Pulmonary fibrosis: Bronchoalveolar cell types and impaired function of alveolar macrophages in experimental silicosis

ENVIRONMENTAL

RESEARCH

27, 226-236

(1982)

Pulmonary Fibrosis: Bronchoalveolar Cell Types and Impaired Function of Alveolar Macrophages in Experimental Silicosisl JAMES H. DAUBER,

MILTON D. ROSSMAN, AND RONALD P. DANIELE

Received

April

10, 1981

To determine the impact of chronic silicosis on lung cell populations and macrophage function, we quantified the types of cells in lavage fluid (bronchoalveolar cells) from guinea pigs with chronic silicosis and tested irr vi/w the function of alveolar macrophages. The fractions of neutrophils and giant cells in bronchoalveolar cells of silicotic animals were increased while the fraction of macrophages was slightly decreased compared to those of controls (intratracheal saline). The fractions of both lymphocytes and eosinophils were similar in the two groups. The majority of macrophages from silicotic lungs did not adhere to a glass surface. In addition, the random and stimulated migration of the macrophages from the silicotic lung was subnormal. However. adherent macrophages from silicotic animals spread on glass in both the absence and presence of a potent stimulant. N-formylmethionyl phenylalanine, as did ceils from controls. These results suggest that the types of cells recovered by lavage from the silicotic lung mirror the inflammatory reaction in the pulmonary parenchyma and that the function of some alveolar macrophages in the silicotic lung is impaired.

INTRODUCTION

Silica is toxic for peritoneal macrophages in vitro (Allison et nl., 1966), and even in sublethal doses it impairs intracellular killing of tubercle bacilli (Allison and D’Arcy Hart, 1968). Presumably, silica which deposits in the lung exerts similar effects on alveolar macrophages, but information about the properties of these cells in chronic silicosis is limited. The function of at least some alveolar macrophages in the silicotic lung may be abnormal. Mice with severe silicosis do not clear inhaled bacteria as efficiently as normal mice (Goldstein ef al., 1969). This impairment may be due to reduced phagocytosis and killing by alveolar macrophages. Some alveolar macrophages from silicotic rats appear to be injured, since they have fragile cell membranes (Miller and Kagan, 1977). In addition, these cells have decreased membrane IgG receptor activity. On the other hand, there is evidence that some macrophages in lungs exposed to silica are stimulated, since these cells exhibit enhanced IgG receptor activity (Miller and Kagan, 1977), spreading, phagocytosis, and killing (Davis et al., 1980). ’ These investigations were supported by Young Investigator Awards IR23-HL-21134 (Dr. and lR23-HL-24500 (Dr. Rossman), Research Career Development Award 1K04-HL-00210 Daniele), and Grant ROl-HL-23877 from the National Heart, Lung and Blood Institute. 226 0013-9351/82/010226-11$02.00/O Copyright All rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

Dauber) (Dr.

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Recently, we developed a model of chronic silicosis in the guinea pig (Dauber et nl., 1980). To determine the impact of this form of silicosis on the function of alveolar macrophages, we recovered lung cells by lavage and measured the ability of the lavaged macrophages to adhere, spread, and migrate in vitro. We also quantified the types of cells recovered by lavage to ascertain if changes in bronchoalveolar cell populations reflect the pulmonary inflammatory response in silicosis as reported for other interstitial lung diseases (Reynolds et al., 1977; Weinberger et al., 1978). We found that the proportions of neutrophils and multinucleated macrophages (giant cells) were increased in the lavage fluid from silicotic animals compared to controls. In addition, the adherence and migration of macrophages from the silicotic lung were deficient, but cells which adhered appeared to spread normally. These results indicate that experimental silicosis is associated with impairment of at least two basic macrophage functions and that the types of cells recovered by lavage mirror the lung’s inflammatory response in this disorder. MATERIALS

AND METHODS

Inductiorl of Experimental Silicosis Experimental silicosis was produced in outbred Hartley strain guinea pigs by a single intratracheal injection of 50 mg of sized SiO, particles suspended in 1 ml of saline. Control animals received only saline (saline control). The preparation of silica, handling of animals, and the histologic and biochemical abnormalities produced have been described in detail elsewhere (Dauber et al., 1980). Control and silicotic animals in this study were sacrificed 6 months after the intratracheal injection. Quantification

of Bronchoalveolnr

Cell Populations

Under adequate pentobarbital anesthesia, a thoracotomy was performed and the animal exsanguinated by direct cardiac puncture. The heart and lungs were removed en bloc and the trachea was cannulated with a plastic catheter. In six experimental and five control animals, the left mainstem bronchus was clamped and the right lung only was lavaged. The left lung was saved for histologic evaluation. The method of lavage was described previously (Dauber et al., 1980). Briefly, IO- to 15ml aliquots of cold Ca2+, Mg 2f-free Hanks’ balanced salt solution (MHBSS; GIBCO, Grand Island, N.Y.) were injected and aspirated with a syringe. The lungs were gently massaged after the aliquot was withdrawn. Lavage was continued until 100 ml of lavage fluid was collected. In an additional four experimental and three control animals, the right and left lungs were lavaged simultaneously with 20 to 30 ml aliquots until 200 ml of lavage fluid was collected. Lavage fluid was centrifuged at SOOg and 4°C for 10 min. The cell pellets were washed twice in cold MHBSS and resuspended in RPM1 1640 (GIBCO). Cell counts were made on a Model Zf Coulter Counter (Coulter Electronics, Hialeah, Fla.). Total cell yield was expressed as the number of cells per 100 ml of lavage fluid recovered. This normalized value was used because the amount of lavage fluid recovered per lung in all animals was approximately 100 ml. Viability was assayed by exclusion of trypan blue. Smears for differential counting were pre-

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pared on a cytocentrifuge (Shandon Southern Corporation, Sewickley, Pa.). Morphologic criteria were used to differentiate cell types on smears stained with Diff-Quik (Harleco, Philadelphia, Pa.). To assist in distinguishing macrophages from lymphocytes, smears were also stained for nonspecific cytoplasmic esterase (Li et al., 1973). To differentiate macrophages from type II pneumocytes, a suspension of bronchoalveolar cells from one silicotic animal was stained with Phosphine 3R (Roboz Surgical Instrument Co., Washington, D.C.) (Mason et al., 1977) and smears of bronchoalveolar cells from three additional silicotic animals were stained for cytoplasmic alkaline phosphatase activity (Fisher and Furia, 1977; Kaplow , 1963). Lymphocyte

Subpopulations

in Bronchoalveolar

Cells

Techniques used for identification of T and B cells have been described in detail elsewhere (Gorenberg and Daniele, 1976). In brief, T cells were identified by their ability to form rosettes spontaneously with unsensitized rabbit erythrocytes, whereas B cells were identified by their ability to form rosettes with sheep erythrocytes coated with antibody and complement (EAC). These assays were performed on cells from five experimental and four saline control animals. Macrophage

Adherence

and Spreading

Alveolar macrophages adhere and spread on glass spontaneously, but there is recent evidence that N-formylmethionyl phenylalanine (FMP) enhances adherence and stimulates elongation and spreading of adherent cells (Rossman et al., 1980a). Therefore, we determined the proportion of macrophages in bronchoalveolar cells of control and experimental animals that adhered to a glass surface in both the absence and presence of FMP. In addition, we quantified the spreading of adherent macrophages which occurred spontaneously and after stimulation with FMP. Adherence and spreading were measured as previously reported (Rossman et al., 1980a). In brief, we placed 5 x lo5 lung cells suspended in 1 ml of RPM1 1640 (GIBCO) plus 10% v/v heat-inactivated fetal calf serum (GIBCO) in each chamber of a two-chamber Lab-Tek tissue culture slide (Miles Laboratories, Inc., Naperville, Ill.). After incubation for l/, 1, 4, or 18 hr with or without FMP, (Sigma Chemical Co., St. Louis, MO.) the culture medium was removed and the number of nonadherent cells counted. The monolayer was stained with Diff-Quik and the proportion of adherent cells that were macrophages was determined. The number of adherent macrophages equaled (number of cells plated - number of nonadherent cells) x (proportion of adherent cells which are macrophages). To quantify spreading, we allowed macrophages to adhere spontaneously for 1 hr as described above. After removing nonadherent cells, we incubated the monolayers in serum-free RPM1 with or without FMP for an additional 1, 2, or 3 hr. At the end of the incubation period, the monolayer was fixed with 2% glutaraldehyde in phosphate-buffered saline and the greatest cell diameter measured using a calibrated micrometer in the ocular lens of a microscope. The mean maximum diameter was determined for 50 consecutive macrophages in each chamber.

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Macrophage Motility To measure motility, we employed blind well chemotactic chambers (Neuroprobe, Bethesda, Md.) as described in detail previously (Dauber and Daniele, 1978). Briefly, 3 x lo5 viable macrophages from control and silicotic animals suspended in Gey’s Balanced Salt Solution (GBSS; GIBCO) plus 2% bovine serum albumin (BSA, Sigma) were placed in the upper compartment, which was separated from the lower compartment by a 5-pm-pore polycarbonate filter (Nucleopore Corp., Pleasanton, Calif.). The lower compartment contained either GBSS alone (random migration) or the chemotaxin N-formylmethionyl phenylalanine (FMP) at an optimal concentration of 10m6 M (directed migration) (Dauber and Daniele, 1978). In some experiments, the concentration of FMP was also made equal (5 x 1OV M) in both compartments to measure stimulated random migration. After 90 min of incubation in humidified air at 37”C, the filters were removed, dried, and stained. The number of cells migrating through the filter in 20 oil immersion fieIds (OIF) was counted. The magnitude of migration was expressed as the mean number of cells per OIF in triplicate filters. Statistics Student’s t test (unpaired) and the Wilcoxon rank sum tests were used to determine the significance of the difference between mean values. RESULTS

Bronchoalveolar Cell Populations The proportion of neutrophils in the lavage fluid of silicotic animals was threefold greater than the value for controls (10 i 1 vs 3 ? 1, mean ? SE, P < 0.001, Student’s t test), while the proportions of both eosinophils and lymphocytes were similar in the two groups (Fig. 1). Although the fraction of macrophages in

Macrophoges

1. Mean 2 1 SE animals. The proportion trols (P < 0.001). The difference compared to FIG.

Neutrophils

Lymphocytes

Eosinophils

values for the proportions of cell types in lavage fluid of silicotic and control of neutrophils was significantly higher in silicotic animals compared to conproportion of macrophages was decreased in the silicotic animals but the that in controls was of borderline significance (0.05 < P < 0.08).

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the lavage fluid of silicotic animals was lower than that of controls (70 t 6% vs 76 ? 2%), the difference was of borderline significance (0.05

2

3

4 NUMBER

5 OF NUCLEI

6

7 PER

?9

CELL

FIG. 2. A histogram of the distribution of nuclei in multinucleated cells from silicotic (KQ and control (0) animals. The percentage of macrophages containing two nuclei was similar in the two groups, but the percentage of cells containing from three to eight nuclei was significantly greater in the silicotic animals (P < 0.05). No cells from controls were found to contain nine or more nuclei.

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the difference was not statistically significant by either test ((68 ? 15 vs 52 f 13) x lo6 cells/100 ml). On the other hand, recovery of neutrophils from the silicotic animals clearly exceeded that from controls ((10.2 -+ 2.8 vs 2.0 -+ 1.1) X IO6 cells/100 ml, 0.02 < P < 0.03, Student’s r test). Viability of bronchoalveolar cells was nearly equal for the two groups (92 2 1% vs 94 ? l%, respectively). Macrophage Adherence

After 1 hr in culture, only one-third of the macrophages from silicotic animals were adherent compared to more than 80% for the controls (Fig. 3). Macrophages from the silicotic animals that were cultured with FMP increased their adherence in a dose-dependent manner (Fig. 3), but the stimulated adherence of these cells was significantly less than that of cells from controls (P < 0.02). After 18 hr of culture in the absence of FMP, however, the proportions of macrophages originally plated that were still adherent were alike for the two groups (36 t 16% vs 36 + 5%, n = 4 and n = 3, respectively).

CONCENTRATION

OF FMP-MOLE/L

FIG. 3. Upper panel: Stimulation of macrophage adherence by FMP. The proportion of adherent macrophages from silicotic (0) and control (0) guinea pigs was measured after 1 hr of culture with cells exposed to the concentration of FMP indicated on the abscissa. The proportion of macrophages from silicotic animals (n = 3) which adhered in FMP (lo-’ M) was significantly greater than the proportion which adhered in the absence of FMP (P < .05). At all concentrations of FMP, adherence of cells from control animals (n = 3) exceeded that of cells from silicotic animals. Lower panel: Stimulation of spreading by FMP. The mean maximum diameter of cells from the silicotic animals incubated in the absence of FMP (n = 3) appears to be less than that of saline controls (12= 3) but the difference is not statistically significant (0.2 < P < 0.25). Cells from silicotic and control animals incubated for 2 hr with FMP demonstrated comparable increases in maximum diameters.

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ROSSMAN,

AND DANIELE

Macrophage Spreading

After 1 hr in culture, adherent cells from silicotic and control animals had similar mean maximum diameters (Fig. 3). The increases in the mean maximum diameter that occurred during the subsequent 2-hr incubation period were comparable for the two groups, suggesting that the cells from silicotic and control animals spread to a similar degree. We have previously shown that FMP causes normal alveolar macrophages to elongate in vitro (Rossman ef al., 1980a). Cells from the silicotic animals also elongated when stimulated by FMP (Fig. 3), and the magnitude of their response was similar to that of cells from the saline controls. Macrophage Motility

Macrophages from silicotic animals did not migrate as well as macrophages from controls in either the absence or presence of a stimulus (FMP). Random migration of macrophages from silicotic animals was about one-half that of controls while directed migration was only one-fifth as great (Fig. 4). Directed migration of cells from silicotic animals was approximately twice as great as stimulated random migration in two experiments (data not shown). This indicated that the enhanced migration occurring in a concentration gradient of FMP was due in part to chemotaxis. DISCUSSION

There is a growing body of evidence which supports the idea that cells obtained by lung lavage mirror the type of inflammation in interstitial lung disease. For instance, the proportion of lymphocytes in bronchoalveolar cells is greater than normal in hypersensitivity pneumonitis (Reynolds et al., 1977), while in idiopathic pulmonary fibrosis, the proportion of neutrophils is increased (Weinberger et al., 1978). In this form of experimental silicosis, there is both granulomatous inflam-

P(O.001

RANDOM

MIGRATION

DIRECTED

MIGRATION

FIG. 4. Comparison of random migration (no stimulus) and directed migration (FMP lo-” M in right compartment) measured in blind well chemotactic chambers for macrophages from control and experimental animals. Note the difference in the scales on the ordinates.

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mation with nodular fibrosis and diffuse interstitial inflammation with septal fibrosis (Dauber er al., 1980).- The granulomas consist almost exclusively of macrophages while the interstitial infiltrate contains not only macrophages but polymorphonuclear leukocytes and lymphocytes as well. Overall, however, the macrophage is the most conspicuous inflammatory cell in the histologic sections. The macrophage was also the predominant cell recovered by lavage from these animals. In addition, the proportion of neutrophils in the lavage fluid was increased compared to that in controls. A similar finding has been reported for silicotic rats (Miller and Kagan, 1977). Thus, the types of cells recovered by lavage appear to mirror the type of inflammation occurring in the lung in experimental silicosis. Although the proportion of macrophages in the bronchoalveolar cells of silicotic animals was similar to that in controls, the proportion of multinucleated macrophages (giant cells) was abnormally high. The origin of giant cells and their role in the inflammatory response are incompletely understood. Chambers (1977) demonstrated that simultaneous phagocytosis of a particle by two macrophages results in fusion of the cells and formation of giant cells in vitro. Since most giant cells from silicotic animals appeared to contain silica, it is possible that these cells arose in this way. The presence of neutrophils on histologic sections and their prevalence in the lavage fluid suggest that neutrophils are continuously recruited to the silicotic lung. The finding of increased numbers of neutrophils and giant cells in the silicotic lung raises the possibility that these cells play a role in the inflammatory and fibrotic response to silica. In contrast to the relatively good correlation between the types of cells recovered by lavage and the type of inflammation in the lung, the number of cells recovered by lavage did not appear to reflect the intensity of the inflammatory response. On average, more cells were recovered from silicotic animals than from controls, but there was enough overlap in individual values for total cells and macrophages in the two groups that a statistically significant difference between mean values could not be demonstrated by either Student’s t test (normal distribution) or Wilcoxon’s rank sum test (nonparametric analysis). We do not have an explanation for the variation in recovery of total cells and macrophages from the silicotic animals but this finding suggests that the number of cells recovered by lavage may not be an accurate estimate of the intensity of the pulmonary inflammatory response in this form of chronic experimental silicosis. Because silica is selectively toxic for macrophages (Allison et al., 1966), it is important to determine the functional capacity of alveolar macrophages in silicosis. In these studies, we examined adherence, spreading, and migration of macrophages from the silicotic lung and the effect of a potent stimulant (FMP) on these functions. The ability to adhere to a surface is a basic property of macrophages. The bond between macrophage and substrate is normally highly resistant to disruption by physical forces, proteolytic digestion, and chelating agents. Macrophages from the silicotic lung did not appear to form a tight bond with the glass substrate. Although FMP enhanced the adherence of these cells, it did not restore it to normal. Thus, it appears that a large population of macrophages from the silicotic lung are injured or altered in a fundamental way.

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Macrophages from the silicotic lung that were able to adhere spread on glass normally and, when exposed to FMP, elongated as well as cells from controls. Thus, the spreading of adherent macrophages from experimental animals seemed normal. Adherence to a substrate is an integral step in the migration of a cell (Stossel, 1978). A cell that does not adhere probably cannot migrate in vitro. The poor adherence of macrophages from the silicotic lung may be one explanation for the decreased magnitude of random motility since the reduction in adherence and in random migration for these cells was similar (about 50% of control). On the other hand, the magnitude of directed migration was reduced even more (about one-fifth of control). This observation is similar to that reported for guinea pigs exposed to silica by inhalation (Miller, 1980). The explanation for the impaired migratory response to FMP is unclear. It is possible that ingested silica interferes with the locomotor apparatus of the cell or impairs the cell’s movement through the filter. An alternate explanation is that a subpopulation of cells can migrate randomly but FMP does not stimulate the migration of these cells. Results of preliminary studies in this laboratory support the contention that macrophages from the silicotic lung may not respond normally to FMP. We found that adherent macrophages from silicotic animals failed to increase membrane IgG receptor activity when exposed to a range of doses of FMP, whereas cells from controls doubled receptor activity (Rossman et al.. 1980b). Thus, even though a subpopulation of macrophages from the silicotic animals adhere and spread in vitro as well as cells from controls, the function of these cells may not be entirely normal since they do not appear to respond appropriately to a macrophage stimulant. Macrophages recovered from sites of inflammation usually exhibit enhancement of many functions and thus are considered to be “activated” (Cohn, 1978). Because the lung in this form of experimental silicosis is clearly inflamed, we expected to find activated macrophages in lavage fluid. We were unable, however, to detect activated cells by the assays employed in these studies, i.e., adherence, spreading, and motility. Other investigators have found activated macrophages in the lavage fluid of animals exposed to silica by inhalation. Alveolar macrophages from pathogen-free rats that received a short-term, high intensity dusting exhibited enhanced spreading, phagocytosis, killing, and oxygen consumption (Davis et nl., 1980). Macrophages from chronically dusted rats have increased membrane IgG receptor activity, another marker for cell activation (Miller and Kagan, 1977). In preliminary studies, however, we found that adherent macrophages from guinea pigs which received an intratracheal injection of silica 6 months earlier have normal IgG receptor activity (Rossman et nl., 1980b). Explanations for these conflicting results include differences in species of experimental animals, route of exposure, amount of silica in the lung at the time the cells were harvested, severity of the response to silica, and methods used to detect activated cells (Cohn, 1978). Further studies are necessary to resolve these differences. In summary, we have shown that the types of cells recovered by lavage from guinea pigs with chronic experimental silicosis mirror the inflammatory response in the lung. The number of cells recovered, however, may not correlate well with the intensity of the interstitial inflammation. The finding of elevated proportions of

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neutrophils and multinucleated cells in the bronchoalveolar cells raises the possibility that these cells, along with the macrophage, play a role in the pathogenesis of silicosis. Finally, the majority of lavaged macrophages failed to adhere and even the adherent macrophages may not have responded to a potent stimulus. Thus, some alveolar macrophages in the siiicotic lung probably function normally, Ixt it is likely that many do not. ACKNOWLEDGMENTS The authors thank Ms. Angel Cassizzi, Ms. Lynette McMillan, Ms. Mary Rosemiller, and, especially, Ms. Lynne Uhl for their assistance in maintaining the animals, preparing cells, and conducting the assays of macrophage function. Ms. Mary McNichol deserves our gratitude for her preparation of the manuscript. We also thank Dr. A. P. Fishman for his critical review of the manuscript.

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