Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCδ. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCδ were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCδ in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.
- alveolar macrophage
- protein kinase Cδ
silica is ubiquitous in the environment as an abundant mineral found in rock, sand, and soil. Silicosis is an occupational disease resulting from acute or chronic high levels of silica exposure that leads to decreased pulmonary function and increased susceptibility to other diseases of the respiratory tract (2). Upon inhalation, silica is deposited in the alveolar spaces and ingested by alveolar macrophages (AM) (5). High exposures of silica have been reported to impair the clearance of silica particles, leading to accumulation of silica-laden macrophages within the lymphatics and interstitial spaces of the lung (5). Furthermore, focal injury of macrophages by silica at these sites has been reported to result in the release of silica particles and repeat triggering of the local inflammatory response (36, 37). Silicosis has also been associated with increased incidence of systemic autoimmune disease, such as systemic lupus erythematosus and scleroderma (25, 26, 39). Patients with silicosis have increased autoantibodies, immunoglobulins, and immune complexes (12). Although some of the enhanced humoral response could be attributed to a nonspecific adjuvant effect, the tendency toward select autoimmune diseases suggests a more specific effect. Using autoimmune-prone New Zealand mixed (NZM) mice, we have previously reported that silica exposure significantly exacerbates the progression of autoimmune disease (6). After silica exposure, NZM mice develop high levels of autoantibodies to nuclear antigen, including histones, as well as develop glomerulonephritis and pulmonary fibrosis within 14 wk (6).
Apoptosis has been reported to play a role in the initiation and progression of systemic autoimmune disease, and silica has been reported to induce a caspase-specific apoptotic response within AM (14, 15, 19, 21, 24, 30, 34). We have previously reported a possible role for apoptosis in the exacerbation of autoimmune disease in NZM mice (27). Autoantibodies from silica-exposed mice specifically recognize apoptotic cells, and there appears to be greater recognition of silica-induced apoptotic cells compared with live, necrotic, or cycloheximide-induced apoptotic cells (27).
PKCδ is a novel PKC family member that has been reported to play a role in the initiation of apoptosis in many cell types (3, 4, 22, 28, 32, 38). PKCδ is activated by diacylglycerol/phorbol esters in a calcium-independent manner as well as by phosphorylation and caspase cleavage (20). PKCδ is activated in neutrophils undergoing spontaneous apoptosis, in H2O2-induced apoptosis, TNF-α-induced apoptosis, Fas-mediated apoptosis, and asbestos-induced apoptosis of alveolar epithelial cells (3, 4, 22, 28, 32, 38). PKCδ has been reported to translocate to the nucleus and mitochondria after activation by the above-mentioned mechanisms, thereby inducing caspase activation (4, 16, 20). Rottlerin has been reported as an inhibitor of apoptosis mediated by PKCδ (4, 16, 17, 20). However, it remains controversial as to whether the inhibition of apoptosis by rottlerin is mediated directly or indirectly through PKCδ. The overall objective of this study was to test the hypothesis that in vivo treatment with rottlerin could significantly affect the exacerbation of systemic autoimmune disease by silica.
MATERIALS AND METHODS
Male and female NZM 2410 mice were obtained from Taconic (Germantown, NY) and maintained in microisolation containers in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council. The animal room was set on 12-h dark/light cycles with food and water provided ad libitum. All protocols for the use of animals were approved by the University of Montana Institutional Animal Care and Use Committee.
BALB/c bone marrow-derived macrophages (BMDM) were generated for in vitro use. BALB/c mice were killed with a lethal injection of Nembutal (200 μl ip), and the femur bones were collected. Recovery of the bone marrow cells was done within a sterile hood, and the cells were flushed from the bones using a 25-gauge needle. The cells were counted and plated at 3 × 107 cells in 75-cm2 flasks. The cells were incubated overnight at 37°C. The non-adherent cells were then collected, allowing removal of stromal cells. The nonadherent cells were then replated in RPMI 1640/10% FCS and 10 ng/ml recombinant mouse macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN). The cells were allowed to differentiate into macrophages for 5–7 days before use. Cell viability was determined to be >90% by trypan blue exclusion before treatments.
Apoptosis was determined by DNA fragmentation in bone marrow-derived macrophages using a TiterTacs Assay (R&D Systems). The manufacturer's protocol was followed. Equal numbers of cells (1 × 105/well) were plated in a 96-well plate and allowed to adhere overnight. Silica was added for 8 h at 500 μg/ml with or without the addition of 1 μg/ml rottlerin 30 min prior. Nontreated cells were used as a negative control, and a nuclease-generated positive control was used. Experiments were repeated three times. The reported values are mean optical density (OD) values from each treatment.
Silica (Min-U-Sil-5, with an average particle size of 1.5–2 μm) was obtained from Pennsylvania Glass Sand (Pittsburgh, PA) and was acid washed, dried, and determined to be free of endotoxin. At 6 wk of age, mice were instilled intranasally with either 30 μl of saline (n = 6) or 30-μl saline suspensions of 1 mg of crystalline silica (n = 6) or 500 μg of TiO2 (n = 6) as previously described. All mice received two instillations 2 wk apart. Control and experimental groups were matched for equal number of male and female mice. AM were collected at 14 wk for use in microarray experiments. A second cohort of mice was used to measure changes in protein levels of PKCδ and received 30 μl of saline (n = 4) or 30-μl saline suspensions of 1 mg of crystalline silica (n = 4) or 500 μg of TiO2 (n = 4). A third cohort of mice was used for in vivo inhibition of PKCδ using rottlerin (AG Scientific, San Diego, CA), a PKCδ-selective inhibitor (11, 17). Rottlerin was given at 10 μg/instillation and diluted in sterile saline; a volume of 30 μl was used for intranasal instillation. Mice received either saline (n = 5), silica (1 mg × 2 instillations) (n = 5), silica (1 mg × 2 instillations) plus rottlerin (10 μg/weekly instillation; n = 5), or rottlerin only (10 μg/weekly instillation; n = 5). Rottlerin was given intranasally 1 day before addition of saline or silica and then again with the instillation of saline or silica at 6 wk of age. Rottlerin was then given once a week until the mice were killed at 14 wk. The saline- and silica-only treated animals were given saline instillations once a week for 14 wk as a control instillation to match the rottlerin instillations. After 14 wk, a time point when the majority of silica-exposed mice developed autoimmune disease as measured by proteinuria and autoantibodies, the mice were killed with a lethal injection of Nembutal (200 μl ip), and blood was collected by cardiac puncture. Clotted blood was centrifuged, and serum was collected and frozen at −20°C until use. AM were harvested from the lung by bronchoalveolar lavage for generation of protein lysates for use in Western immunoblotting, and kidneys were removed and placed in Histochoice fixative (Amresco, Solon, OH).
RNA extraction and amplification.
RNA was extracted from six mouse bronchoalveolar lavage samples per condition (saline, silica, or TiO2 treatment) using TRIzol (Invitrogen, Carlsbad, CA) with an extra chloroform extraction following the initial chloroform step to further remove organics from the final RNA product. Equal amounts of the resulting RNA from each sample within a condition were pooled and further purified and concentrated using RNeasy mini-preps (Qiagen, Valencia, CA). Production of amplified RNA (aRNA) was performed using a RiboAmp kit (Arcturus, Carlsbad, CA).
Microarray target labeling and hybridization.
For each sample, 5 μg of aRNA and 10 μg of Universal Mouse Reference RNA were indirectly labeled with Alexa Fluor 647 or Alexa Fluor 555, respectively, using Superscript II (Invitrogen) reverse transcriptase according to the ARES DNA labeling kit protocol.
Labeled cDNA from reference RNA and aRNA were combined and hybridized overnight in a 25% formamide hybridization buffer to an inhouse cDNA microarray with ∼1,200 different cDNA probes specific to mouse toxicology and immunology genes.
Hybridized arrays were scanned with an Axon GenePix 4000B laser slide scanner and visualized with GenePix Pro 4.0 software. Resulting result and image files were imported into GeneTraffic Duo Microarray Data Management and Analysis software (Iobion Informatics, La Jolla, CA) and normalized to a total 1:1 red to green ratio for the entire slide. Data were filtered based on replicate quality, signal intensity, and signal-to-background ratios of 1.2 or higher. An arbitrary cutoff of 1.5-fold up- or downregulation was used to determine genes of interest.
Kidneys were removed and routinely processed using an automated processor (ThermoShandon, Pittsburgh, PA). The kidneys were embedded in paraffin wax, sectioned 5 μm thick, and then collected on poly-l-lysine-coated slides (Sigma Chemical, St. Louis, MO). The kidney sections were boiled in a 0.01 M sodium citrate buffer for 10 min followed by washes in distilled water and PBS. The kidney sections were then blocked with 4% FBS in PBS. Goat anti-mouse IgG-FITC antibody (1:100; ICN Biomedicals, Irvine, CA) and goat anti-mouse C3-FITC (1:100, ICN Biomedicals) were added for 4 h for the detection of immune complexes and complement deposition. A goat anti-rat IgG antibody (1:100, ICN Biomedicals) was used as an isotype control. Samples were blinded and examined using a confocal microscope.
Proteinuria was measured by Chemstrip 2 GP test strips as described by the manufacturer (Boehringer Mannheim Diagnostics, Indianapolis, IN). Milligram protein per deciliter was measured among groups following the provided scale (0 = negative; trace, 1+ = 30 mg/dl; 2+ = 100 mg/dl; 3+ = 500 mg/dl).
Detection of serum autoantibodies.
Anti-histone autoantibodies were detected using an ELISA kit (Alpha Diagnostics, San Antonio, TX). Sera were diluted 100-fold before assay, and the manufacturer's protocol was followed. The reported values are mean OD values from each treatment group.
Western immunoblots were performed using lysates generated from AM collected by bronchoalveolar lavage. Lavage cells were collected and determined to be between 75 and 90% AM, and an equal number of cells were lysed using a 0.5% Nonidet P-40 detergent (Sigma) with freshly added complete protease inhibitors: 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF (Roche, Indianapolis, IN). Samples were kept on ice during lysis and frozen immediately at −20°C until use. Protein concentrations of lysates were determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA), and equal concentrations of lysates were loaded onto Invitrogen 12% Nu-PAGE gels (Carlsbad, CA) followed by transfer onto nitrocellulose (Bio-Rad). Nitrocellulose blots were probed using an antibody to PKCδ (1:300; BD Transduction Laboratories, San Diego, CA). Purified PKCδ (Oxford Biomedical Research, Oxford, MI) was used as a positive control. Detection of antibody to PKCδ was performed using goat anti-mouse IgG-horseradish peroxidase (1:2,000; Jackson Immunoresearch, West Grove, PA). Detection of positive bands was done using enhanced chemiluminescence reagent (Amersham, Piscataway, NJ), and they were visualized using a VersaDoc imaging system (Bio-Rad). Densitometry was performed using Quantity One software (Bio-Rad) to quantitate differences between bands. Numbers reported are density [intensity (int)/mm2].
Statistical analysis was done using the software package PRISM, version 3.03 (GraphPad, San Diego, CA). Differences among saline-, TiO2-, and silica-treated mouse groups were assessed using one-way ANOVA with Bonferroni posttest. Statistical differences in the development of proteinuria were determined by the nonparametric Kaplan-Meier log-rank comparison. All values are reported as means ± SE; P ≤ 0.05 was considered significant.
PKCδ is upregulated in AM from silica-exposed NZM mice.
Silica exposure has been reported to induce apoptosis in several cell types; however, the signaling cascade leading to the apoptotic response is not fully defined (9, 18, 34). Therefore, AM were collected from silica-exposed NZM mice to screen for changes in apoptotic genes using microarray technology. Using an inhouse microarray chip with 1,200 immunology and toxicology genes, we identified several genes involved in apoptosis that were upregulated 1.5-fold or higher after silica exposure (Table 1). Fourteen weeks after two instillations of 1 mg of silica, the RNA levels of PKCδ were elevated 1.5-fold compared with saline-instilled mice. The nonfibrogenic control particle, TiO2, did not elevate the levels of PKCδ RNA and was similar to the saline control mice. Similar to the RNA results, PKCδ protein levels were also found to be significantly elevated in silica-exposed mice compared with saline- and TiO2-exposed mice (4,921 ± 357 int/mm2 vs. 2,571 ± 333 int/mm2 and 1,805 ± 256 int/mm2; P ≤ 0.05, respectively; Fig. 1). These results indicate that PKCδ RNA and protein levels were both increased long term in AM after silica exposure in NZM mice.
Rottlerin reduces silica-induced apoptosis of BMDM and PKCδ protein levels.
To determine whether rottlerin could block silica-induced apoptosis of macrophages, BMDM were utilized in vitro. Apoptosis of silica-treated BMDM was determined by measuring DNA fragmentation. Figure 2 shows that 500 μg/ml silica induced a threefold increase in apoptotic cells within 8 h compared with an untreated control and was significantly reduced by the addition of rottlerin (1.074 ± 0.084 vs. 0.325 ± 0.073 vs. 0.663 ± 0.091; P ≤ 0.05, respectively). The apparent increase in DNA fragmentation by rottlerin alone was not statistically significant from control (Fig. 2). However, if cells are treated with 500 μg/ml silica for >12 h, rottlerin does not inhibit apoptosis (data not shown). These results demonstrated that rottlerin can reduce or delay apoptosis of silica-treated cells in vitro. Furthermore, PKCδ levels were significantly increased fivefold within 2 h after treatment with 500 μg/ml silica compared with an untreated control (1,766 ± 177 int/mm2 vs. 311 ± 13 int/mm2; P ≤ 0.001, respectively; Fig. 3). TiO2 (250 and 500 μg/ml) did not increase the levels of PKCδ compared with an untreated control (404 ± 93 int/mm2 and 359 ± 40 int/mm2 vs. 311 ± 13 int/mm2, respectively; Fig. 3). To inhibit PKCδ, rottlerin was added to the media of the BMDM culture 1 h before the addition of silica. Figure 3 demonstrates that addition of 1 μg/ml rottlerin inhibited the upregulation of PKCδ by silica at 2 h for the 500 μg/ml silica exposure (302 ± 40 int/mm2 vs. 1,766 ± 177 int/mm2; P ≤ 0.001, respectively). Treatment of BMDM with rottlerin alone did not have an effect on the levels of PKCδ at 2 h (Fig. 3).
Rottlerin treatment decreases levels of PKCδ in NZM mice.
On the basis of the results above supporting the role of PKCδ in silica-induced apoptosis in vitro through the ability of rottlerin to inhibit this process, the effect of rottlerin was examined in vivo. NZM mice were exposed to saline, two instillations of 1 mg of silica, two instillations of 1 mg of silica and 10 μg of rottlerin, or 10 μg of rottlerin alone. Rottlerin instillations were given 1 day before silica instillation and then once a week for 14 wk. Similar to the results in Fig. 1, PKCδ levels in AM, lavaged from the lungs of silica-treated NZM mice at 14 wk, were significantly increased compared with saline control mice (2,887 ± 643 int/mm2 vs. 249 ± 44 int/mm2; P ≤ 0.001, respectively; Fig. 4). However, instillation of rottlerin once a week for 14 wk significantly decreased the levels of PKCδ expression in silica-treated mice compared with mice receiving only silica (606 ± 124 int/mm2 vs. 2,887 ± 643 int/mm2; P ≤ 0.001, respectively; Fig. 4). These results suggest an association between the effects of rottlerin and PKCδ.
Rottlerin decreases severity of kidney disease in silica-treated NZM mice.
Silica exposure in NZM mice has been previously reported to increase proteinuria and immune complex and complement deposition within the kidney (6). In this study, proteinuria levels >500 mg/dl were seen in three out of five (60%) of the silica-treated mice 14 wk postinstillation. However, none of the mice developed >500 mg/dl proteinuria in silica-instilled mice that were also treated with rottlerin once a week for 14 wk (Fig. 5). Proteinuria was observed in one out of five (20%) mice receiving rottlerin-only treatments (Fig. 5). Kidneys from these mice were examined for IgG immune complex and complement C3 deposition. Figure 6 shows increased IgG deposition within the glomerulus of silica-exposed mice compared with the saline control mice, similar to previously reported results (6). In contrast, silica-exposed mice receiving rottlerin instillations once a week for 14 wk showed less extensive IgG deposition within the glomerulus (Fig. 6). Similar staining patterns for complement C3 were also seen (data not shown).
Rottlerin decreases the level of anti-histone autoantibodies in NZM mice.
As previously reported, silica exposure in NZM mice results in a significant increase in the levels of anti-nuclear autoantibodies, with an increase in anti-histone autoantibodies being the most prominent (6). We therefore tested the ability of rottlerin in vivo to affect this indicator of autoimmune disease. A significant increase in anti-histone autoantibodies was observed in silica-exposed NZM mice at 14 wk compared with saline control mice (0.392 ± 0.086 vs. 0.187 ± 0.034; P ≤ 0.05, respectively; Fig. 7). However, instillation of rottlerin significantly decreased the levels of anti-histone autoantibodies within silica-exposed NZM mice compared with mice that received only silica (0.159 ± 0.045 vs. 0.392 ± 0.086; P ≤ 0.05, respectively; Fig. 7). Consequently, rottlerin was effective in blocking the silica-induced increase in anti-histone autoantibodies.
Silica exposure has been reported to induce autoimmune responses and is associated with an increased incidence of systemic autoimmune disease (2, 25, 26). However, the mechanisms leading to these autoimmune responses are not understood. Elucidating these mechanisms may provide a better understanding of other environmentally relevant exposures implicated in triggering autoimmune responses as well as providing direction for novel therapies.
Possible mechanisms of silica-induced autoimmune disease may involve the AM, an immune cell of the lung that is the first line of defense. It has previously been reported that silica exposure leads to a caspase-dependent apoptosis of AM (19, 34). Silica-induced apoptosis of AM leads to release and uptake of silica by other AM, producing a cyclical process of inflammation and cell death (10). This constant inflammation and cellular death may provide excess antigen that is being presented to the immune system, thereby breaking immune tolerance.
Studies by Rosen and Casciola-Rosen (30) have reported concentrated autoantigens within the blebs of apoptotic cells. Lupus autoantigens are represented by structures that are chemically cleaved or modified during apoptosis, and if this material is not removed by noninflammatory processes, apoptotic material could be presented by specialized antigen-presenting cells to induce immune responses (29, 35). In an in vitro system, macrophages were found to prevent immunity to apoptotic material by competing with dendritic cells for uptake of apoptotic blebs, demonstrating the importance of the cell population silica is targeting for injury (1). Consistent with this hypothesis, mice intravenously exposed to apoptotic cellular material have been reported to develop autoantibodies (24).
In this study, a cDNA microarray specific for mouse toxicology and immunology genes was used with RNA from AM collected from silica-exposed NZM mice to analyze for expression changes in genes involved in the apoptotic process. Silica exposure in NZM mice induced several genes involved in apoptosis, including thymosin β10, PKCδ, TNF-α-induced protein-6, and Bcl-2-like protein. The apoptosis-related functions of these genes are reviewed in Brown et al. (8). In this study, we focused on the possible role PKCδ could play in silica-induced apoptosis and autoimmune disease. PKCδ was increased after silica exposure in vivo and remained increased long term, possibly through release of TNF-α (13). Concurrent with this hypothesis, we have previously reported a significant increase in TNF-α levels within the lung lavage fluid of silica-treated NZM mice 14 wk after silica exposure (7).
By measuring DNA fragmentation, we observed that silica induced apoptosis of BMDM, and this process could be delayed by the addition of rottlerin. Silica exposure has previously been reported to induce mitochondrial cytochrome c release, leading to a caspase-mediated apoptosis of macrophages (34). Furthermore, PKCδ has been reported to migrate to the mitochondria after exposure to asbestos and H2O2, resulting in altered mitochondrial membrane permeability, thereby activating caspases (23, 32). Therefore, it appears silica could induce apoptosis by multiple pathways and that rottlerin can delay the onset of apoptosis in macrophages. However, it remains controversial as to whether rottlerin is able to inhibit apoptosis through PKCδ. Soltoff (33) recently reported that rottlerin is a mitochondrial uncoupler and decreases cellular ATP levels, thereby indirectly affecting PKCδ activation. However, in our model, rottlerin ultimately did reduce apoptosis of silica-treated macrophages, but it remains unclear if this was mediated through PKCδ.
To test our hypothesis that in vivo treatment with rottlerin could significantly affect the exacerbation of systemic autoimmune disease by silica, we utilized weekly intranasal instillations of rottlerin to reduce silica-induced apoptosis. We have previously reported that intranasal instillation of silica in autoimmune-prone NZM mice results in a significant exacerbation of autoimmunity 14 wk after instillation as measured by increases in mortality, proteinuria, autoantibodies, and glomerulonephritis (6). NZM mice were chosen for these studies because they typically do not develop high levels of autoantibodies and glomerulonephritis until around 6 mo of age, thereby allowing us to observe any disease acceleration due to silica exposure (31). After silica exposure, NZM mice developed high levels of autoantibodies, and severe immune complex and complement deposition took place within the kidney around 14 wk (6, 31). Similar to the results previously reported, NZM mice in this study that received only silica instillations developed high proteinuria (60% of the mice), high levels of anti-histone autoantibodies, and excess immune complex and complement C3 deposition in the kidneys (6). However, silica-exacerbated systemic autoimmune disease was significantly reversed with weekly rottlerin instillations. Silica-exposed NZM mice receiving weekly instillations of rottlerin did not develop high levels of proteinuria, anti-histone autoantibodies, or severe immune complex or complement C3 deposition within the kidney.
Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of systemic autoimmune disease by silica exposure. Because of the role of PKCδ in apoptosis, it is tempting to speculate that this effect was due to inhibition of apoptosis. It would be interesting to test the effect of pan-caspase inhibitors in this model, but such a study is complicated by the myriad of cellular effects of the caspases, including posttranslational modification of cytokines. Since the exact cellular function(s) of rottlerin are also not entirely clear, an in vivo demonstration of a role of apoptosis inhibition in reducing silica-induced autoimmune disease is very challenging. This study demonstrates an important protective effect of rottlerin in systemic autoimmune disease in this model and sets the stage for future studies on its role in immune modification, possibly with therapeutic implications.
This work was supported by National Institutes of Health Grant ES-04084, COBRE National Center for Research Resources Grant P20-RR-17670, and an American Foundation for Pharmaceutical Education Fellowship (to J. M. Brown).
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