Self-Generated Chemoattractant Gradients: Attractant Depletion Extends the Range and Robustness of Chemotaxis.

Chemotaxis is fundamentally important, but the sources of gradients in vivo are rarely well understood. Here, we analyse self-generated chemotaxis, in which cells respond to gradients they have made themselves by breaking down globally available attractants, using both computational simulations and experiments. We show that chemoattractant degradation creates steep local gradients. This leads to surprising results, in particular the existence of a leading population of cells that moves highly directionally, while cells behind this group are undirected. This leading cell population is denser than those following, especially at high attractant concentrations. The local gradient moves with the leading cells as they interact with their surroundings, giving directed movement that is unusually robust and can operate over long distances. Even when gradients are applied from external sources, attractant breakdown greatly changes cells' responses and increases robustness. We also consider alternative mechanisms for directional decision-making and show that they do not predict the features of population migration we observe experimentally. Our findings provide useful diagnostics to allow identification of self-generated gradients and suggest that self-generated chemotaxis is unexpectedly universal in biology and medicine.

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

Bacterial metallothionein, PmtA, a novel stress protein found on the bacterial surface of Pseudomonas aeruginosa and involved in management of oxidative stress and phagocytosis.

Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in homeostasis and protection against heavy metal toxicity and oxidative stress. The opportunistic pathogen, Pseudomonas aeruginosa, expresses a bacterial MT known as PmtA. Utilizing genetically modified P. aeruginosa PAO1 strains (a human clinical wound isolate), we show that inducing pmtA increases levels of pyocyanin and biofilm compared to other PAO1 isogenic strains, supporting previous results that pmtA is important for pyocyanin and biofilm production. We also show that overexpression of pmtA in vitro provides protection for cells exposed to oxidants, which is a characteristic of inflammation, indicating a role for PmtA as an antioxidant in inflammation. We found that a pmtA clean deletion mutant is phagocytized faster than other PAO1 isogenic strains in THP-1 human macrophage cells, indicating that PmtA provides protection from the phagocytic attack. Interestingly, we observed that monoclonal anti-PmtA antibody binds to PmtA, which is accessible on the surface of PAO1 strains using both flow cytometry and enzyme-linked immunosorbent assay techniques. Finally, we investigated intracellular persistence of these PAO1 strains within THP-1 macrophages cells and found that the phagocytic endurance of PAO1 strains is affected by pmtA expression. These data show for the first time that a bacterial MT (pmtA) can play a role in the phagocytic process and can be found on the outer surface of PAO1. Our results suggest that PmtA plays a role both in protection from oxidative stress and in the resistance to the host's innate immune response, identifying PmtA as a potential therapeutic target in P. aeruginosa infection. IMPORTANCE: The pathogen Pseudomonas aeruginosa is a highly problematic multidrug-resistant (MDR) pathogen with complex virulence networks. MDR P. aeruginosa infections have been associated with increased clinical visits, very poor healthcare outcomes, and these infections are ranked as critical on priority lists of both the Centers for Disease Control and Prevention and the World Health Organization. Known P. aeruginosa virulence factors have been extensively studied and are implicated in counteracting host defenses, causing direct damage to the host tissues, and increased microbial competitiveness. Targeting virulence factors has emerged as a new line of defense in the battle against MDR P. aeruginosa strains. Bacterial metallothionein is a newly recognized virulence factor that enables evasion of the host immune response. The studies described here identify mechanisms in which bacterial metallothionein (PmtA) plays a part in P. aeruginosa pathogenicity and identifies PmtA as a potential therapeutic target.

Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages.

Chronic inhalation of crystalline silica is an occupational hazard that results in silicosis due to the toxicity of silica particles to lung cells. Alveolar macrophages play an important role in clearance of these particles, and exposure of macrophages to silica particles causes cell death and induction of markers of apoptosis. Using time-lapse imaging of MH-S alveolar macrophages, a temporal sequence was established for key molecular events mediating cell death. The results demonstrate that 80 % of macrophages die by apoptosis and 20 % by necrosis by clearly distinguishable pathways. The earliest detectable cellular event is phago-lysosomal leakage, which occurs between 30 and 120 min after particle uptake in both modes of death. Between 3 and 6 h later, cells undergoing apoptosis showed a dramatic increase in mitochondrial transmembrane potential, closely correlated with activation of both caspase-3 and 9 and cell blebbing. Externalization of phosphatidyl serine and nuclear condensation occurred 30 min-2 h after the initiation of cell blebbing. Cells undergoing necrosis demonstrated mitochondrial membrane depolarization but not hyperpolarization and no caspase activation. Cell swelling followed the decrease in mitochondrial membrane potential, distinguishing necrosis from apoptosis. All cells undergoing apoptosis followed the same temporal sequence, but the time lag between phago-lysosomal leakage and the other events was highly variable from cell to cell. These results demonstrate that crystalline silica exposure can result in either apoptosis or necrosis and each occurs in a well-defined but temporally variable order. The long time gap between phago-lysosomal leakage and hyperpolarization is not consistent with a simple scenario of phago-lysosomal leakage leading directly to cell death. The results highlight the importance of using a cell by cell time-lapse analysis to investigate a complex pathway such as silica induced cell death.

Chemotaxis: Under Agarose Assay.

The unicellular eukaryotic amoeba, Dictyostelium discoideum, represents a superb model for examining the molecular mechanism of chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folic acid. Under starved conditions, they lose their sensitivity to pterins and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including ease of growth, genetic tractability, and the conservation of mammalian signaling pathways. In this chapter, we describe the use of the under-agarose chemotaxis assay to understand the signaling pathways controlling directional sensing and motility in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect conserved pathways involved in eukaryotic chemotaxis.

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.

Teaching a biology laboratory course using Dictyostelium.

The Dictyostelium discoideum model system is a powerful tool for undergraduate cell biology teaching laboratories. The cells are biologically safe, grow at room temperature and it is easy to experimentally induce, observe, and perturb a breadth of cellular processes making the system amenable to many teaching lab situations and goals. Here we outline the advantages of Dictyostelium, discuss laboratory courses we teach in three very different educational settings, and provide tips for both the novice and experienced Dictyostelium researcher. With this article and the extensive sets of protocols and tools referenced here, implementing these labs, or parts of them, will be relatively straightforward for any instructor.

Micro-Acoustic-Trap (µAT) for microparticle assembly in 3D.

Acoustic tweezers facilitate the manipulation of objects using sound waves. With the current state of the technology one can only control mobility for a single or few microparticles. This article presents a state of the art system where an Acoustic Lens was used for developing a Micro-Acoustic Trap for microparticle assembly in 3D. The model particles, 2 µm diameter polystyrene beads in suspension, were driven via acoustic pressure to form a monolayer at wavelength-defined distances above the substrate defined by the focal point of an Acoustic Lens The transducer was driven at 89 MHz, mixed with 100 ms pulses at a repetition rate of 2 Hz. Beyond a threshold drive amplitude sufficient to overcome Brownian motion, this led to 2D assembly of the microparticles into close-packed rafts >80 µm across (∼5 wavelengths of the carrier wave and >40 particles across). This methodology was further extended to manipulation of live Dictyostelium discoideum amoebae. This approach therefore offers maneuverability in controlling or assembling micrometer-scale objects using continuous or pulsed focused acoustic radiation pressure.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

The phagocytosis of crystalline silica particles by macrophages.

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.

Actin binding domains direct actin-binding proteins to different cytoskeletal locations.

BACKGROUND: Filamin (FLN) and non-muscle alpha-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell. RESULTS: We have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and alpha-actinin (GFP-alphaA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-alphaA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and alpha-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant. CONCLUSION: These observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.

Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcgammaR, phagocytosis.

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

Automated real-time measurements of leukocyte chemotaxis.

We have previously described an automated system (ECIS/taxis) for measuring chemotactic movement of Dictyostelium amoebae in a folic acid gradient [Hadjout, N., Laevsky, G., Knecht, D.A. and Lynes, M.A., 2001. Automated real-time measurement of chemotactic cell motility. Biotechniques 31, 1130-1138.]. In the ECIS/taxis system, cells migrate in an under-agarose environment, and their position is monitored by determining the impedance change caused by cells crawling onto the surface of an electrode. In this report, we show that chemotaxis of primary and immortalized leukocytes in response to complement (C5a) could be measured using the ECIS/taxis system. Several modifications to the design of the target electrode were tested, and a linear electrode perpendicular to the direction of movement was found to increase the sensitivity and reliability of the assay. Using the optimized ECIS/taxis assay, the dose response of neutrophils and WBC 265-9C cells was established and compared to the Boyden chamber assay. The ECIS/taxis assay system can be used to compare the movement of different cell types, to assess the effect of complex chemotactic gradients, or to determine the effects of pharmaceuticals on chemotactic motility.

Single Cell Analysis of Phagocytosis, Phagosome Maturation, Phagolysosomal Leakage, and Cell Death Following Exposure of Macrophages to Silica Particles.

Chronic inhalation of silica in various occupational settings results in the development of silicosis, a disease characterized by lung fibrosis. Uptake of silica particles by alveolar macrophages results in cell death and this is one of the contributing factors to the development of silicosis. We have characterized the uncoated or protein-coated (non-opsonized) and Fc receptor-mediated (antibody-opsonized) routes of silica phagocytosis and toxicity. Numerous microscopy techniques and fluorescent probes are outlined in this chapter to carefully measure particle uptake, by macrophages, phagosome maturation, phagosomal reactive oxygen species generation, phagolysosomal leakage, and cell death.

Under-agarose chemotaxis of Dictyostelium discoideum.

In the vegetative state, Dictyostelium amoebae are chemotactic toward pterins released by bacteria, whereas during multicellular development, they become chemotactic to endogenously produced cAMP. A variety of assays have been used to visualize and quantify chemotactic movement. Under-agarose chemotaxis provides a simple and flexible assay that permits high-resolution imaging and quantification of the motility behavior of individual cells and populations by both transmitted light and fluorescence microscopy. The assay requires cells to deform a solid but flexible matrix; therefore, it also provides a way to measure defects in the ability of mutant cells to move in these restrictive conditions.

Metallothionein mediates leukocyte chemotaxis.

BACKGROUND: Metallothionein (MT) is a cysteine-rich, metal-binding protein that can be induced by a variety of agents. Modulation of MT levels has also been shown to alter specific immune functions. We have noticed that the MT genes map close to the chemokines Ccl17 and Cx3cl1. Cysteine motifs that characterize these chemokines are also found in the MT sequence suggesting that MT might also act as a chemotactic factor. RESULTS: In the experiments reported here, we show that immune cells migrate chemotactically in the presence of a gradient of MT. This response can be specifically blocked by two different monoclonal anti-MT antibodies. Exposure of cells to MT also leads to a rapid increase in F-actin content. Incubation of Jurkat T cells with cholera toxin or pertussis toxin completely abrogates the chemotactic response to MT. Thus MT may act via G-protein coupled receptors and through the cyclic AMP signaling pathway to initiate chemotaxis. CONCLUSION: These results suggest that, under inflammatory conditions, metallothionein in the extracellular environment may support the beneficial movement of leukocytes to the site of inflammation. MT may therefore represent a "danger signal"; modifying the character of the immune response when cells sense cellular stress. Elevated metallothionein produced in the context of exposure to environmental toxicants, or as a result of chronic inflammatory disease, may alter the normal chemotactic responses that regulate leukocyte trafficking. Thus, MT synthesis may represent an important factor in immunomodulation that is associated with autoimmune disease and toxicant exposure.

Macrophages phagocytose nonopsonized silica particles using a unique microtubule-dependent pathway.

Silica inhalation leads to the development of the chronic lung disease silicosis. Macrophages are killed by uptake of nonopsonized silica particles, and this is believed to play a critical role in the etiology of silicosis. However, the mechanism of nonopsonized-particle uptake is not well understood. We compared the molecular events associated with nonopsonized- and opsonized-particle phagocytosis. Both Rac and RhoA GTPases are activated upon nonopsonized-particle exposure, whereas opsonized particles activate either Rac or RhoA. All types of particles quickly generate a PI(3,4,5)P3 and F-actin response at the particle attachment site. After formation of a phagosome, the events related to endolysosome-to-phagosome fusion do not significantly differ between the pathways. Inhibitors of tyrosine kinases, actin polymerization, and the phosphatidylinositol cascade prevent opsonized- and nonopsonized-particle uptake similarly. Inhibition of silica particle uptake prevents silica-induced cell death. Microtubule depolymerization abolished uptake of complement-opsonized and nonopsonized particles but not Ab-opsonized particles. Of interest, regrowth of microtubules allowed uptake of new nonopsonized particles but not ones bound to cells in the absence of microtubules. Although complement-mediated uptake requires macrophages to be PMA-primed, untreated cells phagocytose nonopsonized silica and latex. Thus it appears that nonopsonized-particle uptake is accomplished by a pathway with unique characteristics.

Silica particles cause NADPH oxidase-independent ROS generation and transient phagolysosomal leakage.

Chronic inhalation of silica particles causes lung fibrosis and silicosis. Silica taken up by alveolar macrophages causes phagolysosomal membrane damage and leakage of lysosomal material into the cytoplasm to initiate apoptosis. We investigated the role of reactive oxygen species (ROS) in this membrane damage by studying the spatiotemporal generation of ROS. In macrophages, ROS generated by NADPH oxidase 2 (NOX2) was detected in phagolysosomes containing either silica particles or nontoxic latex particles. ROS was only detected in the cytoplasm of cells treated with silica and appeared in parallel with an increase in phagosomal ROS, as well as several hours later associated with mitochondrial production of ROS late in apoptosis. Pharmacological inhibition of NOX activity did not prevent silica-induced phagolysosomal leakage but delayed it. In Cos7 cells, which do not express NOX2, ROS was detected in silica-containing phagolysosomes that leaked. ROS was not detected in phagolysosomes containing latex particles. Leakage of silica-containing phagolysosomes in both cell types was transient, and after resealing of the membrane, endolysosomal fusion continued. These results demonstrate that silica particles can generate phagosomal ROS independent of NOX activity, and we propose that this silica-generated ROS can cause phagolysosomal leakage to initiate apoptosis.

Silica-induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity.

Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate-treated silica still induced lysosomal permeability (by FITC-dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.