TRPV1 receptors mediate particulate matter-induced apoptosis

N. Agopyan,1 J. Head,2 S. Yu,2 and S. A. Simon1,2

Departments of 1Anesthesiology and 2Neurobiology, Duke University Medical Center, Durham, North Carolina 27710

Submitted 2 September 2003 ; accepted in final form 30 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity.

inflammation; acid-sensitive ion channels; TRPV1; human airway epithelial cells; sensory neurons; cAMP; necrosis; apoptosis; air pollution; capsaicin; acid


AIR POLLUTION HAS BEEN DEEMED a major public health issue ever since epidemiological studies have found a linear correlation between the concentration of airborne particulate matter (PM) and mortality associated with respiratory and cardiovascular disorders (18, 21, 22, 50). Although the specific mechanisms underlying increases in mortality are not known, it is generally accepted that PM exposure can exacerbate preexisting respiratory illnesses and also enhance the development of new diseases (3, 4, 14, 17, 53-56, 72). For example, in mice, tracheal instillation of bentonite, oil fly ash, metal salts, and ambient air particles enhanced, by >50%, mortality from infection (28), suggesting a reduced immune function. In a significant percentage of the population, air pollutants also caused hypersensitivity by elevating immune response to allergens to produce a variety of allergic diseases such as chronic obstructive pulmonary disease or asthma (18, 54, 62). Despite many studies regarding the health effects of PMs, a single mechanism underlying the clinical symptoms following PM exposure is still not identified, most likely, due to the heterogeneity in environmental PM composition.

PM consists of a very wide range of solid or liquid particles of various sizes that are small enough to remain suspended for long periods in the atmosphere (32, 43, 59, 70). PM has many sources including soil, combustion of fossil fuels, and industrial discharges, which may contain metals, solvents, aromatic hydrocarbons, endotoxins, and other chemicals that can modulate specific immune function (32, 37). Of the heavy metals found in PM, cadmium, vanadium, chromium, lead, and nickel have been shown to decrease antibody formation, antigen processing, and lymphocyte proliferation in experimental animals (37, 47). Organic compounds such as benzene, trichloro-ethylene, dioxins, phenols, organotonins, and diester phorbol compounds, which are found in the atmosphere, have also been shown to alter immune function (58). Both organic and heavy metal PM components were reported to induce proinflammatory effects and oxidative stress due to their ability to generate reactive oxygen species (ROS) (30, 39). However, the intermediary events between PM binding to the cell membrane and the generation of ROS and oxidative stress remain unknown.

Although the above mentioned studies provide arguments for the epidemiological findings and offer clues for the mechanisms of these effects, additional work is required to identify how PM binding to the cell surface gives rise to effects that could lead to PM-induced toxicity (14, 53, 55, 69, 72). In previous studies, to determine some of the mechanisms and receptors involved in environmental PM-induced toxicity, we and others used synthetic acid aerosols [carboxylate-modified particles (PCs)], which are similar to environmental PMs in size and surface charge density but are devoid of exogenous organics, endotoxins, and heavy metals (1, 2, 27, 46, 49, 64, 66). Applying PCs to both airway epithelia and sensory neurons resulted in the activation of vanilloid receptors (TRPVs) and likely acid-sensitive ion channels (ASICs) that produced increases in calcium influx and subsequently apoptosis (1, 2, 49). Such synthetic particles were also shown to cause an increase in cytokine release (66, 71), an increase in the microvasculature permeability (27), and an increase in pulmonary inflammation as a result of their ability to enhance capsaicin- and carbachol-induced release of substance P and histamine from C-fibers and mast cells (46).

The aim of this study was to better understand the mechanisms underlying PM-induced toxicity in human airway epithelial cells and sensory neurons and determine whether PMs, like synthetic particles, produce cytotoxicity (apoptosis/necrosis) by activating proton-gated ion channels. We chose to use residual oil fly ash (ROFA), which is a highly reactive PM that contains heavy metals, and volcanic ash from Mount St. Helens (MSHA), which is a representative of PMs with low potency (24). Their effects were tested on primary cultures of human bronchial/tracheal epithelial cells [normal human bronchial epithelial (NHBE)], human distal airway epithelial cells [small airway epithelial cells (SAEC)], and sensory neurons from wild-type and TRPV1-/- mice. In cells that were activated by acid and capsaicin, it was found that the adhesion of either ROFA or MSHA to the plasma membrane induced a sustained calcium influx that resulted in apoptosis, but not necrosis. Apoptosis was completely blocked by removal of extracellular calcium or by inhibiting TRPV1 receptors and was not present in sensory neurons in TRPV1-/- mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Care of animals conformed to standards established by the National Institutes of Health. All animal protocols were approved by the Duke University Institutional Animal Care and Use Committee.

Cell Culture

Primary NHBE and SAEC cells, as well as all the basal media and supplements, were obtained from Clonetics (San Diego, CA). Because NHBE and SAEC cells become irreversibly contact inhibited, they were either subcultured or used before they reached 80% confluence between passages 4 and 5. As described previously (1), NHBE cells were maintained in bronchial/tracheal epithelial basal medium, whereas SAEC cells were maintained in SAEC basal medium. For imaging studies, 1 day before use, the cells were plated on 20-mm-diameter fibronectin-coated glass coverslips (Carolina Biological Supply, Burlington, NC).

Trigeminal ganglion neurons. TRPV1 wild-type and knockout (-/-) mice were the generous gift of Dr. David Julius of the University of California at San Francisco. A total of 20 wild-type and 24 TRPV1(-/-) neurons were used. As described previously (2), trigeminal ganglion (TG) neurons were dissected and collected in modified Hanks' balanced salt solution (mHBSS) that contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 D-glucose (pH adjusted to 7.4 with NaOH). After being washed in mHBSS, the ganglia were cut into small pieces and incubated at 37°C for 30-50 min in mHBSS with 0.1% collagenase (type Xl-S). We dissociated individual cells by triturating the tissue through a fire-polished glass pipette, followed by a 10-min incubation at 37°C in 10 µg/ml DNase I (type lV) in F-12 medium (Life Technologies, Gaithersburg, MD). After washing them three times with F-12, we cultured the cells in DMEM supplemented with 10% fetal bovine serum. The cells were plated on poly-D-lysine-coated glass coverslips (15-mm diameter) and cultured at 37°C in a water-saturated atmosphere with 5% CO2. All experiments were carried out at room temperature (22-25°C). We measured cell diameters using a calibrated eyepiece. Cells with projected soma diameters of <=25 µm were defined as small, and those whose projected soma diameters were >25 µm were defined as large.

Calcium Imaging

NHBE, SAEC, and TG cells were loaded in serum-free culture medium for 45 min at 37°C with fura 2-AM (Molecular Probes, Eugene, OR). Before imaging, cells were washed three times with HBSS and left for 20 min at room temperature in the dark. Images were acquired with a General Electric SIT camera and processed with an Argus-10 image processor (Hamamatsu, Japan). Data acquisition and off-line analysis were performed with Axon Imaging Workbench 2 (AIW2.2; Axon Instruments, Foster City, CA). Cells were illuminated at 340 and 380 nm, and their fluorescent emission was detected at 510 nm. For each experiment, a background image was obtained at each wavelength (340 nm, 380 nm) and was subtracted from subsequent measurements of cells. Background subtracted data were presented as the 340:380 ratio. A response to 1 µM capsaicin, pH 5.0, and PMs was defined to have occurred when the 340:380 ratio was >0.1 and was sustained for >1 s.

Cells were tested for their responsiveness to both pH 5.0 and 1 µM capsaicin. We did not test the cells for PM sensitivity when they were sensitive only to pH 5.0 but not to 1 µM capsaicin (~60% of cell population).

PMs (100 µg/ml) were injected into the bath solution where they eventually settled onto the cells, with the numbers adhering to the plasma membrane increasing with time. ROFA and MSHA aggregated in the bath (HBSS) solution (see Fig. 1).



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Fig. 1. Interaction of particulate matter (PM) with human lung cells. Shown are examples of PMs of Mount St. Helens ash (MSHA) and residual oil fly ash (ROFA) in contact with NHBE and SAEC cells. Differential interference contrast (DIC) images of NHBE (A and B) and SAEC (C and D) cells illustrate the interaction of MSHA (A and C) and ROFA (B and D) with the plasma membranes. The images were acquired before the local perfusion was turned on, hence both bound and unbound PMs can be visualized. Middle insets: images of synthetic particles of 0.02, 0.1, 0.2, 0.5, and 1 µm in size are given as a calibration for the PMs and cells.

 

For analyzing PM-induced changes, we counted all cells as being PM bound in which at least one particle was visually observed to be in physical contact with the plasma membrane. Cells that were not treated with PM but kept in culture for the same period were used as controls. To test for the possibility of secondary activation as a consequence of released substances from PM-bound cells, we also used cells in the PM-treated group, which did not have visibly adhering particles, as internal controls, but only for this purpose. Nevertheless, all the comparisons and statistical analysis were performed between the treated and untreated groups. In our preparation, particles >0.02 µm in size could be visualized (see Fig. 1). We confirmed physical contact of the PMs with cells from images obtained before and after turning on the local perfusion. When the local perfusion was turned on, some of the PMs dissociated from the cells, a process that was considered as wash and is denoted in the figures with upward-pointing arrowheads (e.g., cf. Fig. 2D). At the end of each series of experiments, 100 µM ionomycin (IO) was added to the bath solution to produce maximal increases in [Ca2+]i. However, when it was important to observe other cells that were not in the original field and that did not have bound PMs, this protocol was not followed. Consequently, responses were not normalized with respect to IO.



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Fig. 2. ROFA and MSHA induce sustained calcium influxes in NHBE and SAEC cells. The calcium imaging data in all 4 panels are similar and represent cells that are activated by both pH 5.0 and 1 µM capsaicin (Cap, {circ}) and cells that are not activated by these agonists ({bullet}). In all these cells ROFA or MSHA were bound (downward-pointing arrowheads). A: response of SAEC cells to pH 5.0, 1 µM Cap, and to ROFA binding (downward-pointing arrow). Also shown is that 10 µM capsazepine (CPZ) application diminished ROFA-induced calcium influx. B: responses of a Cap- and pH 5.0-sensitive SAEC cell. Perfusion of 10 µM CPZ and 20 µM amiloride inhibited the ROFA-induced calcium increase. Replacing the CPZ- and amiloride-containing buffer with one containing CPZ alone restored some of the ROFA-induced response. When CPZ was also removed from the buffer, ROFA-induced response recovered. Ionomycin (IO, 100 µM), perfused at the end of the experiment, induced calcium influx in both Cap- and pH 5.0-sensitive and -insensitive cells. C: response of a both Cap- and pH 5.0-sensitive and -insensitive NHBE cells to the reversible binding of MSHA. Upon the unbinding of MSHA from the cell membrane (upward-pointing arrow) the response slowly decreased. The cell remained responsive to Cap and IO, indicating the reversibility of the PM changes. D: response of NHBE cells. As before, CPZ and amiloride perfusion reversibly blocked the response to MSHA. For clarity, only data points at every 15 s are presented.

 

PM-Induced cAMP Measurements

cAMP was quantified by enzyme-linked immunosorbent assay following the manufacturer's protocol (Amersham, Piscataway, NJ). Initially, NHBE and SAEC cells were placed in 96-well plates at a density of 1 x 105 cells/ml and allowed to reach 80% confluence. Under this condition, their concentration was 5-7 x 105 cells/ml, or 30-50 µg protein/well. Just before adding PMs, the culture medium was changed. To test the involvement of TRPV1 receptors in cAMP production, we incubated cells for 20 min at 37°C in buffer with or without 10 µM capsazepine (CPZ), a TRPV1 antagonist (13). After the addition of 100 µg/ml PMs, the cells were incubated for 6 h. This incubation time was chosen because previous work showed that cytokine release was maximal and synthetic acidic aerosol-induced apoptosis was relatively small (1, 49). Data reported are from eight independent experiments performed in triplicate.

Measurements of Apoptosis and Necrosis

NHBE and SAEC cells were treated with either ROFA or MSHA for 2, 6, 24, and 48 h. TG neurons were treated with either ROFA or MSHA for 24 h. Within 15 min after injection of the PMs (100 µg/ml) into the culture medium, they sank and usually adhered to the cells (cf. Fig. 1). After the prescribed incubation periods, the culture medium was gently removed and replaced with 500 µl of mHBSS. To ensure that the time spent in culture per se was not the cause of apoptosis/necrosis, in parallel, we subjected control cells to the same procedures, only omitting PMs. As noted, cells that did not contact the PMs in the treated dishes were also used as internal controls to differentiate between direct (receptor activation due to PM binding) and indirect effects (chemicals released from cells as a consequence of PM binding). To differentiate between calcium influx and calcium release from internal stores, some experiments were performed using mHBSS buffer in which calcium was omitted (called "calcium-free"). We have previously established that in this medium these cells survive and do not exhibit apoptosis or necrosis (1).

Apoptosis and necrosis was measured by using Alexa Fluor 488-conjugated annexin V (1:10 final dilution) and propidium iodide (10 µg/ml) labeling (Molecular Probes), respectively (1). For imaging, cells on coverslips were loaded with the dyes, incubated in the dark for 15 min at room temperature, and then washed several times with the buffer. They were maintained in mHBSS in the dark before being transferred to the recording chamber. Fluorescent samples were viewed under a Zeiss Axioscope fluorescence microscope equipped with fluorescein and rhodamine filters. The images were acquired with a Hamamatsu digital camera (Hamamatsu, Japan). The intensity of auto fluorescence, which was determined at the beginning of each experiment by imaging the fluorescence of several nonapoptotic cells, was subtracted from the fluorescence intensity of apoptotic cells ({Delta}F). Because in the absence of apoptosis there was no fluorescence (F0 having a pixel intensity value of zero), we chose to report the absolute fluorescence ({Delta}F ranging from 0 to 255 intensity units) rather than the percent increase in fluorescence ({Delta}F/F0). A response was defined when {Delta}F was >=10 intensity units. Cells that were both apoptotic and necrotic were considered as necrotic and removed from the apoptotic pool.

Chemicals

Stock solutions (10 mM) of capsaicin and CPZ (Tocris, Ellisville, MO) were made in 100% ethanol and DMSO, respectively. A 100 mM stock solution of amiloride (Sigma, St. Louis, MO) and 1 mM stock solution of the protein kinase A (PKA) inhibitor KT-5720 (Tocris) were prepared in DMSO. Before the experiment, capsaicin, CPZ, amiloride, and KT-5720 were diluted in the buffer to final concentrations of 1, 10, 20, and 2 µM, respectively. At these final concentrations, neither DMSO nor ethanol had any effect on the assays used in this study. ROFA and MSHA, which were a gift from Dr. Daniel Costa of the Environmental Protection Agency (Research Triangle Park, NC), were prepared as 1.2 mg/ml dispersions.

Statistical Analysis

Statistical analysis was performed using the Sigma Plot software. Data are reported as means ± SE. Statistical differences between different treatment groups were established using the Student's paired t-test and a 95% confidence interval.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium Imaging

Human lung cells. NHBE and SAEC. Calcium imaging studies were performed on individual NHBE and SAEC cells before and after exposure to ROFA or MSHA (Fig. 1). In 100% of capsaicin (1 µM)- and acid (pH 5.0)-sensitive cells, the binding of one or more particles of either ROFA or MSHA induced an increase in [Ca2+]i (Table 1 and Fig. 2, A-D). Cells that were unresponsive to 1 µM capsaicin were not activated upon the binding of either ROFA or MSHA (Fig. 2). PM-induced increases in [Ca2+]i remained elevated for as long as the PMs were bound to the plasma membrane. When the cells were washed, if and when the PMs disjoined from the plasma membrane, [Ca2+]i slowly decreased back to baseline (Fig. 2, A and C).


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Table 1. Percentages of lung epithelial cells that showed an increase in [Ca2+]i

 

ROFA- or MSHA-induced increases in [Ca2+]i were inhibited by the coapplication of 10 µM CPZ together with 20 µM amiloride. CPZ-induced inhibition was equally effective when it was applied either before or after PMs were bound to the membrane (Fig. 2). Upon removal of both of these antagonists, PM-induced increases in [Ca2+]i were reactivated (Fig. 2, B and D). To test whether in the presence of CPZ alone other PM-induced pathways could be activated, we replaced the buffer containing CPZ and amiloride with the same buffer containing only 10 µM CPZ. Under this condition, only 26 ± 5% of the PM-mediated response recovered, indicating that most of the PM-induced responses arise from the activation of CPZ-inhibitable receptors.

Sensory neurons from TRPV1(+/+) and (-/-) mice. To validate the hypothesis that TRPV1 receptors are activated by environmental PMs, we measured the differences in ROFA and MSHA-induced [Ca2+]i increases in sensory neurons obtained from TRPV1(+/+) and TRPV1(-/-) mice. Figure 3 shows the responses of PM binding to neurons obtained from TRPV1(+/+) and TRPV1(-/-) mice. All capsaicin- and pH 5.0-sensitive neurons from TRPV1(+/+) mice responded to ROFA or MSHA binding with an increase in [Ca2+]i. Capsaicin- and pH 5.0-insensitive neurons from the same group of cells were responsive to the binding of ROFA. In neurons from TRPV1(-/-) mice that were activated by pH 5.0, PM binding produced a very small increase in [Ca2+]i (Fig. 3, n = 25 neurons). In pH-insensitive TG neurons from TRPV1(-/-) mice, the binding of ROFA or MSHA to the plasma membrane failed to induce increases in [Ca2+]i (Fig. 3).



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Fig. 3. PMs fail to induce calcium influx in sensory neurons from TRPV1(-/-) mice. A: trigeminal ganglion (TG) neurons from TRPV1(+/+) mice. The results from a calcium-imaging study illustrate the response of a larger and smaller diameter neuron. The small diameter Cap- and acid-responsive neurons also responded to binding of ROFA by increases in intracellular calcium. The recovery from ROFA unbinding was not due to either cell death or dye quenching as 100 µM IO induced a further increase in calcium response. The larger neuron that was unresponsive to Cap and acid ({bullet}) was also unresponsive to ROFA but was activated by IO. B: TG neurons from TRPV1(-/-) mice. Neither a smaller-diameter (<25 µm, {circ}) nor a larger-diameter (>25 µm) neuron responded to Cap or to bound ROFA. The smaller-diameter neuron was, however, responsive to pH 5.0. Both neurons responded to 100 µM IO. Bars in insets are 20 µm.

 

PM-Induced Increase in cAMP

PM-induced activation of TRPV1 receptors, like those of acid, are of pathological interest because their response does not desensitize, even in the presence of extracellular calcium (Figs. 2 and 3 and Ref. 42). Because the activation of the cAMP-PKA pathway has been shown to remove desensitization of TRPV1 receptors (10), we tested whether PM would increase cAMP levels in human lung epithelial cells. In SAEC and NHBE cells, both ROFA and MSHA (100 µg/ml) produced statistically significant increases in cAMP (Fig. 4). This increase was completely (100%) blocked by 10 µM CPZ, indicating that PM-induced increases in cAMP is mediated through TRPV1 receptor activation.



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Fig. 4. PMs increase cAMP in human airway epithelial cells. Histograms show how ROFA (A and B) and MSHA (C and D) (100 µg/ml) significantly increased the concentration of cAMP (pmol/mg of protein) in SAEC and NHBE cells. The PM-induced increase in cAMP was significantly inhibited In the presence of 10 µM CPZ. Data represent means ± SE. *Statistical significance, P < 0.005.

 

Apoptosis

Human lung cells. To determine whether adhering PMs would trigger apoptosis and/or necrosis, we incubated NHBE and SAEC cells with either ROFA or MSHA for 2, 6, 24, and 48 h. In both of these cell types, incubation with either ROFA or MSHA induced apoptosis in a time-dependent manner. For both PMs, apoptosis peaked after a 24-h exposure (Fig. 5). After 24- and/or 48-h PM incubations, none of the cells that were subjected to the same treatment without the addition of PMs were apoptotic: NHBE (459) and SAEC (514). Furthermore, none of the cells within the treatment group that did not have visibly bound ROFA or MSHA displayed apoptosis: NHBE (227/227) and SAEC (282/282).



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Fig. 5. TRPV1 receptors mediate PM-induced apoptosis. Human airway epithelial cells (A-D): graphs illustrating how the proportion of apoptotic NHBE (blue) and SAEC (green) cells increased with incubation time in ROFA (A) and MSHA (C). Corresponding panels B and D show that 10 µM CPZ inhibited 95% of ROFA- or MSHA-induced apoptosis, respectively. The remaining apoptotic cells were inhibited by 20 µM amiloride (Ami). Mouse sensory neurons (E, F): E: superimposed DIC and fluorescence images of TG neurons from TRPV1(+/+) and TRPV1(-/-) mice taken 24 h after ROFA and MSHA treatment. It is seen by the fluorescence produced by annexin V binding that only the smaller-diameter neurons from TRPV1(+/+) mice were apoptotic. Note that despite the adhesion of numerous PMs (arrowheads), the larger-diameter neuron from a TRPV1(+/+) mouse was not apoptotic. In contrast, neither the small nor the large diameter neurons from TRPV1(-/-) mice were apoptotic, despite having PM bound to their membrane. Bar = 10 µm. F: summary of the ROFA- and MSHA-induced mouse apoptosis measurements.

 

To determine whether PM-induced activation of protongated receptors are involved in the induction of apoptosis, we treated the NHBE and SAEC cells with 10 µM CPZ 20 min before exposing them for 24 h to ROFA or to MSHA. We found that this treatment almost completely (95-98%) blocked ROFA-and MSHA-induced apoptosis in both cell types (Fig. 5, B and D). The small remaining PM-induced apoptosis was blocked by amiloride (Fig. 5, B and D), suggesting the presence of other receptors, most likely ASICs (8, 34, 38).

TG neurons from TRPV1(+/+) and TRPV1(-/-) mice. To unequivocally demonstrate the role of TRPV1 receptors on PM-induced apoptosis, we repeated the above experiments using sensory neurons from TRPV1(+/+) (a total of 257 neurons) and TRPV1(-/-) mice (a total of 187 neurons). Apoptosis was induced in 76% (25/33)1 of PM-bound small-diameter (<25 µm) neurons from TRPV1(+/+) mice that we tested after a 24-h incubation of either ROFA or MSHA (100 µg/ml). Neither the PM-bound larger-diameter (>25 µm) neurons (n = 52/52) nor the neurons that did not have PM bound to their membrane (n = 172/172) were apoptotic. In contrast, none of the PM-bound smaller-diameter neurons (n = 59/59) from TRPV1(-/-) mice was apoptotic (Fig. 5, E and F). We also found that similar to TRPV1(+/+) neurons, none of the PM-bound larger-diameter neurons (38/38) nor the neurons that did not have PM bound to their plasma membrane (90/90) were apoptotic (Fig. 5E).

PM Does Not Induce Necrosis

In contrast to PM-induced apoptosis, we found that PM-induced necrosis was negligible. That is, at 2, 6, 12, and 24 h, <1% of the cells were necrotic. Specifically, at 24 h, only 21/301 NHBE- and 26/332 SAEC ROFA-bound cells and 2/235 NHBE- and 19/447 SAEC MSHA-bound cells were necrotic. Neither CPZ nor removal of external calcium blocked the small percentage of cells that were necrotic (data not shown). Similarly, after 24 h of PM exposure, TG neurons from either TRPV1(+/+) (257/257) or TRPV1(-/-) (187/187) mice did not exhibit necrosis.

PM-Induced Apoptosis Depends on the Influx of Calcium

To determine whether PM-induced apoptosis was dependent on the influx of extracellular calcium, SAEC and NHBE cells were incubated with ROFA for 24 h in calcium-free HBSS. A total of 418 (214 bound) NHBE and 611 (398 bound) SAEC cells were examined in the presence of ROFA, and 407 (203 bound) NHBE and 418 (205 bound) SAEC cells were examined in the presence of MSHA. Neither the PM-bound cells nor the unbound cells were apoptotic, suggesting that Ca2+ influx is necessary to produce PM-induced apoptosis. The percentage of necrotic cells at 24 h as reported above was not affected by the removal of extracellular calcium.

PM-Induced Activation of PKA Delays Apoptosis

To determine whether PKA that is presumably activated by PM-induced cAMP (Fig. 4) may contribute to apoptosis, we inhibited PKA by incubating the airway epithelial cells with 2 µM KT-5720 (61). A total of 600 SAEC and 408 NHBE cells were studied at 24 h posttreatment. As seen in Fig. 6, in the control group, which was not treated with PM, inhibition of PKA greatly increased the extent of apoptosis (NHBE: 46 ± 5%, n = 103; SAEC: 44 ± 5%, n = 120; compare with Fig. 5). However, in the PM-treated group, >90% of the cells were apoptotic in the presence of KT-5720 (NHBE: 91 ± 5% of MSHA-bound and 97 ± 10% of ROFA-bound cells; SAEC: 96 ± 6% of MSHA-bound and 93 ± 4% of ROFA-bound cells). The increase in apoptosis seen with both PKA inhibition alone and PM treatment in the presence of PKA inhibitor is dependent on the influx of calcium, because apoptosis was not observed in a calcium-free buffer (280/280 NHBE and 347/347 SAEC cells).



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Fig. 6. Role of calcium influx and PKA activation in PM-induced apoptosis. Graphs illustrating how the proportion of apoptotic NHBE (solid bars) and SAEC (open bars) cells decreased at 24 h of ROFA (A) and MSHA (B) incubation in the absence of calcium and increased in the presence of PKA inhibitor KT-5720.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The data presented show that PM-induced apoptosis in sensory neurons and human epithelial cells lining the airways arises from a sustained calcium influx through TRPV1 receptors and that apoptosis can be virtually eliminated by removing extracellular calcium or by blocking TRPV1 receptors.

Comparison of ROFA, MSHA, and PCs

In this study two environmental PMs were used: ROFA and MSHA. ROFA is a highly reactive PM that contains, among other compounds, heavy metals that have been attributed to many of its health effects (12, 24, 36). MSHA is a representative PM that also contains proinflammatory metals but has a relatively low potency (26). We showed that when equal access to the cells is provided, ROFA and MSHA are equally potent in inducing cellular toxicity (Fig. 5). More interestingly, we demonstrated that environmental PMs act similar to synthetic particles made from negatively charged polymers that do not have exogenous materials bound to them and are thought to act by virtue of their surface properties that act to increase the proton concentration near proton-gated receptors such as TRPV1s and ASICs (1, 2, 49, 64, 66). In this regard, PMs act like protons that activate TRPV1 receptors in TG neurons in the sense that the response does not (rapidly) desensitize, even in the presence of extracellular calcium (42).

Despite the similar responses evoked by PMs and the synthetic particles, we found that upon wash the natural PMs more readily disjoin from the plasma membrane than the PCs (compare the responses shown in Refs. 1 and 2 with Fig. 2). Possible reasons for this behavior include different surface charge densities and/or different geometries that could result in a smaller area of contact. Another difference that we observed in studying cytotoxicity between PMs and PCs was that only PCs were endocytosed. However, we cannot rule out the possibility that we could not detect endocytosed particles smaller than 0.02 µm (assuming that they did not aggregate), although investigators using electron microscopy were able to find ultrafine PMs in the cells (39).

The low biological potency of MSHA may arise either from its relative inability to diffuse to the deeper areas of the lungs (perhaps because of the larger size particles) or because its surface reactivity is masked by the nonreactive volcanic components during ash formation (31). Nevertheless, in vitro, MSHA is biologically active as it was found to induce increases in cytokine release in airway epithelial cells (35). That is, to summarize our findings insofar as PM-induced cytotoxicity through the activation of TRPV1 receptors, ROFA, MSHA, and synthetic particles behaved similarly. This should, however, not be interpreted to imply that organics, endotoxins, and heavy metals are unimportant in producing adverse health effects, it simply means that under the conditions performed in these experiments involving apoptosis and necrosis, they do not appear to be necessary components.

PMs Induce Apoptosis

The data presented provide direct evidence that both ROFA and MSHA induce a time-dependent apoptosis in both the airway epithelial cells and sensory neurons (Fig. 5). Numerous studies have shown that chronic exposure to pollutants (23) results in the damage of lung epithelial barrier (6, 47) and in mitochondrial damage (29, 39, 40), the latter of which is well known for its role in apoptosis induction (11). However, the mechanism underlying PM-induced apoptosis remains unknown. One hypothesis is that PM-induced ROS damage the mitochondrion (39) and thus give rise to apoptosis. However, since antioxidants have been shown to be ineffective in blocking PM-induced apoptosis (7, 40), one may conclude that ROS is a byproduct rather than the trigger that initiates a cascade of events following PM binding.

Mechanisms Underlying PM-Induced Apoptosis

TRPV1 activation and calcium influx. We found that TRPV1 activation triggers PM-induced apoptosis. This was demonstrated both by the inhibition of apoptosis with CPZ in lung cells and by the lack of apoptosis found in neurons from TRPV1(-/-) mice (Fig. 5). The possibility that apoptosis arises from cytokines or other compounds (metals) released from the PM-bound cells into the media was eliminated by the observation that cells that were adjacent to apoptotic cell but were not in contact with PMs were not apoptotic.

We have previously documented with electrophysiological recordings that PCs can induce inward currents and sustained calcium influx through activation of both TRPV1 and amiloride-sensitive (perhaps ASIC) receptors (2, 51). In this regard, RT-PCR studies have shown TRPV1, ASIC1a, and ASIC3 mRNA expression in human airway epithelial cells (1). However, despite a higher relative expression of ASIC1a in airway epithelial cells, both PM (present study)- and PC-induced calcium influx and apoptosis (1) are mediated mainly through the activation of TRPV1 receptors. In both of the lung cell types studied, the contribution of the amiloride-sensitive component to calcium response was ~30% of the PM-induced calcium increase (Fig. 2). This finding is consistent with the lower calcium permeability of ASIC1a [PNa/PCa = 3-18.5 (16, 67)] than of TRPV1 receptors [PCa/PNa = 8/1 (13, 68)], or alternatively a fewer number of functional ASIC receptors. These differences could explain why ASIC receptors do not appear to be as crucial as TRPV1 receptors in PM-induced apoptosis in these in vitro experiments. That is, cells may be capable of compensating for this lower calcium flux mediated through PM-activated ASIC channels and thereby prevent apoptosis.

Activation of TRPV1 receptors has previously been shown to lead to cell death (15, 44, 45, 48). This can occur as a consequence of cell swelling leading to membrane lysis (i.e., necrosis). However, our finding that PM-induced necrosis was negligible compared with PM-induced apoptosis argues against this pathway, at least within 24-48 h (Fig. 5 and text). Increased calcium influx can also induce cell death (apoptosis) by activating calcium-dependent proteases, lipases, and nucleases and other calcium-dependent proteins in the apoptotic pathway (15, 25, 44, 45). In both airway epithelial cells and sensory neurons, PMs induce a sustained calcium influx, which is dependent on the presence of functional TRPV1 receptors (Figs. 2 and 3; also see Refs. 1 and 2). The findings that, in the absence of external calcium, PM exposure eliminated apoptosis in human airway epithelial cells and that PM-induced calcium influx and apoptosis were absent in sensory neurons from TRPV1(-/-) mice support the hypothesis that TRPV1-mediated calcium loading leads to apoptosis. Indeed, capsaicin-induced TRPV1-mediated increases in intracellular calcium lead to mitochondrial calcium accumulation and thereby disruption of mitochondrial function (5, 19, 20, 33, 45, 48).

In nociceptive neurons, several studies reported the existence of two TRPV1 pools, one of which is on the plasma membrane and the other on the endoplasmic reticulum (48, 63). Capsaicin activation of either population was shown to elevate intracellular calcium in a CPZ-sensitive manner. It was suggested that the presence of TRPV1 receptors on intracellular organelles would give rise to further calcium release and thus serve as a signal amplifier for weak stimuli. Such a mechanism could also be present in the airway epithelia and may explain the sustained nature of PM-induced increase in [Ca2+]i. However, if such an internal TRPV1 pool is functional in the airway epithelia, then even in the absence of external calcium, one should still be able to observe a PM- or capsaicin-induced increase in [Ca2+]i, which would (eventually) lead to apoptosis. Both PM-induced increase in [Ca2+]i and apoptosis being absent in calcium-free solution argue against the involvement of a store-operated TRPV1 in the cellular effects of PMs in airway epithelia, at least after a 24-h incubation. In this regard, PM-induced IL-6 release was also dependent on the presence of extracellular calcium (57, 65).

Our observation that, even though initially only 30% of the epithelial cells were both acid and capsaicin sensitive (Table 1) and yet after 24 h of PM exposure, 60-70% of them exhibited CPZ-sensitive apoptosis may appear to contradict the conclusion that activation of TRPV1 receptors is essentially responsible for PM-induced apoptosis. However, exposure to PMs has been shown to increase the expression levels of TRPV1 mRNA in airway epithelial cells (66), suggesting that functional TRPV1 receptors may be upregulated by PM, as they have been shown to be by other inflammatory mediators (9, 60). This upregulation in TRPV1 gene expression may also explain the time-dependent increase in apoptosis.

Apoptosis: Roles of cAMP and PKA

Because capsaicin (41) and negatively charged particles (1, 2) both induce an increase in cAMP in neurons or airway cells, it follows that neuronal and epithelial TRPV1 receptors share this common signal transduction pathway. Here we have shown that PMs, such as ROFA and MSHA, also produce a CPZ-sensitive increase in cAMP in airway epithelial cells (Fig. 4). PM-induced cAMP-PKA activation may act to remove TRPV1 desensitization (10), thus permitting a sustained calcium influx (Fig. 2), and paradoxically delay PM-induced apoptosis. This latter statement is concluded because inhibition of PKA increased both endogenous and PM-mediated apoptosis, suggesting that cAMP-PKA-dependent processes aid in the delay of calcium cytotoxicity. This finding is consistent with previous studies, which showed that elevation of cAMP caused a delay in apoptosis (51, 52). Because apoptosis is not observed in a calcium-free environment, whether or not PKA is inhibited or PM is bound, it follows that the influx of calcium is the initial trigger in the cascade of events resulting in apoptosis.

In summary, we provided functional evidence that activation of TRPV1 channels is a mechanism underlying PM-induced apoptosis in epithelial cells and sensory neurons. We suggest that sustained calcium influx via PM-activated TRPV1 channels induce apoptosis. These data suggest that pharmacological manipulation of TRPV1 receptors will be a powerful tool in blocking PM-induced inflammation and toxicity.


    ACKNOWLEDGMENTS
 
We thank Sherry Larson and Deanna Gould of Duke Culture Facility for growing and maintaining the human epithelial cells.

GRANTS

This work was sponsored by National Institute of Environmental Health Sciences Grant ES-09844-01A2.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Simon, Duke Univ. Medical Center, Dept. of Neurobiology, PO Box 3209, Bryan Research Bldg., Rm. 427E, Research Dr., Durham, NC 27710 (E-mail: sas{at}neuro.duke.edu).

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.

1 Although neurons were seeded from two mice, the number of TG neurons adhering to the coverslips was very low. Hence, the scarcity of neurons made finding a neuron bound to the injected PMs even harder, which is reflected in smaller sample size. Back


    REFERENCES
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 DISCUSSION
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