Combined SPA/bleomycin effect on cytokines by THP1 cells; Impact of surfactant lipids on this effect

Combined SP-A/bleomycin effect on cytokines by THP-1

cells; Impact of surfactant lipids on this effect

Weixiong Huang1, Guirong Wang1, David S. Phelps2, Hamid Al-Mondhiry3,

and Joanna Floros 1,2

Departments of 1Cellular and Molecular Physiology, 2Pediatrics, and 3Medicine,

Penn State College of Medicine, Hershey, PA 17033, USA

Running Title: SP-A/bleomycin and Cytokines

Corresponding author

Joanna Floros, Ph.D.

Departments of Cellular and Molecular Physiology, H166

Penn State College of Medicine

500 University Drive

Hershey, PA 17033, USA

Telephone: (717) 531-6972

Fax: (717) 531-7667

E-mail: [email protected]

Copyright 2002 by the American Physiological Society.

AJP-Lung Articles in PresS. Published on February 22, 2002 as DOI 10.1152/ajplung.00434.2001

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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 TNF-α, 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 ribonuclease protection assay and synergistic by ELISA. 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.

Keywords: chemotherapeutic agent, ELISA, synergistic effect, Ribonuclease Protection Assay

(RPA)

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Introduction

Bleomycin is a group of glycopeptides isolated from Streptomyces verticillus. Although it 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

macrophage, in the fibrotic process. Bleomycin induces inflammatory cells from human and

animal lung to secrete multifunctional cytokines, such as TNF-α, IL-1β, IL-8, and 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 NF-kB, which may result from the increase of reactive oxygen species by

bleomycin (26). NF-kB 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).

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Pulmonary surfactant is essential for normal lung function. Surfactant protein A (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 a time-dependent manner (19; 36; 45). Similar effects are seen in other cells of

monocytic origin from both rats and humans (17; 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 (16). 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 into 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 (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 of 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 was 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

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lipids (30; 38). Increased SP-A content in rat 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 following 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% fetal calf serum (FCS; Summit Biotechnology, Ft. Collins, CO)

at 37oC in an atmosphere of 5% CO2. The cells were split periodically and used at passages 8-15

in the various experiments. After differentiation with 10-8 M Vitamin D3 for 72h, 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 pellet were stored at –80oC until assayed.

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Bleomycin and native human SP-A

Bleomycin (Blenoxane; Bristol-Myers Squibb Co, Princeton, NJ) solutions were prepared

immediately before use with endotoxin-free saline (American Pharmaceutical Partners, Inc. Los

Angeles, CA). Lipopolysaccharide (LPS) was not detected in the stock solution of bleomycin at

a bleomycin concentration of 5 U/ml (1U=1mg) 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 twice in a freshly prepared

buffer (20 mM n-Octyl β-D-Glucopyranoside, 10mM HEPES, 150 mM NaCl, pH 7.4). After

pelleting, the insoluble protein was dissolved in 5 mM Tris-HCl, pH 7.5 and dialyzed for 48h

against the same buffer. The dialyzed solution was centrifuged (210,000 × g, 4oC, 30 min) and

the supernatant containing SP-A was collected and stored at –80oC. The purified protein was

examined by two-dimensional gel electrophoresis followed by western blotting and silver

staining, and was found to be greater than 98% pure. Protein concentration was determined with

the micro bicinchoninic acid method (Pierce, Rockford, IL) with RNase A as a standard. SP-A

was stored at -80oC. Endotoxin content was determined with the QCL-1000 Limulus amebocyte

lysate assay (Biowhittaker; Walkersville, MD). This test indicated that SP-A used in this study

contained <0.1 pg LPS/10µg SP-A.

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

After differentiation with 10-8 M vitamin D3 for 72h, 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

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plates. For dose-response study, cells were stimulated with bleomycin at concentrations ranging

from 0-100 mU/ml. Time-dependent secretion of cytokines following bleomycin treatment was

studied from 0-24h with 5 and 50 mU/ml of bleomycin. In experiments where 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 4h or 6h for the ELISA assay of cytokine production and cells were harvested at

2h or 4h 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 non-cardiogenic 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 4h time point was done.

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 35mg phospholipids/ml of 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

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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 + bleomycin for 15min at 37oC before addition to the THP-1

cells. Cells were incubated for 4h 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

versus the corresponding absorbance.

Analysis of cytokine mRNA

Total RNA was isolated from THP-1 cells at 2h or 4h 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 ribonuclease protection assay (RPA).

RiboQuantTM Ribonuclease RPA Starter Package and a Customized Human Template Set

(Pharmingen, San Diego, CA) 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, anti-sense RNA probes. These can be

hybridized to TNF-α, IL-1β, and IL-8 mRNA. Templates for the L32 and GAPDH

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housekeeping genes were also included to allow for normalization of sampling or technical error.

Aliquots of 2µg of total RNA were hybridized with radiolabeled probes at 56oC for 16h. 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 ± SEM. Data were analyzed using SigmaStat statistical

software. For each experiment, statistical treatment included a one way analysis of variance

(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 0-100 mU/ml, which spans

a relevant pharmacological dose (33). As shown in Figure 1, a bleomycin concentration as low

as 0.5 mU/ml increased both TNF-α and IL-8 levels (panels A and B), but a higher concentration

of bleomycin (50 mU/ml) was needed to increase the IL-1β level significantly (panel C).

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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 control (panel D).

Figure 2 illustrates the time-dependent secretion (0-24h) 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 0h

(starting point) was low. The increase of TNF-α levels was usually detected at 3h following

bleomycin treatment and quickly reached a maximum by 4 to 6h depending on the dose selected

(Fig. 2A). The content of TNF-α subsequently decreased and returned to background levels at

24h. Initially, IL-8 had a similar response pattern to TNF-α, with respect to its increase and peak

response time, but after reaching maximal level at around 5h, IL-8 didn’t show a significant

decline until 24h (Fig. 2B). The level of IL-1β increased much later (Fig. 2C) than that of TNF-

α and IL-8, and reached a peak at 10h 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 24h test period.

The 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 of SP-A was chosen rather than the dose of 50 µg/ml we have used in

previous experiments (19; 45), as the low dose may better identify synergistic or additive effects

of the two substances. As shown in Figure 3A, TNF-α values induced by SP-A (10 µg/ml) alone

and bleomycin (5 mU/ml) alone were 86.6 ± 11.5 pg/ml and 45.9 ± 10.6 pg./ml, respectively.

But the combined treatment increased the level to 201.7 ± 34.3 pg/ml. High concentration of

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bleomycin (50 mU/ml) alone induced a TNF-α level of 82.1 ± 17.3 pg/ml, while 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. 3B). Because IL-1β reached a

maximum value at a later time point than TNF-α and IL-8 did, we measured its level at 6h after

treatment. The means of IL-1β levels (Fig.3C) 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.

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 2h and 4h separately. LPS-treated

groups (0.1 ng/ml) were included as a positive control. After 2h of incubation (Fig.4A), TNF-α

and IL-8 mRNA significantly increased as compared to the control (p<0.05). When the cells

were treated with SP-A + 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 + 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 2h.

The effect of SP-A and bleomycin on IL-1β mRNA expression at 4h (Fig. 4B) were greater than

that at 2h. A significant increase of IL-1β mRNA expression was observed when SP-A

treatment was combined with bleomycin compared to bleomycin alone or SP-A alone. The IL-8

mRNA level (2.47 ± 0.75) following SP-A + bleomycin treatment was significantly higher

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(p<0.05) than that with SP-A alone (1.26 ± 0.14) or bleomycin alone (1.02 ± 0.16). For TNF-α at

4h, a significant difference was observed between SP-A + 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.

When SP-A (10 µg/ml) was preincubated with bleomycin (50mU/ml) 15min 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).

The inhibitory effect of Infasurf on SP-A and bleomycin-induced cytokine production and

mRNA expression.

We examined the ability of surfactant lipids to modulate cytokine level. As shown in Fig. 6,

Infasurf had no effect on TNF-α level in the absence of SP-A and bleomycin. 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 800µg/ml. Infasurf decreased the TNF-α level induced by SP-A + 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 pg/ml and 41.5 ± 0.7 pg/ml at 200 µg/ml and 800 µg/ml

of Infasurf, respectively.

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

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µg/ml), but at the same time bleomycin-induced TNF-α mRNA level was not significantly

changed. In SP-A + 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.

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 ribonuclease protection assay 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

ribonuclease protection assay 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

methods) acquire macrophage-like phenotype. Undifferentiated THP-1 cells, on the other hand,

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respond minimally to SP-A (19) and to bleomycin (data not shown). The response pattern of

TNF-α time course observed in this study is similar but not identical to that of clinical

observations about 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 to 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, 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 level of TNF-α and IL-1β induced by bleomycin treatment (33), it is possible that

the THP-1 cells 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

non-cardiogenic 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

(16; 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

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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 due to removal of the proinflammatory stimulus

provided by the pathogen.

While SP-A clearly enhances many aspects of host-defense function, these different lines

of evidence prevent it from being easily classified as pro-inflammatory or anti-inflammatory. It

is possible that its role changes at different stages of the inflammatory response, as has been

proposed recently for NF-kB (20).

In the present study, we showed that SP-A in 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 2h after treatment (Fig. 4A). 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), while the differences between LPS and SP-A in TNF-α or IL-8 mRNA are only

around 2-fold. 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).

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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 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 to either

protein-free synthetic surfactant or to 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 due to the association of “lipid-free” SP-A with surfactant lipids since the

bleomycin effect on cytokine 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 + bleomycin

effect on TNF-α production (not shown).

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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-kB

(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-kB 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-kB (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 4h 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 + bleomycin shown in cytokine protein production was

much greater than that observed in mRNA level indicates that various post-transcriptional and

post-translational 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-cells is unclear. The carbohydrate recognition domain (CRD) of SP-A may

18

interact with bleomycin which is a group of glycopeptides. We speculate that in experiments

where SP-A and bleomycin are added to the cells simultaneously, these agents exert their effect

through different moieties and that the different pathways converge and cause increased cytokine

gene expression. To distinguish whether the synergistic effect is due to the binding of SP-A to

bleomycin or to independent action of SP-A and bleomycin, we performed preincubation

experiments. We observed that the TNF-α level was decreased following 15 min preincubation

of SP-A with bleomycin before addition to cells. The reduced effect seen when SP-A and

bleomycin are pre-incubated 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 complication observed during chemotherapy.

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Acknowledgments

The authors thank Susan DiAngelo and Todd M. Umstead for their expert technical

assistance. This work was supported by NIH 1R01 ES09882-01, R37 HL34788 and the Julia

Cotler Hematology Research Fund

20

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Legends:

Figure 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 4h

or 6h. TNF-α (Panel A) and IL-8 (Panel B) levels in culture medium at 4h incubation and IL-1β

(Panel C) at 6h were quantified by ELISA. The effect of Ara-C on TNF-α level was examined at

4h (panel D). Data are derived from 5 separate experiments except the Ara-C experiments which

are from two experiments in triplicate. Results are given as means ± SEM. The indicated values

(*) are significantly different (p<0.05) from points obtained in the absence of bleomycin.

Figure 2. Time course of cytokine production after bleomycin treatment. Differentiated THP-1

cells were incubated in the presence of 0, 5, 50 mU/ml bleomycin for the indicated time. TNF-α

(Panel A), IL-8 (Panel B) and IL-1β (Panel C) levels in culture medium were quantified by

ELISA.

Figure 3. The combined effect of SP-A and bleomycin on cytokine production by THP-1 cells.

Differentiated THP-1 cells were stimulated with SP-A (10µg/ml) and bleomycin (BLM 5, 50

mU/ml) simultaneously for 4h or 6h. TNF-α (Panel A) and IL-8 (Panel B) in culture medium at

4h incubation and IL-1β (Panel C) at 6h were quantified. Data are derived from 5 separate

experiments, and the results are given as means ± SEM. The indicated 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.

27

Figure 4. The 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 (BLM 5

mU/ml) simultaneously. After a 2 h (Panel A) or a 4 h (Panel 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 2µg of total cell RNA was normalized to mRNA for

ribosomal protein L32. Data shown in Panel A and B were derived from 4 and 3 separate

experiments, respectively. Results were given as means ± SEM. The indicated values are

significantly different (p<0.05) from points treated with SP-A (*) and bleomycin (^) alone,

respectively. LPS (0.1ng/ml) treatment was included as a positive control.

Figure 5. The effect of preincubation of SP-A and bleomycin (BLM) on TNF-α production.

Differentiated THP-1 cells were stimulated with SP-A (10 µg/ml) and bleomycin (50 mU/ml)

that were either preincubated together for 15 min at 37oC before adding them to the cells, or were

not preincubated. Data were derived from 4 separate experiments. Results were given as means

± SEM. The indicated values (*) are significantly different from points without preincubation.

Figure 6. The effect of surfactant lipids (Infasurf) on SP-A and bleomycin induced TNF-α

production. Infasurf at indicated concentration was preincubated separately with SP-A

(10µg/ml), bleomycin (5mU/ml), and SP-A + bleomycin for 15min at 37oC before addition to

THP-1 cells. Incubation was continued for 4 h. Cells were incubated for 4h 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 three experiments. Results are

28

given as means ± SEM. The indicated values (*) are significantly different (p<0.05) from points

obtained in the absence of Infasurf.

Figure 7. The effect of surfactant lipids (Infasurf) on SP-A and bleomycin induced TNF-α and

IL-8 mRNA expression. Infasurf (Infa; 400µg/ml) was preincubated separately with SP-A

(10µg/ml), bleomycin (5mU/ml), and SP-A + bleomycin 15min at 37oC before addition to THP-

1 cells. Cells were incubated for 4h after treatment. Cells were processed for quantification of

cytokine mRNA by RPA. The mRNA levels of TNF-α and IL-8 in 2µg of total RNA was

normalized to the mRNA of the housekeeping gene GAPDH. Data shown are from 3

experiments. Results are given as means ± SEM. The indicated values (*) are significantly

different (p<0.05) from points obtained in the absence of Infasurf. BLM: bleomycin.