E L S E V I E R Biochimica et Biophysica Acta 1256 (1995) 305-309

BB Biochi~ic~a et Biophysica A~ta

Effect of Escherichia coli lipopolysaccharide on surfactant secretion in primary cultures of rat type II pneumocytes

Carmen Romero, Enrique Benito, Marfa A. Bosch *

Department q['Biochemistrv and Molecular Biology, Faculty qf Chemistry, Unicersidad Complutense, 28040-Madrid, Spain

Received 22 March 1994; revised 31 May 1994; accepted 13 February 1995

Abstract

The purpose of this study is to evaluate the effect of the Escherichia coli lipopolysaccharide on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, in primary cultures of rat alveolar type II pneumocytes. Lipopolysaccharide stimulated phosphatidylcholine secretion in a time- and dose-dependent manner. At a concentration of 200/zg/ml, lipopolysaccharide stimulated the release of phosphatidylcholine 4-fold over the basal secretory rate, and the concentration producing half the maximal response was 20 /xg/ml. The stimulatory effect of lipopolysaccharide on phosphatidylcholine secretion was additive to that of the protein kinase C activator TPA, which is a potent stimulator of surfactant secretion. Lipopolysaccharide did not activate protein kinase C, which suggests that stimulation of phosphatidylcholine secretion by the endotoxin was through a mechanism independent of protein kinase C activation.

Keywords: Pulmonary surfactant; Endotoxic shock; Lipopolysaccharide; Type II pneumocyte" Phosphatidylcholine secretion

1. Introduct ion

Lipopolysaccharides (LPS), endotoxins from Gram- negative bacteria, are known to be involved in the patho- genesis or induction of endotoxaemia and septic shock. These bacterial compounds exhibit a broad spectrum of biological effects in mammals, the lung being one of the target organs of their actions [1].

The physiopathologic similarity of adult respiratory dis- tress syndrome (ARDS) secondary to sepsis and endotoxin induced pulmonary abnormalities has provided extensive descriptive information confirming the role of bacterial endotoxin as a factor initiating the heterogeneous pul- monary changes in ARDS. Some of these changes are mediated by endotoxins acting directly on secreted surfac- tant or indirectly through pulmonary alveolar type II cells [2].

Pulmonary surfactant, a complex mixture of lipids and proteins, is synthesized by the alveolar type II cells, where it is stored intracellularly in lamellar bodies and ultimately secreted by the process of exocytosis [3,4]. Phosphatidyl- choline (PC) accounts for over 80% of surfactant phospho-

* Corresponding author. Fax: +34 1 3944159.

0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 3 3 - X

lipids [5], being their disaturated species largely responsi- ble for the surface tension-lowering properties of surfac- tant [6]. Secretion of PC by purified cultures of isolated type II cells has been used as an in vitro model to study the regulation of surfactant secretion which can be influ- enced by a variety of physiological and pharmacological agents [7-9]. One agent that has been demonstrated to stimulate surfactant secretion is the turnout promoter 12- O-tetradecanoylphorbol- 13-acetate (TPA) [ 10], which is a direct activator of protein kinase C (PKC) [11].

Protein kinase C covers a family of serine- and threo- nine-specific protein kinases that play an important role in cellular signalling [12,13]. Many reports have described an apparent translocation of PKC from the cytosol to the membrane in response to treatments activating the enzyme [14]. Phorbol esters, such TPA, share some common bio- logical activities with LPS. This fact has led to suggest that some of the biological effects of LPS may be mediated by its interaction with PKC [15].

In the present study, we have examined the effect of Escherichia coli lipopolysaccharide alone or in combina- tion with the direct activator of protein kinase C TPA in the secretory response of type II cells. LPS appears to be an activator of surfactant secretion by an unknown mecha- nism independent of PKC activation.

306 C. Romero et aL / Biochimica et Biophysica Acta 1256 (1995) 305-309

2. Materials and methods

2.1. Animals and materials

Male adult Wistar rats (Charles River, Spain) weighing 200-250 g were used in all the experiments. The experi- ments described were performed in adherence to the CEE (86/609) and Ministerio de Agricultura (Spain, BOE 223/1988) guidelines for care and use of laboratory ani- mals. Elastase and DNase I were obtained from Boehringer Mannheim. Trypsin, 12-O-tetradecanoylphorbol- 13-acetate (TPA), rabbit IgG and chemical reagents were supplied by Sigma Chemical Co. (St Louis, MO, USA). Earle's bal- anced salt solution was supplied by Flow Laboratories (Irvine, Ayrshire, UK); newborn calf-serum and Dulbecco's modified Eagle's medium (DMEM) were obtained from Gibco Europe (Paisley, Renfrewshire, UK). [methyl- 14 C]Choline chloride and [3/-32 P]ATP were purchased from Amersham International (Amersham, Bucks, UK). Percoll was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Lipopolysaccharide form Escherichia coli 0111:B4 was supplied by Difco (MI, USA). Az6o/A24o was determined for purity verification.

2.2. Isolation and culture of type H cells

Type II cells were isolated from rat lungs as described previously [16], with some modifications: the addition of trypsin (75 /xg/ml) and DNase I (100/xg/ml) to improve yield and minimize cell clumping, and a further purifica- tion step by differential adherence to plates coated with IgG as described by Dobbs et al. [17]. The freshly isolated cells were plated at a density of 1.5 • l 0 6 cells per dish on 35-mm plastic dishes and cultured in 1.5 ml DMEM containing 10% fetal bovine serum, streptomycin (100 /xg/ml) and penicillin (100 units/ml) for 18-20 h at 37°C in a humidified atmosphere of 90% air/10% CO 2. At this stage at least a 90% of the attached cells were type II pneumocytes as determined by alkaline phosphatase stain [ 18].

2.3. Phosphatidylcholine secretion

[methyl-~4C]Choline choride (2 /xCi/ml) was included in the medium during the overnight culture of the cells. At the end of this period the medium was removed and the cells rinsed three times with fresh serum and antibiotic-free DMEM to remove [ 14 C]choline and unattached cells. Fresh DMEM was added and the incubation continued for 30 min after which the secretagogues were added. TPA was dissolved in dimethylsulfoxide (DMSO), and lipopoly- saccharide (LPS) was suspended in DMEM. The final concentration of DMSO in the culture medium was 1% and this amount was also added to the media of the corresponding control groups. After the predetermined time has elapsed, the medium was aspirated and the attached

cells lysed with ice-cold water. The medium was cen- trifuged at 200 × g for 10 min to remove any floating cells. Lipids were extracted from both the cell extract and the medium with a mixture of chloroform and methanol by the method of Bligh and Dyer [19] and separated by two-dimensional thin-layer chromatography on silica gel G plates. PC fractions were identified by exposing the plates to iodine vapour and the incorporated radioactivity was measured in a Beckman LS-3801 scintillation counter.

Secretion of phosphatidylcholine is expressed as the amount of [14C]phosphatidylcholine in the medium as a percentage of the total content in cells plus medium. The percentage of [~4C]phosphatidylcholine secreted during the 30 rain equilibration period was subtracted from all sam- ples.

2.4. Protein kinase C assay

Alveolar type II cells in primary culture prepared as described above were incubated with LPS (20 ~g /ml ) or TPA (10 -5 M) for 30 min at 37 ° C. After washing the plates with PBS, the cells were scraped with trypsin/EDTA (0.05%:0.02% (w/v) in PBS), washed twice with DMEM and then were sonicated in 50 mM Tris-HC1 buffer (pH 7.5), containing 2 mM EDTA, 2 mM EGTA, 0.3% w / v /3-mercaptoethanol, 10 mM benzamidine, 20 /zg/ml leu- peptin and 50 /xg/ml phenylmethylsulfonyl fluoride (0.5 ml per eight dishes) for 1 min. The homogenates were centrifuged for 10 rain at 1000 X g to eliminate cell debris and nuclei and then the supernatant was centrifuged for 60 min at 100000 X g to obtain the cytosolic and particulate fractions. The cytosolic fraction was directly assayed for protein kinases. The particulate fraction was resuspended

6

W ,~ 4 Ld

3

2

1 I ! i

0 50 100 150 200

k P S ( ~ g / m l )

Fig. 1. Effect o f LPS on phosphat idylchol ine secretion as a function o f

LPS concentrat ion. Alveolar type II cells prelabelled with [methyl-

t4C]choline for 19 h were incubated for 90 min with different concentra-

tions o f LPS, and [14C]PC was measured as a marker o f surfactant

secretion as described in Section 2. Each point represents the mean

value + S.E. (bars) of triplicate samples f rom four different experiments.

Statistical s ignif icance compared with control cells: * P < 0.05, ' * P <

0.01, * * " P < 0.001.

C. Romero et al. / Biochimica et Biophysica Acta 1256 (1995) 305-309 307

in 0.5 ml of the same buffer containing 0.03% Triton X-100, sonicated, and incubated for 30 min at 4 ° C follow- ing centrifugation for 60 min at 100 000 × g. The super- natant thus obtained was subjected to protein kinase C assay.

Protein Kinase C was assayed by using an Amersham's protein kinase C enzyme assay system. Briefly, this system is based upon the PKC catalysed transfer of the y-phos- phate group from adenosine-5'-triphosphate to a peptide. The phosphorylated peptide is separated on binding paper. After washing the paper, the extent of phosphorylation may be detected in a Beckman Gamma 5500 scintillation counter.

2.5. Other procedures

The rate of lactate dehydrogenase release into the medium was determined to assess cellular integrity. The cells were cultured as in the secretion experiments but in the absence of radioactive material, after which lactate dehydrogenase activity in the cells and media was assayed by measuring the disappearance of NADH at 340 nm [20]. Protein values were determined by the Lowry et al. method [21].

2.6. Statistics and data analysis

Type II cells isolated from four rats were pooled in each experiment and distributed among the various control and treatment groups. In the secretion experiments three dishes were used for each group. These were processed separately and the values averaged to yield a single data point per group per experiment. In the PKC assay eight dishes were used per group and PKC was assayed in duplicate in

• CONTROL ~ LPS

z 4 ~ ~

o_ 3

W m 2

1

I I I i i I

30 60 90 120 150 180 min

Fig. 3. Effect of LPS on phosphatidylcholine secretion as a function of time. After a 30 rain preincubation period, cells prelabelled with [methyl- 14C]choline were incubated with ( v ) or without (O) 20 p,g/ml LPS for the designated time. The percentage of total cellular [14 C]PC secreted into the medium was determined as described in Section 2. Each point represents the mean value ___ S.E. (bars) of triplicate samples from four different experiments. Statistical significance compared with control cells: * P<O.05, ~* P<0.01 , *** P<0.001.

cytosolic and particulate fractions, and the values averaged to yield a single data point per group per experiment. Data from at least four experiments were averaged and the groups compared statistically with Student's t-test for paired samples.

3. Results

Lipopolysaccharide stimulated [~4C]phosphatidylcholine secretion by type II cells in a concentration-dependent

9

8

7 Z

o 6 I - - h i

ac 5 f J h i

u~ 4

3

1

I

10 -10 1 0 - g

I I P I L

10 - e 10 - 7 1 0 - s 1 0 - s 1 0 - 4 1 0 - 3

TPA(M)

Fig. 2. Effect of TPA on phosphatidylcholine secretion as a function of TPA concentration. Alveolar type II cells prelabelled with [methyl- ~4C]choline for 19 h were incubated for 90 min with different concentra- tions of TPA, and [J4C]PC was measured as a marker of surfactant secretion as described in Section 2. Each point represents the mean value + S.E. (bars) of triplicate samples from four different experiments. Differences are significant with respect to control values (P < 0.001).

z 8 * ~ / ~

ILl

2

CONTROL LPS TPA TPA+LPS

Fig. 4. Effect of LPS and TPA on phosphatidylcholine secretion. After a 30 min preincubation period, cells were incubated in the absence (control) or presence of 20 /~g/m] LPS, or with TPA (10 5 M), or with TPA + LPS for 2 In. The percentage of total cellular [14 C]PC secreted into the medium was determined as described in Section 2. Each column represents the mean value _+ S.E. (bars) of triplicate samples from four different experiments. Statistical significance compared with control cells:

P < 0.001.

308 C. Romero et al. / Biochimica et Biophysica Acta 1256 (1995) 305-309

Table 1

Influence of LPS and TPA on the rate of lactate dehydrogenase release by type ll cells

Treatment Incubation time (min) % LDH release h I

Non-treated cells 90/180 2.52 _+ 0.35 LPS (20/zg/ml) 180 2.48 _+ 0.50 LPS (200/~g/ml) 90 2.61 _+ 0.12 TPA 10 4 M 90 2.82 _+ 0.25 LPS (20 g g / m l ) + TPA 10 5 M 120 2.98 + 0.62

Type lI cells were cultured overnight, washed and then incubated in fresh medium with and without the indicated LPS and TPA concentrations for different time periods, after which LDH activity in cells and media was measured. The data are means _+ S.E. of triplicate samples from at least four different experiments. The rate of LDH release in the treated cells was not significantly different from that in non-treated control cells.

manner in the range 2 /~g/ml-200 /zg/ml (Fig. 1) and the concentration of LPS required to produce half-maximal response was 20 /zg/ml. The higher LPS concentration assayed, 200 /~g/ml, enhanced secretion by 4-fold over the basal rate, this value being similar to that obtained with the addition of 10-SM of the protein kinase C activator TPA (Fig. 2), which is a potent stimulator of surfactant secretion [10]. The release of [14C]PC by type II cells in primary culture was linear with time (Fig. 3), and the basal rate of secretion was approximately 1% of total cellular PC per hour. This rate of secretion was similar to that reported by Dobbs et al. [17]. Addition of 20 /zg /ml LPS to the cell culture media stimulated secretion in a time-dependent manner (Fig. 3), the maximal accumulation of secreted [14C]PC at 90 rain being 1.5-fold greater than the constitu- tive secretion (1.44 _+ 0.14%). The release of [HC]PC in- duced by LPS decreased from its maximal value after 2 h, whereas the constitutive release of [J4C]PC remained con- stant during the period of study. Leakage of cytosolic enzymes such as lactate dehydrogenase provides a suitable and quantitative test to assess cellular integrity, under the assumption that a cell with a permeable membrane has

% 100

90

80

70

60

50

40

30

20

10

SOLUBLE ~ PARTrCULATE

CONTROL TPA

Fig. 5. Distribution of protein kinase C activity

LPS

in soluble and particulate fractions of alveolar type II cells. The cells were incubated with or without LPS (20 /zg/ml) or TPA (10 -5 M) for 30 rain. Activities in cytosolic and particulate fractions are expressed as the percentage with respect to total PKC activity. Each column represents the mean value_+ S.E. (bars) of duplicate samples from four different experiments. Statisti- cal significance compared with control cells: " P < 0.001.

suffered severe, irreversible damage. As shown in Table l, LPS and TPA were not cytotoxic, since they did not significantly increase the rate of LDH release over that in control cells.

Phosphatidylcholine secretion in type II cells can be stimulated by direct activators of protein kinase C such TPA, which cause a translocation of protein kinase C from the cytoplasmic fraction to the particulate fraction [l 1]. This phenomenon is assumed to be an initial event in the action of TPA [14], from which it has been inferred that protein kinase C plays a role in stimulating surfactant secretion. While the mechanism for LPS stimulation is unknown, it should be possible that its action should be mediated by a process dependent on PKC activation.

The possible interaction between TPA and LPS in the stimulatory response was tested by adding the two agents simultaneously. In these experiments the concentration of TPA used, 10 5 M, was expected to elicit maximal PC secretion (Fig. 2) without being cytotoxic. As shown in Fig. 4, when LPS and TPA were both included in the culture media PC-secretion was stimulated by 5.8-fold over the basal rate, this effect adding to those of LPS (l.5-fold) and TPA (4-fold). The additive effect on the stimulatory response of LPS and the PKC activator TPA suggests that the two agents are acting through different mechanisms.

To confirm this suggestion, the activation of PKC was assayed in type II cells treated with LPS or TPA. Fig. 5 shows that 86% of the PKC activity was recovered in the cytosolic fraction in non-treated control cells, whereas 50% of the activity was translocated to the particulate fraction in TPA-treated cells. However, LPS did not pro- mote translocation, which suggests that the effects of LPS on PC secretion are not mediated by a process dependent on PKC activation.

4. Discussion

Administration of bacterial endotoxins to several species causes a lung injury similar to that seen in patients with septic shock and adult respiratory distress syndrome that is associated to alveolar surfactant disruption [2]. Because type lI pneumocytes develop crucial functions in lung

C. Romero et al. / Biochimica et Biophysica Acre 1256 (1995) 305-309 309

physiology, they have been the subject of numerous stud- ies concerning the pulmonary surfactant and related pathologies. In the present study we showed that LPS stimulated surfactant secretion from cultured type II cells in a time- and dose-dependent manner without causing any cell damage. The ability of the higher LPS concentration assayed to induce surfactant secretion is similar to that obtained with the addition of the potent stimulator of surfactant secretion TPA. The stimulatory response elicited by TPA is related to the activation of protein kinase C, in such a way that TPA causes translocation of protein kinase C from cytosolic fraction to the particulate fraction [11].

If PKC activation is an initial event in the stimulation of surfactant secretion, it should be possible that LPS action should be mediated by a PKC-dependent process. When type II cells were incubated concomitantly with TPA and LPS, the effect on surfactant secretion was additive, and the treatment of type II cells with 20 /xg/ml LPS that stimulated phosphatidylcholine secretion did not promote any change in the distribution of PKC between cytosolic and particulate fractions. These data suggest that the stimu- lation of surfactant secretion by LPS occurs through a mechanism not mediated by PKC activation, that can amplify the stimulatory action of TPA.

Although the mechanism by which LPS stimulates phosphatidylcholine secretion remains to be elucidated, it could be related to the alterations in plasma membrane permeability and fluidity and phosphatidylcholine biosyn- thesis observed in previous in vitro studies performed with isolated type II cells [16]. These alterations are the result of the LPS-cellular membrane interaction. Studies on surface binding and subcellular distribution after cellular uptake of LPS performed in vitro by immunolabelling methods and electron microscopy [22] have shown that the transport of endotoxin aggregates across the cytoplasm could be related to intracellular structures such as microtubules. If some kind of intracellular association between LPS and micro- tubules occurs and causes a disruption of the microtubular network, the subsequent intracellular effects of this disor- ganization could explain many of the complex cellular events observed in LPS-mediated processes.

In summary, the present study has demonstrated that LPS appears to be a potent activator of surfactant secretion by an unknown mechanism independent of PKC activa- tion. The relevance of this mechanism of activation to the in vivo state remains to be determined. Nevertheless, LPS stimulation of cultured type II cells provides interesting

new data that can help us to understand the primary mechanism of bacterial lipopolysacchafide toxicity in type II cells.

Acknowledgements

This work was supported by research grant PM90-0037 from Direccirn General de Investigaci6n Cientffica y Trcnica (MEC, Spain). C.R. is greatly indebted to Com- plutense University for her research fellowship.

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