Toxicology, 11 (1978) 353--359 © Elsevier/North-Holland Scientific Publishers Ltd.
A L V E O L A R MACROPHAGE FUNCTION IN NICKEL DUST EXPOSED RABBITS
CONNIE JARSTRAND, MARGOT
LUNDBORG
a, A N N A W I E R N I K and P E R C A M N E R a
Department of Clinical Bacteriology, Serafimer Hospital and Karolinska Institute, Stockholm and aDepartment of Environmental Hygiene, National Environment Protection Board and Karolinska Institute, Stockholm (Sweden) (Received September 13th, 1978) (Revision received November 19th, 1978) (Accepted November 23rd, 1978)
SUMMARY
8 rabbits were exposed to metallic nickel dust (2 m g / m 3, o f which about half was respirable) for 4 weeks. The lungs were lavaged and the macrophages were collected. In comparison with 8 control rabbits, a significant increase was n o t e d in the nickel exposed rabbits as c o n c e r n e d the weight and density of the lungs, the size variation of the lung cells, the phagocytosis of silver coated particles, and the metabolic acitivity as measured by NBT reduction. The last m e n t i o n e d increase was recorded during basal conditions as well as during phagocytosis. The NBT r e d u c t i o n during phagocytosis was significantly correlated with the degree of phagocytosis o f silver coated particles in both control and exposed rabbits. It is suggested that the exposure to nickel dust has unspecifically activated the macrophages perhaps by increased production of phospholipids.
INTRODUCTION
In earlier studies, clear effects on alveolar macrophages have been seen in rabbits exposed to 0.5--2 m g / m 3 o f metallic nickel dust for 4 weeks (7 h / d a y and 5 days/week) [1,2]. Approximately half of the dust was respirable in the sense that it penetrated a preseparator, in which particles larger than about 7 p m were trapped. Macrophages f r o m the animals exposed to nickel dust had a higher phagocytic activity and a larger size variation than the controls. Ultrastructurally, t h e y had numerous slender microviUi and long protrusions from the cell surface and t h e y contained laminated structures. Abbreviation: NBT, nitroblue tetrazolium.
353
The lung wash was rich in an amorphous substance which probably contained phospholipids. The effects of nickel had at least some features in c o m m o n with alveolar lipoproteinosis, which has been described in rats after exposure to quartz dust [3]. One purpose of the study to be described here was to estimate the metabolic activity of macrophages from nickel exposed rabbits by the nitroblue tetrazolium (NBT) test. The state of formazan is an indirect measurement o f the oxidative metabolism of the cells [4]. A n o t h e r purpose was to compare, in the same macrophages, the values for engulfment of silver coated particles with the values for reduction of NBT during phagocytosis. Although quite different tests, both should reflect the uptake of particles or bacteria. In order to make sure t h a t the conditions for the reaction to nickel dust would be the same as in earlier studies, recordings were also made of the size variations of the macrophages as well as the weights and densities of the lungs. MATERIALS AND METHODS
Exposure to nickel dust 16 male rabbits, weight range 1.6--2.6 kg, were used, 8 controls and 8 for exposure to nickel dust during 4--5 weeks, 5 days/week, 6 h/day. The nickel dust was produced from metallic nickel powder (320 Mesh) with an aerosol generator described by HellstrSm [ 5]. The dust was sucked into exposure chambers with a volume of 0.6 m 3 and ventilated with filtered air, 6 m3/h [6]. The nickel concentration was measured daffy by sucking the air through a millipore filter with and w i t h o u t placing a preseparator (Cassella T13043--2) in f r o n t of the filter. The volume of air was registered and the filter was weighed. The average concentration of nickel dust was 2.0 mg/m 3, standard deviation 1.0 mg/m 3. A b o u t half of the dust penetrated the preseparator.
Collection o f macrophages and estimation o f their size The alveolar macrophages were collected according to a modification of the m e t h o d of Myrvik et al. [1,7,8]. The viability of the macrophages was tested by staining with eosin-y. A smear of the cells from the lung wash was air-dried, immediately fixed in m e t h a n o l and stained with a Giemsa solution. Micrographs were taken and size distribution was estimated by measuring the diameters of 100--200 cells from each rabbit.
Phagocytosis o f silver coated particles Detailed descriptions of the m e t h o d s of producing particles and of the m e t h o d for phagocytosis have been given earlier [6,8,9]. A suspension of 4 t~m silver coated Teflon particles (6 × 103 particles/mm 3) in 75% Parker 199 solution of 25% autologous rabbit serum was added to Leighton tubes containing a monolayer of macrophages. After 1.5 h of exposure at 37°C, photographs were taken in a light microscope and the number of ingested particles/macrophage (200--300 macrophages/rabbit) was counted. 354
NBT-test 0.5 ml macrophages suspension (6 × 106 cells/ml Eagle's m e d i u m ) and 0.3 ml autologous serum or pooled h u m a n AB 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 t u b e s while to the other was added 0.45 ml NBT-solution together with 0.05 ml of a saline suspension of heatkilled E. coli containing a b o u t 10 ~° bacteria/ml. The tubes were incubated for 30 min under c o n t i n u o u s agatiation in a 37°C water-bath. The reaction was then interrupted b y the addition of 1 ml 0.5% HC1. The tubes were centrifuged at 1000 g for 15 min, the supernatant decanted and the cell pellet washed twice with 2 ml 0.85% NaC1 solution. The remaining cell pellet was extracted for 1 h with 1 ml dimethylsulfoxide under agitation in a water-bath at 70°C. Thereafter, the optical density (O.D.) of the extract was determined in a Vitatron manual DCP s p e c t r o p h o t o m e t e r at 572 nm. RESULTS
The lavage fluid from 1 control rabbit (C IV) and from 1 e x p o s e d rabbit (E III) contained b l o o d macroscopically. Both rabbits were excluded from the experiment. In the nickel dust exposed rabbits, the lavage fluid had an o p a q u e appearance. In all rabbits the p r o p o r t i o n of non-viable mcrophages was less than 3%. In Table I the weights and densities of the lungs are given. T h e y were significantly higher in the e x p o s e d animals (t-test: P < 0.01). The variances of the sizes of the macrophages are also given. In the exposed rabbits the variances were significantly larger than in the controls (Mann-Whitney test: P < 0.001). In Table II the numbers of phagocytized 4 ~m silver coated Teflon particles/macrophage are given. The macrophages from the exposed rabbits phagocytized significantly more particles than those from the control rabbits (t-test: P < 0.01). In the NBT-tests pooled h u m a n serum was used in the first 2 pairs of animals, 2 exposed rabbits (E I, E I I ) and 2 controls (C I and C II). In the second pair autologous serum was used in parallel. With human serum the NBT-reduction was n o t increased u p o n stimulation with E. coil, for which reason autologous rabbit serum was used in subsequent parts of the experiment. (See Table II). In Table II the NBT-reduction " a t rest" as well as u p o n stimulation with E. coli is shown. In the experiments where autologous rabbit serum was used, the r e d u c t i o n " a t rest" as well as the reduction in the presence of E. coli was significantly higher in the macrophages from t h e e x p o s e d rabbits than in those from the controls (t-test: P < 0.01 and P < 0.05, respectively). Also when pooled h u m a n serum was used, the NBT activities "at rest" and u p o n stimulation were higher in the macrophages from the exposed rabbits than in those from the controls, see Table II. When autologous serum was used the difference b e t w e e n the NBT-reduction "at rest" and u p o n stimulation was significant in both control and e x p o s e d rabbits (paired t-test: P < 0.01 and P < 0.02, respectively). 355
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Z
A L V E O L A R MACROPHAGE FUNCTION IN NICKEL DUST EXPOSED RABBITS
CONNIE JARSTRAND, MARGOT
LUNDBORG
a, A N N A W I E R N I K and P E R C A M N E R a
Department of Clinical Bacteriology, Serafimer Hospital and Karolinska Institute, Stockholm and aDepartment of Environmental Hygiene, National Environment Protection Board and Karolinska Institute, Stockholm (Sweden) (Received September 13th, 1978) (Revision received November 19th, 1978) (Accepted November 23rd, 1978)
SUMMARY
8 rabbits were exposed to metallic nickel dust (2 m g / m 3, o f which about half was respirable) for 4 weeks. The lungs were lavaged and the macrophages were collected. In comparison with 8 control rabbits, a significant increase was n o t e d in the nickel exposed rabbits as c o n c e r n e d the weight and density of the lungs, the size variation of the lung cells, the phagocytosis of silver coated particles, and the metabolic acitivity as measured by NBT reduction. The last m e n t i o n e d increase was recorded during basal conditions as well as during phagocytosis. The NBT r e d u c t i o n during phagocytosis was significantly correlated with the degree of phagocytosis o f silver coated particles in both control and exposed rabbits. It is suggested that the exposure to nickel dust has unspecifically activated the macrophages perhaps by increased production of phospholipids.
INTRODUCTION
In earlier studies, clear effects on alveolar macrophages have been seen in rabbits exposed to 0.5--2 m g / m 3 o f metallic nickel dust for 4 weeks (7 h / d a y and 5 days/week) [1,2]. Approximately half of the dust was respirable in the sense that it penetrated a preseparator, in which particles larger than about 7 p m were trapped. Macrophages f r o m the animals exposed to nickel dust had a higher phagocytic activity and a larger size variation than the controls. Ultrastructurally, t h e y had numerous slender microviUi and long protrusions from the cell surface and t h e y contained laminated structures. Abbreviation: NBT, nitroblue tetrazolium.
353
The lung wash was rich in an amorphous substance which probably contained phospholipids. The effects of nickel had at least some features in c o m m o n with alveolar lipoproteinosis, which has been described in rats after exposure to quartz dust [3]. One purpose of the study to be described here was to estimate the metabolic activity of macrophages from nickel exposed rabbits by the nitroblue tetrazolium (NBT) test. The state of formazan is an indirect measurement o f the oxidative metabolism of the cells [4]. A n o t h e r purpose was to compare, in the same macrophages, the values for engulfment of silver coated particles with the values for reduction of NBT during phagocytosis. Although quite different tests, both should reflect the uptake of particles or bacteria. In order to make sure t h a t the conditions for the reaction to nickel dust would be the same as in earlier studies, recordings were also made of the size variations of the macrophages as well as the weights and densities of the lungs. MATERIALS AND METHODS
Exposure to nickel dust 16 male rabbits, weight range 1.6--2.6 kg, were used, 8 controls and 8 for exposure to nickel dust during 4--5 weeks, 5 days/week, 6 h/day. The nickel dust was produced from metallic nickel powder (320 Mesh) with an aerosol generator described by HellstrSm [ 5]. The dust was sucked into exposure chambers with a volume of 0.6 m 3 and ventilated with filtered air, 6 m3/h [6]. The nickel concentration was measured daffy by sucking the air through a millipore filter with and w i t h o u t placing a preseparator (Cassella T13043--2) in f r o n t of the filter. The volume of air was registered and the filter was weighed. The average concentration of nickel dust was 2.0 mg/m 3, standard deviation 1.0 mg/m 3. A b o u t half of the dust penetrated the preseparator.
Collection o f macrophages and estimation o f their size The alveolar macrophages were collected according to a modification of the m e t h o d of Myrvik et al. [1,7,8]. The viability of the macrophages was tested by staining with eosin-y. A smear of the cells from the lung wash was air-dried, immediately fixed in m e t h a n o l and stained with a Giemsa solution. Micrographs were taken and size distribution was estimated by measuring the diameters of 100--200 cells from each rabbit.
Phagocytosis o f silver coated particles Detailed descriptions of the m e t h o d s of producing particles and of the m e t h o d for phagocytosis have been given earlier [6,8,9]. A suspension of 4 t~m silver coated Teflon particles (6 × 103 particles/mm 3) in 75% Parker 199 solution of 25% autologous rabbit serum was added to Leighton tubes containing a monolayer of macrophages. After 1.5 h of exposure at 37°C, photographs were taken in a light microscope and the number of ingested particles/macrophage (200--300 macrophages/rabbit) was counted. 354
NBT-test 0.5 ml macrophages suspension (6 × 106 cells/ml Eagle's m e d i u m ) and 0.3 ml autologous serum or pooled h u m a n AB 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 t u b e s while to the other was added 0.45 ml NBT-solution together with 0.05 ml of a saline suspension of heatkilled E. coli containing a b o u t 10 ~° bacteria/ml. The tubes were incubated for 30 min under c o n t i n u o u s agatiation in a 37°C water-bath. The reaction was then interrupted b y the addition of 1 ml 0.5% HC1. The tubes were centrifuged at 1000 g for 15 min, the supernatant decanted and the cell pellet washed twice with 2 ml 0.85% NaC1 solution. The remaining cell pellet was extracted for 1 h with 1 ml dimethylsulfoxide under agitation in a water-bath at 70°C. Thereafter, the optical density (O.D.) of the extract was determined in a Vitatron manual DCP s p e c t r o p h o t o m e t e r at 572 nm. RESULTS
The lavage fluid from 1 control rabbit (C IV) and from 1 e x p o s e d rabbit (E III) contained b l o o d macroscopically. Both rabbits were excluded from the experiment. In the nickel dust exposed rabbits, the lavage fluid had an o p a q u e appearance. In all rabbits the p r o p o r t i o n of non-viable mcrophages was less than 3%. In Table I the weights and densities of the lungs are given. T h e y were significantly higher in the e x p o s e d animals (t-test: P < 0.01). The variances of the sizes of the macrophages are also given. In the exposed rabbits the variances were significantly larger than in the controls (Mann-Whitney test: P < 0.001). In Table II the numbers of phagocytized 4 ~m silver coated Teflon particles/macrophage are given. The macrophages from the exposed rabbits phagocytized significantly more particles than those from the control rabbits (t-test: P < 0.01). In the NBT-tests pooled h u m a n serum was used in the first 2 pairs of animals, 2 exposed rabbits (E I, E I I ) and 2 controls (C I and C II). In the second pair autologous serum was used in parallel. With human serum the NBT-reduction was n o t increased u p o n stimulation with E. coil, for which reason autologous rabbit serum was used in subsequent parts of the experiment. (See Table II). In Table II the NBT-reduction " a t rest" as well as u p o n stimulation with E. coli is shown. In the experiments where autologous rabbit serum was used, the r e d u c t i o n " a t rest" as well as the reduction in the presence of E. coli was significantly higher in the macrophages from t h e e x p o s e d rabbits than in those from the controls (t-test: P < 0.01 and P < 0.05, respectively). Also when pooled h u m a n serum was used, the NBT activities "at rest" and u p o n stimulation were higher in the macrophages from the exposed rabbits than in those from the controls, see Table II. When autologous serum was used the difference b e t w e e n the NBT-reduction "at rest" and u p o n stimulation was significant in both control and e x p o s e d rabbits (paired t-test: P < 0.01 and P < 0.02, respectively). 355
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