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Reference: Ultrafine Particles Cause Cytoskeletal Dysfunctions in Macrophages. Mőller, W., Hofer, T., Siesenis, A., Karg, E., and Heyder. J. Toxicol. Appl. Pharmacol. 182, 197-207. 2002.

· Alveolar macrophages:

o Alveolar macrophages (AM) were obtained from beagles by bronchoalveolar lavage (BAL). The AM were isolated by routine methods. Total cell numbers, cell differentials, and cell viability were calculated. AM were incubated in supplemented buffered media during UFP treatment for 24 h.

o J774A.1 macrophages, a mouse AM line from a BALB/c/NIH mouse, were incubated in the same medium used above for AM from dogs.

· Magnetic particle binding assay:

o AM and spherical ferromagnetic microparticles were incubated together for 24 h before the addition of of FP or any drugs. This time period allowed phagocytosis of most of the magnetic beads and adherence of the cells.

· Cytoskeletal drugs and ultrafine test particles:

o Microfilaments were disrupted with Cytochalasin D, known to inhibit AM phagocytosis.

o Microtubuli were disrupted with Colchicine or Nocodazole.

o Particles used were:

§ Fine TiO₂ (220 nm diameter, 6m²/g surface area

§ Ultrafine TiO₂ (20 nm diameter, 48 m²/g surface area

§ Ultrafine carbon black (51 nm diameter, 30²m/g)

§ Ultrafine carbon black (12 nm, 30²m/g)

§ Ultrafine elemental carbon (90 nm, 600²m/g)

§ Diesel exhaust particles (DEP) (120 nm, 108²m/g)

§ Urban dust (? Diameter,? surface area)

Particles and macrophages were incubated together in various concentrations for times up to one day. The organic components of diesel exhaust and urban dust were extracted to study their role.

· The effect of UFP’s on the AM cytoskeleton was in part determined by measurement of such mechanical cell properties as cytoplasmic twisting, relaxation, phagosome twisting and cell stiffness. This are only briefly described below and can be described mathematically:

o Magnetic twisting cytometry: This was used to measure relaxation and mechanical cell properties after macrophages ingested magnetic particles.

o Stocastic phagosome motion or relaxation: This is a result of phagosomes moving along microfilaments in the cytoskeleton with energy provided by ATP. This is part of the normal intracellular transport system. Motor proteins couple phagosomes to the filaments. The relaxation process involved an two phase exponential decay.

o Magnetic phagosome twisting: When magnetic particles are aligned by a magnetic field, the mechanical characteristics of the cytoplasm can be defined. A term defined as "cell stiffness" was considered to be an integral description of cytoskeletal mechanics.

· Cell proliferation and capacity for phagocytosis are both important functions of macrophages for proper defense of the respiratory system. Changes in these functions after UFP treatment indicate UFP toxicity. Phagocytosis was measured by the uptake of 1.0-um fluorescent diameter latex beads. Flow cytometry was used to determine the number of beads per cell as based on the fluorescence spectrum. Changes in cell proliferation were measured with J774A.1 cells. A decrease in cell concentration indicated a decrease in cell proliferation from UFP treatment.

· Changes in cell necrosis and apoptosis were also measured after UFP treatment as indicators of toxicity. Apoptotic cells could be differentiated from necrotic cells with two-wavelength flow cytometry and dyes. Fluorescence microscopy was used for transmission images of cells to be classified as apoptotic or necrotic or both.

· All UFP drug measurements were repeated with 5 different probes and separate controls.

· Phagosome motion and magnetic particle twisting in J774A.1 macrophages:

o 24 hours of incubation of J774A.1 from the macrophage cell line with UFPs at the lowest concentration used (32 ug/ml) caused no effect on either measurement of cytoskeletal function above.

o EC90, DEP and urban dust caused the cytoskeleton to become thicker and relaxation to become slower.

o Relaxation impairment is decreased and cytoskeletal stiffness is increased again when urban dust and DEP are washed in dichloromethane (DC1M).

· Phagosome motion and magnetic particle twisting in beagle dog macrophages:

o There does not to be an effect on the parameters measured here after 24 h incubation of BD-AM with particles at the lowest concentration of particles used.

o Ultrafine TiO₂ resulted in cytoskeletal stiffness proportional to particle concentration.

o The most impressive increase in relaxation was proportional to the EC90 particles concentration. There was a loss of cell adherence maximal after incubation with the highest concentration of EC90 particles.

o Elemental carbon was associated with increased relaxation compared to carbon black.

o All types of fine particles and UFP incubated with beagle dog macrophages demonstrate increased relaxation with increased particle surface area.

· Cell proliferation and phagocytosis related to UFPs:

o Urban dust decreases cell proliferation the most in the macrophage cell line. UFP, ufTiO₂, fTiO₂ also decrease cell division.

o Swelling of cells may also occur with exposure to UFPs in the cell line. The resulting increased cell diameter and increased cytoplasmic stiffness and decreased cytoplasmic relaxation are related.

o Phagocytosis of latex beads also decreases with increasing numbers of cytoplasmic UFP in the cell line. This phenomenon does not correlate with decreased cell proliferation.

o Phagocytosis in BD-AM is not affected by TiO₂ although it is by C and DEP.

· UFP-induced apoptosis and necrosis:

o 24 h incubation of macrophages from both sources caused both increased apoptosis and necrosis as measured by flow cytometry. The degrees of each cell function measured depended upon the particle type and macrophage source. In some cases increased particle surface area is related to decreased cell viability.

o Cell viability was also assessed by fluorescence microscopy. This technique shows more viable cells for both control cells and cells treated with UFPs than did flow cytometry

· UFP effects on phagosome motion and magnetic particle twisting:

o Magnetic twisting cytometry (MTC) measures two different aspects of phagosome transport, relaxation and twisting. Successful phagosome transport also requires intracellular Ca⁺⁺. Thus cytotoxicity can be caused by a variety of malfunctions. Microfilament damage seems to cause cytotoxicity after ufTiO₂ and EC90. Microtubule damage resulting in accelerated relaxation occurs after P90 exposure.

o Higher concentrations of UFPs than are found in the ambient environment are needed to cause cytoskeletal damage as measured by MTC. The authors speculate that high amounts may still accumulate in the lungs because of the chemical stability of C and long-term inhalation scenarios.

· Changes in capacity for phagocytosis by UFP:

o Phagocytosis in J774A.1 macrophages is inhibited more strongly than it is in BD-AM after 24 h incubation with UFP. Changes in phagocytosis also correlate with the type of UFP ingested. However there is no apparent correlation between cytomagnetometric data and phagocytosis although both are properties of cytoskeleton functioning. The authors postulate that the reason for the above may be that different surface composition of different UFP’s may affect the multitude of cell surface receptors differently.

o The authors also emphasize that they did not find 100 ug/ml TiO₂ to alter phagocytosis in BD-AM although other investigators showed that 1 ug/ml/10⁶ aggregates of UF carbon could alter phagocytosis. These results could not be explained.

o Diesel exhaust was the strongest inhibitor of phagocytosis in both types of macrophages regardless of the preparation of the particles. Results of the examination of urban dust in this experimental system also led to the conclusion that cytoskeletal dysfunctions measured were not solely caused by organics adsorbed onto ultrafine particle surfaces.

· Apoptosis and necrosis by UFP:

o Below is as measured by flow cytometry:

§ Loss of cell viability observed cannot explain the degree of toxic effects in the cytoskeleton leading to dysfunction of the cytoskeleton.

§ The BD-AM have higher viability than does the macrophage cell line. They appear to be able to endure conditions of isolation and incubation better than does the cell line.

§ Both types of AM’s show decreasing viability as the UFP surface area increases.

§ There may be an additional surface effect that was not measured causing differences between TiO₂ and carbon particles.

§ Most nonviable cells were necrotic.

o As observed with fluorescence microscopy, there were more viable cells under similar incubation conditions. Several possible explanations involving technique were given for this observation.