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The Journal of Immunology, 1999, 162: 6200-6208.
Copyright © 1999 by The American Association of Immunologists

Bleomycin Stimulates Lung Fibroblasts to Release Neutrophil and Monocyte Chemotactic Activity

Akemi Takamizawa*, Sekiya Koyama1,*,{dagger}, Etsuro Sato*, Takeshi Masubuchi*, Keishi Kubo*, Morie Sekiguchi*, Sonoko Nagai{dagger} and Takateru Izumi{dagger}

* First Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan; and {dagger} Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-ß Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-ß in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bleomycin is one of anti-tumor antibiotics produced by Streptomyces verticullis, discovered in 1966 by Umezawa et al. (1). Although it has been used in the treatment of a variety of neoplasma, bleomycin-induced pneumonitis or pulmonary fibrosis sometimes becomes fatal (2). The incidence of bleomycin-induced pneumonitis varies from 3 to 40% (3, 4, 5, 6, 7, 8), and fatalities have been reported to be 1–15% (5, 7, 9, 10) of patients who receive this agent. Bleomycin-induced pneumonitis is dose dependent and involves pulmonary inflammatory responses characterized by increases in mononuclear cells, neutrophils, fibroblast proliferation, and collagen synthesis (11, 12).

Sequestration of peripheral blood neutrophils and monocytes within the lung is characteristic of a number of acute and chronic pulmonary diseases (13, 14, 15, 16, 17). The presence of neutrophils is determined by the local generation of chemotactic agents, which direct neutrophil migration from the vascular compartment to the alveolar space along chemotactic gradients. The alveolar macrophages are also derived predominantly from differentiated peripheral blood monocytes and to a limited extent from local macrophages replication (18, 19, 20). Although elicited neutrophils and macrophages serve a vital role in the host defense against a number of organisms, the presence of increased numbers of activated neutrophils and macrophages can lead to excessive tissue injury via the overzealous elaboration of inflammatory cytokines, proteolytic enzymes, and oxygen radicals (14, 21). Substantial investigation has focused on the alveolar macrophages as a primary source of chemotactic factors (22, 23, 24). However, neutrophil chemotactic activity (NCA)2 and monocyte chemotactic activity (MCA) has been found to be produced by endothelial cells (25), fibroblasts (26), and pulmonary epithelial cells (27, 28, 29).

The fibroblast is the principal cell of most connective tissues and is involved in constituting collagenous and noncollagenous components of the extracellular matrix. This synthetic activity serves an important structural function by providing a frame network for organ integrity. In addition to this traditionally accepted function, recent studies have demonstrated that fibroblasts not only serve to maintain the connective tissue but are important participants in the orchestration of acute and chronic inflammation. In this context, fibroblasts released monocyte chemoattractant protein-1 (MCP-1), GM-CSF, and TGF-ß in response to IL-1, TNF-{alpha}, and platelet-derived growth factor, suggesting the contribution to certain disease states (30, 31, 32, 33, 34, 35, 36). Therefore, the fibroblast, because of its anatomical location, is in a pivotal position to participate in and direct bidirectional communications between interstitial and vascular events.

Although airway epithelial cells and alveolar macrophages may play a role in inflammatory cell migration from the interstitium to the alveolar and bronchial spaces in response to bleomycin, the underlying mechanism of inflammatory cell migration from the vascular compartment to the interstitium remains to be elucidated. The role of human lung fibroblasts (HLFs) in inflammatory cell recruitment from the vascular compartment to the interstitium in response to bleomycin is uncertain. The purpose of the present investigation is to determine whether lung fibroblasts could participate in the recruitment of inflammatory cells into the lungs. Specifically, the possibility of lung fibroblasts to release NCA and MCA in response to bleomycin is evaluated. The results demonstrate that HLF can release NCA and MCA in response to bleomycin, including IL-8, G-CSF, MCP-1, GM-CSF, and TGF-ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of HLF

We used fetal HLF (lung, diploid, human, passage 27), which is an established cell line and commercially available (American Type Tissue Culture Collection, Manassas, VA). HLF were suspended at 1.0 x 106 cells/ml in F-12 supplemented penicillin (50 u/ml; Life Technologies, Grand Island, NY), streptomycin (50 µg/ml; Life Technologies), fungizone (2 µg/ml; Life Technologies), and 10% FCS (Life Technologies). HLF suspensions (3 ml) were added to a 30-mm diameter tissue culture dish (Corning, Corning, NY) and were cultured at 37°C in a 5% CO2 atmosphere. After 4–6 days in culture, the cells had reached confluence, and then the culture medium was replaced with 2 ml of medium supplemented as above and was incubated 1 day further.

Exposure of HLF to bleomycin

Medium was removed from cells by washing twice with serum-free F-12, and cells were incubated in the presence and absence of bleomycin. To determine the dose- and time-dependent release of NCA and MCA, the cultures were incubated at various concentrations of bleomycin (0, 0.01, 0.1, 1.0, and 10 µg/ml; Sigma, St. Louis, MO) for 12, 24, 48, 72, and 96 h at 37°C in a humidified 5% CO2 atmosphere. Bleomycin did not cause HLF injury (no deformity of cell shape, no detachment from tissue culture dish, and >95% of cells were viable by trypan blue exclusion) after 72 h incubation at 10 µg/ml. However, bleomycin at 100 µg/ml caused substantial HLF cytotoxicity after 24 h incubation. The supernatant fluids were then harvested and stored at -80°C until assayed. At least six separate HLF supernatant fluids were harvested for each experimental condition.

Measurement of NCA and MCA

Polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Boyum (37). Briefly, 15 ml of venous blood was obtained from healthy volunteers, then sedimented with 3% dextran in isotonic saline for 45 min to separate the white blood cells from RBC. The leukocyte-rich upper layer was collected, and neutrophils were separated from mononuclear cells by Ficoll-Hypaque density centrifugation (Histopaque 1077; Sigma). Contaminating RBC were removed by using lysing solution consisting of 0.1% KHCO3 and 0.83% NH4Cl. The suspension was then centrifuged at 400 x g for 5 min and washed three times in HBSS (Biofluids, Rockville, MD). The resulting cell pellet, as determined by trypan blue and erythrosin exclusion, consisted of >96% neutrophils and >98% viable cells. The cells were suspended in Gey’s balanced salt solution (Life Technologies) containing 2% BSA (Sigma) at pH 7.2 to give a final concentration of 3.0 x 106 cells/ml. This suspension was used for the neutrophil chemotaxis assay.

Mononuclear cells for the chemotaxis assay were obtained from normal human volunteers by Ficoll-Hypaque density centrifugation to separate RBC and neutrophils from mononuclear cells. The mononuclear cells were harvested at the interface. The suspension was then centrifuged at 400 x g for 10 min and washed three times in HBSS. The preparation routinely consisted of 30% large monocytes and 70% small lymphocytes determined by morphology and {alpha}-naphthyl acetate esterase staining (Sigma) with >98% viability as assessed by trypan blue and erythrosin exclusion. The cells were suspended in Gey’s balanced salt solution containing 2% BSA at pH 7.2 to give a final concentration of 5.0 x 106 cells/ml. The suspension was then used for the monocyte chemotaxis assay.

The chemotaxis assay was performed by a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD), as has been described previously (38). The bottom wells of the chamber were filled with 25 µl of fluid containing the chemotactic stimulus or media in duplicate. A 10-µm thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA), with a pore size of 3 µm for neutrophil chemotaxis and 5 µm for monocyte chemotaxis, was placed over the bottom wells. The silicon gasket and upper pieces of the chamber were applied, and 50 µl of the cell suspension was placed into the upper wells above the filter. The chambers were incubated in humidified air in 5% CO2 at 37°C for 30 min for neutrophil chemotaxis and 90 min for monocyte chemotaxis. After incubation, the chamber was disassembled, and nonmigrated cells were wiped away from the filter. The filter was then immersed in methanol for 5 min, stained with Diff-Quik (American Scientific Product, McGlaw Park, IL), and mounted on a glass slide. Cells completely migrated through the filter were counted by using light microscopy in 10 random high power fields (magnification, x1000) per well.

To ensure that monocytes, but not lymphocytes, were the primary cells that migrated in the monocyte chemotaxis assay, some membranes were stained with {alpha}-naphthyl acetate esterase according to the manufacturer’s directions (Sigma).

To determine whether the migration was due to movement along a concentration gradient (chemotaxis) or stimulation of random migration (chemokinesis), a checkerboard analysis was performed with HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of bleomycin (39). To do this, various dilutions of HLF supernatant fluids (1:1, 1:4, 1:16, 1:64, and 1:256) were placed below the membrane and above the membrane with target cells.

Partial characterization of NCA and MCA

Partial characterization of NCA and MCA released from HLF was performed with supernatant fluids harvested after 72 h at the concentration of 10 µg/ml of bleomycin. Sensitivity to proteases was tested by incubating the supernatant fluids with trypsin (100 µg/ml; Sigma) for 30 min at 37°C 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 F-12 used for the cell culture before the chemotaxis assay. Both extracted and extractant materials were evaluated for chemotactic activity. Heat sensitivity was determined by maintaining a supernatant fluid at 98°C for 15 min.

Molecular sieve column chromatography of the chemotactic activity

To determine the approximate m.w. of the released chemotactic activity in the supernatant fluids harvested at 72 h in response to 10 µg/ml of bleomycin, molecular sieve column chromatography was performed using Sephadex G-200 (Pharmacia, Piscataway, NJ). At a flow rate of 6 ml/h, HLF culture supernatant fluid was eluted with PBS, and fractions after the void volume were evaluated for NCA and MCA in duplicate.

Effects of metabolic inhibitors on the release of NCA and MCA

The effects of nonspecific lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA, 100 µM; Sigma) and diethylcarbamazine (DEC, 1 mM; Sigma) and 5-lipoxygenase inhibitor AA-861 (100 µM; Takeda Pharmaceutical, Tokyo, Japan) on the release of NCA and MCA were evaluated in response to 10 µg/ml of bleomycin after 72 h incubation. To further examine the involvement of protein synthesis in the release of chemotactic activity, cycloheximide (20 µg/ml; Sigma) was added to inhibit protein synthesis (40). At these concentrations, NDGA, DEC, and AA-861 inhibited the release of leukotriene B4 (LTB4) in HLF cultures and did not cause cytotoxicity to HLF after 72 h incubation.

Effects of LTB4 receptor antagonist on NCA and MCA

Because the release of NCA and MCA was blocked by 5-lipoxygenase inhibitors, and because both NCA and MCA were extracted into ethyl acetate, LTB4 receptor antagonist (ONO 4057, ONO Pharmaceutical, Tokyo, Japan) at the concentration of 10-5 M was used to evaluate the involvement of LTB4 as NCA and MCA in the crude supernatant fluids and in the column chromatography-separated lowest m.w. (41, 42).

Measurement of LTB4 in the supernatant fluids

The concentration of LTB4 in the supernatant fluids was measured by RIA as previously described (43). Anti-LTB4 serum, [5,6,8,9,11,12,14,15-[3H] (N)]-LTB4, and synthetic LTB4 were purchased from Amersham (Arlington Heights, IL). Briefly, ethanol and supernatants mixture were centrifuged at 5500 x g at 0°C. At a temperature of 37°C, the supernatants were evaporated under N2 gas to remove ethanol. To each sample, 10 ml of distilled water was added. These samples were acidified to pH 4.0 with 0.1 N hydrochloric acid and applied to Sep-pak C18 columns (Waters Associates, Milford, MA). The columns were washed with a 10-ml mixture of distilled water and 20 ml of petroleum ether, then eluted with 15 ml of methanol. These eluates were dried with N2 gas at 37°C, then redissolved in 20 µl of methanol and 180 µl of RIA buffer (50 mM Tris-HCl buffer containing 0.1% (w/v) gelatin, pH 8.6). [3H]-LTB4 was diluted in RIA buffer (0.1 ml, containing ~4000 dpm) and mixed with 0.1 ml of standards or samples in disposable siliconized tubes. Anti-LTB4 serum, diluted by RIA buffer (0.1 ml), was added to siliconized tubes to give a total incubation volume of 0.4 ml. The mixture was incubated at 4°C for 18 h. Free LTB4 was absorbed onto dextran-coated charcoal. The supernatant, containing the Ab-bound LTB4 was decanted into scintillation counter following centrifugation for 15 min at 2000 x g. Scintillation fluid (Aquazol 2; New England Nuclear, Boston, MA) was added, and radioactivity was counted by a scintillation counter (Tricarb-3255; Packard, Downers Grove, IL) for 4 min.

Effects of polyclonal Abs to IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES

Because the results of partial characterization and the effects of metabolic inhibitors suggested the involvement of peptides as NCA and MCA, we assessed chemokines known as NCA and MCA. The neutralizing Abs to IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES (Genzyme, Cambridge, MA) were added to the HLF supernatant fluids, which were harvested at 72 h in response to 10 µg/ml of bleomycin at the suggested concentrations to inhibit these cytokines and incubated for 30 min at 37°C. Then, these samples were used for chemotactic assay. As we have previously reported that these Abs inhibited each chemokine-induced NCA and MCA, and that each Ab did not influence the neutrophil and monocyte chemotaxis induced by activated serum or FMLP (44, 45, 46). As a negative control, we used nonimmune IgG, which did not have any influences on bleomycin-conditioned medium.

Measurement of IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES in the supernatant fluids

The concentrations of IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES in HLF supernatant fluids cultured for 72 h at the concentration of 10 µg/ml of bleomycin were measured by ELISA according to the manufactures’ direction. GM-CSF and RANTES kits were purchased from Amersham (Buckinghamshire, England), and the minimum concentration detected by these methods was 2.00 pg/ml for GM-CSF and 15.6 pg/ml for RANTES. IL-8, MCP-1, and TGF-ß kits were purchased from R & D Systems (Minneapolis, MN), and the minimum detectable concentration of IL-8, MCP-1, G-CSF, and TGF-ß was 10.0 pg/ml, 31.3 pg/ml, and 0.31 ng/ml, respectively. G-CSF (chemiluminescence enzyme immunoassay method) kit was obtained from Chugai Pharmaceutical (Tokyo, Japan), and the minimum detectable concentration of G-CSF was 1.0 pg/ml.

Evaluation of IL-8, G-CSF, MCP-1, and GM-CSF mRNA expressions

The protein secretions of IL-8, G-CSF, MCP-1, and GM-CSF were augmented by bleomycin. RT-PCR was used to evaluate mRNA expression of IL-8, G-CSF, MCP-1, and GM-CSF in HLF in response to 10 µg/ml bleomycin after 6 h incubation. Total RNA was extracted from HLF as previously described (47). One microgram of total RNA was reverse-transcribed into cDNA with a cDNA synthesis kit (Boeringer Mannheim, Mannheim, Germany) and then amplified for 27 or more cycles in Perkin-Elmer Gene Amp PCR System 9600 (denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 30 s; Perkin-Elmer, Norwalk, CT). IL-8, G-CSF, MCP-1, and GM-CSF sense, anti-sense, and probe used in the present study were as follows: GM-CSF sense, 5'-TGA ACC TGA GTA GAG ACA CTG C-3'; anti-sense, 5'-TGA CAA GCA GAA AGT CCT TCA G-3'; probe, 5'-ATG TTT GAC CTC CAG GAG CCG ACC TGC CTA-3'; L-8 sense, 5'-AAC ATG ACT TCC AAG CTG GC-3'; anti-sense, 5'-ACT GGC ATC TTC ACT GAT TC-3'; probe, 5'-TTG AGA GTG GAC CAC ACT GCG CCA ACA CAG-3'; MCP-1 sense, 5'-TAG CAG CCA CCT TCA TTC CC-3'; anti-sense, 5'-CAG GTG GTC CAT GGA ATC CTG AA-3'; probe, 5'-GTG CAG AGG CTC GCG AGC TAT AGA A-3'; G-CSF sense, 5'-GCT TAG AGC CAA GTG AGG AAG-3'; anti-sense, 5'-AGG TGG CGT AGA ACG CGG TA-3'; probe, 5'-ACC CAG GGT GCC ATG CCG GCC TTC GCC TCT-3'; ß-actin sense, 5'-TGA CCC AGA TCA TGT TTG AG-3'; anti-sense, 5'-TCA TGA GGT AGT CAG TCA GG-3'; probe, human cDNA probe.

Preliminary studies indicated that more than 27 cycles were subsaturating for mRNA tested and thus was appropriate for comparison of relative levels of mRNA between groups. PCR products were separated by electrophoresis on 3% agarose gel and were visualized by 32P-labeled exposure. PCR band densities were determined by NIH Image (National Institutes of Health, Bethesda, MD) on unaltered, computer-scanned images. ß-actin mRNA, which has been shown not to change by stimulation, was measured in both normal and stimulated RNA samples at each point, using the same cDNA that was analyzed for cytokines. Integrated OD measurements of 10 separate ß-actin samples did not vary >10% from the mean integrated OD, which is an indication of expected variation resulting from experimental technique.

Statistics

In experiments where multiple experiments were made, differences between groups were tested for significance using one-way ANOVA with Fisher’s multiple range test applied to data at specific time and dose points. In experiments where single measurement was made, the differences between groups were tested for significance using the paired Student’s t test. In all cases, a value of p < 0.05 was considered significant. Data in figures and tables were expressed as means ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Release of NCA and MCA from HLF

HLF released NCA and MCA in a dose-dependent manner in response to bleomycin (Fig. 1Go, A and B). The lowest doses of bleomycin to stimulate HLF were 1 µg/ml for neutrophils and 0.1 µg/ml for monocytes. Increasing concentrations of bleomycin progressively increased the release of NCA and MCA up to 10 µg/ml. At the concentration of 100 µg/ml, NCA and MCA dropped because bleomycin caused cytotoxicity to HLF after 24 h.



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  		FIGURE 1. The dose-dependent release of NCA (A) and MCA (B) in response to bleomycin from HLF monolayers after 72 h incubation (n = 8). Chemotactic activity is on the ordinate, and the concentration of bleomycin is on the abscissa. Values are expressed as means ± SE. *, p < 0.05 compared with supernatant fluids without bleomycin; #, p < 0.001 compared with supernatant fluids without bleomycin.

 
Although HLF released NCA and MCA constitutively, HLF further released NCA and MCA in response to bleomycin in a time-dependent manner (Fig. 2Go, A and B). The release of NCA was significant after a 12-h exposure to bleomycin (Fig. 2GoA), and MCA was significant after 48 h (Fig. 2GoB). The release of chemotactic activity increased even at 96 h. Bleomycin itself did not show any chemotactic activities for neutrophils and monocytes (data not shown).



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  		FIGURE 2. The time-related release of NCA (A) and MCA (B) in response to 10 µg/ml of bleomycin, and the baseline release of NCA and MCA from HLF monolayers (n = 8). Chemotactic activity is on the ordinate, and the incubation time is on the abscissa. Closed squares express with bleomycin and closed circles express without bleomycin. Values are expressed as means ± SE. *, p < 0.05 compared with medium alone; **, p < 0.01 compared with medium alone; #, p < 0.001 compared with medium alone; §, p < 0.01 compared with supernatant fluids without bleomycin; §§, p < 0.001 compared with supernatant fluids without bleomycin.

 
Checkerboard analysis revealed that the HLF supernatant fluids stimulated by bleomycin induced neutrophil migration in the presence of a gradient across the membrane in a concentration-dependent manner (Table IGo). Thus the migration of neutrophils was consistent with chemotactic rather than chemokinetic. In contrast, monocyte migration was induced slightly in the absence of gradient (Table IIGo). Then, monocyte migration was predominantly chemotactic rather than chemokinetic.


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  		Table I. Checkerboard analysis of NCA in HLF supernatant fluids1

 

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  		Table II. Checkerboard analysis of MCA in HLF supernatant fluids1

 
Confirmation that the migrated cells were monocytes was provided by the following lines of evidence: 1) >90% of the migrated cells appeared to be monocytes morphologically by light microscopy; 2) >90% of the migrated cells were esterase positive; and 3) lymphocytes purified by allowing the monocytes to attach to plastic and tested in the chemotaxis assay yielded 0–20% of the chemotactic activity of the monocyte preparation.

Partial characterization of NCA and MCA

The NCA and MCA were heterogeneous in character. NCA was partially sensitive to heat, predominantly extracted by ethyl acetate, and partly digested by trypsin (p < 0.05; Fig. 3GoA). MCA was partially sensitive to heat, extracted by ethyl acetate, and predominantly digested by trypsin (p < 0.001, Fig. 3GoB).



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  		FIGURE 3. Partial characterization of NCA (A) and MCA (B) obtained from HLF supernatant fluids harvested after 72 h incubation in response to 10 µg/ml of bleomycin (n = 8). Chemotactic activity is on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SE. EA, ethyl acetate. *, p < 0.05 compared with crude supernatant fluids; #, p < 0.001 compared with crude supernatant fluids.

 
Molecular sieve column chromatographic findings of the released chemotactic activities

Molecular sieve column chromatography using Sephadex G-200 revealed that NCA was heterogeneous in size (Fig. 4GoA). At least three peaks of NCA were separated by column chromatography with the estimated m.w. before and after cytochrome c (m.w. 12,300), as well as an additional peak that eluted after quinacrine (m.w. 450). The released MCA was also heterogeneous (Fig. 4GoB). At least three peaks of MCA seemed to be separated by column chromatography, with the estimated m.w. before cytochrome c, as well as an additional peak that eluted after quinacrine.



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  		FIGURE 4. Molecular sieve column chromatographic findings of released NCA (A) and MCA (B) from HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of bleomycin. The data presented was representative of four experiments. NCA and MCA are on the ordinate, and fraction numbers are on the abscissa. Closed squares express with bleomycin, and closed circles express without bleomycin. The m.w. markers BSA (66,000), cytochrome c (12,300), and quinacrine (450) are indicated by arrows.

 
We conducted the column chromatographic separation on the heat-treated samples. As was shown in the Fig. 3Go, heat inactivated 60–70% of NCA and MCA. After heat inactivation, the chemotactic activity in the high m.w. disappeared, although the lowest m.w. peaks also decreased to 70%. Thus, higher m.w. materials were not a carrier for LTB4.

Inhibition of the release of chemotactic activity by metabolic inhibitors

The supernatant fluids incubated with 10 µg/ml of bleomycin in the presence of NDGA, DEC, and AA-861 showed a decrease in the release of NCA and MCA (p < 0.01; Fig. 5Go, A and B). Cycloheximide inhibited the release of both NCA (p < 0.001; Fig. 5GoA) and MCA (p < 0.001; Fig. 5GoB).



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  		FIGURE 5. The inhibition of the release of NCA (A) and MCA (B) by 1 mM DEC, 100 µM NDGA, AA-861, and 20 µg/ml cycloheximide in response to 10 µg/ml bleomycin for 72 h incubation (n = 8). Chemotactic activity is on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SE. **, p < 0.01 compared with untreated supernatant fluids; #, p < 0.001 compared with untreated supernatant fluids.

 
Inhibition of NCA and MCA by LTB4 receptor antagonists

Both NCA and MCA of crude samples were significantly inhibited by the addition of the LTB4 receptor antagonist, ONO 4057, by about 70% for NCA and 40% for MCA (p < 0.01; Fig. 6Go, A and B). ONO 4057 also inhibited the column chromatography-separated lowest m.w. peak of NCA and MCA (about 80% for NCA and 60% for MCA). LTB4 receptor antagonist at a concentration of 10-5 M completely inhibited the neutrophil migration in response to 10-7 M LTB4, but showed no inhibitory effects on FMLP and endotoxin-activated serum-induced neutrophil and monocyte chemotaxis (data not shown).



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  		FIGURE 6. The inhibition of NCA (A) and MCA (B) in the HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of bleomycin by LTB4 receptor antagonist (n = 8). Chemotactic activity is on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SE. **, p < 0.01 compared with crude supernatant fluids; #, p < 0.001 compared with crude supernatant fluids.

 
Release of LTB4 from HLF

The measurement of LTB4 in the supernatant fluids by RIA revealed that HLF released significant amount of LTB4 in the baseline culture condition. However, the addition of bleomycin at the concentration of 10 µg/ml for 72 h did not induce LTB4 release from HLF [270 ± 20 pg/ml (control) vs 244 ± 7 pg/ml (bleomycin)].

Inhibition of NCA and MCA by polyclonal Abs to IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES

Because HLF had the potential to release chemokines, and because chemokines released from HLF might be responsible for NCA and MCA, we used polyclonal blocking Abs to IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and RANTES. Among these Abs, anti-IL-8 and G-CSF Abs inhibited NCA (p < 0.05; Fig. 7GoA). Anti-MCP-1, GM-CSF, and TGF-ß Abs attenuated MCA (p < 0.05; Fig. 7GoB). In contrast, RANTES Ab did not inhibit MCA. We evaluated the effect of IL-8, G-CSF, MCP-1, GM-CSF, and TGF-ß Abs on the column chromatography-separated NCA and MCA. These Abs also inhibited the chemotactic activities at the corresponding m.w. chemotactic peak about 60–80%.



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  		FIGURE 7. The effects of blocking Abs on NCA (A) and MCA (B) of HLF supernatant fluids in response to 10 µg/ml of bleomycin for 72 h incubation (n = 8). Chemotactic activity is on the ordinate, and the experimental groups are on the abscissa. Values are expressed as means ± SE. *, p < 0.05 compared with untreated supernatant fluids; **, p < 0.01 compared with untreated supernatant fluids; #, p < 0.001 compared with untreated supernatant fluids.

 
We have previously reported that fibroblasts constitutively release monocyte chemokinetic activity (48). The released chemoattractants were MCP-1, GM-CSF, LTB4, and TGF-ß. The proportion of chemotactic potential for MCP-1, GM-CSF, and TGF-ß to attract monocytes was almost similar with bleomycin-stimulated conditioned medium. However, the proportion of LTB4 in the total MCA became lower by the stimulation of bleomycin. In neutrophil chemotaxis, the proportion of LTB4-induced neutrophil chemotaxis was higher than that in monocyte chemotaxis in bleomycin-stimulated medium. However, the IL-8 and G-CSF in the unstimulated fibroblasts medium explained only 5–10% of the NCA as assessed by Ab experiments (data not shown). Thus, MCP-1, GM-CSF, TGF-ß, IL-8, and G-CSF were produced from HLF by bleomycin stimulation as NCA and MCA.

The release of IL-8, MCP-1, G-CSF, GM-CSF, TGF-ß, and RANTES from HLF by bleomycin

The measurement of chemotactic cytokines by ELISA revealed that bleomycin at the concentration of 10 µg/ml for 72 h incubation stimulated the release of IL-8 and G-CSF as NCA (p < 0.001; Table IIIGo) and GM-CSF, MCP-1, and TGF-ß (p < 0.05; Table IIIGo). In contrast, RANTES was not detected in HLF supernatant fluids.


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  		Table III. The release of cytokines from HLF in response to bleomycin

 
Augmentation of IL-8, GM-CSF, and MCP-1 mRNA expression by bleomycin

Bleomycin treatment of HLF for 6 h resulted in the augmented expression of IL-8, GM-CSF, and MCP-1 mRNA (Fig. 8Go). However, the expression of G-CSF was not detected under unstimulated and stimulated conditions after 40 cycles of amplification by PCR.



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  		FIGURE 8. The augmentation of IL-8, GM-CSF, and MCP-1 mRNA expression by 10 µg/ml bleomycin after 6 h incubation in HLF monolayers. Data presented is representative of four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that bleomycin stimulated HLF to release NCA and MCA in a dose- and time-dependent manner. Partial characterization and molecular sieve column chromatography revealed the heterogeneity of NCA and MCA. Anti-IL-8 and G-CSF Abs and LTB4 receptor antagonist inhibited NCA. Anti-MCP-1, GM-CSF, and TGF-ß Abs and LTB4 receptor antagonist inhibited MCA. Although, LTB4 was released constitutively, the releases of IL-8, G-CSF, MCP-1, GM-CSF, and TGF-ß were induced by bleomycin. These data suggest that an interaction between lung fibroblasts and bleomycin may modulate inflammatory cell recruitment from the vasculature to the interstitium after bleomycin exposure through the generation of chemotactic cytokines.

Fujita et al. (49) reported that the concentrations of bleomycin in blood and cancer tissue in human administrated at the dose of 15 mg/body i.v. were 0.8 µg/ml and 0.4 µg/ml. Although bleomycin is predominantly secreted from the kidney, the activity of bleomycin-inactivating enzyme (bleomycin hydrolase) is weak at skin, lung, and squamous cell carcinoma tissue. Therefore, the high concentration of bleomycin at these tissues is observed and thought to have a relationship with the tissue cytotoxicity. Thus the concentration of 0.1–1 µg/ml of bleomycin found to stimulate HLF in the present study may be relevant to the clinical concentration of bleomycin in the lung tissue.

Bleomycin is reported to stimulate T cells and alveolar macrophages, leading to pulmonary inflammatory responses characterized by an increase in leukocyte infiltration, fibroblast proliferation, and collagen synthesis. Inflammatory responses that recruit and activate large numbers of leukocytes often involve specific chemotactic mediators (50). In this context, previous studies have shown that dermal and synovial fibroblasts can release soluble chemotactic factors that direct the migration of neutrophils and monocytes in response to TNF, IL-1, and platelet-derived growth factor (30, 32, 33, 51). The present study demonstrated that HLF released NCA and MCA constitutively and further in response to bleomycin. Therefore, lung fibroblasts may modulate their local immunologic environment by releasing chemotactic activity for both neutrophils and monocytes, and may contribute to the lung inflammation in addition to T lymphocytes and alveolar macrophages in response to bleomycin.

It is reported that lung fibroblasts have the potential to release IL-8 and G-CSF in response to TNF or IL-1 ß (52). In the present study, the blocking Abs to IL-8 and G-CSF attenuated NCA similarly about 40%. Bleomycin significantly stimulated the release of IL-8 and G-CSF from HLF. Thus, HLF released IL-8 and G-CSF as NCA in response to bleomycin.

Wang et al. (53) reported that the concentration of G-CSF as NCA was 7–70 ng/ml. Although the concentration of G-CSF detected in the HLF supernatant fluids was less than that reported. We performed neutrophil chemotaxis by using human recombinant G-CSF. The chemotactic concentration of G-CSF as NCA was from 10 to 100 pg/ml (44). The discrepancies of the G-CSF concentration as neutrophil chemotactic factor may be due to the difference of neutrophil separation. Because the blocking Abs of G-CSF inhibited both total NCA in the supernatant fluids and NCA in the column chromatography-separated peaks, the contribution of G-CSF to NCA may be a direct chemoattractant rather than the activation of neutrophils.

The identification of MCA released from HLF is not complete. However, the trypsin sensitivity of MCA along with the inhibition of the release by cycloheximide treatment suggests that the activity is at least partly dependent on protein synthesis (54). MCA was attenuated by Abs to MCP-1, GM-CSF, and TGF-ß. The concentrations of MCP-1, GM-CSF, and TGF-ß in the supernatant fluids reached the concentrations as MCA (55, 56, 57). Thus HLF at least partly released MCP-1, GM-CSF, and TGF-ß as responsible MCA.

HLF have the potential to release MCP-1, GM-CSF, and TGF-ß. However, the predominant MCA was GM-CSF rather than MCP-1 and TGF-ß. The release of GM-CSF from HLF in response to bleomycin was striking compared with the release from type II epithelial cells or bronchial epithelial cells (data not shown). Because GM-CSF is one of a group of glycoproteins that has the ability to stimulate the in vitro proliferation and differentiation of macrophage progenitor cells (58, 59), the augmented release of GM-CSF from fibroblasts in response to bleomycin suggests that fibroblasts may be profoundly concerned with macrophage recruitment, differentiation, and proliferation in the lung interstitium rather than epithelial cells.

The extractability of NCA and MCA into ethyl acetate along with the inhibition of release by NDGA, DEC, and AA-861 suggests that the activity is composed of lipoxygenase product. The NCA and MCA was inhibited by LTB4 receptor antagonist. Although, the release of LTB4 from HLF in response to bleomycin was not significant compared with control, the concentration of LTB4 reached the chemotactic range of neutrophils and monocytes. Thus, LTB4 may be one of the important chemoattractants for neutrophils and monocytes released from fibroblasts constitutively.

Although bleomycin stimulated the release of many cytokines from lung fibroblasts, it did not augment the release of LTB4 by RIA. Because NCA and MCA were inhibited by LTB4 receptor antagonist, and because the column chromatographic profiles showed the increase in lowest m.w., we expected the augmented release of LTB4 from HLF. However, the release of LTB4 was not significant. The exact mechanisms for bleomycin to stimulate fibroblasts resulting in the release of cytokines are uncertain, and the mechanism of activation or synthesis of 5-lipoxygenase in fibroblasts is also unclear. We speculate that the stimulatory potential of bleomycin is not enough for the activation or synthesis of 5-lipoxygenase in fibroblasts compared with other stimulus, which induced LTB4 release from fibroblasts. However, it might be possible that bleomycin induced the release of 12- or 15-hydroxyeicosa tetraenoic acid, which were NCA and MCA, instead of LTB4, and this may explain the augmentation of the lowest chemotactic peaks.

In conclusion, bleomycin stimulated HLF to release NCA and MCA. The released activities were chemotactic by checkerboard analysis. The released NCA and MCA by bleomycin were IL-8, G-CSF, MCP-1, GM-CSF, TGF-ß, and LTB4. These results suggest that lung fibroblasts may play a role in the inflammatory cell recruitment by releasing chemotactic activity in response to bleomycin.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Sekiya Koyama, First Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi Matsumoto, 390 Japan. Back

2 Abbreviations used in this paper: NCA, neutrophil chemotactic activity; MCA, monocyte chemotactic activity; MCP, monocyte chemoattractant protein; HLF, human lung fibroblast; NDGA, nordihydroguaiaretic acid; DEC, diethylcarbamazine; LTB4, leukotriene B4. Back

Received for publication May 5, 1998. Accepted for publication March 1, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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