The inflammatory cytokines tumor necrosis factor α and interleukin-1β β stimulate phosphatidylcholine secretion in primary cultures of rat type II pneumocytes Enrique Benito and María A. Bosch Department of Biochemistry and Molecular Biology, Faculty of Chemistry, Universidad Complutense, Madrid, Spain Received 29 September 1997; accepted 13 April 1998
Abstract Tumor necrosis factor α and interleukin-1β increase surfactant secretion in type II pneumocytes in a time- and dose-dependent manner. This stimulatory effect was additive to that of lipopolysaccharide, suggesting that cytokines and lipopolysaccharide may exert their actions through different signal transduction pathways. Tumor necrosis factor α and interleukin-1β did not modify the increase on phosphatidylcholine secretion induced by the direct protein kinase C activator tetradecanoylphorbol 13-acetate, whereas this effect was inhibited by the protein kinase C inhibitors bisindolylmaleimide (2 × 10–6M) and 1-(5-isoquinolinylsulphonyl)-2-methyl piperazone (10–4M). In addition, the stimulatory effect of tumor necrosis factor α and interleukin-1β was not suppressed by the intracellular Ca2+ chelator BAPTA (5 × 10–6M) or by KN-62 (3 × 10–5M), a specific inhibitor of Ca2+-calmodulin-dependent protein kinase. These results suggest that tumor necrosis factor α or interleukin-1β stimulate phosphatidylcholine secretion via protein kinase C activation in a Ca2+ -independent manner. (Mol Cell Biochem 189: 169–176, 1998) Key words: pulmonary surfactant, lipopolysaccharide, TNFα, IL-1β, Type II pneumocytes, phosphatidylcholine secretion
Introduction Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) are macrophage/lymphocyte-derived cytokines endowed with a broad spectrum of immunoregulatory, metabolic and proinflammatory activities. These cytokines seem to be of particular importance in the pathophysiology of septic shock as it has been shown that administration of TNFα or/and IL-1β to experimental animals mimics the pathophysiological changes observed during the septic shock course [1, 2, 3, 4]. In the lung, TNFα and IL-1β are secreted by pulmonary macrophages after endotoxin exposure , and, among other cellular types, type II pneumocytes may represent a target for those molecules . Type II pneumocytes synthesize, store and secrete surfactant [7, 8], a complex mixture of lipids and
proteins that reduces the tension at the air-alveolar interface in the lung and provides for alveolar stability. Phosphatidylcholine (PC) accounts for over 80% of surfactant phospholipids , being their disaturated species largely responsible for the surface tension lowering properties of surfactant . PC secretion is a regulated process and in isolated type II cells can be induced by physiological and other agents [11, 12], that act via at least three signal transduction mechanisms involving the activation of different protein kinases . Alterations in type II pneumocyte function, including surfactant biosynthesis and secretion, may have a significant role in the pathogenesis of sepsis-induced lung injury. At this respect it has been demonstrated that lipopolysaccharide (LPS) is an activator of surfactant secretion by an unknown mechanism independent of protein kinase C (PKC) activation .
Address for offprints: M.A. Bosch, Departamento de Bioquímica y Biología Molecular, Facultad de CC. Químicas, Universidad Complutense, 28040-Madrid, Spain
170 TNFα and IL-1β actions are mediated by different, specific high-affinity cell surface receptors that are expressed on virtually all cell types. The signal mechanisms by which these cytokines exert their biological effects have not been fully elucidated. At this respect, some possible mechanisms such as the release of arachidonic acid from phospholipids  or PKC activation via diacyIglycerol generated from PC by phosphatidylcholine-specific phospholipase C (PC-PLC) [16, 17], have been reported. The purpose of the present study was to examine the effect of TNFα and IL-1β in the secretory response of type II cells and the signal transduction pathways involved in this effect. Both cytokines appear to be activators of surfactant secretion by a mechanism dependent on PKC activation. The involvement of PKC in TNFα and IL-1β-stimulation of surfactant secretion was further supported by data from studies on the effects of specific inhibitors for signal transduction pathways, on the induction of PC biosynthesis.
Materials and methods Animals and materials Male adult Wistar rats (Charles River, Spain) weighing 200– 250 g were used. The experiments described here were performed in adherence to the CEE (86/609) and Ministerio de Agricultura (Spain, BOE 223/1988, 265/1990) guidelines for the care and use of laboratory animals. Elastase, DNase I, TNFα and IL-1β were obtained from Boehringer Mannheim (Germany). The specific activity of IL-1β was 5 × 107 U/mg, being 1 unit the amount of IL that is required to support halfmaximal stimulation of DNA synthesis with mouse C3H/HeJ thymocytes in the presence of saturating amounts of human IL-2. Trypsin, 12-O-tetradecanoylphorbol 13-acetate (TPA), rabbit IgG, newborn calf-serum, Dulbecco’s modified Eagle’s medium (DMEM), lipopolysaccharide from E. coli 0111:B4, Earle’s balanced salt solution, EGTA, thapsigargin (TSG), DMSO, propidium iodide and terbutaline were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Indo-1/AM and BAPTA-AM were purchased from Molecular Probes (Leiden, Netherlands). 4-Br-A23187 ionophore and bisindolylmaleimide were supplied by Calbiochem (La Jolla, CA, USA). H-7 and KN-62 inhibitors were obtained from ICN Pharmaceuticals, Inc (Costa Masa, CA, USA). [Methyl14 C]choline chloride was purchased from Amersham International (Amersham, Bucks, UK). Percoll was obtained from Pharmacia Biotech (Uppsala, Sweden). Isolation and culture of type II pneumocytes Type II cells were isolated from rat lungs as described previously , with some modifications: The addition of
trypsin (75 µg/ml) and DNase I (100 µg/ml) to improve yield and minimize cell clumping, and a further purification step by differential adherence to plates coated with IgG as described by Dobbs et al. . The freshly isolated cells were plated at a density of 106 cells per well on a 12-well tissueculture plate (Cultek) and cultured in 1 ml DMEM containing 10% fetal bovine serum, streptomycin (100 µg/ml) and penicillin (100 units/ml) for 20 h at 37°C in 5% CO2 in an air water-saturated atmosphere. At this stage at least a 90% of the attached cells were type II pneumocytes as determined by alkaline phosphatase stain , and their viability was 95% as determined by exclusion of trypan blue.
Phosphatidylcholine secretion [Methyl-14C]Choline choride (2 µCi/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 antibiotic and serum-free DMEM to remove [14C]choline and unattached cells. The cells on the dishes were then equilibrated for 30 min in fresh DMEM, after which the medium was changed and the test agents were added. The incubation was continued for 90 min, except for time course experiments as indicated. All inhibitors were added 10 min before the addition of activators. Some agents, were dissolved in DMSO before addition to DMEM. The final concentration of DMSO in the culture medium was 0.1% and this amount was also added to the media of the corresponding control dishes. At the end of the incubation period, the medium was aspirated and the attached cells lysed with ice-cold water. The spent medium was centrifuged 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  and separated by two-dimensional thin-layer chromatography on silica gel G plates. PC fractions were identified by exposing the plates to iodine vapor and the incorporated radioactivity was measured in a Beckman LS-3801 scintillation counter. Secretion of phosphatidylcholine is expressed as the percentage of [ 14C]phosphatidylcholine in the medium relative to the total amount (cells plus medium).
Intracellular [Ca2+] measurement by flow cytometry Isolated type II pneumocytes (106 cells/ml) were incubated at 37°C for 45 min with 6 µM indo-l/AM in a modified Krebs-Ringer buffer (containing 10 mM HEPES pH 7.4, 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose) or without Ca2+ (the same without CaCl2 and with 1 mM EGTA).
171 After indo-1 loading, cells were treated with 5 ng/ml of TNFα or 50 U/ml of IL-1β during 30 min. Controls without cytokines were also performed. In order to measure the intracellular [Ca2+], the fluorescence of indo-1 loaded cells was excited by a 5W laser turning to 345–365 nm and the emitted fluorescence was measured with a 395/25 band pass filter in a FACStar Plus Becton Dickinson flow cytometer. Cell viability, monitored by addition of propidium iodide (PI; 0.005% in phosphate buffered saline) to stain the DNA of dead cells, was 85–90% in control and cytokine-treated cells. The effect of 4-Br-A23187 ionophore on intracellular calcium content, was assayed after incubation of indo-1 labeled cells (106 cells/ml) with 3 µM ionophore for 2 min. Lactate dehydrogenase assay The rate of lactate dehydrogenase release into the medium was determined to assess cellular integrity. After the secretion experiments, lactate dehydrogenase activity in the cells and medium was assayed by measuring the disappearance of NADH at 340 nm . The lactate dehydrogenase activity released into the medium did not exceed 1% of the total cellular content in all experiments.
Statistics and data analysis Type II cells isolated from three rats were pooled in each experiment and distributed among the various control and treated groups. In the secretion experiments three wells were used for each group. They were processed separately 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.
Results Both TNFα and IL-1β stimulated the secretion of PC from type II cells in a concentration and time-dependent manner. As shown in Fig. 1, TNFα and IL-1β increased PC secretion at concentrations higher than 2 ng/ml for TNFα and 20U/ml for IL-1β, reaching their maximum values with 80 ng/ml of TNFα (161% increase vs. controls) and with 500 U/ml of IL-1β (138% increase vs. controls). The concentrations of cytokines required to produce half-maximal response were 5 ng/ml of TNFα and 50 U/ml IL-1β (Fig. 1). At these concentrations both cytokines enhanced PC secretion at least up to 120 min (Fig. 2). Phosphatidylcholine secretion in type II cells was stimulated up to the same level by LPS, TNFα, IL-1β and TNFα
plus IL-1β (Fig. 3). When either TNFα or IL-1α or both plus LPS were included in the culture media, PC-secretion was stimulated by 2.6-fold over the basal rate, being this effect additive to those of TNFα (1.35-fold) or IL-1β (1.29-fold) and LPS (1.23-fold) (Fig. 3). This additive effect suggest that the LPS and cytokines are acting through different mechanisms. At this respect, we have demonstrated in a previous work , that LPS activated PC secretion by an unknown mechanism independent of protein kinase C (PKC) activation. Thus, these cytokines may act via PKC, or by activation of a cAMP-dependent protein kinase (PKA) or a Ca2+ calmodulindependent protein kinase (Ca-CM-PK) that also stimulate PC secretion in type II cells. To evaluate these possibilities we have assayed the effect of TPA, which is a direct activator of PKC, terbutaline, a beta adrenergic agonist that increases cellular cAMP levels and the calcium ionophore A23187 that promotes calcium influx into the cell which in turn activates a Ca-CM-PK. We have shown (Fig. 4) that TNFα and IL-1β did not produce any change in the PC secretion induced by TPA, but they had an additive effect with terbutaline, and enhanced the stimulation due to the calcium ionophore A23187 although without additive effect. To evaluate whether the TNFα and IL-1β effects are mediated via activation of PKC or Ca-Cm-PK, we investigated their actions in the presence of two inhibitors of PKC, bisindolylinaleimide (BIM) (2 × 10–6M) and 1-(5isoquinolinylsulphonyl)-2-methyl piperazone (H-7) (10–4M), and a specific inhibitor of Ca-Cm-PK, KN-62 (3 ×10–5M). Both BIM and H-7 inhibited the TNFα- and IL-1β-stimulated increase in PC secretion (Table 1), as they do when added to TPA-treated cells. KN-62 did not affect the stimulatory effect of secretagogues except for calcium ionophore A23187 and thapsigargin (TSG), an endoplasmic reticulum Ca-ATPase inhibitor, which lost their stimulatory capacity only in the presence of KN-62. The stimulatory effect of terbutaline is reverted by H-7 that inhibits not only PKC activity but also PKA (Table 1). To examine whether intracellular Ca2+ is involved in the stimulatory effect of TNFα and IL-1β, we compared the afore mentioned effect with the effects of TSG (endoplasmic reticulum Ca-ATPase inhibitor) and BAPTA-AM (intracellular Ca2+ chelator). TSG increased PC secretion and added with cytokines increased their stimulatory effect, although without additive effect (Fig. 5). BAPTA-AM, an acetoxymethylester of a double aromatic analogue of EGTA, can cross plasma membranes and is hydrolysed by cellular esterases to BAPTA, which has high selectivity for Ca2+ over Mg2+ . BAPTA suppressed the stimulatory effect of TSG and A23187, but had no effect on the PC secretion mediated by cytokines (Fig. 5). Finally, in order to evaluate the participation of this second messenger in their stimulatory effect, we have measured the possible changes in the levels of intracellular Ca2+ in type II pneumocytes treated with cytokines. The emitted fluore-
Fig. 1. Effect of TNFα and IL-1β on phosphadylcholine secretion as a function of concentration. Type II pneumocytes were labeled with [methyl14 C]choline chloride (2 µCi/ml) during overnight culture, washed, equilibrated for 30 min in 5% CO 2 in air and exposed to indicated concentrations of TNFα and IL-1β for 90 min. Incubations were terminated by rapid aspiration of the media and the attached cells lysed with ice cold water. Lipids were then extracted and separated by two dimensional TLC. Secretion of phosphatidylcholine is expressed as the percentage of [14C]PC in the medium relative to the total amount (cells plus medium). Each point represents the mean value ± S.D. (bars) of triplicate samples from four different experiments. *p < 0.05 vs. control (¡).
Fig. 2. Time course of TNFα and IL-1β-stimulated phosphadylcholine secretion. Type II pneumocytes were treated as described in Fig. 1 with 5 ng/ml TNFα or 50 U/ml IL- 1β and incubated for the indicated periods of time. PC secretion is expressed as in Fig. 1. Each point represents the mean value ± S.D. (bars) of triplicate samples from four different experiments. *p < 0.05 vs. control.
Fig. 3. Effect of TNFα, IL-1β and LPS on phosphatidylcholine secretion. Type II pneumocytes were incubated for 90 min either without (CON, control) or with LPS (20 µg/ml), TNFα (5 ng/ml), IL-1β (50 U/ml), or with different combinations of these agonists, and PC secretion expressed as indicated in Fig. 1. Each column represents the mean value ± S.D. (bars) of triplicate samples from four different experiments. *p < 0.05, **p < 0.001 vs. control.
scence light of the indo-1 dye shifts to shorter wavelengths on calcium binding . This shift is detectable by fluorescence measurement (FL) at 395 ± 12.5 mn, for Ca2+-bound
indo-1. A light increase of this parameter was observed between 0–10 min (Fig. 6), and this increase occurs irrespective of the presence of Ca2+ in the culture medium.
Fig. 4. Effect of TNFα, IL-1β and other secretagogues on phosphadylcholine secretion. Type II pneumocytes prelabeled with [14C]choline were cultured either without (CON, control) or with TNFα (5 ng/ml), IL-1β (50 U/ml), TPA (10–5M), terbutaline (10–5M), A23187 ionophore (10–6M), or with combinations of TNFα or IL-1β with the other secretagogues for 90 min. The data, expressed as percentage of PC secretion vs. control, are the mean ± S.D. (bars) of triplicate samples from four different experiments. *p < 0.05, **p < 0.005.
174 Table 1. Effect; of the inhibitors BIM H-7 and KN-62 on stimulated phosphatidylcholine secretion by TNFα, IL-1β and other secretagogues
Type II pneumocytes were labeled with [methyl-14C]choline chloride (2 µCi/ml) during overnight culture, washed, equilibrated for 30 min in 5% CO2 in air. Before exposure to secretagogues, cells were preincubated for 10 min with or without BIM (2 × 10–6M), H-7(10–4M) or KN-62 (3 × 10–5M) inhibitors, then cells were treated with TNFα (5 ng/ml), IL-1β (50 U/ml), TPA (10–5M), terbutaline (10–5M), A23187 (10–7M) or TSG (10–7M) for 90 min. Incubations were terminated by rapid aspiration of the media and the attached cells lysed with ice cold water. Lipids were then extracted and separated by two dimensional TLC. Secretion of phosphatidylcholine is expressed as the percentage of [14C]PC in the medium relative to the total amount in cells plus medium. Inhibitors did not alter the basal secretion in nonstimulated cells (3.4 ± 0.3%). The data are the mean ± S.D. of triplicate samples from four different experiments. *p < 0.01 vs. without inhibitor.
Discussion Bacterial LPS is a potent activator of alveolar macrophages resulting in the release of immune mediators such as TNFα and IL-1β . These mediators are major effector molecules in the pathogenesis of septic shock and adult respiratory distress syndrome [1, 25] that is associated to alveolar surfactant disruption. Because type II pneumocytes develop crucial functions in lung physiology, the isolation and primary culture of this type of cells allow to carry out studies concerning the pulmonary surfactant and related pathologies. In the present study we showed that TNFα and IL-1β stimulated PC secretion from cultured type II pneumocytes in a time- and dose-dependent manner. Rates of labeled choline into cellular PC and LDH release were not affected by TNFα and IL-1β, then the stimulatory effect on PC secretion is likely to be a direct effect rather than one secondary to synthesis or cellular injury. The stimulatory effect of TNFα and IL-1β was additive to that observed with LPS, which stimulates surfactant secretion by an unknown mechanism independent of PKC activation , and with terbutaline, which stimulates surfactant
Fig. 5. Effect of BAPTA on the phosphadylcholine secretion stimulated by TNFα, IL-1β and other seeretagogues. Type II pneumocytes prelabeled with [14C]choline were incubated with or without BAPTA-AM (5 × 10–6M) for 15 min, and then incubated with TNFα (5 ng/ml), IL-1β (50 U/ml), A23187 ionophore (10–6 M), TSG (10–7 M), TSG plus TNFα or IL-1β, or TSG plus A23187 for 90 min. The data, expressed as percentage of PC secretion vs. non-stimulated cells (control), are the mean ± S.D. (bars) of triplicate samples from four different experiments.*p < 0.05 vs. TNFα, IL-1β- and A23187-stimulated cells. ∇p < 0.05 vs. BAPTA nontreated cells.
Fig. 6. Effect of TNFα and IL-1β on intracellular [Ca 2+] as a function of time. Isolated type II pneumocytes were incubated at 37°C for 45 min with 6 µM indo-1/AM in a modified Krebs-Ringer buffer with or without Ca2+. After indo-1 loading, cells were incubated with 5 ng/ml TNFα or 50 U/ml IL-1β. The effect of A23187 ionophore on intracellular calcium content, was assayed by incubation of indo-1 labeled cells with 3 µM ionophore for 2 min. Emitted fluorescence light was determined at indicated times by flow cytometry at 395 ± 12.5 nm (FL). FL was evaluated with the LYSIS II Program of Becton Dickinson as a measure of the intracellular [Ca2+] vs. time. Each point represents the mean value ± S.D. (bars) of four experiments. ● With Ca2+ plus TNFα or IL-1β; ¡ without Ca2+ plus TNFα or IL-1β; o with Ca2+ plus A23187; ∇without Ca2+ plus 23187.
secretion by increasing intracellular cAMP, which in turn activates a cAMP-dependent protein kinase (PKA) . Otherwise these cytokines did not alter the increase on PC secretion due to TPA, which is a direct activator of PKC . Taken together, these data suggest the involvement of PKC in TNFα and IL-1β-stimulated PC secretion. This involvement was further supported by using a strategy of specific inhibitors for signal transduction pathways, which demonstrated that the inhibitors of PKC, BIM and H-7, reverted the TNFα and IL-1β-stimulated PC secretion to basal values, having the same effect on TPA stimulated secretion. Elevated levels of diacyIglycerol (DAG) and calcium can both independently activate protein kinase C , then, further studies were performed to evaluate the implication of both second messengers in the stimulatory effect of TNFα and IL-1β on PC secretion. The reported almost additive effect of A23187 ionophore and TSG on the stimulatory response of cytokines, suggested that it was mediated via PKC activation by increasing DAG, which is a well-established activator of PKC , rather than via the activation of Ca2+ channels. This possibility was supported by the observation that BAPTA and KN-62 did not inhibited the stimulatory effects of TNFα and IL-1β on PC secretion, whereas these
inhibitors reverted the stimulatory effects of A23187 and TSG. The poor participation of Ca2+ in the stimulatory effect of cytokines was confirmed by measuring the possible changes in the intracellular Ca2+ levels in type II pneumocytes treated with cytokines. The light and transient increase in Ca2+ levels that was observed in media with or without calcium, pointed to its intracellular origin. Two different mechanisms have been described in the generation of DAG, involving either phospholipase D or C enzymatic cleavage. The latter catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield DAG and inositol 1,4,5-trisphosphate (IP3), that initiates Ca2+ mobilization from intracellular stores . At this respect a possible implication of IP 3 in the increase of cytosolic Ca2+ levels could explain the effect almost additive of A23187 and TSG on the stimulatory response of cytokines. In conclusion, we have shown that TNFα and IL-1β stimulate surfactant secretion via PKC activation in a Ca2+independent manner, being this effect additive to that observed with LPS, which stimulates surfactant secretion by an unknown mechanism independent of PKC activation. The implication of two different signal transduction pathways in the stimulatory effect of cytokines and LPS may explain some
176 of the differences observed between shock induced by LPS and shock induced by cytokines and indicates that some other mediators may play a role in the pathogenesis of endotoxic shock.
Acknowledgements This work was supported by research grants PB94-0244 from Dirección General de, Investigación Cientifíca y Técnica (MEC, Spain) and PR218-94-5677 from Complutense University of Madrid. Enrique BENITO is greatly indebted to Complutense University for his research fellowship.
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