Macrophage functions after exposure to nonfibrous mineral dusts.

The effects of the standard dusts, electrocorundum, and Dorentruper quartz (DQ12), as well as mine dusts have been tested in guinea pig lung macrophage cultures. The parameters compared were: release of lactic dehydrogenase for demonstration of plasma membrane permeability and production of lactic acid as an indicator of carbohydrate metabolism. In addition to the dose-dependent toxicity of different mine, coal and other mineral dusts, we studied the influence of cell culture media and the supplement of fetal calf serum (FCS) and bovine serum albumin (BSA) upon the cell toxicity in this test system. We demonstrated a protective effect of FCS and BSA on dusts of low and medium toxicity, while dusts of high toxicity, like DQ12, were not influenced in their toxicity.


Introduction
Lung macrophages play an important role in the pathogenesis of inflammation (1) and the development of fibrosis (2) after the inhalation of dusts. The exact mechanism of diseases caused by mixed dusts like mine dusts is still obscure. It seems to be clear that quartz and other forms of crystalline silica are significant in the evolution of pneumoconiosis. However, Seemayer et al. (3) demonstrated that quartz alone, in the mass concentrations usually found in coal mine dusts, exerts no detectable cytotoxic effects on guinea pig alveolar macrophages in vitro. Furthermore, Schlipko6ter et al. (4) demonstrated a protective effect of anthracite against the cytotoxic potential of quartz in the in vitro system. In the past, differing results were obtained using macrophages from different sources (3,(5)(6)(7)(8)(9)(10)(11) and diverse parameters and culture conditions.

Cell Cultures of Alveolar Macrophages
Unstimulated guinea pig and rat lung macrophages were obtained by pulmonary lavage with heparinized (8 U/mL) Ca2+and Mg+free phosphate buffered saline (PBS) (5). The resulting cell suspension was washed three times in heparin-free PBS, *Hygiene-Institute for the Justus-Liebig University, Giessen, Federal Republic of Germany. adjusted to 1.0 x 106 cells/mL and transferred into the cavities of multiwell plates (Falcon-Plastics No. 3008) in Minimum Essential Medium (MEM). After 2 hr, after cell attachment at 37°C in 5% COP5°/ air, the MEM was removed, and the dusts were applied in different media with and without fetal calf serum (FCS) and bovine serum albumin (BSA) in different concentrations.
Lactic dehydrogenase and lactic acid were determined as described elsewhere (12).

Coal Mine and Control Dusts
Coal mine dust samples of four different coal mines in North-Rhine-Westfalia (NRW) and two dusts from the Sarre (S) mining area of FRG were tested. The physicochemical data of these dusts are listed in Table 1l (13). Dorentruper quartz (DQt2) and electrocorundum with similar size distributions served as control dusts.
After weighing and sterilizing all dusts, samples were suspended in the suitable medium by ultrasound and applied in a concentration of 100 pg/106 cells after cell attachment.
Results and Discussion Table 2 shows lactic dehydrogenase (LDH) release and lactic acid production of guinea pig lung macrophages 20 hr after exposure to 100 pg dust/106 cells in the presence of 5% FCS, 0.25°h BSA and without any supplement. After incubation without addition of FCS of BSA, the results demonstrate a wide spectrum of toxicity in comparison to electrocorundum and DQ,2, as measured by LDH release. No correlation between quartz content and cytotoxicity can be observed. In the case of lactic acid production, no great differences can be observed. In two samples which are not toxic with regard to LDH release, the lactic acid production is even enhanced in comparison to the corundum control.
In the presence of 5%/ FCS and 0.25%/ BSA, only two of the six dusts which are toxic in the absence of any supplement exhibit a slight toxicity, i.e., increased LDH release when cultured with O.250/ BSA. Lactic acid production is not influenced by any of these dust samples, while the two nontoxic samples even enhanced carbohydrate metabolism. Table 3. It demonstrates that an incubation time of 44 hr in the presence of FCS is not sufficient for the cells to abolish the protective affect of macromolecules in comparison to the exposure without FCS. When decreasing the concentration of FCS to 3.2 or 1% only dust V220 exhibits an increased toxicity (Fig. 1 In further experiments we tested the influence of dust concentration. At the higher concentrations of 200 and 300 pg/106 cells in the presence of 1% serum, there is as slight increase of toxicity with respect to LDH (Fig. 2) and lactic acid (Fig. 3). 321 321 *.FCS H621 H520 321 32-1 32 1 0120 N320 V220 In further investigations we tested lower concentrations of FCS and BSA, and, with dusts showing a significant toxicity in serum-free medium, we found a FCS and BSA dose-dependent protective effect (Figs. 4 and 5). To test the possibility that macro-phages are able to abolish this protective effect, we incubated rat lung macrophages in the presence of three different dosages of FCS over a period of 40 hr (Fig. 6). The protective effect of 0. 5   The results show that small quantities of protein molecules, like serum or BSA, have a considerable protective effect on the toxicity of coal mine dusts, while the toxicity of the control dust DQ12 is not or only slightly influenced by these protein concentrations; with serum concentrations of 30-40%, however (not demonstrated here), a significant decrease of DQ12 toxicity can be observed. Under these circumstances it appears important to use protein-free medium for the testing of coal mine dusts when employing long incubation times.

The influence of incubation time under these standard conditions is documented in
Earlier investigations (12) have demonstrated that the presence of Bactopepton with a molecular weight between 900 and 9000 in McCoy's 5 A Medium (Seromed, Munich, FRG) does not influence the toxicity of coal mine dusts in comparison to Minimum Essential Medium (MEM) and Medium NCTC 109. The fact that silica, such as DQ,2, is not influenced by proteins may be attributed to the effect of the large free silica surface in contrast to the natural coal mine dusts.
In the future it may be possible that the interpretation of the influence of proteins with different molecular weights on the toxicity of coal mine dusts gives further information about free silica or other toxic surfaces. In this context, the possible alteration of the surface in the course of dust preparation, e.g., the effect of sterilization and ultrasound treatment, should also be discussed and investigated.