in long protrusions and microvilli (Fig. 1). The cytoplasm contained many lamellar bodies and lipid droplets. The cells also had polymorphic nuclei and well developed nucleoli. The Golgi zones were p r o n o u n c e d as was the granular endoplasmic reticulum. Many cells tended to accumulate in clusters and several cells with 2 or more nuclei, so-called "giant cells", were seen. However, 3 of the animals showed less response than the others and many macrophages in all rabbits appeared normal. When macrophages from unexposed rabbits were treated with surfactant from nickel-treated ones, the preparations contained, outside the cells, accumulations of lameUated material, cell debris and dead cells. These dead cells probably came from the surfactant fraction because they contained large amounts of lamellated material. Macrophages from the unexposed
b Fig. 1. Alveolar macrophage from a nickel-treated rabbit. The cytoplasm contains numerous lamellated bodies (LB) of different density. The surface is rich in microvilli and protrusions (x 6800).
172
rabbits contained lameUar bodies, although n o t as frequently as the macrophages from the nickel-treated rabbits (Fig. 2). The debris and lamellated material were phagocytized by the macrophages (Fig. 3). There were several cells accumulating around the surfactant material and the cells had protrusions and broad zones in close apposition. The surfactant was seen as lamellated structures, lamellae arranged in concentrical whorls, intermingled with a lattice-like net (Fig. 3). Similar but less pronounced reactions were seen in the preparations consisting of surfactant from unexposed rabbits and macrophages from the same kind of animals. The surfactant did n o t contain much debris and lamellated structures and fewer cells in the preparations contained lamellar bodies and phagosomes. In the macrophages exposed to surfactant t w o main types of lamellar bodies were present. One t y p e was characterized by electron dense, concentrically arranged whorls. The other t y p e was very electron lucent and had few lamellae (Fig. 4). The control macrophages incubated without surfactant appeared mostly normal as compared to our previous studies of alveolar macrophages [8,11]. Some cells contained occasional lamellar bodies and had many lysosomal structures {Fig. 5}.
Fig. 2. A l v e o l a r m a c r o p h a g e f r o m a n u n t r e a t e d r a b b i t , i n c u b a t e d for 3 h w i t h s u r f a c t a n t f r o m a n i c k e l - t r e a t e d one. T h e cell c o n t a i n s l a m e l l a t e d b o d i e s (LB) a n d t h e surface has p r o t r u s i o n s . Debris sticks to t h e cell surface ( a r r o w ) (× 8 5 0 0 ) .
173
Fig. 3. Macrophage from an untreated rabbit is sticking to a cluster of surfactant material, from a nickel-treated one, with protrusions from cell surface. Surfactant material is seen in a cleft between 2 protrusions (arrow) (x 20 400).
Size In 2 out of 3 animals examined the mean of the diameter and the mean of the variances of the diameter of the macrophages were larger, 10% and 35%, respectively, after incubation with surfactant from nickel-exposed animals than after incubation w i t h o u t this substance or with surfactant from unexposed rabbits.
Fig. 4. Fusion of 2 lamellated structures (arrow) of different density (× 20 400).
174
5 Fig. 5. Alveolar macrophage, from an untreated rabbit, incubated without surfactant. Several lysosomal structures are seen in the cytoplasm (x 8500).
Function The NBT-reduction at rest and with E. coli stimulation of the macrophages from 9 unexposed rabbits was significantly higher after preincubation for 3 h with surfactant from exposed rabbits than after incubation without this substance for the same time (P < 0.01). Incubation of macrophages from 3 o u t of the 9 control rabbits with surfactant from unexposed rabbits did n o t give a change of any importance in the NBT-values (Table I). Macrophages from the 9 nickel-exposed rabbits showed, without any incubation, NBT-values of the same magnitude as the corresponding cells of unexposed rabbits after incubation with the surfactant from nickeltreated animals (Table I). The attachment as well as ingestion of particles by macrophages from 7 untreated animals was significantly higher after preincubation with the surfactant from exposed rabbits for 3 h (P < 0.01) (Table II) than after incubation w i t h o u t this substance for the same time. Surfactant from unexposed rabbits had no influence on phagocytosis.
175
TABLE I NBT-REDUCTION OF MACROPHAGES FROM UNEXPOSED RABBITS AFTER 3 h I N C U B A T I O N IN V I T R O W I T H O U T A N D W I T H S U R F A C T A N T FROM EXPOSED AND UNEXPOSED RABBITS Values represent amount of formazan produced by 3 × 104 macrophages during 0.5 h, expressed as optical densities. Mean values for the NBT-reduction of the macrophages from the nickel-intubatedrabbits were at rest 0.27 and with E.coli stimulation 0.55. No surfactant
Surfactant from exposed rabbits
Surfactant from unexposed rabbits A t rest
E. coli stimulated
0.25 0.19 0.19 0.21±0.03
0.38 0.35 0.26 0.33±0.06
A t rest
E. coli stimulated
A t rest
E. coli stimulated
0.20 0.19 0.14 0.24 0.27 0.20 0.17 0.24 0.12 0 . 2 0 ± 0.05 a
0.32 0.35 0.33 0.33 0.33 0.25 0.24 0.37 0.25 0.31 ± 0 . 0 5
0.27 0.25 0.21 0.49 0.47 0.31 0.23, 0.45 0.20 0.32 ± 0.12
0.30 0.35 0.53 0.62 0.52 0.40 0.25 0.61 0.38 0 . 4 4 ± 0.13
a M e a n ± S.D.
T A B L E II NUMBER OF INGESTED AND ATTACHED YEAST PARTICLES/MACROPHAGE F R O M U N E X P O S E D R A B B I T S A F T E R 3 h I N C U B A T I O N IN V I T R O W I T H O U T A N D WITH SURFACTANT FROM EXPOSED AND UNEXPOSED RABBITS No surfactant
Surfactant from exposed rabbits
Surfactant from unexposed rabbits
Ingest particles/ macrophage
Attached particles/ macrophage
Ingested particles/ macrophage
Attached particles/ macrophage
Ingested particles/ macrophage
Attached particles/ macrophage
1.3 0.8 0.6 1.2 1.9 1.0 1.0 1.1±0.4 a
1.0 2.9 2.3 2.2 1.5 1.0 0.7 1.7±0.8
0.9 0.9 0.9 1.9 2.3 1.9 1.8 1.5±0.6
2.8 3.3 3.9 2.2 1.7 1.2 0.9 2.3±1.1
0.5 1.5 1.9 1.0 1.1 1.2±0.5
2.1 1.7 1.6 1.0 1.0 1.5±0.5
a M e a n + S.D.
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DISCUSSION
The exposure to nickel dust was in this study simplified and consequently different from that previously used. However, the macrophages washed o u t from the nickel-intubated rabbits showed the same type of changes that was typical for such cells from rabbits after inhalation of nickel dust for I month. These cells had increased size variation and phagocytic capacity. Ultrastructurally, the cytoplasm contained numerous lamellar structures and the surface appeared activated in the sense that it was rich in microviUi and protrusions. These macrophages also had enhanced metabolic activity as measured by the NBT-reduction. Several publications have established that an elevated NBT-reduction can be a sign of activation in mononuclear phagocytes [ 16,17]. In the lungs of the intubated rabbits, however, a variation between them in the response to the nickel was observed and many macrophages looked apparently normal. This is most certainly due to the uneven distribution of the nickel caused by the intubation procedure. In this study macrophages from normal unexposed rabbits were incubated for 3 h with surfactant from nickel-intubated ones. After this time morphologic changes were seen that were less pronounced b u t of the same kind as noted in the nickel-exposed rabbits. The 2 types of lamellar bodies most likely represent surfactant in different stages of decomposition. Also the functional changes of the macrophages after incubation with the surfactant for 3 h were the same as in the nickel-exposed rabbits e.g. there was an increased NBT-reduction and an increased capacity to engulf particles. Although both surfactants compared to the in vivo situation were administered in excess, the one from unexposed rabbits did not give any morphological or functional changes. This indicates that the change of the surfactant by nickel exposure is qualitative rather than quantitative. The 2 surfactants used in this study were only compared by volume and n o t by biochemical analysis. However, previous studies of surfactant from rabbits exposed for 1 m o n t h showed increased amounts of phospholipid, especially phosphatidylcholine and phosphatidylinositol [18]. It is probable that the surfactants from the exposed animals used in this study were changed in the same way. Further, various works have shown that different lipids and fatty acids can be incorporated in the cell membrane and cause functional changes of the cell [19,20]. An activation of macrophages by lipids has also been demonstrated [21]. Therefore it is probable that the morphological and functional changes of the macrophages recorded in this and previous studies, is due to the phospholipids of the surfactant. The substance might be both engulfed by the macrophages and incorporated in the cell membrane. ACKNOWLEDGEMENTS
We are grateful to Marina Nyborg, Margot Lundborg and Karin WidskiSldOlsson for skilful technical assistance.
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