Abstract
Rationale: Recently, models of macrophage activation have been revised. Macrophages stimulated with Th2 cytokines have been classified as alternatively activated.
Objectives: This article examines the expression and regulation of CC chemokine ligand 18 (CCL18), a marker of alternative activation, by human alveolar macrophages (AMs).
Methods: AM were obtained from bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis, sarcoidosis, or hypersensitivity pneumonitis (n = 69) and healthy volunteers (n = 22). Expression of CCL18 was determined by quantitative reverse transcriptase–polymerase chain reaction, in situ hybridization, flow cytometry, and immunohistochemistry, respectively.
Measurements and Main Results: Spontaneous CCL18 production by BAL-derived cells was markedly increased in patients with pulmonary fibrosis and correlated negatively with pulmonary function test parameters. CCL18 gene expression and protein production were up-regulated in normal AMs after Th2 cytokine stimulation and/or coculture with human lung fibroblasts. Native collagen significantly up-regulated CCL18 expression in normal AMs activated with Th2 cytokines via a mechanism mediated by β2-integrin/ scavenger receptor(s). Culture supernatants of AMs from patients with idiopathic pulmonary fibrosis increased collagen production by normal lung fibroblasts partly mediated via CCL18.
Conclusions: Our findings suggest that AMs from patients with pulmonary fibrosis disclose a phenotype of alternative activation and might be a part of a positive feedback loop with lung fibroblasts perpetuating fibrotic processes.
Pulmonary fibrosis is the final stage of various pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP), and sarcoidosis (SAR) (1). Currently available therapeutic approaches have little impact on pulmonary fibrotic processes. A new paradigm regarding the pathogenesis of pulmonary fibrosis has recently been proposed (2). IPF is viewed to be the result of an abnormal wound-healing process in which aberrant cross-talk between fibroblasts and epithelial cells promotes chronic fibroproliferation (2–5). This paradigm change has resulted in a debate regarding the role of inflammation in the pathogenesis of IPF (6, 7). Pulmonary macrophages are known to play a crucial role in the development of SAR and HP (8–10). Earlier studies suggest a profibrotic role of macrophages and their products, such as fibronectin, insulin-like growth factor, or platelet-derived growth factor in IPF (11, 12). In the present study, we reevaluated the contribution of alveolar macrophages (AMs) to fibrosing processes of the lung.
Numerous recent studies have documented a dominant role for interleukin 13 (IL-13) in the pathogenesis of pulmonary fibrosis (13–15). These studies are of particular interest to pulmonary macrophage biology and the pathogenesis of pulmonary fibrosis because macrophages activated in the context of type 2 cytokines represent “alternatively activated” macrophages. Alternatively activated alveolar macrophages (aaAMs) produce high levels of fibronectin and promote fibrogenesis in fibroblastoid cells (16). The induction of arginase during the alternative activation of macrophages favors polyamine and proline biosynthesis, promotion of cell growth, collagen formation, and tissue repair (17). aaAMs release IL-10, IL-1 receptor antagonist, and a chemokine named alternative macrophage activation–associated CC chemokine-1 (18, 19). This chemokine is also known as macrophage inflammatory protein-4, dendritic cell chemokine-1, pulmonary and activation-regulated chemokine (PARC), and CC chemokine ligand 18 in accordance with the international chemokine nomenclature classification (20–23). The receptor for CCL18 or a rodent homolog has not been identified. There are reports that CCL18 attracts naive T cells, B cells, activated monocytes, and dendritic cells but not granulocytes (24, 25). Despite the fact that CCL18 was discovered and cloned from human lung, little is known about the regulation of CCL18 expression or its possible role in pulmonary diseases (26).
We postulated that AMs involved in fibrotic processes in the lung express an alternatively activated phenotype, characterized by CCL18 production. Furthermore, we hypothesized that AM-produced CCL18 might be directly involved in the pathogenesis of pulmonary fibrosis in interstitial lung disease (ILD) by augmenting collagen production by pulmonary fibroblasts.
Our data show that (1) normal human AMs spontaneously express and produce CCL18, (2) AMs are the main source of CCL18 in the lung, and (3) increased production of CCL18 by AMs is associated with pulmonary fibrosis. In addition, we observed that fibroblast contact and exposure to native collagen increase the spontaneous CCL18 release of AMs, indicating an alternatively activated phenotype, and we demonstrate that this phenotype up-regulates collagen production by lung fibroblasts (FBR) partly mediated via the production of CCL18. Hence, our data suggest the existence of a positive feedback loop between AMs and fibroblasts promoting collagen production in patients with pulmonary fibrosis. Some of the results of these studies have been previously reported in the form of an abstract(s) (27, 28).
Informed consent was obtained from each subject. Fifteen patients with IPF, diagnosed according to a recently published consensus statement (1), were subjected to bronchoscopic evaluation. An open lung biopsy was obtained from 7 of 15 patients. All patients were histologically classified as suffering from usual interstitial pneumonia (UIP), which is a prerequisite for diagnosis of IPF. In addition, high-resolution computer tomography (HRCT)-thorax scans from all patients were classified as UIP-pattern (29). Forty-six patients with SAR were included. All patients displayed typical histology as shown by transbronchial or video-assisted thoracoscopic biopsy. The SAR group was further divided according to the radiologic type of disease by a plain chest X-ray examination (30). There were 16 patients with SAR type I (SAR I), 15 patients with SAR type II (SAR II), eight patients with SAR type III (SAR III), and seven patients with SAR type IV (SAR IV). Eight patients with HP were studied. All patients with HP had a typical history and typical clinical, laboratory, bronchoalveolar lavage (BAL), and HRCT findings (31, 32). Twenty-two volunteers served as a healthy control group. The study was approved by the local ethics committee. Pulmonary function tests were routinely performed with a standard methodology according to the American Thoracic Society criteria (33). The diffusing lung capacity for carbon monoxide (DlCO) was measured by a single-breath method using a gas mixture of 0.2% CO and 8% helium and corrected for hemoglobin. Data are presented as percent of predicted value.
BAL was performed using a standard technique. BAL-derived cells were processed and cultured as previously described (34) (for further details, see the online supplement).
See the online supplement.
The lung tissue was sliced and washed three times in phosphate-buffered saline at 4°C. The washed slices were placed into six-well plates containing Quantum (PAA, Pasching, Austria) and 1% penicillin/streptomycin solution. The slices were maintained in the medium at 37°C in a 5% CO2 incubator for 2 to 3 wk. The cells that grew from these tissue slices were serially passaged several times in 150-cm2 cell culture flasks to yield pure populations of lung FBRs. All primary fibroblast cell lines were used at passages 4 to 5 in the experiments described herein. The fibroblast cell lines were assessed by cell morphology, immunostaining for vimentin, and fluorescence-activated cell sorter (FACS) analysis. Stable vimentin expression was detected in 99% of cells. Lung slices were also incubated in sterile dispase solution at 37°C for 45 min and processed for type II alveolar epithelial cell (AEC-II) isolation as previously described (35). Human airway epithelial cell–like lines A549 and BEAS-2B were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Human cell lines MonoMac1, MonoMac6 (obtained from DSMZ), and normal human peripheral blood monocytes (> 97% of CD14+ cells, positive selection by MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) were used as controls for AMs. Cell viability was ⩾ 95% in all cell cultures as determined by trypan blue exclusion.
AMs were treated with recombinant human cytokines IL-2, IL-7, IL-9, and IL-15 (10 ng/ml); IL-4, IL-13, and IL-10 (0.1–100 ng/ml); transforming growth factor (TGF)-1β (1–100 ng/ml); and CCL18 (100–1000 ng/ml) to assess the effect of stimulation on CCL18 expression and production (all cytokines were from PeproTech, London, UK). AMs were grown in 24-well plates (5 × 105 cells/well, 1 × 106 cells/ml) in a humidified atmosphere containing 5% CO2 at 37°C for 24 h. RPMI 1640 medium (Gibco, Karlsruhe, Germany) with 10% fetal calf serum and 1% penicillin/streptomycin solution was used for these experiments. In some experiments for fibroblast coculture, cells were separated by polycarbonate membrane transwell inserts (0.4-μm pore size membrane; Corning Inc., Corning, NY) in Dulbecco's modified Eagle medium (DMEM) supplemented with 1% penicillin/streptomycin (DMEM-CM; Gibco). In addition, AMs were cultured on collagen-coated plastic surfaces (collagen-R; Serva, Heidelberg, Germany; 100 μg/ml, 37°C, overnight incubation) in the presence of conditioned medium from FBR (diluted 1:1 with fresh DMEM-CM) in the presence of IL-4, IL-13, or IL-10 (10 ng/ml of each). In some experiments, collagen-coated wells were treated with collagenase from Clostridium histolyticum (Sigma-Aldrich, Deisenhofen, Germany) at a concentration of 200 μg/ml, 37°C, for 18 h. The direct effect of collagen on CCL18 expression and production was assessed in AMs cultured in serum-free medium for human macrophages (M-SFM; Gibco). AMs were cultured on collagen-coated surfaces (20–400 μg/ml) for 24 h. To stimulate FBRs with AM culture supernatants, normal human FBRs were seeded in a 24-well culture plate at a density of 0.5 × 106 cells/well in 250 μl Quantum supplemented with 1% penicillin/streptomycin solution. After 2 h of cell culture, serum-free AM supernatants (250 μl) or control medium were added. For control, TGF-β (10 ng/ml) and CCL18 (1,000 ng/ml) were added. AM supernatants were used with and without preincubation with neutralizing CCL18 antibodies (10 μg/ml; R&D Systems Europe, Wiesbaden, Germany). FBRs were harvested after 48 h of cell culture. Cell-free supernatants were harvested at the end of the culture period. Cells were lysed with Trizol reagent. Cell-free supernatants and cell lysates were frozen and kept at −70°C before ELISA assay and RNA isolation.
See the online supplement.
Hepes-glutamic acid buffer mediated organic solvent protection effect (HOPE)–fixed, paraffin-embedded lung tissue specimens were prepared as previously described (35). Tissues from five patients with IPF, three patients with SAR, and five control subjects were analyzed. Immunohistochemistry and in situ hybridization (ISH) were performed as previously described (35, 36). Freshly isolated BAL-derived cells from healthy volunteers and patients with ILD were fixed in 4% paraformaldehyde and stained as recently described (37) (for further details, see the online supplement).
See the online supplement.
See the online supplement.
CCL18 expression in lung tissue was evaluated by ISH and immunohistochemical staining from patients with IPF or SAR and from control lungs. ISH using a digoxygenin (DIG)-labeled cDNA probe detected specific signals for CCL18 mRNA primarily in intraalveolar macrophages in each of the lung tissue preparations studied (Figures 1A and 1B). Enhanced CCL18 expression was noticed in areas of inflammatory reactions and surrounding fibrotic areas in IPF/UIP (Figure 1A). Positive signals for CCL18 mRNA were also observed in hyperplastic AEC-II, which were typically localized to areas adjacent to fibrotic changes (Figure 1A). Fibroblastic foci and bronchiolar and small lung parenchymal vessels were always negative for CCL18 mRNA (Figure 1A and data not shown). Specific signals were not detected in control preparations in which specific DNA probes were substituted by hybridization buffer (Figures 1A and 1B, insets). The expression pattern of the immunoreactive protein paralleled the mRNA observations. Figures 1C and 1D illustrate many cells revealing an intense staining for CCL18 in IPF/UIP-affected lungs. Lung tissue samples were always negative for CCL18 staining when incubated with nonimmune murine sera or when the primary antibody was omitted (Figures 1C–1F, insets). Immunohistochemical staining for CCL18 was markedly reduced in normal lungs when compared with tissues from fibrotic lungs (Figure 1E). Immunohistochemical staining for CCL18 was weaker in SAR-affected lungs (SAR II-III) compared with tissues from patients with IPF/UIP, but was higher than in normal lung tissues (Figure 1F).
Figure 1. Localization of CCL18 mRNA and CCL18-immunoreactive protein in the lung tissue biopsies obtained from patients with idiopathic pulmonary fibrosis (IPF/UIP), sarcoidosis (SAR), and control subjects. (A and B) Representative photomicrographs of in situ hybridization for CCL18. The insets demonstrate the absence of the signal in preparations where CCL18 probe was omitted (negative control). CCL18 mRNA is highly expressed within the epithelium, interstitium, and intraalveolar cells of lung tissue from patients with IPF/UIP (A), whereas tissue from control lung displays a positive signal only in the free-located macrophages within the alveoli (B). (C–F) Immunohistochemistry for CCL18. Inserts demonstrate the absence of the signal in preparations where primary anti-CCL18 antibody was omitted (negative control). Strong CCL18 immunoreactivity can be seen within the interstitium and in the intraalveolar-located cells in IPF/UIP (C and D) and a weaker CCL18 immunoreactivity in the inflammatory cells and in giant cells in lung tissues from patients with sarcoidosis (F). The control lung showed no detectable signal (E). Bars = 100 μm.
[More] [Minimize]To confirm the data from ISH and immunohistochemistry, we isolated lung FBRs and AEC-II from human normal lung tissue. AMs were obtained from BAL-derived cell preparations from healthy volunteers. Levels of CCL18 mRNA expression and protein production were determined in each of these cell types. CCL18 mRNA expression was evaluated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in freshly isolated AMs and cultured lung FBRs, AEC-IIs, peripheral blood monocytes, and human cell lines of macrophage-like cells (MonoMac1, MonoMac6) and airway epithelial like cells (BEAS-2B and A549). Freshly isolated AMs constitutively expressed high levels of CCL18 mRNA (Figures 2A and 2B). In contrast to AMs, CCL18 mRNA was not detected in A549 and BEAS-2B cells, and minimal amounts of CCL18 message were detected in peripheral blood monocytes cultured for 24 h, in human primary lung FBRs, and in AEC-IIs, MonoMac1, and MonoMac6. The trace of CCL18 message observed in the freshly isolated AEC-IIs and FBRs is likely due to AM contamination. CCL18 mRNA expression corresponds well with protein production in all tested cell types. Among all cell types tested, only AMs displayed consistently high levels of CCL18 protein. Spontaneous release of CCL18 by AMs was time dependent. CCL18 accumulated in the conditioned culture medium (CCM) from AM culture during the first 72 h (Figure 2C). CCL18 is also expressed in human peripheral blood monocytes. However, the level of protein production was lower when compared with AMs. The levels of CCL18 in the CCM from FBRs, AEC-IIs, and MonoMac6 were near the lower detection limit of the ELISA and were nondetectable in CCM from MonoMac1, A549, and BEAS-2B (Figure 2). Thus, human normal AMs spontaneously express and produce CCL18 during in vitro culture.
Figure 2. CCL18 mRNA expression and protein production of normal human alveolar macrophages (AM), peripheral blood monocytes (Mon), lung fibroblasts (FBR), alveolar epithelial cells type II (AEC-II), and A549, BEAS-2B, MonoMac1, and MonoMac6 cell lines. (A) Data from real-time polymerase chain reaction (PCR) with CCL18-specific oligonucleotide primers and cDNA obtained from the indicated cell types. (B) Relative levels of CCL18 mRNA expression and protein production in the indicated cells. (C) Time-dependent spontaneous release of CCL18 by normal AMs. Data are (mean ± SEM of triplicate experiments; shown is one out of three (*p < 0.0001 compared with 12 and 24 h).
[More] [Minimize]Spontaneous ex vivo CCL18 production was significantly increased in BAL-derived cells from patients suffering from IPF/UIP, HP, SAR III, and SAR IV (Figure 3A). CCL18 levels were significantly higher in patients with IPF/UIP when compared with patients with SAR I–III and healthy volunteers (p < 0.0001). However, no difference was found between SAR IV and IPF/UIP. The CCL18 concentrations produced by BAL-derived cells from patients with IPF/UIP were extraordinarily high: levels in some patients' BAL-derived cell cultures exceeded 200 ng/ml × 106 cells/24 h. BAL-derived cells from patients with HP produced less CCL18 than BAL-derived cells from patients with IPF/UIP (p < 0.0001); however, the level of CCL18 production was approximately 10-fold higher than in BAL-derived cells from healthy volunteers or patients with SAR I (Figure 3A). BAL-derived cells from patients with SAR IV produced significantly higher levels of CCL18 than BAL-derived cells from patients with SAR I, II, and III (Figure 3A). In the group of patients with SAR, CCL18 levels produced by BAL-derived cells increased in parallel with the increase of fibrotic transformation of the lung as estimated by chest X-ray (Figure 3A). The percentages of CCL18-positive BAL-derived cells and CCL18-specific relative fluorescence intensities were significantly higher in patients with IPF/UIP compared with healthy control subjects. Thus, the significant increase in CCL18 production of BAL-derived cells from patients with IPF/UIP was associated with an increase in the percentage of CCL18-producing cells within the BAL-derived cell population and an increase in CCL18 protein production per cell (Figure 3B).
Figure 3. Spontaneous CCL18 production of bronchoalveolar lavage (BAL)–derived cells from patients with interstitial lung diseases. (A) CCL18 levels in supernatants from 24-h cultured BAL-derived cells: healthy volunteers (n = 22, Control), sarcoidosis subgroups according to the chest X-ray type (SAR I, n = 16; SAR II, n = 15; SAR III, n = 8; SAR IV, n = 7), idiopathic pulmonary fibrosis (IPF/UIP, n = 14), and hypersensitivity pneumonitis (HP, n = 9) given as box plots. Horizontal lines represent median and 25th and 75th percentiles; small lines characterize 10th and 90th percentiles; *p < 0.05; **p < 0.005 compared with control. (B) Flow cytometric analysis of CCL18 staining by AMs. Relative fluorescence intensity (RFI) and percentage of CCL18-positive cells in freshly isolated AMs from a healthy volunteer and a patient with IPF are shown (one representative experiment out of five). (C) Negative correlation between DlCO (% predicted value) and CCL18 concentrations in BAL-derived cell supernatants from all patients and control subjects.
[More] [Minimize]CCL18 was present in unconcentrated BAL fluid (BALF); concentrations as high as 20 ng/ml (data not shown) were noted. Levels of CCL18 in BALF were highest in patients with IPF and SAR IV. Levels of CCL18 in BALF of patients with HP were lower than CCL18 levels from patients with IPF (p = 0.007); however, these levels were higher than CCL18 levels in BALF from healthy volunteers or patients with SAR I (p < 0.001). Thus, CCL18 is differentially up-regulated in AMs from fibrotic lung tissues when compared with normal AMs.
We obtained a highly positive statistical correlation of CCL18 concentrations in BAL-derived cell culture supernatants and BALF (r = 0.64, p < 0.0001) and a significant negative correlation between BAL-derived cell CCL18 production and DlCO (r = −0.61, p < 0.0001; Figure 3C). Thus, the levels of spontaneous CCL18 release by AMs correlate with the severity and/or extent of the fibrotic process in the lung.
A Th2 cytokine profile predominates in diseases associated with pulmonary fibrosis. Therefore, we investigated the effects of Th2-associated cytokines on CCL18 gene expression and protein production by human AMs. IL-4, IL-13, and IL-10 significantly up-regulated CCL18 production by AMs (Figure 4). This effect was observed in a time- and concentration-dependent manner (data not shown). The most pronounced stimulatory effect on CCL18 expression and protein production from AMs was noted with the combination of IL-4 or IL-13 and IL-10 (Figure 4). The synergistic effects of IL-4 and IL-10 stimulation were about threefold higher compared with each cytokine alone and about 10-fold higher compared with nonstimulated control subjects. Other cytokines known to operate through the IL-4 receptor complex (a heterodimer comprised of the IL-4Rα chain and the IL-2Rγ chain), such as IL-2, IL-7, IL-9, and IL-15, had no influence on CCL18 mRNA expression or protein release in AMs (data not shown).
Figure 4. CCL18 production by normal AMs, nonstimulated and activated with IL-4, IL-13, and/or IL-10 in vitro. Demonstrated are the CCL18 up-regulatory effects of IL-4, IL-13, IL-10, and their combinations (10 ng/ml) in normal AMs. Data are means ± SEM from one representative triplicate experiment out of five independent experiments (*p < 0.0001 compared with control and TGF-β–stimulated cells; §p < 0.005 compared with IL-13/IL-10–activated cells).
[More] [Minimize]Granulocyte-macrophage colony–stimulating factor and TGF-β did not significantly up-regulate CCL18 mRNA expression or protein production by normal AMs at concentrations of 10 to 100 ng/ml (Figure 4). Stimulation of normal AMs with human recombinant CCL18 protein (10–1,000 ng/ml) had no influence on CCL18 mRNA expression or protein production (data not shown).
To our knowledge, no studies have evaluated the regulatory capacities of FBRs on CCL18 gene expression and/or protein secretion. Therefore, we analyzed CCL18 production by nonstimulated and IL-4/IL-10–stimulated AMs during coculture with human primary lung FBRs (Figure 5A). AM/FBR cocultures resulted in a significant increase in CCL18 production compared with AMs alone. The increased production was dependent on the number of FBRs in the coculture system. Increased CCL18 levels were noted with increases in the numbers of FBRs in coculture. The up-regulatory effects of AM/FBR coculture were more evident when AMs were activated with IL-4/IL-10 (Figure 5A). When FBRs were separated from AMs by a transwell membrane, 50% of the coculture-induced CCL18 protein secretion was abolished in IL-4/IL-10–activated AMs, and approximately 30% was abolished in nonstimulated cells (Figure 5A). Nevertheless, CCL18 levels in the membrane-separated cocultures were significantly higher when compared with AM cultures alone. The transwell effect was dependent on the AM/FBR ratio and on the state of activation (Figure 5A). Lung FBRs cultured alone and stimulated with IL-4/IL-10 or left untreated were negative for CCL18 (Figures 2 and 5B). In control experiments, autologous peripheral blood CD3+ lymphocytes, primary AEC-II, A549, or BEAS-2B cells were cocultured with AMs in the same conditions and cell–cell ratio as FBRs. CCL18 production by nonstimulated or IL-4/IL-10–activated AMs in coculture with these types of cells was not significantly different when compared with CCL18 produced by AMs in monoculture. Human lung FBRs fixed with 4% paraformaldehyde did not display any up-regulatory capacity on CCL18 production by AMs. Thus, human normal lung FBRs spontaneously produce a soluble and contact mediated factor(s) that has a “priming” effect on CCL18 production in normal human AMs.
Figure 5. Secretion of CCL18 mediated by the interaction of normal lung fibroblasts (FBR) and AMs. (A) FBRs were seeded in culture plates, and after 2 d AMs were directly layered on nonstimulated FBR monolayers or placed in transwell inserts and activated with IL-4/IL-10. Supernatants were collected after 24 h of coculture, and CCL18 was measured by ELISA. Data are means ± SEM from one triplicate experiment out of five using five different FBR lineages and five different AM preparations from different healthy individuals *p < 0.001 compared with AM alone, and **p < 0.005 compared with cytokine-activated cells in the Transwell inserts. (B) Representative photomicrographs demonstrating immunohistochemical localization of CCL18 protein in AM and FBR coculture. Staining with anti-CCL18 antibody demonstrates a strong CCL18 signal associated with AMs, which markedly increases after IL-4/IL-10 stimulation. No staining was observed in nonstimulated and IL-4/IL-10–stimulated FBRs. There was no specific staining in preparations incubated with control IgG (not shown). Bars = 100 μm.
[More] [Minimize]Collagen is one of the proteins most abundantly produced by FBRs. We hypothesized that collagen might play a role in the up-regulation of CCL18 production in AM/FBR cocultures. To evaluate the direct effect of collagen on CCL18 mRNA expression and protein production, normal human AMs were placed in collagen free M-SFM medium and cultured in 24-well plates coated with collagen at different concentrations. Collagen directly induced CCL18 mRNA expression of AMs in a concentration-dependent manner (Figure 6A). Specific message accumulation was accompanied by an increased release of protein into CCM compared with CCM from cells incubated without collagen. IL-4/IL-10–activated AMs released significantly higher amounts of protein in the culture with and without collagen compared with the relevant control subjects. Culture of normal human AMs on bovine fibronectin– or human fibrinogen–coated plastic surfaces at the same concentrations as was used for collagen did not produce up-regulation of CCL18 mRNA expression or protein release. Collagenase treatment of collagen-coated wells before AM culture completely abrogated the up-regulatory effect of collagen on CCL18 expression and release (data not shown).
Figure 6. Direct effect of native collagen on CCL18 mRNA expression and protein production of normal AMs cultured in serum-free medium. (A) A dose-dependent effect of collagen on CCL18 mRNA accumulation (table of threshold cycles and calculation of the relative mRNA expression). Similar results were obtained for CCL18 protein production by ELISA (data not shown). (B) The effects of AM/FBR coculture, collagen, and conditioned culture medium from nonstimulated normal lung FBRs (FBR-CCM) on CCL18 mRNA expression and protein production by normal human AMs, nonactivated or IL-4– and IL-4/IL-10–activated in vitro (means ± SEM, one triplicate experiment out of three with AMs from three different healthy individuals; *p < 0.001 compared with control [without collagen], and **p < 0.0001 compared with nonstimulated cells). (C) Effects of anti–β-integrins blocking monoclonal antibodies (mAbs) and dextran sulfate (DS) on CCL18 release by normal AMs cultured on a collagen-coated surface and activated with IL-4/IL-10. AMs were preincubated with CD29-, CD18-blocking mAbs or isotype-control, or with 100 μg/ml of DS or chondroitin sulfate (CS) and placed into collagen-coated wells. Parallel cultures were activated with IL-4/ IL-10 (10 ng/ml). Data are means ± SEM of triplicates from one representative experiment out of three with AMs from three different healthy individuals (*p < 0.01, compared with the respective control subjects).
[More] [Minimize]To further validate a role for soluble factors released from human lung FBRs, we analyzed CCL18 levels in the supernatants of AMs after the addition of CCM obtained from 5- to 7-d passages of eight different human lung FBR lineages or AMs cultured on collagen-coated plates (Figure 6B). The presence of collagen and FBR-CCM duplicated the effects of AM/FBR coculture (Figure 6B). The effect of IL-4/IL-10 activation was more pronounced in the FBR-CCM/collagen cultures when compared with AM/FBR coculture. The combination of collagen, FBR-CCM, and IL4/IL10 activation was the most efficient regimen for up-regulating CCL18 mRNA expression in normal AMs (Figure 6B). These experiments demonstrate that native collagen increases CCL18 mRNA expression and protein production by AMs.
β1- and β2-integrins and scavenger receptors play important roles in macrophage binding to extracellular matrix protein and their activation (38). We hypothesized that these receptors are involved in collagen-mediated CCL18 mRNA expression. Normal AMs were preincubated with blocking monoclonal antibodies (mAbs) for several integrin subunits or a nonspecific blocker of scavenger receptors (dextran sulfate [DS]). Controls were made up of AMs in CM alone, AMs in CM containing an isotope-matched murine IgG, or AMs in CM containing chondroitin sulfate. CD29 (β1-integrin subunit)-blocking mAbs (clone P4C10) had no effect on CCL18 levels in noncytokine-stimulated or IL-4/IL-10–activated AMs (Figure 6C). In contrast, CD18 (β2-integrin subunit)-blocking mAbs (clone P4H9-A11) significantly decreased the up-regulatory effect of collagen on CCL18 production by nonstimulated AMs and almost completely abrogated the collagen effect on IL-4/IL-10–activated cells compared with the isotype-matched IgG control (Figure 6C). Two mAbs that recognize CD11b and CD11c subunits (clones ICRF44 and B-ly6) had weak inhibitory effects (not statistically significant), and anti-CD11a (clone HA111) had no effect on the collagen-induced up-regulation of CCL18 production by nonstimulated and IL-4/IL-10–activated AMs (data not shown). DS had no effect on the CCL18 up-regulation in nonstimulated AMs but strongly inhibited collagen-induced CCL18 production in IL-4/IL-10–activated cells compared with chondroitin sulfate (control for DS; Figure 6C). Anti-CD18 mAbs and DS inhibited the accumulation of CCL18 mRNA in nonstimulated and IL-4/IL-10–stimulated AMs. Similar results were obtained for CCL18 mRNA expression (data not shown) and with AMs cultured in serum-free CM on collagen-coated plastic surfaces (data not shown). Thus, native collagen directly activates normal human AMs by triggering a specific set of surface molecules.
BAL-derived cells from 22 patients with IPF/UIP, HP, or SAR (SAR IV) and from 10 healthy volunteers were fixed with paraformaldehyde and stained with a collagen type I antibody for further FACS analysis. AMs from patients with ILD displayed significantly more collagen type I at their surface than healthy volunteers (p < 0.001; Figure 7A). This finding gives evidence that AMs from patients with ILD have more direct contact with collagen type I than AMs from healthy volunteers.
Figure 7. (A) Flow cytometric analysis of collagen type I staining by AMs. Two original registrations are shown of BAL-derived cells from a control subject and a patient with IPF/UIP. AMs from patients with ILD (IPF/UIP, HP and SAR IV, n = 22) disclosed statistically significant more collagen type I staining than AMs from control subjects (n = 10). (B and C) Effect of conditioned culture medium of AMs on collagen type I A1 mRNA expression (B) and protein production (C) by human normal lung fibroblasts. Data are expressed as means ± SEM of three AM culture supernatants from three different patients with IPF/UIP and from three different healthy volunteers. Parallel culture supernatants were used with or without preincubation with neutralizing CCL18 antibodies. Shown is one representative measurement out of three experiments with different human normal lung fibroblasts. FBR = fibroblasts cultured alone; CCM AM_IPF = conditioned culture medium of AMs from patients with IPF/UIP; CCM AM_C = conditioned culture medium of AMs from control subjects; αCCL18 = neutralizing CCL18 antibody was added (10 μg/ml). * p < 0.001 versus FBR; § p < 0.001 versus FBR+CCM AM_C; $ p < 0.001 versus FBR+ CCM AM_IPF + αCCL18.
[More] [Minimize]Cell culture supernatants generated from AMs of patients with IPF/UIP increased collagen type I production by normal human lung FBRs significantly more than cell culture supernatants from AMs of healthy volunteers. Data were obtained by measurement of collagen type I A1 mRNA expression in RT-PCR and collagen type I protein content in an ELISA system. Increase in collagen type I A1 mRNA expression and collagen type I production by CCM from AMs of patients with IPF/UIP was higher than in nonstimulated FBRs or FBRs stimulated with TGF-β, CCL18, or CCM from AMs of healthy volunteers (Figures 7B and 7C). Preincubation (20 min) of AM culture supernatants with neutralizing CCL18 antibodies inhibited increase in collagen type I production induced by CCM from AMs of patients with IPF/UIP. Blocking CCL18 with neutralizing antibodies abrogated induced collagen type I A1 mRNA expression by 93% (RT-PCR) and collagen type I production by 64% analyzed by ELISA (Figures 7B and 7C).
The pathology of pulmonary fibrosis is characterized by aberrant repair with fibroproliferation and deposition of extracellular matrix leading to progressive loss of lung function (2, 3, 6). The pathogenesis remains uncertain. Molecular, cellular, and histopathologic studies have identified numerous mediators, including growth factors, proteases, antiproteases, and cytokines and chemokines associated with cellular trafficking, inflammation, immunity, and angiogenesis, that are involved in the production and deposition of extracellular matrix proteins in the lung (2, 3, 6–12, 39–43). Our studies show markedly increased gene expression and protein levels of CCL18 in BAL and BAL-derived cell cultures from patients with diverse pulmonary fibrotic diseases, including IPF/UIP, SAR, and HP. Our data document that alternative activation of AMs is a feature shared by all of the fibrotic lung diseases that we studied. CCL18 gene expression in AMs from healthy volunteers and protein production is up-regulated by Th2 cytokine stimulation, coculture with human lung FBRs, and native collagen. Alternatively, activated AMs obtained from patients with pulmonary fibrosis increase collagen production by normal lung FBRs; the increased collagen production is mediated, in part, by AM CCL18 production. These findings identify a positive feedback loop between AMs and FBRs in which AM-produced CCL18 induces increased collagen production and FBR-produced collagen increases AM CCL18 production.
BAL-derived cells from IPF/UIP, SAR IV, and HP spontaneously produce significantly higher levels of CCL18 than BAL-derived cells obtained from normal healthy control subjects. BAL-derived cells from patients with IPF/UIP produced up to 100-fold more CCL18 than those of healthy individuals. BAL-derived cells from patients with SAR who have evidence of pulmonary fibrosis by chest X-ray (SAR IV) also produced significantly higher concentrations of CCL18 compared with patients with SAR who have little or no evidence of fibrotic processes within the lung (SAR I–III). The increase in CCL18 production noted in BAL-derived cells from patients with IPF/UIP was due to an increase in the percentage of CCL18 producing AMs in BAL-derived cell populations and the amount of protein produced by each cell. Thus, we observed a dramatic up-regulation of CCL18 production in all of the fibrotic lung diseases that we studied.
CCL18 was expressed and produced by intraalveolar and interstitial macrophages in fibrotic lungs. However, CCL18 expression was also seen in hyperplastic AEC-II cells, which surround fibroblastic foci and line collapsing alveoli. In contrast, neither CCL18 mRNA nor protein was found within fibroblastic foci, endothelial cells, or bronchiolar cells. Minimal amounts of CCL18 mRNA was detected in human primary lung FBRs and AEC-IIs isolated from histologically normal lungs, but neither of these cell types produced detectable amounts of protein in vitro. In addition, airway epithelial cell–like lines A549 and BEAS2B were negative for CCL18 message and protein. The minimal amounts of CCL18 mRNA noted in primary cell isolates are likely due to minimal macrophage contamination of these cells. However, we cannot exclude that cellular interactions between AEC-IIs and other cells are necessary for CCL18 expression in vitro because this was demonstrated for the in vitro induction of inducible nitric oxide synthase in alveolar macrophages (44). These data document that alveolar and interstitial macrophages are the major source of CCL18 in fibrotic lungs. Changes in the pulmonary microenvironment and cellular interactions during fibrotic processes may cause differentiative events in AEC-IIs that enable these cells to produce CCL18, although we could not observe CCL18 production by freshly isolated AEC-IIs in vitro.
Our findings differ from a previous study that reported that CCL18 expression was higher in patients with HP than in patients with IPF/UIP (26). In this study, CCL18 expression was assessed by real-time PCR analysis of whole lung tissue only. BAL-derived cells were not analyzed, and CCL18 protein levels were not measured. In a previous study regarding CCL18 production in SAR (45), no difference in CCL18 mRNA expression by BAL-derived cells from patients with SAR was observed when compared with BAL-derived cells from normal volunteers. Our results reproduced this study as far as SAR I is concerned; however; they differ from this study in that our SAR-II and SAR-III patients had significantly higher concentrations of CCL18 than control patients. In the previous study, the majority of patients were SAR-I and SAR-II; however, they included no SAR-IV patients, who show the highest levels of CCL18 in our studies. Differences in analysis and the inclusion of patients with stage IV SAR in our study may account for our different results.
Our studies provide strong support for the concept that overproduction of CCL18 in the lung plays a role in pulmonary fibrotic disease processes. A significant negative correlation exists between CCL18 levels and DlCO. Previous studies suggested a role for CCL18 as a chemoattractant for lymphocytes in HP; however, no correlation was detectable between BAL lymphocyte differential counts and CCL18 (data not shown). This discrepancy might be due to the fact that Pardo and colleagues (26) measured CCL18 expression by immunohistochemistry and counted lymphocytes in tissue. Fibrotic tissue remodeling in patients with HP is a common finding, especially in patients with chronic HP. In our study, we observed in patients with chronic HP the highest levels of CCL18 production. Based on our findings, we postulate that CCL18 in HP is more associated with fibrotic tissue remodeling than T-cell migration.
No previous study has evaluated the regulation of CCL18 gene expression and protein production by human AMs. IL-4, IL-13, and IL-10 significantly increase CCL18 production in normal human AMs in a time- and dose-dependent manner. TGF-β had no effect on AM CCL18 mRNA expression or protein production. IL-10 has significant synergistic up-regulatory effects on AM CCL18 mRNA expression and protein production when combined with IL-4 or IL-13. These findings are of particular importance because a persistent imbalance in expression of Th2 versus Th1 cytokines exists in the lungs of patients with pulmonary fibrosis (46–49). Because CCL18 is regarded as a marker of alternatively activated macrophages (18, 19, 21, 22), we conclude that AMs from patients with pulmonary fibrosis–associated diseases display a phenotype of alternative activation. Our findings correspond with data concerning the expression of other aaAM-associated products in pulmonary fibrosis. In 1993, Janson and colleagues (51) demonstrated increased levels of IL-1 receptor antagonist, now known as another marker of aaAMs, in BALF from patients with pulmonary fibrosis. Furthermore, in animal models of pulmonary fibrosis, it was shown that two other chemokines associated with the aaAM phenotype, CCL17 and CCL22, are significantly up-regulated. However, although the existence of aaAMs has been widely accepted based on in vitro models (18, 19, 21, 22), their relevance in vivo has not been well described. The results mentioned in this article are the first proof of the evolution of this alternative phenotype of AMs (aaAMs) in patients with fibrotic lung diseases.
Our studies of fibrotic lung tissue demonstrate close proximity of CCL18 producing AMs and abnormal accumulations of fibroblasts. Significant AM–lung FBR interactions occurred in vitro insofar as lung FBRs significantly enhanced CCL18 production by AMs. This effect was synergistic, with the stimulatory effect being observed with Th2 cytokines. In contrast, airway epithelial cells had no effect on CCL18 by AMs. The increases in CCL18 production noted in AM/FBR cocultures were the result of quantitative and qualitative changes in CCL18 expression patterns in AMs but not in FBRs. Formalin-fixed FBRs did not alter CCL18 production; accordingly, we believe that its effect is not dependent on direct cell membrane–associated receptor interactions. The studies show that a soluble mediator(s) produced by FBRs can up-regulate CCL18 production by AMs.
Contact between extracellular matrix molecules and monocytes can trigger intracellular events; binding of monocytes to type I collagen significantly increases the production of superoxide anion by monocytes (52). Contact between native collagen and AMs in serum-free media increased CCL18 mRNA expression and protein release in a concentration-dependent manner. This direct inductive effect of collagen on AM CCL18 production was dramatically increased when IL-4 and IL-10 were added, demonstrating a strong synergistic interaction between collagen type I and Th2 cytokines. AMs from patients with pulmonary fibrosis likely interact directly with collagen in vivo. AMs from patients with advanced fibrotic disease have collagen on their surfaces, as evidence by strong positive staining with antibodies specific for collagen type I. Soluble collagen was present in FBR culture supernatants. Thus, soluble collagen produced by lung FBRs has potent up-regulatory effects on CCL18 mRNA expression and protein production by normal AMs and IL-4/IL-10 aaAMs. These studies are the first to document that collagen can serve as a “priming” signal for alternative activation of macrophages.
The effects of collagen induced CCL18 up-regulation are mediated via interactions between collagen and β2-integrins and scavenger receptors on AMs. Antibodies to CD18 (β2-integrin subunit) significantly decreased up-regulatory effects of collagen on CCL18 production. Antibodies to CD29 (β1-integrin subunit) had no effect. mAbs to CD11b and CD11c subunits had weak inhibitory effects. Antibodies to CD11a had no effect on collagen-induced effects on CCL18 production. Scavenger receptors such as CD36 and CD163 are markers for aaAMs (21, 22, 53). DS, which was described as a scavenger receptor blocker in macrophages (38), was a potent inhibitor of collagen-induced CCL18 production.
Soluble mediators produced by AMs obtained from patients with IPF/UIP increased type I collagen production by normal FBRs in vitro. This effect was in large part mediated by CCL18 because antibodies to CCL18 blocked the effect of AM supernatants on collagen production. Recently, Atamas and colleagues (54) have shown that CCL18 enhances collagen production by lung FBRs. A profibrotic role of macrophages in patients with IPF has been discussed (11, 12). However, a mediator-enhancing collagen production that is directly produced by macrophages in high amount was not known. Because TGF-β was thought to be mainly produced by alveolar epithelial cells in pulmonary fibrosis, recent research was focused on the abnormal cross-talk between alveolar epithelial cells and FBRs. With our findings in mind, we propose a positive feedback loop, involving aaAMs and their specific product (CCL18) on one side and lung FBRs and their specific product (collagen) on the other side, causing a vicious circle perpetuating pulmonary fibrosis. Because our data also demonstrate that the stimulation of AMs by native collagen in combination with Th2 cytokines exhibits a dramatic synergistic increase in CCL18 production, we postulate that the presence of increased Th2 cytokines in the lungs of patients with pulmonary fibrosis will amplify the effect of the described vicious circle. In this context, even minor T-cell inflammation might have a deep impact on fibrogenesis, as suggested by Gross and colleagues (43). In addition, our data obtained in patients with SAR show that even in a disease that is described as a classical Th1 immune response with the evolution of fibrotic remodeling at the chest X-ray, the phenotype of macrophages changes toward a phenotype that is known to be induced by Th2 cytokines in macrophages. However, our data do not demonstrate the presence of Th2 cytokines in these diseases, but we demonstrate that other factors like collagen might be capable of inducing such a macrophage phenotype. In aggregate, our studies implicate a role for aaAMs and their specific product, CCL18, in fibrotic remodeling of the lung.
Our studies define a mechanism for the perpetuation of pulmonary fibrotic responses. Th2 cytokines potently induce the specific aaAM product CCL18. AMs, which are in close proximity to sites of pulmonary injury undergoing repair, release CCL18, which stimulates fibroblast production of collagen. Proximate aaAMs that bind to this collagen type I via β2-integrins and scavenger receptors further increase their CCL18 production, creating a self-perpetuating vicious cycle of augmented, continuous alternative macrophage activation and excessive collagen production by FBRs. Our studies demonstrate the presence of elements of this positive feedback loop in fibrotic diseases believed to be mediated by specific T-cell responses (HP and SAR) and in IPF/UIP where the contribution of immunologic/inflammatory responses is controversial. Our findings provide support for the hypothesis that a type 2 immune response, represented by aaAMs, plays a crucial role in the perpetuation of fibroproliferative processes in diffuse parenchymal lung diseases. The presence of a shared pathologic mechanism for perpetuation of fibroproliferative responses in immunologically different diseases (55) offers hope for novel therapeutic approaches directed toward interrupting alternative activation of macrophages or toward blockade of its specific profibrotic product, CCL18.
The authors thank S. Hahn, S. Bock, H. Kuehl, and S. Kamenker for skillful technical assistance.
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