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Supernatants from quartz dust treated human macrophages stimulate cell proliferation of different human lung cells as well as collagen-synthesis of human diploid lung fibroblasts in vitro
Toxicology Letters 96,97 (1998) 85 – 95
Supernatants from quartz dust treated human macrophages stimulate cell proliferation of different human lung cells as well as collagen-synthesis of human diploid lung fibroblasts in vitro H. Olbru¨ck a,b,*, N.H. Seemayer a, B. Voss c, M. Wilhelm b,c b
a Medical Institute of En6ironmental Hygiene at the Uni6ersity Du¨sseldorf, Auf’m Hennekamp 50, D-40225 Du¨sseldorf, Germany Institute of Hygiene, Social- and En6ironmental Medicine, Uni6ersity Bochum, Uni6ersita¨tsstrasse 150, D-44780 Bochum, Germany c Professional Associations Research Institute for Occupational Medicine (BGFA), Bu¨rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany
Abstract Silicosis is a chronic lung disease, which is caused by inhalation of silica-containing dusts, leading to pulmonary fibrosis. Alveolar macrophages play a key-role in defence against these particles entering the lung. As a result of phagocytosis, the macrophages release mediators, which are involved in various processes of inflammation and immunological defence mechanisms. We established an in-vitro test system composed of human macrophages, human pneumocyte type II cells (line A-549), human diploid lung fibroblasts (line Wi38) and human tracheobronchial epithelial cells (line BEAS-2B). With this model, we were able to study the influence of various cytokines, produced by the macrophages, on cell proliferation and collagen synthesis (only fibroblasts) of the cells in our test-system. In this report, we will summarize data obtained from our in-vitro test system on two cytokines, which are thought to be important in pathogenesis of lung fibrosis: insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-b). © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Macrophages; Collagen synthesis; Fibroblasts; Cytokines
1. Introduction The long-term inhalation of dust, which contains crystalline silica can cause irreversible lung damage and silicosis. Silicosis is characterised by an increase in lung collagen content (Davis, 1986; * Corresponding author.
Crouch, 1990; Richards et al., 1991; Kane, 1995). This fibrotic reaction destroys the lung parenchyma and impairs lung function (Kane, 1995). In the absence of inhibitory signals, the continued production of cytokines promotes connective tissue synthesis which results in alterations of lung tissue. Heppelston and Styles, (1967) were able to demonstrate, that peritoneal macrophages release
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after quartz dust phagocytosis mediators, which stimulate chicken fibroblasts to an increased collagen synthesis. The lung macrophages are the primary target cell for the inhaled dust. They phagocytise the quartz dust and are able to release beside reactive oxygen species mediators, which can influence the proliferation of adjacent cells as well as the collagen synthesis of human lung fibroblasts. Bittermann et al. (1982) reported that macrophages produce after incubation with different particulates a factor, which was named ‘alveolar-macrophage-derived-growth-factor’ (AMDGF). Later this factor was identified as insulin-like growth factor-1 (IGF-1) (Rom et al., 1988). IGF-1 has a molecular weight of 7.5 kD and is bound under normal conditions in 99% to monospecific binding proteins called the IGFbinding proteins (Rosenfeld and Gargosky, 1996). These proteins bind to IGF-1 and IGF-2 with a high affinity and serve as carriers of IGFmolecules, prolonging the average mean-time of this growth factor in human serum from unbound 8 – 12 min to 2–4 h of bound (Rosenfeld and Gargosky, 1996). Recent results obtained from our test system show that human macrophages release after quartz dust phagocytosis a cytokine, which stimulates quiescent human fibroblasts (Seemayer and Braumann, 1985; Seemayer et al., 1987, 1988; Hu¨bner and Seemayer, 1989; Seemayer, 1989; Seemayer and Maly, 1990; Olbru¨ck et al., 1998; Seemayer et al., 1997), as well as pneumocyte type II-like cells (line A-549) (Thiel et al., 1992; Griwatz et al., 1993; Thiel et al., 1993; Thiel-Griwatz et al., 1993; Griwatz et al., 1994, 1995; Olbru¨ck et al., 1998; Seemayer et al., 1997) and human tracheobronchial epithelial cells (line BEAS-2B) (Olbru¨ck et al., 1997, 1998) under serum-free conditions to enhanced cell proliferation. We could show, that quartz dust exposed human macrophages synthesise IGF-1 and a specific binding protein. Furthermore, the stimulation of cell proliferation of human tracheobronchial epithelial cells was markedly reduced by coincubation with an antibody against human IGF-1. In contrast to the stimulation of cell proliferation by IGF-1, the incubation of lung fibroblasts (line Wi38) and pneumocyte-type-II-like cells (line A-549) with transforming growth factor-beta
(TGF-b) did not increase cell proliferation (Olbru¨ck, unpublished results). TGF-b is synthesised by human macrophages (Khalil et al., 1989; Kelley, 1990; Kovacs, 1991; Vanhee et al., 1994, 1995; Vignola et al., 1996). There are two forms of TGF-b, a latent and an active form. Latent TGFb is bound to a protein complex and therefore inactive (Roberts and Sporn, 1988; Lyons and Moses, 1990). By proteolytic cleavage (Lyons and Moses, 1990; Lyons et al., 1990) or acidification (Ikeda et al., 1987; Mitchen et al., 1995) latent TGF-b can be activated. Active TGF-b can induce collagen synthesis of human fibroblasts (Roberts and Sporn, 1988; Khalil et al., 1989; Fine et al., 1990; Kelley, 1990; Kovacs, 1991, Phillips et al., 1992; Border and Noble, 1994; Butt et al., 1995; Vanhee et al., 1995; Jagirdar et al., 1996). We tested, if human macrophages exposed to quartz dust synthesise TGF-b and if collagen synthesis of human lung fibroblasts is enhanced by incubation with conditioned medium from quartz dust exposed human macrophages in our test-system.
2. Materials and methods
2.1. Quartz dust We used Do¨rentruper quartz dust grinding no. 12 (DMT-Essen, Germany) with a diameter B5 mm as standard quartz dust (Robock, 1973). The quartz dust was resuspended in a concentration of 0.5 mg/ml in Hanks balanced salt solution (HBSS, Biochrom, Germany) and sonified (Sonifier B-12, Branson Sonic Power Company, USA) (Griwatz and Seemayer, 1995).
2.2. Culti6ation of human monocytes/macrophages The isolation of human monocytes from enriched peripheral blood samples and the maturation to human macrophages has been described in detail elsewhere (Seemayer and Braumann, 1985; Seemayer et al., 1987). In brief, human monocytes separated in a Ficoll-Hypaque-gradient (Biochrom) were cultivated in tissue culture flasks. After 10–12 days, the cells revealed characteristics
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of macrophages and were refed with serum-free medium. At the same time, the macrophages were incubated with quartz dust at non-toxic concentrations (e.g. 30 mg/ml). The conditioned medium was collected after 24 and 48 h and frozen at − 20°C until further analysis.
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medium but DMEM supplemented with 0.15% BSA. At the same time, the test samples were added to the cultures. After 6 days we counted the cells in the Coulter counter.
2.5. Bioassay for insulin-like growth factor-1 (IGF-1)
2.3. Culture of human lung fibroblasts (line Wi38) The cell line Wi38 was obtained from Serva, Germany and cultured in Dulbeccos Modified Eagle Medium (DMEM, Boehringer Mannheim, Germany) supplemented with 10% fetal calf serum (FCS, Seromed, Germany). The cells were subcultured every 7 days in a ratio of 1:2. For measurement of collagen synthesis, the cells were seeded at a density of 80000 cells into the wells of a 24-well plate (Becton-Dickinson, Germany) in DMEM containing 10% FCS. After the cells reached confluency (48 h), the cells were washed twice with PBS (Sigma, Germany) and refed with DMEM without serum, but 0.15% (w/v) bovine serum albumin (BSA, Boehringer Mannheim) and 50 mg/ml ascorbic acid. Then the conditioned medium from quartz-exposed human macrophages was added. After a cultivation period of 7 days, the conditioned medium from the fibroblast cultures was collected and frozen until collagen measurement. The cell number was determined by counting the cells in a Coulter counter (Model Zb, Coulter Electronics, Germany).
2.4. Culture of human tracheobronchial epithelial cells (line BEAS-2B) The cell line BEAS-2B (Reddel et al., 1988) was a kindly gift from Professor C.C. Harris (National Cancer Institute, Bethesda, MD). The cells were cultivated in serum-free medium HC3 (Hornberg et al., 1993). This medium contained equal parts of HAM F12 (Biochrom), CMRL 1066 (Biochrom), growth factors and conditioned medium of NIH-3T3 cells. The cells were subcultured every 3–4 days. For cell proliferation assay, 3000 cells were inoculated into wells of a 24-well plate in HC3-medium. After 24 h, the cells were washed twice with PBS and then refed with HC0medium, containing no conditioned NIH-3T3-
We used the human mammacarcinoma cell-line MCF-7 (Soule et al., 1973) for the IGF-1-bioassay (Van Zoelen, 1990; Van Zoelen et al., 1993). This cell-line is cultured in DMEM containing 5% FCS. 5000 logarithmically growing cells were inoculated into wells of a 24-well plate. After 24 h the medium was changed to serum-free DMEM containing 0.15% bovine serum albumin (BSA). After the medium-change, the cells were not longer able to proliferate and were arrested in G0/G1-part of the cell cycle (Van Zoelen et al., 1993). By addition of insulin-like growth factor-1 (IGF-1) or insulin-like growth factor-2 (IGF-2) the cells are restimulated to cell division. Other cytokines like epidermal growth factor (EGF), transforming growth factor-beta (TGF-b) or platelet-derived growth factor (PDGF) are not stimulating cell division of this cell line (Van Zoelen et al., 1993). After a culture period of 4 days with conditioned medium from quartz dust exposed macrophages, the cell number was determined by counting the cells in a Coulter counter.
2.6. Bioassay for transforming growth factor-beta (TGF-b) TGF-b was measured utilising the highly specific bioassay with mink lung cells of the line Mv1Lu (NBL-7) (Ikeda et al., 1987; Mitchen et al., 1995) obtained from the European Collection of Cell Cultures (ECACC, UK). Cell proliferation of this cell line is inhibited in serum-containing medium dose-dependently to TGF-b. The cells were cultured routinely in DMEM containing 10% FCS. For the TGF-b bioassay, 2500 cells were inoculated into wells of a 24-well plate in 10% DMEM. After 24h, the cells were washed two times with PBS and then refed with DMEM containing 2% FCS. At this point, we added the conditioned medium from quartz dust exposed
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human macrophages to the cultures. In order to differentiate between active and total TGF-b, we tested each sample under two different conditions. First, the conditioned medium was left without any treatment to measure free TGF-b. Secondly, the conditioned medium was treated with 1 N HCl for 10 min at room temperature to release the latent TGF-b out of the binding complex. This samples were then neutralised with 1 N NaOH and added to the Mv1Lu (NBL-7)-cultures. The cells were counted in a Coulter counter after 72 h.
2.7. Neutralisation-experiments with specific antibodies against IGF-1 and TGF-b Conditioned medium from quartz dust exposed human macrophages was incubated at 37°C with a monoclonal antibody against IGF-1 (Biomol, Germany) for 2 h before the mixture was added to cell cultures. The neutralisation of TGF-b was performed with a specific antibody against human TGF-b1 (Becton Dickinson).
2.8. Radioimmunoassay for the N-terminal propeptide of collagen type-I (PINP) The N-terminal propeptide of collagen type I (PINP) was measured utilising a specific radioimmunoassay (Orion Diagnostica, Finland). The basic principle of the test is, that an unknown amount of PINP in the conditioned medium of fibroblasts is mixed with a standard amount of radiolabelled PINP. After the addition of a highly specific antibody against human PINP, the unlabelled PINP from the conditioned medium and the radiolabelled PINP are allowed to compete for the limited binding sites of the antibody. The amount of bound radiolabeled antigen is reversely proportional to the amount of the unlabelled antigen in the reaction mixture. After separating the free antigen from the antigen – antibody complex the remaining radioactivity is counted in a g-counter. In brief, 50 ml conditioned medium was mixed with 200 ml of radiolabelled PINP and 200 ml of PINP antibody. After 2 h at 37°C a separationreagent was pipetted to each sample and mixed.
After a short incubation period at room temperature the samples were centrifuged. The supernatant was discarded and radioactivity of the sedimented antigen–antibody complex was measured in a g-counter.
2.9. Anion exchange chromatography of conditioned medium from quartz dust exposed human macrophages Conditioned medium from quartz dust exposed human macrophages was concentrated 100-fold utilising ultrafiltration-devices (Vivascience, England) with a molecular weight separation limit of 30 kD. The concentrated fraction larger than 30 kD was equilibrated with running buffer (20 mM Tris–HCl, pH 8,5) before applying to the anion exchange column (Q-Sepharose 16/10 HiLoadColumn, Pharmacia, Germany). The concentrated conditioned medium was fractionated with a linear NaCl-gradient from 20 mM Tris–HCl pH 8.5 to 20 mM Tris–HCl pH 8.5 1 M NaCl. Fractions of 5 ml (flow rate: 2.5 ml/min) were collected and frozen at − 20°C until further analysis.
2.10. Determination of IGF-binding proteins utilising ligand blotting We used the human IGF-1 Western ligand blotting kit (Schu¨tzdeller, Germany) to analyse fractions from anion-exchange chromatography experiments for the presence of binding proteins for IGF. The fractions were concentrated 5-fold and the proteins were separated under non-denaturing conditions in a 7.5% SDS-polyacrylamid gel (Laemmli, 1970). The gel was equilibrated in Towbins transfer-buffer (Towbin et al., 1979; Towbin and Gordon, 1984) for 10 min before the proteins were blotted onto a PVDF-membrane (NEN, Germany). After a quenching and blocking reaction, the membrane was incubated overnight with biotinylated IGF-1, which binds to the binding sites of the IGF-binding proteins. After incubation with a streptavidin–peroxidase complex, the blots were developed with peroxidase-substrate. The blots were scanned and stored at − 20°C.
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Fig. 1. Stimulation of cell proliferation of human tracheobronchial epithelial cells (line BEAS-2B) by incubation with a supernatant (SN) from quartz dust exposed human macrophages and inhibition of this mitogenic effect by coincubation with a antibody against human insulin-like growth factor-1 (anti-IGF).
3. Results
3.1. Synthesis and release of insulin-like growth factor-1 (IGF-1) by quartz dust exposed human macrophages Human macrophages, which have phagocytised quartz dust DQ-12, secrete a cytokine into the serum-free culture medium, which stimulates quiescent human fibroblasts (Seemayer and Braumann, 1985; Seemayer et al., 1987; Hu¨bner and Seemayer, 1989; Olbru¨ck et al., 1997; Seemayer et al., 1997), human pneumocyte type II-like cells (line A-549) (Griwatz et al., 1993, 1995; Olbru¨ck et al., 1998; Seemayer et al., 1997) and human tracheobronchial epithelial cells (line BEAS-2B) (Olbru¨ck et al., 1997, 1998) to increased cell proliferation. Recent experiments utilising ultrafiltration to determine the molecular mass of the proliferation factor from conditioned medium from quartz dust exposed human macrophages revealed that the molecular mass of this cytokine is \30 and 50 kD (Olbru¨ck et al., 1998), respectively. The ultrafiltration fraction \50 kD stimulated the MCF-7 indicator cell line for IGF-1 and
IGF-2 to cell proliferation. This stimulation could be reduced to 50% with a specific antibody against human IGF-1 (Olbru¨ck et al., 1997). IGF1 is a polypeptide of 70 amino-acid residues with a molecular weight of 7.5 kD. The high molecular weight of the proliferation factor synthesised by human macrophages after quartz phagocytosis in comparison to the low molecular weight of IGF-1 could be explained by the involvement of monospecific binding proteins, which have been described for IGF in human serum (Baxter, 1988; Clemmons, 1992, 1993; Binoux, 1995; Rosenfeld and Gargosky, 1996) The binding proteins act as a storage site for this growth factor, extending the half-time in serum from 8–12 min to 2–4 h (Rosenfeld and Gargosky, 1996). We were able to show (Fig. 1), that stimulation of cell proliferation of human tracheobronchial epithelial cells (line BEAS-2B) incubated with conditioned medium of quartz dust exposed human macrophages could be reduced dose-dependently with a specific antibody against IGF-1. Furthermore, we investigated in our test system the possible association with a specific binding protein of the IGF-1 secreted by human macrophages utilising Western ligand
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blotting. As our results demonstrate we were able to detect an IGF-binding protein of : 48 kD (Fig. 2).
3.2. Synthesis and release of transforming growth factor-b by quartz dust exposed human macrophages We tested the conditioned medium from quartz dust exposed human macrophages for the presence of TGF-b with the highly sensitive bioassay utilising Mv1Lu (NBL7)-cells (Ikeda et al., 1987; Mitchen et al., 1995). We could demonstrate that conditioned medium from human macrophages contained transforming growth factor-b after quartz dust phagocytosis (Fig. 3). Approximately 24 pg/ml active TGF-b was detected. Nearly the same amount was released by acidification of the conditioned medium, representing the total amount of latent and active TGF-b.
Collagen synthesis of human lung fibroblasts (line Wi38) was stimulated by addition of conditioned medium from quartz dust exposed human macrophages to the fibroblast cultures (Fig. 4). The collagen synthesis—measured as the release of the N-terminal propeptide of collagen type I (PINP)—was rising from 103.64 to 110.12 mg/ 100000 cells (n= 4, P= 0.08). Incubation of the conditioned medium with an antibody against human IGF-1 stimulated the collagen-biosynthesis to 127.04 mg/100000 cells. Results indicate that IGF-1 has no direct stimulating effect on collagen biosynthesis. We were able to show that the PINP-release after incubation of the conditioned medium with an antibody against human TGFb1 was declining from 110.12 mg/100000 cells nearly to control levels (105.19 mg/100000 cells, n= 4, P= 0,06). Coincubation of the conditioned medium with both antibodies revealed, that it was possible to reduce the elevated PINP release observed by addition of the anti-IGF-antibody with the TGF-b-antibody from 127.04 to 115.33 mg/ 100000 cells (n= 4, P= 0.02).
4. Discussion
Fig. 2. SDS-PAGE (7.5%) of active fractions obtained after anion-exchange chromatographie (Q-Sepharose) of concentrated cell culture supernatant from quartz dust exposed human macrophages. On the right side you see the results of a insulin-like growth factor-1 (IGF-1) Western ligand blotting. One band of : 48 kD binds the biotinylated IGF-1.
Proliferation of lung cells is controlled by complex interactions between endocrine and autocrine/paracrine growth factors. Somatomedins (insulin-like growth factor-1, insulin-like growth factor-2) may act as autocrine and paracrine regulators of lung cell proliferation. It is assumed that the activity of IGF is regulated by monospecific IGF-binding proteins (Baxter, 1988; Clemmons, 1992, 1993; Binoux, 1995; Bonner and Brody, 1995), which bind IGF with a high affinity and serve as a reservoir for this growth factor. Little is known concerning the involvement of IGF-1 in the pathogenesis of silicosis. Vanhee et al. (1995) presented data concerning the synthesis of different cytokines by human alveolar macrophages obtained from bronchioalveolar lavage of control individuals and patients with simple pneumoconioses and progressive massive fibrosis. The release of IGF-1 from explanted human alveolar macrophages was elevated in the individuals suffering from progressive massive fibrosis, in con-
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Fig. 3. Transforming growth factor-beta (TGF-b)-bioassay utilising Mv1Lu (NBL-7) cells of serum-free supernatants (SN) from quartz dust exposed human macrophages (dilution 1:4). We were able to find :24 pg/ml active TGF-b (SN 1:4, pH 7). Nearly the same amount could be activated by acidification (SN 1:4, pH 7-2-7) and represents the amount of total TGF-b ( :48 pg/ml).
trast to levels from patients with simple pneumoconiosis which were similar to control levels. Rom et al. (1988) showed, that human alveolar macrophages are capable to synthesise IGF-1. This is in good agreement with the report from Chen et al. (1994), who demonstrated that alveolar macrophages obtained from rats which were intratracheally injected with quartz dust synthesised IGF, stimulated fibroblasts to cell replication. Seemayer et al. (1987) and Hu¨bner and Seemayer (1989) reported the stimulation of cell replication of human lung fibroblasts (line Wi38) and named the factor responsible for this stimulation ‘fibroblast proliferation factor’. A similar stimulation of cell proliferation was observed by Griwatz et al. (1995) with quiescent human pneumocyte type-II-like cells of the line A-549. The factor responsible for the effect in both cell systems seems to be the same. Arguments are the molecular weight \30 kD and the thermal stability (1 h, 56°C). Recent results (Olbru¨ck et al., 1998) obtained by ultrafiltration of conditioned medium from quartz dust exposed human macrophages clearly demonstrated that the factor synthesised by quartz dust exposed human
macrophages responsible for cell proliferation is \ 50 kD. Furthermore, we were able to show that human macrophages produce the insulin-like growth factor-1 (Olbru¨ck et al., 1997) and that cell proliferation of the human tracheobronchial epithelial cell line BEAS-2B could be inhibited by addition of a specific antibody against human IGF-1. Utilising Western ligand blotting, we could detect, in conditioned medium from quartz dust exposed human macrophages, a specific binding protein of : 48 kD for IGF-1. On binding conditions, this complex has a molecular weight \ 50 kD (IGF-1: 7.5 kD, binding protein. 48 kD). This is in good agreement with the observed stimulation of cell proliferation by the fraction \ 50 kD of conditioned medium from quartz dust exposed human macrophages and the inhibition of cell proliferation by an antibody against human IGF-1 (Olbru¨ck et al., 1997). Results indicate that human macrophages in our test system secrete IGF-1 and a specific IGF-binding protein into the serum-free cell culture medium. As Rosenfeld and Gargosky (1996) reported, nearly 99% of the IGF-1 is associated with binding proteins.
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Fig. 4. Measurement of the release of N-terminal propeptide of collagen type-I (PINP) by human lung fibroblasts (line Wi38) after incubation with a 1:4 diluted supernatant (SN) from quartz dust exposed human macrophages alone or with antibodies against human insulin-like growth factor-1 (IGF-1) and/or transforming growth factor-beta (TGF-b).
Silicosis is characterised by fibroblast proliferation and an accelerated collagen content of the lung (Davis, 1986). One cytokine which has the capability to induce collagen synthesis of human lung fibroblasts is transforming growth factor-b (Ignotz and Massague, 1987; Roberts and Sporn, 1988; Kovacs, 1991; Maquart et al., 1994; Kovacs and DiPietro, 1994; Vanhee et al., 1995). TGF-b is also synthesised by macrophages (Khalil et al., 1989; Kelley, 1990; Broeckelmann et al., 1991). We could demonstrate that conditioned medium from quartz dust exposed human macrophages contained TGF-b. Furthermore, we could show that human lung fibroblasts incubated with the conditioned medium secreted higher amounts of the N-terminal propeptide of collagen type I (PINP) into the culture medium, typical for collagen biosynthesis. One factor suspected for this augmented collagen synthesis is TGF-b. We could clearly demonstrate that the PINP-release of fibroblasts is reduced to control levels by addition of a TGF-b-antibody to the conditioned medium from quartz dust exposed human
macrophages. This is in good agreement with results obtained by different authors, who demonstrated a higher collagen synthesis to addition of TGF-b (Ignotz and Massague, 1987; Varga et al., 1987; Reed et al., 1994). Furthermore, we could show that collagen synthesis (measured as release of PINP) is rising by co-incubating the cells with conditioned medium from quartz dust exposed human macrophages in the presence of an antibody against human IGF-1. Green and Goldberg (1963) showed that cell replication and collagen synthesis are two processes, which cannot run parallel, but successively. By inhibition of the IGF-1-mediated cell proliferation in presence of a specific antibody there are more resting cells, which are able to synthesise collagen. In conclusion, human macrophages incubated with quartz dust synthesise different cytokines among there is insulin-like growth factor-1 and transforming growth factor-b. IGF-1 seems to be responsible for cell proliferation, whereas TGF-b stimulates collagen synthesis of human lung fibroblasts.
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TGF-b activity found in prostate cell conditioned medium. In vitro Cell. Dev. Biol.-Anim. 31, 692–697. Olbru¨ck, H., Maciuleviciute, L., Seemayer, N.H., 1998. The effect of supernatants from quartz dust treated human macrophages on cell proliferation and collagen synthesis of human diploid lung fibroblasts in vitro. In: Mohr, U., Dungworth, D.L., Brain, J.D., Driscoll, K.E., Grafstro¨m, R.C., Harris, C.C. (Eds.), Relationships between Respiratory Disease and Exposure to Air Pollution, ILSI Press, pp. 389 –393. Olbru¨ck, H., Maciuleviciute, L., Seemayer, N.H., 1997. Stimulierung der Zellproliferation von menschlichen Lungenfibroblasten, Pneumozyten Typ-II- und Tracheobronchialzellen durch U8 bersta¨nde Quarzstaub-exponierter humaner Makrophagen. Atemw. Lungenkrankh. 23, 367 – 369. Phillips, C.L., Tajima, S., Pinnell, S.R., 1992. Ascorbic acid and transforming growth factor-b1 increase collagen biosynthesis via different mechanisms: Coordinate regulation of Proa1(I) and Proa1(III) collagens. Arch. Biochem. Biophys. 295, 397–403. Reddel, R.R., Ke, Y., Gerwin, B.I., McMenamin, M.G., Lechner, J.F., Su, R.T., Brash, D.E., Park, J.-B., Rhim, J.U.S., Harris, C.C., 1988. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48, 1904 – 1909. Reed, M.J., Vernon, R.B., Abrass, I.B., Sage, E.H., 1994. TGF-b1 induces the expression of type-1 collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors. J. Cell. Physiol. 158, 169 – 179. Richards, R.J., Masek, L.C., Brown, R.F.R., 1991. Biochemical and cellular mechanisms of pulmonary fibrosis. Toxicol. Pathol. 19, 526–539. Roberts, A.B., Sporn, M.B., 1988. Transforming growth factor-b. Adv. Cancer Res. 51, 107–145. Robock, K., 1973. Standard quartz DQ12 B 5 mm for experimental pneumoconiosis research projects in the Federal Republic of Germany. Ann. Occup. Hyg. 16, 63– 66. Rom, W.N., Basset, P., Fells, G.A., Nukiwa, T., Trapnell, B.C., Crystal, R.G., 1988. Alveolar macrophages release an insulin-like growth factor 1-type molecule. J. Clin. Invest. 82, 1685 – 1693. Rosenfeld, R.G., Gargosky, S.E., 1996. Assays for insulinlike growth factors and their binding proteins: Practicalities and pitfalls. J. Pediatr. 128, 52–57. Seemayer, N.H., Braumann, A., 1985. Investigations on cytotoxic effects of quartz DQ12 and coal mine dusts on human macrophages in vitro. Silicosis Rep. North-Rhine Westfalia 15, 301 – 320. Seemayer, N.H., Maly, E.R., 1990. Release of a fibroblast proliferation factor from human macrophages in vitro treated with quartz dust DQ12 or coal mine dust. In: Proceedings of the 7th International Pneumoconioses
Conference. DHHS (NIOSH) publ no 90-108 part II. US Department of Health and Human Services, pp. 926 – 929. Seemayer, N.H., Braumann, A., Maly, E., 1987. Development of an in vitro test system with human macrophages and fibroblasts for analysis of the effect of quartz dusts and coal mine dusts. I. Formation of a fibroblast proliferation factor. Silicosis Rep. North-Rine Westfalia 16, 143 – 155. Seemayer, N.H., Behrendt, H., Maly, E., Hu¨bner, K., 1988. The role of quartz and coal mine dust induced mediators from human macrophages in pathogenesis of silicosis. J. Aerosol Sci. 19, 1129 – 1132. Seemayer, N.H., Olbru¨ck, H., Griwatz, U., 1997. Biological and biochemical characterisation of a ‘proliferation factor’ from quartz dust-treated human macrophages. Ann. Occup. Hyg. 41 (Suppl. 1), 426 – 433. Seemayer, N.H., 1989. The role of a ‘fibroblast proliferation factor’ in the pathogenesis of anthracosilicosis II. Biological characterization of a ‘fibroblast proliferation factor’ formed by human macrophage cultures following exposure to quartz dust and coal mine dust. Silicosis Rep. North Rhine Westfalia 17, 199 – 208. Soule, H.D., Vazquez, J., Long, A., Albert, S., Brennan, M., 1973. A human cell line from pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51, 1409 – 1413. Thiel, U., Jung, B., Seemayer, N.H., Idel, H., 1992. The role of mediators in the pathogenesis of silicosis. I. Stimulation of cell proliferation of human pneumocytes type II (line A-549) induced by supernatants of quartz dust exposed human macrophages. Pathobiology 60 (S1), 34. Thiel, U., Seemayer, N.H., Idel, H., 1993. The role of cytokines in the pathogenesis of pneumoconiosis. In: Hurych, J., Lesage, m., David A. (Eds.), Proceedings of the VIIIth International Conference on Occupational Lung Diseases. International Labour Office (ILO) Genf pp. 1148 – 1154. Thiel-Griwatz, U., Jung, B., Seemayer, N.H., Idel, H., 1993. Neue Erkenntnisse u¨ber die Bedeutung von Zytokinen in der Pathogenese der Silikose. I. Die Wirkung von Zytokinen in Kulturu¨bersta¨nden quarzstaubexponierter Makrophagen auf Pneumozyten Typ-II-Zellen. Atemw. Lungenkrankh. 19, 396 – 397. Towbin, H., Gordon, J., 1984. Immunoblotting and dot immunoblotting: Current status and outlook. J. Immunol. Methods 72, 313 – 340. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Van Zoelen, E.J.J., Delaey, B., van der Burg, B., Huyleboeck, D., 1993. Detection of polypeptide growth factors: application of specific bio-assays and PCR technology. In: McKay und, I., Leigh, I. (Eds.), Growth
H. Olbru¨ck et al. / Toxicology Letters 96,97 (1998) 85–95 Factors: A Practical Approach. Oxford University Press, Oxford, pp. 13 – 34. Van Zoelen, E.J.J., 1990. The use of biological assays for detection of polypeptide growth factors. Prog. Growth Factor Res. 2, 131 – 152. Vanhee, D., Gosset, P., Wallaert, B., Tonnel, A.B., 1994. Role of macrophage-derived cytokines in coal-workers pneumoconiosis. Ann. New York Acad. Sci. 725, 183– 192. Vanhee, D., Gosset, P., Boitelle, A., Wallaert, B., Tonnel, A.B, 1995. Cytokine and cytokine network in silicosis
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and coal workers pneumoconiosis. Eur. Respir. J. 8, 1 – 9. Varga, J., Rosenbloom, J., Jimenez, S.A., 1987. Transforming growth factor beta (TGF-b) causes a persistent increase in steady-state amounts of type I and III collagen and fibronectin mRNAs in normal human and dermal fibroblasts. Biochem. J. 247, 597 – 604. Vignola, A.M., Chanez, P., Chippara, G., Merendino, A., Zinnanti, E., Bousquet, J., Bellia, V., Bonsignore, G., 1996. Release of transforming growth factor-beta (TGFb) and fibronectin by alveolar macrophages in airway diseases. Clin. Exp. Immunol. 106, 114 – 119.
Supernatants from quartz dust treated human macrophages stimulate cell proliferation of different human lung cells as well as collagen-synthesis of human diploid lung fibroblasts in vitro H. Olbru¨ck a,b,*, N.H. Seemayer a, B. Voss c, M. Wilhelm b,c b
a Medical Institute of En6ironmental Hygiene at the Uni6ersity Du¨sseldorf, Auf’m Hennekamp 50, D-40225 Du¨sseldorf, Germany Institute of Hygiene, Social- and En6ironmental Medicine, Uni6ersity Bochum, Uni6ersita¨tsstrasse 150, D-44780 Bochum, Germany c Professional Associations Research Institute for Occupational Medicine (BGFA), Bu¨rkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany
Abstract Silicosis is a chronic lung disease, which is caused by inhalation of silica-containing dusts, leading to pulmonary fibrosis. Alveolar macrophages play a key-role in defence against these particles entering the lung. As a result of phagocytosis, the macrophages release mediators, which are involved in various processes of inflammation and immunological defence mechanisms. We established an in-vitro test system composed of human macrophages, human pneumocyte type II cells (line A-549), human diploid lung fibroblasts (line Wi38) and human tracheobronchial epithelial cells (line BEAS-2B). With this model, we were able to study the influence of various cytokines, produced by the macrophages, on cell proliferation and collagen synthesis (only fibroblasts) of the cells in our test-system. In this report, we will summarize data obtained from our in-vitro test system on two cytokines, which are thought to be important in pathogenesis of lung fibrosis: insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-b). © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Macrophages; Collagen synthesis; Fibroblasts; Cytokines
1. Introduction The long-term inhalation of dust, which contains crystalline silica can cause irreversible lung damage and silicosis. Silicosis is characterised by an increase in lung collagen content (Davis, 1986; * Corresponding author.
Crouch, 1990; Richards et al., 1991; Kane, 1995). This fibrotic reaction destroys the lung parenchyma and impairs lung function (Kane, 1995). In the absence of inhibitory signals, the continued production of cytokines promotes connective tissue synthesis which results in alterations of lung tissue. Heppelston and Styles, (1967) were able to demonstrate, that peritoneal macrophages release
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after quartz dust phagocytosis mediators, which stimulate chicken fibroblasts to an increased collagen synthesis. The lung macrophages are the primary target cell for the inhaled dust. They phagocytise the quartz dust and are able to release beside reactive oxygen species mediators, which can influence the proliferation of adjacent cells as well as the collagen synthesis of human lung fibroblasts. Bittermann et al. (1982) reported that macrophages produce after incubation with different particulates a factor, which was named ‘alveolar-macrophage-derived-growth-factor’ (AMDGF). Later this factor was identified as insulin-like growth factor-1 (IGF-1) (Rom et al., 1988). IGF-1 has a molecular weight of 7.5 kD and is bound under normal conditions in 99% to monospecific binding proteins called the IGFbinding proteins (Rosenfeld and Gargosky, 1996). These proteins bind to IGF-1 and IGF-2 with a high affinity and serve as carriers of IGFmolecules, prolonging the average mean-time of this growth factor in human serum from unbound 8 – 12 min to 2–4 h of bound (Rosenfeld and Gargosky, 1996). Recent results obtained from our test system show that human macrophages release after quartz dust phagocytosis a cytokine, which stimulates quiescent human fibroblasts (Seemayer and Braumann, 1985; Seemayer et al., 1987, 1988; Hu¨bner and Seemayer, 1989; Seemayer, 1989; Seemayer and Maly, 1990; Olbru¨ck et al., 1998; Seemayer et al., 1997), as well as pneumocyte type II-like cells (line A-549) (Thiel et al., 1992; Griwatz et al., 1993; Thiel et al., 1993; Thiel-Griwatz et al., 1993; Griwatz et al., 1994, 1995; Olbru¨ck et al., 1998; Seemayer et al., 1997) and human tracheobronchial epithelial cells (line BEAS-2B) (Olbru¨ck et al., 1997, 1998) under serum-free conditions to enhanced cell proliferation. We could show, that quartz dust exposed human macrophages synthesise IGF-1 and a specific binding protein. Furthermore, the stimulation of cell proliferation of human tracheobronchial epithelial cells was markedly reduced by coincubation with an antibody against human IGF-1. In contrast to the stimulation of cell proliferation by IGF-1, the incubation of lung fibroblasts (line Wi38) and pneumocyte-type-II-like cells (line A-549) with transforming growth factor-beta
(TGF-b) did not increase cell proliferation (Olbru¨ck, unpublished results). TGF-b is synthesised by human macrophages (Khalil et al., 1989; Kelley, 1990; Kovacs, 1991; Vanhee et al., 1994, 1995; Vignola et al., 1996). There are two forms of TGF-b, a latent and an active form. Latent TGFb is bound to a protein complex and therefore inactive (Roberts and Sporn, 1988; Lyons and Moses, 1990). By proteolytic cleavage (Lyons and Moses, 1990; Lyons et al., 1990) or acidification (Ikeda et al., 1987; Mitchen et al., 1995) latent TGF-b can be activated. Active TGF-b can induce collagen synthesis of human fibroblasts (Roberts and Sporn, 1988; Khalil et al., 1989; Fine et al., 1990; Kelley, 1990; Kovacs, 1991, Phillips et al., 1992; Border and Noble, 1994; Butt et al., 1995; Vanhee et al., 1995; Jagirdar et al., 1996). We tested, if human macrophages exposed to quartz dust synthesise TGF-b and if collagen synthesis of human lung fibroblasts is enhanced by incubation with conditioned medium from quartz dust exposed human macrophages in our test-system.
2. Materials and methods
2.1. Quartz dust We used Do¨rentruper quartz dust grinding no. 12 (DMT-Essen, Germany) with a diameter B5 mm as standard quartz dust (Robock, 1973). The quartz dust was resuspended in a concentration of 0.5 mg/ml in Hanks balanced salt solution (HBSS, Biochrom, Germany) and sonified (Sonifier B-12, Branson Sonic Power Company, USA) (Griwatz and Seemayer, 1995).
2.2. Culti6ation of human monocytes/macrophages The isolation of human monocytes from enriched peripheral blood samples and the maturation to human macrophages has been described in detail elsewhere (Seemayer and Braumann, 1985; Seemayer et al., 1987). In brief, human monocytes separated in a Ficoll-Hypaque-gradient (Biochrom) were cultivated in tissue culture flasks. After 10–12 days, the cells revealed characteristics
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of macrophages and were refed with serum-free medium. At the same time, the macrophages were incubated with quartz dust at non-toxic concentrations (e.g. 30 mg/ml). The conditioned medium was collected after 24 and 48 h and frozen at − 20°C until further analysis.
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medium but DMEM supplemented with 0.15% BSA. At the same time, the test samples were added to the cultures. After 6 days we counted the cells in the Coulter counter.
2.5. Bioassay for insulin-like growth factor-1 (IGF-1)
2.3. Culture of human lung fibroblasts (line Wi38) The cell line Wi38 was obtained from Serva, Germany and cultured in Dulbeccos Modified Eagle Medium (DMEM, Boehringer Mannheim, Germany) supplemented with 10% fetal calf serum (FCS, Seromed, Germany). The cells were subcultured every 7 days in a ratio of 1:2. For measurement of collagen synthesis, the cells were seeded at a density of 80000 cells into the wells of a 24-well plate (Becton-Dickinson, Germany) in DMEM containing 10% FCS. After the cells reached confluency (48 h), the cells were washed twice with PBS (Sigma, Germany) and refed with DMEM without serum, but 0.15% (w/v) bovine serum albumin (BSA, Boehringer Mannheim) and 50 mg/ml ascorbic acid. Then the conditioned medium from quartz-exposed human macrophages was added. After a cultivation period of 7 days, the conditioned medium from the fibroblast cultures was collected and frozen until collagen measurement. The cell number was determined by counting the cells in a Coulter counter (Model Zb, Coulter Electronics, Germany).
2.4. Culture of human tracheobronchial epithelial cells (line BEAS-2B) The cell line BEAS-2B (Reddel et al., 1988) was a kindly gift from Professor C.C. Harris (National Cancer Institute, Bethesda, MD). The cells were cultivated in serum-free medium HC3 (Hornberg et al., 1993). This medium contained equal parts of HAM F12 (Biochrom), CMRL 1066 (Biochrom), growth factors and conditioned medium of NIH-3T3 cells. The cells were subcultured every 3–4 days. For cell proliferation assay, 3000 cells were inoculated into wells of a 24-well plate in HC3-medium. After 24 h, the cells were washed twice with PBS and then refed with HC0medium, containing no conditioned NIH-3T3-
We used the human mammacarcinoma cell-line MCF-7 (Soule et al., 1973) for the IGF-1-bioassay (Van Zoelen, 1990; Van Zoelen et al., 1993). This cell-line is cultured in DMEM containing 5% FCS. 5000 logarithmically growing cells were inoculated into wells of a 24-well plate. After 24 h the medium was changed to serum-free DMEM containing 0.15% bovine serum albumin (BSA). After the medium-change, the cells were not longer able to proliferate and were arrested in G0/G1-part of the cell cycle (Van Zoelen et al., 1993). By addition of insulin-like growth factor-1 (IGF-1) or insulin-like growth factor-2 (IGF-2) the cells are restimulated to cell division. Other cytokines like epidermal growth factor (EGF), transforming growth factor-beta (TGF-b) or platelet-derived growth factor (PDGF) are not stimulating cell division of this cell line (Van Zoelen et al., 1993). After a culture period of 4 days with conditioned medium from quartz dust exposed macrophages, the cell number was determined by counting the cells in a Coulter counter.
2.6. Bioassay for transforming growth factor-beta (TGF-b) TGF-b was measured utilising the highly specific bioassay with mink lung cells of the line Mv1Lu (NBL-7) (Ikeda et al., 1987; Mitchen et al., 1995) obtained from the European Collection of Cell Cultures (ECACC, UK). Cell proliferation of this cell line is inhibited in serum-containing medium dose-dependently to TGF-b. The cells were cultured routinely in DMEM containing 10% FCS. For the TGF-b bioassay, 2500 cells were inoculated into wells of a 24-well plate in 10% DMEM. After 24h, the cells were washed two times with PBS and then refed with DMEM containing 2% FCS. At this point, we added the conditioned medium from quartz dust exposed
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human macrophages to the cultures. In order to differentiate between active and total TGF-b, we tested each sample under two different conditions. First, the conditioned medium was left without any treatment to measure free TGF-b. Secondly, the conditioned medium was treated with 1 N HCl for 10 min at room temperature to release the latent TGF-b out of the binding complex. This samples were then neutralised with 1 N NaOH and added to the Mv1Lu (NBL-7)-cultures. The cells were counted in a Coulter counter after 72 h.
2.7. Neutralisation-experiments with specific antibodies against IGF-1 and TGF-b Conditioned medium from quartz dust exposed human macrophages was incubated at 37°C with a monoclonal antibody against IGF-1 (Biomol, Germany) for 2 h before the mixture was added to cell cultures. The neutralisation of TGF-b was performed with a specific antibody against human TGF-b1 (Becton Dickinson).
2.8. Radioimmunoassay for the N-terminal propeptide of collagen type-I (PINP) The N-terminal propeptide of collagen type I (PINP) was measured utilising a specific radioimmunoassay (Orion Diagnostica, Finland). The basic principle of the test is, that an unknown amount of PINP in the conditioned medium of fibroblasts is mixed with a standard amount of radiolabelled PINP. After the addition of a highly specific antibody against human PINP, the unlabelled PINP from the conditioned medium and the radiolabelled PINP are allowed to compete for the limited binding sites of the antibody. The amount of bound radiolabeled antigen is reversely proportional to the amount of the unlabelled antigen in the reaction mixture. After separating the free antigen from the antigen – antibody complex the remaining radioactivity is counted in a g-counter. In brief, 50 ml conditioned medium was mixed with 200 ml of radiolabelled PINP and 200 ml of PINP antibody. After 2 h at 37°C a separationreagent was pipetted to each sample and mixed.
After a short incubation period at room temperature the samples were centrifuged. The supernatant was discarded and radioactivity of the sedimented antigen–antibody complex was measured in a g-counter.
2.9. Anion exchange chromatography of conditioned medium from quartz dust exposed human macrophages Conditioned medium from quartz dust exposed human macrophages was concentrated 100-fold utilising ultrafiltration-devices (Vivascience, England) with a molecular weight separation limit of 30 kD. The concentrated fraction larger than 30 kD was equilibrated with running buffer (20 mM Tris–HCl, pH 8,5) before applying to the anion exchange column (Q-Sepharose 16/10 HiLoadColumn, Pharmacia, Germany). The concentrated conditioned medium was fractionated with a linear NaCl-gradient from 20 mM Tris–HCl pH 8.5 to 20 mM Tris–HCl pH 8.5 1 M NaCl. Fractions of 5 ml (flow rate: 2.5 ml/min) were collected and frozen at − 20°C until further analysis.
2.10. Determination of IGF-binding proteins utilising ligand blotting We used the human IGF-1 Western ligand blotting kit (Schu¨tzdeller, Germany) to analyse fractions from anion-exchange chromatography experiments for the presence of binding proteins for IGF. The fractions were concentrated 5-fold and the proteins were separated under non-denaturing conditions in a 7.5% SDS-polyacrylamid gel (Laemmli, 1970). The gel was equilibrated in Towbins transfer-buffer (Towbin et al., 1979; Towbin and Gordon, 1984) for 10 min before the proteins were blotted onto a PVDF-membrane (NEN, Germany). After a quenching and blocking reaction, the membrane was incubated overnight with biotinylated IGF-1, which binds to the binding sites of the IGF-binding proteins. After incubation with a streptavidin–peroxidase complex, the blots were developed with peroxidase-substrate. The blots were scanned and stored at − 20°C.
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Fig. 1. Stimulation of cell proliferation of human tracheobronchial epithelial cells (line BEAS-2B) by incubation with a supernatant (SN) from quartz dust exposed human macrophages and inhibition of this mitogenic effect by coincubation with a antibody against human insulin-like growth factor-1 (anti-IGF).
3. Results
3.1. Synthesis and release of insulin-like growth factor-1 (IGF-1) by quartz dust exposed human macrophages Human macrophages, which have phagocytised quartz dust DQ-12, secrete a cytokine into the serum-free culture medium, which stimulates quiescent human fibroblasts (Seemayer and Braumann, 1985; Seemayer et al., 1987; Hu¨bner and Seemayer, 1989; Olbru¨ck et al., 1997; Seemayer et al., 1997), human pneumocyte type II-like cells (line A-549) (Griwatz et al., 1993, 1995; Olbru¨ck et al., 1998; Seemayer et al., 1997) and human tracheobronchial epithelial cells (line BEAS-2B) (Olbru¨ck et al., 1997, 1998) to increased cell proliferation. Recent experiments utilising ultrafiltration to determine the molecular mass of the proliferation factor from conditioned medium from quartz dust exposed human macrophages revealed that the molecular mass of this cytokine is \30 and 50 kD (Olbru¨ck et al., 1998), respectively. The ultrafiltration fraction \50 kD stimulated the MCF-7 indicator cell line for IGF-1 and
IGF-2 to cell proliferation. This stimulation could be reduced to 50% with a specific antibody against human IGF-1 (Olbru¨ck et al., 1997). IGF1 is a polypeptide of 70 amino-acid residues with a molecular weight of 7.5 kD. The high molecular weight of the proliferation factor synthesised by human macrophages after quartz phagocytosis in comparison to the low molecular weight of IGF-1 could be explained by the involvement of monospecific binding proteins, which have been described for IGF in human serum (Baxter, 1988; Clemmons, 1992, 1993; Binoux, 1995; Rosenfeld and Gargosky, 1996) The binding proteins act as a storage site for this growth factor, extending the half-time in serum from 8–12 min to 2–4 h (Rosenfeld and Gargosky, 1996). We were able to show (Fig. 1), that stimulation of cell proliferation of human tracheobronchial epithelial cells (line BEAS-2B) incubated with conditioned medium of quartz dust exposed human macrophages could be reduced dose-dependently with a specific antibody against IGF-1. Furthermore, we investigated in our test system the possible association with a specific binding protein of the IGF-1 secreted by human macrophages utilising Western ligand
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blotting. As our results demonstrate we were able to detect an IGF-binding protein of : 48 kD (Fig. 2).
3.2. Synthesis and release of transforming growth factor-b by quartz dust exposed human macrophages We tested the conditioned medium from quartz dust exposed human macrophages for the presence of TGF-b with the highly sensitive bioassay utilising Mv1Lu (NBL7)-cells (Ikeda et al., 1987; Mitchen et al., 1995). We could demonstrate that conditioned medium from human macrophages contained transforming growth factor-b after quartz dust phagocytosis (Fig. 3). Approximately 24 pg/ml active TGF-b was detected. Nearly the same amount was released by acidification of the conditioned medium, representing the total amount of latent and active TGF-b.
Collagen synthesis of human lung fibroblasts (line Wi38) was stimulated by addition of conditioned medium from quartz dust exposed human macrophages to the fibroblast cultures (Fig. 4). The collagen synthesis—measured as the release of the N-terminal propeptide of collagen type I (PINP)—was rising from 103.64 to 110.12 mg/ 100000 cells (n= 4, P= 0.08). Incubation of the conditioned medium with an antibody against human IGF-1 stimulated the collagen-biosynthesis to 127.04 mg/100000 cells. Results indicate that IGF-1 has no direct stimulating effect on collagen biosynthesis. We were able to show that the PINP-release after incubation of the conditioned medium with an antibody against human TGFb1 was declining from 110.12 mg/100000 cells nearly to control levels (105.19 mg/100000 cells, n= 4, P= 0,06). Coincubation of the conditioned medium with both antibodies revealed, that it was possible to reduce the elevated PINP release observed by addition of the anti-IGF-antibody with the TGF-b-antibody from 127.04 to 115.33 mg/ 100000 cells (n= 4, P= 0.02).
4. Discussion
Fig. 2. SDS-PAGE (7.5%) of active fractions obtained after anion-exchange chromatographie (Q-Sepharose) of concentrated cell culture supernatant from quartz dust exposed human macrophages. On the right side you see the results of a insulin-like growth factor-1 (IGF-1) Western ligand blotting. One band of : 48 kD binds the biotinylated IGF-1.
Proliferation of lung cells is controlled by complex interactions between endocrine and autocrine/paracrine growth factors. Somatomedins (insulin-like growth factor-1, insulin-like growth factor-2) may act as autocrine and paracrine regulators of lung cell proliferation. It is assumed that the activity of IGF is regulated by monospecific IGF-binding proteins (Baxter, 1988; Clemmons, 1992, 1993; Binoux, 1995; Bonner and Brody, 1995), which bind IGF with a high affinity and serve as a reservoir for this growth factor. Little is known concerning the involvement of IGF-1 in the pathogenesis of silicosis. Vanhee et al. (1995) presented data concerning the synthesis of different cytokines by human alveolar macrophages obtained from bronchioalveolar lavage of control individuals and patients with simple pneumoconioses and progressive massive fibrosis. The release of IGF-1 from explanted human alveolar macrophages was elevated in the individuals suffering from progressive massive fibrosis, in con-
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Fig. 3. Transforming growth factor-beta (TGF-b)-bioassay utilising Mv1Lu (NBL-7) cells of serum-free supernatants (SN) from quartz dust exposed human macrophages (dilution 1:4). We were able to find :24 pg/ml active TGF-b (SN 1:4, pH 7). Nearly the same amount could be activated by acidification (SN 1:4, pH 7-2-7) and represents the amount of total TGF-b ( :48 pg/ml).
trast to levels from patients with simple pneumoconiosis which were similar to control levels. Rom et al. (1988) showed, that human alveolar macrophages are capable to synthesise IGF-1. This is in good agreement with the report from Chen et al. (1994), who demonstrated that alveolar macrophages obtained from rats which were intratracheally injected with quartz dust synthesised IGF, stimulated fibroblasts to cell replication. Seemayer et al. (1987) and Hu¨bner and Seemayer (1989) reported the stimulation of cell replication of human lung fibroblasts (line Wi38) and named the factor responsible for this stimulation ‘fibroblast proliferation factor’. A similar stimulation of cell proliferation was observed by Griwatz et al. (1995) with quiescent human pneumocyte type-II-like cells of the line A-549. The factor responsible for the effect in both cell systems seems to be the same. Arguments are the molecular weight \30 kD and the thermal stability (1 h, 56°C). Recent results (Olbru¨ck et al., 1998) obtained by ultrafiltration of conditioned medium from quartz dust exposed human macrophages clearly demonstrated that the factor synthesised by quartz dust exposed human
macrophages responsible for cell proliferation is \ 50 kD. Furthermore, we were able to show that human macrophages produce the insulin-like growth factor-1 (Olbru¨ck et al., 1997) and that cell proliferation of the human tracheobronchial epithelial cell line BEAS-2B could be inhibited by addition of a specific antibody against human IGF-1. Utilising Western ligand blotting, we could detect, in conditioned medium from quartz dust exposed human macrophages, a specific binding protein of : 48 kD for IGF-1. On binding conditions, this complex has a molecular weight \ 50 kD (IGF-1: 7.5 kD, binding protein. 48 kD). This is in good agreement with the observed stimulation of cell proliferation by the fraction \ 50 kD of conditioned medium from quartz dust exposed human macrophages and the inhibition of cell proliferation by an antibody against human IGF-1 (Olbru¨ck et al., 1997). Results indicate that human macrophages in our test system secrete IGF-1 and a specific IGF-binding protein into the serum-free cell culture medium. As Rosenfeld and Gargosky (1996) reported, nearly 99% of the IGF-1 is associated with binding proteins.
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Fig. 4. Measurement of the release of N-terminal propeptide of collagen type-I (PINP) by human lung fibroblasts (line Wi38) after incubation with a 1:4 diluted supernatant (SN) from quartz dust exposed human macrophages alone or with antibodies against human insulin-like growth factor-1 (IGF-1) and/or transforming growth factor-beta (TGF-b).
Silicosis is characterised by fibroblast proliferation and an accelerated collagen content of the lung (Davis, 1986). One cytokine which has the capability to induce collagen synthesis of human lung fibroblasts is transforming growth factor-b (Ignotz and Massague, 1987; Roberts and Sporn, 1988; Kovacs, 1991; Maquart et al., 1994; Kovacs and DiPietro, 1994; Vanhee et al., 1995). TGF-b is also synthesised by macrophages (Khalil et al., 1989; Kelley, 1990; Broeckelmann et al., 1991). We could demonstrate that conditioned medium from quartz dust exposed human macrophages contained TGF-b. Furthermore, we could show that human lung fibroblasts incubated with the conditioned medium secreted higher amounts of the N-terminal propeptide of collagen type I (PINP) into the culture medium, typical for collagen biosynthesis. One factor suspected for this augmented collagen synthesis is TGF-b. We could clearly demonstrate that the PINP-release of fibroblasts is reduced to control levels by addition of a TGF-b-antibody to the conditioned medium from quartz dust exposed human
macrophages. This is in good agreement with results obtained by different authors, who demonstrated a higher collagen synthesis to addition of TGF-b (Ignotz and Massague, 1987; Varga et al., 1987; Reed et al., 1994). Furthermore, we could show that collagen synthesis (measured as release of PINP) is rising by co-incubating the cells with conditioned medium from quartz dust exposed human macrophages in the presence of an antibody against human IGF-1. Green and Goldberg (1963) showed that cell replication and collagen synthesis are two processes, which cannot run parallel, but successively. By inhibition of the IGF-1-mediated cell proliferation in presence of a specific antibody there are more resting cells, which are able to synthesise collagen. In conclusion, human macrophages incubated with quartz dust synthesise different cytokines among there is insulin-like growth factor-1 and transforming growth factor-b. IGF-1 seems to be responsible for cell proliferation, whereas TGF-b stimulates collagen synthesis of human lung fibroblasts.
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