Effects of instilled fibrogenic particles on the clonal growth of murine pulmonary alveolar macrophages.

Murine pulmonary alveolar macrophages (PAM) form macrophage colonies in vitro with colony-stimulating factors, which stimulate the clonal growth of radioresistant alveolar colony-forming cells (AL-CFC). The toxic effects of fibrogenic mineral dust particles on AL-CFC were investigated after intratracheal instillation into mice. Exposure to either crocidolite asbestos or silica (Min-u-sil) induced a significant depletion of AL-CFC as well as a decrease in PAM recovery compared to either untreated or titanium dioxide-exposed animals. Such effects were also noted with different doses (50-200 micrograms/animal) of instilled particles. The plating efficiency of AL-CFC was depleted in PAM exposed to fibrogenic particles in vitro, but not when exposed to nonfibrogenic titanium dioxide particles. These results indicate the toxic effects of fibrogenic dust particles on the clonal growth of PAM, cells which play a role in the clearance of inhaled particles from the lung and in subsequent pathologic processes.


Introduction
Pulmonary alveolar macrophages (PAM) have an essential role in lung clearance and in defense mechanisms against inhaled particles (1). Changes in the number of PAM and in their functions may therefore affect the subsequent pathologic processes in the lung. The recovery ofPAM by bronchoalveolar lavage has been reported to be reduced after the inhalation ofradioactive particles (2) and asbestos fibers (3). However, it is still uncertain whether this decrease in the numbers of recovered PAM is due to the direct effects of radiation or to the toxicity of particles on a stem cell population. Normally, PAM are maintained in a steady state either by self-renewal (4,5) or by clonal growth ofa putative stem cell: alveolar colony-forming cells (AL-CFC). These cells have the capacity to replicate and form macrophage colonies in vitro by culturing with colony-stimulating factors (CSFs) (6,7).
The present study was designed to compare in vitro the toxic effects of instilled fibrogenic asbestos or silica particles on murine AL-CFC with those effects induced by nonfibrogenic titanium dioxide (T102). We also compared the effects of in vitro exposures to particles with different toxicity.

Materials and Methods
Groups of C3H/He female mice (8-10 weeks old) were instilled intratracheally (IT) with 0.1 mLphosphate-buffered saline containing either Ti02, crocidolite asbestos (CR; Uion International Centre Cancer standard reference preparation), or a microcrystalline form of silica (Min-u-sil; The Pennsylvania Glass Sand Co., Pittsburgh). Test animals, together with untreated controls, were kept under barrier conditions for 40 days after instillation. We obtained bronchoalveolar cells (BAC) from animals by repeated bronchoalveolar lavage as described previously (8). The total numbers of nucleated cells in BAC preparations were determined by a Coulter counter (Model ZM). We made differential counts from cytocentrifuge preparations stained with Giemsa and by indirect inumunofluorescence using anti-asialo GMI (AsGM1) antibody specific for mouse PAM. To detect AL-CFC, 1 x 104 BAC were plated in triplicate in 1 mL of 0.3% agarose medium, supplemented with 10% fetal bovine serum (FBS) and 500 U/mL rGM-CSF in 35-mm culture dishes. These were incubated for 21 days at 37°C in a humidified environment of5 % CO2 in air. We then fixed the cultures and stained them with Giemsa to score the number of colonies containing 30 or more cells. For in vitro exposures, fresh PAM were incubated with 100 ztg/mL of particles in suspension at 37°C for 1 hr and assayed For the clonal growth. 'Groups of mice were untreated (control) or exposed intratracheally to 200 Ag of each particle preparation, and bronchoalveolar cells were harvested on day 30. "Total recovery of cells was counted by a Coulter counter, and the number of each cell type was enumerated by the proportion (%). cData represent mean ± SD of six animals in each group. *Significant difference compared to the control, p < 0.05.

Recovery of PAM from Mice Instilled with Various Dusts
The numbers of BAC recovered from untreated control mice were in the range of 3.0-5.0 x 105 of which 90-95 % were PAM or AsGM -positive macrophages (M+), and 5% or less were lymphocytes and polymorphonuclear leukocytes (PMN). The numbers of BAC and PAM recovered by lavage were reduced significantly both in the asbestosand silica-exposed animals during the 40-day period after instillation, whereas those from T102-exposed animals were not reduced significantly. Numbers of PMN and multinucleated Mb were increased significantly both in the asbestosand silica-exposed animals as compared to the control and Ti02-exposed mice ( Table 1). The proportions of particle-laden PAM ("dusted" M+~) were 60-70% in TiO2-exposed animals, 30-60% in silica-exposed animals, and 20-40% in asbestos-exposed animals.
Cloning Efficiency of AILCFC Exposed to Dusts in Vivo When normal BAC (mostly PAM) were incubated in agarose medium containing rGM-CSF for 21 days, the plating efficiency for macrophage colonies (AL-CFC) was about 1.8-2.0%. Compared to the untreated animals, the plating efficiency was considerably reduced with cells from both the asbestosand silica-exposed animals, although the reduction was not significant in Ti02-exposed animals (Fig. IA). As shown in Figure IB, the number ofAL-CFC with cells recovered from T102-exposed animals was not much affected by doses within the range 50-200 ytg. However, the effect ofdose was much more marked with cells from animals exposed to silica and asbestos.
As compared to the control, the plating efficiency of AL-CFC was much lower (about 0.3-0.4% per total BAC plated and about 0.4-0.5 % per total PAM plated) both in the groups of asbestosand silica-exposed animals ( Table 2). The plating efficiency of AL-CFC was not significantly different in Ti02-exposed animals from the control.

Cloning Efficiency of Cells Exposed to Dusts in Vitro
To test the effects ofdust particles with a range oftoxicity, normal BAC were exposed invitro to 100 jAg/mL ofeachtype ofdust in suspension at 37°C. After 1 hr, cells that had incorporated or bound particles were separated on a Percoll gradient solution (density 1.074), washed, resuspended, and incubated in agarose medium containing rGM-CSF. As shown in Figure 2, the number of AL-CFC after exposure to TiO2 particles did not differ from the untreated control. Both asbestos (crocidolite) and silica (Min-u-sil and alpha quartz) depleted the plating efficiency of AL-CFC severely. Fly ash also suppressed colony formation, but not as much as either crocidolite or silica.

Discussion
In murine PAM, there is a subpopulation (AL-CFC) with proliferative capacity in vitro. AL-CFC have been recognized as more radioresistant (9) and lass sensitive to hydrocortisone acetate than hemopoietic macrophage progenitors (10)  the significance ofAL-CFC in the maintenance or self-renewal of PAM in the lung. The reduction of PAM recovered from asbestosor silica-exposed animals reflects the impairment of PAM to sustain their own stem cells. After instillation of asbestos or silica, AL-CFC, a putative stem cell population, was depleted to 10% or less of the normal level, and this could account for the observed decreased in numbers ofPAM. Because ofthe long turnover time (11), the population size of PAM will decline after losing the capacity for clonal growth. On the contrary, TiO2 particles failed to induce a significant reduction in numbers of PAM and in the plating efficiency of AL-CFC, although a high proportion of PAM had phagocytized particles.
After the exposure to TiO2 particles in vitro, AL-CFC were detected with almost equal plating efficiency as in the untreated controls, although silica and asbestos depressed the plating efficiency ofAL-CFC. These results may indicate the toxicity of fibrogenic mineral dust particles on the clonal growth of PAM, although both the specific toxic mechanism and how such acutephase damage is related to late-developing fibrogenesis is unknown. In conclusion, toxic effects of fibrogenic mineral dusts on AL-CFC may lead to an impairment of PAM to function in the clearance ofparticles from the lung and in subsequent pathologic processes.