FASEB J. Avanti Polar Lipids
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-095299.
 (The FASEB Journal. 2008;22:2053-2063.)
© 2008 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-095299v1
22/6/2053    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beamer, C. A.
Right arrow Articles by Holian, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beamer, C. A.
Right arrow Articles by Holian, A.

Silica suppresses Toll-like receptor ligand-induced dendritic cell activation

Celine A. Beamer1 and Andrij Holian

Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana, USA

1Correspondence: Center for Environmental Health Sciences, School of Pharmacy and Allied Health Sciences, 32 Campus Dr., Skaggs Bldg., Rm. 285A, University of Montana, Missoula, MT 59812-1552, USA. E-mail: celine.beamer{at}umontana.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation of silica, without evidence of silicosis, is believed to predispose individuals to bacterial infections and impair respiratory immune functions. Silica may alter the sensitivity of antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), to other types of infection; however, the exact nature of these exchanges remains uncertain. The purpose of the present study is to characterize the effect of silica exposure on innate pulmonary defense mechanisms following Toll-like receptor (TLR) ligand-induced activation using DCs as a model APC and determine whether these signals act in synergy or opposition to one another. Using C57Bl/6 mice, pattern recognition receptor expression on DCs was examined in vitro and in vivo using flow cytometry, and the activation state of pulmonary and granulocyte-macrophage colony-stimulating factor-derived DCs was assessed in response to silica in combination with TLR ligands (lipopolysaccharide, cytosine-phosphate-guanine, or polyinosinic:polycytidylic acid) using flow cytometry and measurement of cytokine production. In this study, silica attenuated TLR ligand-dependent DC activation with regards to accessory molecule expression as well as nitric oxide and inflammatory cytokine production. Furthermore, silica’s ability to modulate TLR ligand-dependent DC activation did not appear to be dependent on the class A scavenger receptors. Taken together, silica’s ability to alter susceptibility to infection may be due to impaired inflammatory responses and reduced antibacterial activity.—Beamer, C. A., Holian, A. Silica suppresses Toll-like receptor ligand-induced dendritic cell activation.


Key Words: pattern recognition receptor • costimulatory molecule • TLR4 • MARCO


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SILICOSIS IS AN OCCUPATIONAL lung disease resulting from the inhalation of silica particles over prolonged periods of time, which causes chronic inflammation and progressive pulmonary fibrosis as well as systemic immune dysfunctions (1 , 2) . Although the prevalence of silicosis has been decreasing in developed countries because of occupational health and safety standards, it remains commonplace in developing countries. While the association between silicosis and tuberculosis (TB) has been known for many years, recent reports suggest that exposure to silica, without evidence of silicosis, predisposes individuals to TB infection (3 , 4) . Moreover, silicosis was associated with not only increased risk of reactivity of latent TB infection but also with persistent difficulty in treating active infections (4) , such that this increased risk of TB is lifelong, even if silica exposure ceases (5) . Although inconclusive, silica may alter the sensitivity of antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), to other types of infection (6 7 8) . Two decades ago, Zimmerman et al. (9) reported that silica decreased the phagocytic capacity of macrophages and neutrophils. In vivo, silica enhanced lipopolysaccharide (LPS)-induced production of interleukin (IL)-1β, tumor necrosis factor (TNF)-{alpha}, and IL-12 release by peritoneal cells (10 , 11) ; whereas, in vitro, silica suppressed LPS-induced IL-1β and IL-6 production (12) . In addition, silica rendered mice either resistant (13) or hyperresponsive to subsequent LPS challenge in vivo (10) .

Inhalation of bacteria, virus, protein antigen, or other environmental contaminants, including silica, induces a wave of DC recruitment into the respiratory tract (14 , 15) . DCs capture and kill pathogens, followed by processing, transport, and presentation of antigen, whereon they act as a bridge between innate and adaptive immunity (16) . This complex sequence of DC activation is characterized by up-regulation of numerous molecules involved in antigen presentation, cell-cell interactions, and costimulation, as well as cytokine and chemokine receptors (17) . Moreover, DCs mediate protection against pulmonary tuberculosis infection in mice, and elevated numbers of DCs have been detected in lung cancer tissue and sarcoidosis (18) . Disruption of DC activation may be important to the pathogenesis of many respiratory disorders, including infections, lung cancer, asthma, and smoking-related diseases (19 , 20) .

A crucial event for initiation and regulation of the proper immune response is the initial discrimination of the pathogen challenge. To accomplish this, DCs express on their surface a number of innate receptors—the so-called pattern recognition receptors (PRRs)—which bind to unique pathogen-associated molecular patterns (PAMPs) expressed by several related pathogens but not host cells (21) . The ever-growing list of PRRs includes toll-like receptors (TLRs), different subfamilies of C-type lectins, β2-integrins, and class A scavenger receptors (SRs). Of these, TLRs are among the most well-studied receptors, and it is well known that TLR ligand recognition induces cytokine production and DC activation (22) . Of the 10 TLRs discovered, TLR2, TLR4, and TLR5 are expressed on the cell surface, where they can be activated by PAMPs expressed on the surface of pathogens. The prototypical TLR4 ligand is LPS, a major component of the outer membrane of gram-negative bacteria. In addition, bacterial DNA and oligodeoxynucleotides carrying the cytosine-phosphate-guanine (CpG) motif, as well as double-stranded RNA from viruses are also recognized by TLRs and exhibit immunostimulatory activity on DCs (23) .

Although previous reports clearly established a link between silicosis and TB, indicating that silica exposure alters susceptibility to infection, the underlying cellular and molecular mechanisms remain elusive. We hypothesize that silica predisposes individuals to infection and impairs pulmonary immune functions due to deleterious effects on APC functions. The present study characterized DC activation in vitro in response to coincident silica and pathogen exposure and determined whether these signals act in synergy or opposition to one another.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and genotyping
Breeding pairs of C57Bl/6 mice were originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA); while breeding pairs of CD204–/–, MARCO–/–, and MARCO/CD204–/– (maintained on C57Bl/6 background) mice were kindly provided by Dr. Lester Kobzik (Harvard School of Public Health). Genotyping was carried out as described previously (24) . All mice were maintained in the University of Montana specific pathogen-free (SPF) laboratory animal facility, and allowed food and water ad libitum. Cages, bedding, and food were sterilized by autoclaving and mice were handled with aseptic gloves at all times. All animal use procedures were in accordance with the U.S. National Institutes of Health and University of Montana Institutional Animal Care and Use Committee guidelines.

Bone marrow-derived dendritic cells (BMDCs)
Bone marrow was aspirated from the femurs and tibiae of C57Bl/6 mice (6–8 wk) using a 3 ml syringe filled with RPMI culture media (25 , 26) and seeded in tissue culture flasks. Following overnight stromal cell elimination, 20 x 106 nonadherent cells were transferred to new flasks, including murine recombinant granulocyte-macrophage colony-stimulating factor (R&D Systems Minneapolis, MN, USA) (final concentration=33 ng/ml). By 8 days, cells were fully differentiated, >75% confluent, and immunopositive for DC characteristics (14) . Viability was determined to be >90% by trypan blue exclusion staining prior to experimental manipulations. Nonadherent BMDCs were removed, and the flasks were gently washed and lightly scraped to recover adherent BMDCs.

Stimulation
BMDCs were seeded at 106 cells/ml/well of a 6-well plate and immediately exposed in suspension to media, crystalline silica (25–100 µg/ml, PA Glass Sand Corporation, Pittsburgh, PA, USA), or titanium dioxide (50 µg/ml, Fischer Scientific, Houston, TX, USA), plus or minus LPS (endotoxin, 1 µg/ml, Sigma, St. Louis, MO, USA), CpG (0.5 µM; Invivogen, San Diego, CA, USA), or polyinosinic:polycytidylic acid (Poly I:C; a synthetic analog of dsRNA; 250 µg/ml; Invivogen) and allowed to incubate for 24 h at 37°C. Silica (Min-U-Sil-5, average particle size 1.5–2 µm) was acid washed, dried, and determined to be free of endotoxin by Limulus assay (data not shown; Cambrex, Walkersville, MD, USA). Particulates were suspended in media and sonicated for 1 min. Following stimulation, BMDCs were lightly scraped within the spent culture media, centrifuged, and the supernatant and cells separated for analysis. Additional experiments included pretreatment with silica for 1 h prior to TLR ligand exposure or pretreatment with LPS, CpG, or Poly I:C for 1 h prior to silica exposure. The alternate stimulus was then added and the cells incubated for an additional 23 h.

Flow cytometry
BMDCs were harvested as described above and nonspecific antibody binding blocked with purified rat anti-mouse CD16/CD32 (BD Pharmingen, San Jose, CA, USA) diluted 1:100 in 30 µg of rat IgG (Jackson ImmunoResearch, West Grove, PA, USA). One µg of monoclonal antibodies specific to CD40 PE, CD86 APC, CD45 APC-Cy7 (BD Pharmingen), CD54 Pacific Blue (Biolegend, San Diego, CA, USA), major histocompatibility complex class II (MHC class II) FITC, TLR4 PE (eBioscience, San Diego, CA, USA), CD36, and MARCO (Serotec Raleigh, NC, USA) were added and incubated for 30 min on ice. Five µl of 7-amino-actinomycin D (7-AAD; BD Pharmingen) was added directly to the cells and incubated 10–15 min at room temperature. Cells were washed, resuspended in polyclonal antibody (1% bovine serum albumin, 0.01% sodium azide in PBS) and analyzed immediately. Cell acquisition and analysis was performed on a FACS Aria flow cytometer using FACS Diva software (version 4.1.2; BD Biosciences, San Jose, CA, USA). A live cell gate was established using 7-AAD negative cells and 100,000 events captured. Compensation of the spectral overlap for each fluorochrome was calculated using anti-rat/hamster Ig compensation beads (BD Biosciences).

Antigen uptake assay
BMDCs were isolated and incubated for 1 h with silica or titanium dioxide in suspension. Subsequently, 0.5 µl Alexa 488-conjugated ovalbumin (OVA), LPS, or LDL (Invitrogen, Carlsbad, CA, USA) were added and phagocytosed for 2 h. The cells were washed twice with PBS, and the median fluorescence intensity (MFI) of ingested antigen assessed by flow cytometry and 50,000 events collected.

Cytokine ELISA, lactate dehydrogenase (LDH), and nitrite assay
Acellular supernatants resulting from experiments described above were collected and analyzed for IL-1β, IL-6, IL-10, IL-12, IL-13, IFN{gamma}, TNF-{alpha}, and activated TGF-β production using murine cytokine ELISA kits according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). In addition, 50 µl of tissue culture supernatants was analyzed for LDH (Biovision, Mountain View, CA, USA) or nitrite levels according to the Griess method (Promega, Madison, WI, USA).

Preparation of lung cell suspensions
Lungs were ascetically removed, minced, and incubated in RPMI containing 1 mg/ml collagenase IA (Sigma, St. Louis, MO, USA) at 37°C for ~90 min. Tissue was disrupted through a 70 µm cell strainer (BD Biosciences) and enzymatic action terminated with excess RPMI. White cells were isolated by centrifugation over a 40–70% Percoll gradient (27) and enumerated using a Coulter counter. Nonspecific antibody binding was blocked, and cells were incubated with CD11c APC and anti-APC magnetic beads and isolated using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec, Auburn, CA, USA). Purity was confirmed by flow cytometry and determined to be ≥85% CD11chi. Pulmonary DCs were seeded at 106 cells/ml/well and exposed to silica or titanium dioxide, plus or minus LPS (1 µg/ml) and allowed to incubate for 24 h. Cells and cell-free supernatants were collected and analyzed as indicated.

Data analysis
For each parameter, the values for individual mice were averaged and the SD and SE calculated. The significance of the differences between the exposure groups was determined by t test or 1-way or 2-way ANOVA, in conjunction with Bonferroni’s posthoc analysis and Tukey’s test for variance, where appropriate. All ANOVA models were performed with Prism software, version 4 (GraphPad, San Diego, CA, USA). A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-dependent effects of silica
After DCs encounter pathogens, TLR-mediated activation results in increased surface expression of MHC class II and other accessory molecules, which are essential to T-cell priming (16) . To test whether coincident exposure of BMDCs to endotoxin (LPS, the prototypical TLR4 ligand) and silica (a known scavenger receptor ligand) (28) resulted in altered expression of cell surface molecules, BMDCs were stimulated with LPS (1 µg/ml) and varying concentrations of silica (25–100 µg/ml) for 24 h. Flow cytometry confirmed that LPS increased the expression of MHC class II, CD40, CD54, and CD86 on BMDCs compared with unstimulated cells as measured by MFI (Fig. 1 ). However, LPS-induced BMDC activation was reduced by coexposure with silica at all concentrations examined, and 100 µg/ml was the most effective dose (Fig. 1) . Although a slight increase in LDH was observed, indicating cell damage in response to exposure, the differences detected in TLR-induced activation were not attributed to excessive cell death because the MFI analysis was performed on live cells (gated as 7-AAD negative; data not shown).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Figure 1. Silica decreased LPS-induced costimulatory molecule expression in a dose-dependent manner in BMDCs. LPS increased expression of MHC class II, CD40, CD54, and CD86 compared with the unstimulated BMDCs. This increased accessory molecule expression was dose-dependently reduced by silica. Of the 3 concentrations examined, 100 µg/ml of silica was the most potent. n = 3–5, in triplicate; error bars = SEM; *P < 0.05 vs. media-only control; #P < 0.05 vs. LPS-only control.

Nitric oxide (NO) production
Playing diverse roles in inflammation and innate immune responses (29) , NO is a major mediator of innate immune responses because of its involvement in bactericidal activity (30) and may play a role in the regulation of cytokine synthesis (31) . To investigate the impact of silica exposure on NO production in response to 1 µg/ml LPS, 0.5 µM CpG, or 250 µg/ml Poly I:C, the levels of nitrite (NO2–), which is one of two primary, stable, and nonvolatile breakdown products of NO, were measured. As expected, only trace amounts of NO2– were detected in the acellular supernatants collected from unstimulated BMDCs. These values were not altered by 100 µg/ml of silica, although titanium dioxide (50 µg/ml, nonfibrogenic particle control at a comparable surface area) resulted in a slight increase in NO2– (Fig. 2 A). As anticipated, exposure to LPS, CpG, or Poly I:C increased NO2– to varying degrees compared with unstimulated BMDCs (Fig. 2A ). However, coincident exposure to silica and LPS, CpG, or Poly I:C decreased TLR ligand-induced NO2– production compared with the TLR ligand alone (Fig. 2A ). Although a trend toward a reduction in NO2– levels was observed with concomitant silica and Poly I:C exposure, this decline was not statistically significant compared with Poly I:C alone. Coincident exposure of BMDCs to titanium dioxide and TLR ligands similarly reduced the induction of NO2– levels by LPS and CpG but not Poly I:C compared with TLR ligand alone, though these decreases were moderate compared with silica’s ability to reduce NO2– (Fig. 2A ). Although an increase in LDH was observed on exposure to silica plus TLR ligand compared with unstimulated BMDCs or the TLR ligand alone, the differences were not attributed to excessive cell death because of the modest changes observed (Fig. 2B ). Furthermore, cell viability measured using the exclusion dye 7-AAD revealed minor changes between unstimulated cells and any of the culture conditions examined (Fig. 2C ).


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		Figure 2. Silica decreased LPS-, CpG-, and Poly I:C-induced nitrite production (A), increased LDH release (B), and showed limited effects on cell viability (C). Although silica had no effect, titanium dioxide, LPS, CpG, and Poly I:C increased nitrite levels compared with unstimulated BMDCs. This increase in nitrite by TLR ligand was reduced by concomitant silica and titanium dioxide exposure (A). Silica increased LDH whereas titanium dioxide decreased LDH. LPS but not CpG or Poly I:C decreased LDH release compared with unstimulated BMDCs. Coexposure to LPS, CpG, or Poly I:C and silica resulted in increased LDH release compared with TLR ligand alone. (B) Viability assays revealed nominal differences in the percentage of viable cells based on 7-AAD exclusion (C). n = 3–5, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only; P < 0.05 vs. silica only.

Accessory molecule expression
To investigate whether silica modulates TLR ligand-induced expression of accessory molecules, CD40, CD86, CD54, and MHC class II were analyzed by flow cytometry following coculture with LPS, CpG, or Poly I:C. Neither silica nor titanium dioxide alone exhibited any appreciable effects on the expression of accessory molecules, although silica reduced CD54 expression compared with unstimulated BMDCs (Fig. 3 ). As anticipated, LPS, CpG, and Poly I:C increased MHC class II, CD40, CD54, and CD86 expression on BMDCs compared with unstimulated cells (Fig. 3) . Consistent with the observed effect on NO2– levels, concomitant exposure to 100 µg/ml silica attenuated TLR ligand-induced accessory molecule expression compared with TLR ligand alone (Fig. 3) . In contrast, 50 µg/ml titanium dioxide had no effect (Fig. 3) . Notably, this inhibitory effect of silica on LPS-, CpG-, or Poly I:C-induced BMDC activation occurred even when cells were pretreated for 1 h with TLR ligand prior to the addition of silica to the culture and vice versa (data not shown).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Figure 3. Silica but not titanium dioxide inhibited LPS-, CpG-, or Poly I:C-induced accessory molecule expression. Neither silica nor titanium dioxide exposure caused appreciable changes in the expression of MHC class II (A), CD40 (B), CD54 (C) or CD86 (D). In contrast, LPS, CpG, and Poly I:C increased the expression of all 4 molecules on BMDCs compared with unstimulated BMDCs. These increases were reduced by coexposure to silica but not titanium dioxide compared with TLR ligand alone. n = 3–5, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only; P < 0.05 vs. silica only.

To examine whether silica-attenuated accessory molecule expression on BMDCs was relevant to primary cells, the expression of accessory molecules was examined on freshly isolated CD11c+ pulmonary DCs. FACS analysis profiles showed that CD11c+ cells isolated from collagenase-digested lungs of C57Bl/6 mice using MACS bead technology exhibited > 85% purity and were immunopositive for the surface markers CD11b, CD86, and MHC class II, consistent with a pulmonary DC phenotype (14) . Similar to the results observed with BMDCs, concomitant exposure of pulmonary DCs with silica but not titanium dioxide, and LPS, CpG, or Poly I:C for 24 h in culture reduced the expression of the accessory molecules: CD40 (21.9%), CD86 (23.1%), and MHC class II (31.7%) relative to TLR ligand alone.

Class A scavenger receptor-deficient BMDCs
Of the class A scavenger receptors, both CD204 and MARCO have previously been implicated in silica binding, signaling, and toxicity in macrophages (28 , 32) . To explore the possible role of class A scavenger receptors in mediating silica-dependent attenuation of LPS-induced activation, BMDCs were simultaneously prepared from CD204–/–, MARCO–/–, MARCO/CD204–/–, and wild-type C57Bl/6 mice, and cells were concomitantly stimulated as described. In the presence of silica or titanium dioxide alone, no differences were observed with regard to CD40 MFI compared with unstimulated BMDCs in any of the 4 strains examined (Fig. 4 ). Consistent with C57Bl/6 wild-type BMDCs, exposure of CD204–/–, MARCO–/–, and MARCO/CD204–/– BMDCs to 1 µg/ml LPS increased CD40 MFI compared with unstimulated BMDCs (Fig. 4) . Concomitant exposure of MARCO–/– and MARCO/CD204–/– but not CD204–/– BMDCs to 100 µg/ml silica and LPS resulted in reduced CD40 expression compared with LPS alone (Fig. 4) . In contrast to wild-type BMDCs, exposure of CD204–/–, MARCO–/–, and MARCO/CD204–/– BMDCs to 50 µg/ml titanium and LPS resulted in reduced CD40 expression compared with LPS alone (Fig. 4) . These results were confirmed by pretreating C57Bl/6 wild-type BMDCs for 1 h with an anti-MARCO blocking antibody (10 µg/ml final concentration; Fig. 5 ) and examining for expression of cell surface molecules. Exposure to the anti-MARCO blocking antibody alone increased expression of MHC class II, CD40, CD54, and CD86 compared with unstimulated cells. Moreover, silica continued to reduce LPS-induced expression of accessory molecules in the presence of anti-MARCO blocking antibody (Fig. 5) . Because C57Bl/6 mice express an aberrant form of CD204 that is not recognized by the anti-CD204 2F8 blocking antibody, these experiments were not performed.


Figure 4
View larger version (9K):
[in this window]
[in a new window]

  		Figure 4. BMDCs generated from class A scavenger receptor-deficient and wild-type mice increase CD40 expression in response to LPS stimulation. Silica but not titanium dioxide diminished LPS-induced CD40 MFI in C57Bl/6 wild-type BMDCs (A). In contrast, using MARCO–/– BMDCs, both silica and titanium dioxide reduced LPS-dependent induction of CD40 MFI (B). Titanium but not silica attenuated LPS-induced CD40 MFI in CD204–/– BMDCs (C). Using MARCO/CD204–/– BMDCs, silica but not titanium dioxide reduced LPS-induced expression of CD40 (D). n = 3–5, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only.


Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		Figure 5. Pretreatment with anti-MARCO blocking antibody showed no effect on the ability of silica to modulate MHC class II and accessory molecule expression in response to LPS stimulation. Anti-MARCO blocking antibody (10 µg/ml) alone increased the expression of MHC class II, CD40, CD54, and CD86 compared with unstimulated BMDCs. Although anti-MARCO blocking antibody reduced LPS-induced CD40 MFI, it had no effect on induction of MHC class II, CD54, or CD86 expression. Anti-MARCO blocking antibody also exhibited no effect on silica’s ability to attenuate LPS-induced expression of MHC class II (A), CD40 (B), CD54 (C), or CD86 (D). n = 3, in triplicate; error bars = SEM. *P < 0.05 vs. media only; #P < 0.05 vs. LPS only; Ab = antibody.

Cytokine production
Synergistic induction of cytokine production has been observed for DCs activated by combinations of PRR ligands (22) . To determine whether the observed changes in DC activation correlated with differential cytokine secretion, acellular supernatants were assayed for both Th1 (IL-1β, IL-6, TNF-{alpha}, and IFN{gamma}) and Th2 (IL-10, IL-12, IL-13, and TGF-β) cytokines. Silica alone increased IL-1β yet decreased activated TGF-β levels compared with unstimulated BMDCs (Fig. 6 A, E, respectively). In contrast, titanium dioxide had no effect on cytokine production (Fig. 6) . LPS-, CpG-, or Poly I:C-induced activation resulted in elevated levels of IL-6, IL-10, and TNF-{alpha} compared with unstimulated BMDCs (Fig. 6B-D ) (33 , 34) . Coexposure to silica and LPS, CpG, or Poly I:C resulted in increased IL-1β and TNF-{alpha} compared with silica or TLR ligand alone (Fig. 6) . In contrast, coexposure to silica and LPS, CpG, or Poly I:C reduced levels of IL-6, IL-10, and activated TGF-β compared with TLR ligand alone (Fig. 6) . No changes were observed in the production of IFN{gamma}, IL-12, or IL-13 in response to LPS, CpG, or Poly I:C and/or silica and titanium dioxide (data not shown).


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Figure 6. Silica exhibits distinct effects on LPS-, CpG-, and Poly I:C-induced cytokine production by BMDCs. Silica and CpG but not titanium dioxide, LPS, or Poly I:C increased IL-1β production compared with unstimulated BMDCs. Potent synergy was observed between silica and LPS, CpG, and Poly I:C with ~10-fold increased IL-1β compared with TLR ligand or silica alone (A). Neither silica nor titanium dioxide increased IL-6 production compared with unstimulated BMDCs. In contrast, LPS and CpG increased IL-6 production, although Poly I:C did not. While coexposure to LPS plus silica or CpG plus silica reduced IL-6 production, coexposure to Poly I:C plus silica increased IL-6 production compared with TLR ligand alone (B). Silica and titanium dioxide failed to influence TNF-{alpha} release, whereas LPS, CpG, or Poly I:C increased TNF-{alpha} compared with unstimulated BMDCs. These TNF-{alpha} levels were augmented by coexposure to silica but not titanium dioxide (C). Levels of IL-10 were below the limits of detection in silica-, titanium dioxide-, and Poly I:C-treated wells. In contrast, stimulation with either LPS or CpG resulted in appreciable amounts of IL-10 detected. These levels were reduced by coincident exposure to silica but not titanium dioxide (D). Levels of activated TGFβ were reduced by silica but not titanium dioxide exposure compared with unstimulated BMDCs. Only CpG increased levels of activated TGFβ compared with unstimulated BMDCs. Coexposure to TLR ligands in conjunction with silica decreased TGF-β compared with TLR ligand alone (E). n = 3–5, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only; P < 0.05 vs. silica only.

Endocytic activity and pattern recognition receptor expression
The capacity of DCs to phagocytose various fluorescent particles is a well documented phenomenon in vitro (35 , 36) , and, 2 decades ago, Zimmerman et al.(9) reported that silica decreased the phagocytic capacity of macrophages and neutrophils. Therefore, the ability of BMDCs to endocytose Alexa488-conjugated OVA, LDL, and LPS following silica or titanium dioxide exposure was measured. Preliminary experiments demonstrated that background MFI of BMDCs was not altered by silica or titanium dioxide (data not shown). The data shown in Fig. 7 A revealed a trend toward silica decreasing the phagocytic capacity of BMDCs similar to earlier reports in macrophages (Fig. 7A ) (9) . Experiments performed to assess whether exposure to LPS, CpG, or Poly I:C affects the ability of BMDCs to interact with silica or titanium dioxide demonstrated increased side scatter (SSC) profiles (indicative of particle binding) compared with unstimulated BMDCs. These SSC profiles were not altered by LPS, CpG, or Poly I:C (Fig. 7B ). To assess whether PRR expression on BMDCs was altered by particles, BMDCs were exposed to silica and titanium dioxide, and the expression of TLR4 and MARCO was examined. TLR4+ and MARCO+ BMDCs appeared to be mutually exclusive populations (Fig. 7C , ovals). Neither silica nor titanium dioxide affected the percent of BMDCs expressing TLR4 or MARCO, nor the MFI of those cells which are TLR4+ or MARCO+ (data not shown). Because it was not known precisely how applicable this in vitro model is to DC populations in vivo, particularly those located in subepithelial and interstitial compartments such as the lung, the distribution of TLR4 and MARCO was analyzed using freshly isolated pulmonary DCs. Analysis of the flow cytometry data yielded FACS plots of TLR4+ vs. MARCO+ pulmonary DCs similar to those of in vitro-derived BMDCs (Fig. 7C ); however, because of the inability to isolate and purify sufficient quantities of pulmonary DCs, the distribution of TLR4 and MARCO in response to silica or titanium dioxide treatment was not examined.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		Figure 7. Neither silica nor titanium dioxide affects the phagocytosis of fluorescently labeled LPS, OVA, or LDL (A). Similarly, LPS, CpG, or Poly I:C stimulation does not alter the uptake of silica or titanium dioxide (B). Representative FACS plots demonstrate similar expression of TLR4 vs. MARCO with regard to BMDCs and pulmonary DCs (C). n = 3, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only; P < 0.05 vs. silica only.

APC-T cell interaction
To determine any functional relevance of the observed reductions in costimulatory molecule expression and cytokine production in response to coexposure of silica and LPS, the ability of DCs to process and present OVA antigen to naive CD4+ T cells isolated from OT-II-transgenic mice in the presence of silica alone or silica plus LPS was measured. Results demonstrated enhanced APC activity as measured by increased production of IFN{gamma} by T cells following BMDC exposure to silica but not media or titanium dioxide, which was not affected by LPS (Fig. 8 A). These data are consistent with a previously published report which showed increased APC activity of bone marrow-derived macrophages exposed to silica (27) , although with BMDCs no effects were observed on IL-10, IL-12, or IL-13 production (data not shown). Experiments whereby BMDCs were sterile, sorted into either TLR4+ or MARCO+ fractions (Fig. 7C , representative FACS plots), revealed that it is the TLR4+ fraction (Fig. 8B ), and not the MARCO+ fraction, of BMDCs that is primarily responsible for the observed increase in APC activity, as measured by increased IFN{gamma} production by T cells (Fig. 8C ).


Figure 8
View larger version (7K):
[in this window]
[in a new window]

  		Figure 8. Silica increased APC activity as measured by production of IFN{gamma} by T cells at 48 h. In the presence of OVA antigen, the production of IFN{gamma} by CD4+ T cells is increased by exposure of BMDCs to silica but not titanium dioxide. Coexposure to LPS plus silica showed no significant difference compared with silica alone (A). Sterile sorting of BMDCs into TLR4+ MARCO lo/neg (B) or MARCO+ TLR4lo/neg (C) subpopulations demonstrated that the TLR4+ MARCO lo/neg BMDCs were responsible for the observed induction of IFN{gamma} production by T cells. n = 2, in triplicate; error bars = SEM; *P < 0.05 vs. media.

CD45 signaling
Intracellular signaling pathways involved in regulation of cellular responses induced by silica have received considerable attention (37 , 38) . These responses appear to be regulated through activation of a range of intracellular enzymes and transcription factors. Moreover, silica-induced activation of Src family kinases (SFKs) appears to be crucial to the induction of cytokines and chemokines, and subsequent inflammation (39) , by regulating the activity of extracellular regulated kinase (ERK)1/2. However, little is known still about how these events may be initiated. Because CD45 provides positive regulatory control of SFKs (40) and silica reportedly activates SFKs (39) , the ability of silica to alter expression of CD45 in a dose- and time-dependent manner were quantified. LPS did not modify the cell surface expression of CD45 compared with unstimulated BMDCs (Fig. 9 A). In contrast, in response to increasing concentrations of silica in the presence of LPS for 24 h in culture, the MFI of CD45 was significantly increased (Fig. 9A ). The maximal dose of silica alone or in combination with LPS compared with unstimulated BMDCs demonstrated peak expression of CD45 occurs at 24 h, although increases in CD45 expression were observed as early as 2 h (Fig. 9B ).


Figure 9
View larger version (13K):
[in this window]
[in a new window]

  		Figure 9. Silica induced a dose- and time-dependent increase in CD45 expression as measured by flow cytometry. LPS did not alter CD45 expression levels compared with unstimulated BMDCs; however, the addition of silica resulted in a dose-dependent increase in CD45 expression (A). Silica increased CD45 expression at both 2 and 24 h but not 0.5, 1, or 4 h. Similarly, coexposure to LPS and silica resulted in increased CD45 expression at 2, 4, and 24 h compared with unstimulated BMDCs (B). n = 3, in triplicate; error bars = SEM; *P < 0.05 vs. media only; #P < 0.05 vs. LPS only.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Given that silica predisposes individuals to bacterial infection, the overall purpose of this study was to examine the interactions between silica (a known scavenger receptor ligand) and three typical TLR ligands, LPS, CpG oligonucleotide, and Poly I:C, in granulocyte-macrophage colony-stimulating factor BMDCs and freshly isolated pulmonary DCs. The results from this study suggest that silica interferes with the ability of myeloid-derived DCs to appropriately respond to LPS, CpG, or Poly I:C in a dose-dependent manner by down-regulating the expression of accessory molecules such as MHC class II, CD40, CD54, and CD86, as well as the bactericidal production of NO. These results appear to be specific to silica because similar effects are not observed with the nonfibrogenic control particle titanium dioxide. Although silica attenuated LPS-, CpG-, or Poly I:C-induced expression of accessory molecules and NO production, its effect on cytokine expression was not universal, indicating a divergence in signaling pathways between induction of accessory molecules and cytokines.

Studies established an important role for DCs in stimulating pulmonary immune responses; however, the functional activity of DCs in vivo to activate Th1- or Th2-mediated lymphocyte responses remains unclear, primarily because of the complexities required to isolate sufficient quantities of cells for biological assays and lack of DC specific markers (41 , 42) . Although freshly isolated pulmonary DCs most closely resemble lung DCs in situ (41) and are believed to be phenotypically and functionally distinct (43) from DCs in other tissues, BMDCs have been widely used and are an accepted model of DCs because of the relative simplicity of the isolation procedure, the high number of cells yielded, and the consistency of the model system’s response to immune activation.

Class A scavenger receptor-deficient BMDCs were morphologically and phenotypically indistinguishable from C57Bl/6 wild-type (data not shown), indicating that these receptors are not essential for generation or survival of DCs in vitro. In addition, no alteration in either the frequencies or phenotype of respiratory DCs was detected in these mice (data not shown), similar to previously published reports using CD204–/– mice (44) , which likewise implies that the receptors are not essential to DC generation or survival in vivo. Class A scavenger receptor-deficient mice were utilized to demonstrate that silica’s ability to modulate TLR ligand-induced activation may not be entirely dependent on either CD204 or MARCO. These findings suggest that although MARCO and CD204 may be important in the binding and toxicity of silica by macrophages, this mechanism may be redundant and compensated for by other PRRs on DCs.

The distribution of PRRs was established using BMDCs as well as freshly isolated pulmonary DCs. The salient findings from these experiments were that MARCO was expressed on fewer DCs compared with TLR4 (3:1), and that the TLR4+ and MARCO+ subpopulations appeared to be mutually exclusive both in vitro and in vivo. Further analysis revealed that the expression levels of TLR4 and MARCO on BMDCs were not significantly altered by either silica or titanium dioxide exposure. Although there is generalized down-regulation of antigen uptake receptors with maturation, this condition is not absolute. Maturation stimuli were reported to up-regulate DEC-205 (45) , and bacterial activation was reported to enhance MARCO transcripts on DCs (46) . In our studies, PRR expression was performed on resting, unchallenged mice maintained under SPF conditions. Therefore, we cannot rule out the possibility that the distribution and relative expression levels of the receptor on DCs may be altered as a consequence of infection or other immunological challenge in vivo.

T-cell immunity is triggered and maintained by professional APCs via differential cytokine production and membrane presentation of accessory molecules (47 , 48) . Silica increased APC activity as measured by IFN{gamma} but not IL-13 production by T cells, suggestive of a Th1 type of response in cells derived from strains of mice on the C57Bl/6 background. These findings are similar to previously published results using bone marrow-derived macrophages derived from Balb/c mice (27) yet differ in the lack of Th2 cytokine production in response to silica, possibly because of inherent differences between C57Bl/6 and Balb/c mice. FACS sorting revealed that this activity may be mediated by TLR4+ and not MARCO+ BMDCs. DC subpopulations have been hypothesized to exhibit unique or differential properties that facilitate their precise roles within the immune system, yet this theory remains controversial because of the lack of supporting data of the specialized activities of identified subsets (49) . TLR4+ and MARCO+ subsets of DCs may represent unique populations of DCs responsive to diverse ligands in vivo. In the case of the lung, respiratory DCs stimulate Th2 responses on antigen challenge both in vitro and in vivo (50 51 52) . Moreover, pulmonary DCs are a potent source of IL-10, a cytokine crucial to the inhibition of Th1 responses and the maintenance of tolerance within the lungs (52) . It should be noted that silica does act on other APCs that increase Th2 cytokines (27) ; therefore, it is not just silica acting on DCs alone that may alter cytokine tolerance within the lungs. The results presented herein expand on these findings and demonstrate that the Th2-promoting potential of silica may be due to its ability to reduce Th1 differentiation resulting from down-regulation of accessory molecules rather than the promotion of Th2 differentiation per se.

Expression of CD45 was demonstrated on myeloid DCs in response to silica alone and in combination with TLR ligands, suggesting it may be a signaling mechanism. Mechanistically, CD45 negatively regulates NF-{kappa}B activation, TNF-{alpha}, and IL-6 cytokine production triggered by CpG or Poly I:C, and the response to inflammatory cytokines (53) . Although the exact means remains unknown, it is possible that silica-induced expression of CD45 modulates inflammatory signaling by dephosphorylating an adaptor protein and/or Src kinases, which is an intriguing possibility given that tyrosine kinase-deficient mice show attenuated responses to TLR ligands (54) . Clarifying the mechanisms by which silica affects pulmonary DCs is crucial to understanding how silica alters susceptibility to infection.

An important unresolved question is how, given the common signaling pathways shared by many PRRs, discriminatory signals are transmitted from the receptor to the cell nucleus. Such interactions between different types of PRRs augment, inhibit, or synergize with the functions of either participating ligand (22) . Most PRRs have similar signaling pathways, activate NF-{kappa}B, and contribute positively to inflammation. However, there is almost certainly negative regulation of PRRs to protect the host against uncontrolled inflammation or immune pathology, but, so far, knowledge in this area remains limited. It may be that silica signaling occurs through a pathway involved in the negative regulation of TLR. Evidence has begun to emerge from human DC work supporting the concept that engagement of specific PRRs may trigger anti-inflammatory pathways (55) . Further dissection of these pathways will illuminate the nature of these exchanges and may reveal novel therapeutic targets for immune modulation and rational drug design.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the U.S. National Institutes of Health (ES-015294) and the National Center for Research Resources (COBRE P20 RR17670) to A.H., and the National Research Service Award fellowship (ES-013044) to C.A.B.

Received for publication October 3, 2007. Accepted for publication November 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Parks, C. G., Cooper, G. S., Nylander-French, L. A., Sanderson, W. T., Dement, J. M., Cohen, P. L., Dooley, M. A., Treadwell, E. L., St Clair, E. W., Gilkeson, G. S., Hoppin, J. A., Savitz, D. A. (2002) Occupational exposure to crystalline silica and risk of systemic lupus erythematosus: a population-based, case-control study in the southeastern United States. Arthritis Rheum. 46,1840-1850[CrossRef][Medline]
  2. Ding, M., Chen, F., Shi, X., Yucesoy, B., Mossman, B., Vallyathan, V. (2002) Diseases caused by silica: mechanisms of injury and disease development. Int. Immunopharmacol. 2,173-182[CrossRef][Medline]
  3. Cowie, R. L. (1994) The epidemiology of tuberculosis in gold miners with silicosis. Am. J. Respir. Crit. Care Med. 150,1460-1462[Abstract]
  4. Rees, D., Murray, J. (2007) Silica, silicosis and tuberculosis. Int. J. Tuberc. Lung Dis. 11,474-484[Medline]
  5. Bang, K. M., Weissman, D. N., Wood, J. M., Attfield, M. D. (2005) Tuberculosis mortality by industry in the United States, 1990–1999. Int. J. Tuberc. Lung Dis. 9,437-442[Medline]
  6. Antonini, J. M., Roberts, J. R., Yang, H. M., Barger, M. W., Ramsey, D., Castranova, V., Ma, J. Y. (2000) Effect of silica inhalation on the pulmonary clearance of a bacterial pathogen in Fischer 344 rats. Lung 178,341-350[CrossRef][Medline]
  7. Saeki, H., Ishii, T. (1996) Effect of silica treatment on resistance to Babesia rodhaini infection in immunized mice. Vet. Parasitol. 61,201-210[CrossRef][Medline]
  8. Antonini, J. M., Yang, H. M., Ma, J. Y., Roberts, J. R., Barger, M. W., Butterworth, L., Charron, T. G., Castranova, V. (2000) Subchronic silica exposure enhances respiratory defense mechanisms and the pulmonary clearance of Listeria monocytogenes in rats. Inhal. Toxicol. 12,1017-1036[CrossRef][Medline]
  9. Zimmerman, B. T., Canono, B. P., Campbell, P. A. (1986) Silica decreases phagocytosis and bactericidal activity of both macrophages and neutrophils in vitro. Immunology 59,521-525[Medline]
  10. Rao, T. D., Frey, A. B. (1998) Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function. Immunol. Invest. 27,181-199[CrossRef][Medline]
  11. Vogel, S. N., English, K. E., O'Brien, A. D. (1982) Silica enhancement of murine endotoxin sensitivity. Infect. Immun. 38,681-685[Abstract/Free Full Text]
  12. Hwang, M., Park, K. K., Chang, Y. C., Choo, Y. A., Jeon, J. P., Shin, I. H., Lee, T. S. (2007) Silica-ceramic suppresses the expression of proinflammatory cytokines induced by lipopolysaccharide in macrophages. J. Biomed. Mater. Res. A 80,513-519[Medline]
  13. Hanna, P. C., Acosta, D., Collier, R. J. (1993) On the role of macrophages in anthrax. Proc. Natl. Acad. Sci. U. S. A. 90,10198-10201[Abstract/Free Full Text]
  14. Beamer, C. A., Holian, A. (2007) Antigen Presenting Cell Population Dynamics During Murine Silicosis. Am. J. Respir. Cell Mol. Biol. 37,729-738[Abstract/Free Full Text]
  15. McWilliam, A. S., Napoli, S., Marsh, A. M., Pemper, F. L., Nelson, D. J., Pimm, C. L., Stumbles, P. A., Wells, T. N., Holt, P. G. (1996) Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli. J. Exp. Med. 184,2429-2432[Abstract/Free Full Text]
  16. Macagno, A., Napolitani, G., Lanzavecchia, A., Sallusto, F. (2007) Duration, combination and timing: the signal integration model of dendritic cell activation. Trends Immunol. 28,227-233[CrossRef][Medline]
  17. Saalmuller, A. (2006) New understanding of immunological mechanisms. Vet. Microbiol. 117,32-38[CrossRef][Medline]
  18. Demedts, I. K., Brusselle, G. G., Vermaelen, K. Y., Pauwels, R. A. (2005) Identification and characterization of human pulmonary dendritic cells. Am. J. Respir. Cell Mol. Biol. 32,177-184[Abstract/Free Full Text]
  19. Robbins, C. S., Dawe, D. E., Goncharova, S. I., Pouladi, M. A., Drannik, A. G., Swirski, F. K., Cox, G., Stampfli, M. R. (2004) Cigarette smoke decreases pulmonary dendritic cells and impacts antiviral immune responsiveness. Am. J. Respir. Cell Mol. Biol. 30,202-211[Abstract/Free Full Text]
  20. Lambrecht, B. N., Prins, J. B., Hoogsteden, H. C. (2001) Lung dendritic cells and host immunity to infection. Eur. Respir. J. 18,692-704[Abstract/Free Full Text]
  21. Medzhitov, R., Janeway, C. A., Jr (2002) Decoding the patterns of self and nonself by the innate immune system. Science 296,298-300[Abstract/Free Full Text]
  22. Trinchieri, G., Sher, A. (2007) Cooperation of Toll-like receptor signals in innate immune defence. Nat. Rev. Immunol. 7,179-190[CrossRef][Medline]
  23. Kaisho, T., Akira, S. (2002) Toll-like receptors as adjuvant receptors. Biochim. Biophys. Acta 1589,1-13[Medline]
  24. Dahl, M., Bauer, A. K., Arredouani, M., Soininen, R., Tryggvason, K., Kleeberger, S. R., Kobzik, L. (2007) Protection against inhaled oxidants through scavenging of oxidized lipids by macrophage receptors MARCO and SR-AI/II. J. Clin. Invest. 117,757-764[CrossRef][Medline]
  25. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R. M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176,1693-1702[Abstract/Free Full Text]
  26. Wan, H., Dupasquier, M. (2005) Dendritic cells in vivo and in vitro. Cell. Mol. Immunol. 2,28-35[Medline]
  27. Migliaccio, C. T., Hamilton, R. F., Jr, Holian, A. (2005) Increase in a distinct pulmonary macrophage subset possessing an antigen-presenting cell phenotype and in vitro APC activity following silica exposure. Toxicol. Appl. Pharmacol. 205,168-176[CrossRef][Medline]
  28. Hamilton, R. F., Jr, Thakur, S. A., Mayfair, J. K., Holian, A. (2006) MARCO mediates silica uptake and toxicity in alveolar macrophages from C57BL/6 mice. J. Biol. Chem. 281,34218-34226[Abstract/Free Full Text]
  29. Bogdan, C. (2001) Nitric oxide and the immune response. Nat. Immunol. 2,907-916[CrossRef][Medline]
  30. Delclaux, C., Azoulay, E. (2003) Inflammatory response to infectious pulmonary injury. Eur. Respir. J. Suppl. 42,10s-14s[Medline]
  31. Coleman, J. W. (2002) Nitric oxide: a regulator of mast cell activation and mast cell-mediated inflammation. Clin. Exp. Immunol. 129,4-10[CrossRef][Medline]
  32. Beamer, C. A., Holian, A. (2005) Scavenger receptor class A type I/II (CD204) null mice fail to develop fibrosis following silica exposure. Am. J. Physiol. 289,L186-L195
  33. Samarasinghe, R., Tailor, P., Tamura, T., Kaisho, T., Akira, S., Ozato, K. (2006) Induction of an anti-inflammatory cytokine, IL-10, in dendritic cells after toll-like receptor signaling. J. Interferon Cytokine Res. 26,893-900[CrossRef][Medline]
  34. Lee, M. S., Kim, Y. J. (2007) Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu. Rev. Biochem. 76,447-480[CrossRef][Medline]
  35. Kaisho, T., Akira, S. (2004) Pleiotropic function of Toll-like receptors. Microb. Infect. 6,1388-1394[CrossRef][Medline]
  36. Kaisho, T., Akira, S. (2006) Toll-like receptor function and signaling. J. Allergy Clin. Immunol. 117,979-987[CrossRef][Medline]
  37. Ding, M., Huang, C., Lu, Y., Bowman, L., Castranova, V., Vallyathan, V. (2006) Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway. Am. J. Physiol. 290,L291-L297
  38. Choi, J. K., Lee, S. G., Lee, J. Y., Nam, H. Y., Lee, W. K., Lee, K. H., Kim, H. J., Lim, Y. (2005) Silica induces human cyclooxygenase-2 gene expression through the NF-kappaB signaling pathway. J. Environ. Pathol. Toxicol. Oncol. 24,163-174[CrossRef][Medline]
  39. Ovrevik, J., Lag, M., Schwarze, P., Refsnes, M. (2004) p38 and Src-ERK1/2 pathways regulate crystalline silica-induced chemokine release in pulmonary epithelial cells. Toxicol. Sci. 81,480-490[Abstract/Free Full Text]
  40. Palacios, E. H., Weiss, A. (2004) Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23,7990-8000[CrossRef][Medline]
  41. Swanson, K. A., Zheng, Y., Heidler, K. M., Zhang, Z. D., Webb, T. J., Wilkes, D. S. (2004) Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function. J. Immunol. 173,4875-4881[Abstract/Free Full Text]
  42. Tsoumakidou, M., Tzanakis, N., Papadaki, H. A., Koutala, H., Siafakas, N. M. (2006) Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid. Immunol. Cell Biol. 84,267-273[CrossRef][Medline]
  43. Swanson, K. A., Zheng, Y., Heidler, K. M., Mizobuchi, T., Wilkes, D. S. (2004) CDllc+ cells modulate pulmonary immune responses by production of indoleamine 2,3-dioxygenase. Am. J. Respir. Cell Mol. Biol. 30,311-318[Abstract/Free Full Text]
  44. Becker, M., Cotena, A., Gordon, S., Platt, N. (2006) Expression of the class A macrophage scavenger receptor on specific subpopulations of murine dendritic cells limits their endotoxin response. Eur. J. Immunol. 36,950-960[CrossRef][Medline]
  45. Martinez del Hoyo, G., Martin, P., Arias, C. F., Marin, A. R., Ardavin, C. (2002) CD8alpha+ dendritic cells originate from the CD8alpha- dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 up-regulation. Blood 99,999-1004[Abstract/Free Full Text]
  46. Granucci, F., Petralia, F., Urbano, M., Citterio, S., Di Tota, F., Santambrogio, L., Ricciardi-Castagnoli, P. (2003) The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia. Blood 102,2940-2947[Abstract/Free Full Text]
  47. Lenschow, D. J., Walunas, T. L., Bluestone, J. A. (1996) CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14,233-258[CrossRef][Medline]
  48. Wang, S., Chen, L. (2004) Co-signaling molecules of the B7-CD28 family in positive and negative regulation of T lymphocyte responses. Microb. Infect. 6,759-766[CrossRef][Medline]
  49. Diebold, S. S., Montoya, M., Unger, H., Alexopoulou, L., Roy, P., Haswell, L. E., Al-Shamkhani, A., Flavell, R., Borrow, P., Reis e Sousa, C. (2003) Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424,324-328[CrossRef][Medline]
  50. Lambrecht, B. N., De Veerman, M., Coyle, A. J., Gutierrez-Ramos, J. C., Thielemans, K., Pauwels, R. A. (2000) Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation. J. Clin. Invest. 106,551-559[Medline]
  51. Stumbles, P. A. (1999) Regulation of T helper cell differentiation by respiratory tract dendritic cells. Immunol. Cell Biol. 77,428-433[CrossRef][Medline]
  52. Stumbles, P. A., Thomas, J. A., Pimm, C. L., Lee, P. T., Venaille, T. J., Proksch, S., Holt, P. G. (1998) Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity. J. Exp. Med. 188,2019-2031[Abstract/Free Full Text]
  53. Piercy, J., Petrova, S., Tchilian, E. Z., Beverley, P. C. (2006) CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells. Immunology 118,250-256[CrossRef][Medline]
  54. Jefferies, C. A., Doyle, S., Brunner, C., Dunne, A., Brint, E., Wietek, C., Walch, E., Wirth, T., O'Neill, L. A. (2003) Bruton’s tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4. J. Biol. Chem. 278,26258-26264[Abstract/Free Full Text]
  55. Chieppa, M., Bianchi, G., Doni, A., Del Prete, A., Sironi, M., Laskarin, G., Monti, P., Piemonti, L., Biondi, A., Mantovani, A., Introna, M., Allavena, P. (2003) Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program. J. Immunol. 171,4552-4560[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
A. E. Seif, D. M. Barrett, M. Milone, V. I. Brown, S. A. Grupp, and G. S. D. Reid
Long-term protection from syngeneic acute lymphoblastic leukemia by CpG ODN-mediated stimulation of innate and adaptive immune responses
Blood, September 17, 2009; 114(12): 2459 - 2466.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
fj.07-095299v1
22/6/2053    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beamer, C. A.
Right arrow Articles by Holian, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beamer, C. A.
Right arrow Articles by Holian, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS