Cyclophosphamide stimulates lung fibroblasts to release neutrophil and monocyte chemoattractants

Sekiya Koyama1,2, Akemi Takamizawa2, Etsuro Sato2, Takeshi Masubuchi2, Sonoko Nagai3, and Takateru Izumi3

1 Pulmonary Section, The National Chuushin Matsumoto Hospital, Matsumoto 399-0021; 2 The First Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto 390-8621; and 3 Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8057, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene B4 receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene B4 receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide.

interstitial lung disease; interleukin-8; granulocyte colony-stimulating factor; monocyte chemoattractant protein-1; granulocyte-macrophage colony-stimulating factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYCLOPHOSPHAMIDE IS AN ALKYLATING AGENT used in several conditions including fibrosis associated with collagen vascular diseases and cryptogenic fibrosing alveolitis (11). A report (5) indicated that, paradoxically, cyclophosphamide may induce interstitial pulmonary inflammation and fibrosis. Cyclophosphamide has been associated with pulmonary toxicity in ~1% of the cases, manifested most frequently as interstitial pneumonitis and pulmonary fibrosis (5). However, little is known about the mechanism and risk factors of its pulmonary toxicity (5). Neither specific lung histopathological findings nor definite relationships with risk factors have been established (5, 18). Lung damage from cyclophosphamide can occur at any time after the start of treatment and is not dose dependent (24).

Sequestration of peripheral blood neutrophils and monocytes within the lung is characteristic of a number of acute and chronic pulmonary diseases (1, 10). 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 macrophage is also derived predominantly from peripheral blood monocyte migration and, to a limited extent, from local replication (3). 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 tissue injury via the excessive elaboration of inflammatory cytokines, proteolytic enzymes, and oxygen radicals (10, 23). Substantial investigation has focused on alveolar macrophages as a primary source of chemotactic factors (9). However, neutrophil (NCA) and monocyte (MCA) chemotactic activities have been found to be produced by endothelial cells (26), fibroblasts (27), and pulmonary epithelial cells (25).

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 (15-17, 22) 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 (MCP)-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-beta in response to interleukin (IL)-1beta , tumor necrosis factor-alpha , and platelet-derived growth factor, suggesting the contribution to certain disease states (15-17, 22). Recently, Takamizawa et al. (28) reported that bleomycin stimulates human lung fibroblasts (HLFs) to release NCA and MCA including IL-8, granulocyte colony-stimulating factor (G-CSF), MCP-1, and GM-CSF. Therefore, the fibroblast, because of its anatomic location, is in a pivotal position to participate in and direct bidirectional communication between interstitial and vascular events in drug-induced pulmonary inflammation and fibrosis.

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 cyclophosphamide, the underlying mechanism of inflammatory cell migration from the vascular compartment to the interstitium remains to be elucidated. The role of HLFs in inflammatory cell recruitment from the vascular compartment to the interstitium in response to cyclophosphamide is uncertain. The purpose of the present investigation was to determine whether HLFs could participate in the recruitment of inflammatory cells into the lungs. Specifically, the possibility of HLFs to release NCA and MCA in response to cyclophosphamide was evaluated. The results demonstrate that HLFs can release NCA and MCA, including IL-8, G-CSF, MCP-1, and GM-CSF, in response to cyclophosphamide.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Culture of HLFs. We used fetal HLFs (lung, diploid, human, passage 27), which are an established cell line and commercially available (American Type Culture Collection, Manassas, VA). HLFs were suspended at 1.0 × 106 cells/ml in F-12 medium supplemented with penicillin (50 U/ml; GIBCO BRL, Life Technologies, Grand Island, NY), streptomycin (50 µg/ml; GIBCO BRL), Fungizone (2 µg/ml; GIBCO BRL), and 10% fetal calf serum (GIBCO BRL). 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 incubated for 1 more day.

Exposure of HLFs to cyclophosphamide. The medium was removed from the cells by two washes with serum-free F-12 medium, and the cells were incubated in the presence and absence of cyclophosphamide. To determine the dose- and time-dependent release of NCA and MCA, the cultures were incubated with various concentrations of cyclophosphamide (0, 1.0, 10, and 100 µg/ml; Sigma, St. Louis, MO) for 12, 24, 48, and 72 h at 37°C in a humidified 5% CO2 atmosphere. Cyclophosphamide (10 µg/ml) did not cause HLF injury (no deformity of cell shape, no detachment from the tissue culture dish, and >95% of cells viable by trypan blue exclusion) after 72 h of incubation. However, cyclophosphamide at 100 µg/ml caused substantial HLF cytotoxicity after 24 h of 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 with the method of Boyum (4). 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 (GIBCO BRL) containing 2% bovine serum albumin (Sigma), pH 7.2, at a final concentration of 3.0 × 106 cells/ml. This suspension was used for the neutrophil chemotaxis assay.

Mononuclear cells for the chemotaxis assay were obtained from normal human volunteers, and red blood cells and neutrophils were separated from mononuclear cells by Ficoll-Hypaque density centrifugation (28). The preparation routinely consisted of 30% large monocytes and 70% small lymphocytes as 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% bovine serum albumin, pH 7.2, at a final concentration of 5.0 × 106 cells/ml. The suspension was then used for the monocyte chemotaxis assay.

The chemotaxis assay was performed in a 48-well microchemotaxis chamber (NeuroProbe, Cabin John, MD) as previously described (7). 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 were placed in 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 Products, McGaw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted with light microscopy in 10 random high-power fields (×1,000)/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 to stimulation of random migration (chemokinesis), checkerboard analysis was performed with HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of cyclophosphamide (31). 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 to evaluate cell migration with various concentration gradients between the upper well and the lower well. By using a variety of concentration gradients, we determined that cell migration with the concentration gradient was chemotactic and migration without the concentration gradient was chemokinetic.

Molecular-sieve column chromatography of the chemotactic activity. To determine the approximate molecular mass of the released chemotactic activity in the supernatant fluids harvested at 72 h in response to 10 µg/ml of cyclophosphamide, molecular-sieve column chromatography was performed with Sephadex G-200 (Pharmacia, Piscataway, NJ). At a flow rate of 6 ml/h, the 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 the nonspecific lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA; 100 µM; Sigma) and diethylcarbamazine (DEC; 1 mM; Sigma) and the 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 cyclophosphamide after 72 h of 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. At these concentrations, NDGA, DEC, and AA-861 inhibited the release of leukotriene (LT) B4 in HLF cultures (14) and did not cause cytotoxicity to HLFs after 72 h of incubation.

Effects of a LTB4 receptor antagonist on NCA and MCA. Because release of NCA and MCA was blocked by 5-lipoxygenase inhibitors and because both NCA and MCA were extracted into ethyl acetate, a LTB4 receptor antagonist (ONO-4057; ONO Pharmaceuticals, Tokyo, Japan) at a 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 molecular mass fractions.

Measurement of LTB4 in the supernatant fluids. The concentration of LTB4 in the supernatant fluids was measured with a radioimmunoassay as previously described (21).

Effects of polyclonal antibodies to IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and regulated on activation normal T cell expressed and secreted. Because the effects of metabolic inhibitors suggested the involvement of peptides as NCA and MCA, we assessed chemokines known as NCA and MCA. The neutralizing antibodies to IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and regulated on activation normal T cell expressed and secreted (RANTES; Genzyme, Cambridge, MA) were added to the HLF supernatant fluids that were harvested at 72 h in response to 10 µg/ml of cyclophosphamide at the suggested concentrations to inhibit these cytokines and were incubated for 30 min at 37°C. Then these samples were used for chemotactic assay. It has been previously reported (13, 16, 28) that these antibodies inhibited each chemokine-induced NCA and MCA and each antibody did not influence the neutrophil and monocyte chemotaxis induced by activated serum or formyl-methionyl-leucyl-phenylalanine. As a negative control, we used nonimmune IgG; nonimmune IgG did not have any influence on cyclophosphamide-conditioned medium.

Measurement of IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and RANTES in the supernatant fluids. The concentrations of IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and RANTES in HLF supernatant fluids cultured for 72 h with 10 µg/ml of cyclophosphamide were measured by ELISA according to the manufacturer's directions. GM-CSF and RANTES kits were purchased from Amersham, 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-beta kits were purchased from R&D Systems (Minneapolis, MN), and the minimum detectable concentrations of IL-8, MCP-1, and TGF-beta were 10.0 pg/ml, 31.3 pg/ml, and 0.31 ng/ml, respectively. G-CSF (CLEIA) kit was obtained from Chugai Pharmaceutical (Tokyo, Japan), and the minimum detectable concentration of G-CSF was 1.0 pg/ml.

Statistics. In experiments where multiple experiments were performed, differences between groups were tested for significance with one-way analysis of variance, with Fisher's multiple range test applied to data at specific time and dose points. In experiments where a single measurement was made, the differences between groups were tested for significance with Student's paired t-test. In all cases, a P value < 0.05 was considered significant. Data are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Dose-dependent release of neutrophil (NCA; A) and monocyte (MCA; B) chemotactic activities in response to cyclophosphamide (CPM) from human lung fibroblast (HLF) monolayers after 72 h of incubation. Values are means ± SE; n = 8 monolayers. * P < 0.05 compared with supernatant fluids without CPM. ** P < 0.001 compared with supernatant fluids without CPM.

Although HLFs released NCA and MCA constitutively, HLFs further released NCA and MCA in response to cyclophosphamide in a time-dependent manner (Fig. 2). The release of NCA and MCA was significant after 24 h of exposure to cyclophosphamide (Fig. 2). The release of chemotactic activity increased even at 72 h. Cyclophosphamide itself did not show any chemotactic activity for neutrophils and monocytes (data not shown).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Time-related release of NCA (A) and MCA (B) in response to 10 µg/ml of CPM () and baseline release (without CPM; ) of NCA and MCA from HLF monolayers. Values are means ± SE; n = 8 monolayers. ** P < 0.01 compared with medium alone. # P < 0.01 compared with supernatant fluids without CPM.

Checkerboard analysis revealed that the HLF supernatant fluids stimulated by cyclophosphamide induced neutrophil migration in the presence of a gradient across the membrane in a concentration-dependent manner (Table 1). Thus the migration of neutrophils was consistent with chemotactic rather than chemokinetic activity. In contrast, HLF supernatant fluids stimulated with cyclophosphamide induced monocyte migration in the presence of a gradient across the membrane in a concentration-dependent manner. But monocyte migration was induced slightly in the absence of gradient (Table 1). Thus monocyte migration was predominantly chemotactic and partly chemokinetic.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Checkerboard analysis of neutrophil and monocyte chemoattractant activities in HLF supernatant fluids

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.

Inhibition of the release of chemotactic activity by metabolic inhibitors . The supernatant fluids incubated with 10 µg/ml of cyclophosphamide in the presence of NDGA, DEC, and AA-861 showed a decrease in release of NCA and MCA (P < 0.01; Fig. 3). Cycloheximide inhibited the release of both NCA and MCA (P < 0.001; Fig. 3). The combination of NDGA and cycloheximide almost completely inhibited the release of both NCA and MCA (P < 0.001; Fig. 3).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibition of the release of NCA (A) and MCA (B) in response to 10 µg/ml of CPM combined with 1 mM diethylcarbamazine (DEC), 100 µM nordihydroguaiaretic acid (NDGA), 100 µM AA-861, or 10 µg/ml cycloheximide (CYCLO) after 72 h of incubation. F-12, F-12 medium (control). Values are means ± SE; n = 8 monolayers.* P < 0.01 compared with untreated supernatant fluids. ** P < 0.001 compared with untreated supernatant fluids.

Molecular-sieve column chromatography findings of the released chemotactic activities. Molecular-sieve column chromatography with Sephadex G-200 revealed that NCA was heterogeneous in size (Fig. 4A). At least three peaks of NCA with estimated molecular masses before and after cytochrome c (molecular mass 12,300 Da) and an additional peak after quinacrine (molecular mass 450 Da) were separated by column chromatography. The released MCA was also heterogeneous (Fig. 4B). At least three peaks of MCA seemed to be separated by column chromatography: two peaks with the estimated molecular masses before cytochrome c and an additional peak after quinacrine.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Molecular-sieve column chromatographic findings of released NCA (A) and MCA (B) from HLF cell supernatant fluids harvested at 72 h in response to 10 µg/ml of cyclophosphamide. Data are from 1 of 4 experiments. Nos. and arrows on top, molecular mass (in Da) and position, respectively, of chemotactic peak of indicated markers.

We carried out the column chromatography separation on heat-treated samples. Heat inactivated 60-70% of NCA and MCA. After heat inactivation, the chemotactic activity in the high molecular mass peak disappeared, although the lowest molecular mass peaks also decreased to 70%. Thus higher molecular mass materials were not carriers for the lowest molecular mass materials.

Inhibition of NCA and MCA by LTB4 receptor antagonists. Both NCA and MCA of the crude samples were significantly inhibited by addition of the LTB4 receptor antagonist ONO-4057 (~70% for NCA and 40% for MCA; P < 0.01; Figs. 5 and 6). ONO-4057 also inhibited the column chromatography-separated lowest molecular mass peak of NCA and MCA (~80% for NCA and 60% for MCA). The LTB4 receptor antagonist at concentrations of 10-5 to 10-9 M inhibited the neutrophil migration dose dependently in response to 10-7 M LTB4 but showed no inhibitory effects on formyl-methionyl-leucyl-phenylalanine- and endotoxin-activated serum-induced neutrophil and monocyte chemotaxis (data not shown).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Inhibition of NCA in the HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of CPM combined with anti-interleukin (IL)-8 antibody, anti-granulocyte colony-stimulating factor (G-CSF) antibody, or leukotriene (LT) B4 receptor antagonist. Values are means ± SE; n = 8 monolayers. * P < 0.01 compared with crude supernatant fluids. ** P < 0.001 compared with crude supernatant fluids.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition of MCA in the HLF supernatant fluids harvested at 72 h in response to 10 µg/ml of CPM combined with anti-monocyte chemoattractant protein (MCP)-1 antibody, anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody, anti-regulated on activation normal T cell expressed and secreted (RANTES) antibody, anti-transforming growth factor (TGF)-beta antibody, or LTB4 receptor antagonist. Values are means ± SE; n = 8 monolayers. * P < 0.01 compared with untreated supernatant fluids. ** P < 0.001 compared with untreated supernatant fluids.

Release of LTB4 from HLFs. The measurement of LTB4 in the supernatant fluids by radioimmunoiassay revealed that HLFs released a significant amount of LTB4 in the baseline culture condition. However, the addition of cyclophosphamide at a concentration of 10 µg/ml for 72 h did not induce LTB4 release from HLFs [252 ± 20 (control) vs. 277 ± 6 (cyclophosphamide) pg/ml].

Inhibition of NCA and MCA by polyclonal antibodies to IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and RANTES. Because HLFs had the potential to release chemokines and because chemokines released from HLFs might be responsible for NCA and MCA, we used polyclonal blocking antibodies to IL-8, G-CSF, MCP-1, GM-CSF, TGF-beta , and RANTES. Among these antibodies, anti-IL-8 and anti-G-CSF antibodies inhibited NCA (P < 0.01; Fig. 5). Anti-MCP-1 and anti-GM-CSF antibodies attenuated MCA (P < 0.001; Fig. 6). In contrast, anti-RANTES and anti-TGF-beta antibodies did not inhibit MCA. We evaluated the effect of anti-IL-8, anti-G-CSF, anti-MCP-1, and anti-GM-CSF antibodies on the column chromatography-separated NCA and MCA. These antibodies also inhibited the chemotactic activities at the corresponding molecular mass chemotactic peaks ~60-80%. Nonimmune IgG was used to evaluate the effect of nonspecific antibody. Nonimmune IgG did not attenuate the NCA and MCA in the same cyclophosphamide-conditioned medium.

The combination of anti-IL-8 and anti-G-CSF antibodies and the LTB4 receptor antagonist inhibited the neutrophil chemotactic response even less than F-12 conditioned medium but not completely (P < 0.01; Fig. 5). The combination of anti-MCP-1 and anti-GM-CSF antibodies and the LTB4 receptor antagonist also similarly inhibited the monocyte chemotactic response even less than the F-12 conditioned medium (P < 0.01; Fig. 6).

The release of IL-8, MCP-1, G-CSF, GM-CSF, TGF-beta , and RANTES from HLFs with cyclophosphamide. The measurement of chemotactic cytokines by ELISA revealed that cyclophosphamide at a concentration of 10 µg/ml for 72 h of incubation stimulated the release of IL-8 and G-CSF as NCA (P < 0.001; Table 2) and GM-CSF and MCP-1 as MCA (P < 0.05; Table 2). In contrast, RANTES was not detected in HLF supernatant fluids. The concentrations of cytokines from HLFs in response to cyclophosphamide also showed an increase in the corresponding molecular mass column fractions (Table 2).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Release of cytokines from HLFs in response to cyclophosphamide and corresponding molecular mass column fractions


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The incidence of pulmonary disease associated with cycloheximide is estimated to be <1%. Lung damage from cyclophosphamide can occur at any time after the start of treatment and is not dose dependent (5). Cases have been reported after administration of as little as 150 mg of cyclophosphamide (8), and the period between administration of the drug and the onset of symptoms has varied from 2 wk (6) to 13 yr (12). When the total dose does not exceed 10 g, regression is usually seen after withdrawal of the drug (18). However, the mechanism of pulmonary toxicity is uncertain. Allergic reaction was suggested by Batist and Andrews (2). Morse et al. (20) have shown that in mice, a single dose of cyclophosphamide (100 mg/kg) produced irreversible pulmonary fibrosis, characterized by an atypical histological picture, increased content of hydroxyproline in the lungs, and decreased lung volume and compliance. The changes were amplified in animals exposed to 70% oxygen. Two types of alveolar macrophages were demonstrated, and the authors suggested that the macrophages stimulate fibroblast proliferation. But the role of lung fibroblasts in inflammatory cell recruitment from the vascular compartment to the interstitium in response to cyclophosphamide is undetermined.

It has been reported (17, 28) that lung fibroblasts have the potential to release IL-8 and G-CSF in response to tumor necrosis factor-alpha , IL-1beta , or bleomycin. In the present study, the blocking antibodies to IL-8 and G-CSF attenuated NCA similarly (~40%). Cyclophosphamide significantly stimulated the release of IL-8 and G-CSF from HLFs. Thus the release of IL-8 and G-CSF as NCA may at least partly explain neutrophil infiltration to the interstitium in response to cyclophosphamide.

Wang et al. (29) 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 (13). The discrepancies in G-CSF concentration as a neutrophil chemotactic factor may be due to the difference in neutrophil separation. Because the blocking antibodies to 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 as a direct chemoattractant rather than as the activation of neutrophils.

The identification of MCA released from HLFs is not complete. However, the inhibition of the release by cycloheximide treatment suggests that the activity is at least partly dependent on protein synthesis. MCA was attenuated by antibodies to MCP-1 and GM-CSF. The concentrations of MCP-1 and GM-CSF in the supernatant fluids reached those levels as MCA (28, 30). Thus HLFs released MCP-1 and GM-CSF at least partly as responsible MCA.

HLFs release MCP-1 and GM-CSF. However, the predominant MCA was GM-CSF rather than MCP-1 and TGF-beta . GM-CSF is one of a group of glycoproteins that have the ability to stimulate the in vitro proliferation and differentiation of macrophage progenitor cells (19). Thus the augmented release of GM-CSF from fibroblasts in response to cyclophosphamide suggests that fibroblasts may be profoundly concerned with macrophage recruitment, differentiation, and proliferation in the lung interstitium rather than in epithelial cells.

The inhibition of release by NDGA, DEC, and AA-861 suggests that the activity is composed of a lipoxygenase product. However, NDGA has the potential to inhibit the release of IL-8. NCA and MCA were inhibited by the LTB4 receptor antagonist. Although the release of LTB4 from HLFs in response to cyclophosphamide was not significant compared with the control value, the concentration of constitutively released LTB4 reached the chemotactic range of neutrophils and monocytes (14). Koyama et al. (14) previously reported that the LTB4 receptor antagonist and MCP-1 antibody inhibited the monocyte chemotactic response in the baseline condition and that HLFs constitutively release MCP-1 and LTB4. Thus it is no wonder that the combination of antibodies and LTB4 receptor antagonist inhibited the neutrophil and monocyte chemotactic responses less than the F-12 conditioned medium. Although the molecular-sieve column profiles did not show a striking increase in the lowest molecular mass peak in both neutrophil and monocyte migration, the LTB4 receptor antagonist abolished the lowest molecular mass chemotactic activity. Thus LTB4 may be one of the important chemoattractants released from fibroblasts constitutively for neutrophils and monocytes.

Cyclophosphamide stimulated the release of many cytokines from lung fibroblasts. The exact mechanisms for cyclophosphamide 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 cyclophosphamide is not enough for the activation or synthesis of 5-lipoxygenase in fibroblasts compared with other stimuli that induced LTB4 release from fibroblasts. However, it might be possible that cyclophosphamide induced the release of 12-hydroxyeicosatetraenoic acid (HETE) and/or 15-HETE, which were NCA and MCA. 12-HETE and/or 15-HETE in combination with LTB4 may explain the augmentation of the lowest chemotactic peaks.

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


    FOOTNOTES

Address for reprint requests and other correspondence: S. Koyama, Pulmonary Section, The National Chuushin Matsumoto Hospital, 811 Kotobuki Toyooka, Matsumoto 399-0021, Japan (E-mail: koyama{at}ka3.so-net.ne.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 February 2000; accepted in final form 9 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adamson, IYR, and Bowden DH. Role of monocytes and interstitial cells in the generation of alveolar macrophages. II. Kinetic studies after carbon loading. Lab Invest 42: 518-524, 1980[ISI][Medline].

2.   Batist, G, and Andrews JL. Pulmonary toxicity of antineoplastic drugs. JAMA 246: 1449-1453, 1981[Abstract].

3.   Blusse van Oud Albas, A, Mattie H, and van Furth R. A quantitative evaluation of pulmonary macrophage kinetics. Cell Tissue Kinet 16: 211-219, 1983[ISI][Medline].

4.   Boyum, A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 97: 77-89, 1968.

5.   Cooper, JAD, White DA, and Matthay RA. Drug-induced pulmonary disease. Part 1: cytotoxic drugs. Am Rev Respir Dis 133: 321-340, 1986[ISI][Medline].

6.   Dohner, VA, Ward HP, and Standord RE. Alveolitis during procarbazine, vincristine and cyclophosphamide therapy. Chest 62: 636-639, 1972[Medline].

7.   Falk, W, Goodwin RH, Jr, and Leonard EJ. A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 33: 239-247, 1980[ISI][Medline].

8.   Ginsberg, SJ, and Comis RL. The pulmonary toxicity of antineoplastic agents. Semin Oncol 9: 34-51, 1982[ISI][Medline].

9.   Hunninghake, GW, Gadek JE, Fales HM, and Crystal RG. Human alveolar macrophage-derived chemotactic factor for neutrophils. Stimuli and partial characterization. J Clin Invest 66: 473-483, 1980[ISI][Medline].

10.   Hunninghake, GW, Garrett KC, Richerson HB, Fantone JC, Ward PA, Rennard SI, Bitterman PB, and Crystal RG. Pathogenesis of the granulomatous lung disease. Am Rev Respir Dis 130: 476-496, 1984[ISI][Medline].

11.   Johnson, MA, Kwan S, Snell NJC, Darbyshire JH, and Turner-Warwick M. Randomised controlled trial comparing prednisolone in combination in cryptogenic fibrosis alveolitis. Thorax 44: 280-288, 1989[Abstract].

12.   Karim, FWA, Ayash RE, Allam C, and Salem PA. Pulmonary fibrosis after prolonged treatment with low-dose cyclophosphamide. Oncology 40: 174-176, 1983[ISI][Medline].

13.   Koyama, S, Sato E, Masubuchi T, Takamizawa A, Kubo K, Nagai S, and Izumi T. Alveolar type II-like cells release G-CSF as neutrophil chemotactic activity. Am J Physiol Lung Cell Mol Physiol 275: L687-L693, 1998[Abstract/Free Full Text].

14.   Koyama, S, Sato E, Masubuchi T, Takamizawa A, Nomura H, Kubo K, Nagai S, and Izumi T. Human lung fibroblasts release chemokinetic activity for monocytes constitutively. Am J Physiol Lung Cell Mol Physiol 275: L223-L230, 1998[Abstract/Free Full Text].

15.   Larsen, CG, Zachariae CO, Oppenheim JJ, and Matsushima K. Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin 1 or tumor necrosis factor. Biochem Biophys Res Commun 160: 1403-1408, 1989[ISI][Medline].

16.   Le, J, Weinstein D, Gubler V, and Vilcek J. Induction of membrane associated interleukin-1 by tumor necrosis factor in human fibroblasts. J Immunol 138: 2137-2142, 1987[Abstract/Free Full Text].

17.   Leizer, T, Cebon J, Layton JE, and Hamilton JA. Cytokine regulation of colony-stimulating factor production in cultured human synovial fibroblasts. I. Induction of GM-CSF and G-CSF production by interleukin-1 and tumor necrosis factor. Blood 76: 1989-1996, 1990[Abstract].

18.   Mark, JG, Lehimgar-Zadeh A, and Ragsdale BD. Cyclophosphamide pneumonitis. Thorax 33: 89-93, 1978[Abstract].

19.   Metcalf, D. The granulocyte-macrophage colony-stimulating factors. Science 229: 16-22, 1985[ISI][Medline].

20.   Morse, CC, Sigler C, Lock D, Hakkinen PJ, Haschek WM, and Witschi HP. Pulmonary toxicity of cyclophosphamide: a 1-year study. Exp Mol Pathol 42: 251-260, 1985[ISI][Medline].

21.   Poubelle, PE, Borgeat P, and Rola-Pleszczynski M. Assessment of leukotriene B4 synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods. J Immunol 139: 1273-1277, 1987[Abstract/Free Full Text].

22.   Rollins, BJ, Stier P, Ernst T, and Wong GG. The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol 9: 4689-4695, 1989.

23.   Sibille, Y, and Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis 141: 471-501, 1990[ISI][Medline].

24.   Spector, JI, Zimbler H, and Ross JS. Early-onset cyclophosphamide-induced interstitial pneumonitis. JAMA 242: 2852-2854, 1979[Abstract].

25.   Standiford, TJ, Kunkel SL, Basha MB, Chensue WS, Lynch JP, Toews GB, Westwick J, and Strieter RM. Interleukin-8 gene expression by a pulmonary epithelial cells. A model for cytokine networks in the lung. J Clin Invest 86: 1945-1953, 1990[ISI][Medline].

26.   Strieter, RM, Kunkel SL, Showell HJ, Remick DG, Phan SH, Ward PA, and Marks RM. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta. Science 243: 1467-1469, 1989[ISI][Medline].

27.   Strieter, RM, Showell HJ, Remnick DG, Lynch JP, III, Genord M, Raiford C, Eskandari M, Marjs RM, and Kunkel SL. Monokine-induced neutrophil chemotactic factor gene expression in human fibroblasts. J Biol Chem 264: 10621-10626, 1989[Abstract/Free Full Text].

28.   Takamizawa, A, Koyama S, Sato E, Masubuchi T, Kubo K, Sekiguchi M, Nagai S, and Izumi T. Bleomycin stimulates human lung fibroblasts to release neutrophil and monocyte chemotactic activity. J Immunol 162: 6200-6208, 1999[Abstract/Free Full Text].

29.   Wang, JM, Chen ZG, Colella S, Bonilla MA, Welte K, Bordignon C, and Mantovani A. Chemotactic activity of recombinant human granulocyte colony-stimulating factor. Blood 72: 1456-1460, 1988[Abstract].

30.   Wang, JM, Colella S, Allavena P, and Mantovani A. Chemotactic activity of human recombinant granulocyte-macrophage colony-stimulating factor. Immunology 60: 439-444, 1987[ISI][Medline].

31.   Zigmond, SH, and Hirsch JG. Leukocyte locomotion and chemotaxis. New methods for evaluation, and demonstration of a cell-derived chemotactic factor. J Exp Med 137: 387-410, 1973[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 280(6):L1203-L1211
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society