Upsala J Med Sci 102: 199-210, 1997 

Hyaluronan Production in vitro by Fetal Lung Fibroblasts and 
Epithelial Cells Exposed to Surfactants of N-acetylcysteine 

H. Johnsson,’.* P. Heldin,* G. Sedin’ and T. C. Laurent2 
Departments of ‘Pediatrics, and =Medical and Physiological C h e m i s t q  

Uppsalu University, Uppsala, Sweden 

ABSTRACT 

Fetal human lung fibroblasts and feline lung epithelial cells were exposed to either a 

surfactant or N-acetylcysteine in various concentrations for 24-48 hours, after which the 

hyaluronan concentration in the culture medium was determined. Most of the experi- 

ments showed no stimulatory effect of either artificial or natural surfactant on hyaluronan 

synthesis. N-acetylcysteine 5-1 00 mg/mL induced progressive stimulation of hyaluronan 

synthesis by human fetal lung fibroblasts, resulting in a maximum hyaluronan concentra- 

tion six times that released by unexposed cells. A slight increase in hyaluronan synthe- 

sis was also observed after exposure of feline fetal lung epithelial cells to N-acetyl- 

cysteine 50-1 00 pg/mL. 

INTRODUCTION 

The hyaluronan concentration in the lung decreases during the last fifth of normal fetal 

development, with the lowest value immediately before term (1). The production of 

hyaluronan increases during pulmonary disease in infants and adults, and as an early 

response to lung injury in animal models (2, 3, 4). It also increases as a reaction to 

hyperoxia (5,6). The in vifro hyaluronan production is known to be enhanced by several 

inflammatory mediators (7). 

In adult patients with inflammatory diseases of the lungs, increased hyaluronan 

concentrations have been observed both in interstitial lung tissue and in broncho- 

alveolar lavage fluid (8). In the adult respiratory distress syndrome (ARDS), charac- 

terized by non-cardiogenic pulmonary edema and inactivation of surfactant as a result of 

leakage of plasma proteins into the air spaces from areas of epithelial disruption (9), 

accumulation of hyaluronan parallels the development of edema (1 0 , l l ) .  

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In infant respiratory distress syndrome (IRDS), a frequent complication of preterm, 

birth, there is a reduction of surfactant activity, due to a combination of surfactant defi- 

ciency and surfactant inactivation by plasma proteins (9, 12). This results in increased 

permeability of the alveolar wall, hyaline membrane formation, and increased interstitial 

lung water (13). The hyaluronan concentration in lung extracts has been shown to 

increase with increasing severity of IRDS in premature monkeys, and to decrease with 

surfactant treatment (2). 

Surfactants, which are used to treat infants with IRDS by direct instillation into the 

airways, must not be assumed to be inert substances with a singular capacity to alter 

surface tension and gas exchange properties of the lung (14). Arnon e t a /  (1 5) reported 

that surfactant treatment of newborns with IRDS increased the total white cell count and 

the number of macrophages in bronchoalveolar lavage fluid, while Gerdes ef a/ (16) 

found no increase in neutrophil elastase in tracheal aspirates after surfactant treatment. 

The effects of surfactants in cellular cultures have mostly been studied in macro- 

phages and monocytes, where inhibition of effects of inflammatory mediators has 

generally been found (17,18). A decrease in the phagocytic function of these cells has 

also been noted in some studies (19,20). Fewer reports have dealt with the effects on 

fibroblasts, but surfactants have been shown in vifro to both stimulate and inhibit 

fibroblast proliferation (21), to have effects on the surfactant metabolism in exposed 

cells (22), and to inhibit synthesis of DNA and inflammatory mediators in normal human 

lung fibroblasts (23). Surfactants may also affect tracheal epithelial cells by increasing 

the membrane potential, and by slightly (<lo%) increasing the ciliary beat frequency in a 

dose-dependent manner (1 8). 

Several surfactants are in clinical use, and these are either animal lung extracts (e.g. 

CurosurfG9; 20) or artificial products (e.9. Exosurf@; 24). Apart from the surfactant itself, 

surfactant preparations may contain other components that may have effects of their 

own. Some preparations from animal sources have contained platelet-activation factor 

(PAF), which has been associated with neonatal disease and increased leukotriene 

production (25). Tyloxapol and cetyl alcohol (detergent and spreading agent, respec- 

tively), which are used in Exosurf, can function as antioxidants in vifro, and their in vivo 
instillation is associated with reduction of lung edema, of oxidized tissue products, and 

of mortality after hyperoxia (1 4). 

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N-acetylcysteine is frequently used in ventilator-treated infants and applied directly 

into the airways, usually by nebulization. It is primarily used for its mucolytic effect (26), 

but it may also act as a free oxygen radical scavenger (27), prevent the production of 

cytokines by stimulated fibroblasts (28), and prolong the human fibroblast life span (29). 

In the airway, any instilled drug primarily affects the epithelial lining cells, but epithelial 

cells and underlying fibroblasts closely interact. For example it is proposed that the 

prenatal steroid-induced production of surfactant in preterm infants may be due to a 

direct effect of steroids on fibroblasts, with subsequent release of a stimulatory factor 

that influences the lung epithelial cells (30). In addition, fetal fibroblasts differ in some 

respects from adult cells; for instance the inhibition of normal hyaluronan synthesis seen 

after a cell culture has grown to confluence is less pronounced in fetal than in adult cells 

(31). Fetal cells, therefore, may yield in vifro results that more closely reflect the events 

in the perinatal period in vivo. 

The aim of the present study was to determine whether addition of surfactant or N- 

acetylcysteine to cultures of fetal lung cells would affect their hyaluronan production. 

Two brief reports have been presented, one preliminary communication based on the 

first two experiments (32), and a report on the results of surfactant exposure (33). 

METHODS 

Three drugs were tested: a) Exosurf@, a synthetic surfactant composed of dipalmitoyl- 

phosphatidyl-choline (DPPC, colfosceril palmitate) in cetyl alcohol (I-hexadecanol) and 

tyloxapol [4-(1 ,I ,3,3-tetramethylbutyI) phenol polymerized with formaldehyde and oxi- 

rane], from Glaxo Wellcome, Goteborg, Sweden, b) Curosurf @(a porcine lung extract 

containing lipids and surfactant-associated proteins B and C 80 mg/mL in DPPC), from 

Serono, Geneva, Switzerland, and c) Acetylcystein NM PharmaO (N-acetylcysteine 200 

mg/mL in 0.5 mg/mL EDTA, and ascorbic acid and sodium hydroxide q.s. ad pH 7) from 

NM Pharma, Stockholm, Sweden. All preparations are in clinical use. 

16-980148, 



Two types of cells, obtained from the European Collection of Cell Cultures, Salisbury, 

UK, were used. Human fetal fibroblasts (WI -38, from approximately the 12th gestational 

week, passage (P) 14-1 7) were cultured in Dulbecco’s modified Eagle medium (DMEM), 

and feline fetal lung epithelial cells (AKD, P 24) were cultured in Ham’s F12 medium 
with 1 % non-essential amino acids. To both culture media 4 mM L-gluthamine, penicillin 

120 IU/mL, and streptomycin 100 pg/mL were added. Cells were grown in incubators in 

5% CO, at 37OC in a humidified atmosphere. 

Fibroblasts (30-40 000) and epithelial cells (200 000) were first grown in 12-well 

dishes in 0.5 mL culture medium containing 10% fetal bovine serum (FBS) for 24 hours. 

Following one wash with medium containing O.lor 0.25% FBS (starvation medium), the 

cells were cultured in 0.5 mL “starvation medium” for 48 hours, in order to obtain basal 

conditions. Then 0.5 ml of fresh starvation medium, containing one of the three tested 

drugs at indicated concentrations, was added to duplicate or quadruplicate wells. The 

tested concentrations were: Exosurf 5 pg - 40 mg/mL, Curosurf 5 pg - 20 mg/mL, and 
N-acetylcysteine 5 pg - 120 mg/mL. Plain starvation medium served as control and me- 

dium containing 10% FBS as reference. The cultures were then incubated for another 

24 or 48 hours. The hyaluronan concentrations in the culture media were determined 

with a radiometric assay kit (HA 50, Pharmacia, Uppsala, Sweden) (34). Mean hyaluro- 

nan values 5 SEM were calculated for each drug concentration. The values were then 

divided by the number of cells and the drug exposure time. Since the absolute hyaluro- 

nan values differed substantially between the different experiments, the results were 

finally expressed in percent of the control value (starvation medium only). The coeffi- 

cients of variation (the standard deviation divided by the mean) (35) within groups were 

calculated. 

RESULTS 

Cells were tested in six instances between December 1993 and April 1995, with some 

variation in drug concentration, duration of exposure, and % FBS added to the starva- 

tion media (see method). In one experiment fetal feline epithelial cells were used, and in 

the other five experiments only fetal human fibroblasts. 

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The following letters are used to denote the different experiments: 

A. Fibroblasts, P14, drug exposure 48 hours, 30 000 cells/well. 

B. Fibroblasts, P16, drug exposure 24 hours, 25 000 cells/well. 

C. Fibroblasts, P16-18, drug exposure 24 hours, 30 000 cells/well. 

D. Fibroblasts, P I  7, drug exposure 24 hours, 30 000 cells/well. 

E. Epithelial cells, P24, drug exposure 48 hours, 200 000 cells/well, also indicated by*.) 

F. Fibroblasts, P17, drug exposure 48 hours, 30 000 cellslwell. 

For each experiment, the mean hyaluronan concentration/cell/24 hour drug exposure 

was calculated and expressed as percent of the control (starvation medium alone). As 
an example, the result of the first experiment with exposure to Exosurf is shown in Table 

1. The rest of the results are summarized in Figures 1-3. 

Table 1. Hyaluronan (HA) concentration in cell culture medium. Results from experiment 

A with human fetal lung fibroblasts, passage 14, exposed to Exosurf or fetal bovine 

serum (FBS) only for 48 hours, 30 000 cells/well. 

Exosurf FBS n HAlwelI HAlcel ll24h HAlcel V24h 
mglml mean f SEM mean mean 

nglml pg % of control 

03.38 0.1% 4 433.9 k 17.4 7.2 67.1 
06.75 0.1% 4 382.6 f 36.2 6.4 59.1 
13.50 0.1% 4 320.3 k 22.5 5.3 49.5 
27.00 0.1% 4 295.8 f 14.2 4.9 45.7 

0 0.1% 4 647.0 f 21.8 10.8 100.0 
0 10% 4 1444.9 f 45.3 24.1 223.3 

In general, the results varied considerably between different experiments, with a mean 

coefficient of variation of 0.17 (Exosurf 0.18, Curosurf 0.14, N-acetylcysteine 0.19). 

203 



200 
180 

/x 

20 

0 I ~ Tpppp 
0 10 20 30 40 

Exosurf mglml 

Figure 1 A. Hyaluronan (HA) in culture medium in relation to Exosurf exposure. Sum- 

mary of 4 experiments (A-D) with human fetal fibroblasts. 

Exosurf pglml 

Figure 1 B. Hyaluronan (HA) in culture medium in relation to Exosurf exposure. Sum- 
mary of 2 experiments, E* with feline fetal epithelial cells, and F with human fetal fibro- 

blasts. 

After exposure to Exosurf, no increase in the hyaluronan concentration in the culture 

medium, compared to the control (starvation medium alone), was found in three experi- 

ments with fibroblasts (A,C,F) and one with epithelial cells (E). In two experiments with 

fibroblasts (B,D) a progressive increase to 188% and 141%, respectively, was noted 

after exposure to Exosurf 2-40 mg/mL (Fig. I). 

204 



180 

- 160 
$ 140 
- 

s 120 
5 

100 

80 

2 60 
Q) 40 

c 

L - 
$ 20 

+A 
-0-B 

-A-C 

+D 1 

1 _ _  0 1  ~~~ I -  ~ I 

0 5 10 15 20 

Curosurf rnglml 

Figure 2 A. Hyaluronan (HA) in culture medium in relation to Curosurf exposure. Sum- 

mary of 4 experiments (A-D) with human fetal fibroblasts. 

Curosurf pg/ml 

Figure 2 B. Hyaluronan (HA) in culture medium in relation to Curosurf exposure. Sum- 

mary of 2 experiments, E* with feline fetal epithelial cells, and F with human fetal fibro- 

blasts. 

Following Curosurf exposure, three experiments with fibroblasts (A,C,F) and one with 

epithelial cells (E) showed no increase in the hyaluronan concentration compared to the 

control (starvation medium alone). In two experiments with fibroblasts (B,D) maximal 

increases to 172 and 122%, respectively, were observed with Curosurf at 1 and 5 

mg/mL. Higher concentrations yielded hyaluronan values below the control level (Fig. 2). 

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600 

2 500 
0 400 

5 300 

- 
c 
C 
0 

0 
u- 

r 
d 

0 
2 200 - 
3 100 

0 7 I- 1 I ~- -- 
0 20 40 60 80 100 120 

N -acetylcystei ne mglml 

+A 
-0-B 
+C 

+D 
I+F 

Figure 3 A. Hyaluronan (HA) in culture medium in relation to N-acetylcysteine exposure. 

Summary of 5 experiments (A-D, F) with human fetal fibroblasts. 

= 200 e 
150 

c1 
S 

c 
0 

E 100 
z 
d 
N 
: - 50 

0 50 100 150 200 

N-acetylcysteine pg/ml 

Figure 3 B. Hyaluronan (HA) in culture medium in relation to N-acetylcysteine exposure. 

Summary of 2 experiments, E* with feline fetal epithelial cells, and F with human fetal 
fibroblasts. 

After exposure to N-acetylcysteine, one experiment with fibroblasts (A) showed no in- 

crease in the hyaluronan concentration compared to the control (starvation medium 

alone), while in four experiments with fibroblasts (B,C,D,F) a mainly progressive in- 

crease to 185-589 YO was noted with N-acetylcysteine 5-100 mg/mL. In the experiment 
with epithelial cells (E*), there was an increase to 171-185% after exposure to N-acetyl- 

cysteine 50-100 pg/mL, but only to 118% after 200 pg/mL (Fig. 3). 

206 



DISCUSSION 

Our findings do not indicate that hyaluronan synthesis by fetal lung epithelial cells or 

fetal lung fibroblasts is stimulated by either artificial or natural surfactant in the concen- 

tration ranges 5 pg/mL - 40 mg/mL, and 5 pg/mL - 20 mg/mL, respectively. This is in 

accordance with the finding by Juul e t a /  (2) that in vivo surfactant treatment of IRDS re- 
duces the hyaluronan concentration in the lung, and with the report by Thomassen et al 

(23) that surfactants inhibit in vitro synthesis of inflammatory mediators in normal human 
lung fibroblasts. Oxidants are involved in the signal transduction pathways for cytokine 

secretion, and the inhibitory effect of surfactant supports a proposed antioxidant effect 

(14). 
Progressive stimulation of hyaluronan synthesis by fetal human lung fibroblasts was 

induced by N-acetylcysteine in the doserange 5-100 mg/mL, and fetal feline lung epithe- 

lial cells responded with increased hyaluronan synthesis after addition of N-acetyl- 

cysteine in the doserange 50-1 00 pg/mL. This could have some clinical implications, 

since N-acetylcysteine is commonly used in concentrations of 20 - 200 mg/mL and ad- 
ministered directly as a fluid into the airway or by nebulizer. Thus, the treatment may 

result in in vivo concentrations comparable to those used in our in vitro experiments. 
In a preliminary communication (32) we reported a moderate increase in the hyaluro- 

nan concentration after in vifro stimulation of fibroblasts with Exosurf or Curosurf . The 
present results, based on several additional experiments, show a more varied picture, 

with no stimulatory effect of surfactant preparations in most of the experiments. The 

results of addition of Curosurf in concentrations above 20 mg/mL have now been omit- 

ted, as the volume of the drug did not allow for sufficient culture medium. Our prelimi- 

nary finding of increased hyaluronan concentrations after exposure to N-acetylcysteine 

has been confirmed in the present study. 

We conclude that surfactants used for treatment of IRDS have only minor effects on 

the hyaluronan production by fetal pulmonary epithelial cells and fibroblasts in vitro, and 
that exposure to N-acetylcysteine results in increased synthesis of hyaluronan by fetal 

pulmonary fibroblasts, but only in a moderate increase in fetal epithelial cells. 

ACKNOWLEDGEMENTS 

This work was supported by the Swedish Medical Research Council (grants 19X- 
4998, K97-03X, and 03X-4) and the Gillbergska Foundation, Uppsala, Sweden. 

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Oxford, UK, 1995. 

Correspondence to: 
Hans Johnsson, MD 
Department of Pediatrics, Uppsala University Children's Hospital 
S-751 85 Uppsala, Sweden 

209