Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

Abstract

Background: Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter). Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP) can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis.

Methods: In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively), such as elemental carbon (EC90), commercial carbon (Printex 90), diesel particulate matter (DEP) and urban dust (UD), were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 microg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively.

Results: Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 microg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA) suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only.

Figure 1

Magnetic twisting device to measure relaxation and twisting (10 sec) of aligned ferromagnetic microparticles ingested by macrophages and detection by an array of magnetic fluxgate sensors (Förster devices, Förster GmbH, Reutlingen, Germany).

Figure 2

Measurement of stochastic intracellular phagosome transport (relaxation, A) and cytoskeletal stiffness (B) by Magnetic Twisting Cytometry (MTC). Control and cytochalasin D (CyD) probes do not contain UFP. Other probes show co-incubation with 100 μg/ml P90 without serum (w/o FCS) and with 100 μM Verapamil or 20 μM BAPTA-AM for 4 hours.

Figure 3

Relaxation of ingested magnetic particles (A, relative decay after 5 min, b5, normalized to control probes without particles) and mechanical integrity (B, stiffness, mean between low and high stress) of J774A.1 macrophages after 4 h incubation with different types of 100 μg ultrafine particles/ml/million cells under different incubation conditions, such as without serum (w/o FCS), 100 μM Verapamil, 20 μM BAPTA-AM or 25 μM W-7 (normalized values +/- SD; N = 5; **: p < 0.01, *: p < 0.05).

Figure 4

Relaxation of ingested magnetic particles (A, relative decay after 5 min, b5, normalized to control probes w/o particles) and mechanical integrity (B, stiffness, mean between low and high stress) of J774A.1 macrophages after 4 h incubation with different types of 100 μg ultrafine particles/ml/million cells under different incubation conditions, such as without (w/o) and with 5% serum (wFCS), 1% bovine serum albumin (BSA) or 200 μM Nacystelin (NAL); normalized values +/- SD; N = 5; **: p < 0.01, *: p < 0.05.

Figure 5

Cell viability (PI exclusion) of J774A.1 macrophages after 4 hours of incubation without (control) and with 320 μg UFP/ml/million under different incubation conditions. A): without serum (w/oFCS), 100 μM Verapamil, 20 μM BAPTA-AM and 25 μM W-7. B): w/oFCS, with 5% serum (wFCS), 1% bovine serum albumin (BSA) or 200 μM Nacystelin (NAL); mean +/- SD, **: p < 0.01, *: p < 0.05.