Published online before print December 3, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
pathway in macrophages and its potential role in silica-induced pulmonary fibrosisCenter for Environmental Health Sciences, University of Montana, Missoula, Montana, USA
1Correspondence: University of Montana, Center for Environmental Health Sciences, 285C Skaggs Building, Missoula, MT 59812, USA. E-mail: christopher.migliaccio{at}umontana.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and IFN-
. Previous studies determined that Th2 mediators (i.e., IL-13) are vital to development of pulmonary fibrosis. The present study, using in vivo and in vitro techniques, compares silica exposures between Balb/c and Th2-deficient mice in an effort to determine the link between Th2 immunity and silicosis. In long-term experiments, a significant increase in fibrosis and activated interstitial macrophages was observed in Balb/c but not IL-4R–/– mice. Additionally, a significant increase in Ym1 mRNA levels, a promoter of Th2 immunity, was determined in the interstitial leukocyte population of silica-exposed Balb/c mice. To elucidate the effects of silica on macrophage function, bone marrow-derived macrophages (BMdM) were exposed to particles and assayed for T cell (TC) stimulation activity. As a control, Ym1 mRNA expression in Balb/c BMdM was determined using IL-4 stimulation. In the in vitro assay, a significant increase in TC activation, as defined by surface markers and cytokines, was observed in the cultures containing the silica-exposed macrophages in wild-type and IL-4R–/– mice, with one exception: IL-4R–/– BMdM were unable to induce an increase in IL-13. These results suggest that crystalline silica alters cellular functions of macrophages, including activation of TC, and that the increase in Th2 immunity associated with silicosis is via the IL-4R-Ym1 pathway.
Key Words: mouse • alternatively activated • T cell • lung
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A well-documented result of exposure to silica dust is pulmonary fibrosis, referred to as silicosis, which presents as fibrotic lesions and a decreased lung capacity. The exact etiology remains undetermined, but alveolar macrophages (AM) are considered to play a vital role in the process [6
]. Studies in our laboratory and others have shown an increase in, and role of, Th1 factors subsequent to silica exposure, including IFN- and TNF-, which may mediate an initial inflammation. However, studies about Th2 cytokines, including IL-4 and IL-13, and their respective receptors have indicated a key role of these factors in the generation of fibrosis. The transition from a Th1-dominated inflammatory response to one predominantly Th2 in nature appears to be a key element in the generation of a profibrotic environment [7
8
9
10
11
]. This suggests that the interplay between silica-exposed macrophages and lymphocytes is a key to silicosis.
Historically, the Th1/Th2 dogma has centered on TC subsets and their cytokine production. Recently, subsets of other cell types have been described, including dendritic cells (DC) [12
13
14
] and macrophages [15
, 16
]. One macrophage subtype, the alternatively activated macrophage (aaMac), is defined as having a regulatory and/or protective phenotype that is a result of activation by IL-4, IL-13, and/or glucocorticoids [17
18
19
]. This pathway of activation results in production of a more Th2-type panel of mediators and an increase in antigen presentation function. The aaMac has been associated with Th2 immune responses including pulmonary inflammation (asthma, allergy) and parasitic infections [20
, 21
]. The generation of this subtype has, in in vitro and in vivo experiments, been shown to require signaling through the IL-4R and STAT6 pathways [22
23
24
25
].
One protein that is considered a marker of the aaMac is the secreted protein Ym1, and Ym1 mRNA and protein are consistently up-regulated in Th2 immune responses and are linked with scavenger receptor-A and membrane-activated complex 1 macrophage markers. Ym1 is chitinase-like in structure and has Th2-promoting activity [24 , 26 ]. Both potential functions are supported by experiments linking the protein and cells to parasitic infections and epithelial remodeling as seen in pulmonary as well as olfactory inflammation [27 ]. Recently, Arora et al. [28 ] suggested a direct link between the expression of the Ym1 protein in DC and the differentiation of Th2 TC. In those studies, a cholesterol-lowering statin generated a change in DC phenotype that resulted in Ym1 production and the promotion of Th2 immunity.
The above observations lead to the hypothesis that the Th2-dominated fibrosis associated with crystalline silica exposure is dependent on the IL-4R-Ym1 pathway in APC. Silica-exposed bone marrow-derived macrophages (BMdM) possess an altered capacity to activate TC that may include a switch from Th1 to Th2, and this may be a key component to the Th2-dominated fibrotic process in silicosis. Based on our previous work, suggesting a connection between APC activity and silica-induced pulmonary fibrosis [29
], we analyzed a variety of parameters in an in vitro APC assay to determine the effect of silica on TC activation and specifically, the potential for affecting the Th1/Th2 balance.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice
Balb/c and IL-4R–/– (Balb/c background) mice (Jackson Laboratory, Bar Harbor, ME, USA) were used for all in vivo studies, as well as the generation of BMdM. DO11.10 mice were used for the isolation of transgenic TCs to use in the in vitro assays described below. Animals were housed in microisolators on a 12:12-h light-dark cycle. The mice were maintained on an OVA-free diet and given deionized water ad libitum. The University of Montana Institutional Animal Care and Use Committee (Missoula, MT, USA) approved all animal procedures. Euthanasia was performed by i.p. injection of a lethal dose of pentobarbital sodium.
Isolation of interstitial macrophages
Cells from the interstitial spaces of the lung were isolated by mincing collagenase-treated tissue. Minced lung preparations were performed according to the following protocol. Lungs were removed, finely minced, and incubated 2 h in collagenase buffer (
5 ml/lung) at 37°C. Collagenase buffer consisted of 1 mg/ml collagenase type IA (Sigma Chemical Co., St. Louis, MO, USA) in media (RPMI, Mediatech, Herndon, VA, USA; 10% FBS, Gibco, Grand Island, NY, USA; 1% Pen/Strep, Gibco). Post-collagenase treatment, multiple cell populations were isolated by gradient centrifugation (Percoll, GE Healthcare, Piscataway, NJ, USA). The band containing leukocytes (macrophages, DC, granulocytes, and lymphocytes) was collected and washed with PBS. Cells were then enumerated and aliquoted for analysis by flow cytometry.
BMdM
BMdM were generated as described previously [30
]. Briefly, wild-type Balb/c and IL-4R–/– mice were killed and hind legs removed for BM isolation. BM was flushed from the femur and tibia with media. All cells were incubated in T75 culture flasks with 20 ml media overnight for stromal cell elimination. Aspirated cells were aliquoted to new T75 flasks (1.5x107 cells/flask) in 20 ml media and 40 µl M-CSF (10 ng/ml stock, R&D Systems, Minneapolis, MN, USA). Cultures continued for 7–10 days with re-feeding every 3–4 days. Generated macrophages were assessed by flow cytometry for expression of CD11b and F4/80 (>90% were positive).
Silica exposure
Silica exposure of BMdM from Balb/c and IL-4R–/– mice (Jackson Laboratory) was performed with cells in suspension in 1.5 ml microfuge tubes. In 1 ml vol, 1 x 106 cells and 200 µg silica in 40 µl PBS were incubated in suspension with end-over-end tumbling for 1 h at 37°C. In vitro exposure controls included PBS (no particles) and titanium dioxide (TiO2; Fischer Scientific, Denver, CO, USA), where 100 µg TiO2 is used to account for a larger surface area compared with silica. For in vivo exposures, the mice were anesthetized using 0.1 cc ketamine (1:4 in sterile PBS). Mice were then instilled intranasally (i.n.) with 30 µl specified treatment, which included PBS alone or 1 mg crystalline silica in PBS. The treatment regimen consisted of two instillations at Days 0 and 14.
Analysis of collagen content
Total collagen of the lung tissue was quantified by analysis of hydroxyproline, an amino acid unique to collagen. Briefly, lung tissue was excised, transferred to preweighed tubes, and frozen immediately in liquid nitrogen. The lung tissue was weighed prior to homogenization using a Tissue Tearor in 1 ml 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, which was quantified by spectrophotometry. Hydroxyproline content for each sample was determined by triplicate analysis to provide a mean value.
Histology
Silica-induced inflammation and fibrosis were assessed in Balb/c wild-type and IL-4R–/– mice. Following treatment with silica or PBS, lungs were removed and stored overnight in histochoice and then processed for paraffin embedding. The tissue was sectioned at 7 µm, and adjacent sections were stained with Gomori’s Trichrome using a Thermo Shandon automated stainer (Shandon, Pittsburgh, PA, USA) to visualize collagen content, as well as the cellular architecture of the lung. The extent of lung injury was scored by two independent researchers on a scale of zero for normal lung to four for severe distortion of structure. The major criteria examined included interstitial thickening of alveolar or bronchiolar walls, collagen deposition, and inflammatory cell infiltration.
Flow cytometry
Analysis of cell surface marker expression was performed using a FACSAria (BD Biosciences, San Jose, CA, USA) system. Briefly, cells were resuspended in FACS buffer (0.1% BSA+0.5% sodium azide) and blocked for 
10 min on ice with anti-CD16/32 (FcBlock, BD Biosciences). Cells were then incubated with fluorescently labeled antibodies at previously determined optimal dilutions for 30 min on ice. Antibodies against the TC activation markers [CD25, clone 7D4; CD44, clone 1M7; CD62 ligand (CD62L), clone MEL-14], as well as the macrophage marker CD11b (clone M1/70) were obtained from BD Biosciences. Detection of the macrophage marker F4/80 (clone CI:A3-1) was obtained from Caltag Laboratories (Burlingame, CA, USA). Intracellular cytokine assessment was performed using the anti-mouse IFN- mAb (clone XMG1.2, eBioscience, San Diego, CA, USA) or anti-mouse IL-13 (#AF-413-NA, R&D Systems) followed by a PerCP-Cy5.5-conjugated anti-goat secondary antibody. Cells were washed with 10% FBS and then transferred to filter-top polypropylene tubes (BD Labware, Franklin Lakes, NJ, USA) for analysis. Data analysis was performed using FACSDiva software (BD Biosciences), and histogram overlays were generated with FlowJo software (Tree Star Inc., Ashland, OR, USA).
mRNA analysis
The relative levels of Ym1 and β-actin mRNA were assessed by QuantiGene Reagent System 2.0 (Panomics, Fremont, CA, USA) and RT-PCR. The QuantiGene Reagent System was used to quantify the level of Ym1 expression in interstitial pulmonary leukocytes. As described above, interstitial immune cells were isolated from lungs by collagenase treatment and gradient centrifugation. For mRNA analysis, all steps after the collagenase treatment were performed at 4°C. Using the TRIzol protocol, RNA was isolated from each sample and stored at –80°C until analysis. The QuantiGene system uses branched DNA (bDNA) for signal amplification [31
32
33
]. The PubMed accession numbers were sent to Panomics (Chi3l3: NM_009692; β-actin: NM_007393), where specific probe sets were designed. For the assay, 10 µl RNA dilution from each sample was loaded into a 96-well plate and hybridized (overnight at 55°C) to the probe set, and subsequently, oligonucleotides were fixed to the bottom of the wells for capture. The signal is amplified via bDNA molecules conjugated with alkaline phosphatase and luminescence generated following addition of substrate. Luminescence was detected and measured by the SpectraMax GenesisXS plate reader. The luminescence was reported as relative light units normalized to the housekeeping gene β-actin RNA.
RT-PCR was performed on RNA isolated from BMdM cultures, where BMdM were generated as described above with 1 x 106 cells/ml seeded in T25 culture flasks. Individual flasks were treated with 10 ng/ml cytokine (IFN-, IL-4, or none) overnight. Cells were washed extensively with PBS following each treatment. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to the flasks (1 ml/flask), and the cells were scraped and transferred to 1.5 ml Eppendorff tubes. RNA isolation was performed according to the TRIzol protocol. Briefly, methanol extraction was followed by isopropanol precipitation and resuspension in RNase-free buffer. Where necessary, mRNA was amplified using oligo(dT) primers. PCR amplification consisted of the following conditions: 1 min denaturation at 95°C, 30 s annealing at 55°C, and 1 min extension at 72°C for 30 cycles. Primers (Integrated DNA Technologies, Coralville, IA, USA) for PCR analysis were as follows: β-actin sense (5'-ACACTGTGCCCATCTACGAG-3'), antisense (5'-TCAACGTCACACTTCATGATG-3'); Ym1 sense (5'-GGGCATACCTTTATCCTGAG-3'), antisense (5'-CCACTGAAGTCATCCATGTC-3').
APC assay
Macrophages were assayed for antigen-presenting capability using a modified in vitro assay described previously [34
]. Briefly, macrophages obtained via BM were incubated with silica, TiO2, or PBS in 1.5 ml tubes at 37°C for 1 h. The standard silica concentration was 200 µg/ml with 1 x 106 cell/ml. Following silica exposure, the cells were aliquoted into 96-well plates at a density of 1 x 105 cells/well (100 µl/well) plus 50 µl media, with or without OVA (chicken OVA, Grade V, Sigma Chemical Co.; 10 mg/ml final concentration). Plates were then incubated (cells+OVA) for 3 h at 37°C. During this incubation, TC were prepared from the spleen of DO11.10 mice using a TC enrichment kit (SpinSep, StemCell Technologies, Vancouver, BC, Canada), where >75% of recovered cells were CD3+ cells expressing the transgenic TCR (data not shown). TC were added to the 96-well plate following OVA incubation at a concentration of 4 x 105 cells/well (100 µl/well) for a final ratio of 4:1 (TC:APC). For controls, coculture wells were set up with anti-CD3 (2C11 mAb at a final concentration of 5 µg/ml) or media alone in lieu of OVA antigen. Plates were incubated at 37°C for 24 or 48 h and then centrifuged, and supernatants were collected for assay. TC cytokine levels were determined by ELISA (DuoSet kits, R&D Systems).
Cell proliferation assay
Macrophages and TC were set up in the APC assay described above, and TC proliferation was assessed by [3H]thymidine (Amersham, Piscataway, NJ, USA) incorporation. On Day 3 of the assay 1 µCi [3H]thymidine was added to each well. Following 16 h incubation, wells were harvested (Dynatech Automash 2000 Cell Harvester, LabTech Intl., UK) and monitored for proliferation by radioactivity (LS6500 multipurpose scintillation counter, Beckman Coulter, Inc., Fullerton, CA, USA).
Statistical analysis
The values for individual samples/experiments were averaged, and the SEM was calculated. The significance of the differences between the groups was determined by t-test or one-way ANOVA (greater than two groups), followed by Newman-Keuls post-hoc test for pair-wise mean comparisons. For histology scoring using subjective scaling, the analysis included a Mann-Whitney test to determine differences between median scores. Inter-rater reliability of the scoring was calculated using Cronback’s Alpha. All calculations were performed with Prism software except for Cronback’s Alpha, which was calculated with SPSS (Chicago, IL, USA). A P value (type I error) of <0.05 was considered statistically significant.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
–/– mice versus wild-type mice–/–) were instilled with silica. Mice were treated with silica or PBS at Days 0 and 14 and analyzed after 18 weeks for inflammation and fibrosis. At 18 weeks post-treatment, lungs were excised, and hydroxyproline content was assessed as a measure of fibrosis. As shown in Figure 1A
, wild-type mice treated with silica had a concomitant increase in collagen and organ wet weight (data not shown), whereas silica-treated IL4R–/– animals had no significant change in either parameter as compared with PBS controls. In wild-type and null mice treated with PBS, there were only background levels of collagen and no visible inflammation (Fig. 1B
and 1D)
by histology. Although there was extensive inflammation in the Balb/c and IL-4R–/–silica-treated mice, there was qualitatively less fibrosis in the IL-4R–/– tissue (Fig. 1C
and 1E)
. In addition, using a scoring system of one to four for intensity of inflammation or collagen expression, each mouse was analyzed and scored, and the average of the scores was used for statistical calculations. Using Cronback’s Alpha, the inter-rater reliability of the scoring had a value of 0.928 and was determined to be statistically significant (P<0.001). The analysis indicated a significantly (P<0.01) lower amount of fibrosis in the null mice (Fig. 1F)
, as determined by collagen staining with Gimori’s Trichrome, in addition to a statistically, significantly lower amount of inflammation (P<0.05). These data suggest that Th2 immunity is instrumental in the generation of pulmonary fibrosis following silica exposure and that it is specifically linked to the IL-4R–/– pathway.
 View larger version (50K): [in a new window] |
Figure 1. Comparison of pulmonary inflammation and fibrosis in silica-treated IL-4R–/– mice and controls. Balb/c and IL-4R–/– mice were used for these experiments with five to six mice per treatment group. Mice were treated i.n. with silica or PBS as described, and lungs were harvested 18–24 weeks after the first instillation. At 18 weeks, lungs were excised, weighed, and homogenized for hydroxyproline analysis. Hydroxyproline content was assessed by reaction with chloramine T. Hydroxyproline values are expressed in micrograms per milliliter, where whole lungs were processed in 1 ml. In the silica-treated, wild-type Balb/c mice, there was a significant increase in hydroxyproline levels (**, P<0.01) as compared with PBS controls (A), whereas there were no significant changes in the silica-treated IL4R–/– mice (A). Significance was determined using a t-test. At 24 weeks, lungs were excised and embedded in paraffin for sectioning, with three sections per slide. Three slides per mouse (for a total of nine sections/mouse) were stained with Gomori’s Trichrome for analysis of collagen deposition. The four panels (B–E) are representative sections from each treatment group. The PBS-treated wild-type and null mice (B and D) had no inflammation and expected levels of background collagen (blue) as indicated by the arrows. Silica treatment of wild-type Balb/c mice (C) resulted in massive inflammation (arrowheads) and increased collagen deposition or fibrosis (arrows); whereas silica treatment of IL-4R–/– mice also resulted in massive inflammation (E, arrowheads), there were markedly lower levels of collagen deposition. In the whisker-and-box graph (F), all sections were scored by two independent researchers on a scale of one to four for each parameter (collagen and inflammation). The values between the two reviewers were averaged, and the median of the averages for the five mice in a group was plotted. The null mice had significantly (P<0.01) less fibrosis and less (P<0.05) inflammation than observed in wild-type mice. Mann-Whitney nonparametric analysis was used to determine significance. Original magnification for all slides was 100x.
|
25%) was maintained for up to 100 days (Fig. 2A
). Although the increase observed in the Th2-deficient IL-4R–/– mice also was elevated significantly at Day 3, it significantly decreased through the next several time-points (Fig. 2B)
. These data indicate a correlation between this cellular subset and silica-induced pulmonary fibrosis and suggest that this activated phenotype may play a role in the silicotic process.
 View larger version (17K): [in a new window] |
Figure 2. APC-like macrophage subset alterations following silica exposure in wild-type Balb/c and IL-4R–/– mice. (A) The significant increase in the MHC Class II+ interstitial macrophage (IM) subset in Balb/c mice (*, P<0.05; **, P<0.01; and ***, P<0.001) following silica exposure as compared with PBS controls. This increase is maintained over an extended period of time, from Day 7 to Day 100 with no significant difference between the time-points. Although there is an initial increase of this subtype in the IL-4R–/– mice at Day 3, the increase is decreased significantly over the next several time-points, where Days 14, 28, and 100 are significantly lower (#, P<0.05) than the level observed at Day 3 (B). Although the level at Day 100 is higher (P<0.05) than the PBS control, it is still continuing the trend of decreasing. Values are an average of three to five mice ± SEM.
|
null mice had levels below that of control samples, and wild-type mice treated with crystalline silica generated a significant increase in Ym1 mRNA expression with a greater than fivefold increase in signal (all samples were normalized to β-actin). These results suggest that in mice that develop fibrosis from silica exposure, there is an early increase in Th2-promoting aaMac, of which Ym1 is a marker, and that despite similar, early increases in the active interstitial macrophages in both strains, they appear to have fundamental phenotypic differences.
 View larger version (16K): [in a new window] |
Figure 3. Early expression of Ym1 mRNA in interstitial cells following silica exposure. Mice (Balb/c and IL-4R–/–) were instilled with PBS or crystalline silica. Two weeks post-instillation, interstitial pulmonary cells were purified, and the mRNA was isolated for analysis. Using the QuantiGene Assay system, Ym1 mRNA was semiquantified by normalizing to β-actin mRNA expression and comparing treated samples with controls. Normal background level of Ym1 mRNA is depicted by the broken line and was set at the average value of the PBS-treated Balb/c mice. Values for the PBS- and silica-treated IL-4R–/– mice were below the normal background level. However, there was a significant (*, P<0.05) increase in the silica-treated Balb/c mice of the Ym1 mRNA, which was a greater than fivefold increase in expression. Values are an average of three to five mice ± SEM.
|
and IL-13 were increased significantly following silica treatment of APC [29
] and provide further evidence that crystalline silica enhances the APC activity of macrophages.
 View larger version (11K): [in a new window] |
Figure 4. TC proliferation assessment using [3H]thymidine incorporation in DO11.10 TC following coculture with Balb/c BMdM. TC were pulsed on Day 2 of culture and harvested 16 h later for analysis of radioactivity incorporation. In wild-type cocultures, there is a significant (**, P<0.01) increase in TC proliferation in silica-treated cultures as compared with PBS and TiO2 controls. Values are an average of 10–13 separate experiments ± SEM.
|
 View larger version (31K): [in a new window] |
Figure 5. Flow cytometric analysis of TC activation markers following coculture with Balb/c BMdM. Following 2 days of coculture with BMdM, CD4+ TC were isolated and stained with antibodies against several surface makers for flow analysis. Activation controls are seen in the middle and bottom rows with macrophages + TC and "no OVA" or anti-CD3 ("CD3") stimulation. In the presence of PBS or TiO2 controls, all TCs are CD25+ when activated with OVA in the APC assay but not in silica-treated cultures (top, left panel). Activated TC in PBS or TiO2 control cultures have down-regulated CD62L expression (high-to-low), and the TC in silica-treated cultures express high levels of CD62L (top, middle panel) comparable with that observed in nonstimulated controls. Activated TCs in PBS or TiO2 control cultures have up-regulated CD44 expression, and the TC in silica-treated cultures express levels of CD44 (top, right panel) comparable with that observed in nonstimulated controls. Histograms in the above figure are representative of at least five separate experiments. CD44-APC, CD44-allophycocyanin.
|
and IL-13 following TC isolation and analysis. As expected, the pattern reported previously [29
] was likewise observed here (data not shown), where there was increased release of both cytokines. These results indicate that the increase in TC cytokines is not a result of an increase in the numbers of activated TC but an increase in individual TC activation.
 View larger version (57K): [in a new window] |
Figure 6. Representative samples of activated versus unactivated TC in the APC assay. The dot-blots depict two distinct populations of TC, where the unactivated population is negative for CD25 and low for CD44, and the activated is CD25+CD44hi. The number displayed in the plot represents the percentage of the gated population (activated). The same level of activated cells is observed for all samples in the stimulation controls (Mac+TC+CD3 and Mac+TC+no stim). In the right column, the percentage of activated cells in the PBS and TiO2 controls is comparable with that of the anti-CD3 stimulation control cultures (>90%), whereas the silica-treated cultures with OVA stimulation are roughly half (49.7%). The plots in this figure are representative of at least five separate experiments.
|
or IL-13. Cytokine levels were analyzed in CD25+ and CD25– TC by flow cytometry at 24 h of culture (Fig. 7
), where CD25 expression represented the activated TC phenotype. The fluorescent levels in the CD25– cells were consistently low among all treatment groups, suggesting that this is a negative signal, and the IL-13-PerCP-Cy5.5 (Fig. 7
, upper) and IFN--APC (Fig. 7
, lower) peak mean values in the CD25+-activated population from silica cultures were significantly higher than the levels of control cultures. CD25+ TC exclusively produced both cytokines, as observed by intracellular staining. Silica enhanced this level of cytokines when compared with controls. These results demonstrate that activated CD4+ TC are the primary producers of IL-13 and IFN- in this assay, and they are sensitive to silica exposure.
 View larger version (21K): [in a new window] |
Figure 7. IL-13 and IFN- production in activated versus unactivated TC. In this figure, cells were analyzed for intracellular levels of IFN-. TC isolated from APC cocultures were stained with anti-CD3, anti-CD25, and anti-IFN- or anti-IL-13 to identify the relative levels of the respective cytokines in activated and unactivated cells among the treatments. As described in the previous figure, >90% of TC were positive for CD25 in the control cultures (PBS and TiO2), whereas <50% were CD25+ in the silica cultures. The CD25– cells from each treatment group were consistently low for each group and considered negative based on isotype control (data not shown). Although the per-cell expression or mean fluorescence intensity (MFI) for CD25+ cells in each group was positive, the CD25+ cells from silica-treated cultures expressed a significantly higher level of IL-13 (***, P<0.001) and IFN- (*, P<0.05) than PBS and TiO2 controls. Values are the average of five experiments ± SEM. Significance was determined using a one-way ANOVA.
|
generates a classical phenotype and no Ym1 expression. In Figure 8
, the IL-4-treated BMdM expressed the marker, and the classic phenotype was negative. However, BMdM not treated with cytokines were found to express Ym1 mRNA. A qualitative comparison using the β-actin control suggests that the untreated BMdM expressed the mRNA at lower but detectable levels. These data suggest that BMdM possess a basal level capacity for the aaMac phenotype and that the expression of Ym1 in treated and untreated BMdM proves an ability of these APC to promote a Th2 phenotype in TC.
 View larger version (32K): [in a new window] |
Figure 8. Expression of aaMac marker Ym1 by IL-4-treated BMdM, which were treated with IL-4, IFN-, or no cytokine overnight. The cultures were washed, and mRNA was isolated with TRIzol and analyzed using RT-PCR. Strong levels of Ym1 mRNA were detected in IL-4-treated but not IFN--treated cells. There were detectable levels of Ym1 in untreated BMdM, suggesting a basal level of production before further differentiation via cytokine activation/stimulation. For each sample, β-actin mRNA levels were assessed for normalization and assay control. These gels are representative of three separate experiments.
|
–/– mice–/–) were used for the APC assay. Previous work demonstrated a significant increase in IFN- and IL-13 release from silica-exposed, wild-type cocultures [29
]. However, although there was a similar, silica-induced increase in IFN- release from DO11.10 TC cocultured with wild-type (Balb/c) control or IL-4R–/– BMdM (Fig. 9
) and activated with soluble OVA antigen, there was an absence of the expected increased release of IL-13. Despite the cytokine profile differences, the IL-4R–/– cultures generated levels of proliferation significantly greater than PBS or TiO2 controls (data not shown), as seen in the wild-type cocultures (Fig. 4)
. These results suggest a role of the IL-4R pathway in the ability of macrophages to promote Th2 immunity.
 View larger version (13K): [in a new window] |
Figure 9. Cytokine analysis of DO11.10 TCs in cocultures with IL-4R–/– BMdM or Balb/c controls. The bar graph illustrates the percent change relative to PBS control as a result of variability of absolute values between assays. Following 2 days of coculture with DO11.10 TC, supernatants were collected and assayed by ELISA for release of Th1 (IFN-) and Th2 (IL-13) cytokines. APC (BMdM) were activated with soluble antigen (OVA) to present to TC. In silica-treated samples, the IFN- levels were increased significantly (*, P<0.05) in wild-type and null cultures, and the IL-13 levels were increased significantly by silica treatment in the Balb/c cultures but not the IL-4R–/– cultures. Although silica- and TiO2-treated IL-4R–/– cultures had significantly increased IFN- over PBS controls, silica-treated cultures were increased significantly above TiO2 levels. Statistical analysis was performed using a one-tailed, one-sample t-test compared with zero and adjusted for multiple comparisons. Values are an average of three to five separate experiments ± SEM.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A common theme in immunological research, including the present work, is determining the respective role(s) of Th1 and Th2 mediators in chronic inflammation. In silicosis, increases in Th1 and Th2 factors have been implicated [7
8
9
, 38
]. Currently, one hypothesis is that an initial Th1-mediated inflammation gives way to a Th2-dominated response that is required for the development of fibrosis. Although i.n. instillation of Balb/c mice with crystalline silica results in inflammation and fibrosis, instillation of mice lacking the ability to generate a Th2 response (IL-4R–/–) resulted in a significantly lower level of pathology (Fig. 1)
. And the results in Figures 2
and 3
, where there was an early increase in the expression of Ym1 in the interstitial leukocytes of silica-treated, wild-type mice and no expression in the null mice, despite initial increases in the active macrophage in both strains, further support the idea that aaMac, via the Ym1-IL4R pathway, are contributing to the Th2-dominated fibrosis. In addition, aaMac have been found to possess a diminished capacity to respond to intracellular bacteria [39
], which correlates with an increased tuberculosis susceptibility in patients with silicosis [40
, 41
]. Because of the importance of macrophages in pulmonary immunity and the documented interaction of silica and AM, these results suggest there is a potential role for the Th2-associated aaMac subset in this disease.
Ym1, a well-described marker of the aaMac, is a chitinase-like protein [21
, 22
, 25
, 42
], associated with Th2 immunity, as well as tissue remodeling [21
, 27
, 43
]. In addition to the detection of Ym1 mRNA in wild-type BMdM (Fig. 8)
, recent TC activation studies have found that DC can also express Ym1 and that this protein, as stimulated through the IL-4R pathway, plays a role in the differentiation of Th2 cells [28
]. Using IL-4R–/– BMdM, silica-treated cocultures produced a significant increase in IFN- but not IL-13 (Fig. 9)
. A potential explanation of these results is the inability of IL-4R–/– cells to produce Ym1. Taken together, these observations suggest that silica, via the IL-4R pathway, activates Th2 immunity, an effect that may be critical in the generation of silicosis.
Activation of TC by an APC occurs via a coordinated series of events. Antigen is processed and presented on MHC molecules on the surface of APC to the TCR of the TC. For full activation of the TC, costimulation via accessory molecules and/or cytokines is also required. TC activation can be defined by cytokine production, clonal proliferation, and the expression of specific activation markers. Previously, we reported a significant increase in the production of IL-13 and IFN- when TC were activated by silica-exposed APC [29
], and the present studies determined that these increased levels were primarily from the activated TC population. Primary (AM) and BMdM behaved identically in our experiments. However, the mechanism underlying increased APC activity remains to be determined. We propose that a silica-induced increase in APC activity may play a role in silicosis.
To better characterize the effects of silica-treated BMdM on TC activation, TC were phenotypically analyzed by flow cytometry. In our studies, CD25 and CD44 [44
] were used to discern activated and unactivated cells. Activated TC express CD25 (IL-2R) and have increased expression levels of CD44, an adhesion molecule [45
, 46
]. When BMdM were exposed to silica, they generated a significantly lower number of activated TC (Fig. 5)
. This suggests that although there is a significant increase in TC proliferation and cytokine production, these outcomes are derived from a smaller number of activated TCs. Therefore, silica-treated APC appear to have a capacity to increase the per-cell activation level of T lymphocytes.
Although the vast majority of the literature points to a vital role of Th2 immunity in the generation of pulmonary fibrosis, there are exceptions depending on model and strain. A recent study by Misson et al. [47
] concluded that the IL-4R subunit and Th2 immunity in general are not required for silica-induced fibrosis. In this study, the IL-4R–/– mice on a C57Bl/6 background developed fibrosis after silica exposure. In addition to C57Bl/6 mice being described as Th1-prone and Balb/c mice as Th2-prone [48
49
50
51
], there is evidence in the literature describing differences in fibrosis, depending on the model. In hepatic fibrosis models, Th2 immunity was required in the Balb/c strain for pathology, and C57Bl/6 mice displayed a Th1 response [35
, 52
]. In the Th2-dominanted OVA asthma model, Balb/c mice present with a significantly higher level of collagen deposition than C57Bl/6 mice [53
]. Furthermore, each strain responds uniquely in pulmonary fibrosis models depending on the method of induction. In bleomycin models, the Balb/c are considered fibrosis-resistant [54
55
56
57
58
], and the use of cyclophosphamide results in fibrosis in Balb/c but not C57Bl/6 mice [59
]. However, the use of silica as a fibrotic agent has generated fibrosis in both strains, and Th2 immunity has been implicated in the fibrotic process [10
, 11
, 35
, 54
, 60
61
62
63
]. In addition, IL-13 has been implicated in FITC-induced pulmonary fibrosis [64
]. All these studies suggest that a silicosis model using the Th2-dominant Balb/c strain is highly likely to require Th2 components such as the IL-4R subunit in the fibrosis process and that strain differences can readily explain the C57Bl/6 results.
The current study was designed to examine the link between APC activity and silicosis. In addition, as Th2 immunity has been described as a vital component to the development of pulmonary fibrosis, we tested the ability of BMdM to generate the Th2-promoting molecule Ym1. In addition, BMdM from IL-4R–/– mice were used to further assess the effects of silica on Th2-mediated immunity. The ability of BMdM to express Ym1 mRNA before and after treatment with IL-4 as well as the lack of Th2 promotion in silica-exposed IL-4R–/– BMdM suggest a link between particle exposure and signaling through specific surface molecules. Although IL-4R–/– cells appear to react to silica similar to wild-type cells, the major difference is in the ability to boost the Th2 response, which further suggests a link between crystalline silica and an increase in Th2 immunity. As the Th1 responses between the two types of BMdM in our study were identical, and Th2-deficient mice have been reported to generate increased, Th1-mediated inflammation, this further suggests that the potential role of Th2 immunity in silicosis is to down-regulate the initial Th1 inflammation, which may be key to generating fibrosis in the lungs. Future studies are needed to assess these parameters of APC activity in vivo, as well as potential molecular characterization of the silica-induced changes in wild-type versus IL-4R–/– macrophages.
Received August 10, 2007; revised October 18, 2007; accepted October 22, 2007.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-producing lung lymphocytes in mouse silicosis Am. J. Respir. Cell Mol. Biol. 20,813-824-based Th1 response in thoracic lymph nodes Am. J. Physiol. Lung Cell. Mol. Physiol. 278,L1221-L1230+, and double-negative Peyer’s patch dendritic cells J. Immunol. 166,4884-4890 gene expression by a STAT6 and CCAAT/enhancer-binding protein-dependent mechanism J. Immunol. 170,1789-1796 upregulates adenosine 2b (A2b) receptor expression and signaling in intestinal epithelial cells: a basis for A2bR overexpression in colitis Cell. Mol. Life Sci. 62,2647-2657[CrossRef][Medline] 2 during a T helper cell type 2-dominant immune response J. Exp. Med. 197,687-701 promotes phospholipase C1 activation, Ca(2+) signaling, and cortactin-cytoskeleton function leading to keratinocyte adhesion and differentiation J. Biol. Chem. 279,29654-29669 receptor mRNA after silica and bleomycin exposure and protection from lung injury in double receptor knockout mice Am. J. Respir. Cell Mol. Biol. 20,825-833
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
