Different particle determinants induce apoptosis and cytokine release in primary alveolar macrophage cultures

Abstract

Background: Particles are known to induce both cytokine release (MIP-2, TNF-alpha), a reduction in cell viability and an increased apoptosis in alveolar macrophages. To examine whether these responses are triggered by the same particle determinants, alveolar macrophages were exposed in vitro to mineral particles of different physical-chemical properties.

Results: The crystalline particles of the different stone types mylonite, gabbro, basalt, feldspar, quartz, hornfels and fine grain syenite porphyr (porphyr), with a relatively equal size distribution (< or = 10 microm), but different chemical/mineral composition, all induced low and relatively similar levels of apoptosis. In contrast, mylonite and gabbro induced a marked MIP-2 response compared to the other particles. For particles of smaller size, quartz (< or = 2 microm) seemed to induce a somewhat stronger apoptotic response than even smaller quartz (< or = 0.5 microm) and larger quartz (< or = 10 microm) in relation to surface area, and was more potent than hornfels and porphyr (< or = 2 microm). The reduction in cell viability induced by quartz of the different sizes was roughly similar when adjusted to surface area. With respect to cytokines, the release was more marked after exposure to quartz < or = 0.5 microm than to quartz < or = 2 microm and < or = 10 microm. Furthermore, hornfels (< or = 2 microm) was more potent than the corresponding hornfels (< or = 10 microm) and quartz (< or = 2 microm) to induce cytokine responses. Pre-treatment of hornfels and quartz particles < or = 2 microm with aluminium lactate, to diminish the surface reactivity, did significantly reduce the MIP-2 response to hornfels. In contrast, the apoptotic responses to the particles were not affected.

Conclusion: These results indicate that different determinants of mineral/stone particles are critical for inducing cytokine responses, reduction in cell viability and apoptosis in alveolar macrophages. The data suggest that the particle surface reactivity was critical for cytokine responses, but contributed less to cell death for the types of particles tested. The size-dependent variations, specially in cytokine release, seem not to be explained only by particle surface area.

Figure 1

MIP-2 response and cell death in rat alveolar macrophages after exposure to quartz particles of different sizes and to amorphous microsilica. The macrophages were exposed to quartz (≤ 0.5 μm, ≤ 2.0 μm, ≤ 10 μm) and microsilica (≤ 0.3 μm) for 20 h as described in Materials and Methods. A, B: MIP-2, concentration-effect curve. C, D: Cell viability, concentration-effect curve. E, F: Apoptosis, concentration-effect curve. In A, C, E the data are related to the mass of the particles, and in B, D, F to the surface area of the particles. The MIP-2 responses were analysed by ELISA. The cell viability was measured by exclusion of PI in a fluorescence microscope. The apoptosis was analysed by flow cytometry after staining by Hoechst 33258. The results represent the mean +/- SEM of three independent experiments.

Figure 2

DNA-fragmentation induced by quartz of different sizes and concentrations. The macrophages were exposed to various quartz concentrations for 20 h. The DNA-fragmentation was measured as described in the Materials and Methods. The results represent a typical of three experiments.

Figure 3

MIP-2 response and apoptosis induced by various concentrations of stone particles. The macrophages were exposed to mylonite, gabbro, feldspar, basalt and quartz (≤ 10 μm) for 20 h. A, B) show MIP-2 responses and C, D) Apoptosis. In A and C the MIP-2 responses and apoptosis, respectively, were related to the mass of the particles, and in B and D to the surface areas of the particles. The MIP-2 release was analysed by ELISA, and apoptosis by flow cytometry after Hoechst 33258 staining. The results represent the mean +/- SEM of three independent experiments.

Figure 4

Cytokine responses and cell death induced by hornfels and porphyr particles of different sizes: Comparison to quartz. A) TNF-α responses, B) MIP-2 responses, C) Cell viability and D) Apoptosis. The macrophages were exposed to ≤ 2.0 μm and ≤ 10 μm particles of porphyr, hornfels and quartz for 8 h (TNF-α) or 20 h (MIP-2, viability and apoptosis). The cytokine release was analysed by ELISA, cell viability by the ability to exclude PI as assessed by fluoroscense microscopy and apoptosis by flow cytometry after Hoechst 33258 staining. The data are related to the surface area of the particles. The results represent the mean +/- SEM of three independent experiments.

Figure 5

The effect of surface modification of particles on MIP-2 release, TNF-α release and apoptosis. A) MIP-2 release, B) TNF-α release and C) Apoptosis. Hornfels-2 and quartz-2 particles were treated in the absence or presence of aluminium lactate as described in Materials and Methods, and thereafter added to the cultured macrophages for 20 h. The MIP-2 and TNF-α release were analysed by ELISA, and apoptosis by flow cytometry after Hoechst 333258 staining. The results represent the mean +/- SEM of 3–5 independent experiments. *Significant reduction in MIP-2 release in aluminium lactate-coated versus non-coated hornfels-2 particles (p ≤ 0.05).

Figure 6

Relationship between the particles abilities to induce MIP-2 release and apoptosis. The figure displays the relative abilities of all the tested particles to induce MIP-2 release plotted against their abilities to induce apoptosis. The values are based on AUCs of relative increases in particle-induced MIP-2 release and apoptosis from the concentration-effect curves in Figs. 1A, 1E, 3A, 3C, 4B and 4D (concentration range: 0–600 μg/ml). Each point depicts the mean of 3–7 independent experiments. The figure also displays the result from a correlation analysis of the relative abilities to induce MIP-2 release versus apoptosis, as well as the linear regression line for the same data set with 95% confidence intervals.