Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium
© Möller et al; licensee BioMed Central Ltd. 2005
Received: 17 May 2005
Accepted: 04 October 2005
Published: 04 October 2005
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.
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 μ g 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.
Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μ g/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.
KeywordsUltrafine particles cytoskeleton stiffness viscoelasticity phagosome transport relaxation intracellular calcium
Epidemiological studies suggest increased health risks (decreased lung function, increased morbidity and mortality) after exposure to environmental particles [1, 2]. Recent epidemiological studies show, that not only the mass of inhaled urban particles (i.e. PM10 or PM2.5 ≡ mass of all particles being smaller than 10 μ m or 2.5 μ m, respectively) is associated with increased morbidity and mortality, but also the number of particles with even higher significance [3, 4]. Because of the smaller mass of ultrafine (diameter < 100 nm) compared to fine particles, they contribute little to the total mass, but are important in the overall number of particles. Most urban particles result from combustion processes; therefore the dominant fraction contains ultrafine carbonaceous particles. These particles are less soluble and therefore can reside in the lung for longer times. Larger particles and bacteria are phagocytized by alveolar and airway macrophages and dendritic cells within hours and further digested, keeping the lung surface in a sterile condition (under physiological conditions). UFP are not only phagocytosed by alveolar macrophages, but can enter epithelial cells, can penetrate into the circulation, being further redistributed to other organs of the body . Therefore, UFP can reach sites in the body far away from the site of entrance, where they can cause inflammatory reactions, in contrast to larger particles [6, 7]. With decreasing particle size the particle surface increases in relation to the particle volume or particle mass. Particles forming aggregates being composed of subunits of much smaller size can further enhance their surface area in relation to the mass (specific surface area). Because of their high specific surface area, UFP can catalyze chemical reactions, which may induce chronic inflammation. Animal studies and in vitro cell studies have shown that high concentrations of ultrafine particles can induce inflammatory processes and increased calcium transients [8–13], can induce the production of reactive oxygen species (ROS, like H2O2, O2 -) [14–16], can induce oxidation of cytoskeletal proteins [17, 18] and cytoskeletal dysfunctions [19, 20], or can act as vehicles to transport toxic gasses and substances to the lung periphery .
Phagocytic cells in the lung, such as macrophages, polymorphonuclear leukocytes and dendritic cells play a key-role in the defense-reaction. They ingest foreign materials, digest bacteria and viruses, and present antigens in order to trigger specific (immunological) defense mechanisms [22–24]. Macrophages reside in the alveoli and chemotactic gradients direct the macrophages within minutes to the site of particle deposition. During phagocytosis the particles are incorporated into a membranous vesicle and ingested into intracellular phagosomes, which fuse with lysosomes . Phagolysosomes are acidic (pH ≈ 5) and contain reactive oxygen species (H2O2) [26, 27]. They are the site where most bacteria and fungi are digested. Non-digestible particles are retained in the lung for longer periods of time, for example iron oxide tracer particles have a clearance half-time of 120 days in healthy non-smoking subjects . UFP are not only engulfed by phagocytosis and therefore must not necessarily reside in phagolysosomes, which may imply much longer residence times in the lung. The cytoskeleton of the AM is crucially involved in the phagocytic defense reactions, including locomotion and cell migration, phagocytosis, intracellular transport, phagosome-lysosome fusion and signal transduction [29–31]. The cytoskeleton consists of three different filamentous structures. Microfilaments (actin) are involved in dynamic processes of the cell, like crawling and phagocytosis. Microtubuli are involved in cell shape and intracellular vesicle transport. Intermediate filaments contribute to the static part of the cytoskeleton. Every filamentous structure has its own family of motor proteins being responsible for the transport of molecules and vesicles . Those transport processes require energy in the form of ATP [33, 34]. The cytoskeleton is sensitive to ROS and oxidative stress, due to the presence of thiol groups located on the actin microfilaments which are sensitive to oxidation, leading to cross linking and reduced motility [18, 35], and the cytoskeletal dynamics is regulated by cytoplasmic calcium.
We have developed protocols to study cytoskeleton associated functions in macrophages in vivo and in vitro, such as phagosome transport, mechanical integrity (viscoelastic properties, stiffness) and phagocytosis, using ferromagnetic microparticles [36–38]. In in vitro studies the magnetic microparticles (1.8 μ m diameter) are incubated for 24 hours together with cultivated macrophages, after which more than 95% are ingested. The particles are then magnetized and aligned in a short magnetic field pulse, and can be detected by a magnetic field sensor. Intracellular phagosome transport causes stochastic disorientations of the particles, which results in a decay of the remanent magnetic field of the cell probe (relaxation). In addition mechanical viscoelastic properties of the cytoskeleton can be investigated by twisting the micromagnets in a weak magnetic field. This method is called 'Magnetic Twisting Cytometry' (MTC) [39, 40] and has been used to investigate the role of the different cytoskeletal structures in macrophage function after co-incubation with cytoskeletal drugs (Cytochalasin D, Nocodazole ). Additionally it has been shown that MTC can monitor cytotoxicity of GaAs-particles in vivo in lung macrophages of animals [41, 42] and in vitro caused by ultrafine particles .
In this study, we have investigated the role of intracellular calcium transients produced by environmentally relevant fine and ultrafine model particles in the induction of adverse reactions on the cytoskeleton of macrophages, using the MTC technique. Cultivated macrophages from the cell line J774A.1 were incubated with increasing amounts of particles. Intracellular phagosome transport (relaxation), stiffness of the cytoskeleton and cell viability was recorded. Particles on the basis of carbon black (CB) having different chemical compositions and impurities, and a wide range of diameters (12 nm - 1.5 μ m) and specific surface areas (≈ 1 m2/g - 600 m2/g) were tested.
Materials and methods
J774A.1 macrophages originate from a BALB/c/NIH mouse  and were obtained from the German Collection of Animal Cell Cultures (Tumorbank, DKFZ Heidelberg, Germany). Cells were grown in RPMI 1640 Medium (Sigma, Taufkirchen, Germany), supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin, 100 μ g/ml streptomycin, 2.5 μ g/ml amphotericin and 0.3 g/L-glutamine in NaHCO3. The cell culture grows with a doubling time of 2 days and was sub-cultured every 4 days. In cytotoxicity studies cells were incubated with particles and with Calcium agonists in serum-free medium. In addition cells were incubated with 5% FCS to test the possible cytotoxic effect of lack of serum in the medium.
Magnetic particle binding assay
A number of 0.2 million macrophages were incubated with 10 μ g of 1.8 μ m spherical ferromagnetic microparticles in glass vials (12 mm outer diameter) for 24 hours prior to adding calcium antagonists or ultrafine particles. This ensured the adherence of the macrophages and more than 95% phagocytosis of the magnetic beads. Before the addition of UFP or calcium antagonists, non-adherent cells and free particles were removed by washing with medium. Ferromagnetic 1.8 μ m magnetite microparticles (beads) were prepared with narrow size variation (geometric standard deviation < 1.1) and spherical shape , which is important for data analysis using mathematical models to estimate the viscous and elastic properties of the cytoskeleton. The particles were not further coated in order to avoid the activation of specific cell surface receptors. It is reported that the non-specific Scavenger-receptor mediates phagocytosis of the magnetite particles .
Ultrafine test-particles and calcium modulating drugs
Fine and ultrafine test particles used in the study together with their physical properties, such as count median diameter and specific surface area. Elemental carbon particles (EC90) were prepared in own laboratory (GSF-IHB, ).
Specific surface area
Printex90 (carbon, P90)
Elemental carbon (EC90)
Diesel exhaust particles (DEP)
Urban dust (UD)
1.5 μ m
≈ 1 m2/g++
Particles were incubated at concentrations of 100 and 320 μ g/ml/million cells for 4 h in serum-free medium, or in combination with calcium antagonists, such as Verapamil (100 μ M, Ca2+ channel blocker), BAPTA-AM (20 μ M, Ca2+ chelator), or W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride, 25 μ M, calmodulin inhibitor). The concentration of the Ca-antagonists was obtained from previous studies and tested for non-toxic responses in own preliminary studies. The antioxidant Nacystelin (NAL, 200 μ M) was also included. Nacystelin (NAL), a thiol antioxidant compound that is a lysinated derivative of N-acetyl cysteine (NAC), possesses potent mucolytics capacities and has been shown to inhibit reactive oxygen species effects . It has the advantage of having a neutral pH compared with NAC, which is acidic, and thus can be administered to the airways without the local airway irritation that occurs with NAC. Additionally the role of serum (FCS, 5%) or bovine serum albumin (BSA, 1%) in the medium was investigated. In some experiments long-term incubation (24 hours) was carried out to compare with previous data.
Cell viability was tested by the propidium iodide (PI) exclusion test. Necrotic cells allow the penetration of PI into the cell and the nucleus, where the fluorescent dye can be visualized. After 10 min PI incubation with adherent cells, the probes were analyzed by fluorescence microscopy, using an inverted microscope (Axiovert 25, Zeiss GmbH, Jena, Germany) together with a mercury-arc lamp and a filter system for the detection of propidium iodide (PI) dye and a digital camera system (CoolSnap-pro, Mediacybernetics Inc., Silver Spring, MD, USA). Digital images were recorded and processed using Image Pro software package (Mediacybernetics Inc., Silver Spring, MD, USA). For every probe a transmission image was recorded as well as a fluorescence image. The transmission image was used to count the total number of cells in a region of interest (ROI), defined by a grid overlay and covering about 80–100% of the image. Each ROI covered at least 100 cells. In the second fluorescence image the number of PI positive cells was analyzed in the same ROI, allowing the fraction of dead cells (PI+) to be assessed.
Magnetic twisting cytometry
Stochastic phagosome motion (relaxation)
Magnetic phagosome twisting
Application of a weak magnetic field BTW induces twisting of a magnetic dipole particle and allows investigating cytoplasmic rheology and mechanical integrity [36, 53]. Particles being suspended in a viscosity η rotate in an external twisting force according to Newton's law:
where dθ/dt is the shear rate and σ is the applied shear stress ( , M = remanent magnetization of particles, κ = rotational shape factor). In case of elasticity the applied stress is proportional to the elastic deformation (strain θ) and we get with the elasticity modulus ν Hook's law:
σ = νθ (2)
Strain was estimated from the measurement of the cell field B(t) according to θ(t) = arccos B(t)/B 0. First the particles were magnetized and aligned parallel to the field sensors (Figure 2B). After 20 sec relaxation the twisting field was applied for 10 sec duration and viscoelastic recoil was recorded for another 3 minutes. Viscoelastic recoil does not force the dipoles back to the undisturbed relaxation curve, indication of viscous shear, which can reflect a permanent deformation (break of filamentous interactions) of the cytoskeletal structure. Figure 2B shows the change of particle alignment during magnetic field twisting together with the elastic recoil in comparison to a relaxation curve recorded without any external forces to the magnetic beads. Permanent deformation is characterized by the difference in complete elastic recoil and undisturbed relaxation, as illustrated in Figure 2B. Cell stiffness was estimated as the ratio between mean stress and strain after a constant twisting duration of 10 sec. This analysis of particle twisting does not view specific viscous or elastic properties. Therefore this parameter provides an integral description of the cytoskeletal mechanical properties.
Influence of UFP on phagosome motion and on magnetic particle twisting
Summary of effects of 100 μ g particles/ml/million cells on phagosome transport (relaxation) together with effect of co-incubation with different Ca-modulating drugs (100 μ M Verapamil, 20 μ M BAPTA-AM or 25 μ M W-7) as well as medium with serum (wFCS), with bovine serum albumin (BSA), or with 200 μ M Nacystelin (NAL); inhib. – inhibition, n.e. – no effect, accel. – acceleration; N = 5; **: p < 0.01, *: p < 0.05.
Summary of effects of 100 μ g particles/ml/million cells on cytoskeletal stiffness together with effect of co-incubation with different Ca-modulating drugs (100 μ M Verapamil, 20 μ M BAPTA-AM or 25 μ M W-7) as well as medium with serum (wFCS), with bovine serum albumin (BSA), or with 200 μ M Nacystelin (NAL); stiff. – stiffening, n.e. – no effect; N = 5; **: p < 0.01, *: p < 0.05.
Influence of serum, BSA and Nacystelin on phagosome motion and on magnetic particle twisting
Figure 4 shows the results of serum and of BSA on phagosome transport and on cytoskeletal stiffness after co-culture with P90 and EC90 (summary in Table 2 and Table 3). Both, 5% FCS and 1% BSA are able to inhibit the particle induced retardation of relaxation (Figure 4A) and the increase in cell stiffness (Figure 4B). The antioxidant Nacystelin does not inhibit the particle induced cytoskeletal dysfunctions. Serum, BSA and Nacystelin do not influence any of the effects of DEP or UD, therefore, the data are not shown.
Influence on cell viability
Discussion and conclusion
Necrosis caused by UFP
Figure 5 shows both the cytotoxic potential of the particles in inducing cell necrosis used in the study, together with the Ca-modulating antagonists, particularly after 4 hours incubation time. The ultrafine carbon black particles (P90 and EC90) had the most significant effect on cell viability, with a doubling of the fraction of dead cells after 4 hours and a triplication after 24 hours. EC90 particles caused more dead cells due to the higher specific surface area compared to P90. FCS and BSA both reduce the cytotoxic potential of P90 and EC90. In the presence of FCS or BSA, the particle surface may become coated with proteins, which may shield reactive sites on the particle surface and reduced the potential of causative oxidative reactions. Although serum free medium allows clearer experimental conditions in case of receptor dependent responses, the serum co-culture may be closer to physiological conditions in the lung, where surfactant coats and opsonizes the particles. On the other hand the inhibitory effects of the Ca-antagonists would not have been seen with the presence of serum in the medium. Long-term incubation of J774A.1 macrophages without serum induced a decrease in cell viability, showing that this by itself is a non-physiological condition. Interestingly, cell viability could be enhanced by the presence of serum or BSA in the medium. But for short time periods (4 hours), the absence of serum did not injure the cells.
DEP and UD produce less cytotoxicity compared to the pure carbon black particles, despite other reactive material may be present, like transition metals, and adsorbed organic substances. One reason for the lower cytotoxicity of DEP and UD may be the lower specific surface area. In a previous study we could show cytoskeletal dysfunctions caused by DEP and UD after 24 hours incubation time . In this study different incubation conditions were used, where particles were dispersed in medium containing serum. It was shown that the particles form larger aggregates without serum proteins in the medium, causing particle clusters in the μ m-size range . Such clusters are subject to different mechanisms of uptake and processing by the cells (phagocytosis versus endocytosis for smaller particles), although both are actin based mechanism. Phagocytosed materials are processed via the lysosomal digestion route, which is not the case after endocytosis. In addition the specific surface area is smaller for aggregated particles. It was also shown that organics adsorbed to the particle surface can lower the specific surface area, can shield reactive sites; therefore make the particles less toxic than plain particles . We suggest here that the organic substances are stable when bound to the surface, in conjunction with low concentrations in the medium. The bound organics on the particles may cause a surface coating, which can shield reactive sites of the carbonaceous core.
Influence of UFP on phagosome motion and on magnetic particle twisting
MTC studies investigate the transport of micron-sized phagosomes from two different perspectives. Relaxation directly monitors the coupling dynamics between phagosomes and the cytoskeleton  while phagosome twisting examines the mechanical integrity (viscoelastic properties, stiffness) of the cytoskeletal filaments, which are linked to the phagosomes . Phagosome transport requires an intact cytoskeleton, intact phagosomes including motor proteins and energy (ATP) [33, 57]. Additionally, intracellular calcium plays an important role for cytoskeletal functions and intracellular signaling in relation to immunological and non-immunological defense reactions [58, 59]. Cytotoxic reactions can involve the energy metabolism of the cell, intracellular calcium transients , the dynamics of cytoskeletal filaments and the transport mechanisms of phagosomes [41, 42]. A previous study has shown that destroying microfilaments by cytochalasin D resulted in a retarded relaxation and an increased stiffness [37, 53]. Colchicine, which disrupts microtubuli, results in an accelerated relaxation and in a moderately increased stiffness. Figure 2A illustrates the retardation of relaxation caused by 100 μ g/ml P90, and the modulation by Verapamil and BAPTA-AM together with the retardation produced by 4 μ M Cytochalasin D. In comparison to the effects induced by the cytoskeletal drugs the dysfunctions produced by the carbonaceous UFP suggests destructions of microfilamentous structures and hence dysfunctions of the cytoskeleton. This is also in agreement with other studies which showed impairment of the phagocytic capacity (an actin based mechanism) after incubation with different types of UFP [19, 20].
Intracellular calcium plays a central role in the modulation of the defense mechanisms causing inflammation. Further studies have shown that ufCB causes a transient increase of intracellular calcium, which can be inhibited by the calcium channel blocker Verapamil [13, 60]. This was reflected in this study by the inhibition of ufCB induced cytoskeletal dysfunctions in the presence of Verapamil. Interestingly, the intracellular calcium chelator BAPTA-AM did not induce the expected suppression of cytoskeletal dysfunction. The reason for this lack of effect is not clear but suggests that not only the level of intracellular calcium is responsible for the ufCB induced dysfunctions.
Calmodulin (CaM) is a ubiquitous calcium binding protein that can bind to and regulate a multitude of different protein targets by affecting many different cellular functions. CaM mediates processes such as inflammation, metabolism, apoptosis muscle contraction and cellular movement. It has been shown that calmodulin has a direct impact on actin polymerization [61–63], acto-myosin binding and cross linking [64, 65]. CaM is thought to activate the myosin light chain kinase (MLCK) and CaM kinase II by displacement of their auto inhibitory domains. Many of the proteins that bind CaM are unable to bind calcium themselves and as such use CaM as a calcium sensor and signal transducer. The fact that the CaM-inhibitor W-7 can suppress most of the cytotoxic reactions caused by ufCB co-culture shows that the calcium dependent signaling pathway is crucial for the cytotoxic effect and cytoskeletal dysfunctions. The lack of suppression after co-culture with EC90 particles may reflect their higher specific surface area.
The antioxidant Nacystelin did not inhibit the UFP induced dysfunctions of the cytoskeleton. Oxygen radicals cause an oxidation and depletion of cytoskeletal proteins (thiols), disruption of actin filaments and the inhibition of F-actin formation, and an actin cross linking . The actin system is the most sensitive constituent of the cytoskeleton to the oxidant attack. Nacystelin can inhibit the thiol oxidation , but did not result in a suppression of UFP induced cytoskeletal dysfunctions. This shows that the thiol oxidation may not be the only response to UFP exposure (besides the modulation of the calcium metabolism), but the cross linking of actin filaments primarily may describe the retarded relaxation and the increased stiffness of the cytoskeleton.
The specific surface area of the particles is discussed to be a significant parameter relating to cytotoxic responses of ultrafine particles [16, 67]. Our studies in part support this hypothesis. Cell viability seems to correlate with the specific surface area of the particles, where EC90 induces more dead cells compared to P90 or to UD and DEP. Impairment of relaxation and stiffening of the cytoskeleton seem not to depend on the specific surface area, but W-7 could inhibit the particle induced impairments in P90, but not in EC90, which has the higher specific surface area. Besides the different surface characteristics of DEP and UD compared to EC90 and P90, the lack of cytoskeletal dysfunctions of these materials seems to be in part induced by the smaller specific surface area.
List of abbreviations
- UFP :
- ufCB :
ultrafine carbon black
- PM2.5 :
particle mass with aerodynamic diameter <= 2.5 μ m
elemental carbon particles, 90 nm mobility diameter
commercial carbon particles, Printex 90 (Degussa)
- DEP :
diesel exhaust particles
- UD :
(N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride, 25 μ M, calmodulin inhibitor
- CaM :
- BAPTA-AM :
- Verap :
- BSA :
bovine serum albumin
- FCS :
fetal calf serum
- ATP :
- PI :
- ROI :
region of interest
- MTC :
magnetic twisting cytometry
- RMF :
remanent magnetic field of cell/magnetic particle probe
- b1 = B(1 min)B0 RMF :
after 1 min relaxation
- b5 = B(5 min)B0 RMF :
after 1 min relaxation
This work was supported by the CEC under contract FIGD-CT-2000-00053.
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