Bleomycin stimulates lung fibroblast and epithelial cell lines to
Bleomycin stimulates lung fibroblast and epithelial cell lines to
Copyright #ERS Journals Ltd 2000 European Respiratory Journal ISSN 0903-1936 Eur Respir J 2000; 16: 951±958 Printed in UK ± all rights reserved Bleomycin stimulates lung fibroblast and epithelial cell lines to release eosinophil chemotactic activity E. Sato*, S. Koyama**, R.A. Robbins* Bleomycin stimulates lung fibroblast and epithelial cell lines to release eosinophil chemotactic activity. E. Sato, S. Koyama, R.A. Robbins. #ERS Journals Ltd 2000. ABSTRACT: The presence of eosinophils in the lungs of patients with pulmonary fibrosis correlates with poor prognosis or resistance to therapy. Furthermore, eosinophils localize to areas undergoing active fibrosis. It was hypothesized that a human lung fibroblast (HFL-1) and a human lung epithelial cell line (BEAS-2B) might release eosinophil chemotactic activity (ECA) in response to bleomycin, a chemotherapeutic agent associated with pulmonary fibrosis. HFL-1 and BEAS-2B cells were cultured in the presence of bleomycin and their supernatant fluids evaluated for ECA by means of a Boyden chamber method. HFL-1 and BEAS-2B cells released ECA in a dose- and time-dependent manner in response to bleomycin, and partial characterization revealed that the ECA was heterogeneous. ECA release from HFL-1 and BEAS-2B cells was significantly reduced by a leukotriene B4 (LTB4) receptor antagonist and an antibody directed against granulocyte-macrophage colony-stimulating factor. HFL-1 cells released LTB4, eotaxin, and GM-CSF constitutively, and BEAS-2B cells released LTB4, eotaxin, regulated on activation, normal T-cell expressed and presumably secreted, and GMCSF constitutively. In both cases, the release of GM-CSF was significantly increased in response to bleomycin. These data suggest that lung fibroblasts and epithelial cells may modulate eosinophil recruitment into the lung in bleomycin-induced pulmonary fibrosis. Eur Respir J 2000; 16: 951±958. Several studies have suggested that eosinophils may play a role in pulmonary fibrosis. An enhanced number of eosinophils is often found in the interstitium and alveolar spaces in lung biopsy specimens obtained from patients with pulmonary fibrosis, including those induced by bleomycin [1±3]. The influx of eosinophils into the pulmonary compartment has also been demonstrated in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis, and a higher number of eosinophils in BALF has been related to a poor response to therapy and shorter survival [4±6]. In animal models of bleomycin-induced pulmonary fibrosis, infiltration by eosinophils precedes and parallels the development of the fibrotic lesion . The concept that eosinophils can contribute to lung injury is not new . Mechanisms of eosinophil influx into the lungs have increasingly emphasized the role of bronchial epithelial cells and lung fibroblasts in releasing a variety of eosinophil chemoattractants, including eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5, regulated on activation, normal T-cell expressed and presumably secreted (RANTES) and leukotriene B4 (LTB4) [9±12]. In vitro studies have demonstrated that migration of eosinophils is induced by these chemoattractants [13, 14], and in vivo studies show the importance of cytokine mobilization in *Research Service, Southern Arizona Veterans Health Care System, and Dept of Medicine, University of Arizona, Tucson, AZ, USA. **First Dept of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan. Correspondence: R.A. Robbins, Research Health Care Group, Tucson Veterans Administration Medical Center, 3601 S. 6th Avenue, Tucson, AZ 85723, USA. Fax: 1 5206291801 Keywords: Chemotaxis eosinophil eotaxin granulocyte-macrophage colonystimulating factor leukotriene B4 regulated on activation, normal T-cell expressed and presumably secreted Received: October 20 1999 Accepted after revision July 20 2000 promoting airway eosinophil recruitment in pulmonary fibrosis . Consistent with the importance of cytokines, the levels of IL-5, GM-CSF and RANTES in BALF and messenger ribonucleic acid expression in lung were increased in idiopathic pulmonary fibrosis patients and bleomycin-induced pulmonary fibrosis animal models [16±19]. Antibodies directed against IL-5 or tumour necrosis factor-a caused a significant reduction in lung eosinophilia and fibrosis in bleomycin-induced pulmonary fibrosis [20, 21]. Furthermore, transfer of a GM-CSF gene with high expression into rat lung induced eosinophilia and fibrosis in rat models . Bleomycin is one of the antitumour antibiotics produced by Streptomyces verticullis . Bleomycin-induced fibrosis limits the usefulness of the drug, and bleomycininduced fibrosis resembling the human disease process has been produced in a variety of animal models [24, 25]. On the basis of these observations, definition of the mediators and cytokines that participate in the recruitment of eosinophils into the lungs in bleomycin-induced pulmonary fibrosis was attempted. The results demonstrate that a human lung fibroblast (HFL-1) and a human lung epithelial cell lines (BEAS-2B) release eosinophil chemotactic activity (ECA), but, of the chemotactic factors assayed, only GM-CSF release was significantly increased by bleomycin. 952 E. SATO ET AL. Materials and methods Culture of human foetal lung fibroblasts and BEAS-2B cells The human foetal lung fibroblast cell line HFL-1 (diploid, passage 14), was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). This cell line was initiated from the lung tissue of a 16±18week-old human foetus . The morphology of HFL-1 cells is fibroblast-like and they retain features of normal lung fibroblasts including collagen and fibronectin production . BEAS-2B, a human bronchial epithelial cell line transformed by the hybrid adenovirus simian vacuolating virus 40, was also purchased from the ATCC . The HFL-1 and BEAS-2B cells were suspended at 1.06106 cells.mL-1 in Ham's F-12 or Dulbecco modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with penicillin (50 mg.mL-1; Gibco), streptomycin (50 mg.mL-1; Gibco) and 10% heat-inactivated foetal bovine serum. Cell suspensions (3 mL) were added to 30-mm-diameter tissue culture dishes (Corning, Corning, NY, USA) and cultured under a 5% CO2 atmosphere at 378C. After 2±3 days in culture, the cells reached confluence and the culture medium was replaced with 2 mL medium supplemented as above and incubated one further day. In order to confirm the results obtained with the HFL-1 and BEAS-2B cell lines, the cell lines CCL-202 and A549 (both purchased from the ATCC), which are a human lung fibroblast cell line and a human lung epithelial cell line respectively, were also studied. These cell lines were grown in DMEM under conditions similar to the HFL-1 cells until confluent. Exposure of lung HFL-1 and BEAS-2B cells to bleomycin Culture medium was removed from cells by washing twice with serum-free medium, and the cells were incubated in the presence and absence of bleomycin. In order to determine the dose- and time-dependent release of ECA, the cultures were incubated at various concentrations of bleomycin (0, 0.01, 0.1, 1.0 and 10 mg.mL-1, 1.5 U=1 mg; Sigma, St Louis, MO, USA) in a humidified 5% CO2 atmosphere for 12, 24, 48 and 72 h at 378C. Cell injury was evaluated by microscopy (cell shape and detachment from tissue culture dish) and trypan blue exclusion. The supernatant fluids were then harvested and stored at -808C until assay. At least six separate HFL-1 and BEAS-2B cell supernatant fluids were harvested for each experimental condition. Measurement of eosinophil chemotactic activity Eosinophils were isolated using a modified method of HANSEL et al.  with magnetic cell separation. Briefly, venous blood anticoagulated with 130 mM trisodium citrate was obtained from normal human volunteers and diluted with phosphate-buffered saline (0.9% saline, pH7.1) in a 1:1 ratio. Diluted blood was overlaid on an isotonic Percoll solution (density 1.082 g.mL-1; Sigma) which was then centrifuged for 30 min at 1,0006g at 48C in a Beckman TJ-6 centrifuge (Beckman Coulter, Fullerton, CA, USA). The supernatant and mononuclear cells at the interface were carefully removed, and red blood cells in the pellet were lysed with two cycles of hypotonic lysis (0.1% KHCO3; 0.83% NH4Cl). Isolated granulocytes were washed twice with piperazine-N, N'-bis (2ethanesulphonic acid (PIPES) buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH7.4) containing 1% defined calf serum (DCS; Hyclone Laboratories, Logan, UT, USA), and an approximately equal volume of anti-CD16 conjugated with magnetic particles (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the cell pellet. After a 60-min incubation on ice, 5 mL PIPES buffer containing 1% DCS were added to the cell/antibody mixture. The resuspended cells were loaded on to the separation column positioned in the magnetic cell separation system (miltenyi) with a strong magnetic field. The cells were eluted three times with 5 mL PIPES buffer containing 1% DCS, according to the manufacturer's instructions. The purity of the eosinophils counted using Randolph's stain was >94%; the viability was >98%. The eosinophils were resuspended in Gey's solution at 2.06106 cells.mL-1 and used for the chemotaxis assay. The chemotaxis assay was performed in a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD, USA) using a modification of a previously described technique . The bottom wells of the chamber were filled with 25 mL fluid containing the chemotactic stimulus or medium in duplicate. A 10-mm-thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA, USA), with a pore size of 5 mm, was placed over the bottom wells. The silicone gasket and upper pieces of the chamber were applied, and 50 mL cell suspension was placed into the upper wells above the filter. The chambers were incubated in humidified air in 5% CO2 for 90 min at 378C. After incubation, the chamber was disassembled and nonmigrated cells were wiped from the filter. The filter were fixed and stained using a modified Wright's stain and mounted on a glass slide. Cells that had completely migrated through the filter were counted using light microscopy in random high-power fields (HPFs, 6400). Partial characterization of eosinophil chemotactic activity Partial characterization of the ECA released from HFL-1 or BEAS-2B cells was performed using supernatant fluids harvested after 72 h at 10 mg.mL-1 bleomycin; the protein or lipid factor in the supernatant fluid was measured. Sensitivity to proteases was tested by incubating the supernatant fluids with trypsin (100 mg.mL-1, Sigma) for 30 min at 378C; this was followed by the addition of a 1.5 M excess of soybean trypsin inhibitor (Sigma) to terminate the proteolytic activity, and then chemotactic activity was evaluated . The lipid solubility was evaluated by mixing the supernatant fluids twice with ethylacetate, decanting the lipid phase after each extraction, evaporating the ethylacetate to dryness and resuspending the extracted material in the medium used for cell culture before the chemotaxis assay. Both extracted materials and the lipid-insoluble phase were evaluated for chemotactic activity. Heat sensitivity was determined by maintaining a supernatant fluid at 988C for 15 min. 953 BLEOMYCIN-INDUCED EOSINOPHIL CHEMOTACTIC ACTIVITY RELEASE LTB4 was measured in the culture supernatant fluids because the released ECA was only partially lipid extractable. The concentrations of LTB4 in HFL-1 or BEAS-2B cell supernatant fluids cultured for 72 h at 10 mg.mL-1 bleomycin were measured using a commercially available enzyme-linked immunosorbant assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The detection limit of this method is 30 pg.mL-1. Measurement of eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage, colony-stimulating factor in the supernatant fluids Several well-characterized protein chemotactic factors were measured in the culture supernatant fluids because the released ECA was partially heat- and protease-sensitive. The concentrations of eotaxin, IL-5, RANTES and GMCSF in HFL-1 or BEAS-2B cell supernatant fluids cultured for 72 h at 10 mg.mL-1 bleomycin were measured using a commercially available ELISA (R&D Systems). The detection limit of these methods was ~15 pg.mL-1. Effects of leukotriene B4 and platelet-activating factor receptor antagonists on eosinophil chemotactic activity LTB4 and platelet-activating factor (PAF) receptor antagonists were used to evaluate the involvement of LTB4 and PAF in the supernatant fluids of cells stimulated with bleomycin, because they were extractable into ethylacetate and LTB4 levels were found to be elevated. An LTB4 receptor antagonist (ONO 4057; ONO Pharmaceutical, Tokyo, Japan) and PAF receptor antagonist (TCV309; Takeda Pharmaceutical, Tokyo, Japan), each at a concentration of 10-5 M, were used to evaluate the involvement of LTB4 and PAF as ECA in the crude supernatant fluids [32, 33]. Analysis In experiments in which multiple measurements were made, differences between groups were tested for significance using the Kruskal-Wallis nonparametric test. In experiments in which a single measurement was made, the differences between groups were tested for significance using the Wilcoxon ranked sum test. In all cases, a p-value of <0.05 was considered significant. Data in figures and tables are presented as meanSEM. Results Release of eosinophil chemotactic activity from lung fibroblast and epithelial cell lines HFL-1 and BEAS-2B cells released ECA in a dosedependent manner in response to bleomycin (fig. 1). The lowest doses of bleomycin to stimulate HFL-1 and BEAS-2B cells were 0.1 and 1 mg.mL-1, respectively. Increasing concentrations of bleomycin up to 10 mg.mL-1 progressively increased the release of ECA. Bleomycin did not cause HFL-1 or BEAS-2B cell injury (no deformity of cell shape, no detachment from tissue culture dish and >95% cell viability by trypan blue exclusion) after 72 h incubation with 10 mg.mL-1 bleomycin. However, bleomycin at 100 mg.mL-1 caused substantial cytotoxicity after 24 h of incubation. a) 35 30 ECA cells·HPF-1 Measurement of leukotriene B4 in the supernatant fluids * * * 25 20 15 10 5 0 Neutralizing antibodies to eotaxin, IL-5, RANTES and GM-CSF (5, 10, 50 and 10 mg.mL-1, respectively, all R&D Systems) were added to the supernatant fluids which were harvested after 72 h at 10 mg.mL-1 bleomycin to inhibit these cytokines and incubated for 30 min at 378C. Then these samples were used for chemotactic assay. As a negative control, nonimmune immunoglobulin G (IgG) was used. These antibodies reduced the ECA of recombinant cytokines (0.02 mg.mL-1 eotaxin, 0.1 ng.mL-1 IL-5, 0.1 mg.mL-1 RANTES, and 0.05 ng.mL-1 GM-CSF (all R&D Systems)) to baseline levels (data not shown). They did not influence the chemotactic response to endotoxin-activated serum and LTB4 (data not shown). b) 25 * * 20 ECA cells·HPF-1 The effects of eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage colony-stimulating factor polyclonal antibodies to eosinophil chemotactic activity 15 10 5 0 0 0.1 0.01 Bleomycin µg·mL-1 1 10 Fig. 1. ± Dose-dependent release of eosinophil chemotactic activity (ECA) from: a) HFL-1; and b) BEAS-2B cells after 72 h incubation with bleomycin (n=8). Data are presented as meanSEM. HPF: high-power field. *: p<0.05 versus supernatant fluids without bleomycin. 954 E. SATO ET AL. a) 35 * ECA cells·HPF-1 30 25 20 * Partial characterization of eosinophil chemotactic activity 15 10 5 0 b) 30 * ECA cells·HPF-1 25 The ECA from HFL-1 or BEAS-2B cells was heterogeneous in character. It was partially sensitive to heat, extracted by ethylacetate and digested by trypsin (p<0.05) (fig. 3). Trypsin and soybean trypsin inhibitor had no effect on the ECA induced by LTB4 (p>0.05). Release of leukotriene B4 from HFL-1 and BEAS-2B cells 20 15 10 5 0 in response to 10 mg.mL-1 bleomycin than when cultured in medium alone (CLL-202: 6.21.1 versus 26.71.5 cells.HPF-1; A549: 143.2 versus 464.5 cells.HPF-1, n=4, p<0.05 for both comparisons). 0 10 20 50 30 40 Incubation time h 60 70 Fig. 2. ± Time course of eosinophil chemotactic activity (ECA) release from: a) HFL-1; and b) BEAS-2B cells in response to 10 mg.mL-1 bleomycin (s ; n=8). * : cultures without bleomycin (n=8). Data are presented as meanSEM. HPF: high-power field. *: p<0.05 versus supernatant fluids without bleomycin. Although HFL-1 and BEAS-2B cells released ECA constitutively, release was further increased in response to bleomycin in a time-dependent manner. The release of ECA was significant after a 48-h exposure to bleomycin for HFL-1 cells and a 72-h exposure for BEAS-2B cells (fig. 2). Bleomycin itself did not show any ECA (data not shown). These results were confirmed in CLL-202 and A549 cells. Both had released significantly more ECA after 48 h a) 35 Measurement of LTB4 level in supernatant fluids by ELISA revealed that HFL-1 and BEAS-2B cells released significant amounts of LTB4 under baseline culture conditions. However, addition of bleomycin at 10 mg.mL-1 for 72 h did not increase LTB4 release significantly from HFL-1 cells (725.164.6 pg.mL-1 in controls versus 821.247.6 pg.mL-1 with bleomycin) or BEAS-2B cells (938.6116.2 pg.mL-1 in controls versus 822.774.2 pg.mL-1 with bleomycin). Release of eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage colony-stimulating factor from HFL-1 and BEAS-2B cells by bleomycin Measurement of chemotactic cytokines by ELISA revealed that incubation with 10 mg.mL-1 bleomycin for 72 h stimulated the release of GM-CSF from HFL-1 and BEAS-2B cells (p<0.05) (table 1). HFL-1 cells released eotaxin and BEAS-2B cells released eotaxin and RANTES under baseline culture conditions; however, addition of bleomycin at 10 mg.mL-1 for 72 h did not increase the release of these factors. b) ECA cells·HPF-1 30 25 * 20 * 15 * * * EA TD * * * LI EA 10 5 0 CSF H LI F12 CSF H TD DMEM Fig. 3. ± Partial characterization of eosinophil chemotactic activity (ECA) obtained from: a) HFL-1; and b) BEAS-2B cells after 72 h incubation with 10 mg.mL-1 bleomycin or medium alone (n=8). HPF: high-power field; CSF: crude supernatant fluid; H: heat-treated supernatant; LI: lipid-insoluble phase; EA: ethylacetate extract; TD: trypsin digest; F12: Ham's F12 medium; DMEM: Dulbecco modified Eagle medium. Data are presented as meanSEM. *: p<0.05 versus CSFs. 955 BLEOMYCIN-INDUCED EOSINOPHIL CHEMOTACTIC ACTIVITY RELEASE Table 1. ± Release of cytokines from HFL-1 and BEAS-2B cells in response to bleomycin HFL-1 Eotaxin pg.mL-1 RANTES pg.mL-1 IL-5 pg.mL-1 GM-CSF pg.mL-1 LTB4 pg.mL-1 BEAS-2B Control Bleomycin Control Bleomycin 691.838.4 ND ND 17.90.3 725.164.6 546.8738.4 ND ND 74.511.2* 821.247.6 25.01.5 62.512.4 ND 19.170.4 938.6116.2 20.221.7 61.413.3 ND 54.610.6* 822.774.2 Data are presented as meanSEM. RANTES: regulated on activation, normal T-cell expressed and presumably secreted; IL-5: interleukin-5; GM-CSF: granulocyte-macrophage colony-stimulating factor; LTB4: leukotriene B4; ND: not determined. *: p<0.05 versus control. Inhibition of eosinophil chemotactic activity release by leukotriene B4 receptor antagonists ECA release from HFL-1 and BEAS-2B cells was significantly inhibited by addition of the LTB4 receptor antagonist, ONO 4057 (p<0.05) (fig. 4). The PAF receptor antagonist, TCV309, did not block ECA release. LTB4 or PAF receptor antagonist at a concentration of 10-5 M inhibited ECA release in response to 10-7 M LTB4 or PAF but showed no inhibitory effects on endotoxin-activated serum-induced eosinophil chemotaxis (data not shown). Inhibition of eosinophil chemotactic activity by polyclonal antibodies to eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage colony-stimulating factor Polyclonal blocking antibodies were used to determine the contribution of identified protein eosinophil chemotactic factors. Among these antibodies, GM-CSF antibodies inhibited ECA release from HFL-1 and BEAS-2B cells (p<0.05) (fig. 5). IL-5 and RANTES antibodies did not show any effect on ECA release. Nonimmune IgG did not have any effect on bleomycin-conditioned medium (data not shown). Discussion The present study demonstrates that bleomycin stimulates HFL-1 and BEAS-2B cells to release ECA in a doseand time-dependent manner. Partial characterization a) 40 revealed the heterogeneity of the ECA. LTB4 and eotaxin were released from HFL-1 cells constitutively. LTB4, eotaxin and RANTES were released from BEAS-2B cells constitutively. However, release of these factors did not increase in response to bleomycin. In contrast, release of GM-CSF from HFL-1 and BEAS-2B cells was increased by bleomycin stimulation. These data suggest that an interaction between fibroblasts or epithelial cells and bleomycin may modulate eosinophil recruitment to the interstitium after bleomycin exposure through the generation of chemotactic cytokines. Eosinophils are known to be a key source of cytokines with inflammatory and fibrosis-promoting activities . Consistent with their potential role in the promotion of fibrosis, eosinophils have been found to localize in areas undergoing active fibrosis, expressing such fibrosispromoting cytokines as transforming growth factor-b1 . Furthermore, the presence of eosinophils in the lungs of patients with idiopathic pulmonary fibrosis correlates with a worse prognosis or resistance to therapy [4±6]. Thus there is compelling evidence to suggest that eosinophils may play an important role in pulmonary fibrosis. Although several types of lung cell can produce chemotactic cytokines, production by airway epithelial cells and fibroblasts may be particularly relevant to pulmonary fibrosis. It is becoming increasingly clear that these cells participate in inflammatory processes by producing a variety of mediators and cytokines, including prostaglandins, PAF, IL-3, IL-5, IL-8, macrophage inflammatory protein 1a, monocyte chemoattractant protein (MCP)-3, MCP-4, RANTES, eotaxin, and GM-CSF b) ECA cells·HPF-1 35 30 25 * 20 * 15 10 5 0 BLEO BLEO + BLEO + ONO 4057 TCV309 F12 BLEO BLEO + BLEO + ONO 4057 TCV309 DMEM Fig. 4. ± Inhibition of eosinophil chemotactic activity (ECA) release from: a) HFL-1; and b) BEAS-2B cells by the leukotriene B4 receptor antagonist, ONO 4057, and the platelet-activating factor receptor antagonist, TCV309 (n=8). As a negative control, cells were incubated with medium alone. Data are presented as meanSEM. *: p<0.05 versus crude supernatant fluids. 956 E. SATO ET AL. a) 40 b) ECA cells·HPF-1 35 30 25 20 * * 15 10 5 0 USF GM-CSF IL-5 USF GM-CSF IL-5 RANTES Eotaxin F12 RANTES Eotaxin DMEM Fig. 5. ± Effects of blocking antibodies on eosinophil chemotactic activity (ECA) from: a) HFL-1; and b) BEAS-2B cells (n=8). All cell cultures were incubated in the presence of 10 mg.mL-1 bleomycin on medium alone prior to the addition of antibodies directed against the indicated cytokines. Data are presented as meanSEM. GM-CSF: granulocyte-macrophage colony-stimulating factor; IL-5: interleukin-5; RANTES: regulated on activation, normal Tcell, expressed and presumably secreted. *: p<0.05 versus untreated supernatant fluids (USFs). [9±12, 35±38]. On the basis of these observations, definition of the mediators involved in bleomycin-induced pulmonary fibrosis was attempted. The cell lines HFL-1 and BEAS-2B were used in the present studies because of the difficulty in obtaining enough samples for experiments from humans. The bleomycin effects were also investigated with another human lung fibroblast cell line (CCL-202, a fibroblast-like cell line derived from normal lung tissue) and A549 cells (type II epithelial cell line derived from an individual with alveolar carcinoma) to confirm the results. Bleomycin stimulated CCL-202 and A549 cells to release ECA. Bleomycin is one of the antitumour antibiotics produced by Streptomyces verticullis, discovered in 1966 by UMEZAWA et al. . It has been used in the treatment of a variety of neoplasms; however, bleomycin-induced pneumonitis or pulmonary fibrosis is sometimes a fatal complication . FUJITA et al.  reported that the concentrations of bleomycin in blood and cancer tissue obtained from humans administrated 15 mg intravenously were 0.8 and 0.4 mg.mL-1. UMEZAWA et al.  reported higher concentrations of bleomycin in the lungs (18 mg. g-1) compared with other organs, which may be related to the lung side-effects of this drug. It is possible that bleomycin concentrations reach these experimental levels around the epithelial cells or fibroblasts in the lung. The results of partial characterization suggest that the ECA released by HFL-1 and BEAS-2B cells is partly composed of lipid. Consistent with these observations, LTB4 concentrations detected in the culture supernatant fluids were in the chemotactic range for eosinophils  and ECA was inhibited by a LTB4 receptor antagonist. Although the release of LTB4 from HFL-1 or BEAS-2B cells in response to bleomycin was not significantly increased compared with controls, LTB4 is one of the chemoattractants for eosinophils released from fibroblasts and epithelial cells constitutively. The results of partial characterization also indicate that the released activities are partially protein in nature. Although eotaxin, RANTES and GM-CSF were detected in the culture supernatant fluids and blocking antibodies decreased ECA, GM-CSF was the only cytokine released in response to bleomycin. These data suggest that GMCSF is an important constituent of the ECA released by HFL-1 and BEAS-2B cells in response to bleomycin. GM-CSF has been shown to activate eosinophil functions including differentiation, chemotaxis, chemotaxis priming, survival, transendothelial migration, expression of CD11b, CD4 and histocompatibility leukocyte antigen DR, and mediator release in vitro [43, 44]. GM-CSF was initially purified from mouse lung conditioned medium, thus indicating that the lung is an important producer of GM-CSF . This cytokine can be produced by a wide spectrum of cell types of the lung, including fibroblasts and epithelial cells, and significantly increased levels are detected in BALF or lung tissues obtained from patients with idiopathic pulmonary fibrosis [18, 19]. Recently XING et al.  reported that overexpression of GM-CSF by means of a gene transfer technique induces eosinophilia and pulmonary fibrotic reactions. GM-CSF induces accumulation of both eosinophils and macrophages in the early stages and irreversible fibrotic reaction later on. In addition, GM-CSF has been found to induce fibrotic responses with accumulation of myofibroblasts after chronic subcutaneous administration of GM-CSF . These findings support the notion of GM-CSF as a fibrogenic cytokine. In conclusion, bleomycin stimulated both HFL-1 and BEAS-2B cells to release eosinophil chemotactic activity. The released eosinophil chemotactic activity was heterogeneous, but granulocyte-macrophage colony-stimulating factor was the only eosinophil chemotactic factor whose concentration was significantly increased by bleomycin stimulation. 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