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Chronic exposure to crystalline silica can lead to the development of silicosis, an irreversible, inflammatory and fibrotic pulmonary disease. Although, previous studies established the macrophage receptor with collagenous structure (MARCO) as an important receptor for binding and uptake of crystalline silica particles in vitro, the role of MARCO in regulating the inflammatory response following silica exposure in vivo remains unknown. Therefore, we determined the role of MARCO in crystalline silica–induced pulmonary pathology using C57Bl/6 wild-type (WT) and MARCO−/− mice. Increased numbers of MARCO+ pulmonary macrophages were observed following crystalline silica, but not phosphate-buffered saline and titanium dioxide (TiO2), instillation in WT mice, highlighting a specific role of MARCO in silica-induced pathology. We hypothesized that MARCO−/− mice will exhibit diminished clearance of silica leading to enhanced pulmonary inflammation and exacerbation of silicosis. Alveolar macrophages isolated from crystalline silica–exposed mice showed diminished particle uptake in vivo as compared with WT mice, indicating abnormalities in clearance mechanisms. Furthermore, MARCO−/− mice exposed to crystalline silica showed enhanced acute inflammation and lung injury marked by increases in early response cytokines and inflammatory cells compared with WT mice. Similarly, histological examination of MARCO−/− lungs at 3 months post–crystalline silica exposure showed increased chronic inflammation compared with WT; however, only a small difference was observed with respect to development of fibrosis as measured by hydroxyproline content. Altogether, these results demonstrate that MARCO is important for clearance of crystalline silica in vivo and that the absence of MARCO results in exacerbations in innate pulmonary immune responses.
Occupational exposure to respirable particles such as crystalline silica is associated with an increase in pulmonary inflammation, which plays a vital role in pathologies such as chronic obstructive pulmonary disease and silicosis (Park et al., 2002). Silicosis is characterized by persistent inflammation, localized fibroblast proliferation, and excess collagen deposition resulting in formation of silicotic nodules in the lung. Silicosis remains a prevalent health problem throughout the world, particularly in developing nations and currently no cure exists (Craighead et al., 1988; Green and Vallyathan, 1996). Although the pathophysiology of silicosis is well characterized, little is known about the molecular mechanisms that initiate and propagate the processes of injury, inflammation and fibrosis.
Alveolar macrophages (AMs) play a central role in crystalline silica–induced inflammation and pulmonary pathologies (Hamilton et al., 2008; Lehnert et al., 1989). These cells function in the recognition, uptake and clearance of particles via the mucociliatary escalator and/or lymphatic systems. They are also purported to be important in mounting an inflammatory response against inhaled particles (Bowden, 1987). The balance between clearance and retention of crystalline silica in the lungs by AM plays an important role in regulating the inflammatory response and silicosis. Previous studies indicate that facilitating the clearance of crystalline silica from the alveolar and interstitial compartments decreases the fibrotic response in the lung (Adamson et al., 1992, 1994). Therefore, unsuccessful clearance of crystalline silica may result in persistent inflammation due to prolonged interaction of particles with both immune and non-immune cell populations such as neutrophils, AM, dendritic cells (DCs), and epithelial cells in the lung. Disruption, of the epithelial lining would not only allow cytokines and growth factors released by AM to reach the interstitium and contribute to the development of silicosis (Merchant et al., 1990), but also, enhance translocation of crystalline silica particles to the interstitial space (Warheit et al., 1997). Once located in the interstitium these particles cannot be easily cleared. The interaction between crystalline silica and interstitial macrophage (IM) initiates a cascade of inflammatory signals that are major contributors to progressive fibrotic development in the lung (Adamson et al., 1991). Therefore, initial recognition and rapid clearance of crystalline silica by AM may be vital to curtail the persistent inflammatory response and development of chronic silicosis.
Previous studies from our laboratory demonstrated that AM recognize and bind crystalline silica particles through Class A scavenger receptors (SRs) expressed on their surface (Hamilton et al., 2006; Thakur et al., 2008). The Class A SRs are pattern recognition receptors that bind a wide variety of ligands including acetylated low density lipoprotein (AcLDL), bacteria, and inhaled particles and are known to play a role in innate immune responses (Murphy et al., 2005; Thakur et al., 2008). To date, five family members have been identified, SRA (splice variants: SRA -I, -II, and -III), MARCO (Macrophage receptor with collagenous structure), CSR1 (cellular stress response 1), SRCL (SR with C-type lectin), and SCARA 5 (class A scavenger receptor 5) (Thakur et al., 2008). Of these, MARCO is the predominant receptor for binding of unopsonized particles such as silica (Arredouani et al., 2004; Thakur et al., 2009). The C-terminal 100 amino acid long cysteine rich (SRCR) domain of MARCO has been established as the binding region for crystalline silica (Thakur et al., 2009). Although, previous work identified a specific role for MARCO in regulation of titanium dioxide (TiO2) induced acute inflammatory response (Arredouani et al., 2004), its physiological role in regulating the inflammatory response against fibrogenic crystalline silica particles has not been shown. We hypothesize that absence of MARCO will diminish the clearance of crystalline silica from the lung leading to increased inflammation and exacerbation of fibrosis. Using, C57Bl/6 wild-type (WT) and MARCO−/− (on a C57Bl/6 background) mice the current study determined the role of MARCO in crystalline silica–induced lung inflammation and fibrosis.
Breeding pairs of C57Bl/6 WT and Balb/c mice were originally purchased from the Jackson Laboratory (Bar Harbor, ME); whereas breeding pairs of MARCO−/− mice on C57Bl/6 background were kindly provided by Dr Lester Kobzik (Harvard School of Public Health, Boston, MA). Age-matched (6–8 weeks) males and females were used for all the studies. Genotyping was carried out as described previously (Dahl et al., 2007). All mice were maintained in the University of Montana specific pathogen-free laboratory animal facility. The mice were maintained on an ovalbumin-free diet and given deionized water ad libitum. The University of Montana Institutional Animal Care and Use Committee approved all animal procedures.
Crystalline silica (Min-U-Sil-5, average particle size 1.5–2 μm), obtained from Pennsylvania sand glass corporation (Pittsburgh, PA), was acid washed, dried and determined to be free of endotoxin by Limulus assay (data not shown) (Cambrex, Walkersville, MD). Titanium dioxide (TiO2) particles were purchased from Fischer Scientific. 4′,6-diamidino-2-phenylindole (DAPI) conjugated amorphous silica particles (1 μm in diameter) were purchased from Postnova Analytics, Inc. (Salt Lake City, UT). Particulates were resuspended in sterile PBS and sonicated 1 min prior to intranasal (i.n.) instillations. C57Bl/6, Balb/c, and MARCO−/− mice were anesthetized with ketamine 80 mg/kg (Fort Dodge Animal Health, Fort Dogdge, IA) and instilled i.n. with either 25 μl of sterile PBS, 1 mg crystalline silica or titanium dioxide (nonfibrogenic particle control) suspended in 25 μl of sterile PBS. Mice were returned to their cages and monitored until mobility returned. Following 3, 7, and 14 days or 4 weeks postinstillations, the mice were euthanized with a lethal dose of sodium pentobarbital (Euthasol).
Lungs were ascetically removed, minced, and incubated in RPMI Mediatech Herndon, VA) containing 1 mg/ml collagenase 1A (Sigma Chemical Co., St Louis, MO) at 37°C for ~90 min. Tissue was mechanically disrupted through 70-μm sterile cell strainer (BD Biosciences, San Jose, CA) and enzymatic action terminated with excess RPMI. White cells were isolated by centrifugation over a 40–70% Percoll (GE Biosciences, Piscataway, NJ) gradient centrifugation (Migliaccio et al., 2005), and enumerated using a Z1 Coulter particle counter (Beckman Coulter, Fullerton, CA). The total leukocyte fraction was collected, washed twice with ice cold sterile PBS (n = 4–8), and stained for flow cytometry or processed for microarray analysis.
Trizol Reagent (Invitrogen Corp. Carlsbad, CA) was added to cells, triturated, and incubated for 10 min at room temperature (RT). Chloroform was added and the solution was mixed by shaking for 30 seconds and incubated at RT for approximately 5 min. Phase separation was performed by centrifugation at 3200 x g for 30 min. Following isopropanol treatment, the samples were then precipitated overnight at −20°C. RNA was pelleted by centrifugation, the supernatant was removed, and 1 ml of 75% ethanol at −20°C prepared with DEPC water per 1 ml of Trizol was added to wash the pellet. Another centrifugation at 3200 × g for 10 min was performed, the supernatant was removed, and the pellets were air-dried. Pellets were resuspended in 100 μl of RNase Free Water from the Qiagen RNeasy Mini kit (Qiagen, Inc., Valencia, CA). Further purification was performed according to the “RNA Cleanup” protocol from the Qiagen RNeasy manual, including the optional DNase treatment. The final elution step was performed with 30 μl of RNase free water. RNA quantity was determined using a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Nano LabChip and reagents to obtain RNA Integrity Numbers and 28S/18S ratios.
RNA amplification and labeling were performed according to the Two-Color Microarray-Based Gene Expression Analysis protocol (Version 5.5, Agilent Technologies, Santa Clara, CA). Arrays used were the Whole Genome Mouse Oligo Microarrays, which are provided in a 4x44k format. A two-color system was used, with either saline-control or silica-exposed samples being hybridized against a Universal Mouse Reference RNA sample (Stratagene, La Jolla, CA). On each 4 × 44k array, two saline samples and two silica samples were hybridized. Hybridization was performed overnight at 65°C in a Robbins Scientific (SciGene, Sunnyvale, CA) hybridization oven with a rotor designed to fit Agilent hybridization chambers. Arrays were washed according to the Agilent protocol, and scanned using an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA). Resulting data files were normalized using a LOWESS algorithm. Normalized data was further analyzed using Microsoft Excel (Microsoft, Seattle, WA), GoMiner (National Center for Biotechnology Information), and PathwayStudio (Ariadne Genomics, Rockville, MD).
Non specific antibody binding of a total of 105 lavagable cells or interstitial cells was blocked by adding purified rat anti-mouse CD16/CD32 BD Pharmingen (San Jose, CA) diluted 1:100 in 30 μg of rat IgG (Jackson ImmunoResearch, West Grove, PA) to each sample prior to staining with fluorochrome-conjugated antibodies. One microgram of monoclonal antibodies specific to CD11c (allophycocyanin), CD11b (PerCP Cy5.5), Gr-1 (allophycocyanin Cy7) (BD Biosciences) major histocompatibility complex Class II (MHC II) (PE, eBiosciences, San Diego, CA), F480 (Pacific blue, Catlag Laboratories, Burlingame, CA) were added and incubated for 30 min on ice. Interstitial cells were also stained with 1 μg of MARCO fluorescein isothiocyanate (Serotec, Raleigh, NC). Cells were washed, resuspended in PAB (PBS buffer with 0.01% sodium azide and 1% fetal calf serum) and analyzed immediately. Cell acquisition and analysis was performed on a FACS Aria flow cytometer using FACS Diva software (version 4.1.2, Beckton Dickinson). Compensation of the spectral overlap for each fluorochrome was calculated using anti-rat/hamster Ig compensation beads (BD Biosciences).
At 24 h following particle instillations, WT and MARCO−/− mice were euthanized and a whole lung lavage was performed by cannulating the trachea and infusing the lungs with sterile PBS four times. Briefly, lavageable cells were pelleted at 400 × g for 5 min, resuspended in 1 ml of PBS, and total recovered cells enumerated using the Coulter counter. The acellular lavage fluid was also collected and frozen at −20°C until further analysis for protein levels (BCA assay, Pierce Rockford, IL) and cytokine concentrations (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, and IL-6; R&D Systems Minneapolis, MN) using kits according to the manufacturer's protocols.
C57Bl/6 (WT) and MARCO−/− mice were i.n. instilled with 25 μl of PBS or 1 mg crystalline silica, once a week for 4 weeks, and allowed to recover for 3 months. Lung weights were determined by weighing unlavaged lungs. The right lobe of the lung was inflated with 1 ml of 4% paraformaldehyde in PBS and postfixed for 24 h at 4°C. Routine histological procedures were used to paraffin embed the lobe. As previously described, five micron sections were cut, mounted on superfrost slides (VWR, West Chester, PA) and stained with Gomori's trichrome (Beamer and Holian, 2005). Five mice per group were examined microscopically and representative images captured with a Nikon E-800 microscope and Nikon DXM 1200 digital color camera using 4× and 40× objectives. The fibrotic condition of the lung was quantitatively assessed by measuring total collagen content of the left lung lobe as indicated by hydroxyproline; an amino acid unique to collagen. Briefly, the left lung lobe was excised, weighed, and immediately frozen in liquid nitrogen. The lung tissue was homogenized using a Tissue tearor in 1 ml of sterile water. An aliquot of lung homogenate was hydrolyzed in 12 N HCl at 110°C for 24 h. The mixture was reacted with chloramine T and Ehrlich's reagent to produce a hydroxyproline-chromophore that was quantified by spectrophotometry at 550 nm. Hydroxyproline content for each lobe was determined by triplicate analysis of the sample to provide a mean value.
For each parameter, the values for individual mice were averaged and the standard error was calculated. The significance of differences between exposure groups was determined by two-way ANOVA, in conjunction with Bonferroni's post hoc analysis, where appropriate. All ANOVA models were performed with Prism Software, version 4 (GraphPad Prism, San Diego, CA). A p value of < 0.05 was considered significant.
MARCO plays an important role in inflammation produced by various bacterial ligands and its expression may be induced on macrophage populations in response to inflammation (Dahl et al., 2007; van der Laan et al., 1999). In order to determine whether crystalline silica exposure altered MARCO expression on PM (AM and IM), microarray and flow cytometric analysis of MARCO expression on lung leukocytes isolated from PBS and crystalline silica–treated mice was assessed. Microarray analysis showed increased MARCO mRNA expression at 4 weeks following silica exposure relative to the PBS controls in pulmonary leukocytes in both C57Bl/6 (1.42-fold) and Balb/c (7.109-fold) mice. To determine whether crystalline silica correspondingly altered the cell surface expression of MARCO and to identify the pulmonary leukocyte populations contributing to the observed changes in MARCO, C57Bl/6, and Balb/c mice were i.n. exposed to PBS, crystalline silica, and TiO2. Of the three previously described macrophage populations (Migliaccio et al., 2005), only F480+/CD11bhi PM showed significant increases in the absolute number of MARCO+ cells at 3, 7, and 14 days following crystalline silica exposure compared with PBS and TiO2 (Figs. 1A–C). However, the expression levels of MARCO per cell as measured by mean channel fluorescence did not change (data not shown). Interestingly, the F480+/CD11blo macrophage population showed no change in MARCO expression in response to crystalline silica; although, the F480lo/CD11bhi macrophage population had no MARCO expression. Taken together, these data show that following exposure to crystalline silica but not TiO2, the number of MARCO+ pulmonary macrophages (AM and IM) increased. These results emphasize the importance of MARCO in crystalline silica–induced pathology.
Having identified a potential role of MARCO in silica-induced inflammation, the biological function of MARCO in crystalline silica recognition and uptake in vivo was determined using C57Bl/6 (WT) and MARCO−/− mice exposed to PBS and crystalline silica particles. CD11c+ lavage cells that expressed low amounts of MHC II were classified as AM, whereas CD11c+ cells with high levels of MHC II expression were classified as DC (Beamer and Holian, 2007). Changes in the side scatter properties of AM, which are indicative of changes in cellular granularity or silica uptake, were measured by flow cytometry (Hamilton et al., 2006). As anticipated, AM from MARCO−/− mice showed attenuated uptake of crystalline silica particles compared with WT mice (Fig. 2A). Increases in mean fluorescence intensity following exposure to particles served as a more sensitive measure for studying particle uptake. Therefore, DAPI conjugated amorphous silica (ASiO2) particles were used to study the role of MARCO in AM mediated uptake of silica particles over time (Fig. 2B). Only WT AM bound the DAPI conjugated ASiO2; although MARCO−/− AM were unable to bind DAPI conjugated ASiO2 particles (Fig. 2B). Together, these results highlight the role of MARCO in silica uptake and clearance from the lung.
Diminished uptake and clearance of crystalline silica particles from the lung by AM leads to increased lung injury marked by protein leakage across alveolar-capillary barrier (Driscoll et al., 1991; Kenyon et al., 2002). To investigate if decreased crystalline silica uptake and clearance by AM leads to increased lung injury and permeability, total protein levels in the lavage fluid from MARCO−/− and WT mice was measured 24 h after i.n. exposure to crystalline silica and PBS (Fig. 3). Although both WT and MARCO−/− mice exposed to crystalline silica showed increased levels of protein in the lavage fluid (38 and 83% increase over PBS, respectively); only MARCO−/− mice demonstrated a statistically significant increase in protein levels in the lavage fluid, indicating enhanced lung injury in the absence of MARCO (Fig. 3).
Infiltration of immune cells such as AM, DC, and neutrophils is an important step in development of pulmonary inflammation following exposure to environmental particles including crystalline silica and enhanced neutrophilia is a classic marker of silica-induced inflammatory response (Lagasse and Weissman, 1996). After crystalline silica exposure, the results demonstrated an increase in the total number of lavageable cells from MARCO−/− mice (2.72-fold) as compared with WT mice (1.95-fold) (Fig. 4A). To identify the cell type contributing to the increased cellularity shown in Fig. 4A, lavage cells from crystalline silica–treated exposed MARCO−/− and WT mice were stained for cell surface markers to differentiate AM (CD11c+/MHC IIlo), DC (CD11c+/MHC IIhi), and neutrophils (CD11b+/GR1+/CD11clo). The absolute number of AM and DC was significantly increased in response to crystalline silica exposure in MARCO−/−, but not WT mice 24-h postinstillation (Figs. 4C and 4D). Similarly, 24-h post–crystalline silica exposure, analysis revealed a significant increase in neutrophil infiltration in both WT and MARCO−/− mice (Fig. 4B); However, MARCO−/− mice demonstrated an enhanced neutrophilia compared with WT (Fig. 4B). These results further strengthen the observation that MARCO plays a role in crystalline silica–induced inflammatory responses.
To determine if the enhanced acute lung injury in MARCO−/− mice correlated with increased expression of early response cytokines, the levels of TNF-α, IL-1β, and IL-6 were measured in lavage fluid from WT and MARCO−/− mice at 24-h postexposure to PBS and crystalline silica. Levels of all three inflammatory cytokines were increased following crystalline silica exposure in WT and MARCO−/− mice compared with their respective PBS controls (Figs. 5A–C). However, levels of IL-6 were augmented in MARCO−/− mice following crystalline silica exposure compared with WT mice (Fig. 5C). The observed differences in cytokine profiles between WT and MARCO−/− mice further substantiate the previous results showing increased injury and inflammatory response (Figs. 3 and and4)4) in MARCO−/− mice following crystalline silica exposure.
Histopatholoical assessment of lung tissue sections from crystalline silica exposed mice was performed to assess whether the acute increase in inflammatory mediators correlated with chronic pathology. WT and MARCO−/− mice were instilled with 1 mg of crystalline silica or 25 μl of PBS, once a week for 4 weeks. Two months later the mice were anesthetized, the lungs were surgically removed and the lung wet weight was assessed and found to be higher in both crystalline silica exposed WT (1.44-fold) and MARCO−/− mice (2.08-fold) as compared with respective PBS-treated mice indicating the presence of either edema or infiltration of inflammatory cells (Fig. 6E). This response was also exacerbated in MARCO−/− mice. Representative sections from PBS exposed WT and MARCO−/− mice showed normal tissue architecture, indicating that the absence of MARCO does not lead to visible gross anatomical changes in the lungs (Figs. 6A and 6C). A typical inflammatory response and thickening of interstitium was observed in crystalline silica–treated WT mice (Fig. 6B and inset). In comparison, MARCO−/− mice demonstrated an increased accumulation of inflammatory cells (Fig. 6D and inset). Along with the increase in lung wet weight, these results indicate that MARCO−/− mice show increased chronic inflammation compared with WT mice, emphasizing the critical role of MARCO in crystalline silica–induced inflammation.
Although these results indicate that MARCO plays a critical role in crystalline silica–induced acute and chronic inflammation, the role of MARCO in the development of pulmonary fibrosis has not previously been explored. Crystalline silica–induced fibrosis in the lungs at 3 months postinstillation was assessed by hydroxyproline quantification of the left lung of WT and MARCO−/− mice. Crystalline silica exposed WT (1.69-fold) and MARCO−/− (2.19-fold) mice exhibited increased levels of hydroxyproline compared with their respective PBS treated mice (Fig. 6F). Crystalline silica–treated MARCO−/− mice exhibited a trend towards worsening of the fibrotic condition of lungs as compared with silica-treated WT mice, however the differences did not reach statistical significance (Fig. 6F). Although, MARCO−/− mice demonstrated a greatly enhanced inflammatory response, MARCO−/− mice showed only a marginal increase in collagen deposition, indicating differences in the complex processes of inflammation and fibrosis.
AMs are the first immune cell to encounter inhaled crystalline silica particles (Warheit et al., 1988). Following recognition and uptake of crystalline silica particles, AM clear some particles from the lung, undergo apoptosis or become activated to secrete various cytokines and growth factors (Iyer and Holian, 1997). These initial steps contribute to inflammatory events, as well as development of silicosis (Bodo et al., 2003; Thakur et al., 2008). An important early step following crystalline silica exposure is AM mediated particle clearance through the mucociliary pathway or lymphatic drainage mechanisms (Adamson et al., 1992; Brody et al., 1982). AM express the SR MARCO, which plays an important role in binding and uptake of crystalline silica particles (Hamilton et al., 2006). The purpose of the current study was to analyze the role of MARCO in crystalline silica clearance in vivo and subsequent inflammation and fibrosis. Results from the current study demonstrated that MARCO expressing pulmonary macrophages (PM) were increased in response to crystalline silica exposure, whereas the nonfibrogenic control particle TiO2 had no effect. Furthermore, MARCO−/− mice exhibited decreased crystalline silica clearance, which contributed to increased lung injury and both acute and chronic inflammation compared with WT mice.
Numerous reports have supported the hypothesis that translocation of crystalline silica loaded AM across the epithelial barrier and retention by IM enhances development of silicosis (Adamson et al., 1991; Zetterberg et al., 2000). In the lung, PM include both AM and IM and play a pivotal role in development of silicosis (Migliaccio et al., 2005; Zetterberg et al., 2000). In order to determine the role of MARCO in development of crystalline silica–induced inflammation and fibrosis, MARCO mRNA levels were quantified in pulmonary macrophages in response to crystalline silica exposure in both Balb/c and C57Bl/6 mice. Microarray analysis at 4 weeks postexposure demonstrated crystalline silica–dependent increases in MARCO mRNA in pulmonary macrophage population (data not shown) in both strains. Similarly, flow cytometric analysis at 3, 7, and 14 days of following crystalline silica exposure showed that C57Bl/6 and Balb/c mice demonstrate increased numbers of MARCO+ PM. It is of interest to note that the increase in MARCO+ PM was more pronounced in Balb/c mice (mRNA levels and earlier induction at 3 days) than C57Bl/6 PM indicating some strain specific changes following crystalline silica treatment. These results might also suggest a preferential role of MARCO in Th2-mediated immunity (Balb/c) as compared with Th1-mediated immunity model (C57Bl/6). Nevertheless, these results highlight the important role of MARCO in crystalline silica–induced inflammation.
The results presented here are specific to crystalline silica, in view of the fact that lung macrophages from mice treated with the nonfibrogenic particle TiO2 showed no change in MARCO expression. Previous studies showed similar results with regards to cytokine profile induced by the two particles. For example, IL-1β induction is unique to crystalline silica as compared with TiO2 (Driscoll et al., 1990; Oghiso and Kubota, 1987). One possibility is that silica directly stimulates MARCO expression on pulmonary macrophages, whereas TiO2 does not. Also, it can be hypothesized that cytokines induced specifically by crystalline silica exposed AM (e.g., IL-1β) can induce MARCO expression on both AM and IM. Consistent with this theory, previous studies have suggested a role of p38 mitogen activated protein kinases (MAPKs) in upregulation of MARCO (Doyle et al., 2004) and both IL-1β and silica are known stimulators p38 MAPK (Ovrevik et al., 2004). Nevertheless, the increased MARCO expression on PM from crystalline silica exposed mice substantiates the important and unique role of MARCO in immune response against fibrogenic silica particles.
To further investigate the exact role of MARCO in vivo and to better define its biological activity during crystalline silica–induced lung inflammation and fibrosis, we analyzed the pulmonary responses to silica in MARCO−/− mice. Analysis of AM from MARCO−/− mice showed diminished capacity for silica binding compared with AM from WT mice. The attenuation of binding and subsequent clearance of silica from the lung may contribute to increased microvascular permeability in MARCO−/− mice as measured by increases in total protein levels in the lavage fluid. These results indicate that diminished clearance of silica particles from the MARCO−/− mice contributes to disruption of alveolar-epithelial barrier and enhanced lung injury.
Similarly, an important first step in acute pulmonary inflammatory response to inhaled silica particles involves an influx of inflammatory cells (Bowden and Adamson, 1984). Staining the lavageable cells for AM, DC, and neutrophil surface markers following crystalline silica exposure demonstrated a significant increase in absolute number of all three cell types. The most profound difference observed was enhanced neutrophila in response to crystalline silica in MARCO−/− mice compared with WT mice. Previous studies from our laboratory reported that another Class A family member, SR-A, is an important player in crystalline silica–induced inflammatory responses since SRA−/− mice similarly developed enhanced neutrophilia and inflammatory response following crystalline silica exposure (Beamer and Holian, 2005). The diminished clearance of crystalline silica from the lungs of MARCO−/− mice may lead to prolonged interaction of crystalline silica particles with the lung epithelial cells causing induction of chemokines such as MIP-2 and KC, which are known to be important for neutrophil infiltration in the lungs (Yan et al., 1998).
In the lungs, the crystalline silica–induced inflammatory response is further orchestrated by proinflammatory cytokines (Rao et al., 2004), which may recruit inflammatory cells into the lung and propagate the inflammatory response as well as promote lung injury (Hamilton et al., 2008). TNF-α, IL-1β, and IL-6 have been extensively studied and shown to be important in pathogenesis of crystalline silica (Driscoll et al., 1990; Srivastava et al., 2002). In the present study, levels of all three cytokines were increased in WT and MARCO−/− mice 24-h post–crystalline silica exposure. Yet only, IL-6 levels from MARCO−/− mice were significantly increased compared with WT mice (Fig. 5C). Furthermore, it has been reported that IL-1β, TNF-α, and IL-6 can mediate inflammatory pathology of autoimmunity and antibodies or receptor antagonists of these inflammatory cytokines are effective therapeutics in autoimmune diseases (Chatzantoni and Mouzaki, 2006; Ishihara and Hirano, 2002). Correspondingly, recent studies have implicated MARCO−/− mice in increased response to self-antigens and have been demonstrated to be at increased risk for development of autoimmune diseases (Wermeling et al., 2007). Taken together, the above results demonstrated that absence of MARCO increases the acute response to crystalline silica exposure.
The increased inflammatory response observed in MARCO−/− mice following acute (24 h) exposure to crystalline silica correlated with an increased chronic inflammatory response (3 months) as evidenced by changes in wet weight and histopathology changes postinstillation. Taken together, these studies support the notion that MARCO is critical for crystalline silica clearance, as well as, for controlling acute and chronic pulmonary inflammation. However, contrary to expectation, 3 months post–crystalline silica exposure both WT and MARCO−/− mice showed increase in fibrotic response or silicosis with only small differences between the two (Fig. 6F). Although, the crystalline silica–treated MARCO−/− mice show increase (2.19-fold) in hydroxyproline content as compared with WT mice (1.69-fold) the differences do not reach significance. Future studies to quantify crystalline silica–induced fibrotic response in MARCO−/− mice at earlier time points will elucidate if the rate of development of silicosis differs between the two types of mice. During the inflammatory response, MARCO can limit both the recruitment of inflammatory cells and the activity of proinflammatory cytokines by clearing harmful crystalline silica particles from the lungs. At later fibrotic stages, however, the uncleared crystalline silica particles in the lung can interact with other pulmonary cell components such as epithelial cells, mesenchymal cells, and lymphocytes causing stimulation and cell injury irrespective of expression of MARCO on AM and DC. Nevertheless, the physiological clearance of crystalline silica particles from the lungs is apparently an important determinant in development of fibrosis (Adamson et al., 1992) and increasing the MARCO-mediated silica clearance during initial stages of exposure may help alleviate the inflammation and fibrotic response induced by crystalline silica.
In summary, the findings of this study highlight the importance of MARCO in crystalline silica–induced pathology. First, crystalline silica, but not TiO2 upregulated MARCO expression on pulmonary macrophages, indicating a specific role for MARCO in crystalline silica–induced inflammation. Second, MARCO plays an important role in clearance of crystalline silica particles from the lung and absence of MARCO leads to enhanced lung injury. Third, MARCO−/− mice exhibited increases in both acute and chronic inflammation following crystalline silica exposure, yet only slightly increased fibrosis. Together, these findings provide evidence of an important role of MARCO in vivo in regulation of crystalline silica–induced inflammatory response and fibrosis.
National Center for Research Resources (P20 RR017670); and the National Institute of Environmental Health Sciences (R01 ES015294).
The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Center for Research Resources, National Institute for Environmental Health Sciences, or National Institutes for Health.