JOURNAL OF PATHOLOGY, VOL.
160: 63-69 ( 1990)
IMPAIRED CHEMOTACTIC RESPONSES OF BRONCHOALVEOLAR LEUKOCYTES IN EXPERIMENTAL PNEUMOCONIOSIS K. DONALDSON, G. M. BROWN, D. M. BROWN, J. SLIGHT. M. D. ROBERTSON AND J. M. G. DAVIS
lnstitutc qf0)c.mputiotinlMedicitie, 8 Ro.rhurgk Place, Edinburgh. EHS YSI., I.. K .
SUMMARY Rats were exposed to clouds of the following pneumoconiotic dusts: quartz, coal-mine dust, and chrysotile asbestos at 10 or 50 mgim? for 8, 32. and 75 days; for comparison. rats were also exposed to the non-pathogenic dust titanium dioxide (TiO,). The bronchoalveolar leukocytes (macrophages and neutrophils) from dust-exposed and control rats were obtained by lavage and tested for their ability to migrate toward zymosan-activated serum. Varying amounts of neutrophils were present depending on the ability of the dust to cause inflammation and the length of exposure. There was a marked loss of chemotactic ability in leukocytes from rats inhaling the pneumoconiotic dusts compared with controls; Ti0,-exposed leukocytes had some impairment of chemotaxis, but this was substantially less than that found with the pneumoconiotic dusts. The loss of chemotactic activity did not correlate with the percentage of neutrophils in the lavage cells except when there were very high levels of neutrophils, and there was substantial impairment of chemotaxis with negligible numbers of neutrophils. showing that macrophage chemotaxis was impaired. A phagocytic burden within the leucocytes was not sufficient alone to inhibit chemotaxis, nor was the loss of chemotactic activity due to occupied receptors. since incubation failed to restore chemotaxis. Loss to chemotactic activity by leukocytes from pneumoconiotic dust-exposed lung could be an important factor in the development of pneumoconiosis. KEY
woms-Pneumoconiosis, coal-mine dust.
bronchoalveolar leukocytes, chemotaxis, phagocytosis, inflammation, quartz.
INTRODUCTION Lavage of the bronchoalveolar space in humans and in laboratory animals has revealed that exposure to dusts commonly associated with pneumoconiosis (e.g., quartz and asbestos) results in recruitment ofleukocytes to the lungparenchyma.'-3 Once accumulated in the bronchoalveolar region, these leukocytes are considered to be important for the development of pathological change through altered functional activities and secretions. The definitive pathological change found in pneumoconiosis is fibrosis of the lung parenchyma, although emphysema has also been reported in the lungs of coal-miners4 and in the lungs of rats exposed to quartz.' Addressee for correspondence: Dr Kenneth Donaldson, Section Head. Cell Biology/Immunology.Cottage 12, City Hospital, Greenbank Drive, Edinburgh, U.K.
0022-341 7/90/010063-07 $05.00 0 1990 by John Wiley & Sons, Ltd.
Not all dusts depositing in the lung cause pneumoconiosis, and in experimental models, different dusts delivered at a similar airborne mass concentration are capable of causing different degrees of inflammatory cell recruitment into the lungs of experimental animals and this probably relates to their differential path~genicity.~ Factors other than those inducing increased numbers and altered secretory activity of leukocytes within the lung could operate in causing a dust to be pathogenic. One possibility is that, following exposure to pathogenic dusts, the bronchoalveolar leukocytes do not migrate as well as in normal lung or in lung exposed to non-pathogenic dust. If this were to be the case, then the pathogenic dusts might not be so well cleared from the lung and dust-activated leukocytes would be more likely to persist, releasing active substances contributing to pathological change. In addition, other aspects of host defence which rely on macrophage chemotaxis could be compromised.
64
K. DONALDSON ET AL.
As part of a study into leukocyte recruitment into the lungs of rats exposed to pneumoconiosisproducing dusts, we examined the chemotactic activity of bronchoalveolar leukocytes lavaged from these animals.
METHOD Rcrts
Syngeneic. PVG rats, SPF maintained and 15 weeks old at commencement of exposure, were used throughout. Minerals The dusts used in this study were (a) titanium dioxide (rutile form), obtained from Tioxide Ltd, Stockton on Tees; (b) the quartz standard DQI 2; (c) coal-mine dust collected from the air of British collieries mining anthracite (A). high rank (coking) coal (H). and low rank (bituminous) coal (L). Airborne coal-mine dust samples were collected on dry Bondina socks mounted in the return airway of a single face at each of the three collieries; full details of the collection procedure are described elsewhere;‘ and (d) chrysotile asbestos (UICC standard sample .A*), Details of the mineralogical and size characteristics of these dust samples are described elsewhere.‘.’ Inhalation exposure
Groups of 48 rats were exposed to airborne dust for 5 daysjweek, 7 hjday in exposure chambers described by Beckett.8 The dusts were dispensed using either Wright or Timbrel1dust dispensers. The concentration of dust in the chamber was monitored as the mass concentration of respirable dust defined by the Johannesburg sampling criterion’ using standard dust samplers. Full details of the exposure system are described elsewhere‘ and the target airborne mass concentrations of 10 or 50 mgjm’ were attained. Rats were exposed for 8,32, or 75 days and then removed from the chamber; on each day, control rats of similar age were also used.
Bronchoaheolar lavage At 8. 32, and 75 days into inhalational exposure, groups of four experimental rats from each exposure group were removed from the chamber and subjected to bronchoalveolar lavage; two control rats maintained in room air were similarly treated. Lungs were exsanguinated and the bronchoalveolar space was lavaged with 4 x 8 ml volumes of saline at 37-C. Bronchoalveolar leukocytes were concentrated by centrifugation, and a differential leukocyte count was performed on MayGrunwald Giemsa-stained cytospin preparations. At each time-point. cells from four dust-exposed rats and two control rats were pooled for use in the chemotaxis assay. Rats exposed to dust by intratracheal instillation, plus controls, were lavaged in the same manner.
Assay of bronchoalveolar leukocyte chemotaxis Chemotaxis was assessed using Blindwell chambers as described elsewhere.‘ Briefly, 200pI of 5 per cent zymosan-activated rat serum were placed in the lower compartment and a filter (Nucleopore, Pleasanton, C A ) placed on top. The top compartment was screwed down and 6 x 10’ bronchoalveolar leukocytes in 4 0 0 ~ 1of RPMI medium (Gibco, Paisley), + 1 per cent BSA (Sigma, Poole) were placed in the upper compartment. The filters used were 5 pm pore size and incubation was for 3.5 h at 37°C in 5 per cent CO, to allow migration of cells through the filter. At the end of the incubation period the filter was removed, washed, stained, and allowed to dry before being mounted on a slide in plastic mountant. Two chambers were set up for each condition and the number of cells that migrated to the underside of the filter in five high-power fields ( x 1000) was assessed for each filter. Statistical analysis of results
Data were analysed by analysis of variance and comparisons were made using a ‘t’-test. RESULTS
Intrat ruched inst illation Rats under general anaesthesia had their trachea exposed by dissection and 0.5ml of sterile saline containing 1 mg of quartz was introduced into the lung; the skin was closed with metal clips and the animals recovered within minutes.
Bronchoalveolar leukocyte response in dust-exposed rats Table I shows the total numbers of leukocytes lavaged from the lungs of rats exposed to the dusts and the percentage of neutrophils (lymphocytes and
65
LEUKOCYTE CHEMOTAXIS I& PNEUMOCONIOSIS
Table I-Percentage ofneutrophils and total leukocytes in bronchoalveolar lavage of rats inhaling different dusts at the indicated airborne mass concentrations for the indicated number of days
Days' exposure
Dust
Airborne mass concentration (mg m')
TiO,
10
Quartz
10
8
32
O/O
PMN
Total
40 PMN
75 0
Total
PMN
Total 4.1 (1.1)
33.5 ( 18.2)
Chrysotile
10
12.55 (4.34)
14.0 ( 1 .2)
6.45 (1.51)
Coal-mine dust H
10
5.8 (1.2) 8.8 (1.2)
0.8 (0.5) 40.2 (9.5)
(1.1)
8.2 (0.8) 11.4 (2.6)
3.2 (5.2) 52.3 ( 17.6)
50 Coal-mine dust A
10
50 Coal-mine dust L
6.6
232.0 (68.7) 8.1 ( 1.4)
50.0 (15.7)
10
12.6
50
(1.7) 39.4
(7.7) Control
Data as mean. with standard deviation in parentheses, of the percentage of PMN or total cells x lo6for four rats. The cell type over and above the neutrophils was predominantly macrophages with less than a total of 5 per cent of lymphocytes, mast cells. basophils. and eosinophils in any population. PMN =Neutrophil.
basophils comprised the remainder and were always less than 5 per cent). The data revealed that inhalation of TiO, had virtually no effect when given at IOmgjm', b i t that at this airborne mass concentration the pneumoconiotic dusts also showed little effect initially, except for chrysotile which rapidly elicited an inflammatory response. Not until towards 32 and 75 days with quartz and coal-mine dust was there increased recruitment and increased proportions of neutrophils, both changes indicative of inflammation. At 50 mg/m' with the coal-mine dusts, these inflammatory effects were earlier in onset and greater in magnitude.
Cliemotaxis versus clientokinesis in leukocyte migration To ensure that chemotaxis was the dominant activity being measured in each sample, and not chemokinesis, we used a modified 'checker board' method. Typical results were as follows (all subsequent data given as mean iSD migration cells/ high power field): spontaneous migration, O.O? 0.0; chemokinesis (measured as migration with 5 per cent zymosan-activated serum in both the upper and the lower compartments) 14.8i6.6; chemotaxis (measured with zymosan-activated serum in the lower compartment), 46.4 4.8. These results
66
K. DONALDSON ET AL.
I
CONTROL
1 .DUST-EXPOYD
0 EZl
a , QUARTZ
T
0
COALMINE OUST
Fig. I-Mean number of migrated cells; high power field of bronchoalveolar leukocytes from control rats and rats inhaling the indicated dusts at I0mg'm'. Open bars show controls and hatched bars shoH dust-exposed. Error bars represent 1 SD. Data obtained by pooling all data from 8, 32, and 75 days. Data derived from two chambers. five high power fieldslchamber at each time-point for each dust. Significant reductions in dustexposed(P
160: 63-69 ( 1990)
IMPAIRED CHEMOTACTIC RESPONSES OF BRONCHOALVEOLAR LEUKOCYTES IN EXPERIMENTAL PNEUMOCONIOSIS K. DONALDSON, G. M. BROWN, D. M. BROWN, J. SLIGHT. M. D. ROBERTSON AND J. M. G. DAVIS
lnstitutc qf0)c.mputiotinlMedicitie, 8 Ro.rhurgk Place, Edinburgh. EHS YSI., I.. K .
SUMMARY Rats were exposed to clouds of the following pneumoconiotic dusts: quartz, coal-mine dust, and chrysotile asbestos at 10 or 50 mgim? for 8, 32. and 75 days; for comparison. rats were also exposed to the non-pathogenic dust titanium dioxide (TiO,). The bronchoalveolar leukocytes (macrophages and neutrophils) from dust-exposed and control rats were obtained by lavage and tested for their ability to migrate toward zymosan-activated serum. Varying amounts of neutrophils were present depending on the ability of the dust to cause inflammation and the length of exposure. There was a marked loss of chemotactic ability in leukocytes from rats inhaling the pneumoconiotic dusts compared with controls; Ti0,-exposed leukocytes had some impairment of chemotaxis, but this was substantially less than that found with the pneumoconiotic dusts. The loss of chemotactic activity did not correlate with the percentage of neutrophils in the lavage cells except when there were very high levels of neutrophils, and there was substantial impairment of chemotaxis with negligible numbers of neutrophils. showing that macrophage chemotaxis was impaired. A phagocytic burden within the leucocytes was not sufficient alone to inhibit chemotaxis, nor was the loss of chemotactic activity due to occupied receptors. since incubation failed to restore chemotaxis. Loss to chemotactic activity by leukocytes from pneumoconiotic dust-exposed lung could be an important factor in the development of pneumoconiosis. KEY
woms-Pneumoconiosis, coal-mine dust.
bronchoalveolar leukocytes, chemotaxis, phagocytosis, inflammation, quartz.
INTRODUCTION Lavage of the bronchoalveolar space in humans and in laboratory animals has revealed that exposure to dusts commonly associated with pneumoconiosis (e.g., quartz and asbestos) results in recruitment ofleukocytes to the lungparenchyma.'-3 Once accumulated in the bronchoalveolar region, these leukocytes are considered to be important for the development of pathological change through altered functional activities and secretions. The definitive pathological change found in pneumoconiosis is fibrosis of the lung parenchyma, although emphysema has also been reported in the lungs of coal-miners4 and in the lungs of rats exposed to quartz.' Addressee for correspondence: Dr Kenneth Donaldson, Section Head. Cell Biology/Immunology.Cottage 12, City Hospital, Greenbank Drive, Edinburgh, U.K.
0022-341 7/90/010063-07 $05.00 0 1990 by John Wiley & Sons, Ltd.
Not all dusts depositing in the lung cause pneumoconiosis, and in experimental models, different dusts delivered at a similar airborne mass concentration are capable of causing different degrees of inflammatory cell recruitment into the lungs of experimental animals and this probably relates to their differential path~genicity.~ Factors other than those inducing increased numbers and altered secretory activity of leukocytes within the lung could operate in causing a dust to be pathogenic. One possibility is that, following exposure to pathogenic dusts, the bronchoalveolar leukocytes do not migrate as well as in normal lung or in lung exposed to non-pathogenic dust. If this were to be the case, then the pathogenic dusts might not be so well cleared from the lung and dust-activated leukocytes would be more likely to persist, releasing active substances contributing to pathological change. In addition, other aspects of host defence which rely on macrophage chemotaxis could be compromised.
64
K. DONALDSON ET AL.
As part of a study into leukocyte recruitment into the lungs of rats exposed to pneumoconiosisproducing dusts, we examined the chemotactic activity of bronchoalveolar leukocytes lavaged from these animals.
METHOD Rcrts
Syngeneic. PVG rats, SPF maintained and 15 weeks old at commencement of exposure, were used throughout. Minerals The dusts used in this study were (a) titanium dioxide (rutile form), obtained from Tioxide Ltd, Stockton on Tees; (b) the quartz standard DQI 2; (c) coal-mine dust collected from the air of British collieries mining anthracite (A). high rank (coking) coal (H). and low rank (bituminous) coal (L). Airborne coal-mine dust samples were collected on dry Bondina socks mounted in the return airway of a single face at each of the three collieries; full details of the collection procedure are described elsewhere;‘ and (d) chrysotile asbestos (UICC standard sample .A*), Details of the mineralogical and size characteristics of these dust samples are described elsewhere.‘.’ Inhalation exposure
Groups of 48 rats were exposed to airborne dust for 5 daysjweek, 7 hjday in exposure chambers described by Beckett.8 The dusts were dispensed using either Wright or Timbrel1dust dispensers. The concentration of dust in the chamber was monitored as the mass concentration of respirable dust defined by the Johannesburg sampling criterion’ using standard dust samplers. Full details of the exposure system are described elsewhere‘ and the target airborne mass concentrations of 10 or 50 mgjm’ were attained. Rats were exposed for 8,32, or 75 days and then removed from the chamber; on each day, control rats of similar age were also used.
Bronchoaheolar lavage At 8. 32, and 75 days into inhalational exposure, groups of four experimental rats from each exposure group were removed from the chamber and subjected to bronchoalveolar lavage; two control rats maintained in room air were similarly treated. Lungs were exsanguinated and the bronchoalveolar space was lavaged with 4 x 8 ml volumes of saline at 37-C. Bronchoalveolar leukocytes were concentrated by centrifugation, and a differential leukocyte count was performed on MayGrunwald Giemsa-stained cytospin preparations. At each time-point. cells from four dust-exposed rats and two control rats were pooled for use in the chemotaxis assay. Rats exposed to dust by intratracheal instillation, plus controls, were lavaged in the same manner.
Assay of bronchoalveolar leukocyte chemotaxis Chemotaxis was assessed using Blindwell chambers as described elsewhere.‘ Briefly, 200pI of 5 per cent zymosan-activated rat serum were placed in the lower compartment and a filter (Nucleopore, Pleasanton, C A ) placed on top. The top compartment was screwed down and 6 x 10’ bronchoalveolar leukocytes in 4 0 0 ~ 1of RPMI medium (Gibco, Paisley), + 1 per cent BSA (Sigma, Poole) were placed in the upper compartment. The filters used were 5 pm pore size and incubation was for 3.5 h at 37°C in 5 per cent CO, to allow migration of cells through the filter. At the end of the incubation period the filter was removed, washed, stained, and allowed to dry before being mounted on a slide in plastic mountant. Two chambers were set up for each condition and the number of cells that migrated to the underside of the filter in five high-power fields ( x 1000) was assessed for each filter. Statistical analysis of results
Data were analysed by analysis of variance and comparisons were made using a ‘t’-test. RESULTS
Intrat ruched inst illation Rats under general anaesthesia had their trachea exposed by dissection and 0.5ml of sterile saline containing 1 mg of quartz was introduced into the lung; the skin was closed with metal clips and the animals recovered within minutes.
Bronchoalveolar leukocyte response in dust-exposed rats Table I shows the total numbers of leukocytes lavaged from the lungs of rats exposed to the dusts and the percentage of neutrophils (lymphocytes and
65
LEUKOCYTE CHEMOTAXIS I& PNEUMOCONIOSIS
Table I-Percentage ofneutrophils and total leukocytes in bronchoalveolar lavage of rats inhaling different dusts at the indicated airborne mass concentrations for the indicated number of days
Days' exposure
Dust
Airborne mass concentration (mg m')
TiO,
10
Quartz
10
8
32
O/O
PMN
Total
40 PMN
75 0
Total
PMN
Total 4.1 (1.1)
33.5 ( 18.2)
Chrysotile
10
12.55 (4.34)
14.0 ( 1 .2)
6.45 (1.51)
Coal-mine dust H
10
5.8 (1.2) 8.8 (1.2)
0.8 (0.5) 40.2 (9.5)
(1.1)
8.2 (0.8) 11.4 (2.6)
3.2 (5.2) 52.3 ( 17.6)
50 Coal-mine dust A
10
50 Coal-mine dust L
6.6
232.0 (68.7) 8.1 ( 1.4)
50.0 (15.7)
10
12.6
50
(1.7) 39.4
(7.7) Control
Data as mean. with standard deviation in parentheses, of the percentage of PMN or total cells x lo6for four rats. The cell type over and above the neutrophils was predominantly macrophages with less than a total of 5 per cent of lymphocytes, mast cells. basophils. and eosinophils in any population. PMN =Neutrophil.
basophils comprised the remainder and were always less than 5 per cent). The data revealed that inhalation of TiO, had virtually no effect when given at IOmgjm', b i t that at this airborne mass concentration the pneumoconiotic dusts also showed little effect initially, except for chrysotile which rapidly elicited an inflammatory response. Not until towards 32 and 75 days with quartz and coal-mine dust was there increased recruitment and increased proportions of neutrophils, both changes indicative of inflammation. At 50 mg/m' with the coal-mine dusts, these inflammatory effects were earlier in onset and greater in magnitude.
Cliemotaxis versus clientokinesis in leukocyte migration To ensure that chemotaxis was the dominant activity being measured in each sample, and not chemokinesis, we used a modified 'checker board' method. Typical results were as follows (all subsequent data given as mean iSD migration cells/ high power field): spontaneous migration, O.O? 0.0; chemokinesis (measured as migration with 5 per cent zymosan-activated serum in both the upper and the lower compartments) 14.8i6.6; chemotaxis (measured with zymosan-activated serum in the lower compartment), 46.4 4.8. These results
66
K. DONALDSON ET AL.
I
CONTROL
1 .DUST-EXPOYD
0 EZl
a , QUARTZ
T
0
COALMINE OUST
Fig. I-Mean number of migrated cells; high power field of bronchoalveolar leukocytes from control rats and rats inhaling the indicated dusts at I0mg'm'. Open bars show controls and hatched bars shoH dust-exposed. Error bars represent 1 SD. Data obtained by pooling all data from 8, 32, and 75 days. Data derived from two chambers. five high power fieldslchamber at each time-point for each dust. Significant reductions in dustexposed(P
