Ambient PM(10) extracts inhibit phagocytosis of defined inert model particles by alveolar macrophages

We used inert melamin particles that are very well defined in size (diameters: 0.5, 1.9, 6.8 microm) to study the effects of ambient particles of <10 microm (PM(10)) on phagocytosis. Dose-response functions were found between the amount of added melamin particle mass and the toxicity. Fine particles (0.5 microm) were more toxic at low and medium amounts of mass per cell added than the larger particles (1.9, 6.8 microm). However, with regard to particle numbers applied per cell, toxicity is reversed, with the largest particles being the most toxic. In the whole dose range tested, the melamin particles used did not stimulate cells to produce oxidative radicals or tumor necrosis factor-alpha (TNF alpha). Flow cytometric analyses visualized the time-dependent melamin particle uptake, which was inhibited by polyinosinic acid (Poly-I), suggesting that phagocytosis is mediated by scavenger-type receptors. Cells exposed to an aqueous PM(10) extract, which also had a dose-dependent toxic potential, were stimulated to produce oxidative radicals, measured as NO(2)(-), and TNF alpha. Combined exposures with the PM(10) extract followed by the inert melamin particles show that the PM(10) extract inhibits inert particle uptake by alveolar macrophages. It is proposed that ambient PM(10), besides being toxic for alveolar macrophages, stimulates them to produce oxidative radicals and the proinflammotry cytokine TNF alpha. Moreover, it inhibits their particle uptake potential. Thus PM(10) appears to promote inflammation and reduce defense.     

AMBIENT PM 10 EXTRACTS INHIBIT PHAGOCYTOSIS OF DEFINED INERT MODEL PARTICLES BY ALVEOLAR MACROPHAGES

We used inert melamin particles that are very well defined in size (diameters: 0.5, 1.9, 6.8 µm) to study the effects of ambient particles of <10 µm (PM 10) on phagocytosis. Dose-response functions were found between the amount of added melamin particle mass and the toxicity. Fine particles (0.5 µm) were more toxic at low and medium amounts of mass per cell added than the larger particles (1.9, 6.8 µm). However, with regard to particle numbers applied per cell, toxicity is reversed, with the largest particles being the most toxic. In the whole dose range tested, the melamin particles used did not stimulate cells to produce oxidative radicals or tumor necrosis factor-α (TNFα) . Flow cytometric analyses visualized the time-dependent melamin particle uptake, which was inhibited by polyinosinic acid (Poly-I), suggesting that phagocytosis is mediated by scavenger-type receptors. Cells exposed to an aqueous PM 10 extract, which also had a dose-dependent toxic potential, were stimulated to produce oxidative radicals, measured as NO 2 -, and TNFα. Combined exposures with the PM 10 extract followed by the inert melamin particles show that the PM 10 extract inhibits inert particle uptake by alveolar macrophages. It is proposed that ambient PM 10, besides being toxic for alveolar macrophages, stimulates them to produce oxidative radicals and the proinflammotry cytokine TNFα. Moreover, it inhibits their particle uptake potential. Thus PM 10 appears to promote inflammation and reduce defense.     

Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (PM2.5) and coarse particles (PM10-2.5) in outdoor and indoor air.

Increased incidence of mortality and morbidity due to cardiopulmonary complications has been found to associate with elevated levels of particulate air pollution (particulate matter with an aerodynamic diameter < 10 microm, PM10 and <2.5 microm, PM2. 5). Lung injury and an imbalance of inflammatory mediators are proposed causative mechanisms, while the toxic constituents may be acidity, transition metals, organic, and biogenic materials. To compare the ability of inhalable fine particles (PM2.5), and coarse particles (PM10-2.5) to cause cell injury and cytokine production in monocytes, dichotomous Andersen samplers were used to collect size-fractionated PM10 for in vitro testing of the particle extracts. Particles from both outdoor and indoor air were collected onto Teflon filters, on nine separate occasions. Each filter was water extracted and each extract assessed for ability to cause cell death, as well as interleukin (IL)-6 and IL-8 production in human monocytes. Significant toxicity and cytokine production was induced by outdoor PM10-2.5, but not by outdoor PM2.5 or the particles collected indoors. Outdoor PM10-2.5 induced 20 times the amounts of IL-6 and IL-8 than the fine particles. Cytotoxicity was inhibited by deferoxamine, a chelator of transition metals, while cytokine production was not. On the other hand, lipopolysaccharide binding protein (LBP) completely inhibited cytokine induction by PM10-2.5, suggesting that gram-negative bacteria and/or endotoxins are components of PM10-2.5. The effective proinflammatory effects of endotoxin on macrophages may upset lung homeostasis while metals-induced cytotoxicity/necrosis may set up inflammation independent of macrophage-derived cytokines.     

Toxicity of airborne dust as an indicator of moisture problems in school buildings

Moisture-damaged indoor environments are thought to increase the toxicity of indoor air particulate matter (PM), indicating that a toxicological assay could be used as a method for recognizing buildings with indoor air problems. We aimed to test if our approach of analyzing the toxicity of actively collected indoor air PM in vitro differentiates moisture-damaged from non-damaged school buildings. We collected active air samples with NIOSH Bioaerosol Cyclone Samplers from moisture-damaged (index) and non-damaged (reference) school buildings (4?+?4). The teachers and pupils of the schools were administered a symptom questionnaire. Five samples of two size fractions [Stage 1 (>1.9?μm) and Stage 2 (1–1.9?μm)] were collected from each school. Mouse RAW264.7 macrophages were exposed to the collected PM for 24?h and subsequently analyzed for changes in cell metabolic activity, production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. The teachers working in the moisture-damaged schools reported respiratory symptoms such as cough (p?=?0.01) and shortness of breath (p?=?0.01) more often than teachers from reference schools. Toxicity of the PM sample as such did not differentiate index from reference building,s but the toxicity adjusted for the amount of the particles tended to be higher in moisture-damaged schools. Further development of the method will require identification of other confounding factors in addition to the necessity to adjust for differences in particle counts between samples.     

Alveolar macrophage cytokine production in response to air particles in vitro: role of endotoxin

The interaction of air particles and alveolar macrophages (AMs) may result in the release of proinflammatory cytokines. Normal mouse AMs were treated with concentrated air particle (CAPs) suspensions in vitro. After 5 h, cytokine release [macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha)] and phagocytosis of ambient air particles were measured. CAPs samples collected from urban air (Boston) on different days were used. The CAPs samples and their soluble and solid components caused significant MIP-2 and TNF-alpha production. Variability in the potency of samples collected on different days was observed. Trace endotoxin was measured in CAPs samples (EU/mg: 2.3 +/- 0.7, mean +/- SE, n = 10). A majority of biologic activity (cytokine induction) and endotoxin content was associated with the solid components. Neutralization of endotoxin by polymyxin B abrogated >80% of TNF-alpha induction by CAPs samples, but inhibited MIP-2 production by only approximately 40%. The trace endotoxin present in CAPs caused much more MIP-2 production than predicted by concentration alone (28 +/- 8-fold increase, n = 9), indicating synergistic interaction with other AM-activating components of the particles. Data suggest that low levels of endotoxin may interact with air particles to activate lung macrophages.     

Alveolar macrophage function in nickel dust exposed rabbits.

8 rabbits were exposed to metallic nickel dust (2 mg/m3, of which about half was respirable) for 4 weeks. The lungs were lavaged and the macrophages were collected. In comparison with 8 control rabbits, a significant increase was noted in the nickel exposed rabbits as concerned the weight and density of the lungs, the size variation of the lung cells, the phagocytosis of silver coated particles, and the metabolic activity as measured by NBT reduction. The last mentioned increase was recorded during basal conditions as well as during phagocytosis. The NBT reduction during phagocytosis was significantly correlated with the degree of phagocytosis of silver coated particles in both control and exposed rabbits. It is suggested that the exposure to nickel dust has unspecifically activated the macrophages perhaps by increased production of phospholipids.     

Study of the mechanism responsible for the elective toxicity of tungsten carbide-cobalt powder toward macrophages.

We have previously demonstrated that tungsten carbide-cobalt powder (WC-Co) is more toxic toward murine macrophages in vitro than pure cobalt metal particles and that the cellular uptake of cobalt is enhanced when the metal is present in the form of WC-Co mixture. The present study was undertaken to assess the possible mechanism(s) of this interaction. We found that solubilization of cobalt in the extracellular milieu was increased in the presence of WC. This phenomenon, however, is not the critical factor explaining the greater toxicity of the WC-Co mixture since increasing the amount of solubilized cobalt in the extracellular medium in the absence of WC did not result in increased toxicity. Moreover, the amount of cobalt solubilized from a toxic dose of WC-Co was insufficient to affect by itself macrophage viability. A toxic effect was only observed when the WC-Co mixture came directly in contact with the cells. The elective toxicity of WC-Co can also not be explained by stimulation of phagocytosis of cobalt metal particles due to the simultaneous presence of other particles (WC) in the extracellular fluid since stimulation of phagocytosis by latex beads or zymosan particles did not amplify the toxicity of cobalt metal particles. These results indicate that the toxicity of the WC-Co mixture does not simply result from an enhanced bioavailability of its cobalt component. This suggests that hard metal dust behaves as a specific toxic entity.     

ALVEOLAR MACROPHAGE CYTOKINE PRODUCTION IN RESPONSE TO AIR PARTICLES IN VITRO: ROLE OF ENDOTOXIN

The interaction of air particles and alveolar macrophages (AMs) may result in the release of proinflammatory cytokines. Normal mouse AMs were treated with concentrated air particle (CAPs) suspensions in vitro. After 5 h, cytokine release [macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor- a (TNF-a)] and phagocytosis of ambient air particles were measured. CAPs samples collected from urban air (Boston) on different days were used. The CAPs samples and their soluble and solid components caused significant MIP-2 and TNF- a production. Variability in the potency of samples collected on different days was observed. Trace endotoxin was measured in CAPs samples (EU/mg: 2.3 +/- 0.7, mean +/- SE, n = 10). A majority of biologic activity (cytokine induction) and endotoxin content was associated with the solid components. Neutralization of endotoxin by polymyxin B abrogated &gt;80% of TNF- a induction by CAPs samples, but inhibited MIP-2 production by only 40%. The trace endotoxin present in CAPs caused much more MIP-2 production than predicted by concentration alone (28 +/- 8-fold increase, n = 9), indicating synergistic interaction with other AM-activating components of the particles. Data suggest that low levels of endotoxin may interact with air particles to activate lung macrophages.     

Ultrafine particles cause cytoskeletal dysfunctions in macrophages

Essential cytoskeletal functions of macrophages are migration, phagocytosis of foreign materials, and intracellular transport and digestion The influence of fine and ultrafine test particles (UFP), such as TiO(2), elemental carbon, commercial carbon black, diesel exhaust particulate matter, and urban dust (UrbD), on cytoskeleton-related functions of macrophages, such as phagocytosis, phagosome transport mechanisms, and mechanical cytoskeletal integrity, were studied by flow cytometry and by cytomagnetometry. Additionally, necrosis and apoptosis caused by the test particles was detected. The diameter of the test particles ranged from 12 to 220 nm and the Brunauer-Emmet-Teller specific surface area ranged from 6 to 600 m(2)/g. Primary alveolar macrophages from beagle dogs (BD-AM), obtained by bronchoalveolar lavage, were used as well as macrophages originating from the cell line J774A.1. For cytomagnetometry studies, spherical 1.8-microm ferromagnetic particles served as probes for cytoskeletal functions and were incubated together with the macrophages 24 h prior to UFP exposure. Macrophages were exposed in vitro with 10-320 microg UFP/ml/10(6) cells up to 24 h. In all experiments, J774A.1 macrophages were more sensitive than BD-AM to UFP exposure. Cytoskeletal dysfunctions evaluated by cytomagnetometry were an impaired phagosome transport and an increased cytoskeletal stiffness and occurred at concentrations of 100 microg UFP/ml/10(6) cells and above, in both BD-AM and J774A.1. Only fine TiO(2) did not show any effect. Urban dust (standard reference material 1649a) and diesel exhaust particles (DEP, standard reference material 1650) caused comparable cytoskeletal dysfunctions to elemental carbon with high specific surface area. Cytoskeletal dysfunctions induced by DEP or UrbD could be reduced after washing the particles by dichloromethane. UFP caused an impaired phagocytosis of 1-microm diameter fluorescent latex beads, inhibited cell proliferation, and decreased cell viability. All recorded cytotoxic parameters showed only weak correlations with the specific surface area or the total number of UFP, which can result from the different types of particles and different surface compositions. UFP cause cytoskeletal toxicity in vitro in macrophages, which can cause cellular dysfunctions, such as impaired proliferation, impaired phagocytic activity, and retarded intracellular transport processes as well as increased cell stiffness and can result in impaired defense ability in the lung.     

Shape Induced Inhibition of Phagocytosis of Polymer Particles

Purpose  To determine if particle shape can be engineered to inhibit phagocytosis of drug delivery particles by macrophages, which can be a significant barrier to successful therapeutic delivery. Methods  Non-spherical polystyrene particles were fabricated by stretching spherical particles embedded in a polymer film. A rat alveolar macrophage cell line was used as model macrophages. Phagocytosis of particles was assessed using time-lapse video microscopy and fluorescence microscopy. Results  We fabricated worm-like particles with very high aspect ratios (>20). This shape exhibits negligible phagocytosis compared to conventional spherical particles of equal volume. Reduced phagocytosis is a result of decreasing high curvature regions of the particle to two single points, the ends of the worm-like particles. Internalization is possible only at these points, while attachment anywhere along the length of the particles inhibits internalization due to the low curvature. Conclusions  Shape-induced inhibition of phagocytosis of drug delivery particles is possible by minimizing the size-normalized curvature of particles. We have created a high aspect ratio shape that exhibits negligible uptake by macrophages. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Trichilia glabra: effect on the phagocytic activity and respiratory burst response of peritoneal macrophages

A diminution in the phagocytic capability and respiratory burst response of murine peritoneal macrophages was observed when these cells were treated `in vitro' with Trichilia glabra leaf aqueous extract. The effect was not observed when non-specific phagocytosis was tested. The extract also inhibited binding of opsonized erythrocytes to macrophages, thus indicating that the observed antiphagocytic effect is possibly due to the failure of opsonized particles to bind to these cells. A low molecular weight fraction is responsible for the reported activities.     

Impairment of alveolar macrophage phagocytosis by ultrafine particles

We investigated whether slowed clearance after exposure to ultrafine particles was due to a failure in alveolar macrophage phagocytosis. This was achieved by measuring the ability of a macrophage cell line (J774.2 MPhi) to phagocytose 2-microg indicator latex beads following 8-h exposures to a number of test particles. Particles utilized were fine titanium dioxide (TiO2), ultrafine titanium dioxide (UTiO2), carbon black (CB), or ultrafine carbon black (UCB). Cytotoxicity of particles was measured by means of MTT activity. In a preliminary study, we assessed the effects of conditioned medium from particle-treated macrophages on the phagocytic ability of naive macrophages. Ultrafine and fine particles had no significant cytotoxic effects on J774.2 MPhi. A significant reduction in the ability of macrophages to phagocytose the indicator beads occurred after exposure to 0.39 microg/mm(2) (p < 0.001) of UCB and 0.78 microg/mm(2) (p < 0.001) of all particle types compared to the control. Furthermore, ultrafine particles were shown to significantly (p < 0.001) impair macrophage phagocytosis at a lower dose than their fine counterparts (0.39 and 0.78 microg/mm(2), respectively). At all doses, UCB resulted in a greater number (p < 0.001) of nonphagocytic macrophages compared to the other test particles. We tested whether a diffusable mediator being released from particle-exposed cells inhibited the phagocytic activity of adjacent macrophages. The conditioned medium from particle-exposed macrophages had no significant effect on the phagocytic ability of macrophages, suggesting that cell-cell contact is responsible for the pattern of failed phagocytosis (data not shown). We have demonstrated that ultrafine particles impair macrophage phagocytosis to a greater extent than fine particles compared on a mass basis. Therefore, we conclude that slowed clearance of particles, specifically the ultrafines, can in part be attributed to a particle-mediated impairment of macrophage phagocytosis.     

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro

Background: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. Methods: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150?nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Results and conclusion: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.     

A role for anti-inflammatory agents and cyclic AMP in regulating fibronectin-mediated phagocytosis

The present study deals with the effects of anti-inflammatory drugs and agents known to elevate intracellular levels of cyclic AMP (cAMP) on plasma fibronectin-mediated (PFn) phagocytosis of radiolabeled, gelatin-coated latex particles (g-Ltx*) by inflammatory macrophages. Monolayers of casein-elicited peritoneal macrophages were preincubated with the specified agents for either 1 or 24 hrs at 37 degrees C prior to the measurements of phagocytosis in the presence of human plasma fibronectin (47 microgram/ml) and heparin (6.7 U/ml). Under these conditions, prostaglandin E1, colchicine, vincristine, and cytochalasin B were all effective in inhibiting g-Ltx* phagocytosis by macrophages in a dose-dependent fashion. More potent inhibition of phagocytosis was manifested by agents known to increase intracellular levels of cAMP in phagocytic cells. Dibutyryl cyclic AMP (dbcAMP), d,1-isoproterenol and aminophylline (10(-5) to 10(-3) M) were all effective in reducing the uptake of g-Ltx* by macrophages. The combination of dbcAMP and aminophylline acted additively. These studies demonstrate that anti-inflammatory drugs and cAMP-elevating agents exert potent inhibitory effects on fibronectin-mediated phagocytosis of gelatin-coated particles by macrophages. Thus, our system provides a suitable in vitro model for further investigations into the humoral regulation of phagocytosis of denatured collagen-coated particles and tissue debris by inflammatory phagocytic cells.     

Release of inflammatory cytokines, cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions.

Several studies have shown that particles of smaller size may be more potent than larger to induce inflammatory and toxic responses in cultured lung cells. However, the relative importance of different size fractions of ambient PM to induce such effects is still not known. In this study, we investigated the potency of different size fractions of urban ambient air particles to induce release of inflammatory cytokines in the human alveolar cell line A549 and primary rat type 2 cells. A mineral-rich ambient air PM10 sample collected in a road tunnel (road PM10) was also included. The coarse fraction of the urban ambient air particles demonstrated a similar or higher potency to induce release of the proinflammatory cytokines IL-8/MIP-2 and IL-6 compared to the fine and ultrafine fractions. The coarse fraction was also the most toxic in both cell systems. In contrast to the A549 cells, no induction of cytokine release was induced by the ultrafine particles in the primary type 2 cells. The mineral-rich road PM10 may be equally or more potent than the various size fractions of the ambient air particles to induce cytokines in both cell types. In conclusion, the coarse fraction of ambient particles may be at least as potent by mass as smaller fractions to induce inflammatory and toxic effects in lung cells.     

Genotoxicity induced by fine urban air particulate matter in the macrophages cell line RAW 264.7.

Recent studies support a participation of fine airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 microm in the effects of air pollutants on health. Particulate matter was collected in an urban area of L'Aquila during the winter 2004. Fine particulate samples were analyzed by X-ray photoelectron spectroscopy (XPS) to determine the chemical inventory of the aerosol particle surfaces and to evaluate the weight of characteristic functional groups of the most frequent carbon-containing organic pollutant compounds (C-C/C-H, C-O/C-N, C=O, COOH). The most important contributor to the mass of fine particulate matter was carbon. The overall purpose of this work was to determine the in vitro toxicity and genotoxicity of fine PM in cultured macrophages (RAW 264.7 cells) since the biological target of inhaled PM are the pulmonary epithelium and resident macrophages. In parallel in vitro toxicity assays were used including cell viability and apoptosis. Genotoxicity was evaluated by the micronucleus (MN) assay. The viability of macrophages was assessed by the MTT method; apoptosis by an ELISA test for programmed cell death (PCD) was determined after RAW 264.7 cells treatment. Concentration of 1, 3 and 10 microg/cm2 of fine particles induced micronuclei in a dose-dependent manner. We also compared the effects of fine PM with those of fine carbon black particles (CB) in similar doses. Fine carbon black particles were consistently less genotoxic than the fine atmospheric particles, suggesting that the contaminants adsorbed on them (i.e. carbon-containing organic compounds in addition to metal oxides and metal salts) are involved in genotoxicity. Fine PM reduced cellular proliferation. Overall, the results presented here demonstrate the utility of in vitro tests in mouse cells for testing genotoxicity of urban air particulate matter.     

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro

Background: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles.

Methods: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150?nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively).

Results and conclusion: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.     

Seasonal variation in the toxicological properties of size-segregated indoor and outdoor air particulate matter

Ambient air particulate matter (PM) as well as microbial contaminants in the indoor air are known to cause severe adverse health effects. It has been shown that there is a clear seasonal variation in the potency of outdoor air particles to evoke inflammation and cytotoxicity. However, the role of outdoor sources in the indoor air quality, especially on its toxicological properties, remains largely unknown. In this study, we collected size segregated (PM_10–2.5, PM_2.5–0.2 and PM_0.2) particulate samples with a high volume cascade impactor (HVCI) on polyurethane foam and fluoropore membrane filters. The samples were collected during four different seasons simultaneously from indoor and outdoor air. Thereafter, the samples were weighed and extracted with methanol from the filters before undergoing toxicological analyses. Mouse macrophages (RAW264.7) were exposed to particulate sample doses of 50, 150 and 300 μg/ml for 24 h. Thereafter, the levels of the proinflammatory cytokine (TNF-α), NO-production, cytotoxicity (MTT-test) and changes in the cell cycle (SubG₁, G₁, S and G₂/M phases) were investigated. PM_10–2.5 particles evoked the highest inflammatory and cytotoxic responses. Instead, PM_2.5–0.2 samples exerted the greatest effect on apoptotic activity in the macrophages. With respect to the outdoor air samples, particles collected during warm seasons had a stronger potency to induce inflammatory and cytotoxic responses, whereas no such clear effect was seen with the corresponding indoor air samples. Outdoor air samples were associated with higher inflammatory potential, whereas indoor air samples had overall higher cytotoxic properties. This indicates that the outdoor air has a limited influence on the indoor air quality in a modern house. Thus, the indoor sources dominate the toxicological responses obtained from samples collected inside house.     

Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages

The role of the glucocorticoid-regulated protein annexin 1 during the process of phagocytosis has been studied using annexin 1 null peritoneal macrophages. Wild type and annexin 1 null macrophages were incubated with several distinct phagocytic targets. No differences were observed in rate or the maximal response with respect to IgG complexes or opsonised zymosan phagocytosis, as assessed by monitoring the production of reactive oxygen species. When annexin 1 null macrophages were incubated with non-opsonised zymosan particles, they exhibited impaired generation of reactive oxygen species, which was linked to a defect in binding of cells to the particles, as determined with fluorescent zymosan. This phenomenon was further confirmed by electron microscopy analysis, where annexin 1 null macrophages internalised fewer non-opsonised zymosan particles. Specific alterations in macrophage plasma membrane markers were observed in the annexin 1 null cells. Whereas no differences in dectin-1 and FcgammaR II/III expression were measured between the two genotypes, decreased membrane CD11b and F4/80 levels were measured selectively in macrophages lacking annexin 1. These cells also responded with an enhanced release of PGE(2) and COX-2 protein expression following addition of the soluble stimulants, LPS and heat-activated IgG. In conclusion, these results suggest that participation of endogenous annexin 1 during zymosan phagocytosis is critical and that this protein plays a tonic inhibitory role during macrophage activation.     

Quantification of microsized fluorescent particles phagocytosis to a better knowledge of toxicity mechanisms

Background: The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments.

Methods: Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production].

Results and conclusion: Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49?±?11%) than for amine 1-µm beads (18?±?5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446?±?7 and 139?±?12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H₂O₂ production.