Published ahead of print on June 12, 2008, doi:10.1165/rcmb.2008-0046OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2008-0046OC The Phagocytosis of Crystalline Silica Particles by Macrophages1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut Correspondence and requests for reprints should be addressed to David A. Knecht, Department of Molecular and Cell Biology, 91 North Eagleville Road, Unit 3125, University of Connecticut, Storrs, CT 06269-3125. E-mail: david.knecht{at}uconn.edu
Silicosis is a chronic lung disease induced by the inhalation of crystalline silica. Exposure of cultured macrophages to crystalline silica leads to cell death; however, the mechanism of cell–particle interaction, the fate of particles, and the cause of death are unknown. Time-lapse imaging shows that mouse macrophages avidly bind particles that settle onto the cell surface and that cells also extend protrusions to capture distant particles. Using confocal optical sectioning, silica particles were shown to be present within the cytoplasmic volume of live cells. In addition, electron microscopy and elemental analysis showed silica in internal cellular sections. To further examine the phagocytosis process, the kinetics of particle uptake was quantified using an assay in which cells were exposed to ovalbumin (OVA)-coated particles, and an anti-OVA antibody was used to distinguish surface-bound from internalized particles. Fc receptor–mediated uptake of antibody-coated silica particles was nearly complete within 5 minutes. In contrast, no OVA-coated particles were internalized at this time. After 30 minutes, 30% of bound silica was internalized and uptake continued slowly thereafter. OVA-coated latex beads, regardless of surface charge, were internalized at a similarly slow rate. These results demonstrate that macrophages internalize silica and that nonopsonized phagocytosis occurs by a temporally, and possibly mechanistically, distinct pathway from Fc receptor–mediated phagocytosis. Eighty percent of macrophages die within 12 hours of silica exposure. Neither OVA coating nor tetramethylrhodamine isothiocyanate labeling has any effect on cell death. Interestingly, antibody coating dramatically reduces silica toxicity. We hypothesize that the route of particle entry and subsequent phagosome trafficking affects the toxicity of internalized particles.
Key Words: silica • phagocytosis • cell death
Silica is the most abundant element on Earth, and crystalline silica dioxide is the predominant form of silica in the environment. As a result, crystalline silica is also the major component of airborne dust generated by wind, manufacturing, or demolition. Chronic inhalation of crystalline silica leads to silicosis, a disease characterized by inflammation and fibrosis of the lung (1). When environmental particles are inhaled, the particulate matter is deposited in the lungs. Silica appears to affect epithelial cells, fibroblasts, and lung macrophages (2–5). Prolonged deposition of particles in the alveoli or bronchioles leads to lung inflammation, the formation of fibrotic scar tissue, and degradation of the mucociliary escalator (1). Even though silicosis is preventable through the use of respirators, this simple expedient is often not used, either through neglect or circumstances, as in the collapse of the World Trade Center (6). Of the 100,000 workers in the United States exposed to crystalline silica, about 250 die each year from silicosis (USDL statistics). Exposure of cells to crystalline silica is known to cause cell death, and it is presumed that this toxicity is the underlying cause of silicosis (1, 4, 7). Although the interaction of silica with cells has been intensively studied, the molecular mechanisms underlying this toxicity are not well understood (8). There is evidence that silica causes the production of free radicals and reactive oxygen species (9–11) and that some cells die due to activation of the apoptotic cascade (12–14). However, the actual mechanism of silica-induced cell death is still unclear. There are three major pathways by which biological particles are recognized and internalized by immune cells. Antibody-coated particles are recognized by the Fc receptor, complement-coated particles by the complement-receptor, and particles such as yeast by the mannose receptor (15, 16). The molecular mechanisms by which cells interact with nonbiological particles such as silica, however, are not clear. One hypothesis is that the scavenger receptor (SR-A) is responsible for binding silica and other uncoated particles (17–20). The three major members of the scavenger receptor family are SR-AI and SR-AII, derived from alternative splicing, and MARCO (macrophage receptor with collagenous structure). These receptors all share collagen-like and cysteine-rich domains in their extracellular regions. At the collagen-like domain, a lysine cluster forms a positive-charged groove to interact with negative-charged particles, such as LDL (21, 22). Silica has a negative surface charge, and this has led to the hypothesis that silica is recognized by a similar mechanism (23, 24). When cells are pretreated with scavenger receptor blockers (24–26), or when alveolar macrophages from an SR-A knockout mouse are exposed to particles (17, 27), binding and toxicity are reduced but not eliminated. These results suggest that there may be multiple mechanisms of binding and uptake of particles. When biological particles bind to the surface of cells, sometimes they are internalized via the process of phagocytosis. Previous work has shown that environmental particles bind to cells, but whether they are phagocytosed is less clear. Electron microscopy of sections of lung tissue and cultured cells shows internal silica particles, but this technique does not allow quantification of uptake or the relationship of uptake to cell death (28, 29). Flow cytometry has been used extensively to characterize the association of opsonized (antibody-coated) and nonopsonized (uncoated or nonspecific protein–coated) particles with cells, but the assays used do not distinguish between surface-bound and internalized particles. Furthermore, much of the work on nonopsonized particles focuses on latex beads and titanium dioxide particles, and less on silica (17, 18, 30–32). Thus, the cellular fate of the silica particles has not been directly determined. This issue is critical to understanding silica toxicity because if particles were mostly on the surface of cells, then toxicity would likely be triggered at the plasma membrane. Internalization would suggest numerous other potential cytoplasmic targets. We have developed a quantitative phagocytosis assay that allows us to compare the internalization of silica and other nonbiological particles with uptake by the Fc receptor–mediated pathway. The results show that nonopsonized particles are internalized slower than opsonized particles, but silica toxicity is dependent upon the route of entry of the particles.
Chemicals All chemicals were from Sigma (Sigma Chemical Co., St. Louis, MO) unless otherwise noted.
Cell Lines
Particle Types
Particle Coating Methods Coating charged latex beads with OVA. Positive- or negative-charged latex beads were coated with OVA according to manufacturer's instructions (Polysciences 238F and 238G protocols) using carbodiimide for carboxy latex beads and gluteraldehyde for amino latex beads. Coating silica and latex beads with antibody. Rabbit anti-chicken egg albumin (Immunology Consultants Laboratory, Newberg, OR) at 0.2 mg/ml in PBS was added to OVA-coated particles and tumbled for 90 minutes at 37°C. For all three protocols, particles were centrifuged and washed three times in PBS before use. Labeling of silica with tetramethylrhodamine isothiocyanate. Two milligrams of crystalline silica was added to 1 ml of PCB containing 50 µg/ml of tetramethylrhodamine isothiocyanate (TRITC)-R (Sigma) and tumbled for 8 hours at 37°C. The particles were then centrifuged and washed with 50 mM NH4Cl, pH 7.25, for 2 hours at 37°C while tumbling to stop the reaction. Particles were then washed six times in PBS and stored in PBS at 4°C.
Live Cell Particle Binding Assay
Confocal Particle Imaging Assay
Electron Microscopy
Quantitative Phagocytosis Assay The antibodies are not membrane permeable in fixed cells, so only external particles become fluorescent. To quantify particle internalization, labeling of particles by antibody was determined visually. The number of surface-bound particles was counted from the fluorescence images, and the total number of particles from the DIC images. The amount of phagocytosis per cell was quantified by subtracting the number of fluorescent particles from the total number of particles to derive the number of internalized particles. At each time point, this number was divided by the total number of particles to derive the percent phagocytosis. The error bars depict standard error from three separate experiments.
Cell Death Assay
Silica-Induced Cell Death Is Time and Concentration Dependent It is well known that silica induces cell death in macrophages, but the cells are usually exposed to concentrations of 50 µg/cm2 or higher. To study the trafficking of individual particles, macrophages must be exposed to lower concentrations of silica to be able to clearly distinguish the number of particles associated with each cell. MH-S and RAW264.7 macrophages were exposed to varying concentrations of silica to determine the lowest concentration at which particles are toxic to cells. At 0, 4, and 10 hours, images were taken to quantify the proportion of dead cells. Concentrations of silica as low as 15 µg/cm2 caused greater than 60% of cells to die by 10 hours (Figure 1 and data not shown). By examining cells treated with low amounts of silica, it appears that cells with more than three to five bound particles usually die, while cells with less than three particles remain alive. Based on these results, a concentration of 15 µg/cm2 of silica was used for all subsequent experiments. This represents an average of eight particles per cell. Latex bead concentrations were adjusted to present cells with similar numbers of particles.
Macrophages Gather Particles by Extending Protrusions To learn how cells interact with silica particles, time-lapse video microscopy of macrophages before and after silica or latex bead exposure was performed. When silica particles are added to the media, they rapidly settle to the bottom of the dish. Some particles land directly on the apical cell surface and remain adhered, while others land on the surface near the cells. After a few hours, there is an increase in the number of particles on the cells and a clear zone is created around each cell as these distant particles are captured (Figure 2A). The clear zones are formed as cells randomly extend membrane protrusions up to about 7 µm away. When these protrusions encounter a particle, the membrane moves underneath the particle and then the particle is either transported along the surface of the extension, or the membrane is retracted, bringing the particle to the cell body (Figures 2B and 2C; see also the movie in the online supplement). Thus, cells actively recruit nearby particles by movement and membrane extension. Once associated with the cell, particles do not appear to be subsequently lost.
Silica Particles Are Phagocytosed by Macrophages Opsonized (antibody-coated) and nonopsonized (uncoated or nonspecific protein–coated) nonbiological particles such as titanium, diesel exhaust, and silica have been extensively studied by flow cytometry to determine their association with macrophages (15, 20, 28–30, 34–36). Although it is clear that macrophages bind quartz silica (17, 24–27), there is little information about the extent and timing of particle phagocytosis. To directly address this question, confocal microscopy was used to visualize the three-dimensional localization of particles associated with cells. The cytoplasm of MH-S macrophages was labeled with 5-chloromethylfluorescien diacetate (CMFDA), and then the cells were exposed to silica that was fluorescently labeled with tetramethylrhodamine. After 1 hour of incubation, confocal optical sectioning of the cells was performed to generate a volumetric data set. Red fluorescent particles were seen surrounded by the green fluorescent cytoplasm of macrophages when re-sectioning of an X-Y-Z-stack was performed (Figure 3A, and movie in the online supplement). Red particles bound to the surface were not surrounded by green cytoplasm. To verify that the fluorescent labeling of the particles was not affecting the uptake process, unlabeled particles were examined using confocal reflection mode. Unlabeled silica particles were also seen surrounded by green cytoplasm, verifying that they are phagocytosed as well (Figure 3B, and movie in the online supplement).
Elemental X-Ray Analysis Confirms the Presence of Silica Particles Inside Macrophages An alternative way to demonstrate the internalization of silica is by electron microscopy (28, 29). MH-S macrophage cells were exposed to silica particles for 2 hours before the cells were fixed, sectioned, and prepared for transmission electron microscopy. Holes were frequently found in the sections of silica-treated cells that are presumed to be due to particles being knocked out of the section by the diamond knife during sectioning. Periodically, a particle remained associated with the section through processing and could be seen in the EM image. Ultrathin sections showed electron dense particles within the cells (Figure 4A). To confirm that this particulate matter was silica, it was subjected to X-ray microanalysis. Both particles 1 and 2 show a characteristic peak at 1.741 keV, indicating the presence of silicon (Figure 4B, and data not shown). Some large silica particles were displaced during the course of sectioning, leading to the appearance of knife marks (Particle 2, Figure 4A). Elemental analysis on a control area (Figure 4A) showed much lower silicon peaks as compared with the particles (Figure 4B), confirming that these peaks do not arise from embedding medium or stain. These data further support the presence of silica within the cells.
Kinetics of OVA-Coated Particle Phagocytosis by Macrophages There has been little investigation into the mechanism by which uptake of nonopsonized particles occurs. Although the confocal and electron microscopy assays confirm that nonopsonized silica particles are taken up by cells, it would be difficult to use these assays to quantify the kinetics of particle internalization. Therefore, an assay was developed that uses the same principle as commonly used for measuring Fc receptor–mediated phagocytosis (37–39). Silica particles were coated with a nonspecific glycoprotein (OVA) and added to cells. Cells were fixed but not permeabilized at various time points and fluorescently stained with an OVA-specific antibody. Since antibodies cannot penetrate intact cells, the antibody only binds to the external particles, allowing internalized and external particles to be distinguished (Figures 5A and 5B). The total number of particles can be determined directly by DIC imaging.
Using this assay, the uptake rate of nonopsonized (OVA-coated particles) and opsonized (the same particles treated with anti-OVA antibody) particles was determined. Consistent with previous work, within 5 minutes almost all antibody-coated latex beads were internalized (38, 40, 41). The same result was found for antibody-coated silica, indicating that the uptake of silica by the Fc receptor is similar to the uptake of other types of opsonized particles. In contrast, there was no internalization of nonopsonized particles at this time. Within 15 minutes, nonopsonized particles had begun to be internalized and, by 30 minutes, there was about 30% uptake of all nonopsonized particle types. Uptake plateaued at about 50% of particles internalized by 180 minutes. Interestingly, OVA-coated carboxy (negative-charged), amino (positively charged) latex beads, and OVA-coated silica were all internalized on a similarly slow time scale (Figure 5C). This result indicates that particle surface charge does not affect the recognition and internalization process (Figure 5C). In addition, the uptake of 1-µm and 3-µm latex beads occurred on a similar time scale (Figure 5C), indicating that the heterogeneous size of the crystalline silica is not a factor in the uptake rate. One potential concern with this internalization assay is that coating particles with OVA might affect the uptake of silica or latex particles. Several control experiments indicate that this is not the case. First, the uptake of unlabeled and TRITC-labeled silica was measured using the confocal microscopy assay. The proportion of both types of nonopsonized silica internalized after 1 hour was consistent with the results found using the OVA antibody assay (data not shown). Second, if soluble ovalbumin was added to the BF media before the addition of uncoated or OVA-coated silica particles, there was no decrease in the number of particles bound per cell nor the amount of particles internalized over time.
Coating Silica Particles with Antibody Dramatically Reduces Their Toxicity
Fc Receptor Cross-Linking Does Not Suppress Silica-Induced Cell Death Since antibody-coated silica suppressed cell death, it is possible that the activation of macrophages by cross-linking of the Fc receptors is sufficient to prevent cell death induced by silica. To test this hypothesis, cells were pretreated with antibody-coated latex beads and then subsequently challenged with silica particles. Different concentrations of antibody-coated 1-µm latex beads or antibody-coated 3-µm spherical silica (not shown) were added to cells to promote cross-linking of the Fc receptors. After 15 minutes, uncoated 3-µm silica was added to these cells and cell death was measured. In all cases, pretreatment of cells with opsonized particles did not prevent silica-induced cell death (Figure 7). Thus, the suppression of toxicity by antibody-coating the silica particles does not appear to be due solely to activation of the macrophages by Fc receptor cross-linking.
The fact that cells interact with environmental particles like silica has been known for some time, and it has been presumed that these particles are internalized. However, the only data that directly demonstrate internalization comes from TEM images of particles in sections of cells exposed to silica (28, 29). This methodology, however, is not amenable to quantitative analysis of the internalization of particles. Most work has used flow cytometric measurement of right angle scatter to detect cell-associated particles (17, 18, 30–32). However, the particles scatter light whether they are inside or outside the cell, so the increase in side scatter essentially measures the total association of particles with cells. One way to estimate the proportion of internalized particles is to treat cells with cytochalasin D, an inhibitor of actin filament elongation that primarily inhibits phagocytosis but not binding of particles (31). Consistent with the idea that some particles are internalized, treatment of cells with cytochalasin D decreased, but did not eliminate, the right angle scatter increase of cells treated with silica (18, 29). To examine to what extent particles are internalized, we have developed assays that allow us to directly observe and quantify the internalization process. Cultured macrophages extend protrusions and move randomly on glass or plastic surfaces. Since particles do not release any chemical signals, it is presumed that, unlike bacteria, macrophages in vivo or in vitro encounter particles accidentally. Whether the particles settle onto the surface or make contact through membrane protrusions, the particles appear to stick avidly to the cell surface. When a pseudopod or lamellipod encounters a particle, the protrusion extends underneath and beyond the particle, and then draws the particle back to the cell body (Figure 2). Particles do not seem to fall off of the cells once they are bound, and we have not seen any evidence of particle exocytosis. We have used several techniques to verify and quantify the internalization of nonopsonized silica and latex particles. Using light microscopy, it is straightforward to visualize the amount and diverse size of crystalline silica bound to each cell. However, to determine if these particles are phagocytosed, several assays were used. Examining the cell volume by confocal optical sectioning or looking at EM thin sections both showed particles inside cells. However, these assays would be cumbersome for routine quantification. Therefore, a third assay was developed using OVA-coated latex beads and silica. These silica particles caused the same cellular toxicity as uncoated silica particles, so the coating process does not seem to markedly alter the silica. Adding soluble OVA to the media before exposure of nonopsonized particles also does not affect binding or internalization, indicating that the OVA does not play a role in the binding or uptake process. Consistent with the other uptake assays, within 60 minutes, 40% of particles were internalized. With this information, previous flow cytometry data can be seen as a measure of total particle association, comprising both internalized and surface bound particles, but the data overestimates the amount of particle internalization. However, it is not yet known if internalization is necessary for toxicity. Two surprising observations have come from direct measurement of the internalization of particles. First, internalization of either nonopsonized silica or latex is much slower than Fc receptor–mediated internalization of either particle type. The number of bound particles per cell remained fairly constant over the course of the assay, regardless of particle type. It is only the internalization of nonopsonized silica and latex particles that was slow, and the flow cytometry assay cannot distinguish between the internal particles and external particles. This may indicate that internalization of nonopsonized particles is, in some way, mechanistically different from Fc receptor–mediated uptake of opsonized particles. It should be noted that we have not measured particle–cell interaction in the presence of any sheer forces, so it is possible that the force of interaction may differ depending upon the particle type. The second key result is that the toxicity of silica is dramatically reduced if the particles are opsonized. This effect is not due to the fact that the particles are coated with protein since coating silica with OVA, bovine serum albumin, casein, or histone IIA had no effect on particle toxicity. It is interesting that coating silica with negative- or positive-charged proteins does not change their toxicity. Thus, it is more likely that the alteration in toxicity is due to targeting the particles to the Fc receptor–mediated uptake pathway. One hypothesis to explain this result would be that the opsonized particles activate the macrophages and that this activation somehow protects them from the toxic effects of silica. When Fc receptors are cross-linked by binding to opsonized particles, Syk and Src tyrosine kinases are phosphorylated and the cell becomes activated leading to an oxidative burst and expression of TNF and MIP-2 (42, 43). To test this hypothesis, cells were activated with antibody-coated latex beads and then exposed to silica. Surprisingly, this pretreatment was not protective. Both Fc receptor activation and silica have been shown to cause an increase in reactive oxygen species (ROS) production by macrophages (11, 18, 35, 36). If ROS were damaging to the cell, then it would seem likely that both particle types would be toxic, and that antibody-coated silica might be even more toxic. Since both particle types lead to ROS production, and only nonopsonized silica is toxic, either ROS does not play a role in the toxicity, or there is something different about the type of ROS, or the timing or location of its production. These results also raise the interesting possibility that the route of entry of particles into the cell affects their subsequent toxicity because some aspect of the signaling pathway associated with nonopsonized particle uptake, or some difference in vesicle trafficking of phagosomes after uptake, alters toxicity. How this relates to the exposure of inhaled particles to mucus, ECM proteins, and serum in the lung is not yet clear. Presumably, these molecules could coat the particles but, like OVA, this may not alter the particle's toxicity. It will be important to establish that alveolar macrophages isolated from bronchoalveolar lavage show the same particle uptake properties as the cultured cell lines that we have used. The mechanism by which the silica particles become bound to the surface of the cell is not clear. It seems unlikely that cells possess a specific receptor for nonbiological materials like silica, titanium, and latex. The literature suggests receptors of the scavenger receptor family-A play a role in silica binding and cell death (17–20). However, it is not clear that these receptors are solely involved since binding and subsequent silica-induced cell death still occur in the absence of these receptors (17, 27). We have found that the binding and rate of internalization of plain, negative-charged, or positive-charged latex beads are all about the same. In addition, nonopsonized silica are internalized at a similarly slow rate. Recently, we have found that manufactured amorphous silica spheres behave in a similar manner to crystalline silica (data not shown). Thus, neither surface charge nor composition of particles has a dramatic effect on binding or internalization. Moreover, these experiments were performed in serum-free media. Therefore, media components are not promoting internalization. These results appear to be inconsistent with the idea of scavenger receptor interacting with a negative-charged surface of the particle, although it is possible that there are negative-charged surface patches even after protein coating. Whether particle internalization is required for cell death is currently unknown. Internalization is prior to any of the known events associated with the cell death pathway (14). It will be important to find a way to block uptake without blocking binding to answer this question. It has been noted that, if silica is killing macrophages, and the macrophages are responsible for eating dying cells, then the lung would likely enter a futile cycle in which silica-exposed macrophages undergo cell death while more macrophages are attracted to the site. Whether internalized silica, which is presumably inside the phagosomal membrane of dying macrophages, is itself toxic to cells is unknown; it will be important to answer this question to get a better understanding of the events that occur in the lung that lead to silicosis.
The authors thank Dr. Andrea K. Hubbard for the Min-U-Sil 5 Crystalline Silica, MH-S macrophage cells, and for many informative discussions; Dr. Laura Machesky for the RAW264.7 macrophage cells; Dr. Carol Norris of the Confocal Microscopy Facility for microscopy assistance; and Dr. Marie Cantino of the Electron Microscopy Facility for elemental analysis assistance.
This work was supported by funding from The Robert Leet and Clara Guthrie Patterson Trust Fund to D.A.K., and from the National Institutes of Health (GM40599 to D.A.K.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1165/rcmb.2008-0046OC on June 12, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form January 23, 2008 Accepted in final form May 12, 2008
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Abstract
CLINICAL RELEVANCE
peak. Similar results were seen for particle 2 (not shown). (C) X- ray spectrum from cytoplasmic region of cell to determine background levels of Si from fixative and embedding material. The peak seen at 1.741 keV was much smaller than that of particle 2.
R or CR3. Nat Cell Biol 2000;2:246–248.