TOXICOLOGICAL SCIENCES 108(2), 462–471 (2009)
Advance Access publication January 16, 2009
Critical Role of MARCO in Crystalline Silica–Induced Pulmonary
Sheetal A. Thakur, Celine A. Beamer, Christopher T. Migliaccio, and Andrij Holian1
The University of Montana, Department of Biomedical and Pharmaceutical Sciences, Center for Environmental Health Sciences, Missoula, MT 59812
Received November 24, 2008; accepted January 13, 2009
Chronic exposure to crystalline silica can lead to the de-
velopment 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 in-
flammatory 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 MARCO2/2mice. Increased numbers of
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
MARCO2/2mice 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. Further-
more, MARCO2/2mice 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 MARCO2/2lungs
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.
Key Words: fibrosis; silicosis; particle clearance; macrophages;
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 de-
position 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
Alveolar macrophages (AMs) play a central role in
crystalline silica–induced inflammation and pulmonary pathol-
ogies (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 trans-
location 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
1To whom correspondence should be addressed at The University of Montana,
Center for Environmental Health Sciences, Department of Biomedical and
Pharmaceutical Sciences, 32 Campus Drive, Skaggs Building Room 280B,
Missoula, MT 59812. Fax: (406) 243-2807. E-mail: email@example.com.
? The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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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), bacte-
ria, 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 physio-
logical 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.
MATERIALS AND METHODS
Mice. 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.
Experimental instillations. Crystalline silica (Min-U-Sil-5, average par-
ticle size 1.5–2 lm), 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 lm
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 ll of sterile PBS, 1 mg crystalline
silica or titanium dioxide (nonfibrogenic particle control) suspended in 25 ll 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).
Isolation of the pulmonary leukocytes. 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-lm 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 ¼ 4–8), and stained for flow cytometry or processed for
RNA isolation. 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 3 g for 10 min was performed, the
supernatant was removed, and the pellets were air-dried. Pellets were
resuspended in 100 ll 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 ll
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.
Microarray analysis. 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 3 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).
Flow cytometry. Non specific antibody binding of a total of 105lavagable
cells or interstitial cells was blocked by adding purified rat anti-mouse CD16/
CD32 BD Pharmingen (San Jose, CA) diluted 1:100 in 30 lg 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 lg 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).
Cytokine ELISA. At24hfollowingparticleinstillations,WTandMARCO?/?
mice were euthanized and a whole lung lavage was performed by cannulating the
MARCO AND SILICA–INDUCED LUNG INFLAMMATION
trachea and infusing the lungs with sterile PBS four times. Briefly, lavageable cells
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]-1b, tumor
necrosis factor [TNF]-a, and IL-6; R&D Systems Minneapolis, MN) using kits
according to the manufacturer’s protocols.
Determination of lung wet weight and histopathological analysis of lung
tissue. C57Bl/6 (WT) and MARCO?/?mice were i.n. instilled with 25 ll 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 43 and 403
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.
Statistical analysis. 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.
Effect of Treatment on the Number of MARCOþPulmonary
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 contrib-
uting to the observed changes in MARCO, C57Bl/6, and Balb/c
mice were i.n. exposed to PBS, crystalline silica, and TiO2. Of
(Migliaccio et al., 2005), only F480þ/CD11bhiPM showed
significant increases in the absolute number of MARCOþcells
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). In-
terestingly, the F480þ/CD11blomacrophage population showed
no change in MARCO expression in response to crystalline
silica; although, the F480lo/CD11bhimacrophage population had
no MARCO expression. Taken together, these data show that
following exposure to crystalline silica but not TiO2, the number
of MARCOþpulmonary macrophages (AM and IM) increased.
These results emphasize the importance of MARCO in
crystalline silica–induced pathology.
MARCO-Mediated Uptake and Clearance of Crystalline
Silica from the Lung
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. CD11cþlavage cells that
expressed low amounts of MHC II were classified as AM,
whereas CD11cþcells 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.
Lung Injury and Inflammation following Crystalline Silica
Exposure in MARCO?/?Mice
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?/?
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).
mice exposed to
THAKUR ET AL.
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 crys-
talline silica and enhanced neutrophilia is a classic marker of
silica-induced inflammatory response (Lagasse and Weissman,
1996). After crystallinesilica exposure,the results demonstrated
an increase in the total number of lavageable cells from
MARCO?/?mice (2.72-fold) as compared with WT mice
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
(CD11cþ/MHC IIlo), DC (CD11cþ/MHC IIhi), and neutrophils
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 neutro-
philia compared with WT (Fig. 4B). These results further
strengthen the observation that MARCO plays a role in
crystalline silica–induced inflammatory responses.
Levels of Early Response Cytokines following Crystalline
Silica Exposure in MARCO?/?Mice
To determine if the enhanced acute lung injury in MARCO?/?
mice correlated with increased expression of early response
cytokines, the levels of TNF-a, IL-1b, and IL-6 were measured in
lavage fluid from WT and MARCO?/?mice at 24-h post-
exposure 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
MARCO expression on C57Bl/6 PM. Three distinct subsets of PM were defined using F480 and CD11b. Of these, the F480þ/CD11bhipopulation showed
increased percent MARCOþcells following crystalline silica (C. Silica) exposure compared with PBS and TiO2. (B) Relative to PBS and TiO2, the percent
MARCOþmacrophages are significantly increased at 7 and 14, but not 3 days following C. Silica treatment. (C) C. Silica treatment in Balb/c mice also lead to
increases in the percent MARCOþPM at 3, 7, and 14 days; whereas TiO2had no effect. The results represent mean ± SE for each group (n ¼ 4–8). **p < 0.01 and
**p < 0.001 compared with PBS;##p < 0.01and###p < 0.001 compared with TiO2.
Expression of MARCOþPM following crystalline silica and TiO2instillation. (A) Representative scatter plot and histograms showing changes in
MARCO AND SILICA–INDUCED LUNG INFLAMMATION
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 4) in MARCO?/?
mice following crystalline silica exposure.
Chronic Inflammation and Fibrosis following Crystalline
Silica Exposure in MARCO?/?Mice
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 ll 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. Represen-
tative 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–
Although these results indicate that MARCO plays a critical
role in crystalline silica–induced acute and chronic inflammation,
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 WT mice were exposed i.n. to 1 mg of crystalline silica (C. Silica) or
25 ll of PBS. After 24 h, AM from WT and MARCO?/?mice showed
increased side scatter. However, AM from MARCO?/?mice exhibited
attenuated binding compared with AM from WT mice. (B) MARCO?/?and
WT mice were i.n. instilled with fluorescent amorphous silica (A. Silica)
particles. At 4, 24, and 72 h, AM were analyzed for increases in mean
fluorescence intensity (MFI) as a marker of uptake. AM from A. Silica–treated
WT mice showed increases in MFI, whereas MARCO?/?mice did not show
any change in MFI. The results are reported as mean values ± SE (n ¼ 5–6).
**p < 0.01 and **p < 0.001 compared with PBS, and ###p < 0.001 compared
with (A). Silica-treated MARCO?/?mice.
Crystalline silica uptake by MARCO?/?AM. (A) MARCO?/?
mice. Lavage fluid from MARCO?/?and WT mice was assessed for changes
in total protein levels. Both WT and MARCO?/?mice showed increases in
protein levels in the lavage fluid however significant changes were observed
only in MARCO?/?mice. The results are reported as mean ± SE (n ¼ 5–6).
**p < 0.01 compared with PBS control.
Lung injury following crystalline silica treatment in MARCO?/?
THAKUR ET AL.
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,
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 nonfibro-
genic control particle TiO2 had no effect. Furthermore,
MARCO?/?mice exhibited decreased crystalline silica clear-
ance, 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
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 MARCOþPM. It is of
interest to note that the increase in MARCOþPM was more
pronounced in Balb/c mice (mRNA levels and earlier induction
at 3 days) than C57Bl/6 PM indicating some strain specific
AM, DC, and neutrophil populations and analyzed by flow cytometry 24 h following exposure. (A) The total number of lavage cells from WT and MARCO?/?
mice increased following crystalline silica instillation. (B) C. Silica exposure resulted in greater recruitment of neutrophils in MARCO?/?mice compared with WT
mice. Similarly, absence of MARCO also lead to pronounced increases in (C) AM and (D) DC populations. The results are represented as mean ± SE (n ¼ 5–6).
*p < 0.05, **p < 0.01, and ***p < 0.001 compared with PBS; and #p < 0.05 compared with WT C. Silica–treated mice.
MARCO?/?mice inflammatory cell infiltration following crystalline silica exposure. Lavage cells were stained for cell surface markers to differentiate
MARCO AND SILICA–INDUCED LUNG INFLAMMATION
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
The results presented here are specific to crystalline silica, in
view of the fact that lung macrophages from mice treated with
the nonfibrogenic particle TiO2showed no change in MARCO
expression. Previous studies showed similar results with regards
to cytokine profile induced by the two particles. For example,
IL-1b 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 TiO2does not. Also, it can
be hypothesized that cytokines induced specifically by crystal-
line silica exposed AM (e.g., IL-1b) 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-1b 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 permeabil-
ity 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 demon-
strated 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 inflamma-
tory responses since SRA?/?
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
mice similarly developed
silica (C. Silica) exposure, the levels of (A) IL-1b (B) TNF-a, and (C) IL-6
were significantly increased in lavage fluid from WT and MARCO?/?mice. In
particular, C. Silica–treated MARCO?/?mice showed enhanced levels of IL-6
compared with C. Silica–treated WT mice. The results are represented as mean
± SE (n ¼ 5–6). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with PBS;
and ##p < 0.01 compared with C. Silica–treated WT mice.
Cytokine response in MARCO?/?mice. After 24-h crystalline
THAKUR ET AL.
(Rao et al., 2004), which may recruit inflammatory cells into the
been extensively studied and shown to be important in
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).
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.
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
evaluated by Gomori’s trichrome staining. Representative sections from WT and MARCO?/?mice treated with PBS (A and C) show normal tissue and cell
architecture and no inflammatory response. In contrast, MARCO?/?crystalline silica (C. Silica)–exposed mice (D) exhibited enhanced infiltration of inflammatory
cells compared with C. Silica–treated WT mice lung sections (B). Magnification bar 25 and 12.5 lm (inset). (E) The wet lung weight of both WT and MARCO?/?
mice was significantly increased 3 months following C. Silica treatment as compared with PBS exposed mice. (F) Hydroxyproline levels were significantly
increased in both WT and MARCO?/?mice 3 months post-C. Silica treatment as compared with PBS treated mice. Also, there was small difference in
hydroxyproline levels between WT and MARCO?/?mice following crystalline silica exposure. Results represent mean ± SE (n ¼ 5–6). *p < 0.05 and ***p <
0.001 compared with PBS; and #p < 0.05 compared with C. Silica–treated WT mice.
Chronic exposure to crystalline silica leads to exacerbated inflammatory response and fibrosis. Chronic inflammatory response in the lung was
MARCO AND SILICA–INDUCED LUNG INFLAMMATION
with only small differences between the two (Fig. 6F). Although,
the crystalline silica–treated MARCO?/?mice show increase
(1.69-fold) the differences do not reach significance. Future
studies to quantify crystalline silica–induced fibrotic response in
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 proinflam-
matory 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
cells, and lymphocytes causing stimulation and cell injury
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
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 inflamma-
tion 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
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.
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