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Am J Physiol Lung Cell Mol Physiol 294: L1137-L1148, 2008. First published April 4, 2008; doi:10.1152/ajplung.00261.2007
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Impact of the Fc{gamma}II-receptor on quartz uptake and inflammatory response by alveolar macrophages

Petra Haberzettl,1 Roel P. F. Schins,1 Doris Höhr,1 Verena Wilhelmi,1 Paul J. A. Borm,1,2 and Catrin Albrecht1

1Particle Research, Institut für Umweltmedizinische Forschung at the Heinrich Heine University, Düsseldorf, Germany; and 2Centre of Expertise in Life Sciences, Hogeschool Zuyd, Heerlen, The Netherlands

Submitted 6 July 2007 ; accepted in final form 30 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the Fc{gamma}II-receptor (Fc{gamma}II-R). Fluorescence immunocytochemistry, demonstrating Fc{gamma}II-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the Fc{gamma}II-R in DQ12 phagocytosis. Fc{gamma}II-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the Fc{gamma}II-R. Consistent with the uptake results, Fc{gamma}II-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-{alpha} release as observed after treatment with DQ12. In conclusion, our results indicate a major role of Fc{gamma}II-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

GTPases; particles; phagocytosis; reactive oxygen species

EXPOSURE TO RESPIRATORY PARTICLES increases the risks of several lung diseases including chronic obstructive pulmonary disease, pneumonia infection, and lung cancer (64, 69). After inhalation of coal or silica particles, the development of fibrotic lung diseases like pneumoconiosis and silicosis has been very well described (15, 66). Accumulation of quartz particles and/or quartz-containing dusts (e.g., coal mine dust) can lead to a persistent inflammatory response that is associated with the development and progression of silicosis (48, 66).

Alveolar macrophages (AM) are considered to be the key players in the initiation and progression of silicosis. Generation of reactive oxygen species (ROS) as well as the activation of the transcription factor NF-{kappa}B are considered to be essential herein. ROS can be generated by the direct interaction of silica particles with the aqueous milieu in the lungs, or indirectly by the ability of particles to activate phagocytes including AM (20). Thereby, the quartz surface potential to induce oxidative stress is directly related to its cytotoxicity (77), whereas the relation to its inflammatory effects seems to be more complicated. Reduction of ROS generation through radical scavengers or particle surface modification using polyvinyl-N-oxide (PVNO) and/or aluminum lactate (AL) diminish the toxic as well as the inflammatory potential of quartz (4, 23, 31). NF-{kappa}B, which is implicated in several diseases like atherosclerosis, type II diabetes, asthma, and cancer as a key factor regulating the expression of abundant inflammatory genes (9, 11, 75) is also considered to drive quartz-induced inflammation (14). Thereby, the quartz-induced activation of the transcription factor requires the activation of a signaling cascade containing different protein tyrosine kinases (PTK) including phosphatidylinositol 3-kinase (PI3K) (14, 35). The quartz-initiated NF-{kappa}B activation effects the formation of inflammatory mediators, e.g., TNF-{alpha} (38, 67). TNF-{alpha} is considered as a key cytokine in the development of silicosis and has shown to be required for the development of silica-induced pulmonary fibrosis (63). Several studies have shown a dose-dependent TNF-{alpha} formation by quartz particles, as well as a direct relation between TNF-{alpha} production and progression of inflammation (8, 10, 21). In addition, we have shown recently the relation between the uptake of quartz particles and the induction of TNF-{alpha} release (36). The synthesis and secretion of a wide range of pro- and anti-inflammatory chemokines and cytokines by AM has, on one hand, a regulatory function in the cellular immune defense, and on the other hand, these mediators couple the native unspecific with the adaptive antigen-specific immunity (24, 29, 44, 68).

AM are in the front line of lung host defense. Besides their regulatory properties, their first outstanding function is clearance of foreign invaders from the lung, which involves binding, ingestion, and digestion of infectious, toxic, or allergenic substances that have passed mechanical barriers of the respiratory tract (55, 78). Elimination of inhaled pathogens can be processed extracellularly within the alveolus by secretion of antimicrobial substances (i.e., oxygen metabolites, lysozymes, or proteases) as well as intracellularly, after phagocytosis into phagolysosomes (24, 29, 44, 68). The phagocytosis process, which has been defined as the active uptake of particles >0.5 µm, is the initial step in the elimination of invaders as well as in the induction of the immune response. Phagocytosis is induced by the interaction of different specialized cell surface receptors and their specific ligands using the actin cytoskeleton as the driving force (49). Among these are classes of specialized pattern recognition receptors, like scavenger receptors (ScR), mannose receptors (MR), and complement receptors (CR), differing in the pathogen recognition motif (pathogen-associated molecular pattern), i.e., opsonin dependent or independent (1, 42, 76). Phagocytosis could further be mediated by the family of Fc receptors (FcR), which recognizes immunoglobulin-coated particles and complexes (1). These various receptors show, besides differences in the morphology of particle binding and subsequent ingestion (e.g., zippering or sinking mechanism), also variations in the mediators involved in the phagocytosis signaling cascade as well as in the receptor-induced immune response (1, 5, 76).

Recently, we have identified quartz uptake as a classic phagocytosis process, forced by the actin framework of the cytoskeleton (36). Therefore, the aim of the present study was to enroll the responsible classic phagocytosis receptor(s) involved in the uptake of quartz particles and its subsequent inflammatory consequences. Phagocytosis was analyzed in quartz-treated NR8383 rat macrophages in the presence or absence of inhibitors of different potentially involved receptors. Furthermore, we examined the morphological characteristics, the signaling cascade, and the inflammatory responses, such as ROS generation and TNF-{alpha} release within the phagocytosis process of quartz particles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and particle treatment. NR8383 cells, a rat alveolar macrophage cell line (American Type Culture Collection, Manassas, VA), were cultured at 37°C and 5% CO2 in Kaihn's modified medium (F12-K Nutrient Mixture; GIBCO, Eggenstein, Germany) supplemented with 15% FCS, 1% penicillin/streptomycin, and 1% glutamine (Sigma-Aldrich, Seelze, Germany). Experiments were performed 3 days after seeding (1.1 x 105 cells/cm2).

Particle treatments for all experiments, except the measurement of ROS generation, were performed in complete cell culture medium. Therefore, macrophages were incubated for 5, 10, 15, or 60 min with freshly prepared DQ12 suspension (Dörentruper quartz, batch 6, mean diameter 0.96 µm, 5 mg/ml) at concentrations of 10 or 40 µg/cm2. DQ12 was baked at 220°C for 16 h to destroy endotoxins. Immediately before treatment of the cells, fresh suspension of DQ12 was either sonicated with medium, or for measurement of respiratory burst, with HBSS+/+ (+Mg2+/+Ca2+, GIBCO) for 5 min (60 W, 35 Hz, waterbath sonicator, Sonorex TK 52; Schaltech, Mörfelden-Walldorf, Germany).

Inhibition experiments, cell preparation, and flow cytometric measurement of DQ12 uptake. Macrophages were preincubated with appropriate inhibitors for 30 min (37°C, 5% CO2) before particle treatment to inhibit different classic phagocytosis receptors, PTKs or GTPases. Only the inhibition of the GTPase Rac1 with NSC23766 [50, 100 µM; Calbiochem, San Diego, CA (30)] was performed by a 24-h preincubation under the same conditions. Inhibition of FcR and CR was performed with the specific Fc{gamma}II-R-antibody (Ab, CD32, 1:200) or CR-Ab [CD11b/c, 1:200, purchased from BD Pharmigen, Heidelberg, Germany (56)]. ScR and MR were blocked using two different receptor agonists to enclose adverse agonist side effects. For ScR, poly-I (polyinosinic acid, 10 µg/ml) or fucoidan (7 µg/ml), and for MR, M-{alpha}-D-MP (methyl-{alpha}-D-mannopyranoside, 200 µg/ml) or mannan [7 µg/ml, Sigma-Aldrich (47, 56)] were used. To verify the antibody specificity for the Fc{gamma}II-R- and CR-Ab, uptake experiments using the same concentrations of the corresponding mouse immunoglobulin G (IgG, IgG1, IgG2a, BD Pharmigen) were performed. Genistein [10, 50 µg/ml, Sigma-Aldrich (16)] was used for inhibition of PTKs, and lovastatin [5, 10, 20, 40, 80 µM, Calbiochem (51, 54)] was used for blocking GTPase function. Additionally, DMSO (0.32%, lovastatin vehicle control) was used as lovastatin solvent control. After 1 h of DQ12 treatment, as described before, macrophages were gently scraped on ice, centrifuged (900 g, 5 min, 4°C), washed twice with 1 ml of HBSS–/– (–Mg2+/–Ca2+, GIBCO), resuspended in 400 µl of HBSS–/–, and kept on ice until measurement.

Particle uptake was analyzed from DQ12-treated cells compared with non-particle-treated controls (0 µg/cm2) using the flow cytometry approach previously described by Haberzettl et al. (36) adopted from Stringer et al. (70). Briefly, particle uptake was determined measuring side scatter angle (SSC) and forward scatter angle (FSC) of 10,000 counts using FACSCalibur (fluorescence-activated cell sorter; Becton Dickinson, Heidelberg, Germany). The SSC, directly related to cell granularity, was utilized as a benchmark of DQ12 uptake, whereas the cell size correlating FSC was used as a cofactor for statistical analysis. The median SSC, as well as the median FSC, were obtained by analyzing gated univariant FSC and SSC histograms from control and quartz-treated cells with Cell Quest 3.3 Software (Becton Dickinson). The median SSC from three independent experiments, each done in triplicate, was used to calculate the means ± SE of cell granularity, which is presented as median SSC in percentage of control.

Immunocytochemistry and fluorescence microscopy. FcR as well as Rac1 immunocytochemistry were performed with cytospin preparations of untreated NR8383 cells or macrophages treated with 10 µg/cm2 DQ12 for 15 min. Therefore, cells were scraped on ice, centrifuged (900 g, 5 min, 4°C), and washed four times with 5 ml of ice-cold PBS (GIBCO). After resuspension in 1 ml of ice-cold PBS supplemented with 2% BSA (Roche, Penzberg, Germany), cells were counted (hematocytometer chamber, Neubauer), and 1 x 106 cells were inserted for each slide preparation (5 min, 300 rpm; Cytospin3, Thermo Shandon, Dreicheich, Germany). Air-dried cytospin preparations were fixed with 3.7% paraformaldehyde/PBS (pH 7.4; Merck, Darmstadt, Germany; 10 min, room temperature) and permeabilized using 0.01% Triton X-100/PBS (Sigma-Aldrich; 3 min, room temperature). After blocking with 1% BSA/PBS (15 min, room temperature), cells were incubated overnight at 4°C with the FcR{gamma}II-Ab (CD32, 1:10, BD Pharmigen) or the Rac1-Ab (1:10, BD Pharmigen) followed by fluorescence-labeled secondary antibody (1:200, MFP488 goat anti-mouse IgG; Molecular Probes, Leiden, Netherlands) incubation (1 h, room temperature). Covering with Immuno-Fluore Mounting Medium (MP Biomedicals) allowed analysis using fluorescence microscopy with evaluation of at least 100 cells for both FcR and Rac1 analysis in three independent experiments (Olympus BX60, Hamburg, Germany).

Transmission electron microscopy. For transmission electron microscopy (TEM) analysis, cells of monolayer cultures were incubated with 10 µg/cm2 DQ12 for 24 h. Therefore, treated cells were washed three times with PBS and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (1 h, 4°C). After postfixation (2% OsO4/0.1 M sodium cacodylate buffer, 1 h, 4°C), cells were en bloc stained with 1.5% uranylacetate dihydrate and phosphotungstic acid, dehydrated in an ethanol series, and embedded in epoxy resin (Epon, Serva, Heidelberg, Germany). Blocks of monolayer samples were cut in ultrathin sections (50 nm, Ultracut E; Leica, Bensheim, Germany) placed on 150 mesh grids and stained with uranylacetate and lead citrate (46) before examination by TEM (STEM CM12; Philips, Eindhoven, Netherlands).

Preparation of cell lysates for affinity precipitation and Western blot analyses. Activation patterns of the GTPases Rac1, Cdc42, and Rho were analyzed by affinity precipitation of the activated GTP-forms (Rac1·GTP, Cdc42·GTP, and Rho·GTP) using EZ-Detect Activation Kit (Pierce). Furthermore, phosphorylation of the kinases Lyn, Syk (spleen tyrosine kinase), and PI3K was analyzed using the same cell lysates of untreated or treated (5, 10, 15, and 60 min, 10 or 40 µg/cm2) cells by Western blotting. Lysates were prepared according to the manufacturer's guidelines of the EZ-Detect Activation Kit with modifications for cells in suspension. Briefly, cells were scraped on ice, centrifuged (900 g, 5 min, 4°C), and washed two times with 10 ml of ice-cold PBS. Lysis was performed with 800 µl of lysis-/binding-/washing buffer (25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 5% glycerol) supplemented with complete protease inhibitor cocktail (Roche, Prenzberg, Germany; 1 tablet/12 ml of lysis-/binding-/washing buffer). After incubation (5 min, 4°C) and centrifugation (16,000 g, 15 min, 4°C), cell lysates were obtained from the supernatant.

After determination of the protein concentrations (BSA standard, 200 µl 1:50 mixture cupper-sulfate and bicinchoninic acid, obtained from Sigma-Aldrich; 30 min, 37°C, OD540 nm) lysate aliquots (20 µg protein for Lyn, total Rac1, total Cdc42, total Rho, or 40 µg of protein for Syk, PI3K) were mixed with Laemmli-sample buffer (250 mM Tris·HCl, pH7.5, Roth, Karlsruhe, Germany; 8% SDS, Serva, Heidelberg, Germany; 40% glycerin, Roth; 0.04% PyroninY, Sigma-Aldrich; 70 µM β-mercaptoethanol, Sigma-Aldrich) or used for precipitation.

Affinity precipitation of activated GTPases Rac1, Cdc42, and Rho. As positive control, 600 µl of lysate was mixed with 10 µl of 0.5 M EDTA (pH 8.0, Sigma-Aldrich) and 5 µl of 10 mM GTP{gamma}S (EZ-Detect Activation Kit, Pierce) and incubated for 15 min at 30°C. After adding 32 µl of 1 M MgCl2 (Sigma-Aldrich) solution, 200 µl of the obtained positive control were inserted for each precipitation (Rac1, Cdc42, and Rho). Precipitation of activated Rac1, Cdc42, and Rho was performed using glutathione-S-transferase (GST)-fusion proteins (Rac1/Cdc42: GST-Pak1-PBD; Rho: GST-Rhotekin-RBD). For precipitation, one SwellGel-immobilized glutathione disc and a volume either according to 20 µg of GST-Pak1-PBD or 400 µg of GST-Rhotekin-RBD was mixed with aliquots of the cell lysate (500 µg protein). For binding, mixtures were incubated for 1 h at 4°C on a vertical shaker. Afterwards, precipitation products were centrifuged (7,200 g/30 s/4°C), washed four times with 400 µl of lysis-/binding-/washing buffer, and 50 µl of SDS-bromphenol blue sample buffer (125 mM Tris·HCl, pH 6.8, 2% glycerol, 4% SDS, 0.05% bromphenol blue, EZ-Detect Activation Kit, Pierce, supplemented with 2.5 µl of β-mercaptoethanol, Sigma-Aldrich) were added.

Western blot analyses. Heat-denatured (5 min, 95°C) cell lysates and precipitation products were separated by SDS-PAGE on 12% gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany; kinases 0.45 µm; total and activated GTPases 0.2µm). Membranes were blocked and incubated with specific antibodies against Rac1 (Pierce, 1:1,000), Cdc42, Rho (Becton Dickinson, 1:250), Phospho-Lyn (Tyr507), Lyn, Phospho-Syk (Tyr525/526), Syk (Cell Signaling Technology, 1:1,000), Phospho-PI3K (Tyr508) (Santa Cruz Biotechnologies, 1:200), or β-tubulin (Sigma-Aldrich, 1:5,000) according to the antibody data sheets. After incubation, membranes were washed three times with TBST (25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.1% Tween, Sigma-Aldrich), incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1 h, room temperature, anti-mouse/rabbit-HRP IgG, Sigma-Aldrich, 1:5,000), and washed again three times with TBST. Signals were detected using ECL Plus Western Blot detection reagent (Amersham, Little Chalfont, UK) by measuring chemiluminescence (Fluor-S-Max, Becton Dickinson) and analyzed using the software Quantity One (Bio-Rad). Results of three independent experiments related to the loading controls β-tubulin or total Lyn are presented as optical density normalized to control (OD normalized, means ± SE).

Flow cytometric and electron paramagnetic resonance analysis of ROS. To investigate the generation of ROS, particle treatment was performed in HBSS+/+. Therefore, the medium was removed and macrophages were washed once with HBSS+/+. Supernatants were centrifuged (10 min, 25°C, 900 g), and the resuspended cell pellets (nonadherent fraction) were added to the adherent cell layers. For recovery, cells were incubated for 30 min (5% CO2, 37°C).

For flow cytometric analysis of ROS generation, cells were preincubated with freshly prepared dihydrorhodamine (DHR) for 30 min at 4.3 µM (DHR 123; Molecular Probes, Leiden, Netherlands) and incubated for 1 h, either with particles (DQ12 suspended in HBSS+/+, 10 or 40 µg/cm2) or with FcR-Ab (5 µg/ml). Afterwards, cells were scraped on ice, centrifuged (900 g, 5 min, 4°C), washed with 1 ml of HBSS–/–, and resuspended in 400 µl of HBSS–/–. To measure the intracellular ROS generation, DHR fluorescence of 10,000 counted cells was detected by the fluorescence (530 nm) detector of the FACSCalibur. After analysis of the data (Cell Quest 3.3. software) of three independent experiments, each done in triplicate, intracellular ROS generation was expressed as median DHR fluorescence (means ± SE).

Electron paramagnetic resonance (EPR) analysis of ROS generation was performed with the cell supernatant using DMPO (5,5-dimethyl-1-pyrroline N-oxide) as spin trap. Therefore, the quartz preparation (DQ12 suspended in HBSS+/+, 10 or 40 µg/cm2) or the FcR-Ab (5 µg/ml) and the spin trap DMPO (0.11 M, Sigma-Aldrich) were added simultaneously and incubated for 1 h at 37°C, 5% CO2. Radical formation was measured using a MiniScope MS100 Spectrometer (Magnettech, Berlin, Germany; instrumental settings: room temperature, microwave frequency = 9.39 GHz, magnetic field = 3,360 G, sweep width = 100 G, scan time = 30 s, number of scans = 3, modulation amplitude = 1.8 G, receiver gain = 1,000). Quantification was carried out on first derivation of EPR signal of the DMPO-OH 1:2:2:1 quartet as the sum of the four amplitudes. The mean of total amplitude values as obtained from five independent experiments was analyzed and expressed as means (EPR signal intensity) ± SE in arbitrary units.

TNF-{alpha} ELISA. After 1 h of treatment either with quartz (0, 10, or 40 µg/cm2) or the FcR-Ab (5 µg/ml), cell-free supernatants of NR8383 cells were obtained by centrifugation and frozen at –80°C until use. All samples were prepared in triplicates for four times. The supernatants were analyzed using a commercial TNF-{alpha} kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer's manual. Calculated data are presented as means ± SE normalized to the control.

Statistical analyses. The median of cell granularity and cell size were chosen as outcome variable from the FACS measurements, and analysis of covariance (SPSS, version 10) was performed to determine significance. In the statistical analyses of the SSC, the median of the FSC was chosen as a covariate, to adjust for the influence of the cell size on the flow cytometric results.

Data derived from Western blotting, EPR, DHR analysis, as well as ELISA, were compared using ANOVA with LSD post hoc testing (SPSS, version 10).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Involvement of distinct classic phagocytosis receptors in the uptake of DQ12. Following up on previous work, showing the actin dependency of quartz particle uptake in NR8383 cells (36), in this study we investigated the participation of different classic phagocytosis receptors. This was done by evaluating the inhibition of receptor particle interaction using specific antibodies (Fc{gamma}R and CR) or agonists (ScR and MR) (Fig. 1). Incubation of NR8383 cells with the inhibitors and/or with DQ12 (10 or 40 µg/cm2) revealed an absence of cytotoxicity as indicated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. Only the MR agonist mannan demonstrated a slight but significant (*P < 0.05) reduction of viability (80% of the control cells, data not shown).


Figure 1
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  		Fig. 1. Flow cytometric analysis of the involvement of phagocytosis receptors on quartz uptake by NR8383 macrophages. A: representative univariant histograms of cell number (counts) and side scatter angle (SSC) of quartz-treated NR8383 cells, either without or with receptor inhibition (vertical panels: without, first; after pretreatment with poly-I, second; fucoidan, third; FcR-Ab, fourth; M-{alpha}-D-MP, fifth; mannan, sixth; CR-Ab, seventh column). B and C: DQ12 uptake as normalized median SSC analyzed by flow cytometry after treatment of NR8383 cells with 10 µg/cm2 (B) or 40 µg/cm2 (C) quartz, without or after pretreatment with poly-I, fucoidan, FcR-Ab, m-{alpha}-D-MP, mannan, or CR-Ab. Data are presented as means ± SE (n = 3) normalized to control in %. Statistical analysis was preceded in a variance analysis using a confidence interval of 95% and the cell size as a cofactor. Significant reduction (*P < 0.05, ***P < 0.001) of DQ12 uptake or significant increase (##P < 0.01) of the median SSC was compared with cells without inhibitory pretreatment. D: DQ12 uptake in NR8383 cells as normalized median SSC values analyzed by flow cytometry after treatment with DQ12 without or after pretreatment with the isotype controls IgG1 and IgG2a. Data are presented as means ± SE (n = 3).

 
The influence of different receptor inhibitors on DQ12 phagocytosis by NR8383 cells was examined using flow cytometry. Results are shown in Fig. 1. Representative gated univariant histograms of cell number (counts) and SSC (side scatter angle) exemplify the increase of granularity due to quartz uptake after 1 h of treatment (Fig. 1A). The first column demonstrates an increase in the number of higher granulated macrophages (SSC) after treatment with an expanding DQ12 dose. Quantification gained an increasing median SSC of ~20% or 70% due to particle phagocytosis 1 h following quartz treatment with 10 µg/cm2 (Fig. 1B) or 40 µg/cm2 (Fig. 1C), respectively. The following columns (Fig. 1A) show the detected SSC distribution after quartz treatment at equal concentrations following inhibition of either ScR (poly-I, fucoidan), FcR (FcR-Ab), MR (m-{alpha}-D-MP, mannan), or CR (CR-Ab). Compared with cells without any pretreatment, no inhibitory changes of SSC distribution were observed after blocking the interaction between ScR and particles, neither with poly-I nor with fucoidan. Actually, at the higher dose (40 µg/cm2), poly-I preincubation significantly elevated the increase in the median SSC compared with cells that were treated with DQ12 alone (Fig. 1C). In contrast, the histograms of cells pretreated with the FcR-Ab, M-{alpha}-D-MP, mannan, or CR-Ab showed alterations of SSC distributions toward smaller SSCs compared with macrophages without inhibition. Thereby, the strongest effects were observed after blocking the FcR. Preincubation with the FcR-Ab after treatment with both quartz doses caused a significant reduction of uptake, ~2.5 fold. Inhibition of MR resulted in a significant reduction of ~5% after the low and 15% after the high dose treatment. Also, CR inhibition was found to provoke similar inhibitory effects on quartz phagocytosis, but the effect reached a statistical significance only with 40 µg/cm2 DQ12 treatment. Control experiments for the application of the FcR and the CR antibody using the corresponding mouse IgGs, IgG1 as well as IgG2a, showed no effect on the uptake of quartz particles (Fig. 1D).

FcR distribution and morphological features during DQ12 phagocytosis in macrophages. To validate the results obtained from the flow cytometric approach, which indicate the important role of the FcR in quartz phagocytosis, immunocytochemical investigations were performed. Representative fluorescent images of FcR immunocytochemistry and corresponding phase-contrast images are presented in Fig. 2, A–D, to visualize characteristic changes of receptor distribution due to quartz phagocytosis. Whereas in untreated cells (A), a uniform receptor distribution is observed, a rearrangement of the receptor within the macrophages is perceived 15 min after treatment with 10 µg/cm2 DQ12 (B). Since such staining patterns are predominantly occurring at sites of particle-cell interaction, and absent at sites where particles do not associate with cells, the appearing fluorescence is not an artifact. This staining pattern was observed in ~80% of cells that were in contact with particles, indicating the involvement of the FcR in DQ12 phagocytosis. Further immunocytochemistry experiments revealed that such changes in staining were not observed upon particle treatment for the MR and CR (data not shown).


Figure 2
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  		Fig. 2. Microscopic analysis of FcR distribution and morphological features of NR8383 macrophages during DQ12 phagocytosis. A–D: representative fluorescence and phase contrast images (magnification x1,000) obtained after FcR immunocytochemistry from cytospin preparations of NR8383 cells without (A and C) or after quartz treatment (15 min, 10 µg/cm2; B and D). Receptor clustering around the nascent phagosome is indicated by arrows. E–G: transmission electron microscopic images of ultrathin section of embedded NR8383 cells treated with 10 µg/cm2 DQ12 for 24 h. Images reflect the morphology of the DQ12 phagocytosis showing the formation of extensive pseudopodia to the particles (E), the generation of the phagosome under a wide spreading membrane extension (F), and the entire internalized phagosome after completed phagocytosis (G).

 
Furthermore, electron microscopic images (Fig. 2, E–G) reflect characteristic morphological changes during phagocytosis of DQ12 particles according to changes as described by the zipper mechanism (1). The first step of phagocytosis, the formation of extensive pseudopodia to the particle which should have been taken up, is shown in E. F demonstrates the generation of the phagosome by membrane closure behind the particle under an intensive membrane extension. The phagocytosis process is completed after the generation of an entire internalized phagosome (G) with a membranous structure.

Quartz particle uptake triggers and requires activation of PTK. To verify the importance of FcR for DQ12 uptake as observed in our present study, we focused on the FcR-specific signaling cascade described by the activation of specific PTKs and GTPases (49, 76). Therefore, we examined initially the activation of the kinases Lyn, Syk, and PI3K in macrophages after DQ12 treatment (Fig. 3).


Figure 3
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  		Fig. 3. Involvement of the protein tyrosine kinases (PTKs) Lyn, PI3K, and Syk in the phagocytosis of quartz particles. A–C: representative Western blots and densitometrical analyses of the phosphorylation of Lyn-(Tyr507) (A), PI3K (p85 unit, Tyr508) (B), and Syk-(Tyr525/526) (C) in quartz-treated NR8383 cells. Data are presented as means ± SE (n = 3) of the normalized optical density (OD) (control = 1) related either to total Lyn or β-tubulin (loading control). Post hoc LSD, *P < 0.05, **P < 0.01. D: DQ12 uptake after inhibition of the tyrosine kinase activity by pretreatment with genistein as analyzed by flow cytometry. Data are presented as means ± SE (n = 3) of the median SSC normalized to control (control = 100%). Post hoc LSD, **P < 0.01.

 
Representative Western blots and corresponding densitometric analysis showed with increasing DQ12 concentrations the dephosphorylation of Lyn at Tyr507, which is equated with the activation of the Src-kinase. Investigations over the time range from 5 to 60 min indicated that the strongest dephosphorylation effect occurred at 5 min (Fig. 3A).

Accordingly, phosphorylation of Syk at Tyr525/526 was examined (Fig. 3B). These investigations provide a trend in the activation of this kinase following 15-min particle treatment in a concentration-dependent manner as shown in the representative Western blot images and the densitometric analysis. In contrast, the expression of total Syk was found to stay constantly (Fig. 3B).

Furthermore, phosphorylation of PI3K at Tyr508 was investigated, showing an increase in the phosphorylation of the p85 unit of PI3K 10 min after treatment with 40 µg/cm2 DQ12 (Fig. 3C).

Additionally, inhibitory studies were performed to investigate the necessity of the PTK activity in the process of quartz phagocytosis. For this purpose, particle uptake was determined by flow cytometry after tyrosine kinases inhibition by genistein, using two different inhibitor concentrations of 10 or 50 µg/ml (Fig. 3D). Pretreatment of NR8383 macrophages with genistein at concentrations of 10 or 50 µg/ml inhibited particle uptake after 1 h of treatment in a concentration-dependent manner of ~15% and 60% with 10 or 40 µg/cm2 quartz, respectively. As such, the level of phagocytosis inhibition after using the high genistein concentration was found to be comparable to the results as obtained by FcR inhibition.

Quartz particle uptake triggers and requires activation and recruitment of GTPases. To analyze the activation pattern of small GTPases during quartz phagocytosis, affinity precipitations of Rac·GTP, Cdc42·GTP, and Rho·GTP were performed with lysates of DQ12-treated cells. Representative Western blots and the results of the corresponding densitometric analysis are shown in Fig. 4. The data demonstrated a dose-dependent increase of the GTP-bound form of Rac, which was significant at the high dose (Fig. 4A) as well as of Cdc42 (Fig. 4B) 15 min after quartz treatment. Following cell treatment for 60 min, the activity of both GTPases went back to the control level. In contrast, Western blot analysis of Rho did not show any activation (Fig. 4C).


Figure 4
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  		Fig. 4. Involvement of GTPases Rac1, Cdc42, and Rho in the phagocytosis of quartz particles. A–C: representative Western blots and densitometrical analyses of the GTPases activity after quartz treatment (15 and 60 min, 10 or 40 µg/cm2) analyzed by Western blotting using affinity precipitation products GTP-forms Rac1·GTP (A), Cdc42·GTP (B), and Rho·GTP (C). Densitometrical results are presented as means ± SE (n = 3). Post hoc LSD, *P < 0.05, **P < 0.01. D and E: DQ12 uptake after inhibition of the GTPase activity by lovastatin (D) or after blocking Rac1 with NSC23766 (E) analyzed by flow cytometry. Data are presented as means ± SE (n = 3) of the median SSC normalized to control (control = 100%). Variance analysis, *P < 0.01, ***P < 0.001.

 
Furthermore, the necessity of the small GTPases for the DQ12 uptake process was investigated using lovastatin or NSC23766. Lovastatin is known to inhibit GTPase activation by blocking their prenylation (51, 54), whereas NSC23766 is known to selectively block Rac1 activation due to binding to the specific GTP/GDP-exchange site of Rac1 (30). Analysis of particle uptake by flow cytometry demonstrated the general involvement of small GTPases by lovastatin preincubation (Fig. 4D) as well as the specific participation of Rac1 using NSC23766 (Fig. 4E) in the quartz particle uptake process. Preincubation of macrophages with various concentrations of lovastatin (10, 20, 40, and 80 µM) as well as NSC23766 (50 and 100 µM) diminished particle uptake in a concentration-dependent manner. Lovastatin pretreatment at the highest concentration (80 µM) showed an inhibitory effect that was similar in extent to that caused by FcR-Ab pretreatment.

The participation of Rac1 in the phagocytosis process of quartz particles was also independently investigated by immunocytochemistry. Representative phase-contrast (Fig. 5, A and B) and fluorescence (C and D) images of Rac1-stained cytospin preparations reveal changes in the Rac1 distribution between macrophages without (A and C) or after 15-min treatment with 10 µg/cm2 DQ12 (B and D). Whereas the fluorescence image of control cells showed a relative uniform distribution of Rac1 over the entire cell (C), particle treatment caused a staining pattern in which Rac1 appears to concentrate around the particle containing nascent phagosome (D), indicative of its participation in the phagocytosis process. These specific staining features were observed in ~70% of cells that interacted with DQ12 particles.


Figure 5
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  		Fig. 5. Participation of Rac1 in the phagocytosis process of quartz particles. A–D: representative phase contrast (A and B) and fluorescence (C and D) images (magnification x1,000) after Rac1 immunocytochemistry of cytospin preparations from untreated NR8383 cells (A and C) or cells after 15-min treatment with 10 µg/cm2 DQ12 (B and D). Recruitment of Rac1 (D) to the nascent phagosome (B) is indicated by arrows.

 
Comparison between DQ12 and FcR-Ab induced inflammatory response. Finally, we compared the inflammatory responses induced by quartz particles and specific receptor ligand binding. Therefore, we examined ROS generation as well as TNF-{alpha} release from the NR8383 cells upon treatment for 1 h with 10 or 40 µg/cm2 DQ12, or alternatively with the specific FcR-Ab (Fig. 6). Data obtained from the flow cytometric measurement (Fig. 6A) showed a dose-dependent trend towards increased intracellular ROS after quartz treatment. The ROS level observed in the cells due to the high quartz dose treatment was similar to those observed after incubation with the FcR-Ab. Similar results were found in the EPR analysis using DMPO spin trapping (Fig. 6B). Finally, the TNF-{alpha} release from macrophages treated with the highest quartz dose was similar to the release observed in FcR-Ab-treated macrophages, although the former effect did not reach a statistical significance (Fig. 6C).


Figure 6
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  		Fig. 6. Comparison of inflammatory response by NR8383 cells upon treatment with quartz particles or FcR antibodies. A and B: intracellular (A) and extracellular (B) ROS generation by NR8383 cells incubated for 1 h with DQ12 (gray bars) or the specific FcR-Ab (darker bars) as measured by flow cytometry (A) and electron paramagnetic resonance (EPR) (B), respectively. Data are presented as means ± SE of the median FL1-height (DHR fluorescence) of 3 independent experiments done in triplicate. Data obtained by EPR are presented as means ± SE of the sum of the EPR signal intensity of 4 independent experiments. C: TNF-{alpha} release from NR8383 cells after 1-h treatment with DQ12 (gray bars) or the specific FcR-Ab (1 h, darker bar) as measured by ELISA. Data are presented as means ± SE normalized to control of 3 independent experiments. Post hoc LSD, **P < 0.01.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Persistent pulmonary inflammation in response to quartz exposure has been demonstrated through a variety of animal studies and is nowadays accepted as a key event in quartz pathogenicity (3, 4, 21, 22, 39, 65). Our previous studies have demonstrated that DQ12 uptake in NR8383 lung macrophages is an actin-dependent classic phagocytosis process whereby actin inhibition was found to have significant impact on particle-induced ROS generation and TNF-{alpha} release. The aim of our current investigation was, therefore, to examine the participation of different classic phagocytosis receptors in quartz particle uptake and their potential responsibility for the inflammatory response. For this investigation, the rat alveolar macrophage cell line NR8383 was used, because our earlier observations concerning their particle uptake rate, compared with primary alveolar macrophages, indicated that this cell line represents a valid model to investigate particle uptake (36).

The first major result was obtained from flow cytometry indicating the significant role of the Fc{gamma}R in quartz particle uptake by alveolar macrophages. The inhibition of this receptor using a blocking antibody resulted in a clear reduction of quartz particle uptake to an extent that could neither be observed after inhibition of other phagocytosis receptors (ScR, MR, or CR) nor could be increased by the simultaneous inhibition of these four classic phagocytosis receptors together (data not shown). Furthermore, the inhibition of the actin component of the cytoskeleton in our previous investigations (36) could not afford a stronger inhibition than that as observed in present study. Also, pretreatment of NR8383 cells with poly-I or fucoidan, to inhibit the ScR, was found not to cause a reduction of DQ12 phagocytosis. In contrast, at the higher quartz dose, poly-I pretreatment led to an increased particle uptake. This indicates that in our experimental setup, the ScR does not participate in quartz uptake in a direct manner in this alveolar macrophage cell line. Our current findings are in contrast to earlier observations, where ScR was identified as a main factor for the uptake of unopsonized particles including quartz, TiO2, or FeO3 (6, 47, 58, 59). The reason for these contrary results might be opsonizing effects due to the fact that we used complete culture medium in our studies. In contrast, Kobzik and coworkers (6, 47, 58) performed their uptake studies in balanced salt solution resulting in the described phagocytosis of unopsonized particles. Under our experimental terms, opsonizing effects by medium constituents including immunoglobulins are likely to occur. Nevertheless, under low-opsonin conditions, as, for instance, in the healthy lung, other pathways may operate, than the one involving CD32. Current results are also in line with a previous study from our lab explaining the increased uptake rate of PVNO-surface-modified quartz particles compared with native particles in NR8383 cells by opsonizing effects (2). Such opsonizing effects have already been shown in earlier studies examining absorption of serum immunoglobulins and components of the complement cascade by silica particles. Plasma-treated crystalline quartz has been shown to bind with low extent to CR3b and highly to IgG, IgA, and IgM. Further on, the increased expression of Fc{gamma}RI, as well as CR3 and CR4 after interferon-{gamma} preconditioning, has been demonstrated to enhance phagocytosis of plasma-treated quartz particles compared with native quartz (37). This corresponds to our current observations on quartz uptake after receptor inhibition (Fc{gamma}R >> CR). Together, these findings indicate that quartz particle uptake by NR8383 cells is mainly processed by the Fc{gamma}R after opsonization with IgG. Notably, the lung surfactant contains, besides lipids, other chemical species, antioxidants, and proteins (and here mainly immunoglobulins) that can interact specifically with inhaled minerals (28). Lung surfactant, and specifically also its main protein component surfactant protein A (SP-A), has, for instance, been known to enhance phagocytosis of bacteria by AM (52, 57). A recent study revealed that SP-A enhances uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake (50).

Despite the fact that these studies support our concept of an Fc{gamma}R-mediated quartz phagocytosis, the participation of MR and CR in the DQ12 uptake and a function of Fc{gamma}R as coreceptor cannot be completely excluded. Synergistic effects have for instance been shown to occur between Fc{gamma}R and CR (34) and more recently between FcR and ScR (43). However, the importance of the FcR for quartz phagocytosis in NR8383 cells was further supported by investigations using immunocytochemistry as well as TEM. Phagocytosis-related changes in receptor distribution show the recruitment of the Fc{gamma}R to the nascent phagosome. The variance in morphology during quartz uptake observed by TEM indicates a membrane-extensive uptake process described as zipper mechanism for the Fc{gamma}R-mediated phagocytosis (1).

To verify the importance of Fc{gamma}R for DQ12 uptake and associated generation of inflammatory mediators, we investigated the quartz-induced signaling cascade activation. Here we focused on downstream signaling molecules, which are described to mediate FcR-dependent phagocytosis (18, 25, 32, 33, 73). The activation of the Src-kinase Lyn, suggested to be responsible for the phosphorylation of the immunoglobulin tyrosine activation motive (ITAM) and clustering of the receptor, has been described as the first step of the signaling pathway during Fc{gamma}R-mediated phagocytosis. Thereby, the Src-kinase gets activated by tyrosine dephosphorylation causing conformational changes releasing the catalytical domain from an intramolecular slope. It is suggested that Src-kinases supported by lipid rafts promote Fc{gamma}R-mediated phagocytosis by phosphorylation of ITAM, transmitting the inactivating phosphate (12, 17, 25, 40, 49, 71). Our present results show a dose-dependent effect of quartz on Lyn dephosphorylation at Tyr507 that is equated with the activation of the Src-kinase. Phosphorylation of ITAM induces the phosphorylation of Syk and is described to be essential for inducing actin recruitment during Fc{gamma}R phagocytosis (18, 45). Processes under the reorganization of the actin cytoskeleton as well as the plasma membrane are critical steps of Fc{gamma}R phagocytosis, because necessary morphological changes, e.g., extensive pseudopodia extension to the particles, engulfment of the particle and its ingestion, were induced. Therefore, activation of the PTK Syk is required to induce the activation of GTPases, responsible for actin recruitment (18) as well as PI3K, sensible for membrane ruffling (19, 27, 32). Activation of PI3K is mediated by activated Syk through the phosphorylation of the regulatory unit p85. This activates the catalytic unit, which allows for its participation in the membrane reorganization process (74, 76). In the present study, we could show effects of DQ12 in NR8383 cells on the phosphorylation of both Syk (Tyr525/526) and the p85 unit of PI3K. The necessity of PTK for DQ12 phagocytosis could be further supported by using its inhibitor genistein, which caused an uptake inhibition similar to that seen by the FcR-Ab.

The activation of GTPases necessary for actin polymerization during the Fc{gamma}R phagocytosis process was subsequently also investigated in our study. Importantly, the specific GTPase activation pattern is conditioned by the involved phagocytosis receptor and varies between Fc{gamma}R, MR, or CR. Our results demonstrate that quartz triggers the activation of the GTPases Rac1 and Cdc42, whereas Rho remains unaffected. This is in line with the literature, where activation of the small GTPases Rac and Cdc42 by Syk, either directly or indirectly, is described as a specific pattern for Fc{gamma}R-mediated phagocytosis (18, 38). In contrast, uptake by CR is mediated under the activation of RhoA and independent of the activation of Syk, Rac, and Cdc42 (13, 53), whereas MR-mediated phagocytosis requires the activation of all three GTPases (79). The contribution of GTPases, especially Rac1 in the DQ12 uptake, could be further shown in our study using flow cytometric approaches by inhibition of their activation using lovastatin or NSC23766, respectively, as well as in the Rac1 immunocytochemistry. Conspicuous is the time dependency of the activation of kinases and GTPases. Activation of Lyn, for instance, shows a maximum as early as 5 min after particle treatment, which is in line with observations showing a maximum of Lyn activity in J774A.1 macrophages 2 min after treatment with TiO2 (60). Together with the overall kinetics of activation as observed with specific downstream signaling proteins, including Syk, PI3K, and the GTPases Rac1 and Cdc42, this further supports the importance of the FcR in quartz phagocytosis.

To investigate the relevance of this specific phagocytosis pathway for the inflammatory response of macrophages, we have investigated the generation or release of inflammatory mediators, specifically ROS and TNF-{alpha} following particle treatment as well as after receptor stimulation with its specific ligand. Here, remarkably similar trends in ROS generation as well as TNF-{alpha} release were found with quartz as well as Fc{gamma}RII antibodies. The explanation for quartz uptake by opsonization of quartz with IgG, which then contributes to the release of inflammatory mediators, is supported by the observation that IgG preincubation of quartz increases its bioreactivity followed by an increased ROS generation in AM (62). ROS generation by different receptors, e.g., Fc{gamma}RII/III or CR3, is catalyzed under phosphorylation of diverse PTKs via the activation of the NADPH-oxidase complex (7, 26, 61, 72) whereby Rac1 represents one unit of this ROS-generating enzyme complex. Furthermore, the activation of the GTPases is necessary for the formation of NADPH oxidase and subsequent superoxide generation, by the induction of actin reorganization (61). The relation between particle uptake and ROS generation was already shown in a previous study, demonstrating the significant reduction of ROS generation as well as TNF-{alpha} release upon inhibition of the actin cytoskeleton using cytochalasin D (36). Along these findings, Imrich and coworkers (41) demonstrated earlier, by the use of various different particles, i.e., quartz, titanium dioxide, and residual oil fly ash, a correlation between the number of internalized particles and the DHR oxidation as measurement for ROS generation. The relation between quartz-induced ROS generation, activation of redox-sensitive transcription factors like NF-{kappa}B, and subsequent TNF-{alpha} release by macrophages is already described by Castranova (14, 15).

In summary, in this study we have demonstrated the involvement of the Fc{gamma}R in the uptake of quartz particles by NR8383 macrophages by showing: 1) inhibition of particle uptake in the presence of FcyRII antibody, 2) Fc{gamma}RII clustering at the particle binding site, and 3) activation of the Fc{gamma}R-specific signaling cascade, involving Lyn, Syk, PI3K, as well as Rac1 and Cdc42. Our findings are summarized in the scheme of Fig. 7 describing Fc{gamma}RII as crucial for the uptake of quartz in our study. The possible relevance of the Fc{gamma}R-mediated quartz particle uptake by AM and its downstream signaling cascade activation for the activation of inflammatory mediator release was revealed by the similarities in ROS generation and TNF-{alpha} release by the AM upon treatment with the Fc{gamma}R-specific ligand (CD32 antibody) and quartz particles. In future studies, the role of other FcR (i.e., CD16, CD64) in quartz-mediated macrophage activation remains to be elucidated as well as their importance compared with other receptors (e.g., CR). It also remains to be investigated to what extent our current in vitro observations hold true for in vivo studies and in realistic human exposure scenario in relation to particle dosimetry and the specific microenvironments (e.g., surfactant, proteins, immunoglobulin content) of healthy vs. inflamed lung.


Figure 7
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  		Fig. 7. Proposed DQ12-induced Fc{gamma}R signaling cascade in macrophages.

 
The further discovery of the specific signaling cascade induced by quartz particle uptake in lung macrophages may help us to understand the molecular mechanisms of particle-macrophage interaction. In addition, such investigations allow for identification of possible molecules that can be targeted in intervention strategies that aim at inhibiting inflammatory mediator release from macrophages without affecting their phagocytosis capacity and hence their crucial clearance function in the lung.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is financially supported by the German Research Council (DFG, IGK738), the Federal Ministry of the Environment (BMU), as well as the Research Commission of the Heinrich Heine University, Duesseldorf.


   ACKNOWLEDGMENTS
 
We are thankful to Dr. U. Kraemer for support with the statistical analysis. We thank Jason Hellmann for help with the English language in this manuscript.

Present address of P. Haberzettl: Institute of Molecular Cardiology, University of Louisville, Louisville, KY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Albrecht, Particle Research, Institut für Umweltmedizinische Forschung at the Heinrich Heine Univ., Auf'm Hennekamp 50, 40225 Düsseldorf, Germany (e-mail: catrin.albrecht{at}uni-duesseldorf.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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