Combined SP-A-bleomycin effect on cytokines by THP-1 cells: impact of surfactant lipids on this effect

Weixiong Huang, Guirong Wang, David S. Phelps, Hamid Al-Mondhiry, Joanna Floros

Abstract

Surfactant protein A (SP-A) plays a role in host defense and inflammation in the lung. In the present study, we investigated the hypothesis that SP-A is involved in bleomycin-induced pulmonary fibrosis. We studied the effects of human SP-A on bleomycin-induced cytokine production and mRNA expression in THP-1 macrophage-like cells and obtained the following results. 1) Bleomycin-treated THP-1 cells increased tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1β production in dose- and time-dependent patterns, as we have observed with SP-A. TNF-α levels were unaffected by treatment with cytosine arabinoside. 2) The combined bleomycin-SP-A effect on cytokine production is additive by RNase protection assay and synergistic by enzyme-linked immunosorbent assay. 3) Although the bleomycin effect on cytokine production was not significantly affected by the presence of surfactant lipid, the additive and synergistic effect of SP-A-bleomycin on cytokine production was significantly reduced. We speculate that the elevated cytokine levels resulting from the bleomycin-SP-A synergism are responsible for bleomycin-induced pulmonary fibrosis and that surfactant lipids can help ameliorate pulmonary complications observed during bleomycin chemotherapy.

  • chemotherapeutic agent
  • enzyme-linked immunosorbent assay
  • synergistic effect
  • ribonuclease protection assay
  • surfactant protein A

bleomycin is a groupof glycopeptides isolated from Streptomyces verticillus. Although bleomycin is an effective antineoplastic agent, bleomycin-induced pulmonary fibrosis sometimes becomes fatal and limits the usefulness of the drug (34, 39). The histological features of pulmonary fibrosis in human and animal studies include inflammatory cell recruitment, fibroblast proliferation, and collagen synthesis (5). A number of studies concerning the pathogenesis of pulmonary fibrosis have focused on the role of inflammatory cells, especially alveolar macrophages, in the fibrotic process. Bleomycin induces inflammatory cells from human and animal lung to secrete multifunctional cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, and transforming growth factor (TGF)-β (8, 9, 13, 33, 48). In a clinical study, TNF-α has been shown to be significantly increased after bleomycin infusion (35).

The mechanism of bleomycin-induced cytokine production is not well understood. The cytotoxic effect of bleomycin is believed to be related to DNA damage that is characterized by the appearance of DNA damage-inducible proteins (25) and apoptosis (29). There is also increased activity of nuclear factor (NF)-κB, which may result from the increase of reactive oxygen species by bleomycin (26). NF-κB is a transcriptional factor that regulates the expression of many cytokine genes (50). Among these, TGF-β is considered to be an important cytokine related to fibroblast proliferation and collagen synthesis (13, 48), and TNF-α is considered to be a central mediator in bleomycin-induced pulmonary fibrosis (27, 28,49). TNF-α receptor knockout mice have been shown to be protected from lung injury after exposure to bleomycin (27,28).

Pulmonary surfactant is essential for normal lung function. Surfactant protein (SP) A, in addition to surfactant-related function (10), plays a role in local host defense and regulation of inflammatory processes (3, 6). SP-A is a collagenous C-type lectin or collectin (24), and its carbohydrate recognition domain (CRD) is involved in binding SP-A to pathogens and promoting phagocytosis of these pathogens by the macrophages (42,43). In the macrophage-like THP-1 cell line, human SP-A stimulates production of TNF-α, IL-1β, IL-8, and IL-6 in a dose- and time-dependent manner (19, 36, 45). Similar effects are seen in other cells of monocytic origin from both rats and humans (16, 19). SP-A-enhanced TNF-α production appears to involve NF-κB activation (14). SP-A also enhances immune cell proliferation (18) and increases expression of some cell surface proteins (17). In addition, SP-A knockout mice show an increased susceptibility to infection (21). A recent in vivo study suggests a role for SP-A in neutrophil recruitment in the lungs of preterm lambs (15). There have also been reports with other systems in which an anti-inflammatory role has been attributed to SP-A (2, 4).

Surfactant lipids (Surfactant TA) can modulate adherence and superoxide production of neutrophils (37). Surfactant lipids inhibit several SP-A-regulated immune cell functions, including stimulation of macrophages (41). Surfactant lipids and SP-A may be counterregulatory, and changes in the relative amounts of surfactant lipids to SP-A may be important in determining the immune status of the lung. Although most SP-A in the normal alveolar space is thought to be lipid-associated, “lipid-free” SP-A could increase if the balance between SP-A and surfactant lipid were altered under certain conditions (31). There is evidence that bleomycin-induced lung injury in animal models is accompanied by qualitative and quantitative changes of surfactant lipids (30,38). Increased SP-A contents in rats has been reported after intratracheal treatment of bleomycin (32, 44).

We hypothesized that lipid-free SP-A, the result of an imbalance of SP-A and surfactant lipids after bleomycin treatment, enhances the effects of bleomycin on proinflammatory cytokine production and may be partly responsible for bleomycin-induced pulmonary fibrosis. In the present study, we examined the effects of SP-A on bleomycin-induced cytokine production and mRNA expression in THP-1 cells.

MATERIALS AND METHODS

Cell culture.

The THP-1 cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640 medium (Sigma, St. Louis, MO) with 0.05 mM 2-mercaptoethanol containing 10% FCS (Summit Biotechnology, Ft. Collins, CO) at 37°C in an atmosphere of 5% CO2. The cells were split periodically and used atpassages 8–15 in the various experiments. After differentiation with 10−8 M vitamin D3 for 72 h, cells were pelleted and washed with cold PBS. The cell pellet was then resuspended in complete RPMI 1640 medium with 10% FCS at a density of 2 × 106 cells/ml in 24-well culture plates and exposed to bleomycin and SP-A. Cell viability was determined by trypan blue exclusion. Under the conditions employed in this study, neither bleomycin nor SP-A appeared to have any effect on the viability of THP-1 cells. Incubations were terminated by pelleting the cells. Supernatants and/or cell pellets were stored at −80°C until assayed.

Bleomycin and native human SP-A.

Bleomycin (Blenoxane; Bristol-Myers Squibb, Princeton, NJ) solutions were prepared immediately before use with endotoxin-free saline (American Pharmaceutical Partners, Los Angeles, CA). Lipopolysaccharide (LPS) was not detected in the stock solution of bleomycin at a bleomycin concentration of 5 U/ml (1 unit = 1 mg) using the method described below.

SP-A was purified from bronchoalveolar lavage of alveolar proteinosis patients with 1-butanol extraction (12). After extraction of whole surfactant with butanol, the pellet was completely dried with a flux of nitrogen gas and then homogenized two times in a freshly prepared buffer (20 mM n-octyl β-d-glucopyranoside, 10 mM HEPES, and 150 mM NaCl, pH 7.4). After pelleting, the insoluble protein was dissolved in 5 mM Tris · HCl, pH 7.5, and dialyzed for 48 h against the same buffer. The dialyzed solution was centrifuged (210,000 g, 4°C, 30 min), and the supernatant containing SP-A was collected and stored at −80°C. The purified protein was examined by two-dimensional gel electrophoresis followed by Western blotting and silver staining and was found to be >98% pure. Protein concentration was determined with the microbicinchoninic acid method (Pierce, Rockford, IL) with RNase A as a standard. SP-A was stored at −80°C. Endotoxin content was determined with the QCL-1000 Limulusamebocyte lysate assay (Biowhittaker, Walkersville, MD). This test indicated that SP-A used in this study contained <0.1 pg LPS/10 μg SP-A.

Stimulation of THP-1 cells with SP-A and bleomycin.

After differentiation with 10−8 M vitamin D3for 72 h, THP-1 cells were pelleted and washed as described above. Cells at a density of 2 × 106 cells/ml were incubated in 24-well culture plates. For dose-response study, cells were stimulated with bleomycin at concentrations ranging from 0 to 100 mU/ml. Time-dependent secretion of cytokines after bleomycin treatment was studied from 0 to 24 h with 5 and 50 mU/ml bleomycin. In experiments in which the combined effects of SP-A and bleomycin were examined, SP-A (10 μg/ml) and bleomycin (5 or 50 mU/ml) were added to cells simultaneously, unless otherwise noted. After treatment, the culture medium was collected at 4 or 6 h for the enzyme-linked immunosorbent assay (ELISA) assay of cytokine production, and cells were harvested for 2 or 4 h for cytokine mRNA analysis.

Cytosine arabinoside (Ara-C, Cytosar-U; Pharmacia & Upjohn, Kalamazoo, MI) was included in some experiments to confirm the specificity of the effect of bleomycin on proinflammatory cytokine production by THP-1 cells. Ara-C is another antineoplastic agent with known cytotoxicity (39). Although it has not been associated with pulmonary fibrosis and cytokine production, it has been shown to cause noncardiogenic pulmonary edema (11). Ara-C, at concentrations ranging from 0.1 to 5 mM in culture medium, was used for evaluating the effect of Ara-C on TNF-α. The conditions employed in the Ara-C experiments were the same as those in bleomycin studies except that only a 4-h time point was used.

Infasurf inhibition of cytokine production.

Infasurf (Forest Pharmaceuticals, St. Louis, MO), an extract of natural surfactant from calf lung, was used as a source of surfactant lipid. Infasurf was supplied by the manufacturer as a suspension containing 35 mg phospholipids/ml sterile saline. Infasurf is predominately phosphatidylcholine and contains ∼2% wt/wt protein that includes SP-B and SP-C, but no SP-A. Infasurf in concentrations ranging from 100 to 800 μg/ml was used in the experiments for ELISA assay of cytokine production, but only a single dose (400 μg/ml) of Infasurf was used in the experiment for mRNA analysis. Infasurf was preincubated separately with SP-A (10 μg/ml), bleomycin (5 mU/ml), and SP-A plus bleomycin for 15 min at 37°C before addition to the THP-1 cells. Cells were incubated for 4 h after the treatment. Culture medium and cell pellets were then collected for ELISA assay and mRNA analysis, respectively.

ELISA assay.

The ELISA assays for TNF-α, IL-8, and IL-1β (OptEIA Human ELISA Sets; Pharmingen, San Diego, CA) were performed according to the instructions recommended by the manufacturer. The ELISA kits were capable of measuring levels of 7.8–500 pg/ml for TNF-α, 6.2–400 pg/ml for IL-8, and 20–1000 pg/ml for IL-1β. A reference curve for each of these cytokines was obtained by plotting the concentration of several dilutions of standard protein vs. the corresponding absorbance.

Analysis of cytokine mRNA.

Total RNA was isolated from THP-1 cells at 2 or 4 h after treatment by using RNeasy Mini Kits (QIAGEN, Valencia, CA) according to the protocol of the RNeasy Mini Handbook. Cytokine mRNA quantification was performed by RNase protection assay (RPA). RiboQuant Ribonuclease RPA Starter Package and a Customized Human Template Set (Pharmingen) were used to analyze TNF-α, IL-1β, and IL-8 mRNA in one assay. The customized template set contains DNA templates that can be used for T7 RNA polymerase-directed synthesis of [α-32P]UTP-labeled antisense RNA probes. These can be hybridized to TNF-α, IL-1β, and IL-8 mRNA. Templates for the L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genes were also included to allow for normalization of sampling or technical error. Aliquots of 2 μg of total RNA were hybridized with radiolabeled probes at 56°C for 16 h. RNase treatment followed, resulting in degradation of single-stranded RNA and free probes. After inactivation and precipitation, protected probes were resolved by a 5% polyacrylamide-urea sequence gel electrophoresis and visualized by autoradiography. Densities of the protected bands were quantified by soft laser densitometry. The mRNA level is expressed as the ratio of the densitometric value of each cytokine mRNA to that of the L32 or GAPDH mRNA.

Statistics.

Values are presented as means ± SE. Data were analyzed using SigmaStat statistical software. For each experiment, statistical treatment included a one-way ANOVA followed by a Student-Newman-Keuls test for pairwise comparison and was judged to be significantly different at P < 0.05.

RESULTS

Dose-response and time course studies of bleomycin effects on stimulation of cytokine production by THP-1 cells.

To study the response of THP-1 cells to bleomycin stimulation, we first performed a dose response and a time course of bleomycin effects on TNF-α, IL-8, and IL-1β levels. The concentrations of bleomycin for the dose-response study ranged from 0 to 100 mU/ml, which spans a relevant pharmacological dose (33). As shown in Fig.1, a bleomycin concentration as low as 0.5 mU/ml increased both TNF-α and IL-8 levels (Fig. 1, Aand B), but a higher concentration of bleomycin (50 mU/ml) was needed to increase the IL-1β level significantly (Fig.1 C). Cytokine production continued to increase as the bleomycin dose was increased to 100 mU/ml. In contrast, the TNF-α level after Ara-C treatment did not differ from that of the control (Fig. 1 D).

Fig. 1.

Dose-response cytokine production after bleomycin treatment by THP-1 cells. Differentiated THP-1 cells were stimulated with the indicated concentration of bleomycin for 4 or 6 h. Tumor necrosis factor (TNF)-α (A) and interleukin (IL)-8 (B) levels in culture medium at 4 h incubation and IL-1β (C) at 6 h were quantified by enzyme-linked immunosorbent assay (ELISA). The effect of cytosine arabinoside (Ara-C) on TNF-α level was examined at 4 h (D). Data are derived from 5 separate experiments except the Ara-C experiments, which are from 2 experiments in triplicate. Results are given as means ± SE. *Values are significantly different (P < 0.05) from points obtained in the absence of bleomycin.

Figure 2 shows the time-dependent secretion (0–24 h) of TNF-α, IL-8, and IL-1β by THP-1 cells in the presence or absence of different bleomycin doses. The basal level of TNF-α at 0 h (starting point) was low. The increase of TNF-α levels was usually detected at 3 h after bleomycin treatment and quickly reached a maximum by 4–6 h, depending on the dose selected (Fig. 2 A). The content of TNF-α subsequently decreased and returned to background levels at 24 h. Initially, IL-8 had a similar response pattern to TNF-α with respect to its increase and peak response time, but after reaching a maximal level at ∼5 h, IL-8 did not show a significant decline until 24 h (Fig.2 B). The level of IL-1β increased much later (Fig.2 C) than that of TNF-α and IL-8 and reached a peak 10 h after bleomycin treatment. Unlike that of TNF-α and IL-8, the level of IL-1β then did not decline but remained elevated over the 24-h test period.

Fig. 2.

Time course of cytokine production after bleomycin (BLM) treatment. Differentiated THP-1 cells were incubated in the presence of 0, 5, or 50 mU/ml bleomycin for the indicated time. TNF-α (A), IL-8 (B), and IL-1β (C) levels in culture medium were quantified by ELISA.

Combined effect of SP-A and bleomycin on cytokine production and mRNA expression by THP-1 cells.

After testing the effects of bleomycin treatment on TNF-α, IL-1β, and IL-8 production by THP-1 cells, we examined the combined effects of SP-A and bleomycin on cytokine production. A dose of 10 μg/ml SP-A was chosen rather than the dose of 50 μg/ml we have used in previous experiments (19, 45), since the low dose may better identify synergistic or additive effects of the two substances. As shown in Fig. 3 A, TNF-α values induced by SP-A (10 μg/ml) alone and bleomycin (5 mU/ml) alone were 86.6 ± 11.5 and 45.9 ± 10.6 pg/ml, respectively, but the combined treatment increased the level to 201.7 ± 34.3 pg/ml. A high concentration of bleomycin (50 mU/ml) alone induced a TNF-α level of 82.1 ± 17.3 pg/ml, whereas the value of the combined effect was 416 ± 61.9 pg/ml. There was a similar response pattern for IL-8 when the combined effects of SP-A and bleomycin were examined (Fig. 3 B). Because IL-1β reached a maximum value at a later time point than TNF-α and IL-8 did, we measured its level 6 h after treatment. The means of IL-1β levels (Fig.3 C) induced by the combined treatment were greater than the sum of the separate means by SP-A or bleomycin alone as we saw with TNF-α and IL-8. SP-A and bleomycin appear to have synergistic effects on TNF-α, IL-1β, and IL-8 production by THP-1 cells.

Fig. 3.

Combined effect of surfactant protein (SP) A and bleomycin on cytokine production by THP-1 cells. Differentiated THP-1 cells were stimulated with SP-A (10 μg/ml) and bleomycin (5 or 50 mU/ml) simultaneously for 4 or 6 h. TNF-α (A) and IL-8 (B) in culture medium at 4 h incubation and IL-1β (C) at 6 h were quantified. Data are derived from 5 separate experiments, and the results are given as means ± SE. *Values are significantly different (P < 0.05) from points with SP-A alone and points with the same dose of bleomycin but without SP-A.

The mRNA levels of TNF-α, IL-1β, and IL-8 were measured by RPA. THP-1 cells were treated with SP-A (10 μg/ml) and/or bleomycin (5 mU/ml) for 2 and 4 h separately. LPS-treated groups (0.1 ng/ml) were included as a positive control. After 2 h of incubation (Fig.4 A), TNF-α and IL-8 mRNA significantly increased compared with the control (P < 0.05). When the cells were treated with SP-A plus bleomycin, the relative intensity of TNF-α mRNA increased from 0.51 ± 0.27 (SP-A alone) to 0.95 ± 0.21 (P < 0.05), but no significant difference was observed between SP-A plus bleomycin and bleomycin alone (0.95 ± 0.21 vs. 0.74 ± 0.20). A similar response pattern was also seen with IL-8 mRNA. Very low levels of IL-1β mRNA were detected in all SP-A- and bleomycin-treated cells, but LPS significantly increased the level of IL-1β mRNA at 2 h. The effects of SP-A and bleomycin on IL-1β mRNA expression at 4 h (Fig. 4 B) were greater than at 2 h. A significant increase of IL-1β mRNA expression was observed when SP-A treatment was combined with bleomycin compared with bleomycin alone or SP-A alone. The IL-8 mRNA level (2.47 ± 0.75) after SP-A plus bleomycin treatment was significantly higher (P < 0.05) than that with SP-A alone (1.26 ± 0.14) or bleomycin alone (1.02 ± 0.16). For TNF-α at 4 h, a significant difference was observed between SP-A plus bleomycin and bleomycin alone, but not between the combined treatment and SP-A alone. These results together indicate an additive effect of SP-A and bleomycin on the TNF-α, IL-1β, and IL-8 mRNA expression.

Fig. 4.

Combined effect of bleomycin and SP-A on cytokine mRNA levels. Differentiated THP-1 cells were stimulated with SP-A (10 μg/ml) and/or bleomycin (5 mU/ml) simultaneously. After a 2-h (A) or a 4-h (B) incubation, the cells were processed for quantification of cytokine mRNA by RNase protection assay (RPA). The mRNA level for TNF-α, IL-1β, and IL-8 in 2 μg of total cell RNA was normalized to mRNA for ribosomal protein L32. Data shown in A and B were derived from 4 and 3 separate experiments, respectively. Results were given as means ± SE. The indicated values are significantly different (P < 0.05) from points treated with SP-A (*) and bleomycin (∧) alone, respectively. LPS (0.1 ng/ml) treatment was included as a positive control.

When SP-A (10 μg/ml) was preincubated with bleomycin (50 mU/ml) 15 min before addition to the cells, the TNF-α level was significantly lower than that without preincubation (Fig.5). However, the synergistic effects on TNF-α production remained. There was a similar response with respect to IL-8 production (data not shown).

Fig. 5.

Effect of preincubation of SP-A and bleomycin on TNF-α production. Differentiated THP-1 cells were stimulated with SP-A (10 μg/ml) and bleomycin (50 mU/ml) and were either preincubated together for 15 min at 37°C before adding them to the cells (Pre-) or were not preincubated. Data were derived from 4 separate experiments. Results were given as means ± SE. *Values are significantly different from points without preincubation.

Inhibitory effect of Infasurf on SP-A and bleomycin-induced cytokine production and mRNA expression.

We examined the ability of surfactant lipids to modulate the cytokine level. As shown in Fig. 6, Infasurf had no effect on the TNF-α level in the absence of SP-A and bleomycin. The SP-A-induced TNF-α level was significantly reduced by Infasurf at 100 μg/ml and was totally inhibited with a higher dose of Infasurf. In contrast, the bleomycin effect was not significantly reduced by Infasurf, even at 800 μg/ml. Infasurf decreased the TNF-α level induced by SP-A plus bleomycin in a dose-dependent pattern. The TNF-α level was significantly decreased from 226.8 ± 35.7 pg/ml in the absence of Infasurf to 109 ± 19.3 and 41.5 ± 0.7 pg/ml at 200 and 800 μg/ml Infasurf, respectively.

Fig. 6.

Effect of surfactant lipids (Infasurf) on SP-A- and bleomycin-induced TNF-α production. Infasurf at indicated concentration was preincubated separately with SP-A (10 μg/ml), bleomycin (5 mU/ml), and SP-A + bleomycin for 15 min at 37°C before addition to THP-1 cells. Cells were incubated for 4 h after treatment. Culture medium was then collected, and TNF-α level was quantified by ELISA. Data shown are from a single experiment in triplicate but are representative of 3 experiments. Results are given as means ± SE. *Values are significantly different (P < 0.05) from points obtained in the absence of Infasurf.

Similar results were obtained when we measured TNF-α and IL-8 mRNA expression by RPA (Fig. 7). TNF-α mRNA expression induced by SP-A was totally inhibited by Infasurf (400 μg/ml), but at the same time the bleomycin-induced TNF-α mRNA level was not changed significantly. In SP-A plus bleomycin treatment, the relative intensity of TNF-α mRNA was decreased from 3.17 ± 0.71 in the absence of Infasurf to 1.14 ± 0.10 in the presence of Infasurf. A similar response pattern was also seen for IL-8 mRNA after Infasurf treatment.

Fig. 7.

Effect of surfactant lipids (Infasurf) on SP-A- and bleomycin-induced TNF-α and IL-8 mRNA expression. Infasurf (Infa; 400 μg/ml) was preincubated separately with SP-A (10 μg/ml), bleomycin (5 mU/ml), and SP-A + bleomycin 15 min at 37°C before addition to THP-1 cells. Cells were incubated for 4 h after treatment. Cells were processed for quantification of cytokine mRNA by RPA. The mRNA levels of TNF-α and IL-8 in 2 μg of total RNA were normalized to the mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. LPS, lipopolysaccharide. Data shown are from 3 experiments. Results are given as means ± SE. *Values are significantly different (P < 0.05) from points obtained in the absence of Infasurf.

DISCUSSION

In the present study, we investigated whether SP-A plays a role in bleomycin-induced inflammation and whether surfactant lipids modulate this process. With the macrophage-like THP-1 cell line, we used RPA and ELISA and observed the following: bleomycin (as has been shown for SP-A) enhances proinflammatory cytokine production by THP-1 cells. The combined bleomycin-SP-A effect on cytokine production is additive by RPA and synergistic by ELISA. No effect on cytokine production is observed by Ara-C, a chemotherapeutic agent that has not been associated with lung inflammation and fibrosis, suggesting that the effect is specific to bleomycin and/or to agents associated with lung inflammation and fibrosis. The surfactant lipids significantly suppress the additive or synergistic effect on cytokine production observed in the presence of both SP-A and bleomycin. These data indicate that surfactant lipids may be useful in the suppression of inflammatory processes induced by SP-A and bleomycin in the lung. This in turn could prevent lung fibrosis, a serious complication of chemotherapeutic agents such as bleomycin.

Bleomycin stimulates THP-1 cells to secrete cytokines in dose- and time-dependent patterns in the present study, as we have observed previously in these cells after treatment with SP-A (19,45). THP-1 cells are of a monocytic origin that, upon vitamin D3 differentiation (as described in materials and methods), acquire a macrophage-like phenotype. Undifferentiated THP-1 cells, on the other hand, respond minimally to SP-A (19) and to bleomycin (data not shown). The response pattern of the TNF-α time course observed in this study is similar but not identical to that of clinical observations about the circulating TNF-α level after bleomycin treatment (35). In THP-1 cells, the TNF-α response is transient, whereas in vivo (35), although it decreases with time, it does not return to basal levels. This may reflect the simple nature of the THP-1 system compared with that in the intact organism. The transient (4–8 h) response of TNF-α is also seen with SP-A and LPS, although other proinflammatory cytokines (IL-1β and IL-8) show a sustained increase (19). Because some differences between the THP-1 cell line and alveolar macrophage were apparent in the kinetics and the level of TNF-α and IL-1β induced by bleomycin treatment (33), it is possible that the THP-1 cell response may not entirely reflect that of alveolar macrophages. However, the data presented in this report demonstrate the usefulness of the THP-1 cell line as a model system for the study of bleomycin-induced cytokine production. Moreover, the THP-1 cells have the advantage of providing a homogeneous cell population for study, whereas primary alveolar macrophages, even from a single subject, vary significantly from one another depending on what they have been exposed to in vivo and the length of time they have been in the alveolus. Ara-C, another chemotherapeutic agent, can also cause pulmonary complications, but this is typically noncardiogenic pulmonary edema (11) rather than inflammation and fibrosis. No effect of Ara-C on cytokine production was observed in THP-1 cells, confirming the specificity of the effect of bleomycin on cytokine production by THP-1 cells and pulmonary toxicity.

It has been previously reported that native human SP-A can stimulate cytokine production in macrophage-like THP-1 cells and that this effect of SP-A can be inhibited by surfactant lipids (17,36). There have also been reports that SP-A can inhibit LPS-induced cytokine production (4). These disparate results have led to a controversy as to whether SP-A is proinflammatory or anti-inflammatory. In several animal models, there are increases in SP-A early in inflammatory processes. These models include neonatal hyperoxia (7), sepsis (23), and preterm ventilated sheep (15). On the other hand, in the SP-A knockout mouse (21) and in a very premature baboon (2) the lack of SP-A appears to cause increased inflammation. In both of these models, there is a significant delay in pathogen clearance, which is likely to prolong the proinflammatory stimulus provided by the pathogen. However, in the presence of SP-A, pathogen clearance is enhanced, and this may result in reduced inflammation because of removal of the proinflammatory stimulus provided by the pathogen.

Although SP-A clearly enhances many aspects of host defense function, these different lines of evidence prevent it from being easily classified as proinflammatory or anti-inflammatory. It is possible that its role changes at different stages of the inflammatory response, as has been proposed recently for NF-κB (20).

In the present study, we showed that SP-A in a low dose (10 μg/ml) significantly increased cytokines at both the protein and mRNA levels. LPS (0.1 ng/ml) was used as a positive control in the present study. A striking difference in IL-1β mRNA expression between LPS- and SP-A-treated cells was observed, especially at 2 h after treatment (Fig. 4 A). The relative intensity of IL-1β mRNA induced by LPS was 18-fold greater than that induced by SP-A (0.92 ± 0.23 vs. 0.05 ± 0.02), whereas the differences between LPS and SP-A in TNF-α or IL-8 mRNA are only around twofold. These data may provide additional evidence that the regulation of proinflammatory cytokine production by SP-A in THP-1 cells occurs by a different pathway than that utilized by LPS (36).

Under normal physiological conditions, most of the SP-A in the alveoli is combined with surfactant lipids in the form of a surfactant lipoprotein complex. Our data suggest that these SP-A-lipid complexes do not affect cytokine production, perhaps because the complexed SP-A is unable to interact directly with immune cells (36). Therefore, it is possible that, if the lipids are reduced in quantity or quality, the stimulatory influence of SP-A could be enhanced. In fact, in bleomycin-induced pulmonary fibrosis, changes in surfactant composition and function have been revealed in animal models (30,38). Studies in rat indicate that there is a significant increase of SP-A but not of surfactant phospholipids in response to bleomycin treatment (32, 44). We showed that, although surfactant lipids by themselves had no effect on cytokine production, Infasurf completely inhibited SP-A proinflammatory function observed in both ELISA and RPA assays of cytokine protein and mRNA, respectively. This result was comparable to that reported previously with Survanta (19). Although both Infasurf and Survanta contain the hydrophobic surfactant proteins SP-B and SP-C, several studies comparing these preparations with either protein-free synthetic surfactant or pure lipids suggest that SP-B and SP-C do not affect cytokine expression (1, 40, 41, 46, 47). Infasurf could significantly inhibit the combined effects of SP-A and bleomycin on cytokines, suggesting involvement of complex mechanisms. This result may be because of the association of lipid-free SP-A with surfactant lipids, since the bleomycin effect on cytokines was not significantly changed by Infasurf, even at the highest dose of 800 μg/ml, further suggesting that these two agents (SP-A and bleomycin) operate through different mechanisms. Infasurf appears to significantly inhibit the LPS and the LPS plus bleomycin effect on TNF-α production (data not shown).

The mechanism of bleomycin-induced cytokine production has not been fully elucidated. It is generally believed to be related to DNA damage (25), apoptosis (29), and activation of NF-κB (50). It has also been demonstrated that bleomycin-induced injury is associated with the generation of reactive oxygen species, particularly superoxide anion (26, 37). We speculate that this mechanism involves the production of reactive oxidants by the bleomycin-treated cells, which in turn activate NF-κB and increase transcription of cytokine genes. Although the mechanism of action of SP-A is not known either, it is likely that it involves interaction with a cell membrane molecule, possibly the C1q receptor (22), activating intracellular events, including the eventual activation of NF-κB (14). When SP-A and bleomycin were added to the cell at the same time, the levels of both TNF-α and IL-8 were higher than the sum of each cytokine induced by SP-A or bleomycin alone. Analysis of mRNA showed that the combined treatment of SP-A and bleomycin exhibited an additive effect on the expression of TNF-α, IL-1β, and IL-8 mRNA. Although bleomycin itself did not induce a significant increase of IL-1β mRNA even at 4 h after treatment, it greatly enhanced the level of IL-1β mRNA after being combined with SP-A. The fact that the combined effect of SP-A plus bleomycin shown in cytokine protein production was much greater than that observed in the mRNA level indicates that various posttranscriptional and posttranslational mechanisms may be involved in bleomycin-induced proinflammatory cytokine production by THP-1 cells in response to SP-A. The details of these mechanisms remain to be determined. The synergistic effect of SP-A and bleomycin on cytokine production in THP-1 cells raises the possibility that SP-A plays a role in bleomycin-induced pulmonary fibrosis.

The underlying mechanism of the combined SP-A and bleomycin effect on cytokine production by THP-1 cells is unclear. The CRD of SP-A may interact with bleomycin, which is a group of glycopeptides. We speculate that, in experiments in which SP-A and bleomycin are added to the cells simultaneously, these agents exert their effect through different moieties, and the different pathways converge and cause increased cytokine gene expression. To distinguish whether the synergistic effect is the result of the binding of SP-A to bleomycin or an independent action of SP-A and bleomycin, we performed preincubation experiments. We observed that the TNF-α level was decreased after 15 min of preincubation of SP-A with bleomycin before addition to cells. The reduced effect seen when SP-A and bleomycin are preincubated may be the result of bleomycin binding to the CRD of SP-A. This may in turn compromise the binding of one or the other of these agents to THP-1 cells and thus interfere with the stimulatory effects.

In summary, we have demonstrated that both SP-A and bleomycin can stimulate production of inflammatory cytokines by THP-1 cells and that there is a synergistic effect when both agents are used. Surfactant lipids significantly suppress the synergistic SP-A-bleomycin effect on cytokine production. We speculate that the significantly elevated cytokine levels resulting from this synergism are responsible for bleomycin-induced pulmonary fibrosis and that surfactant lipids can help ameliorate pulmonary complications observed during chemotherapy.

Acknowledgments

We thank Susan DiAngelo and Todd M. Umstead for expert technical assistance.

Footnotes

  • This work was supported by National Institutes of Health Grants 1R01 ES-09882–01 and R37 HL-34788 and the Julia Cotler Hematology Research Fund.

  • Address for reprint requests and other correspondence: J. Floros, Dept. of Cellular and Molecular Physiology, H166, Penn State College of Medicine, 500 Univ. Dr., Hershey, PA 17033 (E-mail:jfloros{at}psu.edu).

  • 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.

  • First published February 22, 2002;10.1152/ajplung.00434.2001

REFERENCES

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